Epigenomic analysis of multilineage differentiation of human embryonic stem cells.

Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome.

Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.

Spatiotemporal DNA methylome dynamics of the developing mouse fetus.

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited1,2. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.

An atlas of dynamic chromatin landscapes in mouse fetal development.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention.

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, here we report that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. Our data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.

Highly Emissive Lanthanide-Based 0D Metal Halide Nanocrystals for Efficient Ultraviolet Photodetector.

Recently, lanthanide-based 0D metal halides have attracted considerable attention for their applications in X-ray imaging, light-emitting diodes (LEDs), sensors, and photodetectors. Herein, lead-free 0D gadolinium-alloyed cesium cerium chloride (Gd3+-alloyed Cs3CeCl6) nanocrystals (NCs) are introduced as promising materials for optoelectronic application owing to their unique optical properties. The incorporation of Gd3+ in Cs3CeCl6 (CCC) NCs is proposed to increase the photoluminescence quantum yield (PLQY) from 57% to 96%, along with significantly enhanced phase and chemical stability. The structural analysis is performed by density functional theory (DFT) to confirm the effect of Gd3+ in Cs3Ce1- xGdxCl6 (CCGC) alloy system. Moreover, the CCGC NCs are applied as the active layer in UVPDs with different Gd3+ concentration. The excellent device performance is shown at 20% of Gd3+ in CCGC NCs with high detectivity (7.938 × 1011 Jones) and responsivity (0.195 A W-1) at -0.1 V at 310 nm. This study paves the way for the development of lanthanide-based metal halide NCs for next-generation UVPDs and other optoelectronic applications.

Efficacy and safety of MC-003 solution for endoscopic mucosal or submucosal resection: a prospective, multicenter, randomized, triple-blinded, parallel-group, phase III study.

BACKGROUND AND AIMS: The safety and efficacy of solutions for submucosal injection are critical for endoscopic resection of gastric adenomas or early gastric cancers. Although several injectable solutions have been introduced for endoscopic resection, they have some limitations. We aimed to compare the efficacy of the new sodium alginate-based solution MC-003 with that of normal saline (NS; 0.9% sodium chloride). METHODS: In this randomized, triple-blind study, 70 patients were initially enrolled for EMR or endoscopic submucosal dissection (ESD). The main outcomes included the need for additional injections, completion of en bloc resection, and occurrence of adverse events. RESULTS: Each group ultimately included 34 patients. Complete en bloc resections were achieved in all patients (P = 1.000). The MC-003 group had more peri-neoplasm tissue fibrosis (P = .056) and needed fewer additional injections for lesions >15 mm (P = .037), located in the distal portion of the stomach (P = .007), and during ESD procedures (P = .001). The adverse event rate was comparable in both groups. CONCLUSIONS: MC-003 outperformed NS in reducing the need for additional injections during en bloc resection, particularly in larger lesions located in the distal portion of the stomach (where most lesions were found) during ESD procedures, without increasing the incidence of serious adverse events. MC-003 is a promising submucosal injectable solution in real-world clinical settings.

Medication-related incidents in acute care hospitals among different age groups: An analysis of national patient safety report data.

PURPOSE: This study aimed to perform a nationwide analysis of medication errors (MEs) from hospitals using national reporting system data and to compare the ME patterns among different age groups. METHODS: We analyzed medication-related incidents in acute care hospitals reported to the Korean Patient Safety Reporting and Learning System (KOPS), which is a patient safety reporting system, from July 2016 to December 2020. The stages of the medication use process, type of errors, medication class involved in MEs, and degree of harm were analyzed. RESULTS: Among a total of 5071 medication-related incidents, 37.7% (1911 cases) were incidents that caused patient harm and 1.2% caused long-term, permanent, and fatal harm. The proportion of medication-related incidents that resulted in harm was the highest among the <1-year-old age group (67 cases, 51.5%), followed by the elderly (≥ 65 years) (828 cases, 40.9%). The cases leading to patient death were most frequently reported in patients aged ≥65 years. Medication-related incidents occurred mainly in the administration stage (2954 cases, 58.3%), and wrong dose was the most frequently reported ME type. The most prevalent medication class occurring in the 20-64-year age group (256 cases, 11.7%) was 'antibacterials for systemic use', whereas 'contrast media' (236 cases, 11.6%) and 'blood substitutes and perfusion solutions' (98 cases, 19.3%) were the most prevalent drug classes in the ≥65- and <20-year-old age groups, respectively. CONCLUSIONS: It is necessary to establish guidelines for the prevention of medication-related incidents according to the medication use process and patient age group.

