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The relationship between the use of tobacco products and SARS-CoV-2 infection is poorly understood and controversial. Few studies have examined the effect of electronic cigarettes (ECs) on SARS-CoV-2 infection. We tested the hypothesis that EC fluids and aerosols with nicotine promote SARS-COV-2 infection by increasing viral entry into human respiratory epithelial cells. Responses of BEAS-2B cells to JUUL aerosols or their individual constituents were compared using three exposure platforms: submerged culture, air-liquid-interface (ALI) exposure in a cloud chamber, and ALI exposure in a Cultex system, which produces authentic heated EC aerosols. In general, nicotine and nicotine + propylene glycol/vegetable glycerin aerosols increased ACE2 (angiotensin converting enzyme 2) levels, the SARS-CoV-2 receptor; and increased the activity of TMPRSS2 (transmembrane serine protease 2), an enzyme essential for viral entry. Lentivirus pseudoparticles with spike protein were used to test viral penetration. Exposure to nicotine, EC fluids, or aerosols altered the infection machinery and increased viral entry into cells. While most data were in good agreement across the three exposure platforms, cells were more responsive to treatments when exposed at the ALI in the Cultex system, even though the exposures were brief and intermittent. While both nicotine and JUUL aerosols increased SARS-CoV-2 infection, JUUL significantly decreased the effect of nicotine alone. These data support the idea that vaping can increase the likelihood of contracting COVID-19 and that e-liquid composition may modulate this effect.

Oral health is a part of general health and the two are connected in many ways as well as impact each other. Oral diseases are some of the most common chronic diseases of mankind. Diseases such as caries and periodontitis are two of the most common ones affecting the oral cavity. Bacteria associated with these two diseases are Streptococcus mutans and Aggregatibacter actinomycetemcomitans respectively. Our hypothesis is that extracts from guava leaf or matcha tea affect adhesion of A. actinomycetecomitans or S. mutans to human epithelium. The aim of this study is to investigate a DNA-based method for studying attachment of bacteria to epithelial cells. Two different concentrations of the two bacterial species, high and low, were treated with matcha or guava leaf extract and adhesion on the human epithelial cell cultures was analyzed. The results were then analyzed using qPCR-based methods to test the amount of bacterial adhesion to the epithelial cells. Furthermore, the results showed that matcha was more effective for inhibition of bacterial adhesion than guava leaf.In conclusion, our results show that bacterial adhesion of A. actinomycetecomitans and S. mutans to human epithelial cells can be quantified by DNA-based methods, and the adhesion altered by plant extracts.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

The observed sex disparity in bladder cancer (BlCa) argues that androgen receptor (AR) signaling has a role in these malignancies. BlCas express full-length AR (FL-AR), constitutively active AR splice variants, including AR-v19, or both, and their depletion limits BlCa viability. However, the mechanistic basis of AR-dependence is unknown. Here, we depleted FL-AR, AR-v19, or all AR forms (T-AR), and performed RNA-seq studies to uncover that different AR forms govern distinct but partially overlapping transcriptional programs. Overlapping alterations include a decrease in mTOR and an increase of hypoxia regulated transcripts accompanied by a decline in oxygen consumption rate (OCR). Queries of BlCa databases revealed a significant negative correlation between AR expression and multiple hypoxia-associated transcripts arguing that this regulatory mechanism is a feature of high-grade malignancies. Our analysis of a 1600-compound library identified niclosamide as a strong ATPase inhibitor that reduces OCR in BlCa cells, decreased cell viability and induced apoptosis in a dose and time dependent manner. These results suggest that BlCa cells hijack AR signaling to enhance metabolic activity, promoting cell proliferation and survival; hence targeting this AR downstream vulnerability presents an attractive strategy to limit BlCa.

