Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method to Determine Maduramicin in Crayfish (Procambarus clarkii) and Evaluate Food Safety.

Maduramicin (MAD) is widely introduced into aquatic environments and results in the contamination of fish products. Worryingly, the consumption of MAD-contaminated crayfish (Procambarus clarkii) may induce symptoms of Haff disease. In this study, to monitor this potential contamination and to understand the residue and elimination characteristics of MAD in edible tissues of crayfish, a sensitive and efficient ultra-performance liquid chromatography-tandem mass spectrometry method was developed, validated, and applied. After extraction with acetonitrile and purification by solid-phase extraction column, multiple-reaction monitoring mass spectrometry with positive ionization mode was used to determine MAD's residues. The limits of detection and of quantification were 6 µg·kg-1 and 20 µg·kg-1, respectively. The fortified recoveries ranged from 74.2% to 110.4%, with relative standard deviation of 1.2% to 10.1%. Furthermore, MAD was completely eliminated after 3 and 5 days from abdominal muscle and hepatopancreas tissues of crayfish, respectively. The maximum residue limits (MRLs) of MAD respectively was 200 µg·kg-1 in muscle and 600 µg·kg-1 in the hepatopancreas, and its withdrawal time in both edible tissues was 25.8 °C·d. Collectively, the results of this study indicate the proposed method is an efficient tool to evaluate the public health risk associated with crayfish consumption.

Effect of maduramicin on crayfish (Procambius clarkii): Hematological parameters, oxidative stress, histopathological changes and stress response.

Maduramicin, an extensively used anticoccidial drug, has been introduced into environment due to poorly absorbed in the intestine of broiler chicken. To understand the potential ecological toxicity of maduramicin on aquatic organisms, acute and subacute toxicity, hemolymph biochemistry, histopathology and the expressions of drug metabolism and stress response genes of crayfish (Procambius clarkii) were investigated in this study. For the first time, the 96 h median lethal concentration (LC50) of maduramicin on crayfish was 67.03 mgL-1 with a 95% confidence interval (54.06-81.32 mgL-1). Then, the crayfish were exposed to 0.7 mgL-1 (1/100 LC50), 3.5 mgL-1 (1/20 LC50) and 7.0 mgL-1 (1/10 LC50) maduramicin for 28 days. Maduramicin significantly altered biochemical parameters including AST, ALT, CK, LDH and ALP of hemolymph in crayfish at several time points. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of crayfish gills, hepatopancreas and abdominal muscle were significantly decreased or elevated by different concentrations of maduramicin treatment at varying time points. Furthermore, histopathological damage of crayfish gills, hepatopancreas and abdominal muscle were observed in a concentration-dependent manner. The expressions of metabolic and stress response genes (CYP450, GST, COX1, COX2, HSP70 and MT) in hepatopancreas of crayfish were significantly up-regulated by maduramicin (7.0 mgL-1) treatment for 8 h to 7 d, and returned to normal levels after the removal of maduramicin for 3-7 days. In conclusion, our findings demonstrated that environmental exposure of maduramicin threaten to the health of crayfish living in the areas nearby livestock farms or pharmaceutical factory. Crayfish exhibited resistance to the stress of maduramicin via activating drug metabolite and detoxification pathways.

A six-week inspiratory muscle training and aerobic exercise improves respiratory muscle strength and exercise capacity in lung cancer patients after video-assisted thoracoscopic surgery: A randomized controlled trial.

OBJECTIVE: To compare the postoperative outcomes of inspiratory muscle training and aerobic exercise, along with standard care, on lung cancer patients undergoing video-assisted thoracoscopic surgery (VATS). DESIGN: A parallel-group, single-blind randomized clinical trial. SETTING: Thoracic surgery ward and outpatient clinic in a teaching hospital. SUBJECTS: Overall 63 patients underwent VATS were randomly assigned to a triaging (TG, n = 32) or control group (CG, n = 31). A total of 54 patients (TG, n = 26; CG, n = 28) completed the study. INTERVENTION: TG: six-week threshold inspiratory muscle training and aerobic exercise. CG: standard care. MAIN MEASURES: Maximum inspiratory pressure (PImax), maximum expiratory pressure (PEmax) lung expansion volume, and 6-min walking test (6MWT) were performed on the day of chest tube removal (baseline), and 2, 6, and 12 weeks postoperatively. RESULTS: The TG showed significant improvement in PImax at week 6 (71.6 ± 34.9 vs. 94.3 ± 32.8 cmH2O, P = 0.018), PEmax at week 2 (70.9 ± 24.3 vs. 90.9 ± 28.2 cmH2O, P = 0.015) and week 12 (76.1 ± 20.2 vs. 98.6 ± 35.3 cmH2O, P = 0.012), the lung expansion volume at week 2 (1080 ± 433 vs 1457 ± 624 mL, P = 0.02) and week 12 (1200 ± 387 vs 1885 ± 678 mL, P < 0.001), in addition to the 6MWT at week 2 (332 ± 78 vs 412 ± 74 m, P = 0.002), week 6 (360 ± 70 vs 419 ± 60 m, P = 0.007) and week 12 (360 ± 58 vs 402 ± 65 m, P = 0.036). CONCLUSION: A six weeks of inspiratory muscle training and aerobic exercise had improved respiratory muscle strength and aerobic exercise postoperatively in lung cancer patients after VATS as early as 2 weeks.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome.

