Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research.

BACKGROUND: Human induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged. METHODS: To solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids. RESULTS: We established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org. CONCLUSIONS: We have successfully improved the function and convenience of ELCs by utilizing organoid technology.

Establishment of human intestinal organoids derived from commercially available cryopreserved intestinal epithelium and evaluation for pharmacokinetic study.

Human intestinal organoids (HIOs) have been reported to exert their functions in a way that mimics living organs, and HIOs-derived monolayers are expected to be applied to in vitro intestinal pharmacokinetic studies. However, HIOs are established from human tissue, which raises issues of availability and ethics. In the present study, to solve these problems, we have established intestinal organoids using commercially available cryopreserved human intestinal epithelial cells (C-IOs), and compared their functions with biopsy-derived human intestinal organoids (B-IOs) from a pharmacokinetic point of view. Both C-IOs and B-IOs reproduced the morphological features of the intestinal tract and were shown to be composed of epithelial cells. Monolayers generated from C-IOs and B-IOs (C-IO-2D, B-IO-2D, respectively) structurally mimic the small intestine. The C-IOs showed gene expression levels comparable to those of the B-IOs, which were close to those of adult human small intestine. Importantly, the C-IOs-2D showed levels of pharmacokinetics-related protein expression and activity-including cytochrome P450 3A4 (CYP3A4) and carboxylesterase 2 (CES2) enzymatic activities and P-glycoprotein (P-gp) transporter activities -similar to those of B-IOs-2D. This study addresses the difficulties associated with B-IOs and provides fundamental characteristics for the application of C-IOs in pharmacokinetic studies.

Regional Transcriptomics and Proteomics of Pharmacokinetics-Related Genes in Human Intestine.

The intestine is an organ responsible for the absorption and metabolism of orally administered drugs. To predict pharmacokinetics behavior in the small intestine, it is necessary to examine the human intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). In this study, to obtain more accurate expression profiles in various regions of the human intestine, biopsy samples were collected from endoscopically noninflamed mucosa of the duodenum, jejunum, ileum, colon, and rectum from Japanese including Crohn's disease or ulcerative colitis patients, and both RNA-seq and quantitative proteomics analyses were performed. We also analyzed the expression of drug-metabolizing enzymes (cytochromes P450 (CYPs) and non-CYP enzymes), drug transporters, and nuclear receptors. Overall, the mRNA expression levels of these ADME-related genes correlated highly with the protein expression levels. The characteristics of the expression of ADME-related genes differed significantly between the small and large intestines, including the expression levels of CYP enzymes, which were higher and lower in the small and large intestines, respectively. Most CYPs were expressed dominantly in the small intestine, especially the jejunum, but were rarely expressed in the large intestine. On the other hand, non-CYP enzymes were expressed in the large intestine but at lower expression levels than in the small intestine. Moreover, the expression levels of drug metabolizing enzyme genes differed even between the proximal and distal small intestine. Transporters were expressed most highly in the ileum. The data in the present study will enhance understanding of the intestinal ADME of drug candidates and would be useful for drug discovery research.

Gene therapy for spinal muscular atrophy is considerably effective when administered as early as possible after birth.

Introduction: Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by muscle atrophy and progressive muscle weakness. Insurance-approved treatments in Japan include antisense oligonucleotide therapy, gene therapy, and small molecule therapy. The efficacy of these therapies varies depending on the timing of treatment initiation. Case presentation: We report the cases of two infants with SMA born in the same region. Patient 1, who had two copies of SMN2, was born before newborn screening (NBS) was started and received onasemnogene abeparvovec therapy at the age of 4 months. Patient 2, who had three copies of SMN2, was born after the start of NBS and was diagnosed and treated with onasemnogene abeparvovec before symptoms appeared. Unfortunately, Patient 1 became bedridden despite receiving gene therapy, while Patient 2 achieved normal motor development. Discussion: Our findings show that treatment timing is an essential factor affecting patients' motor neurodevelopmental outcomes, although our patients did have differences in the number of copies of SMN2. Therefore, a system should be established to allow all newborns to undergo publicly funded NBS for SMA.

