Dynamics of alternative modes of RNA replication for positive-sense RNA viruses

We propose and study nonlinear mathematical models describing the intracellular time dynamics of viral RNA accumulation for positive-sense single-stranded RNA viruses. Our models consider different replication modes ranging between two extremes represented by the geometric replication (GR) and the linear stamping machine replication (SMR). We first analyse a model that quantitatively reproduced experimental data for the accumulation dynamics of both polarities of turnip mosaic potyvirus RNAs. We identify a non-degenerate transcritical bifurcation governing the extinction of both strands depending on three key parameters: the mode of replication (α), the replication rate (r) and the degradation rate (δ) of viral strands. Our results indicate that the bifurcation associated with α generically takes place when the replication mode is closer to the SMR, thus suggesting that GR may provide viral strands with an increased robustness against degradation. This transcritical bifurcation, which is responsible for the switching from an active to an absorbing regime, suggests a smooth (i.e. second-order), absorbing-state phase transition. Finally, we also analyse a simplified model that only incorporates asymmetry in replication tied to differential replication modes.     

Characterization of microbial communities in a fluidized-pellet-bed bioreactor for wastewater treatment

In order to make clear the microbiological characteristics of the fluidized-pellet-bed bioreactor (FPB) which is a newly developed wastewater treatment device to perform coagulation, particle pelletization, biological degradation and solid-liquid separation in a single unit, the method of denaturing gradient gel electrophoresis (DGGE) was applied in this study paying attention to the microbial diversity of the granular sludge. Spread plate method was also used for enumeration of aerobic bacteria in unit weight of granular sludge. As a result, slight difference was found between the total aerobic bacteria at the bottom, middle, and top sections though the dissolved oxygen (DO) concentration decreased from about 3.5 mg/L at the bottom inlet to 0.23 mg/L at the top of the FPB bioreactor. From the DGGE finger printing, 17 common species were identified from all these sections, and certain specific species were also identified from each section. The comparability of the microbial communities in the three sections was 83.1%, indicating a very stable structure of the microbial communities. The 16 S rRNA sequence analysis results revealed that the 18 operational taxonomic units (OTUs) obtained all belong to Eubacteria. Among them 11 are Proteobacteria, 3 are Actinobacteria, 2 are low G + C gram-positive bacteria and the remaining 2 belong to other bacteria branches. The dominant microbial communities are typical aerobes or facultative anaerobes commonly encountered in conventional activated sludge.     

An Algorithm for Predicting RNA Secondary Structure Including Pseudoknots

A new model and corresponding dynamic programming algorithm are presented to predict RNA secondary structure including pseudoknots in this paper. This algorithm can compute arbitrary planar pseudoknots and one non-planar pseudoknots in O（n5） time and O（n4） space.     

Transmission electron microscopy and scanning force microscopy of poly r(A-U) and poly r(A-U)-ethidium bromide

Transmission electron microscopy and scanning force microscopy of negative-stained, carbon-coated replica and mica-adsorbed preparations of 200 μM poly r(A-U) and 50 μM ethidium bromide/200 μM poly r(A-U) have been employed to evaluate ethidium-induced changes in poly r(A-U) topology. Poly r(A-U) alone exhibits elongated conformations 85–115 nm in length that possess a number of hairpin loops as well as single-stranded domains. While the double-stranded domains are found predominately at the base of the hairpin loops (diameter = 5–30 nm), other rod-like (presumably double-stranded) regions ranging from 25–80 nm in length are present in other portions of the poly r(A-U). In contrast with the poly r(A-U) alone, the EB/poly r(A-U) combination appears as a heterogeneous population of condensed structures whose lengths and widths vary from 12–88 nm and 15–45 nm, respectively. These conformational changes are due to a number of factors, including the displacement of ordered water surrounding the poly r(A-U) and charge shielding of the phosphate groups of the poly r(A-U) upon the binding of the ethidium.     

RNA Relics and Origin of Life

A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial) origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences) that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes) and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells.     

Minor Intron Splicing from Basic Science to Disease

Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.     

pH‐Cleavable Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid‐Based Delivery Systems

Lipid‐based delivery systems are an established technology with considerable clinical acceptance and several applications in human. Herein, we report the design, synthesis and evaluation of novel orthoester nucleoside lipids (ONLs) for the modulation of liposome stability. The ONLs contain head groups with 3′‐orthoester nucleoside derivatives featuring positive or negative charges. The insertion of the orthoester function in the NL structures allows the formation of pH‐sensitive liposomes. ONL‐based liposomes can be hydrolyzed to provide nontoxic products, including nucleoside derivatives and hexadecanol. To allow the release to be tunable at different hydrolysis rates, the charge of the polar head structure is modulated, and the head group can be released at a biologically relevant pH. Crucially, when ONLs are mixed with natural phosphocholine lipids (PC), the resultant liposome evolves toward the formation of a hexadecanol/PC lamellar system. Biological evaluation shows that stable nucleic acid lipid particles (SNALPs) formulated with ONLs and siRNAs can effectively enter into tumor cells and release their nucleic acid payload in response to an intracellular acidic environment. This results in a much higher antitumor activity than conventional SNALPs. The ability to use pH‐cleavable nucleolipids to control the stability of lipid‐based delivery systems represents a promising approach for the intracellular delivery of drug cargos.     

分子模拟研究蛋白质β－折叠区结合随机 RNA 片段

选取来源于大肠杆菌O157： H7全基因组DNA序列随机转录的3条RNA片段,通过RNAComposer进行结构预测,30个核苷酸的RNA片段均形成了特定的空间结构,构象与碱基排列的序列特异性有关。 NPDock可用于RNA－蛋白质相互作用的分子模拟预测,蛋白质的β－结构相对α－螺旋更容易裸露出特定氨基残基的特异性侧链基团,从而易于结合抗原等配体。     

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.