Characterization of the Placenta-Specific 8.1 Gene Function during Zebrafish Embryogenesis

The PLAC8 gene encodes for a small, cysteine-rich protein conserved in vertebrates that is a member of a large family of PLAC8-motif containing proteins with diverse functions in animals, plants, and algae. Recently, high levels of PLAC8 expression have been detected in aggressive colorectal cancer and invasive breast cancer, and potentially contributing to the cancer pathogenesis. However, the molecular and cellular functions of PLAC8 in vertebrate development, homeostasis, and disease remain unclear. To determine the function of PLAC8 under disease and normal conditions, in this thesis work, I identified plac8.1 as a PLAC8 homolog in zebrafish, a vertebrate model system amenable to various embryologic and genetic approaches. Zebrafish plac8.1 was maternally and ubiquitously expressed until four days post fertilization when its transcript became enriched in the gut. During the process of gastrulation, Plac8.1 protein distribution gradually shifted from the cytosol to the cell membrane. At larval stages, Plac8.1 accumulated at the apical region of epithelial cells of the gut and the kidney. This dynamic gene expression and protein localization patterns suggest that Plac8.1 may have functions during zebrafish embryogenesis and organogenesis. In the first part of this thesis, I aimed to address questions concerning the effects of high levels of PLAC8 on cell behavior by overexpressing Plac8.1 during early embryogenesis. Embryos overexpressing Plac8.1 manifested morphological defects starting at early gastrulation. Epiboly as well as convergence and extension gastrulation movements were delayed in plac8.1-overexpressing embryos, a spectrum of phenotypes resembling impaired E-cadherin function in zebrafish. Indeed, E- cadherin levels were significantly reduced in plac8.1-overexpressing embryos by a cell-autonomous and post-transcriptional mechanism. Similarly, PLAC8-overexpressing colon cancer cells exhibited reduced cell surface E-cadherin and many features of epithelial-mesenchymal transition: EMT). Furthermore, knockdown of PLAC8 in colon cancer cells resulted in increased level of E-cadherin. In the second part of this thesis, I aimed to determine the requirement for plac8.1 function during development by employing two loss-of-function approaches. Upon injection of either of two non-overlapping antisense morpholino oligonucleotides: MOs) that effectively reduced Plac8.1 protein levels, embryos displayed an array of defects that phenocopied the class of mutants with defective cilia. Consistently, the motile cilia morphology and motility were impaired in plac8.1 morphants. Moreover, in immunoprecipitation experiments, Plac8.1 bound Cops4, a component involved in ubiquitination regulation. Also Plac8.1 and Cops4 cooperated to regulate motile cilia morphology and motility. As a second loss-of-function approach, I generated loss-of-function allele plac8.1stl33 using Transcription Activator-Like Effector Nucleases: TALENs), a method that utilizes sequence-specific nucleases. Similar to plac8.1 morphants, plac8.1stl33 embryos showed motile cilia morphology and beating function defects. We hypothesize that zebrafish Plac8.1 functions at the ciliary base, possibly by modulating intraflagellar transport through ubiquitination modifications. Our in vivo studies of zebrafish plac8.1 have uncovered pleiotropic functions of plac8.1 during development. In addition, the overexpression experiments shed light on mechanisms underlying human disease. Based on the above results, I propose that Plac8.1 overexpression interferes with regulation of protein stability, whereas Plac8.1 is required for motile cilia morphogenesis and function. Common denominators of these seemingly unrelated phenotypes include the potential connection with the process of ubiquitination, and the enriched apical localization of Plac8.1. Our work can also inform studies of other members of the PLAC8 family of proteins

Identification and characterization of a novel Δ6-fatty acid desaturase gene from Rhizopus arrhizus

AbstractA cDNA sequence putatively encoding a Δ6-fatty acid desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino acids of 52 kDa. The deduced amino acid sequence showed high similarity to those of fungal Δ6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the coding product of the sequence exhibited Δ6-fatty acid desaturase activity by the accumulation of γ-linolenic acid

Mulberry biomass-derived nanomedicines mitigate colitis through improved inflamed mucosa accumulation and intestinal microenvironment modulation

