慢性肺部疾病被列為全球主要致死的重大疾病之一，而造成感染的細菌則是慢性肺部疾病惡化的元兇之一。因慢性肺部疾病病人的肺部細胞受損，過多的痰液累積成為了病原菌形成生物膜的溫床，且因生物膜內有細胞外基質作為保護層，使細菌可以被包覆其中進而避免或降低抗生素的攻擊的影響，再者，因近年來多重抗藥性(Multi-drug resistance, MDR)細菌的出現，使得原先就對抗生素敏感度低的細菌躲藏在生物膜中使抗生素更難發揮藥效，亦成為增加了治療呼吸道慢性病細菌感染上的困難。
　　而為了達到最小抑菌濃度(Minimum inhibitory concentration, MIC)來抑制細菌的生長，抗生素的過度用藥逐漸成為了嚴重的醫療問題之一。針對這樣的臨床問題，現行可供選擇的治療方法仍有限，因此開發一個更有效的治療方法即是目前迫切的臨床需求。
　　作為造成慢性肺部疾病細菌感染的主要細菌之一，本研究使用Acinetobacter baumannii(鮑氏不動桿菌)做為我們的細菌模型進行實驗。我們將兩種傳統治療方式，抗生素與高張食鹽水氣霧治療，結合後作為我們新的治療方式。所謂的高張食鹽水氣霧治療，是一個在臨床上常見用於舒緩肺部疾病病人不適的治療方式，同時在先前的研究中也發現，高張食鹽水氣霧能夠藉由其高滲透壓促使水分由細胞內部分泌至呼吸道表面、以及打斷痰液中的離子交聯而使痰液黏稠度下降。藉由以上的高張食鹽水氣霧治療機制，希望能夠過合併治療法來破壞並降低過度黏稠的痰液，藉此來降低細菌在黏稠痰液中形成生物膜的機率。
　　首先，為了瞭解使用高張食鹽水(7 g/100 mL)合併抗生素之氣霧治療在細菌生存率與生物膜形成上的效率，我們針對培養在2D表面的鮑氏不動桿菌(Acinetobacter baumannii)進行合併氣霧治療的噴灑。結果顯示，高張食鹽水氣霧可以有效減少生物膜的形成，並且抗生素，見大黴素(Gentamicin)與時欣黴素(Acemycin)，在高張食鹽水氣霧的輔助下，細菌存活率明顯下降，因此我們可以了解到高張實驗水氣霧確實可以抑制生物膜的形成並且增加抗生素的藥效。
　　為了再更進一步了解細菌與肺部細胞表面的痰液之間的關係，本研究建立了一個可以藉由調控A549細胞的細胞微球大小來改變細胞微球表面分泌的痰液量的3D模型，藉由此模型來模擬肺部疾病病患呼吸道的微環境。從實驗數據我們發現，當將鮑氏不動桿菌(Acinetobacter baumannii)與A549細胞微球共培養後，附著於細胞球表面的鮑氏不動桿菌(Acinetobacter baumannii)之數量與細胞球表面分泌之痰液量呈正相關，並且，本研究亦發現藉由細菌的貼附後，細菌侵入細胞亦會破壞細胞微球的3D結構，並導致細胞脫落並凋亡。前述之結果皆顯示出痰液對於細菌進行貼附、侵入、與造成細胞凋亡是非常重要的關鍵因子。除此之外，本研究對鮑氏不動桿菌(Acinetobacter baumannii)與A549細胞球共培養的3D模型進行抗生素與高張食鹽水合併氣霧的噴灑，結果顯示在有較多痰液的微環境中，此合併氣霧治療方式可以顯著減少鮑氏不動桿菌(Acinetobacter baumannii)侵入A549細胞球的數量。以上的結果證明此新興的合併治療方式，結合抗生素與高張食鹽水氣霧治療，可以顯著增加抗生素對治療多重抗藥性(Multi-drug resistance, MDR)細菌的藥效，並藉此來降低藥劑量來達到相同的效果。
　　我們的數據證明使用高張食鹽水氣霧治療來增加抗生素藥效之潛力，並且在先前的研究中尚未有類似的研究在探討此種合併新興治療方式在多重抗藥性(Multi-drug resistance, MDR)細菌上的效果。而這項發現也可能可以做為替代的治療方式，來減少抗生素的過度用藥狀況、以及肺部疾病的細菌感染情況。

Chronic pulmonary diseases become a prominent cause of death in the world, and
the bacteria causing respiratory infections plays a major contributing role in disease progression. Because the impaired function of respiratory cells in patients, the excessive accumulation of mucus become a breeding ground for these opportunistic bacteria to form biofilm, which is an assemblage of bacteria enclosed in an extracellular matrix that could prevent bacteria from killing by antibiotic drugs.