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

Macrophages intimately interact with intestinal epithelial cells, but the consequences of defective macrophage-epithelial cell interactions for protection against enteric pathogens are poorly understood. Here, we show that in mice with a deletion in protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in macrophages, infection with Citrobacter rodentium, a model of enteropathogenic and enterohemorrhagic E. coli infection in humans, promoted a strong type 1/IL-22-driven immune response, culminating in accelerated disease but also faster clearance of the pathogen. In contrast, deletion of PTPN2 specifically in epithelial cells rendered the epithelium unable to upregulate antimicrobial peptides and consequently resulted in a failure to eliminate the infection. The ability of PTPN2-deficient macrophages to induce faster recovery from C. rodentium was dependent on macrophage-intrinsic IL-22 production, which was highly increased in macrophages deficient in PTPN2. Our findings demonstrate the importance of macrophage-mediated factors, and especially macrophage-derived IL-22, for the induction of protective immune responses in the intestinal epithelium, and show that normal PTPN2 expression in the epithelium is crucial to allow for protection against enterohemorrhagic E. coli and other intestinal pathogens.

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically restricted, evolutionarily conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light on how spindle orientation influences epidermal stratification.

Squamous cell lung cancer maintains its growth through elevated glucose consumption, but selective glucose consumption inhibitors are lacking. Here, we discovered using a high-throughput screen new compounds that block glucose consumption in three squamous cell lung cancer cell lines and identified 79 compounds that block glucose consumption in one or more of these cell lines. Based on its ability to block glucose consumption in all three cell lines, pacritinib, an inhibitor of FMS Related Receptor Tyrosine Kinase 3 (FLT3) and Janus Kinase 2 (JAK2), was further studied. Pacritinib decreased glucose consumption in squamous cell lung cancer cells in cell culture and in vivo without affecting glucose consumption in healthy tissues. Pacritinib blocked hexokinase activity, and Hexokinase 1 and 2 mRNA and protein expression. Overexpression of Hexokinase 1 blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Overexpression of FLT3 but not JAK2 significantly increased glucose consumption and blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Additional FLT3 inhibitors blocked glucose consumption in squamous cell lung cancer cells. Our study identifies FLT3 inhibitors as a new class of inhibitors that can block glucose consumption in squamous cell lung cancer.

Tuft cells are found in tissues with distinct stem cell compartments, tissue architecture, and luminal exposures but converge on a shared transcriptional program, including expression of taste transduction signaling pathways. Here, we summarize seminal and recent findings on tuft cells, focusing on major categories of function-instigation of type 2 cytokine responses, orchestration of antimicrobial responses, and emerging roles in tissue repair-and describe tuft cell-derived molecules used to affect these functional programs. We review what is known about the development of tuft cells from epithelial progenitors under homeostatic conditions and during disease. Finally, we discuss evidence that immature, or nascent, tuft cells with potential for diverse functions are driven toward dominant effector programs by tissue- or perturbation-specific contextual cues, which may result in heterogeneous mature tuft cell phenotypes both within and between tissues.

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

U2AF1 is one of the most recurrently mutated splicing factors in lung adenocarcinoma and has been shown to cause transcriptome-wide pre-mRNA splicing alterations; however, the full-length altered mRNA isoforms associated with the mutation are largely unknown. To better understand the impact U2AF1 has on full-length isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without a U2AF1 S34F mutation. We identified 49,366 multi-exon transcript isoforms, more than half of which did not match GENCODE or short-read-assembled isoforms. We found 198 transcript isoforms with significant expression and usage changes relative to WT, only 68% of which were assembled by short reads. Expression of isoforms from immune-related genes is largely down-regulated in mutant cells and without observed splicing changes. Finally, we reveal that isoforms likely targeted by nonsense-mediated decay are down-regulated in U2AF1 S34F cells, suggesting that isoform changes may alter the translational output of those affected genes. Altogether, our work provides a resource of full-length isoforms associated with U2AF1 S34F in lung cells.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamicin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical wi

The definitive treatment for end-stage renal disease is kidney transplantation, which remains limited by organ availability and post-transplant complications. Alternatively, an implantable bioartificial kidney could address both problems while enhancing the quality and length of patient life. An implantable bioartificial kidney requires a bioreactor containing renal cells to replicate key native cell functions, such as water and solute reabsorption, and metabolic and endocrinologic functions. Here, we report a proof-of-concept implantable bioreactor containing silicon nanopore membranes to offer a level of immunoprotection to human renal epithelial cells. After implantation into pigs without systemic anticoagulation or immunosuppression therapy for 7 days, we show that cells maintain >90% viability and functionality, with normal or elevated transporter gene expression and vitamin D activation. Despite implantation into a xenograft model, we find that cells exhibit minimal damage, and recipient cytokine levels are not suggestive of hyperacute rejection. These initial data confirm the potential feasibility of an implantable bioreactor for renal cell therapy utilizing silicon nanopore membranes.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.

L'objectiu d'aquest treball és l'estudi de la forma d'una única cèl·lula respectant alguns paràmetres presentats en el funcional d'energia, així com l'anàlisi de sistemes de dues cèl·lules, s'imposin o no condicions de simetria. Es mostren diversos resultats sobre l'existència de valors límit i contrastant els resultats amb els models numèrics., El objetivo de este trabajo es estudiar la forma de una sola célula respetando algunos parámetros presentados en el funcional de energía, así como el análisis de sistemas de dos células, se impongan o no condiciones de simetría. Se muestran diversos resultados abordando la existencia de valores límite, y contrastando los resultados con los modelos numéricos., The aim of this work is to study the shape of a single cell respect some parameters presented in the energy functional, as well as the analysis of systems of two cells, whether symmetrical conditions are imposed or not. It is shown various results addressing the existence of limit values, and contrasting the results with the numerical models.

Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamicin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical

Cell layers eliminate unwanted cells through the extrusion process, which underlines healthy versus flawed tissue behaviors. Although several biochemical pathways have been identified, the underlying mechanical basis including the forces involved in cellular extrusion remains largely unexplored. Utilizing a phase-field model of a three-dimensional cell layer, we study the interplay of cell extrusion with cell-cell and cell-substrate interactions in a flat monolayer. Independent tuning of cell-cell versus cell-substrate adhesion forces reveals that extrusion events can be distinctly linked to defects in nematic and hexatic orders associated with cellular arrangements. Specifically, we show that by increasing relative cell-cell adhesion forces the cell monolayer can switch between the collective tendency towards fivefold, hexatic, disclinations relative to half-integer, nematic, defects for extruding a cell. We unify our findings by accessing three-dimensional mechanical stress fields to show that an extrusion event acts as a mechanism to relieve localized stress concentration.

Coordinated rotational motion is an intriguing, yet still elusive mode of collective cell migration, which is relevant in pathological and morphogenetic processes. Most of the studies on this topic have been carried out on epithelial cells plated on micropatterned substrates, where cell motion is confined in regions of well-defined shapes coated with extracellular matrix adhesive proteins. The driver of collective rotation in such conditions has not been clearly elucidated, although it has been speculated that spatial confinement can play an essential role in triggering cell rotation. Here, we study the growth of epithelial cell colonies freely expanding (i.e. with no physical constraints) on the surface of cell culture plates and focus on collective cell rotation in such conditions, a case which has received scarce attention in the literature. One of the main findings of our work is that coordinated cell rotation spontaneously occurs in cell clusters in the free growth regime, thus implying that cell confinement is not necessary to elicit collective rotation as previously suggested. The extent of collective rotation was size and shape dependent: a highly coordinated disc-like rotation was found in small cell clusters with a round shape, while collective rotation was suppressed in large irregular cell clusters generated by merging of different clusters in the course of their growth. The angular motion was persistent in the same direction, although clockwise and anticlockwise rotations were equally likely to occur among different cell clusters. Radial cell velocity was quite low as compared to the angular velocity, in agreement with the free expansion regime where cluster growth is essentially governed by cell proliferation. A clear difference in morphology was observed between cells at the periphery and the ones in the core of the clusters, the former being more elongated and spread out as compared to the latter. Overall, our results, to our knowledge, provide th

