Molecular variations to the proteome of zebrafish larvae induced by environmentally relevant copper concentrations.

Contaminants are increasingly accumulating in aquatic environments and biota, with potential adverse effects on individual organisms, communities and ecosystems. However, studies that explore the molecular changes in fish caused by environmentally relevant concentrations of metals, such as copper (Cu), are limited. This study uses embryos of the model organism zebrafish (Danio rerio) to investigate effect of Cu on the proteome and amino acid (AA) composition of fish. Wild-type embryos at 24 h post-fertilisation were exposed to Cu (2 µg L-1 to 120 µg L-1) for 96 h and the number of healthy larvae were determined based on larvae that had hatched and did not display loss of equilibrium (LOE). The effect concentrations where Cu caused a 10 % (EC10) or 50 % (EC50) decrease in the number of healthy larvae were calculated as 3.7 µg L-1 and 10.9 µg L-1, respectively. Proteomics analysis of embryos exposed to the EC10 and EC50 concentrations of Cu revealed the proteome to differ more strongly after 48 h than 96 h, suggesting the acclimatisation of some larvae. Exposure to excess Cu caused differentially expressed proteins (DEPs) involved in oxidative stress, mitochondrial respiration, and neural transduction as well as the modulation of the AAs (Proline, Glycine and Alanine). This is the first study to suggest that LOE displayed by Cu-stressed fish may involve the disruption to GABAergic proteins and the calcium-dependent inhibitory neurotransmitter GABA. Moreover, this study highlights that proteomics and AA analysis can be used to identify potential biomarkers for environmental monitoring.

Regulation of non-canonical proteins from diverse origins through the nonsense-mediated mRNA decay pathway.

Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.

Protein-coding potential of non-canonical open reading frames in human transcriptome.

In recent years, proteogenomics and ribosome profiling studies have identified a large number of proteins encoded by noncoding regions in the human genome. They are encoded by small open reading frames (sORFs) in the untranslated regions (UTRs) of mRNAs and long non-coding RNAs (lncRNAs). These sORF encoded proteins (SEPs) are often <150AA and show poor evolutionary conservation. A subset of them have been functionally characterized and shown to play an important role in fundamental biological processes including cardiac and muscle function, DNA repair, embryonic development and various human diseases. How many novel protein-coding regions exist in the human genome and what fraction of them are functionally important remains a mystery. In this review, we discuss current progress in unraveling SEPs, approaches used for their identification, their limitations and reliability of these identifications. We also discuss functionally characterized SEPs and their involvement in various biological processes and diseases. Lastly, we provide insights into their distinctive features compared to canonical proteins and challenges associated with annotating these in protein reference databases.

Combined thioredoxin reductase and glutaminase inhibition exerts synergistic anti-tumor activity in MYC-high high-grade serous ovarian carcinoma.

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

Phosphotyrosine Profiling Using SILAC.

Tyrosine phosphorylation on proteins is an important posttranslational modification that regulates various processes in cells. Mass spectrometry-based phosphotyrosine profiling can reveal tyrosine kinase signaling activity in cells. Using quantitative proteomics strategies such as stable isotope labeling with amino acids in cell culture (SILAC) allows comparison of tyrosine kinase signaling activity across two to -three different conditions. In this book chapter, we discuss the reagents required and a step-by-step protocol to carry out phosphotyrosine profiling using SILAC.

Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles.

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Time Course of Irreversible Electroporation Lesion Development Through Short- and Long-Term Follow-Up in Pulsed-Field Ablation-Treated Hearts.

