Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme

Aminotransferases (ATs) catalyze pyridoxal 5′-phosphate–dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging–based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI–mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime–mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.     

16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进

【背景】在过去的十几年里,基于核糖体RNA基因的扩增子测序技术被广泛用于各种生态系统中微生物群落的多样性检测。扩增子测序的使用极大地促进了土壤、水体、空气等环境中微生物生态的相关研究。【目的】随着高通量测序技术的不断发展和参考数据库的不断更新,针对不同的环境样本的引物选择和改进仍然需要更深入的校验。【方法】本文收集了目前在微生物群落研究中被广泛采用的标记基因扩增通用引物,包括16S rRNA基因扩增常用的8对通用引物和2对古菌引物、9对真菌转录间隔区(internal transcribed spacer,ITS)基因扩增引物,以及18S rRNA基因扩增的4对真核微生物通用引物和1对真菌特异性引物。这些引物中包括了地球微生物组计划(Earth Microbiome Project,EMP)推荐的2对16S rRNA基因扩增引物、1对ITS1基因扩增引物和1对18S rRNA基因扩增引物。采用最近更新的标准数据库对这些引物进行了覆盖度和特异性评价。【结果】EMP推荐的引物依然具有较高的覆盖度,而其他引物在覆盖度及对特定环境或类群的特异性上也各有特点。此外,最近有研究对这些通用引物进行了一些改进,而我们也发现,一个碱基的变化都可能会导致评价结果或扩增产物发生明显变化,简并碱基的引入既可以覆盖更多的物种,但同时也会在一定程度上降低关注物种的特异性。【结论】研究结果为微生态研究中标记基因的引物选择提供了一个广泛的指导,但在关注具体科学问题时,引物的选择仍需数据指导与实验尝试。     

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.     

Molecular evidence suggesting the persistence of residual SARS‐CoV‐2 and immune responses in the placentas of pregnant patients recovered from COVID‐19

ObjectivesRecent studies have shown the presence of SARS‐CoV‐2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high‐risk group for COVID‐19. Based on these findings, we assessed SARS‐CoV‐2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID‐19 and cytokine fluctuations in maternal and foetal tissues.Materials and MethodsRemnant SARS‐CoV‐2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS‐CoV‐2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay.ResultsResidual SARS‐CoV‐2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly.ConclusionsOur study showed that SARS‐CoV‐2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid.     

Lytic replication-defective Kaposi's sarcoma-associated herpesvirus: potential role in infection and malignant transformation

Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G(0)/G(1) apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制

[目的]细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。[方法]首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc² 155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc² 155的胞内药物浓度。[结果]光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc² 155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。[结论]蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

刺激大鼠离断背根外周端对相邻背根电活动的影响

在切断大鼠左侧１２、１３背根后,观察电刺激（刺激参数为０．８－１．２ｍＡ,１００Ｈｚ,０．５ｍｓ,总时程２ｓ）Ｌ２背根外同对Ｌ３背根放电活动的影响。结果表明：连续多次刺激Ｌ２背根可使Ｌ３背根平均放电频率（ＭＤＦ）逐步增加,增加量与刺激次数中于明显直线正相关,各次刺激后的时程分析表明,这种增频作用具有明显的累积效应的后效应,并与刺激前１３背根的活动状态密切相关,刺激前放电活动较强者其增频作用更明显。