Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Enantioselective Rh-Catalyzed Azide-Internal-Alkyne Cycloaddition for the Construction of Axially Chiral 1,2,3-Triazoles.

Significant advances have been achieved for the construction of chiral skeletons containing 1,2,3-triazoles via transition-metal-catalyzed asymmetric azide-alkyne cycloaddition; however, most of them have been limited to terminal alkynes in the synthesis of central chirality via desymmetrization and dynamic/dynamic kinetic resolution. Enantioselective transition-metal-catalyzed azide-internal-alkyne cycloaddition is extremely limited. Moreover, the construction of a challenging five-membered (hetero)biaryl axially chiral molecule via transition-metal-catalyzed asymmetric azide-internal-alkyne cycloaddition is still underexplored. Herein, we first report an atroposelective and atom-economical synthesis of axially chiral 1,4,5-trisubstituted 1,2,3-triazoles, directly acting as core chiral units of challenging five-membered atropisomers, via the enantioselective Rh-catalyzed azide-alkyne cycloaddition (E-RhAAC) of internal alkynes and azides. The reaction demonstrates excellent functional group tolerance, forging a variety of C-C axially chiral 1,2,3-triazoles under mild conditions with moderate to excellent yields (up to 99% yield) and generally high to excellent enantioselectivities (up to 99% ee) along with specific regiocontrol. The origin of regio- and enantioselectivity control is disclosed by density functional theory (DFT) calculations, providing new guidance for the facile construction of axially chiral compounds.

Diabetes-Induced H3K9 Hyperacetylation Promotes Development of Alzheimer's Disease Through CDK5.

The connection between diabetes and Alzheimer's disease (AD) is not fully determined. Hyperphosphorylation of tau protein is mediated by binding and stabilization of truncated p25 with cyclin-dependent kinase-5 (CDK5) in AD. We recently showed that diabetes-associated hyperglycemia increased the CDK5 levels to promote development of AD. Here, we examined the underlying mechanisms. Hyperglycemia and glucose intolerance were induced in rats that had received a low dose of streptozotocin (STZ) and a high fat diet (HFD). Compared to the control rats that received no STZ and normal diet-fed, the STZ + HFD rats exhibited poorer performance in the behavioral test and showed hyperacetylation of H3K9 histone on CDK5 promoter, likely resulting from upregulation of a histone acetyltransferase, GCN5. Inhibition of acetylation of H3K9 histone by a specific GCN5 inhibitor, MB3, attenuated activation of CDK5, resulting in decreased tau phosphorylation in rat brain and improved performance of the rats in the behavior test. Thus, these data suggest that diabetes may promote future development of AD through hyperacetylation of H3K9 histone on CDK5 promoter.

Two highly sensitive and efficient salamo-like copper(II) complex probes for recognition of CN.

Two salamo-like copper(II) complex probes, L1-Cu2+ and L2-Cu2+, were designed and synthesized for sensitive and efficient identification of CN-. UV spectroscopy, high resolution mass spectrometry, RGB analysis and naked eye recognition were performed to explore their recognition mechanisms. High resolution mass spectra indicated that the probes L1-Cu2+ and L2-Cu2+ formed complexes with CN-. The two probes could recognize CN- by the naked eye and the color of the solution changed from light yellow to red. In terms of application, the contents of CN- in the environmental water samples were tested. In addition, the optimal pH ranges for probe detection of CN- were investigated by pH value measurement.

[Effects of sodium salt stress on seed germination of typical annuals in a desert-oasis ecotone of Hexi Corridor, China].

We assessed the effects of different concentrations of salts (0, 40, 80, 120, 160 and 200 mmol·L-1) on the seed germination and re-germination of six typical annuals (Gramineae: Setaria viridis, Chloris virgata and Eragrostis minor; Chenopodiaceae: Bassia dasyphylla, Salsola ruthenica and Corispermum mongolicum) in autumn of current year and next spring, with NaCl and NaHCO3 as neutral sodium salt and alkaline sodium salt. The results showed that NaCl and NaHCO3 significantly affected seed germination of the six species. The inhibition effect of NaHCO3 on seed germination was stronger than NaCl. When the concentration of NaHCO3 reached to 160 mmol·L-1, germination rates of the six species were low. However, when the concentration of NaCl reached to 200 mmol·L-1, the germination rates of the six species were still high. The germination (2.8%-20.0%) and re-germination rates (3.3%-20.0%) in current autumn were much lower than those in next spring, with values of 21.7%-81.6% and 5.0%-41.1%, respectively. In autumn, most of the current year's seeds kept dormancy, but the dormancy weakened in next spring. The salt tolerance of seeds of the six annual species was in the order of C. virgata > S. viridis > S. ruthenica > B. dasyphylla > C. mongolicum > E. minor.