BACKGROUND: Pulsed-field ablation (PFA) is a tissue-selective, nonthermal cardiac ablation modality. A novel PFA ablation system consisted of a multichannel irreversible electroporation generator system and a multielectrode circular irreversible electroporation catheter has been developed for catheter ablation. To understand the progression and immediate impacts of PFA, this study evaluated the subchronic (7±3 day) and chronic (30±3 day) safety and performance of the novel PFA system when simulating pulmonary vein and superior vena cava isolation in a porcine beating heart model. METHODS: Ten swine models were divided into subchronic (n=6) and chronic cohorts (n=4). Lesions were performed within the right and left atrium to conduct right pulmonary veins and superior vena cava isolations, in addition to creating stacked lesions in the left atrium roof and right atrium posterior wall. RESULTS: Acute pulmonary vein and superior vena cava isolation were achieved in 10 out of 10 swine and demonstrated 100% lesion durability in both cohorts, including sustained elimination of electrical activity at the left atrium roof and right atrium posterior wall. Histology demonstrated that all the cardiac sites ablated showed discrete zones of loss of myocardial fibers or smooth muscle cells with preservation of the tissue architecture with resultant fibrocellular replacement, neovascularization, and neocollagen deposition. Mineralization findings were present in association with residual necrotic muscle fibers. Only in 7 days group, areas of mineralization were frequently associated with inflammation. There were no treatment-related changes in other tissues, including complete sparing of the phrenic nerve. CONCLUSIONS: Pulsed-field ablation for pulmonary vein and superior vena cava isolation with the novel PFA system was feasible, safe with myocardial-specific ablative effect. Durable lesions were observed at the target areas. with inflammation phenomena mainly documented at 7 days.

In vivo porcine characterization of atrial lesion safety and efficacy utilizing a circular pulsed-field ablation catheter including assessment of collateral damage to adjacent tissue in supratherapeutic ablation applications.

INTRODUCTION: Pulsed-field ablation (PFA), an ablative method that causes cell death by irreversible electroporation, has potential safety advantages over radiofrequency ablation and cryoablation. Pulmonary vein (PV) isolation was performed in a porcine model to characterize safety and performance of a novel, fully-integrated biphasic PFA system comprising a multi-channel generator, variable loop circular catheter, and integrated PFA mapping software module. METHODS: Eight healthy porcine subjects were included. To evaluate safety, multiple ablations were performed, including sites not generally targeted for therapeutic ablation, such as the right inferior PV lumen, right superior PV ostium, and adjacent to the esophagus and phrenic nerve. To evaluate the efficacy, animals were recovered, followed for 30(±3) days, then re-mapped. Gross pathological and histopathological examinations assessed procedural injuries, chronic thrombosis, tissue ablation, penetration depth, healing, and inflammatory response. RESULTS: All eight animals survived follow-up. PV narrowing was not observed acutely nor at follow-up, even when ablation was performed deep to the PV ostium. No injury was seen grossly or histologically in adjacent structures. All PVs were durably isolated, confirmed by bidirectional block at re-map procedure. Histological examination showed complete, transmural necrosis around the circumference of the ablated section of right PVs. CONCLUSION: This preclinical evaluation of a fully-integrated PFA system demonstrated effective and durable ablation of cardiac tissue and PV isolation without collateral damage to adjacent structures, even when ablation was performed in more extreme settings than those used therapeutically. Histological staining confirmed complete transmural cell necrosis around the circumference of the PV ostium at 30 days.

A simple organic solvent precipitation method to improve detection of low molecular weight proteins.

Mass spectrometry-based proteomics revolutionized global proteomic profiling. Although high molecular weight abundant proteins are readily sampled in global proteomics studies, less abundant low molecular weight proteins are often underrepresented. This includes biologically important classes of low molecular weight proteins including ligands, growth factors, peptide hormones and cytokines. Although extensive fractionation can facilitate achieving better coverage of proteome, it requires additional infrastructure, mass spectrometry time and labour. There is need for a simple method that can selectively deplete high molecular weight abundant proteins and enrich for low molecular weight less abundant proteins to improve their coverage in proteomics studies. We present a simple organic-solvent based protein precipitation method that selectively depletes high molecular weight proteins and enriches low molecular weight proteins in the soluble fraction. Using this strategy, we demonstrate identification of low molecular weight proteins that are generally underrepresented in proteomics datasets. In addition, we show the utility of this approach in identifying functional cleavage products from precursor proteins and low molecular weight short open reading frame proteins encoded by non-coding regions such as lncRNAs and UTRs. As the method does not require additional infrastructure, it can complement existing proteomics workflows to increase detection and coverage of low molecular weight proteins that are less abundant.

