Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged-Notch signaling in fin and limb development.

Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/ß-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged-Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged-Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.

A novel 3D-printed computer-assisted piezocision guide for surgically facilitated orthodontics.

Surgical interventions on the alveolar ridges aimed at facilitating orthodontic tooth movement have been extensively reported. However, unexpected events or complications still occur in daily practice. The purpose of this report was to present a novel 3-dimensional (3D) computer-assisted piezocision guide (CAPG) designed to be translucent for increased visibility, rigid for enhanced support during guidance, and porous for profuse irrigation during procedure. Such a design can function to minimize the risk of surgical complications. In this case, we present a novel 3D-printed CAPG to facilitate a minimally invasive periodontal accelerated osteogenic orthodontics (PAOO) procedure with a guide that provides accuracy, adequate visibility, and greater access for the coolant to reach the surgery site. By navigating the cone-beam computed tomography data, we precisely know the cortical bone thickness, root direction, and interrelations between anatomic structures in an individual situation, which allows us to design our cutting slot for the required length and depth according to the operator's knowledge. Finally, 3D printing was applied, transferring our surgical plan to fabricate the CAPG. Moreover, the well designed pores on the CAPG allow effective irrigation during the piezocision procedure. This minimally invasive procedure was uneventful, and no devitalized tooth or alveolar bone was found.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Baseline Blood Levels of Mucin-1 Are Associated with Crucial On-Treatment Adverse Outcomes in Patients with Idiopathic Pulmonary Fibrosis Receiving Antifibrotic Pirfenidone.

Mucin-1 is a multi-functional glycoprotein expressed by type II alveolocytes and may be detectable in the circulation following pulmonary fibrosis. The prognostic utility of baseline pre-treatment blood levels of mucin-1 in patients with idiopathic pulmonary fibrosis (IPF) receiving antifibrotics has not yet been fully established. We retrospectively studied a cohort of patients (from two hospitals) with IPF who were receiving pirfenidone for >12 weeks. Baseline blood mucin-1 levels were measured via sandwich enzyme-linked immunosorbent assays. We investigated the performance of mucin-1 levels in longitudinally predicting the risks of acute exacerbation of IPF (AE-IPF) and severe adverse outcomes (SAO), including lung transplantation and death. Seventy patients were included; 20 developed AE-IPF; and 31 had SAO during the follow-up period. Patients with baseline mucin-1 levels ≥2.5 ng/mL had enhanced risks of AE-IPF (adjusted hazard ratio [aHR], 14.07; 95% confidence interval [CI], 4.26-46.49) and SAO within 2 years (aHR, 7.87; 95% CI, 2.86-21.70) and anytime during the follow-up (aHR, 4.68; 95% CI, 2.11-10.39). The risks increased across subgroups with increasing mucin-1 levels. Patients in the "mucin-1 ≥ 2.5" group also exhibited an accelerated decline in DLCO. This study supports baseline blood mucin-1 levels as a biomarker for IPF that predicts adverse outcomes during pirfenidone treatment.

Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation.

Backgrounds: Cisplatin is a commonly used chemotherapeutic agent in cancer treatment. However, its high nephrotoxicity limits its therapeutic application and efficacy. Cisplatin induces nephrotoxicity mainly through oxidative stress and inflammation. Reactive oxygen species (ROS) in the kidneys mainly arise from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 2 (NOX2), which is highly upregulated during ischemia-reperfusion injury and diabetes mellitus. However, its role in cisplatin-induced acute kidney injury (AKI) remains unknown. Methods: A 8-10-week-old NOX2 gene-knockout and wild-type mice were injected with 25 mg/kg cisplatin intraperitoneally for experiments. Results: We investigated the role of NOX2 in cisplatin-induced AKI and found that NOX2-mediated ROS production is a key inflammatory mediator of proximal tubular cell injury in cisplatin-induced AKI. NOX2 gene-knockout alleviated cisplatin-induced renal function decline, tubular injury score, kidney injury molecule-1(Kim-1) expression, and interleukin (IL)-6 and IL-1α levels with a reduction of ROS production. Moreover, in cisplatin-induced AKI, intercellular adhesion molecule 1 (ICAM-1) and the chemoattractant CXC ligand 1 (CXCL1) were highly expressed in association with neutrophil infiltration, which were all attenuated by deletion of NOX2. Conclusion: These data indicate that NOX2 aggravates cisplatin nephrotoxicity by promoting ROS-mediated tissue injury and neutrophil infiltration. Thus, appropriate targeting of NOX2/ROS pathway may minimize the risk of cisplatin-induced kidney injury in patients receiving cancer therapy.

