Month: April 2020

For example, changes may occur in DNA methylation, covalent modification of histones, or the activation or silencing of genes by miRNAs. MiRNAs have been shown to modulate cellular differentiation and development, and various hypoxia-responsive miRNAs have been reported in human breast and colon cancers. Although the functional roles of several HRMs have been extensively studied in mammals, the biological roles of miRNAs in fish remain poorly understood. Small freshwater fish models have been used extensively to identify and study biological responses to stresses in the freshwater environment. The marine medaka has been developed as a small fish model for similar studies in the marine environment. However, the miRNA profile of this species is unknown. New technologies such as high throughput RNA sequencing have rapidly increased the rate of miRNA discovery. The high sensitivity of RNA-Seq allows for both the detection of species-specific, conserved miRNAs and the detection of weakly expressed miRNAs. MiRNA discovery has often relied on the mapping of small RNA sequence reads to a reference genome. Because a reference genome is currently not available for O. melastigma, predicting miRNAs in this species is difficult. However, with the development of alignment-type methods such as BLAST for identifying homologs of known miRNAs, it has become possible to predict candidate miRNAs in the marine medaka. This is the first report on the identification and expression profiles of miRNAs in male and female O. melastigma, and also the differential responses of selected miRNAs to hypoxic stress. The results presented here will be invaluable for future transgenerational studies and risk assessment using this model marine fish species. The complete genome sequence of the O. melastigma has yet to be reported; thus, the established methods for novel miRNA discovery that involve mapping to the genome are not yet possible. The primary aim of the present study is to identify the known miRNAs in the organs along the brain-pituitary-gonad axis and the center of detoxification, liver in marine medaka. All of these organs are important for ecotoxicological studies. BLAST-based method was utilized to identify 223 distinct miRNA candidates in the brain, ovary, testis and liver of marine medaka based on homology to reported orthologous miRNAs. Using high-throughput sequencing, we identified unique miRNAs with expression levels ranging from 6 to nearly 27,000 RPM. Thus the methodology was BAY-60-7550 highly sensitive and had sufficient dynamic range to detect miRNAs with both high and low expression levels. Our qRT-PCR analysis further confirmed that the identified O. melastigma miRNAs are authentic miRNAs, which are differentially expressed by specific tissue and sex. The miRNAs were also highly tissue specific, with the majority brain-enriched. Many identified O. melastigma miRNAs in the present study are found to be highly conserved between species and taxa.

S1P receptors are expressed by oligodendrocytes, astrocytes, microglia, and neurons. EAE is attenuated and fingolimod efficacy is lost in conditional null-mutant mice lacking S1P1 by astrocytes, which suggests that S1P receptor agonists may mitigate EAE through functional antagonism of S1P1 expressed by astrocytes. Further, other studies indicate the function of S1P receptors expressed by cells of the oligodendrocyte kinase inhibitors lineage. Oligodendrocytes are myelinating cells of the CNS, which are the principal target of immune attack in MS. Loss of myelin and the failure of remyelination by oligodendrocytes contribute to the functional impairment of patients with MS. Fingolimod-P treatment rescues oligodendrocyte progenitor cells from undergoing apoptosis in a death-inducing environment through S1P1 signaling. S1P or fingolimod-P promotes the survival of mature oligodendrocytes through S1P5. Fingolimod-P enhances remyelination in lysolecithin-induced demyelination in cerebellar slice culture, and an agonist specific for S1P5 shows a trend toward an increase in remyelination. ASP4058 acts agonistically on S1P1 and S1P5 and penetrates the CNS. Thus, the beneficial effects of ASP4058 for treating EAE may involve its direct effect on cells of the CNS. In clinical trials, fingolimod induced adverse events such as reduction of heart rate or mean FEV1. Preclinical findings that bradycardia induced by a nonselective S1P receptor agonist administered to wild-type mice is abolished in S1P3 knockout mice and that an S1P1-selective agonist does not induce bradycardia suggest that S1P3 signaling induces bradycardia. Consistent with these reports, ASP4058, which has weak agonistic activity for S1P3, required a higher dose than fingolimod-P to induce bradycardia in conscious rats. The safety margin between the lymphocyte-reducing effect and the bradycardia-inducing effect was evaluated by determining the plasma or blood concentrations of compounds. The concentrations of fingolimod-P required to change heart rate and to exert the maximal effect on lymphocyte counts in rats were similar. In contrast, the concentration of ASP4058 required to induce bradycardia was more than 30-times higher than that required to induce lymphopenia. These results suggest that ASP4058 presents less risk for adverse cardiovascular events than fingolimod-P because of its low activity for S1P3. Transient bradycardia was observed in healthy humans treated with the S1P1, S1P5 agonist BAF312, which suggest species-specific differences in S1P receptor specificity for first-dose cardiac effects. Therefore, whether or not ASP4058 exerts cardiovascular effects in human remains to be determined. S1P affects airway constriction in a Rho-dependent manner. For example, S1P stimulates contraction of human airway smooth muscle cells and guinea pig tracheal strips and enhances methacholine-induced contraction of guinea pig trachea.

