An integrative platform for detection of RNA 2'-O-methylation reveals its broad distribution on mRNA.

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers.

Transferrin receptor levels and its rare variant are associated with human obesity.

AIM: Iron homeostasis is critical for functional respiratory chain complex of mitochondrial, thus potentially contributing to fat biology and energy homeostasis. Transferrin receptor (Tfrc) binds to transferrin for extracellular iron uptake and is recently reported to be involved in brown fat development and functionality. However, whether TFRC levels and variants are associated with human obesity is unknown. METHODS: To investigate the association of TFRC levels and variants with human obesity, fat biopsies were obtained from surgery. Exon-sequencing and genetic assessments were conducted of a case-control study. For TFRC levels assessment in fat biopsy, 9 overweight and 12 lean subjects were involved. For genetic study, obese (n = 1271) and lean subjects (n = 1455) were involved. TFRC levels were compared in abdominal mesenteric fat of pheochromocytoma patients versus control subjects, and overweight versus lean subjects. For genetic study, whole-exome sequencing of obese and matched control subjects were conducted and analyzed. In addition, the possible disruption in protein stability of TFRC variant was assessed by structural and molecular analysis. RESULTS: TFRC levels are increased in human browning adipose tissue and decreased in fat of overweight patients. Besides, TFRC levels are negatively correlated with body mass index and positively correlated with uncoupling protein 1 levels. Furthermore, a rare heterozygous missense variant p.I337V in TFRC shows a tendency to enrich in obese subjects. Structural and functional study reveals impaired protein stability of the TFRC variant compared to wild-type. CONCLUSIONS: Reduced TFRC levels and its rare variant p.I337V with protein instability are associated with human obesity.

Airway management for a patient with tracheobronchomegaly undergoing lobectomy: a case report.

BACKGROUND: Tracheobronchomegaly (TBM) is a rare disorder mainly characterized by dilatation and malacia of the trachea and major bronchi with diverticularization. This will be a great challenge for airway management, especially in thoracic surgery requiring one-lung ventilation. Using a laryngeal mask airway and a modified double-lumen Foley catheter (DFC) as a "blocker" may achieve one-lung ventilation. This is the first report introducing this method in a patient with TBM. CASE PRESENTATION: We present a 64-year-old man with TBM receiving left lower lobectomy. Preoperative chest computed tomography demonstrated a prominent tracheobronchial dilation and deformation with multiple diverticularization. The most commonly used double-lumen tube or bronchial blocker could not match the distorted airways. After general anesthesia induction, a 4# laryngeal mask was inserted, through which the modified DFC was positioned in the left main bronchus with the guidance of a fiberoptic bronchoscope. The DFC balloon was inflated with 10 ml air and lung isolation was achieved without any significant air leak during one-lung or two-lung ventilation. However, the collapse of the non-dependent lung was delayed and finally achieved by low-pressure artificial pneumothorax. The surgery was successful and the patient was extubated soon after the surgery. CONCLUSIONS: Using a laryngeal mask airway with a modified double-lumen Foley catheter acted as a bronchial blocker could be an alternative method to achieve lung isolation.

Alkaline shear-thinning micro-nanocomposite hydrogels initiate endogenous TGFß signaling for in situ bone regeneration.