Cell layers eliminate unwanted cells through the extrusion process, which underlines healthy versus flawed tissue behaviors. Although several biochemical pathways have been identified, the underlying mechanical basis including the forces involved in cellular extrusion remains largely unexplored. Utilizing a phase-field model of a three-dimensional cell layer, we study the interplay of cell extrusion with cell-cell and cell-substrate interactions in a flat monolayer. Independent tuning of cell-cell versus cell-substrate adhesion forces reveals that extrusion events can be distinctly linked to defects in nematic and hexatic orders associated with cellular arrangements. Specifically, we show that by increasing relative cell-cell adhesion forces the cell monolayer can switch between the collective tendency towards fivefold, hexatic, disclinations relative to half-integer, nematic, defects for extruding a cell. We unify our findings by accessing three-dimensional mechanical stress fields to show that an extrusion event acts as a mechanism to relieve localized stress concentration.

Coordinated rotational motion is an intriguing, yet still elusive mode of collective cell migration, which is relevant in pathological and morphogenetic processes. Most of the studies on this topic have been carried out on epithelial cells plated on micropatterned substrates, where cell motion is confined in regions of well-defined shapes coated with extracellular matrix adhesive proteins. The driver of collective rotation in such conditions has not been clearly elucidated, although it has been speculated that spatial confinement can play an essential role in triggering cell rotation. Here, we study the growth of epithelial cell colonies freely expanding (i.e. with no physical constraints) on the surface of cell culture plates and focus on collective cell rotation in such conditions, a case which has received scarce attention in the literature. One of the main findings of our work is that coordinated cell rotation spontaneously occurs in cell clusters in the free growth regime, thus implying that cell confinement is not necessary to elicit collective rotation as previously suggested. The extent of collective rotation was size and shape dependent: a highly coordinated disc-like rotation was found in small cell clusters with a round shape, while collective rotation was suppressed in large irregular cell clusters generated by merging of different clusters in the course of their growth. The angular motion was persistent in the same direction, although clockwise and anticlockwise rotations were equally likely to occur among different cell clusters. Radial cell velocity was quite low as compared to the angular velocity, in agreement with the free expansion regime where cluster growth is essentially governed by cell proliferation. A clear difference in morphology was observed between cells at the periphery and the ones in the core of the clusters, the former being more elongated and spread out as compared to the latter. Overall, our results, to our knowledge, provide th

We engineered an ultrasensitive reporter of p16INK4a, a biomarker of cellular senescence. Our reporter detected p16INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.

Biodiesel is created through the transesterification of fats/oils and its usage is increasing worldwide as global warming concerns increase. Biodiesel fuel properties change depending on the feedstock used to create it. The aim of this study was to assess the different toxicological properties of biodiesel exhausts created from different feedstocks using a complex 3D air-liquid interface (ALI) model that mimics the human airway. Primary human airway epithelial cells were grown at ALI until full differentiation was achieved. Cells were then exposed to 1/20 diluted exhaust from an engine running on Diesel (ULSD), pure or 20% blended Canola biodiesel and pure or 20% blended Tallow biodiesel, or Air for control. Exhaust was analysed for various physio-chemical properties and 24-h after exposure, ALI cultures were assessed for permeability, protein release and mediator response. All measured exhaust components were within industry safety standards. ULSD contained the highest concentrations of various combustion gases. We found no differences in terms of particle characteristics for any of the tested exhausts, likely due to the high dilution used. Exposure to Tallow B100 and B20 induced increased permeability in the ALI culture and the greatest increase in mediator response in both the apical and basal compartments. In contrast, Canola B100 and B20 did not impact permeability and induced the smallest mediator response. All exhausts but Canola B20 induced increased protein release, indicating epithelial damage. Despite the concentrations of all exhausts used in this study meeting industry safety regulations, we found significant toxic effects. Tallow biodiesel was found to be the most toxic of the tested fuels and Canola the least, both for blended and pure biodiesel fuels. This suggests that the feedstock biodiesel is made from is crucial for the resulting health effects of exhaust exposure, even when not comprising the majority of fuel composition.