Moreover, the emergence of multi-drug resistance(MDR) bacteria recently increase the treatment difficulty. To effectively treat the respiratory infections, antibiotic agents are broadly applied in the treatment.
However, to achieve minimum inhibitory concentration(MIC), overdose of antibiotic drugs become a serious problem in the world. Limited therapeutic options are available for respiratory infectious diseases and the development of effective treatments is urgently needed.
Here, as one of the multi-drug resistance(MDR) opportunistic pathogen which causing the chronic respiratory infections, we choose Acinetobacter baumannii to be our bacterial model to do the following experiments. We combined two traditional therapeutic methods, antibiotic drugs and hypertonic saline aerosol, to be our new therapeutic strategy. Hypertonic saline aerosol, a common therapeutic method used in hospital, was found to alleviate patients’ discomfort and decrease the mucus viscosity by breaking the ion cross-linking in mucus and promoting the water supply to the mucus layer due to the high osmotic pressure of hypertonic saline in the previous study. Through this mechanism of hypertonic saline, this new strategy might enhance the efficacy of antibiotics by destroy the structure of excessive accumulation of mucus and decreasing the biofilm formation.
First, to understand the antibiotics efficacy with hypertonic saline (7 g/100 mL) aerosol carrier on bacteria viability and biofilm formation, we used this aerosol to treat Acinetobacter baumannii on 2D surface. The data showed that HS aerosol could effectively hindered biofilm formation and resulted in a decreased tolerance to gentamicin and acemycin in the Acinetobacter baumannii, which means that this strategy could decrease the biofilm formation and enhance the antibiotic efficacy. To further understand the relationship between bacteria and mucus on pulmonary cells, we established an 3D model, in which we regulate mucus secretion by controlling A549 spheroid size to mimic a microenvironment in a chronic pulmonary respiratory system. In our data, after coculture Acinetobacter baumannii in this 3D model, it showed that the amount of Acinetobacter baumannii staying on the surface of spheroid depends on the amount of mucus secretion. In addition to this, we found that the bacterial invasion would destroy the 3D structure of the spheroid. Both results showed an important role of mucus for bacteria to assemble, invasion, and cause cell apoptosis. Moreover, we treat the antibiotics with HS aerosol on this 3D model and the results indicated that HS aerosol could effectively enhance the efficacy of antibiotics on bacteria even in the microenviroment that including mucus layer. All the data certified that this new strategy could enhance the efficacy of antibiotics on MDR-bacteria, which means achieving the same effects with lower dosage of antibiotic by the strategy of combining antibiotics with hypertonic saline aerosol.
Our findings demonstrate the potential of using HS aerosol carrier to enhance the efficacy of antibiotics, and to our best knowledge, there is no research shows the efficacy of antibiotic drugs combined with hypertonic saline aerosol on MDR bacteria. This may be an alternative therapeutic strategy to eliminate antibiotics overdose and the chronic respiratory infections.

