Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.

MUCIN-4 (MUC4) is a novel tumor antigen in pancreatic cancer immunotherapy.

Pancreatic cancer (PC) is a highly lethal malignancy with a dismal five-year survival rate. This is due to its asymptomatic nature, lack of reliable biomarkers, poor resectability, early metastasis, and high recurrence rate. Limited efficacies of current treatment modalities treatment-associated toxicity underscore the need for the development of immunotherapy-based approaches. For non-resectable, locally advanced metastatic PC, immunotherapy-based approaches including vaccines, antibody-targeted, immune checkpoint inhibition, CAR-T-cells, and adoptive T-cell transfer could be valuable additions to existing treatment modalities. Thus far, the vaccine candidates in PC have demonstrated modest immunological responses in different treatment modalities. The identification of tumor-associated antigens (TAA) and their successful implication in PC treatment is still a challenge. MUC4, a high molecular weight glycoprotein that functionally contributes to PC pathogenesis, is an attractive TAA. It is not detected in the normal pancreas; however, it is overexpressed in mouse and human pancreatic tumors. The recombinant MUC4 domain, as well as predicted immunogenic T-cell epitopes, elicited cellular and humoral anti-MUC4 response, suggesting its ulility as a vaccine candidate for PC therapy. Existence of PC-associated MUC4 splice variants, autoantibodies against overexpressed and aberrantly glycosylated MUC4 and presence of T-cell clones against the mutations present in MUC4 further reinforce its significance as a tumor antigen for vaccine development. Herein, we review the significance of MUC4 as a tumor antigen in PC immunotherapy and discuss both, the development and challenges associated with MUC4 based immunotherapy. Lastly, we will present our perspective on MUC4 antigenicity for the future development of MUC4-based PC immunotherapy.

MUC4 O-GlcNAcylation Regulates the Epithelial Phenotype in Conjunctival Epithelial Cells.

In the present study, our aim was to explore the role of MUC4 in IL-4-stimulated conjunctival epithelial cells and the underlying mechanisms. Human recombinant IL-4 was employed in human conjunctival epithelial cells (HConEpic) cells, and MUC4 shRNA (sh-MUC4) was constructed to explore the functional role of MUC4. The protein level of MUC4, O-GlcNAc transferase (OGT), O-GlcNAc hydrolase (OGA), zonula occludens 1 (ZO-1), gap junction protein beta 2 (GJB2), claudin-8 (CLDN8), and E-cadherin were detected by Western blot in HConEpic cells, the interaction between MUC4 and OGT/OGA was assessed by co-immunoprecipitation (IP) and Western blot in 293T cells. Our results showed that IL-4 significantly up-regulated MUC4 and OGT protein levels in HConEpic cells, while down-regulated OGA protein level. Also, IL-4 down-regulated ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, while which was markedly reversed by sh-MUC4. Additionally, OGT inhibitor significantly reduced MUC4 protein level, and elevated ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, while OGA inhibitor resulted in the opposite results. Furthermore, in addition to the interaction between OGT/OGA and MUC4, Co-IP and Western blot also revealed the alteration of MUC4 O-GlcNAcylation in 293T cells treated with OGT/OGA inhibitor. Above findings suggested that OGT/OGA inhibitor regulated MUC4 protein level by affecting MUC4 O-GlcNAcylation to regulate ZO-1, GJB2, CLDN8, and E-cadherin protein levels in HConEpic cells, which was achieved via inhibiting the interaction between OGT/OGA and MUC4. This study may provide a better understanding of the pathogenesis of allergic conjunctivitis (AC).

An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

Comprehensive clinicopathological characteristics and mucin core protein expression profiles of bronchiolar adenoma.

