Morphological study of TNPO3 and SRSF1 interaction during myogenesis by combining confocal, structured illumination and electron microscopy analysis.

Transportin3 (TNPO3) shuttles the SR proteins from the cytoplasm to the nucleus. The SR family includes essential splicing factors, such as SRSF1, that influence alternative splicing, controlling protein diversity in muscle and satellite cell differentiation. Given the importance of alternative splicing in the myogenic process and in the maintenance of healthy muscle, alterations in the splicing mechanism might contribute to the development of muscle disorders. Combining confocal, structured illumination and electron microscopy, we investigated the expression of TNPO3 and SRSF1 during myogenesis, looking at nuclear and cytoplasmic compartments. We investigated TNPO3 and its interaction with SRSF1 and we observed that SRSF1 remained mainly localized in the nucleus, while TNPO3 decreased in the cytoplasm and was strongly clustered in the nuclei of differentiated myotubes. In conclusion, combining different imaging techniques led us to describe the behavior of TNPO3 and SRSF1 during myogenesis, showing that their dynamics follow the myogenic process and could influence the proteomic network necessary during myogenesis. The combination of different high-, super- and ultra-resolution imaging techniques led us to describe the behavior of TNPO3 and its interaction with SRSF1, looking at nuclear and cytoplasmic compartments. These observations represent a first step in understanding the role of TNPO3 and SRFSF1 in complex mechanisms, such as myogenesis.

Possible Gender-Related Modulation by the ROCK1 Gene in Colorectal Cancer Susceptibility.

AIM: In view of accumulating evidence supporting a pivotal role of the Rho/ROCK pathway in cancer, we investigated Rho-kinase polymorphisms as potential susceptibility factors in colorectal cancer (CRC) in a representative sample of the Italian population. METHODS: DNA obtained from the peripheral blood samples of 137 CRC patients and 141 healthy controls was genotyped for four ROCK1 (rs35996865; rs73963110; rs2127958; rs288980) and five ROCK2 (rs12692437; rs7563468; rs35768389; rs17463896; rs16857265) selected single nucleotide polymorphisms. RESULTS: None of the allelic variants of the nine selected markers was associated with the occurrence of CRC or with the development of regional lymph node metastasis. By contrast, the ROCK1 rs35996865 G variant allele was significantly more frequent in male patients (p = 0.028) than in the control group. CONCLUSION: This finding is, at present, the first that points to a possible gender-related modulation by the ROCK1 gene in CRC susceptibility.

Human Multidrug Resistance 1 gene polymorphisms and Idiopathic Pulmonary Fibrosis.

BACKGROUND: For the first time we tested an association between the human multidrug resistance gene 1 (MDR1) polymorphisms (SNPs) and idiopathic pulmonary fibrosis (IPF). Several MDR1 polymorphisms are associated with pathologies in which they modify the drug susceptibility and pharmacokinetics. MATERIALS AND METHODS: We genotyped three MDR1 polymorphisms of 48 IPF patients and 100 control subjects with Italian origins. RESULTS: No evidence of association was detected. CONCLUSION: There are 50 known MDR1 SNPs, and their role is explored in terms of the effectiveness of drug therapy. We consider our small-scale preliminary study as a starting point for further research.

Colorectal cancer susceptibility: apparent gender-related modulation by ABCB1 gene polymorphisms.