目錄
摘要-------2
Abstract-------5
誌謝-------8
目錄-------10
圖目錄-------13
表目錄-------19
Chapter 1 介紹-------20
1.1研究動機-------20
1.2文獻回顧-------21
1.2.1 呼吸道上皮細胞功能簡介-------21
1.2.2 呼吸道疾病導致之問題-------22
1.2.2.1 呼吸道疾病與過度痰液生成-------22
1.2.2.2 呼吸道細菌感染-------23
1.2.2.3 細菌生物膜形成簡介-------23
1.2.3高張食鹽水噴霧治療-------25
1.2.3.1 目前之臨床研究-------25
1.2.3.2 高張食鹽水氣霧作用機制-------26
1.2.4 目前常見之體外三維系統-------32
1.2.5尚未被滿足之臨床需求-------35
1.2.6 總結-------35
1.3 實驗計畫-------36
Chapter 2 材料與實驗方法-------39
2.1 實驗方法－主題一: 細菌於2D表面之探討-------39
2.1.1 細菌培養-------39
2.1.2 生物膜形成測試-------40
2.1.2.1 結晶紫(Crystal violet)染色測試-------40
2.1.2.2 Air-liquid interface之SEM測試-------40
2.1.3 細菌motility測試-------41
2.1.3.1 於Swarming agar上之測試-------41
2.1.3.2 顯微鏡time-lapse測試-------42
2.1.4 生物膜之EPS分布測試(P/C ratio)-------42
2.1.5 抗生素藥效測試-------43
2.2實驗方法－主題二: 細菌於3D微環境之探討-------44
2.2.1 細胞培養-------44
2.2.1.1 細胞培養於2D培養皿-------45
2.2.1.2 細胞培養於3D微流道晶片-------45
2.2.2 三維微球吸光值測定-------47
2.2.3 微球表面之痰液分泌量化與分析-------47
2.2.3.1 調控細胞微球大小分析痰液分泌-------47
2.2.3.2 細胞間作用與微球痰液分泌之關係測定-------48
2.2.4 氣液介面系統建立(ALI system)-------48
2.2.5 細胞微球與細菌共培養-------50
2.2.6 抗生素與高張食鹽水氣霧治療於細胞微球系統之驗證-------51
2.3 數據統計-------52
2.4 實驗藥品與儀器-------53
Chapter 3 結果與討論-------55
3.1細菌於二維表面之探討-------55
3.1.1 高張食鹽水氣霧治療會降低生物膜之形成-------55
3.1.2 高張食鹽水氣霧治療會改變細菌之運動模式及生物膜組成-------57
3.1.3 高張食鹽水氣霧合併抗生素治療可降低細菌生存率-------62
3.2細菌於三維微環境之探討-------64
3.2.1 三微細胞培養之建立-------64
3.2.2 細胞微球表面之痰液分泌量化-------67
3.2.3 細菌侵入細胞之現象探討-------72
3.2.4 高張食鹽水合併治療於三微系統之效果驗證-------77
Chapter 4 結論-------84
Chapter 5 未來展望-------85
5.1高張食鹽水氣霧治療-------85
5.2晶片建立之微球與氣液介面系統-------85
5.3總結-------86
Chapter 6參考文獻-------87
Chapter 7附錄-------93
7.1細菌於二維表面探討之補充數據-------93
7.2細菌於三維系統探討之補充數據-------96

1. Bartal, M., COPD and tobacco smoke. Monaldi archives for chest disease, 2005. 63(4).
2. Andersen, D.H. and R.G. HODGES, Celiac syndrome: V. genetics of cystic fibrosis of the pancreas with a consideration of etiology. American journal of diseases of children, 1946. 72(1): p. 62-80.
3. Burney, P., D. Jarvis, and R. Perez-Padilla, The global burden of chronic respiratory disease in adults. The International Journal of Tuberculosis and Lung Disease, 2015. 19(1): p. 10-20.
4. Nikaido, H., Multidrug resistance in bacteria. Annual review of biochemistry, 2009. 78: p. 119.
5. Kowalska-Krochmal, B. and R. Dudek-Wicher, The minimum inhibitory concentration of antibiotics: Methods, interpretation, clinical relevance. Pathogens, 2021. 10(2): p. 165.
6. Gupta, P. and A. Asha, The Price we Pay for Overdose of Antibiotics: Is there any Alternative? Infect Dis, 2018. 197(3): p. 435-438.
7. Banerjee, S. and S. McCormack, Acetylcysteine for patients requiring mucous secretion clearance: a review of clinical effectiveness and safety. 2019.
8. Hebestreit, A., U. Kersting, and H. Hebestreit, Hypertonic saline inhibits luminal sodium channels in respiratory epithelium. European journal of applied physiology, 2007. 100(2): p. 177-183.
9. Terlizzi, V., et al., Hypertonic saline in people with cystic fibrosis: review of comparative studies and clinical practice. Italian Journal of Pediatrics, 2021. 47(1): p. 1-7.
10. Wohnhaas, C.T., et al., Cigarette Smoke specifically affects small airway epithelial cell populations and triggers the expansion of inflammatory and squamous differentiation associated basal cells. International journal of molecular sciences, 2021. 22(14): p. 7646.
