[Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna].

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Dual CNN cross-teaching semi-supervised segmentation network with multi-kernels and global contrastive loss in ACDC.

The cross-teaching based on Convolutional Neural Network (CNN) and Transformer has been successful in semi-supervised learning; however, the information interaction between local and global relations ignores the semantic features of the medium scale, and at the same time, the information in the process of feature coding is not fully utilized. To solve these problems, we proposed a new semi-supervised segmentation network. Based on the principle of complementary modeling information of different kernel convolutions, we design a dual CNN cross-supervised network with different kernel sizes under cross-teaching. We introduce global feature contrastive learning and generate contrast samples with the help of dual CNN architecture to make efficient use of coding features. We conducted plenty of experiments on the Automated Cardiac Diagnosis Challenge (ACDC) dataset to evaluate our approach. Our method achieves an average Dice Similarity Coefficient (DSC) of 87.2% and Hausdorff distance ([Formula: see text]) of 6.1 mm on 10% labeled data, which is significantly improved compared with many current popular models. Supervised learning is performed on the labeled data, and dual CNN cross-teaching supervised learning is performed on the unlabeled data. All data would be mapped by the two CNNs to generate features, which are used for contrastive learning to optimize the parameters.

Recent Advances in Investigating Functional Dynamics of Chromatin.

Dynamics spanning the picosecond-minute time domain and the atomic-subcellular spatial window have been observed for chromatin in vitro and in vivo. The condensed organization of chromatin in eukaryotic cells prevents regulatory factors from accessing genomic DNA, which requires dynamic stabilization and destabilization of structure to initiate downstream DNA activities. Those processes are achieved through altering conformational and dynamic properties of nucleosomes and nucleosome-protein complexes, of which delineating the atomistic pictures is essential to understand the mechanisms of chromatin regulation. In this review, we summarize recent progress in determining chromatin dynamics and their modulations by a number of factors including post-translational modifications (PTMs), incorporation of histone variants, and binding of effector proteins. We focus on experimental observations obtained using high-resolution techniques, primarily including nuclear magnetic resonance (NMR) spectroscopy, Förster (or fluorescence) resonance energy transfer (FRET) microscopy, and molecular dynamics (MD) simulations, and discuss the elucidated dynamics in the context of functional response and relevance.

Chromatin Liquid-Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length.

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid-liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.

Human umbilical cord mesenchymal stem cells alleviate acute myocarditis by modulating endoplasmic reticulum stress and extracellular signal regulated 1/2-mediated apoptosis.

Acute myocarditis is a non-ischemic inflammatory disease of the myocardium, and there is currently no standard treatment. Mesenchymal stem cells (MSCs) can alleviate myosin­induced myocarditis; however, the mechanism has not been clearly elucidated. In the present study, the authors investigated the ability of human umbilical cordMSCs (HuMSCs) to attenuate myocardial injury and dysfunction during the acute phase of experimental myocarditis. Male Lewis rats (aged 8 weeks) were injected with porcine myosin to induce myocarditis. Cultured HuMSCs (1x106 cells/rat) were intravenously injected 10 days following myosin injection. A total of 3 weeks following injection, this resulted in severe inflammation and significant deterioration of cardiac function. HuMSC transplantation attenuated infiltration of inflammatory cells and adverse cardiac remodeling, as well as reduced cardiomyocyte apoptosis. Furthermore, it was identified that HuMSC transplantation suppressed endoplasmic reticulum stress and extracellular signal­regulated kinase (ERK)1/2 signaling in experimental autoimmune myocarditis (EAM). The reduced number of TUNEL­positive apoptotic cells in myocardial sections from HuMSC­treated EAM rats compared with control demonstrates HuMSCs' anti­apoptotic function. Based on these data, the author suggested that treatment with HuMSCs inhibits myocardial apoptosis in EAM rats, ultimately protecting them from myocardial damage. The conclusion demonstrated that HuMSC transplantation attenuates myocardial injury and dysfunction in a rat model of acute myocarditis, potentially via regulation of ER stress, ERK1/2 signaling and induction of cardiomyocyte apoptosis.