BACKGROUND: The ATP-binding cassette transporter B1 (ABCB1) gene codes for a membrane efflux pump localized in epithelial cells. Together with other Permeability-glycoproteins in the small and large intestine, its product represents a barrier against xenobiotics, bacterial toxins, drugs and other substances introduced with diet, including carcinogens. The aim of this investigation was to verify the possible contribution of ABCB1 single nucleotide polymorphisms (SNPs) to the genetic risk of colorectal cancer (CRC). RESULTS: DNA obtained from the peripheral blood of 98 CRC patients and 100 healthy controls was genotyped for the three selected SNPs: 1236C > T (rs1128503), 2677G > T/A (rs2032582), and 3435C > T (rs1045642). Molecular data were analyzed to asses allele and haplotype association with CRC. No evidence of an association between ABCB1 alleles and CRC occurrence as a whole was found. However, ABCB1 showed either association with carcinoma of the sigmoid colon, and appeared able to influence the sex ratio among CRC patients. These two effects seemed to act independently based on multivariate analysis. We showed that ABCB1 polymorphisms were able to influence CRC susceptibility related to tumor localization and patient gender. CONCLUSIONS: We suggest that sensitivity to undetermined risk factors could depend on the genetic background of ABCB1 locus, with a mechanism that also depends on patient gender.

Electrospun Poly(L-lactide-co-ε-caprolactone) Scaffold Potentiates C2C12 Myoblast Bioactivity and Acts as a Stimulus for Cell Commitment in Skeletal Muscle Myogenesis.

Tissue engineering combines a scaffold, cells and regulatory signals, reproducing a biomimetic extracellular matrix capable of supporting cell attachment and proliferation. We examined the role of an electrospun scaffold made of a biocompatible polymer during the myogenesis of skeletal muscle (SKM) as an alternative approach to tissue regeneration. The engineered nanostructure was obtained by electrospinning poly(L-lactide-co-ε-caprolactone) (PLCL) in the form of a 3D porous nanofibrous scaffold further coated with collagen. C2C12 were cultured on the PLCL scaffold, and cell morphology and differentiation pathways were thoroughly investigated. The functionalized PLCL scaffold recreated the SKM nanostructure and performed its biological functions, guiding myoblast morphogenesis and promoting cell differentiation until tissue formation. The scaffold enabled cell-cell interactions through the development of cellular adhesions that were fundamental during myoblast fusion and myotube formation. Expression of myogenic regulatory markers and muscle-specific proteins at different stages of myogenesis suggested that the PLCL scaffold enhanced myoblast differentiation within a shorter time frame. The functionalized PLCL scaffold impacts myoblast bioactivity and acts as a stimulus for cell commitment, surpassing traditional 2D cell culture techniques. We developed a screening model for tissue development and a device for tissue restoration.

LGMD D2 TNPO3-Related: From Clinical Spectrum to Pathogenetic Mechanism.

Limb-girdle muscular dystrophies (LGMDs) are clinically and genetically heterogeneous diseases presenting with a wide clinical spectrum. Autosomal dominant LGMDs represent about 10-15% of LGMDs and include disorders due to defects of DNAJB6, transportin-3 (TNPO3), HNRNPDL, Calpain-3 (CAPN3), and Bethlem myopathy. This review article aims to describe the clinical spectrum of LGMD D2 TNPO3-related, a rare disease due to heterozygous mutation in the TNPO3 gene. TNPO3 encodes for transportin-3, which belongs to the importin beta family and transports into the nucleus serine/arginine-rich (SR) proteins, such as splicing factors, and HIV-1 proteins, thus contributing to viral infection. The purpose of this review is to present and compare the clinical features and the genetic and histopathological findings described in LGMD D2, performing a comparative analytical description of all the families and sporadic cases identified. Even if the causative gene and mutations of this disease have been identified, the pathogenic mechanisms are still an open issue; therefore, we will present an overview of the hypotheses that explain the pathology of LGMD D2 TNPO3-related.

Transportin 3 (TNPO3) and related proteins in limb girdle muscular dystrophy D2 muscle biopsies: A morphological study and pathogenetic hypothesis.