11. Yaghi, A., et al., Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects. Respiratory medicine, 2012. 106(8): p. 1139-1147.
12. Noone, A.-M., et al., Cancer Incidence and Survival Trends by Subtype Using Data from the Surveillance Epidemiology and End Results Program, 1992–2013Cancer Incidence and Survival Trends by Subtype, 1992–2013. Cancer Epidemiology, Biomarkers & Prevention, 2017. 26(4): p. 632-641.
13. Vento, S., F. Cainelli, and Z. Temesgen, Lung infections after cancer chemotherapy. The lancet oncology, 2008. 9(10): p. 982-992.
14. Jiang, M., Z. Zhang, and S. Zhao, Epidemiological characteristics and drug resistance analysis of multidrug-resistant Acinetobacter baumannii in a China hospital at a certain time. Polish Journal of Microbiology, 2014. 63(3): p. 275.
15. Rossolini, G. and E. Mantengoli, Treatment and control of severe infections caused by multiresistant Pseudomonas aeruginosa. Clinical Microbiology and infection, 2005. 11: p. 17-32.
16. Oteo, J., et al., Antibiotic resistance in 3113 blood isolates of Staphylococcus aureus in 40 Spanish hospitals participating in the European Antimicrobial Resistance Surveillance System (2000–2002). Journal of Antimicrobial Chemotherapy, 2004. 53(6): p. 1033-1038.
17. Armin, S., et al., Antimicrobial resistance patterns of Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus isolated from patients with nosocomial infections admitted to tehran hospitals. Archives of Pediatric Infectious Diseases, 2015. 3(4).
18. Bodey, G.P., et al., Fever and infection in leukemic patients. A study of 494 consecutive patients. Cancer, 1978. 41(4): p. 1610-1622.
19. Siegel, J.D., et al., Management of multidrug-resistant organisms in health care settings, 2006. American journal of infection control, 2007. 35(10): p. S165-S193.
20. Stoodley, P., et al., Biofilms as complex differentiated communities. Annual Reviews in Microbiology, 2002. 56(1): p. 187-209.
21. Costerton, W., et al., The application of biofilm science to the study and control of chronic bacterial infections. The Journal of clinical investigation, 2003. 112(10): p. 1466-1477.
22. Joo, H.-S. and M. Otto, Molecular basis of in vivo biofilm formation by bacterial pathogens. Chemistry & biology, 2012. 19(12): p. 1503-1513.
23. Macia, M., E. Rojo-Molinero, and A. Oliver, Antimicrobial susceptibility testing in biofilm-growing bacteria. Clinical Microbiology and Infection, 2014. 20(10): p. 981-990.
24. Lu, L., et al., Developing natural products as potential anti-biofilm agents. Chinese medicine, 2019. 14(1): p. 1-17.
25. Cornforth, D.M. and K.R. Foster, Competition sensing: the social side of bacterial stress responses. Nature Reviews Microbiology, 2013. 11(4): p. 285-293.
26. T Garrison, A. and R. W Huigens III, Eradicating bacterial biofilms with natural products and their inspired analogues that operate through unique mechanisms. Current topics in medicinal chemistry, 2017. 17(17): p. 1954-1964.
27. SHIBUYA, Y., P.J. Wills, and P.J. Cole, Effect of osmolality on mucociliary transportability and rheology of cystic fibrosis and bronchiectasis sputum. Respirology, 2003. 8(2): p. 181-185.
28. Kellett, F., J. Redfern, and R.M. Niven, Evaluation of nebulised hypertonic saline (7%) as an adjunct to physiotherapy in patients with stable bronchiectasis. Respiratory medicine, 2005. 99(1): p. 27-31.
29. Elkins, M.R., et al., A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. New England Journal of Medicine, 2006. 354(3): p. 229-240.
30. Cone, R.A., Barrier properties of mucus. Advanced drug delivery reviews, 2009. 61(2): p. 75-85.
31. Boegh, M. and H.M. Nielsen, Mucus as a barrier to drug delivery–understanding and mimicking the barrier properties. Basic & clinical pharmacology & toxicology, 2015. 116(3): p. 179-186.
32. Reeves, E.P., et al., Hypertonic saline in treatment of pulmonary disease in cystic fibrosis. The Scientific World Journal, 2012. 2012.
