A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance.

Lymphangioleiomyomatosis (LAM) is a debilitating, progressive lung disease with few therapeutic options, largely due to a paucity of mechanistic knowledge of disease pathogenesis. Lymphatic endothelial cells (LECs) are known to envelope and invade clusters of LAM-cells, comprising of smooth muscle α-actin and/or HMB-45 positive “smooth muscle-like cells” however the role of LECs in LAM pathogenesis is still unknown. To address this critical knowledge gap, we investigated wether LECs interact with LAM-cells to augment their metastatic behaviour of LAM-cells. We performed in situ spatialomics and identified a core of transcriptomically related cells within the LAM nodules. Pathway analysis highlights wound and pulmonary healing, VEGF signaling, extracellular matrix/actin cytoskeletal regulating and the HOTAIR regulatory pathway enriched in the LAM Core cells. We developed an organoid co-culture model combining primary LAM-cells with LECs and applied this to evaluate invasion, migration, and the impact of Sorafenib, a multi-kinase inhibitor. LAM-LEC organoids had significantly higher extracellular matrix invasion, decreased solidity and a greater perimeter, reflecting increased invasion compared to non-LAM control smooth muscle cells. Sorafenib significantly inhibited this invasion in both LAM spheroids and LAM-LEC organoids compared to their respective controls. We identified TGFβ1ι1, a molecular adapter coordinating protein-protein interactions at the focal adhesion complex and known to regulate VEGF, TGFβ and Wnt signalling, as a Sorafenib-regulated kinase in LAM-cells. In conclusion we have developed a novel 3D co-culture LAM model and have demonstrated the effectiveness of Sorafenib to inhibit LAM-cell invasion, identifying new avenues for therapeutic intervention.

The airway epithelium provides a physical and biochemical barrier playing a key role in protecting the lung from infiltration of pathogens and irritants and is, therefore, crucial in maintaining tissue homeostasis and regulating innate immunity. Due to continual inspiration and expiration of air during breathing, the epithelium is exposed to a plethora of environmental insults. When severe or persistent, these insults lead to inflammation and infection. The effectiveness of the epithelium as a barrier is reliant upon its capacity for mucociliary clearance, immune surveillance, and regeneration upon injury. These functions are accomplished by the cells that comprise the airway epithelium and the niche in which they reside. Engineering of new physiological and pathological models of the proximal airways requires the generation of complex structures comprising the surface airway epithelium, submucosal gland epithelium, extracellular matrix, and niche cells, including smooth muscle cells, fibroblasts, and immune cells. This chapter focuses on the structure-function relationships in the airways and the challenges of developing complex engineered models of the human airway.

Acinetobacter baumannii is an opportunistic pathogen known for high morbidity and mortality. It causes life-threatening infections, such as ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound and urinary tract infections (UTI). Increase in carbapenem resistance exhibited by A. baumannii has accentuated the need for novel targets for effective treatment. Despite the pronounced relevance of PPK2 as a pathogenicity determinant in several pathogens, it has not been explored as a drug target in A. baumannii. The present study was piloted to investigate the substrate binding by A. baumannii PPK2 (AbPPK2), a two-domain Class II polyphosphate kinase 2. A homology model of AbPPK2 was developed and validated for molecular docking of ATP and ADP in the predicted binding pocket. Further analysis of AbPPK2 revealed a set of common residues in the catalytic cleft interacting with ATP and ADP which would be useful for the screening of inhibitors against A. baumannii.