LGMD D2 is a disease caused by TNPO3 mutation. We describe the expression of TNPO3 and selected proteins, likely modified by TNPO3 mutation, in muscle biopsies of affected patients. We also aim to find other genes involved in pathways correlated to TNPO3. Our morphological study on LGMD D2 muscle described the expression of TNPO3 and SRSF1, a splicing factor transported by TNPO3. Moreover, we investigated some sarcomeric and nuclear proteins, likely altered by TNPO3 mutation. Through an in silico approach we tried to identify genes involved in pathways that include, besides TNPO3 and SRSF1, p62 and Murf-1, altered in LGMD D2. In patients' muscles TNPO3 appeared weaker and randomly organized, with sporadic cytoplasmic aggregates positive for TNPO3; both SRSF1 and sarcomeric alpha actinin showed a different expression, while there were no alterations in the expression of the nuclear proteins. The in silico study lead to identify five genes, all coding for proteins responsible for muscle contraction. Our data suggest a possible interference in the morphology and function of myofibrillar network by mutated TNPO3; these findings are supported by the in silico identification of genes involved in muscle contraction that could help to explain the pathogenic mechanisms of LGMD D2.

LGALS4, CEACAM6, TSPAN8, and COL1A2: Blood Markers for Colorectal Cancer-Validation in a Cohort of Subjects With Positive Fecal Immunochemical Test Result.

BACKGROUND: A noninvasive blood test for the early detection of colorectal cancer (CRC) is highly required. We evaluated a panel of 4 mRNAs as putative markers of CRC. MATERIALS AND METHODS: We tested LGALS4, CEACAM6, TSPAN8, and COL1A2, referred to as the CELTiC panel, using quantitative reverse transcription polymerase chain reaction, on subjects with positive fecal immunochemical test (FIT) results and undergoing colonoscopy. Using a nonparametric test and multinomial logistic model, FIT-positive subjects were compared with CRC patients and healthy individuals. RESULTS: All the genes of the CELTiC panel displayed statistically significant differences between the healthy subjects (n = 67), both low-risk (n = 36) and high-risk/CRC (n = 92) subjects, and those in the negative-colonoscopy, FIT-positive group (n = 36). The multinomial logistic model revealed LGALS4 was the most powerful marker discriminating the 4 groups. When assessing the diagnostic values by analysis of the areas under the receiver operating characteristic curves (AUCs), the CELTiC panel reached an AUC of 0.91 (sensitivity, 79%; specificity, 94%) comparing normal subjects to low-risk subjects, and 0.88 (sensitivity, 75%; specificity, 87%) comparing normal and high-risk/CRC subjects. The comparison between the normal subjects and the negative-colonoscopy, FIT-positive group revealed an AUC of 0.93 (sensitivity, 82%; specificity, 97%). CONCLUSION: The CELTiC panel could represent a useful tool for discriminating subjects with positive FIT findings and for the early detection of precancerous adenomatous lesions and CRC.

Systematic large-scale meta-analysis identifies a panel of two mRNAs as blood biomarkers for colorectal cancer detection.

Colorectal cancer (CRC) is the third most common cancer in the world. A significant survival rate is achieved if it is detected at an early stage. A whole blood screening test, without any attempt to isolate blood fractions, could be an important tool to improve early detection of colorectal cancer. We searched for candidate markers with a novel approach based on the Transcriptome Mapper (TRAM), aimed at identifying specific RNAs with the highest differential expression ratio between colorectal cancer tissue and normal blood samples. This tool permits a large-scale systematic meta-analysis of all available data obtained by microarray experiments. The targeting of RNA took into consideration that tumour phenotypic variation is associated with changes in the mRNA levels of genes regulating or affecting this variation.A real time quantitative reverse transcription polymerase chain reaction (qRT- PCR) was applied to the validation of candidate markers in the blood of 67 patients and 67 healthy controls. The expression of genes: TSPAN8, LGALS4, COL1A2 and CEACAM6 resulted as being statistically different.In particular ROC curves attested for TSPAN8 an AUC of 0.751 with a sensitivity of 83.6% and a specificity of 58.2% at a cut off of 10.85, while the panel of the two best genes showed an AUC of 0.861 and a sensitivity of 92.5% with a specificity of 67.2%.Our preliminary study on a total of 134 subjects showed promising results for a blood screening test to be validated in a larger cohort with the staging stratification and in patients with other gastrointestinal diseases.

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors.

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.