Comparative evaluation of endoscopic anti-reflux mucosectomy and stretta radiofrequency ablation in the management of gastroesophageal reflux disease: insights from a retrospective multicenter cohort study.

BACKGROUND: Treatment options for gastroesophageal reflux disease (GERD) that is unresponsive to proton pump inhibitors (PPIs) remain limited. Therefore, we compared the therapeutic effects of anti-reflux mucosectomy (ARMS) and Stretta radiofrequency (SRF) for intractable GERD in over 400 individuals who underwent either procedure. METHODS: We conducted a retrospective study between 2016 and 2023 to evaluate the effectiveness of SRF and ARMS treatments for refractory GERD. The primary measure of success was the change in the GERD questionnaire (GERDQ) score. The secondary outcomes were various GERD-related indicators, including endoscopic Los Angeles (LA) classification, Hill's type-based flap valve grade (FVG), EndoFLIP™ distensibility index (DI), rate of PPI discontinuation, resolution rate of Barrett's esophagus, and incidence of adverse events. RESULTS: The ARMS group included patients with high GERDQ scores, FVG, LA grade, and Barrett's esophagus. Both groups had similar rates of improvements in GERDQ score (P = 0.884) and PPI withdrawal (P = 0.866); however, the ARMS group had significantly more side effects and improvements in the median change in GERDQ score (P = 0.011), FVG (P < 0.001), LA grade (P < 0.001), EndoFLIP™ DI (P < 0.001), and resolution of Barrett's esophagus (P < 0.001). CONCLUSIONS: The ARMS group had a greater GERDQ score improvement than the SRF group but had symptom relief and PPI discontinuation rates similar to those of the SRF group. However, objective measures, including EndoFLIP™ DI and endoscopic evaluations, were better in the ARMS group than in the SRF group.

Endoscopic resection and laparoscopic lymph node dissection for early gastric cancer beyond conventional endoscopic treatment indications: a 10-year outcome study.

BACKGROUND: Endoscopic full-thickness gastric resection (EFTGR) with laparoscopic regional lymph node dissection (LLND) and endoscopic submucosal dissection (ESD) with LLND have been investigated as treatment options for early gastric cancer beyond the absolute indications for ESD. However, comparative studies on the long-term outcomes of these procedures are lacking. This study aimed to analyze and compare the 10-year outcomes of both procedures in a real clinical setting. METHODS: Between January 2009 and December 2013, 28 and 37 patients diagnosed with EGC beyond the absolute indications for ESD were treated with EFTGR with LLND and ESD with LLND, respectively. In both procedures, the dye was injected into the tumor. However, after injection and LLND, EFTGR was performed immediately in the EFTGR with LLND group, whereas LLND was followed by ESD in the ESD with LLND group. The primary endpoint was the 10-year survival rate. RESULTS: The EFTGR with LLND group had one case of local recurrence (3.6%) and mortality (3.6%) each, while the ESD with LLND group had none (0.0% for both); however, the differences were not statistically significant (P = 0.247 for each). Furthermore, there was no significant difference in complications such as ischemia and anastomosis leakage between the groups (P = 0.247). CONCLUSIONS: When the procedures were properly applied, EFTGR with LLND and ESD with LLND did not increase the 10-year mortality in patients with EGC beyond the absolute ESD indications compared with conventional radical gastrectomy.

Da Vinci robot-assisted endoscopic full-thickness gastric resection with regional lymph node dissection using a 3D near-infrared video system: a single-center 5-year clinical outcome.