Correction to: Dgcr8 knockout approaches to understand microRNA functions in vitro and in vivo.

The section: "miRNA­independent functions of DICER" was missed between the section "miRNA­independent functions of DROSHA and DGCR8" and the section "The Dgcr8 knockout strategy to study miRNA functions" in the original publications.

Dgcr8 knockout approaches to understand microRNA functions in vitro and in vivo.

Biologic function of the majority of microRNAs (miRNAs) is still unknown. Uncovering the function of miRNAs is hurdled by redundancy among different miRNAs. The deletion of Dgcr8 leads to the deficiency in producing all canonical miRNAs, therefore, overcoming the redundancy issue. Dgcr8 knockout strategy has been instrumental in understanding the function of miRNAs in a variety of cells in vitro and in vivo. In this review, we will first give a brief introduction about miRNAs, miRNA biogenesis pathway and the role of Dgcr8 in miRNA biogenesis. We will then summarize studies performed with Dgcr8 knockout cell models with a focus on embryonic stem cells. After that, we will summarize results from various in vivo Dgcr8 knockout models. Given significant phenotypic differences in various tissues between Dgcr8 and Dicer knockout, we will also briefly review current progresses on understanding miRNA-independent functions of miRNA biogenesis factors. Finally, we will discuss the potential use of a new strategy to stably express miRNAs in Dgcr8 knockout cells. In future, Dgcr8 knockout approaches coupled with innovations in miRNA rescue strategy may provide further insights into miRNA functions in vitro and in vivo.

A Self-Assembled ZnII-NdIII Heterohexanuclear Dimer Based on a Hexadentate N2O4-Type Ligand and Terephthalic Acid: Synthesis, Structure, and Fluorescence Properties.

A self-assembled ZnII-NdIII heterohexanuclear coordination compound [Zn4Nd2(L)4(bdc)2]·2NO3 based on a hexadentate Salamo-like chelating ligand (H2L = 1,2-bis(3-methoxysalicylideneaminooxy)ethane]) and H2bdc (H2bdc = terephthalic acid) has been synthesized and characterized by elemental analyses, IR and UV/Vis spectra, and X-ray crystallography. Two crystallographically equivalent [Zn2Nd(L)2] moieties lie in the inversion center linked by two (bdc)2- ligands leading to a heterohexanuclear dimer in which the carboxylato group bridges the ZnII and NdIII atoms. The heteropolynuclear 3d-4f coordination compound includes four ZnII atoms, two NdIII atoms, four completely deprotonated (L)2- units, two fully deprotonated (bdc)2- units, and two crystalling nitrate ions. All of the ZnII atoms in the ZnII-NdIII coordination compound possess trigonal bipyramidal geometries and the NdIII atoms possess distorted bicapped square antiprism coordination arrangements. In addition, the fluorescence properties of the ligand and the ZnII-NdIII coordination compound were investigated.

Opposing roles of miR-294 and MBNL1/2 in shaping the gene regulatory network of embryonic stem cells.

Alternative pre-mRNA splicing plays important roles in regulating self-renewal and differentiation of embryonic stem cells (ESCs). However, how specific alternative splicing programs are established in ESCs remains elusive. Here, we show that a subset of alternative splicing events in ESCs is dependent on miR-294 expression. Remarkably, roughly 60% of these splicing events are affected by the depletion of Muscleblind-Like Splicing Regulator 1 and 2 (Mbnl1/2). Distinct from canonical miRNA function, miR-294 represses Mbnl1/2 through both posttranscriptional and epigenetic mechanisms. Furthermore, we uncover non-canonical functions of MBNL proteins that bind and promote the expression of miR-294 targets, including Cdkn1a and Tgfbr2, thereby opposing the role of miR-294 in regulating cell proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). Our study reveals extensive interactions between miRNAs and splicing factors, highlighting their roles in regulating cell type-specific alternative splicing and defining gene expression programs during development and cellular differentiation.

A DGCR8-Independent Stable MicroRNA Expression Strategy Reveals Important Functions of miR-290 and miR-183-182 Families in Mouse Embryonic Stem Cells.