33. Ridley, C., et al., Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin. Journal of Biological Chemistry, 2014. 289(23): p. 16409-16420.
34. Raynal, B.D., et al., Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus. Journal of Biological Chemistry, 2003. 278(31): p. 28703-28710.
35. Matsui, H., et al., Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces. The journal of immunology, 2005. 175(2): p. 1090-1099.
36. Francis, I., et al., Recent advances in lung-on-a-chip models. Drug Discovery Today, 2022.
37. Zhang, M., et al., A 3D human lung-on-a-chip model for nanotoxicity testing. Toxicology research, 2018. 7(6): p. 1048-1060.
38. Huh, D., A human breathing lung-on-a-chip. Annals of the American Thoracic Society, 2015. 12(Supplement 1): p. S42-S44.
39. Huh, D., et al., A human disease model of drug toxicity–induced pulmonary edema in a lung-on-a-chip microdevice. Science translational medicine, 2012. 4(159): p. 159ra147-159ra147.
40. Flemming, H.-C. and J. Wingender, The biofilm matrix. Nature reviews microbiology, 2010. 8(9): p. 623-633.
41. Berry, D., C. Xi, and L. Raskin, Effect of growth conditions on inactivation of Escherichia coli with monochloramine. Environmental science & technology, 2009. 43(3): p. 884-889.
42. Otto, K., H. Elwing, and M. Hermansson, Effect of ionic strength on initial interactions of Escherichia coli with surfaces, studied on-line by a novel quartz crystal microbalance technique. Journal of bacteriology, 1999. 181(17): p. 5210-5218.
43. Pavlovsky, L., J.G. Younger, and M.J. Solomon, In situ rheology of Staphylococcus epidermidis bacterial biofilms. Soft matter, 2013. 9(1): p. 122-131.
44. Kearns, D.B., A field guide to bacterial swarming motility. Nature Reviews Microbiology, 2010. 8(9): p. 634-644.
45. Vijayakumar, S., et al., Biofilm formation and motility depend on the nature of the Acinetobacter baumannii clinical isolates. Frontiers in public health, 2016. 4: p. 105.
46. Ronish, L.A., et al., The structure of PilA from Acinetobacter baumannii AB5075 suggests a mechanism for functional specialization in Acinetobacter type IV pili. Journal of Biological Chemistry, 2019. 294(1): p. 218-230.
47. Guttenplan, S.B. and D.B. Kearns, Regulation of flagellar motility during biofilm formation. FEMS microbiology reviews, 2013. 37(6): p. 849-871.
48. Caiazza, N.C., et al., Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. Journal of bacteriology, 2007. 189(9): p. 3603-3612.
49. Quigg, A., et al., Direct and indirect toxic effects of engineered nanoparticles on algae: role of natural organic matter. ACS Sustainable Chemistry & Engineering, 2013. 1(7): p. 686-702.
50. Bhaskar, P., et al., Production of macroaggregates from dissolved exopolymeric substances (EPS) of bacterial and diatom origin. FEMS Microbiology Ecology, 2005. 53(2): p. 255-264.
51. Tsuneda, S., et al., Extracellular polymeric substances responsible for bacterial adhesion onto solid surface. FEMS microbiology letters, 2003. 223(2): p. 287-292.
52. Nguyen, P.-T., et al., Exopolysaccharide production by lactic acid bacteria: the manipulation of environmental stresses for industrial applications. AIMS microbiology, 2020. 6(4): p. 451.
53. Guo, Y.-S., et al., Bacterial extracellular polymeric substances amplify water content variability at the pore scale. Frontiers in Environmental Science, 2018. 6: p. 93.
54. Zeidler, S., et al., Trehalose, a temperature‐and salt‐induced solute with implications in pathobiology of Acinetobacter baumannii. Environmental microbiology, 2017. 19(12): p. 5088-5099.
55. Harimawan, A., A. Rajasekar, and Y.-P. Ting, Bacteria attachment to surfaces–AFM force spectroscopy and physicochemical analyses. Journal of Colloid and Interface Science, 2011. 364(1): p. 213-218.
56. Davies, D., Understanding biofilm resistance to antibacterial agents. Nature reviews Drug discovery, 2003. 2(2): p. 114-122.
57. Santschi, P.H., et al., Can the protein/carbohydrate (P/C) ratio of exopolymeric substances (EPS) be used as a proxy for their ‘stickiness’ and aggregation propensity? Marine Chemistry, 2020. 218: p. 103734.
