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Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, μ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.

Structural and functional integrities of formulated proteins are key characteristics that provide a better understanding of influencing factors and their adjustment during formulation development. Here, the procedures commonly used for protein analysis were applied and optimized to obtain a higher degree of accuracy, reproducibility, and reliability for the analysis of lysozyme extracts from hot-melt extrudates (HME). The extrudates were prepared with polyethylene glycol 20 000. The test lysozyme HMEs were subjected to extraction procedures and analytical methods following the International Council of Harmonization guidelines for testing the active protein ingredient Q 1 A (R2) in its pure and formulated form. Therefore, reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, and fluorescence-based activity measurements were applied to study lysozyme stability and function after formulation. Long-term accelerated stability studies were performed for the pure and formulated protein. Our findings revealed a high degree of stability for lysozyme toward different temperatures and storage times, confirming that HME is a suitable formulation alternative that preserves lysozyme's properties and stability. The presented methods and workflow are recommended to be exploited for further protein drugs to assess usability and compatibility concerning different pharmaceutical applications., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background: Pulmonary surfactant is a complex mixture of lipids and specific proteins that stabilizes the alveoli at the end of expiration. Mutations in the gene coding for the triphosphate binding cassette transporter A3 (ABCA3), which facilitates the transfer of lipids to lamellar bodies, constitute the most frequent genetic cause of severe neonatal respiratory distress syndrome and chronic interstitial lung disease in children. Hydroxychloroquine can be used as an effective treatment for this rare severe condition., Case Presentation: We report a late preterm Bosnian baby boy (36 weeks) who suffered from a severe form of respiratory distress syndrome with poor response to intensive conventional management and whole exome sequencing revealed homozygous ABCA3 mis-sense mutation. The baby showed remarkable improvement of the respiratory condition after the initiation of Hydroxychloroquine, Azithromycin and Corticosteroids with the continuation of Hydroxychloroquine as a monotherapy till after discharge from the hospital., Conclusion: Outcome in patients with ABCA3 mutations is variable ranging from severe irreversible respiratory failure in early infancy to chronic interstitial lung disease in childhood (ChILD) usually with the need for lung transplantation in many patients surviving this rare disorder. Hydroxychloroquine through its anti-inflammatory effects or alteration of intra-cellular metabolism may have an effect in treating cases of ABCA3 gene mutations.

Public prophylaxis to decrease the emergence of new daily COVID-19 cases is vital. Adjuvant TaibUVID nutritional supplements are promising home-made or hospital-made supplements suggested for rapidly preventing and treating COVID-19 pandemic. We report here a 44 years old male physician who caught COVID-19 infection at hospital in Egypt with confirmed positive nasopharyngeal swab PCR. Ethical committee approval and informed patient's consent were gained before performing this study. Chest X-ray revealed increased bronchovascular markings. Close follow-up was done with no treatment given and he was sent for home isolation. Few days later, he developed progressive non-productive cough and a sense of difficult breathing with no associated fever or chest pain. An antitussive drug was given to him. The patient read about TaibUVID supplements from social media and started to feel improvement after TaibUVID inhalation therapy (using the heated solution of nigella sativa and chamomile five times a day). He also received a home-made TaibUVID nutritional supplement (nigella sativa, chamomile and natural honey) five times daily for four consecutive days. The next day, he was quite better with mild symptoms. Two days later, nasopharyngeal swab PCR was negative while other patients still had positive nasopharyngeal swabs. As few attacks of mild cough and breathing difficulty existed, he was admitted to hospital. A nasopharyngeal swab PCR was done for him again and the result was negative also. Blood gases were normal. He had lymphocytosis (possibly due to TaibUVID effects) that counteract lymphopenia seen in COVID-19 patients. Biochemical and hematological evaluation were quite normal apart from increased serum chloride and lactate dehydrogenase. There was a mild decrease in serum CO 2 and alkaline phosphatase. Chest CT report revealed symmetrically inflated both lungs with non-specific focal nodular infiltrates (scattered in basal and medial lung segments) in left lower lobes with faint ground glass opacities. He was discharged home. Few days later, he was quite improved with no symptoms and returned to his work comfortably. In conclusion, TaibUVID nutritional supplements may be effective in rapidly changing the nasopharyngeal swab PCR from positive to negative. TaibUVID nutritional supplements are advisable as a natural, safe and effective prophylaxis to stop COVID-19 infectiousness, transmission and emergence of new cases. Clinical studies to investigate TaibUVID nutritional benefits are strongly recommended. TaibUVID may be promising and recommended for public prophylaxis to decrease emergence of new COVID-19 cases., Competing Interests: None., (AJBR Copyright © 2020.)

Objectives: To explore the incidence, etiologies, diagnostic methods, treatment options and outcomes in neonates with persistent pulmonary hypertension of the newborn (PPHN) and to identify mortality risk factors in a study from six Asian countries. Methods: A retrospective chart review of patients with documented PPHN from seven centers in six Asian countries (Japan, Kuwait, India, Pakistan, Singapore, and Thailand) between 1 January, 2014 and 31 December, 2016, was performed. Results: A total of 369 PPHN infants were identified. The incidence of PPHN ranged from 1.2 to 4.6 per 1000 live births. The all-cause mortality rate was 20.6% (76 of 369). Meconium aspiration syndrome was the primary cause of PPHN (24.1%). In most cases (84.8%) echocardiography was used to establish the diagnosis of PPHN. Sildenafil was the most commonly used pulmonary vasodilator (51.2%). Multivariate multiple regression analysis indicated gestational age <34 weeks (adjusted odds ratio (OR) = 3.27; 95% CI 1.56-6.74), congenital diaphragmatic hernia (CDH)/lung hypoplasia (LH) (adjusted OR = 6.13 (95% CI 2.28-16.42)), treatment with high frequency oscillation ventilation (HFOV) with or without inhaled nitric oxide (iNO) (adjusted OR = 3.11 (95% CI 1.52-6.34)), and inotropic agents (adjusted OR = 9.43 (95% CI 2.71-32.83)) were independently associated with increased risk of death. Conclusions: The incidence of PPHN in the current study was higher than in western settings. Birth weight, gestational age, CDH/LH, HFOV/iNO, and inotropic agents were significant mortality risk factors.