Dgcr8 knockout cells provide a great means to understand the function of microRNAs (miRNAs) in vitro and in vivo. Current strategies to study miRNA function in Dgcr8 knockout cells depend on transient transfection of chemically synthesized miRNA mimics, which is costly and not suitable for long-term study and genetic selection of miRNA function. Here, we developed a cost-effective DGCR8-independent stable miRNA expression (DISME) strategy based on a short hairpin RNA vector that can be precisely processed by DICER. Using DISME, we found that miR-294 promoted the formation of meso-endoderm lineages during embryonic stem cell differentiation. Furthermore, DISME allowed for a pooled screen of miRNA function and identified an miR-183-182 cluster of miRNAs promoting self-renewal and pluripotency in mouse embryonic stem cells. Altogether, our study demonstrates that DISME is a robust and cost-effective strategy that allows for long-term study and genetic selection of miRNA function in a Dgcr8 knockout background.

Pluripotency-associated miR-290/302 family of microRNAs promote the dismantling of naive pluripotency.

The molecular mechanism controlling the dismantling of naive pluripotency is poorly understood. Here we show that microRNAs (miRNAs) have important roles during naive to primed pluripotency transition. Dgcr8(-/-) embryonic stem cells (ESCs) failed to completely silence the naive pluripotency program, as well as to establish the primed pluripotency program during differentiation. miRNA profiling revealed that expression levels of a large number of miRNAs changed dynamically and rapidly during naive to primed pluripotency transition. Furthermore, a miRNA screen identified numerous miRNAs promoting naive to primed pluripotency transition. Unexpectedly, multiple miRNAs from miR-290 and miR-302 clusters, previously shown as pluripotency-promoting miRNAs, demonstrated the strongest effects in silencing naive pluripotency. Knockout of both miR-290 and miR-302 clusters but not either alone blocked the silencing of naive pluripotency program. Mechanistically, the miR-290/302 family of miRNAs may facilitate the exit of naive pluripotency in part by promoting the activity of MEK pathway and through directly repressing Akt1. Our study reveals miRNAs as an important class of regulators potentiating ESCs to transition from naive to primed pluripotency, and uncovers context-dependent functions of the miR-290/302 family of miRNAs at different developmental stages.

miR-290/371-Mbd2-Myc circuit regulates glycolytic metabolism to promote pluripotency.

Enhanced glycolysis is a main feature of pluripotent stem cells (PSCs) and is proposed to be important for the maintenance and induction of pluripotency. The molecular mechanism underlying enhanced glycolysis in PSCs is not clear. Using Dgcr8-/- mouse embryonic stem cells (ESCs) that lack mature miRNAs, we found that miR-290 cluster of miRNAs stimulates glycolysis by upregulating glycolytic enzymes Pkm2 and Ldha, which are also essential for the induction of pluripotency during reprogramming. Mechanistically, we identified Mbd2, a reader for methylated CpGs, as the target of miR-290 cluster that represses glycolysis and reprogramming. Furthermore, we discovered Myc as a key target of Mbd2 that controls metabolic switch in ESCs. Importantly, we demonstrated that miR-371 cluster, a human homolog of miR-290 cluster, stimulates glycolysis to promote the reprogramming of human fibroblasts. Hence, we identified a previously unappreciated mechanism by which miR-290/371 miRNAs orchestrate epigenetic, transcriptional and metabolic networks to promote pluripotency in PSCs and during reprogramming.

(4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a novel inhibitor of autophagy.

BACKGROUND AND PURPOSE: Autophagy is an important intracellular degradation system, which is related to various diseases. In preliminary experiments we found that D4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline (DMH1) inhibited autophagy responses. However DMH1 also inhibits the signalling pathway activated by bone morphogenetic protein-4 (BMP4). The aim of the present study was to elucidate the inhibitory effects of DMH1 on autophagy and the underlying mechanisms. EXPERIMENTAL APPROACH: The effects of DMH1 on autophagy responses were evaluated in cultures of different cell types and with different stimuli to induce autophagy, using Western blots, transmission electron microscopy and fluorescent microscopy. KEY RESULTS: DMH1 inhibited starvation-induced autophagy in cardiomyocytes, HeLa and MCF-7 cells, without involving the signalling pathway of BMP4. DMH1 inhibited aminoimidazole carboxamide ribonucleotide (AICAR)- and rapamycin-induced autophagy in HeLa and MCF-7 cells. DMH1 reversed starvation- and AICAR-induced inhibition of Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (S6K), and reversed rapamycin-induced inhibition of mTOR and S6K. DMH1 reversed starvation-induced decrease of the phosphorylated form of glycogen synthase kinase-3 in MCF-7 and HT29 cells. Activation of Akt and inhibition of autophagy induced by DMH1 were antagonized by an Akt specific inhibitor or by small interfering RNA for Akt in HeLa cells. CONCLUSION AND IMPLICATIONS: DMH1 inhibited cellular autophagy responses in a range of cell types and the underlying mechanisms include activation of the Akt pathway.