Recruiting endogenous stem cells to bone defects without stem cell transplantation and exogenous factor delivery represents a promising strategy for bone regeneration. Herein, we develop an alkaline shear-thinning micro-nanocomposite hydrogel (10-MmN), aiming to alkaline-activate endogenous TGFß1 and achieve in situ bone regeneration. It contains polyethyleneimine (PEI)-modified gelatin, laponite nanoplatelets (LAP), a bicarbonate buffer with a pH of 10, and gelatin microspheres (MSs). PEI-modified gelatin plays a pivotal role in hydrogel fabrication. It endows the system with sufficient positive charges, and forms a shear-thinning nanocomposite matrix in the pH 10 buffer (10-mN) with negatively charged LAP via electrostatic gelation. For biological functions, the pH 10 buffer dominates alkaline activation of endogenous serum TGFß1 to recruit rat bone marrow stem cells through the Smad pathway, followed by improved osteogenic differentiation. In addition, MSs are incorporated into 10-mN to form 10-MmN, and function as substrates to provide good attachment sites for the recruited stem cells and facilitate further their osteogenic differentiation. In a rat critical-sized calvarial defect model, 10-MmN exhibits excellent biocompatibility, biodegradability, hydrogel infusion and retention in bone defects with flexible shapes and active bleeding. Importantly, it repairs ~95% of the defect areas in 3 months by recruiting TGFßR2+ and CD90+CD146+ stem cells, and promoting cell proliferation, osteogenic differentiation and bone formation. The present study provides a biomaterial-based strategy to regulate alkalinity in bone defects for the initiation of endogenous TGFß signaling, which can be extended to treat other diseases.

G3BP/Rin-Binding Motifs Inserted into Flexible Regions of nsP2 Support RNA Replication of Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus. In infected cells, its positive-sense RNA genome is translated into polyproteins that are subsequently processed into four nonstructural proteins (nsP1 to 4), the virus-encoded subunits of the RNA replicase. However, for RNA replication, interactions between nsPs and host proteins are also needed. These interactions are mostly mediated through the intrinsically disordered C-terminal hypervariable domain (HVD) in nsP3. Duplicate FGDF motifs in the HVD are required for interaction with mammalian RasGAP SH3-binding proteins (G3BPs) and their mosquito homolog Rin; these interactions are crucial for CHIKV RNA replication. In this study, we inactivated G3BP/Rin-binding motifs in the HVD and inserted peptides containing either native or inactivated G3BP/Rin-binding motifs into flexible regions of nsP1, nsP2, or nsP4. Insertion of native motifs into nsP1 or nsP2 but not into the C terminus of nsP4 activated CHIKV RNA replication in human cells in a G3BP-dependent manner. In mosquito cells, activation also resulted from the insertion of inactive motifs after residue 8 or 466 in nsP2; however, the effect was significantly larger when the inserted sequence contained native motifs. Nonetheless, CHIKV mutants harboring mutations in the HVD and containing insertions of native motifs in nsP2 were not viable in mosquito cells. In contrast, mutant genomes containing native motifs after residue 466 or 618 in nsP2 replicated in BHK-21 cells, with the latter mutant forming infectious progeny. Thus, the binding of G3BPs to nsP2 can support CHIKV RNA replication and restore the infectivity of viruses lacking G3BP-binding motifs in the HVD of nsP3. IMPORTANCE CHIKV is a reemerging alphavirus that has spread throughout more than 60 countries and is the causative agent of chikungunya fever. No approved drugs or vaccines are available for the treatment or prevention of CHIKV infection. CHIKV replication depends on the ability of its replicase proteins to interact with host cell factors, and a better understanding of host cell factor roles in viral infection will increase our understanding of CHIKV RNA replication and provide new strategies for viral infection attenuation. Here, we demonstrate that the motifs required for the binding of host G3BP/Rin proteins remain functional when transferred from their natural location in nsP3 to different replicase proteins and may enable mutant viruses to complete a full replication cycle. To our knowledge, this is the first demonstration of interaction motifs for crucial host factors being successfully transferred from one replicase protein to another subunit of alphavirus replicase.

Rapid Gas-Phase Autoxidation of Nicotine in the Atmosphere.

Nicotine is the most abundant alkaloid chemical in smoke emission. In this work, we investigated the gas-phase oxidation mechanism of nicotine initiated by its reactions with the OH radical and ozone. Both initiation reactions start dominantly by hydrogen atom abstractions from the C1, C3, and -CH3 groups of the methylpyrrolidinyl group and form radicals nicotinyl-1, nicotinyl-3, and nicotinyl-6, respectively. The nicotinyl radicals would recombine rapidly with O2, forming RO2 with rapid intramolecular hydrogen-atom transfers (HATs) with rate coefficients from 4 s-1 to greater than 104 s-1. The rapid HATs in peroxy radicals suggest rapid autoxidation of nicotine in the gas phase. Formation of HCNO and HC(O)NH2, being observed in previous studies, arises likely from secondary reactions or photolysis of intermediate products.

