Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Ligand Perception,Activation, and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive

Leucine-rich repeat receptor-like kinases （LRR-RLKs） belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 （BRI1）, a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids （BRs）, which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRIl-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor I （BKI1） were identified by genetic andbiochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

A new bacteria-free strategy induced by MaGal2 facilitates pinewood nematode escape immune response from its vector beetle

Symbiotic microbes play a crucial role in regulating parasite–host interactions; however, the role of bacterial associates in parasite–host interactions requires elucidation. In this study, we showed that, instead of introducing numerous symbiotic bacteria, dispersal of 4th-stage juvenile (J_IV) pinewood nematodes (PWNs), Bursaphelenchus xylophilus, only introduced few bacteria to its vector beetle, Monochamus alternatus (Ma). J_IV showed weak binding ability to five dominant bacteria species isolated from the beetles’ pupal chamber. This was especially the case for binding to the opportunistic pathogenic species Serratia marcescens; the nematodes’ bacteria binding ability at this critical stage when it infiltrates Ma for dispersal was much weaker compared with Caenorhabditis elegans, Diplogasteroides asiaticus, and propagative-stage PWN. The associated bacterium S. marcescens, which was isolated from the beetles’ pupal chambers, was unfavorable to Ma, because it caused a higher mortality rate upon injection into tracheae. In addition, S. marcescens in the tracheae caused more immune effector disorders compared with PWN alone. Ma_Galectin2 (MaGal2), a pattern-recognition receptor, was up-regulated following PWN loading. Recombinant MaGal2 protein formed aggregates with five dominant associated bacteria in vitro. Moreover, MaGal2 knockdown beetles had up-regulated prophenoloxidase gene expression, increased phenoloxidase activity, and decreased PWN loading. Our study revealed a previously unknown strategy for immune evasion of this plant pathogen inside its vector, and provides novel insights into the role of bacteria in parasite–host interactions.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

关于提高水稻三化螟虫现行预测预报办法的意见——试行中期预测预报

近几年来,我们在大面积药剂治螟工作中,深感现行的三化螟虫预测预报办法有提高的必要。当预测预报站向我们预报某日三化螟蛾即将开始盛发时,何日出现发蛾高峰我们不知道。当测报站向我们预报某日即将出现发蛾高峰时,以后会不会还有高峰出现,何日出现,那一个高峰发蛾数量最多,危害最大,我们都不知道。由于不能一次摸清螟虫的     

杭州湾潮滩表层沉积物中磷的分布、赋存形态及生态意义

对杭州湾潮滩表层沉积物样品中的磷进行了总量、形态分级提取及生物有效性分析.结果表明:沉积物中总磷的含量为595.5～1268.8μg·g-1,空间分布特征总体表现为杭州湾北岸＞钱塘江杭州河段＞杭州湾南岸.总磷中有89.3%以无机磷形式存在,钙磷、残留无机态磷、有机磷和铁铝结合态磷是沉积物中磷的主要赋存形态,平均分别占总磷的68.3%、14.6%、10.7%和5.3%.钙磷的分布受到盐度变化影响,自钱塘江河口向外海方向明显增加;铁铝结合态磷则相反,分布朝外海逐步递减.沉积物中的潜在生物有效磷(BAP)含量为43.62～177.84 μg·g-1,平均含量为94.13 μg·g-1.该地区与国内典型的河口和滨岸带潮滩比较,总磷处于相对较高水平.强烈的水动力作用及咸淡水交汇使得环境因子频繁变化,加大了沉积物中磷作为内源向上覆水体释放及造成水体富营养化的可能性.