The potential role of Kv4.3 K+ channel in heart hypertrophy.

Transient outward K+ current (I(to)) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K(+) channel is an important component of I(to). The function and expression of Kv4.3 K(+) channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. Int his review, we summarized the changes of cardiac Kv4.3 K(+) channel in heart diseases and discussed the potential role of Kv4.3 K(+) channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, down regulation of Kv4.3 K(+) channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca(2+)](I), activation of calcineurin and heart hypertrophy/heart failure.However, in canine and human, Kv4.3 K(+) channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K(+) channel/APD/[Ca(2+)](I) pathway, there exits another mechanism of Kv4.3 K(+) channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K(+) channels leads to CaMKII dissociation from Kv4.3­CaMKII complex and subsequent activation of the dissociated CaMKII , which induces heart hypertrophy/heart failure. Upregulation of Kv4.3K(+) channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K(+) channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K(+) channel might be potentially harmful or beneficial to hearts through CaMKII.

Bone morphogenetic protein-4: a novel therapeutic target for pathological cardiac hypertrophy/heart failure.

Bone morphogenetic protein-4 (BMP4) is a member of the bone morphogenetic protein family which plays a key role in the bone formation and embryonic development. In addition to these predominate and well-studied effects, the growing evidences highlight BMP4 as an important factor in cardiovascular diseases, such as hypertension, pulmonary hypertension and valve disease. Our recent works demonstrated that BMP4 mediated cardiac hypertrophy, apoptosis, fibrosis and ion channel remodeling in pathological cardiac hypertrophy. In this review, we discussed the role of BMP4 in pathological cardiac hypertrophy, as well as the recent advances about BMP4 in cardiovascular diseases closely related to pathological cardiac hypertrophy/heart failure. We put forward that BMP4 is a novel therapeutic target for pathological cardiac hypertrophy/heart failure.

Micro-management of pluripotent stem cells.

Embryonic and induced pluripotent stem cells (ESCs and iPSCs) hold great promise for regenerative medicine. The therapeutic application of these cells requires an understanding of the molecular networks that regulate pluripotency, differentiation, and de-differentiation. Along with signaling pathways, transcription factors, and epigenetic regulators, microRNAs (miRNAs) are emerging as important regulators in the establishment and maintenance of pluripotency. These tiny RNAs control proliferation, survival, the cell cycle, and the pluripotency program of ESCs. In addition, they serve as barriers or factors to overcome barriers during the reprogramming process. Systematic screening for novel miRNAs that regulate the establishment and maintenance of pluripotent stem cells and further mechanistic investigations will not only shed new light on the biology of ESCs and iPSCs, but also help develop safe and efficient technologies to manipulate cell fate for regenerative medicine.

3-Methyladenine induces cell death and its interaction with chemotherapeutic drugs is independent of autophagy.

3-Methyladenine (3-MA) is an autophagy inhibitor and has been widely used as a pharmacological tool in the autophagy studies. 3-MA potentiates the chemotherapeutic effects of anticancer drugs, but it is not clear whether the potentiating effects of 3-MA on chemotherapy efficacy comes from the autophagy inhibition or not. The aim of the present work is to identify the relationship between the effects of 3-MA on chemotherapy and the 3-MA-induced autophagy inhibition. The autophagy responses were evaluated by measuring LC3-II level. Cell viability, cell death and cell apoptosis were evaluated by MTT, live and dead assay kit and Tunel staining. Results showed that 3-MA dose-dependently reduced Hela cell viability but did not affect the basal autophagy responses. 3-MA at the concentration that inhibits autophagy induced Hela cell death and apoptosis. 3-MA did not inhibit the increased autophagy responses induced by chemotherapeutic drugs cispcis-diamminedichloroplatinum(II) (CDDP), tamoxifen and 5-fluorouracil (5-FU) in Hela and MCF-7 cells. The synergism or antagonism between 3-MA and chemotherapeutic drugs was dependent on the inhibition ratio of tumor cells. In conclusion, 3-MA itself induces cell death and apoptosis without relationship with autophagy; 3-MA does not inhibit the increased autophagy induced by anti-cancer drugs; the interaction between 3-MA and chemotherapeutic drugs is not related to autophagy.