Oat ß-glucan and L-arabinose synergistically ameliorate glucose uptake in insulin-resistant HepG2 cells and exert anti-diabetic activity via activation of the PI3K/AKT pathway in db/db mice.

Oat ß-glucan (OBG) and L-arabinose (LA) have exhibited positive effects on diabetes and its complications. However, it is unclear whether OBG and LA have a synergistic effect. We investigated the effect of variable compositions (OBG : LA = 1 : 1, 1 : 2, 1 : 4,1 : 6, 1 : 8, 1 : 10, 2 : 1, 4 : 1, 6 : 1, 8 : 1, 10 : 1) on glucose uptake in IR-HepG2 cells induced by dexamethasone (DEX) to find out the optimal composition showing synergistic effects. Furthermore, this study evaluated the anti-diabetic activity of the optimal composition in db/db mice. In vitro, the OBG : LA = 1 : 1 group showed the strongest synergistic effects among the varied compositions, outperforming OBG and LA alone. In vivo, there were more beneficial effects in the OBG : LA = 1 : 1 group compared with the OBG and LA single-dosing groups. OBG : LA = 1 : 1 supplementation markedly decreased the levels of fasting blood glucose (FBG) and insulin (INS) in serum, improved glucose tolerance and insulin sensitivity, lowered blood lipid levels, and reduced liver lipid accumulation. Moreover, the western blot results indicated that the OBG : LA = 1 : 1 group up-regulated the protein expression of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), and phospho-protein kinase B (p-AKT), while down-regulating the protein expression of phospho-phosphorylated insulin receptor substrate-1 (p-IRS1) to enhance insulin transduction in liver tissues. These findings suggest that OBG : LA = 1 : 1 synergistically ameliorated glucose metabolism disorders and alleviated insulin resistance by promoting the PI3K/AKT pathway in the liver.

Clinical Study on the Evaluation of the Condition of Patients with Gastric Tumors and the Choice of Surgical Treatment by Gastric Ultrasonic Filling Method.

OBJECTIVE: To explore the clinical value of the gastric ultrasonic filling method in evaluating the condition of patients with gastric tumors and guiding the selection of treatment methods, provide data support for clinical gastric filling ultrasonography in the evaluation of gastric cancer patients, and provide the basis for the choice of surgical treatment. Methods. This study retrospectively analyzed 50 patients with gastric cancer treated in our hospital from April 2017 to January 2022. All 50 patients were examined by the gastric ultrasound filling method. The TNM staging results of gastric cancer were analyzed with the results of gastroscopic biopsy or postoperative pathological examination as the diagnostic gold standard. Results. The ultrasonic detection rate of 50 patients with gastric cancer was 94.00% (47/50). Among them, 3 cases missed diagnosis were of early intramucosal carcinoma, which were only diagnosed as erosive gastritis. 1 case was located in the gastric body, and the other 2 cases were located in the gastric antrum. Ultrasound assessment of gastric mucosal thickness in T1-T2 stage was 9.8 mm, which was significantly lower than that in T3-T4 stage, which was 17.0 mm (p < 0.05). The diagnostic accuracy of the gastric ultrasound filling method in the diagnosis of T1, T2, T3, and T4 was 41.67%, 57.14%, 96.00%, and 83.33%, respectively. The total diagnostic accuracy of T-stage was 76.00% (38/50). The total judgment rate of too shallow and too deep was 10.00% and 14.00%, respectively. The diagnostic accuracy of the gastric ultrasound filling method was 88.89%, 81.81%, 70.00%, and 82.00%, respectively. The diagnostic accuracy of the gastric ultrasound filling method in the diagnosis of M0 and M1 stages was 100.00%, and the total diagnostic accuracy of the M-stage was 100.00%. The ROC curve drawn by GFUS in the diagnosis of T-stage of gastric cancer had three components: the specificity was the horizontal axis, the sensitivity was the vertical axis, and the area under the curve was 0.978. The difference was statistically significant (p < 0.05). Conclusion. Before the operation of patients with gastric cancer, using the gastric ultrasonic filling method and ultrasonic examination method to diagnose them can timely clarify the clinical stage of patients, so that clinicians can choose the most appropriate operation method according to their clinical stage, which is worthy of popularization and application in clinic.