The therapeutic outcomes of conventional oral medications against ulcerative colitis (UC) are restricted by inefficient drug delivery to the colitis mucosa and weak capacity to modulate the inflammatory microenvironment. Herein, a fluorinated pluronic (FP127) was synthesized and employed to functionalize the surface of mulberry leaf-derived nanoparticles (MLNs) loading with resveratrol nanocrystals (RNs). The obtained FP127@RN-MLNs possessed exosome-like morphologies, desirable particle sizes (around 171.4 nm), and negatively charged surfaces (â 14.8 mV). The introduction of FP127 to RN-MLNs greatly improved their stability in the colon and promoted their mucus infiltration and mucosal penetration capacities due to the unique fluorine effect. These MLNs could efficiently be internalized by colon epithelial cells and macrophages, reconstruct disrupted epithelial barriers, alleviate oxidative stress, provoke macrophage polarization to M2 phenotype, and down-regulate inflammatory responses. Importantly, in vivo studies based on chronic and acute UC mouse models demonstrated that oral administration of chitosan/alginate hydrogel-embedding FP127@RN-MLNs achieved substantially improved therapeutic efficacies compared with nonfluorinated MLNs and a first-line UC drug (dexamethasone), as evidenced by decreased colonic and systemic inflammation, integrated colonic tight junctions, and intestinal microbiota balance. This study brings new insights into the facile construction of a natural, versatile nanoplatform for oral treatment of UC without adverse effects.We are grateful to Dr. J. Sun from University of Oxford for his corrections and improvement of this manuscript. Funding: This study was supported by the National Natural Science Foundation of China (82072060 and 22008201), the Fundamental Research Funds for the Central Universities (SWU-XDPY22006), the Venture & Innovation Support Program for Chongqing Overseas Returnees (2205012980212766), the Natural Science Foundation Project of Chongqing (cstc2020jcyj-msxmX0292), and the Natural Science Foundation Project of Chongqing for Distinguished Young Scholar. Author contributions: W.Y., M.W., X.S., and B.X. designed experiments, supervised the project, and wrote the manuscript draft. W.Y., Y.M., H.X., Z.Z., J.W., C.X., W.S., and E.Z. performed the experiments. R.L.R., S.C.K., M.W., X.S., and B.X. edited and revised the manuscript. All authors have approved the final version of the manuscript. Competing interests: The authors declare that there is no con flict of interest regarding the publication of this article

Investigation of systemic immune-inflammation index, neutrophil/high-density lipoprotein ratio, lymphocyte/high-density lipoprotein ratio, and monocyte/high-density lipoprotein ratio as indicators of inflammation in patients with schizophrenia and bipolar disorder

BackgroundThe systemic immune-inflammation index (SII), system inflammation response index (SIRI), neutrophil/high-density lipoprotein (HDL) ratio (NHR), lymphocyte/HDL ratio (LHR), monocyte/HDL ratio (MHR), and platelet/HDL ratio (PHR) have been recently investigated as new markers for inflammation. The purpose of this research is to use large-scale clinical data to discuss and compare the predictive ability of the SII, SIRI, NHR, LHR, MHR, and PHR in patients with schizophrenia (SCZ) and bipolar disorder (BD), to investigate potential biomarkers.Materials and methodsIn this retrospective, naturalistic, cross-sectional study, we collected the hematological parameter data of 13,329 patients with SCZ, 4,061 patients with BD manic episodes (BD-M), and 1,944 patients with BD depressive episodes (BD-D), and 5,810 healthy subjects served as the healthy control (HC) group. The differences in the SII, SIRI, NHR, LHR, MHR, and PHR were analyzed, and a receiver operating characteristic (ROC) curve was used to analyze the diagnostic potential of these parameters.ResultsCompared with the HC group, the values of the SII, SIRI, NHR, LHR, MHR, and PHR and the levels of neutrophils, monocytes, and triglycerides (TG) were higher in SCZ and BD groups, and levels of platelets, cholesterol (CHO), HDL, low-density lipoprotein (LDL), and apoprotein B (Apo B) were lower in SCZ and BD groups. Compared to the BD group, the values of the SIRI, lymphocytes, monocytes, and HDL were lower and the values of the SII, NHR, PHR, and platelet were higher in the SCZ group. In contrast to the BD-D group, the values of the SII; SIRI; NHR; and MHR; and levels of neutrophils, monocytes, and platelets were higher in the BD-M group, and the levels of CHO, TG, LDL, and Apo B were lower in the BD-M group. The MHR and NHR were predictors for differentiating the SCZ group from the HC group; the SIRI, NHR, and MHR were predictors for differentiating the BD-M group from the HC group; and the MHR was a predictor for differentiating the BD-D group from the HC group. The combination model of the indicators improved diagnostic effectiveness.ConclusionOur study highlights the role of systemic inflammation in the pathophysiology of SCZ, BD-M, and BD-D, the association between inflammation and lipid metabolism, and these inflammation and lipid metabolism indicators showed different variation patterns in SCZ, BD-D, and BD-M

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo

Synthesis and White-Light Emission of ZnO/HfO2: Eu Nanocables

ZnO/HfO2:Eu nanocables were prepared by radio frequency sputtering with electrospun ZnO nanofibers as cores. The well-crystallized ZnO/HfO2:Eu nanocables showed a uniform intact core–shell structure, which consisted of a hexagonal ZnO core and a monoclinic HfO2 shell. The photoluminescence properties of the samples were characterized. A white-light band emission consisted of blue, green, and red emissions was observed in the nanocables. The blue and green emissions can be attributed to the zinc vacancy and oxygen vacancy defects in ZnO/HfO2:Eu nanocables, and the yellow–red emissions are derived from the inner 4f-shell transitions of corresponding Eu3+ ions in HfO2:Eu shells. Enhanced white-light emission was observed in the nanocables. The enhancement of the emission is ascribed to the structural changes after coaxial synthesis

Naked1 Antagonizes Wnt Signaling by Preventing Nuclear Accumulation of β-Catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin