Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

The Pathogenic R5L Mutation Disrupts Formation of Tau Complexes on the Microtubule by Altering Local N-Terminal Structure

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including Progressive Supranuclear Palsy (PSP), Pick's Disease and Alzheimer's disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal disease-associated mutation in Tau, R5L, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine the R5L mutation does not reduce Tau affinity for the microtubule using Total Internal Reflection Fluorescence (TIRF) Microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger order complexes, or Tau patches, at high concentrations of Tau. Using Nuclear Magnetic Resonance (NMR), we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface.

IoT terminal security assessment system based on improved assessment method

The Internet of Things (IoT) technology is widely used and has been improved in research. However, due to the extensiveness of IoT technology, the heterogeneity and diversity of the device structure, the number of attacks against IoT has increased dramatically, so we need a method that can effectively and actively determine safety. Considering the diversity of the terminal structure of IoT, a security method for the IoT terminal based on structural balance, method objectivity, and reliability is currently a challenging task. This paper introduces the idea of rate of change in mathematics into trust analysis, and forms three attribute sets based on trust interval and rate of change: discrete interval, change range, and change frequency. By calculating the above attributes of the entity’s trust value, the entity’s trust situation is obtained, and an overall assessment of the terminal entity’s trust situation is made from the three levels of completeness, accuracy and objectivity. Under the premise of reducing encryption and other means, the above method can evaluate the trust state of the IoT terminal from the perspective of the data, and this evaluation method can provide a basis for the judgment of the IoT terminal more objectively and accurately.

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana

Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated from Holothuria floridana and purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO3−/-COO− molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation–deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-β1,3-d-GalNAc4S6S-β1,4-}-, in which sulfated fucose (FucS) includes Fuc2S4S, Fuc3S4S and Fuc4S residues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.

Sialylation Fine-Tunes Glycoprotein Structural Microheterogeneity Associated with Alzheimer’s Disease as Captured by Native Ion Mobility-Mass Spectrometry

Protein sialylation has been closely linked to many diseases including Alzheimer’s disease (AD) and is broadly implicated in therapeutics in a terminal structure-sensitive manner. However, how sialylation structurally affects mature glycoproteins and how such effect is linked biochemically to AD progression largely remain ill-defined and are, likely beset with the lack of appropriate strategies capable of rapid and in situ manipulation of sialic acids on mature glycoproteins. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based structural probing methodology, enabling well-controlled, synergistic and in situ manipulation of mature glycoproteins and attached sialic acids. Cell viability experiments and IM-MS suggest that the dysregulating effects of transferrin sialylation on the iron-enhanced Aβ cytotoxicity acts through sialylation-dependent Aβ and iron co-importing pathway. Meanwhile, native gel electrophoresis and IM-MS reveal the sialylation-regulated transferrin dimerization tendency. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural micoheterogeneity. Our results may shed new lights on AD-modifying strategies based on sialylation-regulated glycoprotein functions and cytotoxicity.

Sialylation Fine-Tunes Glycoprotein Structural Microheterogeneity Associated with Alzheimer’s Disease as Captured by Native Ion Mobility-Mass Spectrometry

Protein sialylation has been closely linked to many diseases including Alzheimer’s disease (AD) and is broadly implicated in therapeutics in a terminal structure-sensitive manner. However, how sialylation structurally affects mature glycoproteins and how such effect is linked biochemically to AD progression largely remain ill-defined and are, likely beset with the lack of appropriate strategies capable of rapid and in situ manipulation of sialic acids on mature glycoproteins. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based structural probing methodology, enabling well-controlled, synergistic and in situ manipulation of mature glycoproteins and attached sialic acids. Cell viability experiments and IM-MS suggest that the dysregulating effects of transferrin sialylation on the iron-enhanced Aβ cytotoxicity acts through sialylation-dependent Aβ and iron co-importing pathway. Meanwhile, native gel electrophoresis and IM-MS reveal the sialylation-regulated transferrin dimerization tendency. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural micoheterogeneity. Our results may shed new lights on AD-modifying strategies based on sialylation-regulated glycoprotein functions and cytotoxicity.

Changes in Synaptic Terminal Structure in Adolescent Rat During Pregnancy; The Action Potential Propagation and Synaptic Transmission

Synaptic plasticity is a biological system of specific pattern of synaptic activity result in changes in synaptic strength. This influence puberty, pregnancy hormones, sensory experiences, and brain disorders. Long-term synaptic plasticity is accompanied by protein synthesis and trafficking, leading to structural changes of the synapse. Increasing evidence connects the terminal synaptic changes with potential propagation in adolescent and pregnancy. We investigate on the synaptic structural plasticity, which has mainly been studied with in vivo two-photon laser scanning microscopy. We also discuss how a different type of synapses, the multi-contact synapses associated with pregnancy.