58. Panchatcharam, B.S., et al. Staphylococcus aureus biofilm exoproteins are cytotoxic to human nasal epithelial barrier in chronic rhinosinusitis. in International Forum of Allergy & Rhinology. 2020. Wiley Online Library.
59. Gil, C., et al., Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection. Infection and immunity, 2014. 82(3): p. 1017-1029.
60. Azghani, A.O., et al., Pseudomonas aeruginosa outer membrane protein F is an adhesin in bacterial binding to lung epithelial cells in culture. Microbial pathogenesis, 2002. 33(3): p. 109-114.
61. Kapałczyńska, M., et al., 2D and 3D cell cultures–a comparison of different types of cancer cell cultures. Archives of Medical Science, 2018. 14(4): p. 910-919.
62. Vadivelu, R.K., et al., Microfluidic technology for the generation of cell spheroids and their applications. Micromachines, 2017. 8(4): p. 94.
63. Keller, G.M., In vitro differentiation of embryonic stem cells. Current opinion in cell biology, 1995. 7(6): p. 862-869.
64. Sandu, I. and C.T. Fleaca, The influence of gravity on the distribution of the deposit formed onto a substrate by sessile, hanging, and sandwiched hanging drop evaporation. Journal of colloid and interface science, 2011. 358(2): p. 621-625.
65. Barr, J.J., et al., Bacteriophage adhering to mucus provide a non–host-derived immunity. Proceedings of the National Academy of Sciences, 2013. 110(26): p. 10771-10776.
66. Croce, M.V., et al., Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line (A549). Pathology and Oncology Research, 1999. 5(3): p. 197-204.
67. Reid, L., Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis. Thorax, 1960. 15(2): p. 132.
68. Widdicombe, J.H. and J.J. Wine, Airway gland structure and function. Physiological reviews, 2015. 95(4): p. 1241-1319.
69. Riento, K. and A.J. Ridley, Rocks: multifunctional kinases in cell behaviour. Nature reviews Molecular cell biology, 2003. 4(6): p. 446-456.
70. Kasai, Y., et al., ROCK inhibitor combined with Ca2+ controls the myosin II activation and optimizes human nasal epithelial cell sheets. Scientific reports, 2020. 10(1): p. 1-13.
71. Gindele, J.A., et al., Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture. Scientific reports, 2020. 10(1): p. 1-17.
72. Gaddy, J.A., A.P. Tomaras, and L.A. Actis, The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infection and immunity, 2009. 77(8): p. 3150-3160.
73. Grassart, A., et al., Bioengineered human organ-on-chip reveals intestinal microenvironment and mechanical forces impacting Shigella infection. Cell host & microbe, 2019. 26(3): p. 435-444. e4.
74. Decramer, M., et al., Effects of N-acetylcysteine on outcomes in chronic obstructive pulmonary disease (Bronchitis Randomized on NAC Cost-Utility Study, BRONCUS): a randomised placebo-controlled trial. The Lancet, 2005. 365(9470): p. 1552-1560.
75. Yuan, S., et al., Oxidation increases mucin polymer cross-links to stiffen airway mucus gels. Science translational medicine, 2015. 7(276): p. 276ra27-276ra27.
76. Denneny, E., et al., Mucins and their receptors in chronic lung disease. Clinical & translational immunology, 2020. 9(3): p. e01120.
77. Yuta, A. and J.N. Baraniuk, Therapeutic approaches to mucus hypersecretion. Current allergy and asthma reports, 2005. 5(3): p. 243-251.
78. Aldini, G., et al., N-Acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why. Free radical research, 2018. 52(7): p. 751-762.
79. Tomkiewicz, R., A. Biviji, and M. King, Effects of oscillating air flow on the rheological properties and clearability of mucous gel simulants. Biorheology, 1994. 31(5): p. 511-520.
80. Zafarullah, M., et al., Molecular mechanisms of N-acetylcysteine actions. Cellular and Molecular Life Sciences CMLS, 2003. 60(1): p. 6-20.
81. Dinicola, S., et al., N-acetylcysteine as powerful molecule to destroy bacterial biofilms. A systematic review. Eur Rev Med Pharmacol Sci, 2014. 18(19): p. 2942-2948.