First records of powdery mildew fungi (Erysiphales) on medicinal plants in Taiwan.

BACKGROUND: Production of medicinal plants in Taiwan is not only hampered by international market competition, but also lack of knowledge of their pathogens, such as powdery mildew fungi (Erysiphales, Ascomycota). Records of these fungi in Taiwan originate from few researchers for the last one hundred years and are still incomplete. Since powdery mildews in tropical/subtropical environments rarely develop the sexual stages with morphologically diagnostic characteristics, internal transcribed spacer sequences (ITS) of the ribosomal RNA genes obtained from the asexual stages have become important modern tools for species identification. RESULTS: Powdery mildews on medicinal plants from educational and ornamental plantations in Taiwan were identified based on the anamorph morphology and ITS sequences. Four powdery mildews on medicinal plants are new records for Taiwan, Arthrocladiella mougeotii on Lycium chinense, Erysiphe glycines on Pueraria lobata, Erysiphe lespedezae on Bauhinia sp., Desmodium caudatum, and Uraria crinita, and E. lonicerae on Lonicera japonica. Eryngium foetidum is a new host for Erysiphe heraclei hitherto known on other host plants in Taiwan. Eryngium foetidum and Uraria crinita are new host plants for powdery mildews worldwide. Only specific field collection of the pathogens yielded the new records, not checking plant specimens in a phanerogam herbarium. The pathogens did not cause death of the host plants, but appeared to enhance stress by infection of mature leaves. CONCLUSIONS: Taxonomic study of powdery mildews in Taiwan results into new host records of economically important medicinal plants in Taiwan with potential consequences for plant production and quarantine and also shows that host records are quite incomplete worldwide. Although ITS sequences were useful for species identification, the lack of data for several species on the same host genus on the one hand and the low variation between closely related species on the other indicate the need for further study.

Ursolic Acid Induces Apoptotic Cell Death Through AIF and Endo G Release Through a Mitochondria-dependent Pathway in NCI-H292 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Ursolic acid (UA), a triterpene compound present in natural plants, has been shown to induce cytotoxic effects on many human cancer cells through induction of cell-cycle arrest and apoptosis. This study investigated the effects of UA on human lung cancer NCI-H292 cells in vitro. MATERIALS AND METHODS: Flow cytometric assay was used to measure the percentage of cell viability, apoptotic cell death by double staining of annexin V and propidium iodide (PI), production of reactive oxygen species (ROS) and Ca2+, and mitochondriaI membrane potential (Ψm). UA-induced chromatin condensation and DNA fragmentation were examined by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis, respectively. Western blotting was used to examine the changes of apoptosis-associated protein expression in NCI-H292 cells. RESULTS: UA reduced cell viability and induced apoptotic cell death. UA increased Ca2+ production, reduced Ψm, but did not affect ROS production in NCI-H292 cells. UA increased apoptosis-inducing factor (AIF) and endonuclease G in NCI-H292 cells. CONCLUSION: Based on these observations, we suggest UA induces apoptotic cell death via AIF and Endo G release through a mitochondria-dependent pathway in NCI-H292 cells.

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-ß-Hydroxysteroid dehydrogenase /Δ5-4 isomerase (3ß-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3ß-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3ß-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.