Temporal Quantitative Proteomics Reveals Proteomic and Phosphoproteomic Alterations Associated with Adaptive Response to Hypoxia in Melanoma Cells.

Hypoxia is a common feature in various solid tumours, including melanoma. Cancer cells in hypoxic environments are resistant to both chemotherapy and radiation. Hypoxia is also associated with immune suppression. Identification of proteins and pathways that regulate cancer cell survival in hypoxic environments can reveal potential vulnerabilities that can be exploited to improve the efficacy of anticancer therapies. We carried out temporal proteomic and phosphoproteomic profiling in melanoma cell lines to identify hypoxia-induced protein expression and phosphorylation changes. By employing a TMT-based quantitative proteomics strategy, we report the identification and quantitation of >7000 proteins and >10,000 phosphosites in melanoma cell lines grown in hypoxia. Proteomics data show metabolic reprogramming as one of the prominent adaptive responses in hypoxia. We identify several novel hypoxia-mediated phosphorylation changes that have not been reported before. They reveal kinase signalling pathways that are potentially involved in modulating cellular response to hypoxia. In addition to known protein expression changes, we identify several novel proteomic alterations associated with adaptive response to hypoxia. We show that cancer cells require the ubiquitin-proteasome system to survive in both normoxia and hypoxia. Inhibition of proteasome activity affects cell survival and may provide a novel therapeutic avenue to target cancer cells in hypoxia. Our study can serve as a valuable resource to pursue novel candidates to target hypoxia in cancers and improve the efficacy of anticancer therapies.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Protocol for purification and identification of MHC class I immunopeptidome from cancer cell lines.

Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.

An integrated approach for identification of a panel of candidate genes arbitrated for invasion and metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is known for its aggressiveness associated with poor prognosis. The molecular mechanisms underlying the invasion and metastasis are still poorly understood. An improved understanding of these mechanisms shall precede the development of new diagnostic tools and targeted therapies. We report an integrated approach using bioinformatics to predict candidate genes, coupled with proteomics and immunohistochemistry for validating their presence and involvement in OSCC pathways heralding invasion and metastasis. Four genes POSTN, TNC, CAV1 and FSCN1 were identified. A protein-protein interaction network analysis teamed with pathway analysis led us to propose the role of the identified genes in invasion and metastasis in OSCC. Further analyses of archived FFPE blocks of various grades of oral cancer was carried out using TMT-based mass spectrometry and immunohistochemistry. Results of this study expressed a strong communiqué and interrelationship between these candidate genes. This study emphasizes the significance of a molecular biomarker panel as a diagnostic tool and its correlation with the invasion and metastatic pathway of OSCC. An insight into the probable association of CAF's and these biomarkers in the evolution and malignant transformation of OSCC further magnifies the molecular-biological spectrum of OSCC tumour microenvironment.

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii.

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.

Circular Multielectrode Pulsed Field Ablation Catheter Lasso Pulsed Field Ablation: Lesion Characteristics, Durability, and Effect on Neighboring Structures.