A fluorinated peptide with high serum- and lipid-tolerence for the delivery of siRNA drugs to treat obesity and metabolic dysfunction.

Obesity is the major risk factor for metabolic diseases such as fatty liver, hyperlipidemia and insulin resistance. Beige fat has been recognized as a therapeutic target considering its great potential to burn energy. Since the evolutionary discovery of RNA interference and its utilization for gene knockdown in mammalian cells, a remarkable progress has been achieved in siRNA-based therapeutics. However, efficient delivery of siRNA into adipose tissues or differentiated adipocytes is challenging due to high lipid contents in these tissues. Here, we discovered a highly efficient fluoropolypeptide with excellent serum and lipid tolerance for this purpose from a library of amphiphlic polypeptides. The lead material F13-16 exhibited high gene knockdown efficacies in undifferentiated preadipocytes and differentiated adipocytes, as well as adipose tissues. It successfully delivered a siRNA targeting Tle3, an established suppressor gene for energy expenditure, in beige fat, and thereby ameliorated diet-induced obesity and metabolic disorders by increasing energy expenditure and thermogenic capacity. The results demonstrated that fluoropolypeptide is a useful tool for the delivery of siRNA-based therapeutics into adipocyte/adipose tissues for gene therapy.

Comparative study of headspace and headspace single drop microextraction combined with GC for the determination of methanol in wine.

In this work, headspace single-drop microextraction (HS-SDME) method and headspace (HS) method were developed and compared to determine methanol by gas chromatography with flame ionization detector (GC-FID). Several factors influencing extraction efficiency, such as extraction time, temperature, sample volume, stirring rate and extraction solvent were investigated and the optimal conditions could be obtained using 2.0 µL DMF as extractant, 45 °C as heating temperature, 5 min as extraction time, 6 mL sample volume and 1.5 g KCl as addition of salt. The obtained dynamic range of HS-SDME-GC-FID was from 0.05 to 2 mg·L-1 with the limit of detection (LOD) of 0.001 mg·L-1 and that of HS-GC-FID was from 10.0 to 400.0 mg L-1 with LOD of 0.5 mg·L-1. The relative standard deviations (RSD) of HS-SDME-GC-FID was 1.9% (n = 5, C = 0.005 mg·L-1), 4.8%(n = 5, C = 0.02 mg·L-1) and 3.3%(n = 5, C = 0.1 mg·L-1), then the RSD of HS-GC-FID was 4.4%(n = 5, C = 5 mg·L-1), 5.8%(n = 5, C = 20 mg·L-1) and 4.0%(n = 5, C = 40 mg·L-1). Clearly, compared with HS-GC-FID, HS-SDME-GC-FID possessed lower LOD and better reproducibility and both of them were applied to determine methanol in imported wine and the recoveries for the spiked samples were between 83.99 and 117.24%. Overall, HS-SDME approach was confirmed to be a more sensitive and efficient sample pretreatment method and could separate matrix effectively.

Structural properties, antioxidant and immune activities of low molecular weight peptides from soybean dregs (Okara).

In this study, a method for preparing low molecular weight peptides (HPH-VAP) from okara using high-pressure homogenization assisted double enzymes was proposed. In order to explore its advantages, the effects of various methods on protein extraction rate and on the structure, antioxidant and immune properties of peptides were compared. The results showed that the protein extraction rate of this method was increased by 69% and 51% compared with other methods, and the structure only led to changes in the hydrogen bonds between peptide chains. HPH-VAP was screened out through functional characteristics, its structure was identified by HPLC-MS/MS, and further immunological activity analysis was carried out. The results showed that it promoted cell phagocytic ability, NO level and release of cytokines IL-6, IFN- γ, TNF-α. Therefore, this method is an effective and applicable method for industrial preparation of okara peptides, and has a positive effect on the reuse of okara resources.