Establishment of MDR1-knockout human enteroids for pharmaceutical application.

In the drug development process, it is important to assess the contributions of drug-metabolizing enzymes and/or drug transporters to the intestinal pharmacokinetics of candidate compounds. For such assessments, chemical inhibitors are often used in in vitro systems. However, this practice poses two problems: one is the low expression levels of pharmacokinetic-related genes in conventional in vitro systems, such as Caco-2 cells, and the other is the off-target and less-efficient effects of their inhibitors. Here, as a model, we have established human biopsy-derived enteroids deficient in MDR1, a key efflux transporter. The expression levels and activities of other pharmacokinetic-related genes, such as CYP3A4, in the MDR1-knockout (KO) enteroid-derived monolayers were maintained at levels as high as those in the WT enteroid-derived monolayers. The contribution of MDR1 to the cytotoxicity of vinblastine, which CYP3A4 metabolized, was accurately evaluated by using the MDR1-KO enteroid-derived monolayers. In contrast, it could not be evaluated in the WT enteroid-derived monolayers treated by verapamil, a widely used MDR1 inhibitor, due to the off-target effect of verapamil, which also inhibits CYP3A4. The combination of human enteroid-derived monolayers and genome editing technology would be a powerful tool to evaluate the contributions of specific pharmacokinetic-related molecules.

Functional inhibition of MEF2 by C/EBP is a possible mechanism of leukemia development by CEBP-IGH fusion gene.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

Newborn screening for spinal muscular atrophy in Japan: One year of experience.

Spinal muscular atrophy (SMA) is a degenerative neuromuscular disease that causes progressive muscle weakness and atrophy due to loss of the anterior horn cells of the spinal cord. Although effective treatments, such as gene therapy, have emerged in recent years, their therapeutic efficacy depends on a restricted time window of treatment initiation. For the treatment to be effective, it must be started before symptoms of the disease emerge. For this purpose, newborn screening (NBS) for SMA is conducted in many countries worldwide. The NBS program for SMA has been initiated in Japan in several regions, including the Kumamoto Prefecture. We started the NBS program in February 2021 and detected a patient with SMA after screening 13,587 newborns in the first year. Herein, we report our experience with the NBS program for SMA and discuss an issue to be approached in the future.

Protein S-Leu17Pro disrupts the hydrophobicity of its signal peptide causing a proteasome-dependent degradation.

INTRODUCTION: Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T > C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency. MATERIALS AND METHODS: The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry. RESULTS: In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol. CONCLUSION: PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation.

Measurement of reactive oxygen species production by luminol-based assay in Nicotiana benthamiana, Arabidopsis thaliana and Brassica rapa ssp. rapa.

Reactive oxygen species (ROS) are critical for plant biological processes. As signaling molecules, ROS regulate plant growth and development through cell expansion, elongation, and programmed cell death. Furthermore, ROS production is induced by microbe-associated molecular patterns (MAMPs) treatment and biotic stresses, and contributes to plant resistance to pathogens. Thus, MAMP-induced ROS production has been an indicator for plant early immune responses or stress responses. One of widely used methods for the measurement is a luminol-based assay to measure extracellular ROS production with a bacterial flagellin epitope (flg22) as a MAMP elicitor. Nicotiana benthamiana is susceptible to a wide variety of plant pathogenic agents and therefore commonly used for ROS measurements. On the other hand, Arabidopsis thaliana, many of genetical lines of which are available, is also conducted to ROS measurements. Tests in an asterid N. benthamiana and a rosid A. thaliana can reveal conserved molecular mechanisms in ROS production. However, the small size of A. thaliana leaves requires many seedlings for experiments. This study examined flg22-induced ROS production in another member of the Brassicaceae family, Brassica rapa ssp. rapa (turnip), which has large and flat leaves. Our experiments indicated that 10 nM and 100 nM flg22 treatments induced high ROS levels in turnip. Turnip tended to have a lower standard deviation in multiple concentrations of flg22 treatment. Therefore, these results suggested that turnip can be a good material from the rosid clade for ROS measurement.