Sphingosine-1-phosphate is a biologically active sphingolipid, which is involved in the regulation of various physiological functions as well as pathophysiological processes. The concentration of S1P is relatively high in blood but extremely low in tissue interstitium, and an S1P concentration gradient promotes the egress of lymphocytes from secondary lymphoid tissue into the bloodstream. Fingolimod is a nonselective S1P receptor agonist approved by the United States Food and Drug Administration in 2010 as the first oral treatment for relapsing forms of MS. Sphingosine kinase phosphorylates fingolimod in vivo, which then acts as an agonist of four of the five S1P receptors. Fingolimod exerts its immunomodulatory effect, at least in part, by inducing internalization of S1P1 on lymphocytes, which reduces the responsiveness of these cells to the S1P gradient and inhibits egress of lymphocytes from secondary lymphoid tissue. In addition, fingolimod exerts direct effects on S1P receptors expressed on CNS cells, such as S1P1 on astrocytes and S1P5 on oligodendrocytes. In clinical trials, fingolimod treatment was beneficial for patients with MS, and the annualized relapse rate in patients receiving fingolimod was significantly lower than in the patients receiving interferon b-1a, which is an established treatment for relapsing-remitting MS. However, a pooled analysis of two phase 3 studies demonstrated that 6.1% of patients receiving 0.5 mg fingolimod and 11.0% of patients receiving 1.25 mg fingolimod experienced bradycardia after the first dose. Heart rate reduction peaked at 4– 5 h after dosing, and the mean heart rate decreased by 8 and 11 beats per minute at nadir with 0.5 and 1.25 mg, respectively. Further, a reduction in the mean forced expiratory volume in 1 second was observed. The average reduction from baseline in the percentage of predicted FEV1 at month 6 was 8.8% and 2.8% in the patients receiving 5.0 mg and 1.25 mg fingolimod, respectively, as compared with 1.9% in the placebo group. Preclinical data suggest that some of the adverse effects of fingolimod are caused by its interaction with S1P3. We therefore assumed that S1P receptor agonists that do not engage S1P3 might provide a better therapeutic option for lymphocytemediated disease with fewer adverse effects than nonselective S1P receptor agonists such as fingolimod. To address this question, we developed ASP4058 as a novel S1P receptor agonist selective for S1P1 and S1P5. Here we present the in vitro profile of ASP4058, its in vivo effect on peripheral lymphocytes, and its efficacy in MK-1775 experimental autoimmune encephalomyelitis, which is a widely used animal model of MS. We also investigated the effect of ASP4058 on the heart rate and pulmonary function of rats compared with fingolimod. Here we determined the preclinical profiles of ASP4058, a novel S1P1, S1P5 agonist synthesized by Astellas Pharma Inc.

