Biosynthesis of galactomannans found in filamentous fungi belonging to Pezizomycotina.

The galactomannans (GMs) that are produced by filamentous fungi belonging to Pezizomycotina, many of which are pathogenic for animals and plants, are polysaccharides consisting of α-(1→2)-/α-(1→6)-mannosyl and ß-(1→5)-/ß-(1→6)-galactofuranosyl residues. GMs are located at the outermost layer of the cell wall. When a pathogenic fungus infects a host, its cell surface must be in contact with the host. The GMs on the cell surface may be involved in the infection mechanism of a pathogenic fungus or the defense mechanism of a host. There are two types of GMs in filamentous fungi, fungal-type galactomannans and O-mannose type galactomannans. Recent biochemical and genetic advances have facilitated a better understanding of the biosynthesis of both types. This review summarizes our current information on their biosynthesis.

Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells.

APOBEC1 is the catalytic subunit of the complex that edits ApolipoproteinB (ApoB) mRNA, which specifically deaminates cytidine 6666 to uracil in the human transcript. The editing leads to the generation of a stop codon, resulting in the synthesis of a truncated form of ApoB. We have developed a method to quantitatively assay ApoB RNA editing in live cells by using a double fluorescent mCherry-EGFP chimera containing a ∼ 300 bp fragment encompassing the region of ApoB subject to RNA editing. Coexpression of APOBEC1 together with this chimera causes specific RNA editing of the ApoB fragment. The insertion of a stop codon between the mCherry and EGFP thus induces the loss of EGFP fluorescence. Using this method we analyze the dynamics of APOBEC1-dependent RNA editing under various conditions. Namely we show the interplay of APOBEC1 with known interactors (ACF, hnRNP-C1, GRY-RBP) in cells that are RNA editing-proficient (HuH-7) or -deficient (HEK-293T), and the effects of restricted cellular localization of APOBEC1 on the efficiency of the editing. Furthermore, our approach is effective in assaying the induction of RNA editing in Caco-2, a cellular model physiologically capable of ApoB RNA editing.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

Liver lobe and strain difference in gene expression after hydrodynamics-based gene delivery in mice.

Hydrodynamics-based gene delivery (HGD) is a widely recognized technique for delivering exogenous DNA with high efficiency to murine hepatocytes. In this study, we investigated stimulation of exogenous DNA uptake and expression using a commercially available reagent for HGD. We also examined which mouse strain and mouse liver lobe would achieve the best gene delivery performance. Mice were injected with a solution containing reporter plasmid DNA or DNA and a gene delivery reagent. One day after the HGD procedure, liver samples were isolated and subjected to biochemical and histochemical analyses. The reporter plasmid DNA showed the strongest signal when the DNA was dissolved in TransIT-EE Hydrodynamic Delivery Solution (Takara Bio Inc., Shiga, Japan). Evaluation of transgene expression in each hepatic lobe in ICR, C57BL/6N, Balb/cA, and B6C3F1 mice showed that ICR mice exhibited the best gene transfer and that the right median lobe had the highest level of transgene expression. These findings suggest the importance of choice in mouse strains and liver lobes when performing gene-based manipulations of the liver.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Variation of DNA methylation on the IRX1/2 genes is responsible for the neural differentiation propensity in human induced pluripotent stem cells.

Introduction: Human induced pluripotent stem cells (hiPSCs) are useful tools for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, known as differentiation propensity, resulting in reduced reproducibility and increased time and funding requirements for research. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs focusing on DNA methylation, which is the main modulator of cellular properties. Methods: We obtained 32 hiPSC lines and their comprehensive DNA methylation data using the Infinium MethylationEPIC BeadChip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of neural stem cells on day 7 of induction. Using the DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attempted to identify neural differentiation-associated differentially methylated sites. Results: Epigenome-wide unsupervised clustering cannot distinguish hiPSCs with varying differentiation efficiencies. In contrast, HSIC Lasso identified 62 CpG sites that could explain the neural differentiation efficiency of hiPSCs. Features selected by HSIC Lasso were particularly enriched within 3 Mbp of chromosome 5, harboring IRX1, IRX2, and C5orf38 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency and were negatively correlated with gene expression of the IRX1/2 genes, particularly in female hiPSCs. In addition, forced expression of the IRX1/2 impaired the neural differentiation ability of hiPSCs in both sexes. Conclusion: We for the first time showed that the DNA methylation state of the IRX1/2 genes of hiPSCs is a predictive biomarker of their potential for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study may be useful for the selection of suitable undifferentiated hiPSCs prior to differentiation induction.

