Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.     

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases—N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11—as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central “hub” protein that regulates the activity of at least seven methyltransferases.     

长周期地震下风力发电塔架结构地震反应分析

转子-机舱组合体(Rotor-Nacelle Assembly, RNA)又称风机机头,常用的简化模型包括点质量(RNA_M)、偏心点质量(RNA_ME)、偏心点质量-转动惯量(RNA_MEJ)和刚性机舱-刚性叶片(RNA_RB)。该文基于NREL 5MW单桩式海上风机原型,使用Abaqus软件分别建立包含这四种RNA简化模型的风机模型,以刚性机舱-可变形叶片(RNA_FB)风机模型为基准,分析不同的RNA简化模型对风机结构特征频率和地震响应的影响。研究结果表明：对于只涉及支撑结构1阶模态的问题,四种简化RNA模型计算的频率均是准确可靠的;当涉及支撑结构的2阶模态或/和扭转模态时,应采用RNA_MEJ、RNA_RB或RNA_FB模型;当涉及支撑结构的更高阶模态时,应采用RNA_FB模型。对于风机结构地震响应分析,RNA_MEJ与RNA_RB模型计算的结构响应更准确,但它们的大多数结构响应峰值的最大相对偏差均超过了10%,在实际工程应用时应慎重使用这些RNA简化模型。     

Neuronal and Glial Communication via Non-Coding RNAs: Messages in Extracellular Vesicles

Extracellular vesicles (EVs) have been increasingly recognized as essential players in cell communication in many organs and systems, including the central nervous system (CNS). A proper interaction between neural cells is fundamental in the regulation of neurophysiological processes and its alteration could induce several pathological phenomena, such as neurodegeneration, neuroinflammation, and demyelination. EVs contain and transfer complex molecular cargoes typical of their cells of origin, such as proteins, lipids, carbohydrates, and metabolites to recipient cells. EVs are also enriched in non-coding RNAs (e.g., microRNAs, lncRNAs, and circRNA), which were formerly considered as cell-intrinsic regulators of CNS functions and pathologies, thus representing a new layer of regulation in the cell-to-cell communication. In this review, we summarize the most recent and advanced studies on the role of EV-derived ncRNAs in the CNS. First, we report the potential of neural stem cell-derived ncRNAs as new therapeutic tools for neurorepair. Then, we discuss the role of neuronal ncRNAs in regulating glia activation, and how alteration in glial ncRNAs influences neuronal survival and synaptic functions. We conclude that EV-derived ncRNAs can act as intercellular signals in the CNS to either propagate neuroinflammatory waves or promote reparative functions.     

Prophenoloxidase of Odontotermes formosanus (Shiraki) (Blattodea: Termitidae) Is a Key Gene in Melanization and Has a Defensive Role during Bacterial Infection

Melanization mediated by the prophenoloxidase (PPO)-activating system is an important innate immunity to fight pathogens in insects. In this study, the in vitro time-dependent increase in the intensity of melanization and phenoloxidase (PO) activity from the hemolymph of Odontotermes formosanus (Shiraki) challenged by pathogenic bacteria was detected. PPO is one of the key genes in melanization pathway, whereas the molecular characteristics and functions of O. formosanus PPO are unclear. The OfPPO gene was cloned and characterized. The open reading frame of OfPPO is 2085 bp in length and encodes a 79.497 kDa protein with 694 amino acids. A BLASTx search and phylogenetic analyses revealed that OfPPO shares a high degree of homology to the Blattodea PPOs. Moreover, real-time fluorescent quantitative PCR analysis showed that OfPPO is ubiquitously expressed in all castes and tissues examined, with the highest expression in workers and variable expression patterns in tissues of different termite castes. Furthermore, the expression of OfPPO was significantly induced in O. formosanus infected by pathogenic bacteria. Intriguingly, in combination with silencing of OfPPO expression, pathogenic bacteria challenge caused greatly increased mortality of O. formosanus. These results suggest that OfPPO plays a role in defense against bacteria and highlight the novel termite control strategy combining pathogenic bacteria application with termite PPO silencing.     

Clinical Pharmacokinetics of Approved RNA Therapeutics

RNA-mediated drugs are a rapidly growing class of therapeutics. Over the last five years, the list of FDA-approved RNA therapeutics has expanded owing to their unique targets and prolonged pharmacological effects. Their absorption, distribution, metabolism, and excretion (ADME) have important clinical im-plications, but their pharmacokinetic properties have not been fully understood. Most RNA therapeutics have structural modifications to prevent rapid elimination from the plasma and are administered intravenously or subcutaneously, with some exceptions, for effective distribution to target organs. Distribution of drugs into tissues depends on the addition of a moiety that can be transported to the target and RNA therapeutics show a low volume of distribution because of their molecular size and negatively-charged backbone. Nucleases metabolize RNA therapeutics to a shortened chain, but their metabolic ratio is relatively low. Therefore, most RNA therapeutics are excreted in their intact form. This review covers not only ADME features but also clinical pharmacology data of the RNA therapeutics such as drug–drug interaction or population pharmacokinetic analyses. As the market of RNA therapeutics is expected to rapidly expand, comprehensive knowledge will contribute to interpreting and evaluating the pharmacological properties.     

乳酸菌破壁方法的比较研究

本实验旨在探索一种快速、高效、经济的乳酸菌破壁方法。通过甘氨酸破壁法、反复冻融破壁法、溶菌酶破壁法、氧化锆珠破壁法、超声波破壁法以及3种复合破壁法对植物乳杆菌Lactobacillus.plantarum 1.557进行破壁处理,并以革兰氏染色和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）为评价方法,比较破壁效果和破壁时间的差异。此外,通过β-葡萄糖醛酸酶（GUS）活力的测定对氧化锆珠破壁法所提蛋白进行活性检测,通过琼脂糖凝胶电泳对氧化锆珠破壁法所提RNA的质量进行了检测。结果表明:氧化锆珠破壁法优胜于其他几种处理方法,该法所提取的GUS蛋白具有蛋白活性,提取的RNA的OD₂₆₀/OD₂₈₀的比值在1.8².1之间,符合标准的比值,纯度较高,降解程度低。因此,氧化锆珠破壁法适用于快速提取乳酸菌菌体蛋白和总RNA,它是一种快速、高效、经济的乳酸菌破壁方法。      

豆渣RNA及其酶解物对小鼠免疫功能的影响

以脏器指数、迟发性变态反应、脾淋巴细胞增殖反应、抗体生成细胞及巨噬细胞吞噬指数等为检测指标,研究豆渣RNA和豆渣RNA酶解物对小鼠的细胞免疫、体液免疫及非特异性免疫功能的影响,并比较二者免疫调节作用的差异。结果表明,豆渣RNA及其酶解物均能显著增加小鼠脾脏重量,促进脾淋巴细胞增殖能力,提高抗体生成细胞水平,说明豆渣RNA及其酶解物可通过促进机体的细胞免疫和体液免疫而发挥免疫调节作用。其中,豆渣RNA酶解物高剂量组(10.0mg/mL)的效果明显优于豆渣RNA组。