Cervical cancer is a major cause of gynecological related mortalities in developing countries. Cisplatin, a potent chemotherapeutic agent used for treating advanced cervical cancer exhibits side effects and resistance development. The current study was aimed to investigate the repurposing of l-menthol as a potential therapeutic drug against cervical cancer. L-menthol was predicted to be non-toxic with good pharmacokinetic properties based on SwissADME and pkCSM analysis. Subsequently, 543 and 1664 targets of l-menthol and cervical cancer were identified using STITCH, BATMAN-TCM, PharmMapper and CTD databases. STRING and Cytoscape analysis of the merged protein–protein interaction network revealed 107 core targets of l- menthol against cervical cancer. M-CODE identified highly connected clusters between the core targets which through KEGG analysis were found to be enriched in pathways related to apoptosis and adherence junctions. Molecular docking showed that l- menthol targeted E6, E6AP and E7 onco-proteins of HPV that interact and inactivate TP53 and Rb1 in cervical cancer, respectively. Molecular docking also showed good binding affinity of l-menthol toward proteins associated with apoptosis and migration. Molecular dynamics simulation confirmed stability of the docked complexes. In vitro analysis confirmed that l-menthol was cytotoxic towards cervical cancer CaSki cells and altered expression of TP53, Rb1, CDKN1A, E2F1, NFKB1, Akt-1, caspase-3, CDH1 and MMP-2 genes identified through network pharmacology approach.

Acinetobacter baumannii is one of the most significant nosocomial pathogen responsible for substantial morbidity and mortality due to multidrug resistance. Polyphosphates (polyP) are important for the production of virulence factors and stress management in bacteria. PolyP is synthesized by polyphosphate kinase (PPK) which is highly conserved among microorganisms. In this study polyphosphate kinase 1 of A. baumannii ATCC 17978 (AbPPK1) was cloned and expressed in E. coli BL21 (DE3). Enzyme activity of 80 kDa recombinant AbPPK1 and its mutants, AbPPK1H443P and AbPPK1H443P-H601A was 299.86±66, 236.14±13.8 and 121±8.7 μM/min, respectively. Reduced activity of mutants highlighted the importance of active site histidines at position 443 and 601 for AbPPK1 activity. Virtual screening of synthetic and natural compounds and FDA approved drugs allowed the selection of etoposide and genistein which competitively inhibited AbPPK1 at IC50 of 27.53 and 24.38 μM, respectively. These molecules reduced the production of virulence factors in A. baumannii. There was significant (p≤0.05) reduction in biofilm formation and surface motility in A. baumannii ATCC 17978 and the clinical isolates. Genistein in combination with tobramycin further reduced biofilm formation in A. baumannii. Etoposide and genistein led to decrease in survival of A. baumannii under desiccation conditions. RNA polymerase sigma factor, rpoS, critical for survival and virulence, was down-regulated by etoposide and gensitein. The expression of exopolyphosphatase (PPX), polyP degrading enzyme was increased with concomitant reduction in cellular polyP level, critical for the production of virulence determinants. In the absence of PPK orthologue in humans, etoposide and genistein could be sought as novel therapeutic option to supplement the existing antibiotics against A. baumannii.

Acinetobacter baumannii is clinically one of the most significant pathogens, especially in intensive care settings, because of its multidrug-resistance (MDR). Repurposing of high-affinity drugs is a faster and more plausible approach for combating the emergence of MDR and to tackle bacterial infections. This study was aimed to evaluate the approved drugs potentially inhibiting A. baumannii PPK1 (AbPPK1) mediated synthesis of polyphosphates (polyP). Based on virtual screening, molecular dynamic simulation, and CD spectroscopy for thermal stability, two stable ligands, etoposide and genistein, were found with promising contours for further investigation. Following in vitro inhibition of AbPPK1, the efficacy of selected drugs was further tested against virulence traits of A. baumannii. These drugs significantly reduced the biofilm formation, surface motility in A. baumannii and led to decreased survival under desiccation. In addition to inhibition of PPK1, both drugs increased the expression of polyP degrading enzyme, exopolyphosphatase (PPX), that might be responsible for the decrease in the total cellular polyP. Since polyP modulates the virulence factors in bacteria, destabilization of the polyP pool by these drugs seems particularly striking for their therapeutic applications against A. baumannii.

BACKGROUND: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. OBJECTIVE: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. METHODS: 3D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn- MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. RESULTS: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. CONCLUSION: The study provides insights into structural and functional features of novel toxin, Xn- MazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.

Toxin-antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT - a RelB homolog of E. coli. The structure for this toxin-antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein-nucleic acid, protein-protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl β-D-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.