Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Loss-of-Function but Not Gain-of-Function Properties of Mutant TP53 Are Critical for the Proliferation, Survival, and Metastasis of a Broad Range of Cancer Cells.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.

Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer.

Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n = 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n = 30 patients). "Resistant" predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7 weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Genotype-Tailored ERK/MAPK Pathway and HDAC Inhibition Rewires the Apoptotic Rheostat to Trigger Colorectal Cancer Cell Death.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

Lack of direct association between oral mucosal lesions and SARS-CoV- 2 in a cohort of patients hospitalised with COVID-19.

Background: COVID-19 is a disease affecting various human organs and systems, in which the virus seeks to interact with angiotensin-converting enzyme 2 receptors. These receptors are present in the oral cavity, but the direct relationship between such an interaction and possible oral manifestations of COVID-19 is still unclear. Aim: The present study evaluated oral manifestations in a cohort of COVID-19 patients during the period of hospitalisation. Methods: In total, 154 patients presenting moderate-to-severe forms of COVID-19 had their oral mucosa examined twice a week until the final outcome, either discharge or death. The oral alterations observed in the patients were grouped into Group 1 (pre-existing conditions and opportunistic oral lesions) and Group 2 (oral mucosal changes related to hospitalization). Results: Oral lesions found in the patients of Group 1 are not suggestive of SARS-CoV-2 infection as they are mainly caused by opportunistic infections. On the other hand, oral alterations found in the patients of Group 2 were statistically (P < 0.001) related to intubation and longer period of hospitalisation. Conclusion: It is unlikely that ulcerative lesions in the oral cavity are a direct manifestation of SARS-CoV-2 or a marker of COVID-19 progression.

MALDI-TOF mass spectrometry of saliva samples as a prognostic tool for COVID-19.

BACKGROUND: The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate are essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. METHODS: Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. RESULTS: We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. When we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. CONCLUSION: Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a larger dataset is necessary to consolidate the technique.

Cancer Tissue Classification Using Supervised Machine Learning Applied to MALDI Mass Spectrometry Imaging.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.

Low-viscosity matrix suspension culture enables scalable analysis of patient-derived organoids and tumoroids from the large intestine.

Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine.

Frequency and Evolution of Acute Oral Complications in Patients Undergoing Radiochemotherapy Treatment for Head and Neck Squamous Cell Carcinoma.

Despite its effectiveness, radiochemotherapy treatment in the head and neck region is accompanied by acute oral complications such as oral mucositis, dysphagia, xerostomia, and dysgeusia. The aim of this study was to analyze and prospectively assess the frequency and evolution of acute oral complications during radiochemotherapy in patients diagnosed with squamous cell carcinoma in the head and neck region. We have analyzed oral complications of 20 patients during 6 weeks of radiochemotherapy treatment for squamous cell carcinoma. Oral mucositis was evaluated according to the World Health Organization criteria, dysphagia, and dysgeusia according to the National Cancer Institute Common Toxicity Criteria, and xerostomia according to parameters set by the Seminars in Radiation Oncology. Mucositis was first observed in the second week and all patients presented some degree of mucositis in the fourth week of radiotherapy. Xerostomia and dysphagia were initially reported already in the first week of radiotherapy. All patients presented xerostomia in the fourth week; however, dysphagia was observed in all patients, only in the sixth week. Dysgeusia was first observed in the second week, becoming more severe in the third week. Acute oral complications can be observed throughout the treatment, but the third week of radiotherapy seems to represent a critical week, regardless of the grade of the complication. The sixth week presents the worst grades of these complications. Knowledge about the natural course of oral complications during radiotherapy is important to develop better strategies for treatment and improve the patients' quality of life.

Oral shedding of CMV and HSV-1 in hematopoietic stem cell transplantation patients.