Targeting the Autophagy-Lysosome Pathway in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure.

The key biological "drivers" that are responsible for reverse left ventricle (LV) remodeling are not well understood. To gain an understanding of the role of the autophagy-lysosome pathway in reverse LV remodeling, we used a pathophysiologically relevant murine model of reversible heart failure, wherein pressure overload by transaortic constriction superimposed on acute coronary artery (myocardial infarction) ligation leads to a heart failure phenotype that is reversible by hemodynamic unloading. Here we show transaortic constriction + myocardial infarction leads to decreased flux through the autophagy-lysosome pathway with the accumulation of damaged proteins and organelles in cardiac myocytes, whereas hemodynamic unloading is associated with restoration of autophagic flux to normal levels with incomplete removal of damaged proteins and organelles in myocytes and reverse LV remodeling, suggesting that restoration of flux is insufficient to completely restore myocardial proteostasis. Enhancing autophagic flux with adeno-associated virus 9-transcription factor EB resulted in more favorable reverse LV remodeling in mice that had undergone hemodynamic unloading, whereas overexpressing transcription factor EB in mice that have not undergone hemodynamic unloading leads to increased mortality, suggesting that the therapeutic outcomes of enhancing autophagic flux will depend on the conditions in which flux is being studied.

A single allele of the hsa-miR-302/367 cluster maintains human pluripotent stem cells.

Introduction: In a diploid organism, two alleles from a single genetic locus are expressed to generate a normal phenotype. Heterozygous deleterious mutation causes a reduction of functional proteins to a half dose and insufficient amounts of functional proteins can occur to generate an in-normal phenotype, namely haploinsufficiency. Heterozygous deleterious mutation of microRNAs (miRs), non-coding RNAs that regulate the expression level of target transcripts, is still not well understood. The hsa-miR-302/367 cluster is the most abundant and specifically up-regulated miR cluster in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and plays an important role in the maintenance of pluripotency. Methods: We targeted the hsa-miR-302/367 region via a Cas9 nuclease complex with guide RNA and replaced that region with green fluorescent protein (GFP). Using a homologous donor, consisting of left and right arms and GFP, we confirmed deletion of the hsa-miR-302/367 cluster by homologous recombination without cellular destruction by microscopy. We sub-cloned GFP-positive colonies and checked the genotype of each sub-clone by genomic PCR. We then analyzed the pluripotency of heterozygous knockout cells with a hsa-miR-302/367 cluster by assessing cell proliferation ratio, morphology, and undifferentiated marker gene expression. We also used an embryoid body formation assay and transplanted wild-type and heterozygous knockout cells into immune-deficient mice. Furthermore, to analyze the lineage-specific differentiation potential of heterozygous knockout cells, we differentiated both wild-type and heterozygous knockout cells into neural stem cells. Results: Here, we show that the half dose of mature miRs from the hsa-miR-302/367 cluster loci was sufficient for the continued self-renewal of hiPSCs. All GFP-positive clones were revealed to be heterozygous knockout cells, suggesting hsa-miR-302/367 cluster homozygous knockout cells were not maintained. The cell proliferation ratio, morphology, and expression of undifferentiated marker genes were comparable between wild-type and heterozygous knockout of undifferentiated human iPSCs. In addition, we found that heterozygous knockout human iPSCs have the capacity to differentiate into three germ layers, including neural stem cells. Conclusions: Taken together, a single allele of the hsa-miR-302/367 cluster expresses a sufficient amount of miRs to maintain the pluripotent properties of human stem cells.

Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Modifying splice site usage with ModCon: Maintaining the genetic code while changing the underlying mRNP code.