BACKGROUND: Endoscopic full-thickness gastric resection (EFTGR) with regional lymph node dissection (LND) has been used for early gastric cancer (EGC) exceeding the indications for endoscopic submucosal dissection (ESD). The extent of the dissected lymph nodes is crucial. A 3D near-infrared (NIR) video robot system significantly enhances visualization of the lymphatic system. However, this system has not been used in EFTGR with LND. Thus, this study assessed the benefits of the 3D NIR video robot system in a clinical setting. METHODS: Between February 2015 and September 2018, 24 patients with EGC exceeding the indications for ESD were treated with EFTGR and LND using a 3D NIR video system with the da Vinci surgical robot. Indocyanine green (ICG) was injected endoscopically around the tumor, and basin node (BN) dissection around the nodes was examined using the 3D NIR video system of the da Vinci Si surgical robot. Subsequently, robot-assisted EFTGR was performed. The primary outcome was the 5-year survival rate. RESULT: During a 5-year follow-up of all 24 patients, an 80-year-old patient with an ulcer and T2 invasion was lost to follow-up. Among the remaining 23 patients, no mortality or recurrence was observed. CONCLUSION: No metastasis or mortality occurred using the da Vinci robot-assisted EFTGR with LLND and a 3D NIR video system for patients who required radical gastrectomy for EGC in over 5 years. Hence, this may be a safe and effective method for radical gastrectomy; further studies are required confirming its effectiveness.

Isoform Switch of TET1 Regulates DNA Demethylation and Mouse Development.

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.

Bioinformatics and integrated pharmacology network to identify the therapeutic targets and potential molecular mechanism of alpha-lipoic acid on primary ovarian insufficiency.

Women experiencing primary ovarian insufficiency (POI) are more likely to experience infertility, and its incidence is increasing worldwide annually. Recently, the role of alpha-lipoic acid (ALA) in the treatment of POI has been reported. However, details of the potential pharmacological targets and related molecular pathways of ALA remain unclear and need to be elucidated. Thus, this study aims to elucidate the potential therapeutic target and related molecular mechanism of ALA on POI. First, the potential targets of POI and ALA-related targets were downloaded from online public databases. Subsequently, the overlapped target genes between POI and ALA were acquired, and gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) analysis, protein-protein interaction (PPI) networks were performed and constructed. Finally, molecular docking was performed to verify protein-to-protein effect. A total of 152 potential therapeutic targets were identified. The biological processes of the intersecting targets were mainly involved in the cellular response to peptides, response to xenobiotic stimuli, and response to peptide hormones. The highly enriched pathways were the cAMP, PI3K/AKT, estrogen, progesterone mediated oocyte maturation, and apoptosis signaling pathways. The top 10 hub targets for ALA in the treatment of POI were STAT3, STAT1, CASP3, MTOR, PTGS2, CASP8, HSP90AA1, PIK3CA, MAPK1, and ESR1. The binding between ALA and all top hub targets were verified using the molecular docking analysis. In summary, using the systematic integrated pharmacology network and bioinformatics analysis, this study illustrated that ALA participates in the treatment of POI via multiple targets and multiple pathways mechanisms.

EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.

Development of a medication review tool for community-dwelling older adults in Korea.

BACKGROUND: With the aging population, older adults are more likely to receive outpatient care. Therefore, it is necessary to identify drug-related problems (DRPs) and potentially inappropriate medications (PIMs) associated with adverse clinical outcomes in community-dwelling older adults. This study aimed to develop a medication review tool for community-dwelling older adults in Korea. METHODS: We developed the tool using three steps: (i) establishment of a preliminary list by reviewing 21 existing tools, (ii) a two-round Delphi survey to evaluate clinical appropriateness and (iii) a two-round Delphi survey to evaluate applicability. The list was categorized into 23 diseases/conditions with five types of DRPs. The interventions for each item have been described. RESULTS: The preliminary list contained 100 items. The final list contained 81 items, including 17 general PIMs, 26 PIMs under specific disease/conditions, 16 potential drug interactions, 20 potential omissions and 2 PIMs requiring dose adjustment. CONCLUSION: We developed a disease-based explicit medication review tool that can be used in primary care. This tool would assist primary care healthcare providers in identifying inappropriate medication use, which may help reduce adverse clinical consequences in older adults. Further studies are required to validate the clinical efficacy of this tool.

Paeoniflorin Attenuates Lipopolysaccharide-Induced Cognitive Dysfunction by Inhibition of Amyloidogenesis in Mice.