Uncoupling root hair formation and defence activation from growth inhibition in response to damage-associated Pep peptides in Arabidopsis thaliana.

In Arabidopsis thaliana, PROPEPs and their derived elicitor-active Pep epitopes provide damage-associated molecular patterns (DAMPs), which trigger defence responses through cell-surface receptors PEPR1 and PEPR2. In addition, Pep peptides induce root growth inhibition and root hair formation, however their relationships and coordinating mechanisms are poorly understood. Here, we reveal that Pep1-mediated root hair formation requires PEPR-associated kinases BAK1/BKK1 and BIK1/PBL1, ethylene, auxin and root hair differentiation regulators, in addition to PEPR2. Our analysis on 69 accessions unravels intraspecies variations in Pep1-induced root hair formation and growth inhibition. The absence of a positive correlation between the two traits suggests their separate regulation and diversification in natural populations of A. thaliana. Restricted PEPR2 expression to certain root tissues is sufficient to induce root hair formation and growth inhibition in response to Pep1, indicating the capacity of non-cell-autonomous receptor signalling in different root tissues. Of particular note, root hair cell-specific PEPR2 expression uncouples defence activation from root growth inhibition and root hair formation, suggesting a unique property of root hairs in root defence activation following Pep1 recognition.

Comparison of meniscal extrusion and osteophyte formation at the intercondylar notch as a predictive biomarker for incidence of knee osteoarthritis-Data from the Osteoarthritis Initiative.

BACKGROUND: Medial meniscal extrusion (ME) is a biomarker to predict later development of knee osteoarthritis (KOA). On the other hand, we have reported osteophyte formation at the posterior condylar notch of the femur served as a biomarker for the same purpose. The purpose of this study is to compare capacity of the two biomarkers in predicting KOA development. METHODS: Two cohort of knees were established utilizing publicly available data from the Osteoarthritis Initiative (OAI). No OA group (NOA) consisted of knees that were grade 0 or 1 on Kellgren and Lawrence grade (K/L) both at baseline and 48 months later, and pre-radiographic-OA group (PROA) consisted of knees that were grade 0 or 1 at baseline but grade ≥2 48 months later. Baseline MR images were evaluated in terms of ME and osteophyte formation at the posterior condylar notch. ME was evaluated both by meniscus subluxation index (MSI) indicating the ratio of the extruded width of the medial meniscus to the width of medial meniscal body and by the medial radial displacement (MRD) indicating actual extruded width. The size of the osteophyte was assessed using a semi-quantitative whole-organ magnetic resonance imaging score (WORMS). The predictive accuracy of KOA was assessed by the area under the receiver operating characteristic curve (AUC) and optimal cutoff was determined for each parameter. RESULTS: The AUC for MSI was 0.654 (0.561-0.748: 95% CI) and the cutoff value was determined as 17%. That for MRD was 0.677 (0.584-0.770) and the cutoff value was 2.2 mm. The AUC for the WORMS score at the posterior condylar notch was 0.667 (0.579-0.756) and the cutoff value was 2. CONCLUSIONS: Similar predicting capacity of KOA development was found both in ME and osteophyte formation at the posterior condylar notch. Using these simple parameter, mas-screening for KOA development would be possible.

A look at plant immunity through the window of the multitasking coreceptor BAK1.

Recognition of microbe- and danger-associated molecular patterns (MAMPs and DAMPs, respectively) by pattern recognition receptors (PRRs) is central to innate immunity in both plants and animals. The plant PRRs described to date are all cell surface-localized receptors. According to their ligand-binding ectodomains, each PRR engages a specific coreceptor or adaptor kinase in its signaling complexes to regulate defense signaling. With a focus on the coreceptor RLK BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and related SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs), here we review the increasing inventory of BAK1 partners and their functions in plant immunity. We also discuss the significance of autoimmunity triggered by BAK1/SERK4 disintegration in shaping the strategies for attenuation of PRR signaling by infectious microbes and host plants.