OBJECTIVES: To evaluate the oral shedding of herpesviruses in patients undergoing hematopoietic stem cell transplantation (HSCT) and correlate it with oral mucositis (OM). METHODS: Saliva samples were collected before the HSCT and on day D + 8. Multiplex Polymerse Chain Reaction (PCR) was performed to detect herpes simplex virus (HSV)-1 and HSV-2, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Variella-zoster virus (VZV), and human herpesvirus (HHV)-6, HHV-7, and HHV-8. OM was assessed according to WHO criteria. RESULTS: Thirty one patients were enrolled, in which 20 of 31 (64.5%) were males; median age was 50 (21-70) years; 16 of 31 (51.6%) underwent allo-HSCT; and 15 of 31 (48.4%) underwent auto-HSCT. On D + 8, OM grades III and IV were observed in 8 of 31 (25.8%) patients. In the first salivary collection, EBV was found in 24 of 31 (77.4%), followed by HHV-6 (7/31, 22.6%) and HHV-7 (8/31 25.8%). In the second collection, EBV was found in 24 of 27(89%), followed by HSV-1 (8/27, 30%) and CMV, HHV-6, and HHV-7 (5/27, 18.5%, each one). On D + 8, OM grades II and IV were associated with the presence of HSV-1. HSV-1 was also associated with worsening degrees of OM on D + 15. CONCLUSION: The presence of HSV-1 and CMV in oral samples was more frequent on day D + 8 after HSCT. HSV-1 detection was associated with severity and worsening of OM. HSV-1 and CMV seem to be associated with oral dysbiosis due to HSCT.

Differentiation of periapical granuloma from radicular cyst using cone beam computed tomography images texture analysis.

OBJECTIVE: This study aimed to investigate the use of texture analysis for characterization of radicular cysts and periapical granulomas and to assess its efficacy to differentiate between both lesions with histological diagnosis. METHODS: Cone beam computed tomography (CBCT) images were obtained from 19 patients with 25 periapical lesions (14 radicular cysts and 11 periapical granulomas) confirmed by biopsy. Regions of interest were created in the lesions from which 11 texture parameters were calculated. Spearman's correlation analysis was performed and adjusted with Benjamini-Hochberg false discovery rate procedure (FDR <0.005). RESULTS: The texture parameters used to differentiate the lesions were assessed by using a receiver operating characteristic analysis. Five texture parameters were predictive of lesion differentiation for eight positions: angular second moment; sum of squares; sum of average; contrast; correlation. CONCLUSION: Texture analysis of CBCT scans distinguishes radicular cysts from periapical granulomas and can be a promising diagnostic tool for periapical lesions. CLINICAL SIGNIFICANCE: Texture analysis can be used in diagnostic and treatment monitoring to provide supplementary information.

Presence of langerhans cells, regulatory T cells (Treg) and mast cells in asymptomatic apical periodontitis.

Asymptomatic Apical Periodontitis is essentially an inflammatory disease of microbial aetiology. Association and function of the cell components involved, or specific inductive factors and growth mediators associated with development, maintenance and resolution of the periapical lesions are still unknown. The objective of this study was to evaluate the concentration of Regulatory T cells (FoxP3+; Treg), Langerhans cells (CD1a+; LC) and mast cells in asymptomatic apical periodontitis. 73 cases were selected: 30 periapical granulomas, 29 radicular cysts and 14 residual cysts. All groups were submitted to morphological analysis for classification of inflammatory infiltrate and thickness of the epithelial lining as well as to immunohistochemical analysis for detection of LC and Treg cells. Toluidine blue staining was used for detecting mast cells. Analysis showed higher mean numbers of LC (8.2 cells/0.2mm2), and Treg cells in radicular cysts (5.910 cells/0.2mm2). As for mast cells, it was found that radicular cysts had a higher mean number of these cells compared to other periapical lesions (12.68 cells/0.2mm2). The association between thickness of the epithelial lining and inflammatory cells showed that the presence of hypertrophic epithelium in radicular cysts presented higher density of LC. The number of LC and Treg cells play an important role in the control of the inflammatory micro-environment in periapical granulomas and radicular cysts, respectively. The presence of mast cells in radicular cysts may be associated with progression of the lesion. Knowledge regarding the inflammatory cell profile is therefore essential for a better understanding of the pathogenesis of asymptomatic periapical periodontitis.

Defining the susceptibility of colorectal cancers to BH3-mimetic compounds.