These studies used western blot analyses to detect antigenantibody reactions between patient serum and animal inner ear tissues, which can demonstrate the existence of an antigenantibody reaction but provides no information on the identity of the autoantibody. Few studies demonstrated increased proteins in the serum of Meniere’s disease patients that were reported to be related with inflammatory reaction or inner ear disorders by proteomics technique. But, there was no evidence if these materials existed in the inner ear fluid of Meniere’s disease patients. Studies using human inner ear tissue are rare, and no studies have investigated autoimmunity using human inner ear fluid. Meniere’s disease, mass screening-based studies of autoimmune reactions using human inner ear fluid and sera of patients should be conducted. In this study, we tried to provide evidence for the involvement of autoimmunity in Meniere’s disease and identify the candidate antigens that react with autoantibodies, which can suggest diagnostic biomarker candidates for Meniere’s disease. Several studies were performed. First, the protein composition of inner ear fluid from control patients and patients with Meniere’s disease was compared using proteomic analysis. Second, candidate autoantigens that reacted with circulating autoantibodies in patient serum were investigated using protein array. Third, western blots using patient serum and mouse inner ear tissues were performed to investigate whether the circulating autoantibodies reacted with inner ear tissue. The results of this study can provide the basic information for the development of diagnostic biomarkers as well as the understanding of pathologic mechanisms of Meniere’s disease. The pathophysiology of Meniere’s disease is still unknown, and various etiologies have been proposed. One of the proposed etiologies of Meniere’s disease is autoimmunity; this Remdesivir putative etiology is supported by the fact that this disease often occurs bilaterally, responds to glucocorticoids and anti-inflammatory treatments, and is characterized by elevated levels of autoantibodies or circulating immune complexes and antigen-antibody reactions between patient serum and animal inner ear tissues. However, Meniere’s disease does not always occur bilaterally, and experimental studies have only been performed on small numbers of patients or have only targeted a restricted number of autoantibodies or inflammatory markers. Although the number of patients enrolled in this study was small, we found reliable evidence for the inner ear immune/ inflammatory reaction in patients with Meniere’s disease by investigating the protein composition of the inner ear fluid in diseased patients and controls.

Enzymes are likely to belong to one of these common structures. We have collected l for all of the 1000 sequences which tells us how the degree of neutrality of aptamers/ribozymes relates to that of the common structure. Real ribozymes have rather low degree of neutrality because these molecules have been produced by artificial directional selection. Such a decrease in robustness was shown. However, only 48.2% of the considered 305 real sequences have lower l than the median for the random sequences. And 9.1% of the real sequences fall into the upmost decile, i.e. they have a higher l than 90% of random sequences; and 2.2% of the real sequences have higher l than 95% of the random sequences. All in all the distribution of neutralities is not different from the distribution obtained for the random sequences. This is remarkable considering the fact that these ribozymes had been subject to intense directional selection for the required functionality. Although robustness and evolvability are not necessarily in conflict, it is legitimate to ask whether stabilizing selection could increase the robustness of these populations further, as demonstrated in the theory of Remdesivir neutral networks. We have thus exerted stabilizing selection on different molecules that already had a rather high l with population size 500 through 5000 generations. We show the highest degree of neutrality for structure-preserving variants. It is apparent that stabilizing selection can guide robustness to the top 25% or even 5% of the distribution obtained for random sequences. Thus, we can expect that ribozymes in primordial riboorganisms were even more error-resistant than ribozymes evolved in vitro, as they were subject to many generations of stabilizing selection. We have found that the number of 1-step neutral mutants, for short sequences, is an excellent predictor of the error threshold. Other characteristics of structure are not as highly correlated with the error threshold. Maintenance of RNA secondary structure is a good predictor of maintenance of enzymatic activity, but especially around the active site the actual nucleotides presents are also important. In this investigation we have not considered critical sites in our fitness landscape, which would lower the degree of neutrality of sequences. Considering critical sites would most probably not affect the correlation of error threshold with the degree of neutrality. The possibility of estimating the error threshold by available and easily computable characteristic of RNA sequences allows us to assess the replicability of aptamers and ribozymes. We have shown that functional phenotypes are mutationally robust above chance level and that, in effect, most known ribozymes could be replicated.