AIMS: Bronchiolar adenoma (BA) is a novel entity in the 2021 WHO classification of lung tumours. The expression profile of mucin core proteins in BAs is not clear. The aim of this study was to clarify the expression profiles of mucins and to validate the clinicopathologic and molecular features of BAs. METHODS AND RESULTS: We examined the clinicopathological, immunohistochemical, and molecular features of 20 BAs. Our cohort comprised 10 proximal and 10 distal BAs. Only seven of 18 patients (39%) were accurately diagnosed with BA at the time of intraoperative consultation. The frequent genetic alterations were BRAF V600E (35%) and KRAS (30%) mutations, which were mutually exclusive. The expression of MUC1, MUC4, and MUC5B was observed in all cases and that of MUC5AC and MUC6 was observed in nine (45%) and five (25%) cases, respectively. MUC4 was diffusely expressed in 18 cases. In contrast, MUC1, MUC5AC, MUC5B, and MUC6 displayed a patchy expression pattern. These expression patterns were similar to that of bronchiolar epithelium in normal lung tissue. In addition, overexpression of MUC1 and MUC4 on the entire cell surface was not observed in any of the BAs, suggesting their benign nature. CONCLUSION: BA commonly exhibits diffuse MUC4 and patchy MUC1 and MUC5B expression. Its unique expression pattern is probably attributed to mucin expression specific to the bronchiolar epithelium. These results confirm the clinicopathologic and molecular characteristics of BA, including the difficulty in intraoperative frozen section diagnosis and the broad morphologic spectrum of BA derived from the bronchiolar epithelium.

Sclerosing Epithelioid Fibrosarcoma of the Liver in a Pediatric Patient.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare but aggressive sarcoma. We report the first case of hepatic SEF in pediatric patient, which is also the second case in literature. A 17-year-old previously healthy female presented with a liver mass measuring 13.7 cm in greatest dimension and mild elevation of liver enzymes and cancer antigen 19-9. Needle biopsy revealed multiple cores of liver parenchyma mostly replaced by densely hyalinized fibrotic tissue and areas of small-to-medium sized epithelioid cells with eosinophilic and clear cytoplasm. Immunohistochemistry (IHC) demonstrated diffuse strong cytoplasmic staining of MUC4, suggesting a working diagnosis of sclerosing epithelioid fibrosarcoma (SEF)/low-grade fibromyxoid sarcoma (LGFMS). Liver explant demonstrated a well-circumscribed, nodular mass with firm, gray-white cut surface, and similar histopathology as seen in needle biopsy with no convincing evidence suggesting LGFMS. Sequencing panel revealed EWSR1::CREB3L1 gene fusion and confirmed the diagnosis of SEF. Post-operative cancer antigen 19-9 normalized 3 months after transplant; follow-up 3 and 6 months post-transplant imaging at that time showed no concern for disease recurrence.

Utility of MUC4 in the diagnosis of secretory carcinoma of salivary glands.

Salivary gland tumors are diverse in morphology and both benign and malignant tumors may pose diagnostic challenges especially in small biopsies. Secretory carcinoma (SC) is histologically characterized by microcysts, follicles, solid growth pattern and occasional papillary structures, and absence of zymogen granules. SC is molecularly defined by the presence of novel gene fusion ETV6::NTRK3. Among the positive stains (S100 and mammaglobin), MUC4 is now another promising marker for the diagnosis of SC, that would enable the pathologists to exclude other morphologically close simulators. Aim of this study was to report clinicopathological features and assess utility of MUC4 in the diagnosis of SC. MUC4 was performed on 22 cases of SC. Glass slides were reviewed to record morphological patterns and staining of S100, mammaglobin, DOG1 and MUC4. Age ranged from 9 to 63 years with mean age of 34.41 ± 16.28 years. The male: female ratio was 72.7 %:27.3 %. The majority occurred in major salivary glands. A combination of patterns was seen; microfollicles were the most prevalent (90 %) followed by papillary-cystic and macrofollicles. MUC4 was positive in 19/21 (90 %) cases with almost equal number of 2+ and 3+ staining. MUC4 was negative in all cases of acinic cell carcinoma, polymorphous adenocarcinoma, adenoid cystic carcinoma, salivary duct carcinoma, myopepithelioma and myoeithelial carcinoma, cystadenoma and cribriform adenocarcinoma and all except 3 cases of mucoepidermoid carcinoma tested. Overall sensitivity of MUC4 was 95.4 %, specificity 90 %, p-value being <0.01, positive predictive value 87.5 % and negative predictive value 96.4 %. A characteristic cytoplasmic granular pattern was observed in 76.1 % tumors. S100 and mammaglobin were positive in all the performed cases. DOG1 was positive in 6/11 (28.5 %) tumors. In conclusion, MUC4 is a useful addition to a diagnostic immunohistochemical panel for SC, and to distinguish it from close potential mimickers such as acinic cell carcinoma, especially in practice settings where molecular testing is unavailable.

[Alveolar rhabdomyosarcoma: novel surrogate markers associated with oncogenic translocation]. / Al'veolyarnaya rabdomiosarkoma: novye vspomogatel'nye surrogatnye markery onkogennykh translokatsii.

BACKGROUND: Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard¼ for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. OBJECTIVE: Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. MATERIAL AND METHODS: 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. RESULTS: The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). CONCLUSION: The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.

Comprehensive assessments of germline deletion structural variants reveal the association between prognostic MUC4 and CEP72 deletions and immune response gene expression in colorectal cancer patients.

BACKGROUND: Functional disruptions by large germline genomic structural variants in susceptible genes are known risks for cancer. We used deletion structural variants (DSVs) generated from germline whole-genome sequencing (WGS) and DSV immune-related association tumor microenvironment (TME) to predict cancer risk and prognosis. METHODS: We investigated the contribution of germline DSVs to cancer susceptibility and prognosis by silicon and causal inference models. DSVs in germline WGS data were generated from the blood samples of 192 cancer and 499 non-cancer subjects. Clinical information, including family cancer history (FCH), was obtained from the National Cheng Kung University Hospital and Taiwan Biobank. Ninety-nine colorectal cancer (CRC) patients had immune response gene expression data. We used joint calling tools and an attention-weighted model to build the cancer risk predictive model and identify DSVs in familial cancer. The survival support vector machine (survival-SVM) was used to select prognostic DSVs. RESULTS: We identified 671 DSVs that could predict cancer risk. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) of the attention-weighted model was 0.71. The 3 most frequent DSV genes observed in cancer patients were identified as ADCY9, AURKAPS1, and RAB3GAP2 (p < 0.05). The DSVs in SGSM2 and LHFPL3 were relevant to colorectal cancer. We found a higher incidence of FCH in cancer patients than in non-cancer subjects (p < 0.05). SMYD3 and NKD2DSV genes were associated with cancer patients with FCH (p < 0.05). We identified 65 immune-associated DSV markers for assessing cancer prognosis (p < 0.05). The functional protein of MUC4 DSV gene interacted with MAGE1 expression, according to the STRING database. The causal inference model showed that deleting the CEP72 DSV gene affect the recurrence-free survival (RFS) of IFIT1 expression. CONCLUSIONS: We established an explainable attention-weighted model for cancer risk prediction and used the survival-SVM for prognostic stratification by using germline DSVs and immune gene expression datasets. Comprehensive assessments of germline DSVs can predict the cancer risk and clinical outcome of colon cancer patients.

Clinical, pathologic, and molecular analyses of superficial low-grade fibromyxoid sarcoma in two young patients: A rare and deceptive mimic of benignancy.

Low-grade fibromyxoid sarcoma (LGFMS) is a histopathologically deceptive soft tissue neoplasm with bland cytology, which is typically encountered in deep soft tissue of adults. We report two cases of superficial LGFMS in young patients (16 and 21 years old, respectively), which were difficult to diagnose on histopathologic and clinical findings alone. LGFMS commonly mimics benign neoplasms such as cellular neurothekeoma, fibromatosis, neurofibroma, and perineurioma. Malignancies included in the differential diagnosis are soft tissue neoplasms such as dermatofibrosarcoma protuberans and myxofibrosarcoma. A high degree of reported variation in pattern and cellularity among LGFMS further complicates the diagnosis. Careful examination and appropriate immunohistochemistry panels including MUC4 are essential for narrowing the differential diagnosis. Molecular studies for possible FUS translocation can confirm the diagnosis of LGFMS. Sufficient sampling and workup of these lesions are critical, especially in younger patients. Young age and superficial presentation can easily sway dermatopathologists/dermatologists toward an incorrect diagnosis of benignancy.