Alzheimer's disease (AD) is a neurodegenerative disease, associated with progressive cognitive impairment and memory loss. In the present study, we examined the protective effects of paeoniflorin against memory loss and cognitive decline in lipopolysaccharide (LPS)-induced mice. Treatment with paeoniflorin alleviated LPS-induced neurobehavioral dysfunction, as confirmed by behavioral tests, including the T-maze test, novel-object recognition test, and Morris water maze test. LPS stimulated the amyloidogenic pathway-related proteins (amyloid precursor protein, APP; ß-site APP cleavage enzyme, BACE; presenilin1, PS1; presenilin2, PS2) expression in the brain. However, paeoniflorin decreased APP, BACE, PS1, and PS2 protein levels. Therefore, paeoniflorin reverses LPS-induced cognitive impairment via inhibition of the amyloidogenic pathway in mice, which suggests that paeoniflorin may be useful in the prevention of neuroinflammation related to AD.

Intraocular Pressure-Lowering and Retina-Protective Effects of Exosome-Rich Conditioned Media from Human Amniotic Membrane Stem Cells in a Rat Model of Glaucoma.

Glaucoma is one of the most devastating eye diseases, since the disease can develop into blindness and no effective therapeutics are available. Although the exact mechanisms and causes of glaucoma are unknown, increased intraocular pressure (IOP) has been demonstrated to be an important risk factor. Exosomes are lipid nanoparticles secreted from functional cells, including stem cells, and have been found to contain diverse functional molecules that control body function, inhibit inflammation, protect and regenerate cells, and restore damaged tissues. In the present study, exosome-rich conditioned media (ERCMs) were attained via hypoxic culture (2% O2) of human amniotic membrane mesenchymal stem cells (AMMSCs) and amniotic membrane epithelial stem cells (AMESCs) containing 50 times more exosome particles than normoxic culture (20% O2) medium (NCM). The exosome particles in ERCM were confirmed to be 77 nm in mean size and contain much greater amounts of growth factors (GFs) and neurotrophic factors (NFs) than those in NCM. The glaucoma-therapeutic effects of ERCMs were assessed in retinal cells and a hypertonic (1.8 M) saline-induced high-IOP animal model. CM-DiI-labeled AMMSC exosomes were found to readily penetrate the normal and H2O2-damaged retinal ganglion cells (RGCs), and AMMSC-ERCM not only facilitated retinal pigment epithelial cell (RPEC) proliferation but also protected against H2O2- and hypoxia-induced RPEC insults. The IOP of rats challenged with 1.8 M saline increased twice the normal IOP (12-17 mmHg) in a week. However, intravitreal injection of AMMSC-ERCM or AMESC-ERCM (3.9-4.5 × 108 exosomes in 10 µL/eye) markedly recovered the IOP to normal level in 2 weeks, similar to the effect achieved with platelet-derived growth factor-AB (PDGF-AB, 1.5 µg), a reference material. In addition, AMMSC-ERCM, AMESC-ERCM, and PDGF-AB significantly reversed the shrinkage of retinal layers, preserved RGCs, and prevented neural injury in the glaucoma eyes. It was confirmed that stem cell ERCMs containing large numbers of functional molecules such as GFs and NFs improved glaucoma by protecting retinal cells against oxidative and hypoxic injuries in vitro and by recovering IOP and retinal degeneration in vivo. Therefore, it is suggested that stem cell ERCMs could be a promising candidate for the therapy of glaucoma.

Blue Perovskite Nanocrystal Light-Emitting Diodes: Overcoming RuddlesdenPopper Fault-Induced Nonradiative Recombination via Post-Halide Exchange.

Metal halide perovskites (MHPs) have gained traction as emitters owing to their excellent optical properties, such as facile bandgap tuning, defect tolerance, and high color purity. Nevertheless, blue-emitting MHP light-emitting diodes (LEDs) show only marginal progress in device efficiency compared with green and red LEDs. Herein, the origin of the drop in efficiency of blue-emitting perovskite nanocrystals (PNCs) by mixing halides and the genesis of Ruddlesden-Popper faults (RPFs) in CsPbBrX Cl3-X nanocrystals is investigated. Using scanning transmission electron microscopy and density functional theory calculations, the authors have found that RPFs induce possible nonradiative recombination pathways owing to the high chloride vacancy concentration nearby. The authors further confirm that the blue-emitting PNCs do not show RPFs post-halide exchange in the CsPbBr3 nanocrystals. By introducing the post-halide exchange treatment, high-efficiency pure blue-emitting (464 nm) PNC-based LEDs with an external quantum efficiency of 2.1% and excellent spectral stability with a full-width at half-maximum of 14 nm are obtained.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.