Novel targets are required to improve the outcomes for patients with colorectal cancers. In this regard, the selective inhibitor of the pro-survival protein BCL2, venetoclax, has proven highly effective in several hematological malignancies. In addition to BCL2, potent and highly selective small molecule inhibitors of its relatives, BCLxL and MCL1, are now available, prompting us to investigate the susceptibility of colorectal cancers to the inhibition of one or more of these pro-survival proteins. While targeting BCLxL, but not BCL2 or MCL1, on its own had some impact, most (15/17) of the immortalized colorectal cancer cell lines studied were efficiently killed by the combined targeting of BCLxL and MCL1. Importantly, these in vitro findings were confirmed in a xenograft model and, interestingly, in all (5/5) patient derived tumor organoids evaluated. Our results lend strong support to the notion that BCLxL and MCL1 are highly promising targets for further evaluation in efforts to improve the treatment of colorectal cancers.

Tumour infiltrating lymphocyte status is superior to histological grade, DNA mismatch repair and BRAF mutation for prognosis of colorectal adenocarcinomas with mucinous differentiation.

Mucinous colorectal adenocarcinoma (CRC) is conventionally defined by extracellular mucin comprising >50% of the tumour area, while tumours with ≤50% mucin are designated as having a mucinous component. However, these definitions are largely arbitrary and comparisons of clinico-molecular features and outcomes by proportion of mucinous component are limited. A cohort of 1643 patients with stage II/III cancer was examined for tumour mucinous component, DNA mismatch repair (MMR) status, BRAF mutation and tumour infiltrating lymphocytes (TILs). Tumours with ≤50% mucinous component exhibited similar characteristics as mucinous tumours, including association with female gender, proximal location, high grade, TIL-high, defective MMR (dMMR) and BRAF mutation. Proportion of mucinous component did not stratify disease-free survival (DFS). In univariate analysis dMMR status, but not histological grade, stratified survival for mucinous and mucinous component tumours; however, in multivariate analysis dMMR status was not an independent predictor. BRAF mutation prognostic value depended on mucinous differentiation and MMR status, with poor prognosis limited to non-mucinous pMMR tumours (HR 2.61, 95% CI 1.69-4.03; p < 0.001). TIL status was a strong independent predictor of DFS in mucinous/mucinous component tumours (HR 0.40, 95% CI 0.23-0.67; p < 0.001), and a superior predictor of prognosis compared with histological grade, MMR and BRAF mutation. Mucinous component and mucinous stage II/III CRCs exhibit clinico-molecular resemblances, with histological grade and BRAF mutation lacking prognostic value. Prognosis for these tumours was instead strongly associated with TIL status, with the most favourable outcomes in TIL-high dMMR tumours, whilst TIL-low tumours had poor outcomes irrespective of MMR status.

Periodontal disease and detection of human herpesviruses in saliva and gingival crevicular fluid of chronic kidney disease patients.

BACKGROUND: Patients with chronic kidney disease (CKD) have inability to maintain the normal levels of protein metabolism products, blood pressure and hematocrit. Periodontal disease involves an inflammatory destructive process. Identification of opportunistic viruses is extremely important as they are associated with co-morbidities. The objective of this study was to analyse the presence of human herpesviruses in saliva and gingival crevicular fluid (GCF) from patients with CKD. METHODS: One hundred and thirty one individuals were divided depending on the stage of CKD: Group 1 (clearance of creatinine > 75 mL/min) patients with no renal disease (n = 24); Group 2 (clearance of creatinine of 11-75 mL/min) patients with renal disease (n = 67); Group 3 (clearance of creatinine < 10 mL/min) patients on hemodialysis (n = 40). The parameters of periodontal disease were evaluated. The viral detection was assessed by PCR. RESULTS: considering the three groups, the prevalence of herpes simplex virus 1 (HSV-1) were 9% in saliva and 5% in GCF; Epstein-Barr virus 36% in saliva and 39% in GCF; human cytomegalovirus (HCMV) 11% in GCF; varicella zoster virus 6% in saliva and 3% in GCF; of human herpesvirus-6 (HHV-6) 6% in saliva and 2% in GCF; and HHV-7 44% in saliva and 8% in GCF. Of these patients, 46.48% presented with severe periodontitis. A statistically significant association between HSV-1 and HCMV was found in hemodialysis patients and severe periodontitis was also more frequent among them. CONCLUSION: These findings show the importance of evaluating the periodontal disease and detecting herpesviruses in patients with CKD as the inflammatory process observed in these clinical conditions may worsen the course of both periodontal disease and CKD.