Interspinous Ligament Lidocaine and Steroid Injections for the Management of Baastrup's Disease: A Case Series.

STUDY DESIGN: Prospective study. PURPOSE: To examine the long-term effects of interspinous ligament injections of local anesthetics and steroids for the treatment of Baastrup's diseases. OVERVIEW OF LITERATURE: Baastrup's disease is associated with axial low back pains. Baastrup's disease has been more recently described as the "kissing spinous processes" disease. Several authors have reported methods for the diagnosis and treatment of the disease. However, there has been only one report of patients receiving interspinous ligament injections of agents for the treatment of Baastrup's disease. METHODS: Seventeen patients showed severe low back pains between spinous processes at L3-L4 or L4-L5. X-ray imaging, computed tomography, and magnetic resonance imaging revealed kissing spinous processes, consolidation of spinous process, or inflammation of an interspinous ligament. Pain reliefs after lidocaine and dexamethasone administration into interspinous ligament as therapy for low back pains were being examined and followed up. RESULTS: Low back pain scores significantly improved immediately after injection of the agents into interspinous ligaments. At final follow-up (1.4 year), low back pain scores significantly improved as compared with before the treatment. CONCLUSIONS: Findings from the current study indicate that lidocaine and dexamethasone administration into interspinous ligament in patients diagnosed with Baastrup's disease is effective for managing the pain associated with this disease.

Distribution and diversity of anaerobic ammonium oxidation (anammox) bacteria in the sediment of a eutrophic freshwater lake, Lake Kitaura, Japan.

Although the emission of N(2) via anaerobic ammonium oxidation (anammox) is a key process in the elimination of nitrogenous compounds from aquatic environments, little information is available regarding its significance and the relevant microorganisms (anammox bacteria) in eutrophic freshwater lakes. In the present study, the anammox bacteria in the sediment of a eutrophic lake in Japan, Lake Kitaura, were examined using a (15)N-tracer technique to measure their potential anammox activity. Potential anammox activity was localized to the northern region of the lake where a stable supply of both NH(4)(+) and NO(3)(-) existed in the sediment. These results suggest the contribution of anammox bacteria to the total emission of N(2) from sediment in this eutrophic lake to not be negligible. Moreover, selective PCR successfully amplified anammox bacteria-related (Brocadiales-related) 16S rRNA genes from sediment samples in which potential anammox activity was observed. The clone libraries consisted of diverse phylotypes except the genus "Scalindua"-lineages, and the lineages of genus "Brocadia" were dominantly recovered, followed by the genus "Kuenenia"-lineages. Most of them, however, were novel and phylogenetically distinguishable from known Brocadiales species. A unique population of anammox bacteria inhabits and potentially contributes to the emission of N(2) from Lake Kitaura.

Intra-tissue distribution of tetrodotoxin in two marine puffers Takifugu vermicularis and Chelonodon patoca.

Micro distribution pattern of tetrodotoxin (TTX) in several tissues of marine puffers Takifugu vermicularis and Chelonodon patoca was investigated by means of monoclonal antibody-based immunoenzymatic technique under light microscope. In the investigation TTX was visualized at glands in the skin of T. vermicularis, while in C. patoca TTX was detected in succiform cells of the skin section. Similarly, in the ovary section of T. vermicularis TTX was recognized at late peri nucleolus stage, yolk granule stage-I, and yolk granule stage-II of oocytes. The oocytes of late peri nucleolus stage and yolk granule stage-I showed TTX antigen at their nucleus and yolk vesicles, while in yolk granule stage-II TTX was visualized at yolk granules and yolk vesicles. In the ovary of C. patoca TTX was detected in the connective tissues and in the nucleus of some perinucleolar oocytes. In the liver and muscle of C. patoca TTX was found to be distributed in parenchymal hepatocytes and muscle fiber, respectively. This study, however, reveals that intra-tissue distribution of TTX varies in respect of species.