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2.

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.

nsP4 Is a Major Determinant of Alphavirus Replicase Activity and Template Selectivity.

Alphaviruses have positive-strand RNA genomes containing two open reading frames (ORFs). The first ORF encodes the nonstructural (ns) polyproteins P123 and P1234 that act as precursors for the subunits of the viral RNA replicase (nsP1 to nsP4). Processing of P1234 leads to the formation of a negative-strand replicase consisting of nsP4 (RNA polymerase) and P123 components. Subsequent processing of P123 results in a positive-strand replicase. The second ORF encoding the structural proteins is expressed via the synthesis of a subgenomic RNA. Alphavirus replicase is capable of using template RNAs that contain essential cis-active sequences. Here, we demonstrate that the replicases of nine alphaviruses, expressed in the form of separate P123 and nsP4 components, are active. Their activity depends on the abundance of nsP4. The match of nsP4 to its template strongly influences efficient subgenomic RNA synthesis. nsP4 of Barmah Forest virus (BFV) formed a functional replicase only with matching P123, while nsP4s of other alphaviruses were compatible also with several heterologous P123s. The P123 components of Venezuelan equine encephalitis virus and Sindbis virus (SINV) required matching nsP4s, while P123 of other viruses could form active replicases with different nsP4s. Chimeras of Semliki Forest virus, harboring the nsP4 of chikungunya virus, Ross River virus, BFV, or SINV were viable. In contrast, chimeras of SINV, harboring an nsP4 from different alphaviruses, exhibited a temperature-sensitive phenotype. These findings highlight the possibility for formation of new alphaviruses via recombination events and provide a novel approach for the development of attenuated chimeric viruses for vaccination strategies. IMPORTANCE A key element of every virus with an RNA genome is the RNA replicase. Understanding the principles of RNA replicase formation and functioning is therefore crucial for understanding and responding to the emergence of new viruses. Reconstruction of the replicases of nine alphaviruses from nsP4 and P123 polyproteins revealed that the nsP4 of the majority of alphaviruses, including the mosquito-specific Eilat virus, could form a functional replicase with P123 originating from a different virus, and the corresponding chimeric viruses were replication-competent. nsP4 also had an evident role in determining the template RNA preference and the efficiency of RNA synthesis. The revealed broad picture of the compatibility of the replicase components of alphaviruses is important for understanding the formation and functioning of the alphavirus RNA replicase and highlights the possibilities for recombination between different alphavirus species.

The effect of topical application of meloxicam on inflamed dental pulp.

BACKGROUND/PURPOSE: Effective regulation of the inflammatory process is essential for pulp repair and regeneration. Meloxicam has anti-inflammatory activity in systemic administration. The purpose of this study is to observe effects of topically applied meloxicam on inflamed pulp and to explore its potential value in the treatment of pulpitis. MATERIALS AND METHODS: The coronal pulp tissues of rat molars were stimulated with 10 mg/mL lipopolysaccharide (LPS group) and then treated with 500 µmol/L meloxicam (meloxicam group). The untreated pulp tissues were used as the control group. After 3 h of incubation in vitro, the gene expression of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in each group was detected by real-time RT-PCR. The pulp tissues of each group were randomly subcutaneously implanted into nude mice, and 500 µmol/L meloxicam was injected into the subcutaneous pocket of the meloxicam group. Haematoxylin eosin staining, Masson staining and immunohistochemical staining were performed on samples after 3 days and 4 weeks retrieval, respectively. RESULTS: Compared with the LPS group, the mRNA expression levels of TNF-α and IL-6 of the meloxicam group were significantly reduced in vitro. The inflammatory response and cyclooxygenase-2 expression of the meloxicam group were decreased, and osteodentin-like tissue was generated in the pulp cross section of the meloxicam group in vivo. CONCLUSION: The topical application of meloxicam inhibits the inflammatory response of inflamed pulp and further promotes the formation of osteodentin-like tissues but fails to induce the formation of the pulp-dentin complex. Topically applied meloxicam has the potential to regulate pulp inflammation.

