Structure of Rift Valley Fever Virus RNA-Dependent RNA Polymerase.

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus, the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.

Branched DNA-Based Electrochemical Biosensor for Sensitive Nucleic Acids Analysis with Gold Nanoparticles as Amplifier.

A branched DNA-based electrochemical biosensor was designed to sensitively detect specific nucleic acids. On this platform, novel a branched DNA with three sticky ends could be used as a biosensor to sensitively and specifically detect nucleic acids. Meanwhile, we also employed branched DNA-modified AuNPs as a signal amplifier to further improve the sensitivity. Branched DNA sensors, target DNA, and DNA-modified AuNPs formed a sandwich structure to produce an electronic signal for target DNA detection. The reaction primarily involved DNA hybridization without bulky thermal cyclers and enzymes. We proved that the hybridization reaction easily occurred under different conditions, such as the NaCl concentration, reaction time, pH, and temperature, except for a pH lower than 4. The limit of detection could go as low as 0.09 pM (S/N = 3) with excellent specificity and selectivity. There was a correlation curve relationship between the peak current and the logarithm of the target DNA concentration (0.10 pM to 10 nM). The correlation coefficient reached 0.987. The electrochemical platform enables a branched DNA nanostructure to determine nucleic acids for disease diagnosis.

Exon and protein positioning in a pre-catalytic group II intron RNP primed for splicing.

Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA-protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.

Influence of family environment on the development of speech and language in pre-lingually deaf children after cochlear implantation.

OBJECTIVE: To investigate the influence of family environment on the development of speech and language in pre-school children after cochlear implantation. METHODS: A total of 88 pre-lingually deaf children, aged 2-5 years, who received cochlear implantation, were included in this study. All families completed a self-report family environment questionnaire (FES). The deaf children's linguistic progress was assessed by Categories of Auditory Performance (CAP) and Speech Intelligibility Rating (SIR) at 0, 3, 6 and 12 months after implantation. RESULTS: The family environment was the significant factor associated with CAP and SIR at 6 months post-implantation. The children in families with higher levels of Cohesion, Intellectual-Cultural Orientation and the ability to express emotion effectively had better auditory and speech abilities, while children in families with low intimacy and high incompatibility exhibited a delay in the development of auditory speech (P < .05). CONCLUSIONS: The development of speech and language in pre-lingually deaf children after cochlear implantation can be influenced by family environment and parents' roles.

LEARNS Model as Perioperative Education Strategy for Patients with Laryngeal Tumors.

Objective: To evaluate LEARNS model as a perioperative strategy for health education and nursing supervision of patients with laryngeal tumors. Methods: LEARNS scheme based on the best practice guidelines was applied to patients in the observation group: (1) analyze the needs of patients (Listen_L); (2) establish therapeutic partnership (Establish_E); (3) adopt intentional intervention (Adopt_A); (4) reinforce health awareness (Reinforce_R); (5) implement feedback assessment of knowledge (Name_N); (6) strengthen self-management based on community resources (Strengthen_S). In the control group, traditional medical care instructions were provided to the patients by medical staff. Parameters such as anxiety status, treatment compliance, nursing satisfaction, self-care ability, and life quality were compared between the observation and control groups. Results: Upon admission, there was no significant difference in self-care ability and anxiety level between two groups. However, the anxiety level of observation group was significantly lower than that of the control group 1 day before operation and 7 days after operation. Postoperative treatment compliance and nursing satisfaction were also improved in the observation group. In addition, self-care ability and life quality in the observation group were significantly enhanced as compared to the control group. Conclusion: As a mutual learning process between nurses and patients, LEARNS model motivates nurses to assess the needs of patients voluntarily. Furthermore, evidence-based education reinforces the self-care ability and health awareness of the patients. Our data suggests that LEARNS model is of great value in improving the life quality of the patients with laryngeal tumors and nursing satisfaction.

A Convenient and Label-Free Colorimetric Detection for L-Histidine Based on Inhibition of Oxidation of 3,3',5,5'-Tetramethylbenzidine-H2O2 System Triggered by Copper Ions.

L-Histidine (L-His) is an essential amino acid, which is used to synthesize proteins and enzymes. The concentration of L-His in the body is controlled to regulate tissue growth and repair of tissues. In this study, a rapid and sensitive method was developed for colorimetric L-his detection using Cu2+ ions to inhibit the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system. H2O2 can oxidize TMB to oxTMB in the presence of copper, and the change in color from colorless (TMB) to blue (oxTMB) is similar to that observed in the presence of peroxidase. However, because the imidazole ring and carboxyl group of L-His can coordinate with Cu2+ ions to form stable L-His-Cu2+ complexes, the color of the TMB-H2O2 solution remains unchanged after the addition of L-His. Therefore, because L-His effectively hinders the colorimetric reaction of TMB with H2O2, this assay can be used to quantitatively determine the concentration of L-His in samples. Under optimized conditions, our colorimetric sensor exhibited two linear ranges of 60 nM to 1 µM and 1 µM to 1 mM for L-His detection and a detection limit of 50 nM (S/N = 3); furthermore, the assay can be performed within 20 min. Moreover, the proposed assay was used to determine the concentration of L-His in urine samples, suggesting that this convenient and label-free colorimetric method presents promising applications in bioanalytical chemistry and clinical diagnosis.

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM.

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air-water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

Construction of Enantioenriched 9H-Fluorene Frameworks via a Cascade Reaction Involving Remote Vinylogous Dynamic Kinetic Resolution.

The benzylic C-H group of α,α-dicyanoolefins from 3-substituted 1-indanones could be significantly activated via transmission along the aromatic system, thus enabling dynamic kinetic resolution via a traditional reversible deprotonation-protonation process. Enantioenriched 9-substituted 9H-fluorene frameworks were finally constructed through an asymmetric vinylogous Michael addition to nitroolefins, followed by a cascade cyclization and oxidative aromatization process, under the catalysis of a chiral bifunctional thiourea-tertiary amine.