Herpesviruses in the oral cavity of patients subjected to allogeneic hematopoietic stem cell transplantation and its relationship with oral mucositis.

OBJECTIVES: This study was performed to characterise oral shedding of herpesviruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and to investigate its relationship with oral mucositis (OM). MATERIALS AND METHODS: PCR and enzymatic digestion were conducted to identify oral shedding of herpesviruses and its correlation with OM development in 31 patients. The samples were collected at three sites in the oral cavity and at 5 times during follow-up; two additional collections were made from patients who developed ulcerative OM. RESULTS: HSV-1, EBV, CMV, HHV-6A, HHV-6B, and HHV-7 were detected in 4.97%, 16.02%, 4.41%, 2.20%, 3.31%, and 68% of the oral mucosal samples, respectively; 4.41%, 16.57%, 5.52%, 2.20%, 5.52%, and 63.53% of supragingival samples, respectively, and 4.41%, 18.23%, 2.76%, 1.65%, 2.75%, and 35.91% of subgingival samples, respectively. OM was diagnosed in 13 patients. The presence of HHV-7 in C1 (oral mucosa: p = 0.032) and C2 (supragingival: p = 0.009; subgingival: p = 0.002) was significantly increased in patients who developed OM, and patients exhibiting HHV-7 shedding in the oral cavity were 3.32-fold more likely to develop OM. CONCLUSIONS: Patients who developed OM showed higher HHV-7 shedding in the oral cavity at nadir (immediately prior to OM development), suggesting modifications to the inflammatory microenvironment. CLINICAL RELEVANCE: HHV-7 may be involved in oral dysbiosis in HSCT-related OM; enhanced understanding of its role in the pathogenesis of OM may lead to the development of strategies for managing and preventing this common side effect of alloHSCT.

Evaluation of ultraviolet photodissociation tandem mass spectrometry for the structural assignment of unsaturated fatty acid double bond positional isomers.

Fatty acids are a major source of structural diversity within the lipidome due to variations in their acyl chain lengths, branching, and cyclization, as well as the number, position, and stereochemistry of double bonds within their mono- and poly-unsaturated species. Here, the utility of 193 nm UltraViolet PhotoDissociation tandem mass spectrometry (UVPD-MS/MS) has been evaluated for the detailed structural characterization of a series of unsaturated fatty acid lipid species. UVPD-MS/MS of unsaturated fatty acids is shown to yield pairs of unique diagnostic product ions resulting from cleavages adjacent to their C=C double bonds, enabling unambiguous localization of the site(s) of unsaturation within these lipids. The effect of several experimental variables on the observed fragmentation behaviour and UVPD-MS/MS efficiency, including the position and number of double bonds, the effect of conjugated versus non-conjugated double bonds, the number of laser pulses, and the influence of alkali metal cations (Li, Na, K) as the ionizing adducts, has been evaluated. Importantly, the abundance of the diagnostic ions is shown to enable relative quantitation of mixtures of fatty acid isomers across a range of molar ratios. Finally, the practical application of 193 nm UVPD-MS/MS is demonstrated via characterization of changes in the ratios of fatty acid double bond positional isomers in isogenic colorectal cancer cell lines. This study therefore demonstrates the practicality of UVPD-MS/MS for the structural characterization of fatty acid isomers in lipidome analysis workflows.

Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammation-associated colorectal cancer.

Gastrointestinal epithelial cells provide a selective barrier that segregates the host immune system from luminal microorganisms, thereby contributing directly to the regulation of homeostasis. We have shown that from early embryonic development Bcl-G, a Bcl-2 protein family member with unknown function, was highly expressed in gastrointestinal epithelial cells. While Bcl-G was dispensable for normal growth and development in mice, the loss of Bcl-G resulted in accelerated progression of colitis-associated cancer. A label-free quantitative proteomics approach revealed that Bcl-G may contribute to the stability of a mucin network, which when disrupted, is linked to colon tumorigenesis. Consistent with this, we observed a significant reduction in Bcl-G expression in human colorectal tumors. Our study identifies an unappreciated role for Bcl-G in colon cancer.