Dihydroartemisinin overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer.

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is commonly used to treat EGFR-mutant non-small-cell lung cancer (NSCLC). However, acquired resistance to mutant EGFR (T790M) can evolve following osimertinib treatment. High reactive oxygen species (ROS) levels in lung cancer cells can influence heme levels and have an impact on osimertinib resistance. Here, we found that heme levels were increased in osimertinib resistant EGFR-mutant NSCLC cell lines and plasma heme levels were also elevated in osimertinib-treated EGFR-mutant NSCLC patients. The antimalarial drug dihydroartemisinin (DHA), which has anticancer effects and requires heme, was tested to determine its potential to revert osimertinib resistance. DHA downregulated the expression of heme oxygenase 1 and inhibited cell proliferation in osimertinib-resistant EGFR-mutant NSCLC cells (PC9-GR4-AZD1), which was further enhanced by addition of 5-aminolevulinic acid, protoporphyrin IX and hemin. DHA was synergistic with osimertinib in inhibiting cell proliferation and colony formation of all osimertinib-resistant cell lines tested. Combination treatment with osimertinib and DHA also increased the levels of ROS, downregulated the phosphorylation or protein levels of several RTKs that often are overexpressed in osimertinib-resistant EGFR-mutant NSCLC cells, and inhibited tumor growth without toxicity in a PC9-GR4-AZD1 xenograft mouse model. The results suggest that DHA is able to reverse the resistance to osimertinib in EGFR-mutant NSCLC by elevating ROS level and impair heme metabolism.

Interdomain Flexibility of Chikungunya Virus nsP2 Helicase-Protease Differentially Influences Viral RNA Replication and Infectivity.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which has both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of 14 residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible interdomain linker, and there is no direct interaction between the two structured regions. To examine the function of the interdomain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the interdomain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the interdomain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short linker. Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. We also highlight the importance of the flexibility of the interdomain of nsP2 on viral RNA synthesis and infectivity. CHIKV mutants harboring shortened linkers fail to produce infectious virus particles despite showing only relatively mild defects in genomic and subgenomic RNA synthesis. Mutations increasing the length of the interdomain linker have only mild and generally beneficial impacts on virus replication. Thus, our findings link interdomain flexibility with the regulation of viral RNA replication and infectivity of the viral genome.

Cross-utilisation of template RNAs by alphavirus replicases.

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.

A transcriptomic study of probenecid on injured spinal cords in mice.

BACKGROUND: Recent studies have found that probenecid has neuroprotective and reparative effects on central nervous system injuries. However, its effect on genome-wide transcription in acute spinal cord injury (SCI) remains unknown. In the present study, RNA sequencing (RNA-Seq) is used to analyze the effect of probenecid on the local expression of gene transcription 8 h after spinal injury. METHODS: An Infinite Horizon impactor was used to perform contusive SCI in mice. The SCI model was made by using a rod (1.3 mm diameter) with a force of 50 Kdynes. Sham-operated mice only received a laminectomy without contusive injury. The injured mice were randomly assigned into either the control (SCI_C) or probenecid injection (SCI_P) group. In the latter group, the probenecid drug was intraperitoneally injected (0.5 mg/kg) immediately following injury. Eight hours after the injury or laminectomy, the spinal cords were removed from the mice in both groups. The total RNAs were extracted and purified for library preparation and transcriptome sequencing. Differential gene expressions (DEGs) of the three groups-sham, SCI_C and SCI_P-were analyzed using a DESeq software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs were performed using a GOseq R package and KOBAS software. Real-time quantitative reverse-transcriptase polymerase chain reaction was used to validate RNA-Seq results. RESULTS: RNA-Seq showed that, compared to the SCI_C group, the number of DEGs was 641 in the SCI_P group (286 upregulated and 355 downregulated). According to GO analysis, DEGs were most enriched in extracellular matrix (ECM), collagen trimer, protein bounding and sequence specific DNA binding. KEGG analysis showed that the most enriched pathways included: cell adhesion molecules, Leukocyte transendothelial migration, ECM-receptor interactions, PI3K-Akt signaling pathways, hematopoietic cell lineages, focal adhesions, the Rap1 signaling pathway, etc. The sequence data have been deposited into the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA554464).