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome 1, 2 . The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides 3-6 . To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve 7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 Å, presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

Alternate RNA Structures.

RNA molecules fold into complex three-dimensional structures that sample alternate conformations ranging from minor differences in tertiary structure dynamics to major differences in secondary structure. This allows them to form entirely different substructures with each population potentially giving rise to a distinct biological outcome. The substructures can be partitioned along an existing energy landscape given a particular static cellular cue or can be shifted in response to dynamic cues such as ligand binding. We review a few key examples of RNA molecules that sample alternate conformations and how these are capitalized on for control of critical regulatory functions.

Maduramicin induces apoptosis in chicken myocardial cells via intrinsic and extrinsic pathways.

Maduramicin is one of the most extensively used anticoccidial drugs for the treatment of Eimeria spp. infections. However, overdosage, misuse and drug interactions have resulted in the development of ionophore toxic syndrome. Heart and skeletal muscles have been identified as the main target organs of toxicity. In the present study, primary chicken myocardial cells were isolated to investigate the toxicity and underlying mechanisms of maduramicin. Our results showed that maduramicin causes morphological changes and a decrease in the viability of chicken myocardial cells. Annexin V-FITC/PI and 4',6-diamidino-2-phenylindole (DAPI) staining showed a significant increase in the number of apoptotic cells. Furthermore, caspases-3/8/9 were activated at the gene and protein levels and this was accompanied by the upregulation of apoptosis-related genes, including bcl-2, bax, and cytochrome C. Treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (z-VAD-fmk) ameliorated the apoptosis and cytotoxicity. Furthermore, intracellular Ca2+ and reactive oxygen species (ROS) were elevated, whereas mitochondrial membrane potential (MMP) and intracellular glutathione (GSH) decreased with exposure to maduramicin. The antioxidant N-acetyl-cysteine (NAC) had no significant effect on maduramicin-induced cytotoxicity and apoptosis. Taken together, our findings demonstrate that maduramicin is cytotoxic to primary chicken myocardial cells via caspase dependent and independent apoptotic pathways.

Decolorization characteristics and mechanism of Victoria Blue R removal by Acinetobacter calcoaceticus YC210.

Acinetobacter calcoaceticus YC210 has been isolated and its ability to remove Victoria Blue R (VBR) from aqueous solution was assessed. The effects of various factors on decolorization efficiency were investigated in a batch system. The decolorization efficiency was found to be optimal within a pH of 5-7 and increased with VBR concentration up to 450 mg/l with high efficiency (94.5%) in a short time. The decolorization efficiency was significantly affected by cell concentrations. The decolorization of VBR by A. calcoaceticus YC210 followed first order kinetics. The apparent kinetic parameters of the Lineweaver-Burk equation, R(VBR,max) and K(m), were calculated as 6.93 mg-VBR/g-cell/h and 175.8 mg/l, respectively. Based on the biodegradation products, VBR degradation by A. calcoaceticus YC210 involves a stepwise demethylation process to yield partially dealkylated VBR species. To our knowledge, this is the first report using microbes to remove VBR. It clearly demonstrates the dealkylation pathway of VBR degradation.

Analysis of the clinical characters and risk factors of feeding intolerance in neonates / 中国基层医药

Objective To describe the clinical characters and risk factors of feeding intolerance in neonates. Methods The data of all neonates hospitalized in the newborn ward from January 2003 to March 2007 were ana- lyzed.Results The incidence of feeding intolerance was 6.99 % in the term infants and 27.63 % in the premature infants,and this increased in low birth weight and small gestational age infants.The symptoms of feeding intoler- ance.such as vomiting,gastric residual,abdominal distension and defecation difficulty presented usually in the first week of enteral feeding.The neonates with smaller gestational age and lower birth weight often presented with gas- tric residual,abdominal distension and defecation difficulty,but the larger ones with vomiting.The incidence of feed- ing intolerance increased in asphyxia aegrotus.Conclusion The birth weight and gestational age are protection fac- tors of feeding intolerance in neonates,the asphyxia are risk factors.More attention should be paid to vomiting,gas- tric residual,abdominal distension and defecation difficulty of neonates,especially,in the first week of enteral feeding.