Primary sclerosing epithelioid fibrosarcoma of the spine: a single-institution experience.

AIMS: To present our experience on spinal sclerosing epithelioid fibrosarcoma (SEF) and review the existing literature pertaining to SEF of the spine. METHODS AND RESULTS: Six cases of spinal SEF were reviewed, and a literature search of all primary SEFs of the spine was performed. All tumours occurred in adults (median age, 41 years) and were located all along the spine, the lumbar vertebrae being the most commonly involved. All patients presented with pain that they had experienced for months. The mean tumour size at diagnosis was 52 mm. Five tumours showed a spectrum of microscopic features consistent with pure SEF, and one showed a hybrid morphology with areas of low-grade fibromyxoid sarcoma. All were diffusely and strongly positive for mucin 4. Two cases were initially misdiagnosed as epithelioid haemangioendothelioma and aggressive chondroblastoma. Fluorescence in-situ hybridisation showed rearrangements of either FUS or EWSR1 in four cases. Reverse transcription polymerase chain reaction showed the presence of FUS-CREB3L1 and EWSR1-CREB3L1 fusion transcripts in two cases and one case, respectively. Of five patients with follow-up data available, two developed one or more local recurrences and three patients had metastatic disease. Distant metastases were mainly to other osseous locations, followed by lungs and lymph nodes. At last follow-up, three patients had died of disease and one was alive with multiple metastases. CONCLUSIONS: SEF is an aggressive sarcoma that can involve the spine. It is important to recognise the spine as the primary location of SEF, in order to avoid misdiagnosis as more common primary spinal neoplasms, which can impact on therapeutic approaches.

MUC4 is expressed in alveolar rhabdomyosarcoma.

AIMS: Mucin 4 (MUC4) is a transmembrane glycoprotein normally expressed by several human epithelial surfaces, including those of the colon, vagina, and respiratory tract. Although MUC4 overexpression is seen in various carcinomas, its expression among mesenchymal neoplasms is fairly specific to low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma. Having observed unanticipated anti-MUC4 immunoreactivity in rhabdomyosarcoma, we aimed to further characterise its expression. METHODS AND RESULTS: Expression of MUC4 was assessed by immunohistochemistry in a total of 97 rhabdomyosarcomas using formalin-fixed paraffin-embedded tissue sections. MUC4 was expressed by 21 of 26 PAX3/7-FOXO1 fusion-positive cases, wherein immunoreactivity, varying from weak to strong, was present in 20-100% of neoplastic cells. With the exception of one sclerosing rhabdomyosarcoma showing immunoreactivity in 20% of cells, MUC4 was not expressed by embryonal (n = 28), sclerosing (n = 20), or pleomorphic (n = 23) rhabdomyosarcomas. Analysing published gene expression microarray data from a separate cohort of 33 fusion-positive and 25 fusion-negative rhabdomyosarcomas, we found on average a 11.4-fold increased expression in fusion-positive tumours (P = 0.0004). CONCLUSIONS: MUC4 is expressed to a variable extent in the majority of PAX3/7-FOXO1 fusion-positive (alveolar) rhabdomyosarcomas, while expression in other rhabdomyosarcoma subtypes is rare.

MUC4 is a valuable marker for distinguishing secretory carcinoma of the salivary glands from its mimics.

AIMS: Secretory carcinoma (SC) (synonym: mammary analogue secretory carcinoma) is a low-grade salivary gland tumour that occurs in both major and minor salivary glands. SC is known for its wide morphological, architectural and immunohistochemical spectrum, which overlaps with those of several salivary gland neoplasms, including acinic cell carcinoma (AciCC) and intercalated duct-type intraductal carcinoma (IDC) in major salivary glands, and polymorphous adenocarcinoma (PAC) in minor salivary glands. These tumours share with SC some morphological features and SOX10 immunoreactivity; also, with the exception of AciCC, they all coexpress S100 and mammaglobin. METHODS AND RESULTS: We compared MUC4 and mammaglobin expression in 125 salivary gland carcinomas (54 genetically confirmed SCs, 20 AciCCs, 21 PACs, and 30 IDCs) to evaluate the potential of these two markers to differentiate these entities. Moderate to strong diffuse MUC4 positivity was detected in 49 SCs (90.7%), as compared with none of the IDCs and PACs. In contrast, mammaglobin was frequently expressed in SCs (30 of 36 cases; 83.3%), IDCs (24/28; 85.7%), and PACs (7/19; 36.8%). Two of three high-grade SCs lost MUC4 expression in the high-grade tumour component. No significant correlation was found between MUC4 expression and the fusion variant in SC (ETV6-NTRK versus non-ETV6-NTRK). CONCLUSION: The results of our study identify MUC4 as a sensitive (90.7%) and specific (100%) marker for SC, with high positive (100%) and negative (93.4%) predictive values. Thus, MUC4 may be used as a surrogate for SC in limited biopsy material and in cases with equivocal morphology.

Mucins as anti-cancer targets: perspectives of the glycobiologist.

Mucins are highly O-glycosylated glycoproteins that carry a heterogenous variety of O-glycan structures. Tumor cells tend to overexpress specific mucins, such as the cell surface mucins MUC1 and MUC4 that are engaged in signaling and cell growth, and exhibit abnormal glycosylation. In particular, the Tn and T antigens and their sialylated forms are common in cancer mucins. We review herein methods chosen to use cancer-associated glycans and mucins as targets for the design of anti-cancer immunotherapies. Mucin peptides from the glycosylated and transmembrane domains have been combined with immune-stimulating adjuvants in a wide variety of approaches to produce anti-tumor antibodies and vaccines. These mucin conjugates have been tested on cancer cells in vitro and in mice with significant successes in stimulating anti-tumor responses. The clinical trials in humans, however, have shown limited success in extending survival. It seems critical that the individual-specific epitope expression of cancer mucins is considered in future therapies to result in lasting anti-tumor responses.

[MUC4 Silencing Inhibits TGF-ß1-induced Epithelial-mesenchymal Transition VIA the ERK1/2 Pathway in Human Airway Epithelial NCI-H292 Cells].

MUC4 is a predominant membrane-tethered mucin lubricating and protecting the epithelial surface and playing various biological roles in the renewal and differentiation of epithelial cells, cell signaling, cell adhesion, and carcinogenesis. Interestingly, recent studies have demonstrated that MUC4 expression regulates the epithelial-mesenchymal transition (EMT) of cancer cells in ovarian, pancreatic, and lung cancer. However, the effects of MUC4 expression on EMT in human airway epithelial cells are not yet well known. Here, we describe the effects of transforming growth factor beta 1 (TGF-ß1)-induced MUC4 expression on EMT and evaluate its downstream signaling pathway in human airway epithelial cells. In human airway epithelial NCI-H292 cells, exposure to TGF-ß1 induced expression of MUC4, CDH2, VIM and SNAI1 genes and encoded by them proteins, MUC4, N-cadherin, vimentin and Snail, and reduced the level of CDH1 and its product, E-cadherin. In MUC4-knockdown cells, TGF-ß1-induced expression levels of MUC4, CDH2, VIM and SNAI1 and corresponding proteins were suppressed, but CDH1 and E-cadherin levels were not. In addition, TGF-ß1-induced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) was suppressed, but that of Smad2/3, Akt, and p38 was not. The results of this study suggest that MUC4 silencing inhibits TGF-ß1 -induced EMT via the ERK1/2 pathway, and a possible role of MUC4 in the induction of EMT in human airway epithelial cells.

Host genotype and amoxicillin administration affect the incidence of diarrhoea and faecal microbiota of weaned piglets during a natural multiresistant ETEC infection.