BACKGROUND: Pulsed field ablation (PFA) is a nonthermal energy with potential safety advantages over radiofrequency ablation. This study investigated a novel PFA system-a circular multielectrode catheter (PFA lasso) and a multichannel generator designed to work with Carto 3 mapping system. METHODS: A 7.5F bidirectional circular catheter with 10 electrodes and variable expansion was designed for PFA (biphasic, 1800 Volts). This study included a total of 16 swine used to investigate the following 3 experimental aims: Aim 1 examined the feasibility to create a right atrial ablation line of block from the superior vena cava to the inferior vena cava. Aim 2 examined the effect of PFA on lesion maturation including durability after a 30-day survival period. Aim 3 examined the effect of high-intensity PFA (10 applications) on esophageal and phrenic nerve tissue in comparison to normal intensity radiofrequency ablation (1-2 applications). Histopathologic analysis of all cardiac, esophageal, and phrenic nerve tissue was performed. RESULTS: Acute line of block was achieved in 12/12 swine (100%) and required a total PFA time of 14 seconds (interquartile range [IQR], 9-24.5) per line. Ablation line durability after 28±3 days was maintained in 11/12 (91.7%) swine. PFA resulted in transmural lesions in 179/183 (97.8%) sections and a median lesion width of 14.2 mm. High-intensity PFA (9 [IQR, 8-14] application) had no effect on the esophagus while standard intensity radiofrequency ablation (1.5 [IQR, 1-2] applications) resulted in deep esophageal tissue injury involving the muscularis propria and adventitia layers. High-intensity PFA (16 [IQR, 10-28] applications) has no effect on phrenic nerve function and structure while standard dose radiofrequency ablation (1.5 [IQR, 1-2] applications) resulted in acute phrenic nerve paralysis. CONCLUSIONS: In this preclinical model, a multielectrode circular catheter and multichannel generator produced durable atrial lesions with lower vulnerability to esophageal or phrenic nerve damage.

Temperature- and flow-controlled ablation/very-high-power short-duration ablation vs conventional power-controlled ablation: Comparison of focal and linear lesion characteristics.

BACKGROUND: The QDOT MICRO catheter allows temperature- and flow-controlled (TFC) ablation and very-high-power short-duration (vHPSD) ablation. OBJECTIVE: The purpose of this study was to compare lesion characteristics between TFC/vHPSD ablation and standard power-controlled (PC) ablation. METHODS: Lesion characteristics in the right atrium, left atrium, and right ventricle (RV) of 6 sheep were compared between vHPSD (90 W/4 seconds, TC mode with 60°C target using QDOT) and standard radiofrequency settings (PC mode, 30 W/30 seconds with ThermoCool SmartTouch SF). Lesions in the left ventricle (LV) were compared, targeting 50 W for 60-second applications. RESULTS: Forty-six focal atrial lesions, 50 RV focal lesions, and 12 linear lesions were created by vHPSD ablation and PC ablation in each group of 6 animals. vHPSD ablation produced significantly larger focal atrial lesions in length (8.3 [6.4-9.7] mm vs 6.3 [5.2-7.4] mm; P = .0002), width (6.0 [5.3-6.9] mm vs 4.6 [3.8-5.4] mm; P <.0001), and surface area (39.4 [25.4-52.4] mm2 vs 23.6 [16.0-31.1] mm2; P = .0001), with superior transmurality (89.1% vs 69.6%; P = .04) compared to PC ablation. vHPSD ablation produced significantly larger RV lesions in length (7.7 [7.0-8.7] mm vs 6.0 [4.8-6.9] mm; P <.0001), width (6.4 [5.4-7.5] mm vs 4.3 [3.6-5.2] mm; P <.0001), and area (39.4 [29.1-50.1] mm2 vs 19.9 [14.7-25.2] mm2; P <.0001) but similar volume (P = .97) with shallower lesions (2.7 [2.2-3.4] mm vs 3.8 [3.0-4.4] mm; P <.0001). Atrial linear lesions were more homogeneous (P = .02), with fewer gaps in each line (P = .003) with vHPSD ablation. LV focal lesions (15 TFC mode; 21 PC mode) were similar in volume and depth, but lesion size showed less deviation (P <.05) in TFC than PC mode. Fewer steam pops were observed in TFC mode (0% vs 28.6%; P = .03). Hemorrhagic rings around the lesion core were generally smaller with TFC/vHPSD ablation (P <.05). CONCLUSION: TFC/vHPSD ablation produces larger, shallower, more homogeneous, and less hemorrhagic lesions. vHPSD Ablation produces more transmural and contiguous linear lesions compared to PC ablation. LV lesions are more homogeneous with fewer steam pops in TFC ablation.

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

BACKGROUND: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. METHODS: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. RESULTS: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. CONCLUSIONS: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.