Bone morphogenetic protein-4 mediates cardiac hypertrophy, apoptosis, and fibrosis in experimentally pathological cardiac hypertrophy.

Identifying the key factor mediating pathological cardiac hypertrophy is critically important for developing the strategy to protect against heart failure. Bone morphogenetic protein-4 (BMP4) is a mechanosensitive and proinflammatory gene. In this study, we investigated the role of BMP4 in cardiac hypertrophy, apoptosis, and fibrosis in experimentally pathological cardiac hypertrophy. The in vivo pathological cardiac hypertrophy models were induced by pressure-overload and angiotensin (Ang) II constant infusion in mice, and the in vitro model was induced by Ang II exposure to cultured cardiomyocytes. The expression of BMP4 increased in pressure overload, Ang II constant infusion-induced pathological cardiac hypertrophy, but not in swimming exercise-induced physiological cardiac hypertrophy in mice. BMP4 expression also increased in Ang II-induced cardiomyocyte hypertrophy in vitro. In turn, BMP4 induced cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis, and these pathological consequences were inhibited by the treatment with BMP4 inhibitors noggin and DMH1. Moreover, Ang II-induced cardiomyocyte hypertrophy was inhibited by BMP4 inhibitors. The underlying mechanism that BMP4-induced cardiomyocyte hypertrophy and apoptosis was through increasing NADPH oxidase 4 expression and reactive oxygen species-dependent pathways. Lentivirus-mediated overexpression of BMP4 recapitulated hypertrophy and apoptosis in cultured cardiomyocytes. BMP4 inhibitor DMH1 inhibited pressure overload-induced cardiac hypertrophy in mice in vivo. The plasma BMP4 level of heart failure patients was increased compared with that of subjects without heart failure. In summary, we conclude that BMP4 is a mediator and novel therapeutic target for pathological cardiac hypertrophy.

A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree.

Charcot-Marie-Tooth (CMT) disease represents a clinically and genetically heterogeneous group of inherited neuropathies. Here, we report a five-generation family of eight affected individuals with CMT disease type 2, CMT2. Genome-wide linkage analysis showed that the disease phenotype is closely linked to chromosomal region 10p13-14, which spans 5.41 Mb between D10S585 and D10S1477. DNA-sequencing analysis revealed a nonsense mutation, c.1455T>G (p.Tyr485(∗)), in exon 8 of dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1) in all eight affected individuals, but not in other unaffected individuals in this family or in 250 unrelated normal persons. DHTKD1 mRNA expression levels in peripheral blood of affected persons were observed to be half of those in unaffected individuals. In vitro studies have shown that, compared to wild-type mRNA and DHTKD1, mutant mRNA and truncated DHTKD1 are significantly decreased by rapid mRNA decay in transfected cells. Inhibition of nonsense-mediated mRNA decay by UPF1 silencing effectively rescued the decreased levels of mutant mRNA and protein. More importantly, DHTKD1 silencing was found to lead to impaired energy production, evidenced by decreased ATP, total NAD(+) and NADH, and NADH levels. In conclusion, our data demonstrate that the heterozygous nonsense mutation in DHTKD1 is one of CMT2-causative genetic alterations, implicating an important role for DHTKD1 in mitochondrial energy production and neurological development.

[Nonsense-mediated mRNA decay and human monogenic disease].

Nonsense-mediated mRNA decay (NMD) is a widespread quality control mechanism in eukaryotic cells. It can recognize and degrade aberrant transcripts harbouring a premature translational termination codon (PTC), and thereby prevent the production of C-terminally truncated proteins which might be deleterious. Approximately, 30% of human genetic diseases are caused by transcripts containing PTCs. These transcripts are potential targets of NMD. As for monogenic diseases, NMD has effects on the phenotype or mode of inheritance. Here, we explain the mechanism of this surveillance pathway, and take several neuromuscular disorders as examples to discuss its influence for human monogenic diseases. The deeper understanding for NMD will shed light on the nosogenesis and therapies of monogenic diseases.