Enterotoxigenic Escherichia coli (ETEC) is the aetiological agent of postweaning diarrhoea (PWD) in piglets. The SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes have been associated with the susceptibility to ETEC F4 and ETEC F18, respectively. The interplay between the MUC4 and FUT1 genotypes to ETEC infection and the use of amoxicillin in modifying the intestinal microbiota during a natural infection by multiresistant ETEC strains have never been investigated. The aim of this study was to evaluate the effects of the MUC4 and FUT1 genotypes and the administration of amoxicillin through different routes on the presence of diarrhoea and the faecal microbiota composition in piglets naturally infected with ETEC. Seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes-parenteral (P) or oral (O) and a control group without antibiotics (C). Faecal scores, body weight, presence of ETEC F4 and F18 were investigated 4 days after the arrival in the facility (T0), at the end of the amoxicillin administration (T1) and after the withdrawal period (T2). The faecal bacteria composition was assessed by sequencing the 16S rRNA gene. We described that MUC4 and FUT1 genotypes were associated with the presence of ETEC F4 and ETEC F18. The faecal microbiota was influenced by the MUC4 genotypes at T0. We found the oral administration to be associated with the presence of diarrhoea at T1 and T2. Furthermore, the exposure to amoxicillin resulted in significant alterations of the faecal microbiota. Overall, MUC4 and FUT1 were confirmed as genetic markers for the susceptibility to ETEC infections in pigs. Moreover, our data highlight that group amoxicillin treatment may produce adverse outcomes on pig health in course of multiresistant ETEC infection. Therefore, alternative control measures able to maintain a healthy faecal microbiota in weaners are recommended.

Immunohistochemical expression of MUC4 in different grades of head and neck squamous cell carcinoma.

OBJECTIVE: To determine immunohistochemical expression of Mucin 4 in head and neck squamous cell carcinoma and its different histological grades among patients reporting to various tertiary care hospitals in an urban setting. METHODS: The descriptive study was conducted at the Department of Oral Pathology / Morbid Anatomy and Histopathology, University of Health Sciences, Lahore, Pakistan, from January to July 2017 and comprised cases of head and neck squamous cell carcinoma. Histological diagnosis and grading was done for each case. Haematoxylin and eosin stain followed by immunohistochemistry was done. Relation of Mucin 4 expression with tumour types was explored. SPSS 20 was used for statistical analysis. RESULTS: Of the 63 samples, 40(63.5%) were from male patients. The overall mean age of the patients was 53±3.77 years. Mucin 4 expression was positive in 47(74.6%) cases. Of them, 16(34%) had grade 1 tumour, 28(59.6%) had grade 2 and 3(6.4%) had grade 3 tumour. There was a significant relation (p=0.03) between tumour grades and intensity of Mucin 4 expression. CONCLUSIONS: Upregulation of Mucin 4 in tumour tissue with no expression in normal epithelium was found and loss of Mucin 4 expression with increase in tumour grade was noted.

MUC4 expression in meningiomas: under-recognized immunophenotype particularly in meningothelial and angiomatous subtypes.

AIMS: MUC4 is a transmembrane glycoprotein that plays a role in cell growth signalling and is expressed in various epithelial tissues. Gene expression profiling and immunohistochemical analyses revealed that MUC4 is also constantly and specifically expressed in low-grade fibromyxoid sarcomas and sclerosing epithelioid fibrosarcomas among the mesenchymal tumours, and immunohistochemical detection of MUC4 is extremely useful for their diagnoses. In our routine pathological practice, we noticed that meningiomas are also often positive for MUC4, which has not yet been reported previously, despite the extensive scrutiny of its expression in soft tissue tumours. METHODS AND RESULTS: We examined immunohistochemically the expression of MUC4, progesterone receptor (PgR) and somatostatin receptor 2A (SSTR2A) in 140 meningiomas of various histological subtypes and 123 other mesenchymal tumours, including intracranial or sinonasal tumours and peripheral nerve sheath tumours. MUC4 was expressed in 130 meningiomas (92.9%). MUC4 expression was constant and almost diffuse in meningothelial and angiomatous subtypes, whereas it was limited in 5% or fewer tumour cells or absent in 26 of 28 fibrous meningiomas. All other mesenchymal tumours examined were negative for MUC4. PgR and SSTR2A were expressed in 94 (67.1%) and 134 (95.7%) meningiomas, respectively. Five of six SSTR2A-negative meningiomas focally expressed MUC4. CONCLUSIONS: MUC4 is expressed variably but almost consistently in meningiomas, particularly in meningothelial or angiomatous subtypes. Its immunohistochemical detection is useful to distinguish meningiomas from other intracranial or head and neck mesenchymal tumours, particularly those with epithelioid features. Our study could expand a variety of MUC4-positive mesenchymal tumours.