Directional Migration and Odontogenic Differentiation of Bone Marrow Stem Cells Induced by Dentin Coated with Nanobioactive Glass.

INTRODUCTION: This study aimed to use nanobioactive glass (nBG) to guide the directional migration of stem cells and odontogenic differentiation on primary dentin, which are important for the functional regeneration of pulp-dentin tissue. METHODS: Human bone marrow stem cells (BMSCs) were cocultured with 0.5 mg/mL nBG. The cell-biomaterial interaction was monitored using the IncuCyte S3 live cell imaging system (Essen BioScience, Ann Arbor, MI). The adhesion and morphology of BMSCs growing on nBG-coated dentin were assessed at 2 hours and 3 days. The chemotaxis effect of nBG-coated dentin on BMSCs was tested using a 3-dimensional collagen gel model. Subcutaneous transplantation of nBG-treated dentin slices into nude mice was used to investigate cell homing and odontogenic differentiation in vivo. RESULTS: nBG particles showed good biocompatibility, and they were gradually degraded and relocated during interactions with BMSCs. BMSCs had better initial attachment to an nBG-coated dentin surface than to an untreated dentin surface. Cell migration assays showed that nBG-coated dentin induced significantly more cell migration than untreated dentin. An in vivo study revealed that nBG-coated dentin slices facilitated recellularization and revascularization in the root canal and that dentin sialophosphoprotein-positive cells were detected on the surface of the primary dentin. CONCLUSIONS: nBG recruits stem cells to move toward dentin and further promotes cell adhesion and odontogenic differentiation on primary dentin, which help regenerate the biomimetic structure of pulp-dentin tissue.

Comprehensive circular RNA profiling identifies CircFAM120A as a new biomarker of hypoxic lung adenocarcinoma.

BACKGROUND: Hypoxia is crucial in the initiation and progression of tumor metastasis. Circular RNAs (CircRNAs) comprise a novel group of non-coding, RNase R resistant and regulatory RNAs which are generated by 'back-splicing' processes. However, the characterization and function of circRNAs in hypoxic cancer cells remain unknown. METHODS: High throughput RNA-seq assay was performed in lung adenocarcinoma cells (A549) under either normoxic or hypoxic conditions. Bioinformatic analysis of differentially expressed circRNAs was conducted and their target genes were predicted and partially confirmed. RESULTS: Hypoxia increased the expression of hypoxia-inducible factor 1 alpha (HIF-1α) and its downstream genes in A549 cells and enhanced cell migration ability. Comprehensive analysis of global circRNAs expression profiles of A549 identified a total of 558 circRNAs candidates, among which 65 circRNAs were differentially expressed (35 upregulated and 30 downregulated) in hypoxic cancer cells. The difference in their circRNA expressions were compared by computational analysis and circRNA-miRNA networks were constructed. We further characterized one circRNA (hsa_circ_0008193) derived from the FAM120A gene and renamed it as circFAM120A. The expression of circFAM120A, as validated by reverse transcription polymerase chain reaction, was significantly downregulated in both hypoxic A549 and lung cancer tissue from patients with lymph node metastasis. Gene ontology (GO) enrichment analysis and KEGG pathway analysis revealed that circFAM120A may participate in lung cancer development. CONCLUSIONS: CircRNAs profiles were altered in lung adenocarcinoma under hypoxia and circFAM120A may have the potential to be a new biomarker of lung adenocarcinoma hypoxia.