Codon degeneracy of amino acid sequences permits an additional "mRNP code" layer underlying the genetic code that is related to RNA processing. In pre-mRNA splicing, splice site usage is determined by both intrinsic strength and sequence context providing RNA binding sites for splicing regulatory proteins. In this study, we systematically examined modification of splicing regulatory properties in the neighborhood of a GT site, i.e. potential splice site, without altering the encoded amino acids. We quantified the splicing regulatory properties of the neighborhood around a potential splice site by its Splice Site HEXplorer Weight (SSHW) based on the HEXplorer score algorithm. To systematically modify GT site neighborhoods, either minimizing or maximizing their SSHW, we designed the novel stochastic optimization algorithm ModCon that applies a genetic algorithm with stochastic crossover, insertion and random mutation elements supplemented by a heuristic sliding window approach. To assess the achievable range in SSHW in human splice donors without altering the encoded amino acids, we applied ModCon to a set of 1000 randomly selected Ensembl annotated human splice donor sites, achieving substantial and accurate changes in SSHW. Using ModCon optimization, we successfully switched splice donor usage in a splice site competition reporter containing coding sequences from FANCA, FANCB or BRCA2, while retaining their amino acid coding information. The ModCon algorithm and its R package implementation can assist in reporter design by either introducing novel splice sites, silencing accidental, undesired splice sites, and by generally modifying the entire mRNP code while maintaining the genetic code.

High-Fat Diet Enhances Neutrophil Adhesion in LDLR-Null Mice Via Hypercitrullination of Histone H3.

Neutrophil adhesion on the atheroprone femoral artery of high-fat diet-fed low-density lipoprotein receptor-null mice was enhanced more than in wild-type mice. The inhibition of histone H3 citrullination of neutrophils reversed the enhancement of neutrophil adhesion, suggesting that hypercitrullination contributes to enhanced neutrophil adhesion. Furthermore, pemafibrate reduced the citrullination of histone H3 in these mice. Therefore, the hypercitrullination of histone H3 in neutrophils contributes to atherosclerotic vascular inflammation.

HOP protein expression in the hippocampal dentate gyrus is acutely downregulated in a status epilepticus mouse model.

Status epilepticus (SE) is a neurological emergency, and delayed management can lead to higher morbidity and mortality. It is thought that prolonged seizures stimulate stem cells in the hippocampus and that epileptogenesis may arise from aberrant connections formed by newly born cells, while others have suggested that the acute neuroinflammation and gliosis often seen in epileptic hippocampi contribute to hyperexcitability and epilepsy development. Previous studies have identified the expression of homeodomain-only protein (HOP) in the hippocampal dentate gyrus (HDG) and the heart. HOP was found to be a regulator of cell proliferation and differentiation during heart development, while it maintains the 'heart conduction system' in adulthood. However, little is known about HOP function in the adult HDG, particularly in the SE setting. Here, a HOP immunohistochemical profile in an SE mouse model was established. A total of 24 adult mice were analyzed 3-10 days following the SE episode, the 'acute phase'. Our findings demonstrate a significant downregulation of HOP and BLBP protein expression in the SE group following SE episodes, while HOP/Ki67 coexpression did not remarkably differ. Furthermore, coexpression of HOP/S100ß and HOP/Prox1 was not observed, although we noticed insignificant HOP/DCX coexpression level. The findings of this study show no compelling evidence of proliferation, and newly added neurons were not identified during the acute phase following SE, although HOP protein expression was significantly decreased in the HDG. Similar to its counterpart in the adult heart, this suggests that HOP seems to play a key role in regulating signal conduction in adult hippocampus. Moreover, acute changes in HOP expression following SE could be part of an inflammatory response that could subsequently influence epileptogenicity.

Umbilical artery tissue contains p75 neurotrophin receptor-positive pericyte-like cells that possess neurosphere formation capacity and neurogenic differentiation potential.

INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is known as an efficient marker for the prospective isolation of mesenchymal stem cells (MSCs) and neural crest-derived stem cells (NCSCs). To date, there is quite limited information concerning p75NTR-expressing cells in umbilical cord (UC), although UC is known as a rich source of MSCs. We show for the first time the localization, phenotype, and functional properties of p75NTR+ cells in UC. METHODS: Human UC tissue sections were subjected to immunohistochemistry for MSC markers including p75NTR. Enzymatically isolated umbilical artery (UA) cells containing p75NTR+ cells were assessed for immunophenotype, clonogenic capacity, and differentiation potential. To identify the presence of neural crest-derived cells in the UA, P0-Cre/Floxed-EGFP reporter mouse embryos were used, and immunohistochemical analysis of UC tissue was performed. RESULTS: Immunohistochemical analysis revealed that p75NTR+ cells were specifically localized to the subendothelial area of the UA and umbilical vein. The p75NTR+ cells co-expressed PDGFRß, CD90, CD146, and NG2, phenotypic markers of MSCs and pericytes. Isolated UA cells possessed the potential to form neurospheres that further differentiated into neuronal and glial cell lineages. Genetic lineage tracing analysis showed that EGFP+ neural crest-derived cells were detected in the subendothelial area of UA with p75NTR immunoreactivity. CONCLUSIONS: These results show that UA tissue harbors p75NTR+ pericyte-like cells in the subendothelial area that have the capacity to form neurospheres and the potential for neurogenic differentiation. The lineage tracing data suggests the p75NTR+ cells are putatively derived from the neural crest.

Amino acid residue at the 165th position tunes EYFP chromophore maturation. A structure-based design.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (~470 and ~530 nm), and the major peak at ~330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (~85%) and mature chromophore (~15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with ~ 2-fold improved brightness were engineered.

Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing.

Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy.

Optogenetic Stimulation of Vagal Efferent Activity Preserves Left Ventricular Function in Experimental Heart Failure.

Large clinical trials designed to test the efficacy of vagus nerve stimulation (VNS) in patients with heart failure did not demonstrate benefits with respect to the primary endpoints. The nonselective nature of VNS may account for the failure to translate promising results of preclinical and earlier clinical studies. This study showed that optogenetic stimulation of vagal pre-ganglionic neurons transduced to express light-sensitive channels preserved left ventricular function and exercise capacity in a rat model of myocardial infarction-induced heart failure. These data suggested that stimulation of vagal efferent activity is critically important to deliver the therapeutic benefit of VNS in heart failure.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells.

Background & Aims: Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal Na+/H+ exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in an ulcerative colitis-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors. Methods: We assessed NHE8 expression in human CRCs by immunohistochemistry and studied tumor burden in NHE8 knockout (KO) mice using an azoxymethane/dextran sodium sulfate colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NOD scid gamma (NSG) mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by polymerase chain reaction and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of ß-catenin and c-Myc also were analyzed to evaluate Wnt/ß-catenin activation. Results: NHE8 was undetectable in human CRC tissues. Although only 9% of NHE8 wild-type mice showed tumorigenesis in the azoxymethane/dextran sodium sulfate colon cancer model, almost 10 times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared with HT29NHE8 wild type cells. Furthermore, NHE8 deficiency resulted in increased Lgr5 expression in the colon, in HT29-derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/ß-catenin activation. Conclusions: NHE8 might be an intrinsic factor that regulates Wnt/ß-catenin in the intestine.

Drug screening for Pelizaeus-Merzbacher disease by quantifying the total levels and membrane localization of PLP1.

BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is caused by point mutations or copy number changes in the proteolipid protein 1 gene (PLP1). PLP1 is exclusively localized in the myelin sheath of oligodendrocytes. Amino acid-substituted PLP1 protein is unable to fold properly and is subsequently degraded and/or restrictedly translated, resulting in a decrease in the PLP1 protein level and a failure to localize to the membrane. Furthermore, misfolded proteins increase the burden on the intracellular quality control system and trafficking, finally resulting in cell apoptosis. The objective of this study was to identify therapeutic chemicals for PMD by quantifying the total levels and membrane localization of PLP1. METHOD: We established a cell line stably expressing PLP1A243V fused with green fluorescent protein in oligodendrocyte-derived MO3.13 cells. We screened a chemical library composed of drugs approved for central nervous system disorders that increased both the total intensity of PLP1A243V in the whole cell and the cell membrane localization. We analyzed the change in the endoplasmic reticulum (ER) stress and the gene expression of candidate chemicals using a micro-array analysis. Finally, we tested the in vivo effectiveness using myelin synthesis deficient (msd) mice with Plp A243V . RESULTS AND CONCLUSION: Piracetam significantly increased the PLP1A243V intensity and membrane localization and decreased the ER stress. It was also shown to reverse the gene expression changes induced by PLP1A243V in a micro-array analysis. However, in vivo treatment of piracetam did not improve the survival of msd mice (Plp1A243V).