Animals have evolved mechanisms, such as cell competition, to remove dangerous or nonfunctional cells from a tissue. Tumor necrosis factor signaling can eliminate clonal malignancies from Drosophila imaginal epithelia, but why this pathway is activated in tumor cells but not normal tissue is unknown. We show that the ligand that drives elimination is present in basolateral circulation but remains latent because it is spatially segregated from its apically localized receptor. Polarity defects associated with malignant transformation cause receptor mislocalization, allowing ligand binding and subsequent apoptotic signaling. This process occurs irrespective of the neighboring cells' genotype and is thus distinct from cell competition. Related phenomena at epithelial wound sites are required for efficient repair. This mechanism of polarized compartmentalization of ligand and receptor can generally monitor epithelial integrity to promote tissue homeostasis.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

PurposeInvestigate whether adjuvant everolimus, an mTOR inhibitor, improves progression-free survival (PFS) in advanced-stage head and neck squamous cell carcinoma (HNSCC) and provide outcomes related to correlative biological factors associated with disease control.Patients and methodsThis was a prospective, randomized, double-blind phase II trial of patients with advanced-stage HNSCC from 13 institutions who were confirmed disease-free post-definitive therapy and enrolled between December 2010 and March 2015. Patients received adjuvant everolimus or placebo daily (10 mg, oral) for a maximum of 1 year. p16 IHC as a surrogate marker for human papillomavirus infection and whole-exome sequencing were performed. Cox proportional hazard models estimated hazard rates. Log-rank tests evaluated differences in survival. The primary endpoint was PFS. Secondary endpoints and objectives included overall survival (OS) and toxicity assessment.Results52 patients [median (range) age, 58 (37-76) years; 43 men (83%), 9 women (17%)] were randomized to placebo (n = 24) or everolimus (n = 28). PFS favored everolimus, but was not significant [log-rank P = 0.093; HR = 0.44; 95% confidence interval (CI), 0.17-1.17]. There was no difference in OS (P = 0.29; HR = 0.57; 95% CI, 0.20-16.2). Everolimus resulted in significant improvement in PFS for p16-negative patients (n = 31; P = 0.031; HR = 0.26; 95% CI, 0.07-0.97), although subgroup analysis showed no difference for p16-positive patients (n = 21; P = 0.93). Further, PFS was significantly higher in TP53-mutated (TP53mut) patients treated with everolimus compared with placebo (log-rank P = 0.027; HR = 0.24; 95% CI, 0.06-0.95). No treatment difference was seen in patients with TP53 wild-type tumors (P = 0.79).Conclusionsp16-negative and TP53mut patients may benefit from adjuvant treatment with everolimus.

Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

Ciliated cell markers expressed in epithelial ovarian cancers (EOC) are associated with improved survival. We examined the distribution of cells expressing ciliated cell markers in various EOC histologies and stages. Immunohistochemistry and/or multiplex immunofluorescence were used to determine the expression of FOXJ1 and/or CAPS (ciliated cell markers) in tissue microarrays including 4 normal fallopian tubes, 6 normal endometria, 16 cystadenomas, 25 borderline tumors, 21 low-grade carcinomas, and 118 high-grade carcinomas (HGSOC) (46 serous, 29 endometrioid, 30 clear cell, 13 mucinous). CAPS+ cells were observed in normal fallopian tubes and endometria and in ~85% of serous benign and borderline tumors and low-grade carcinomas but only in <40% of HGSOC. mRNA data from an independent cohort showed higher FOXJ1 and CAPS expression in serous borderline tumors and low-grade carcinomas compared to HGSOC. In HGSOC, ciliated cell-positive markers were observed in 52% primary tumors compared to 26% of patient-matched synchronous metastases, and 24% metachronous metastases (p = 0.009). mRNA data from an independent HGSOC cohort showed lower levels of CAPS in metastases than in primary tumors (p = 0.03). Overall, the study revealed that ciliated cells were less common in mucinous EOC, the percentage of ciliated cell marker-positive cases decreased with increasing grade, and the percentage of ciliated cells decreased in HGSOC metastases compared to patient-matched primary tumors.

