Judicious use of low-dosage corticosteroids for non-severe COVID-19: A case report.

Inflammation-mediated lung injury in severe cases of infection with SARS-CoV-2, the aetiological agent of Coronavirus disease 2019 (COVID-19), can lead to respiratory failure and death, and therapies that block or ameliorate lung injury-associated inflammatory "cytokine storms" and progression to acute respiratory distress syndrome (ARDS) are urgently needed. Therapeutic use of corticosteroids for this purpose has been controversial because of conflicting reports on their efficacy and immunosuppressive behaviour. The WHO has strongly recommended treating critical COVID-19 patients with systemic corticosteroid therapy, but recommends against corticosteroid therapy in non-severe COVID-19 disease because of a lack of strong evidence on its efficacy. This retrospective case report describing the successful treatment of a non-severe COVID-19 case in Changchun, China, by judicious administration of corticosteroids using a personalized therapeutic approach was recorded to strengthen the evidence base showing how corticosteroid use in non-severe COVID-19 cases can be safe and efficacious. Alongside supportive care and lopinavir/ritonavir antiviral drugs, a low dosage of methylprednisolone was administered over a short period to attenuate lung inflammation. Regular chest CT scans guided dosage reduction in response to lesion absorption and improved lung condition. Judicious use of corticosteroids safely attenuated disease progression and facilitated rapid and complete recovery.

Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus.

Recombinant plectasin, the first fungus defensin, was expressed in Pichia pastoris and purified, and its physical, chemical and antimicrobial characteristics were studied. Following a 120 h induction of recombinant yeast, the amount of total secreted protein reached 748.63 µg/ml. The percentage of recombinant plectasin was estimated to be 71.79% of the total protein. After purification with a Sephadex G-25 column and RP-HPLC, the identity of plectasin was verified by MALDI-TOF MS. Plectasin exhibited strong antimicrobial activity against the Gram-positive bacteria Staphyloccocusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus suis. At a concentration of 2560 µg/ml, this peptide showed approximately equal activity against S. aureus, S. epidermidis, S. suis, and S. pneumoniae, when compared to 320 µg/ml vancomycin, 640 µg/ml penicillin, 320 µg/ml vancomycin and 160 µg/ml vancomycin, respectively. In addition, plectasin showed anti-S. aureus activity over a wide pH range of 2.0 and 10.0, a high thermal stability at 100 °C for 1h and remarkable resistance to papain and pepsin. The expression and characterization of recombinant plectasin in P. pastoris has potential to treat Streptococcus and Staphyloccocus infections when most traditional antibiotics show no effect on them. Our results indicate that plectasin can be produced in large quantities, and that it has pharmaceutical importance for the prevention and clinical treatment of Staphyloccocus and Streptococcus infections.

Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe.

The lactoferrin cDNA of Kunming mice was isolated by reverse transcription polymerase chain reaction and cloned into vector pET28a(+). Its deduced amino acid sequence was analyzed and compared with lactoferrin of other species. Its secondary and tertiary structure are predicted and modeled by bioinformatics tools online. Then recombinant Kunming mice lactoferrin and its N-lobe were both expressed successfully in the Escherichia coli BL21(DE3) in the form of inclusion bodies. After purification with Ni-NTA His-Bind resin, the yield of recombinant lactoferrin was 17 mg l(-1) with purity of 92.1%, and that of lactoferrin N-lobe was 20 mg l(-1) with purity of 98.5%. The inhibition efficiency of refolded lactoferrin N-lobe against Staphylococcus aureus ATCC 25923 reaches 48.6% at the concentration of 25 micromol l(-1). However, the refolded lactoferrin (12.5 micromol l(-1)) didn't display obvious inhibition activity in the test. The expression of recombinant Kunming mice lactoferrin and its N-lobe will be helpful for the study of lactoferrin on structure, function and application in a mouse model system.

Contribution of bovine lactoferrin inter-lobe region to iron binding stability and antimicrobial activity against Staphylococcus aureus.

The investigation of the recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) demonstrates that the inter-lobe region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against Staphylococcus aureus. rBLfA containing N-lobe (amino acid residues 1-333) and inter-lobe region (residues 334-344) was expressed in Pichia pastoris at shaking flask and fermentor level. The recombinant intact bovine lactoferrin (rBLf) and N-lobe (rBLfN) were expressed in the same system as control. The physical-chemical parameters of rBLfA, rBLfN and rBLf including amino acid residues, molecular weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature than rBLf and rBLfN. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released iron in the pH range of 7.0-3.5, whereas rBLfN lost its iron over the pH range of 7.0-4.0 and iron release from rBLf occurred in the pH range of 5.5-3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5 micromol/L, compared with 12.5 and 25 micromol/L that of rBLfN and rBLf, respectively. These results revealed that S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related positively to the higher anti-S. aureus activity.

Recent advances in lactoferrin research and development during the past two years (2007-2009): in lieu of a preface of the Special Issue Lactoferrin.

This is a short preface of this Special Issue Lactoferrin, it described the major points of key reporters in 'The 9th International Conference on LF Structure, Function and Applications' in Beijing in late Autumn 2009, and the major articles published in this issue. A panorama and the latest advances of lactoferrin R&D during past two years (2007-2009) was tried to extract.

Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3).

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni(2+)-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC(50) of 3.6 and 1.9 microM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method.

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure-activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC(50)=1.56-3.13 microM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 microM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides.

Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris.

Beta-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant beta-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding beta-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, beta-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l(-1). The beta-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized beta-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45 degrees C, respectively.

Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli.

Antimicrobial peptides are promising candidates for therapeutic and industrial application owing to their broad spectrum. In this work, a cost-effective method for expression of a potent antimicrobial peptide, bovine lactoferricin derivative LfcinB15-W4,10, has been developed. The oligonucleotide encoding the peptide was linked to generate different oligomeric oligonucleotide segments containing from one to nine but eight tandem copies which was inserted individually to the E. coli expression vector pET32a. The thioredoxin fusion peptides were successfully expressed and detected with different molecular weight on SDS gel, respectively. Among the monomer and other multimeric peptides, the tetramer was expressed at the highest level. After purification, more than 10 mg of tetramer with 99% purity was obtained from 1 l culture and exhibited similar antimicrobial activity as synthetic LfcinB15-W4,10 monomer. The expression system in this study provides a potential production method for lactoferricin derivatives and other antimicrobial peptides in research and industrial applications.

Cloning of beta-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3).

Beta-1,3-1,4-glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone beta-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml(-1) for 1% (w/v) barley beta-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg(-1) for these two substrates. The expression level of recombinant beta-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40 degrees C, respectively.