Thymoquinone-Induced Tristetraprolin Inhibits Tumor Growth and Metastasis through Destabilization of MUC4 mRNA.

Tristetraprolin (TTP), a well-characterized AU-rich element (ARE) binding protein, functions as a tumor suppressor gene. The purpose of this study was to investigate whether a bioactive substance derived from a natural medicinal plant affects the induction of TTP and to elucidate its mechanism. We examined the effects of natural bioactive materials including Resveratrol (RSV), thymoquinone (TQ) and curcumin on the expression of TTP in cancer cell. TQ derived from a natural plant Nigella sativa increased the expression levels of TTP mRNA and proteins in a dose-dependent manner in gastric and breast cancer cells. TQ-induced TTP increased the instability of MUC4 mRNA by direct binding of TTP to ARE in the 3'UTR of MUC4 mRNA. The induction of TTP by TQ also reduced the proliferation, migration and invasion of cancer cells. The expression of the epithelial-mesenchymal (EMT)-related genes, which were target genes of TTP, was also decreased by the TQ treatment. In the in vivo experiments using mouse melanoma cells, TQ-induced TTP inhibited metastasis of tumor cells. We have found that TQ-induced TTP might inhibit metastasis by reducing tumor cell migration and invasion through destabilization of MUC4 mRNA, which suggest the MUC4 as a novel target to TTP.

Effect of Mucine 4 and Fucosyltransferase 1 genetic variants on gut homoeostasis of growing healthy pigs.

Putative genetic markers have been associated with ETEC F4 (Mucine 4 [MUC4]; MUC4GG;CG as susceptible; MUC4CC as resistant) and F18 (Fucosyltransferase 1 [FUT1]; FUT1GG;AG as susceptible; FUT1AA as resistant) resistances respectively. In this study, 71 post-weaning pigs were followed from d0 (35 days old) to d42 (77 days of age) to investigate the effect of MUC4 or FUT1 genotypes on the mid-jejunal microbiota composition, pigs expression of genes related to inflammation (IL8, GPX2, REG3G, TFF3, CCL20 and LBPI) and glycomic binding pattern profile (Ulex europaeus agglutinin I [UEA] fucose-binding lectin and peanut agglutinin [PNA] galactose-specific), and on blood plasma targeted metabolomics profile, faecal score and performance parameters of growing healthy pigs. The MUC4 and FUT1 resistant genotypes improved the pigs' growth performance and had firmed faecal score susceptible genotypes in d0-d21 period. Pigs with MUC4GG genotype had a higher jejunal expression of genes relate to immune function (CCL20 and REG3G) than MUC4CG and MUC4CC pigs (p < 0.05). MUC4CG pigs had higher expression of TFF3 (implicated in mucosal integrity) than MUC4GG and MUC4CC (p < 0.05). FUT1 influenced the alpha- and beta-jejunal microbial indices. The FUT1AA group had a higher number of operational taxonomic units (OTUs) belonging to Lactobacillus genus, while FUT1GG group had a higher number of OTUs belonging to Veillonella genus. MUC4CC pigs had lower scores for UEA on brush borders and goblet cells in villi than MUC4GG (p < 0.05). FUT1AA pigs had lower UEA positivity and higher PNA positivity on brush borders and goblet cells than FUT1AG and FUT1GG (p < 0.05). Both FUT1 and MUC4 influenced the metabolic profile of healthy pigs. Results highlight the role of MUC4 and FUT1 on pig intestinal homoeostasis and improved the knowledge regarding the potential interaction between host genetics, gut microbiota composition and host metabolism in a healthy status.