The behaviors of infectious bacteria are commonly studied in bulk. This is effective to define the general properties of a given isolate, but insufficient to resolve subpopulations and unique single-microbe behaviors within the bacterial pool. We here employ microscopy to study single-bacterium characteristics among Salmonella enterica serovar Typhimurium (S.Tm), as they prepare for and launch invasion of epithelial host cells. We find that during the bacterial growth cycle, S.Tm populations switch gradually from fast planktonic growth to a host cell-invasive phenotype, characterized by flagellar motility and expression of the Type-three-secretion-system-1. The indistinct nature of this shift leads to the establishment of a transient subpopulation of S.Tm doublets-waist-bearing bacteria anticipating cell division-which simultaneously express host cell invasion machinery. In epithelial cell culture infections, these S.Tm doublets outperform their singlet brethren and represent a hyperinvasive subpopulation. Atop both glass and enteroid-derived monolayers, doublets swim along markedly straighter trajectories than singlets, thereby diversifying search patterns and improving the surface exploration capacity of the total bacterial population. The straighter swimming, combined with an enhanced cell-adhesion propensity, suffices to account for the hyperinvasive doublet phenotype. This work highlights bacterial cell length heterogeneity as a key determinant of target search patterns atop epithelia.

BACKGROUND: Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance. METHODS: hESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein-protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance. RESULTS: RPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication. CONCLUSIONS: We propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at th

Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.

Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work. First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming

Thymic Epithelial Tumors (TETs) arise from epithelial cells of the thymus and are very rare neoplasms comprising Thymoma, Thymic carcinoma, and Thymic Neuroendocrine tumors that still require in-depth molecular characterization. Long non-coding RNAs (lncRNAs) are emerging as relevant gene expression modulators involved in the deregulation of several networks in almost all types of human cancer, including TETs. LncRNAs act at different control levels in the regulation of gene expression, from transcription to translation, and modulate several pathways relevant to cell fate determination under normal and pathological conditions. The activity of lncRNAs is strongly dependent on their expression, localization, and post-transcriptional modifications. Starting from our recently published studies, this review focuses on the involvement of lncRNAs in the acquisition of malignant traits by neoplastic thymic epithelial cells, and describes the possible use of these molecules as targets for the design of novel therapeutic approaches specific for TET. Furthermore, the involvement of lncRNAs in myasthenia gravis (MG)-related thymoma, which is still under investigation, is discussed.

Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work. First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming

KRAS mutants are common in many cancers and wild-type KRAS is essential in development as its absence causes embryonic lethality. Despite this critical role in development and disease, the normal expression pattern of KRAS protein is still largely unknown at the tissue level due to the lack of valid antibodies. To address this issue, we used the mouse model in which the Citrine-KRAS (Cit-K) fusion protein functions as a validated surrogate of endogenous KRAS protein that can be detected on tissue sections by immunolabeling with a GFP antibody. In the embryo, we found expression of KRAS protein in a wide range of organs and tissues. This expression tends to decrease near birth, mainly in mesenchymal cells. During transition to the adult stage, the dynamics of KRAS protein expression vary among organs and detection of KRAS becomes restricted to specific cell types. Furthermore, we found that steady state KRAS protein expression is detectable at the cell membrane and in the cytoplasm and that this subcellular partitioning differed among cell types. Our results reveal hitherto unanticipated dynamics in developmental, tissular, cell-specific and subcellular expression of KRAS protein. They provide insight into the reason why specific cell-types are sensitive to KRAS mutations during cancer initiation.

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.

Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work. First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming

The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth., QC 20220615

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.

Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work. First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects. Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming