In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A.

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion.

BACKGROUND: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages. METHODS: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage. RESULTS: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair. CONCLUSION: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy.

Gender-specific associations between polymorphisms of the circadian gene RORA and cutaneous melanoma susceptibility.

BACKGROUND: Melanoma is the deadliest of skin cancers and has an increasing annual incidence worldwide. It is a multi-factorial disease most likely arising from both genetic predisposition and environmental exposure to ultraviolet light. Genetic variability of the components of the biological circadian clock is recognized to be a risk factor for different type of cancers. Moreover, two variants of a clock gene, RORA, have been associated with melanoma patient's prognosis. Our aim is to test the hypothesis that specific single nucleotide polymorphisms (SNPs) of the circadian clock genes may significantly influence the predisposition to develop cutaneous melanoma or the outcome of melanoma patients. METHODS: We genotyped 1239 subjects, 629 cases of melanoma and 610 healthy controls in 14 known SNPs of seven selected clock genes: AANAT, CLOCK, NPAS2, PER1, PER2, RORA, and TIMELESS. Genotyping was conducted by q-PCR. Multivariate logistic regression was employed for susceptibility of melanoma assessment, modeled additively. Subgroup analysis was performed by gender. For the female subgroup, a further discrimination was performed by age. For prognosis of melanoma assessment, multivariate Cox proportional hazard regression was employed. The Benjamini-Hochberg method was utilized as adjustment for multiple comparisons. RESULTS: We identified two RORA SNPs statistically significant with respect to the association with melanoma susceptibility. Considering the putative role of RORA as a nuclear steroid hormone receptor, we conducted a subgroup analysis by gender. Interestingly, the RORA rs339972 C allele was associated with a decreased predisposition to develop melanoma only in the female subgroup (OR 0.67; 95% CI 0.51-0.88; P = 0.003) while RORA rs10519097 T allele was associated with a decreased predisposition to develop melanoma only in the male subgroup (OR 0.62; 95% CI 0.44-0.87; P = 0.005). Moreover, the RORA rs339972 C allele had a decreased susceptibility to develop melanoma only in females aged over 50 years old (OR 0.67; 95% CI 0.54-0.83; P = 0.0002). None of the studied SNPs were significantly associated with the prognosis. CONCLUSIONS: Overall, we cannot ascertain that circadian pathway genetic variation is involved in melanoma susceptibility or prognosis. Nevertheless, we identified an interesting relationship between melanoma susceptibility and RORA polymorphisms acting in sex-specific manner and which is worth further future investigation.

Spotlight on Circadian Genes and Colorectal Cancer Crosstalk.

Mammalian physiology is regulated by circadian clock through oscillating feedback loops controlling cellular processes and behaviors. Recent findings have led to an interesting connection between circadian disruption and colorectal cancer progression and incidence through controlling the hallmarks of cancer, namely cell cycle, cell metabolism and cell death. Deeper understanding of the circadian mechanisms that define the colorectal cancer pathophysiology is the need of the hour to define a chronotherapy for improving colorectal cancer patient survival. This review identifies the key areas in which circadian genes interact with cellular pathways to modify the outcome with respect to colorectal cancer incidence and progression.

miRNA deregulation targets specific pathways in leiomyosarcoma development: an in silico analysis.

BACKGROUND: MicroRNA (miRNA) mediate post-transcriptional gene repression and are involved in a variety of human diseases, including cancer. Soft tissue sarcomas are rare malignancies with a variety of histological subtypes which may occur virtually anywhere in the human body. Leiomyosarcoma is one of the most common subtypes, shows a smooth muscle phenotype and its cancerogenesis is still unclear. The aim of our study was to investigate the potential role of miRNA differential expression in leiomyosarcoma development. METHODS: We first employed the Sarcoma microRNA Expression Database, a repository that describes the patterns of over 1000 miRNA expression in various human sarcoma types, to identify differentially expressed miRNA comparing leiomyosarcoma and smooth muscle samples. Subsequently, we identified putative target genes of those miRNAs with the TargetScan prediction tool. Finally, we evaluated whether the retrieved pool of putative targets was enriched in genes belonging to specific molecular pathways by means of the Enrichr analysis tool. Protein-protein network analysis was analyzed by means of the STRING web tool. RESULTS: Out of 1120 miRNAs tested, the expression of 301 miRNAs was statistically significantly different between leiomyosarcoma and smooth muscle samples. The hypothetical targets could be predicted for 172 miRNAs. 438 genes were predicted to be the targets with high confidence (cumulative weighted context score cut-off level less than - 1.0) and analyzed for belonging to specific molecular pathways. Pathway analysis suggested that RNA Polymerase III, tRNA functions and synaptic neurotransmission (with special regard to dopamine mediated signaling) could be involved in leiomyosarcoma development. CONCLUSIONS: Our results demonstrate that data mining of publicly available repositories can be useful to suggest molecular pathways underlying the pathogenesis of rare tumors such as leiomyosarcoma.

Understanding the good and poor cell targeting activity of gold nanostructures functionalized with molecular units for the epidermal growth factor receptor.

Nanostructures can strongly interact with cells or other biological structures; furthermore when they are functionalized with targeting units, they are of great interest for a variety of applications in the biotechnology field like those for efficient imaging, diagnosis and therapy and in particular for cancer theranostics. Obtaining targeting with good specificity and sensitivity is a key necessity, which, however, is affected by the complexity of the interactions between the nanostructures and the biological components. In this work we report the study of specificity and sensitivity of gold nanoparticles functionalized with the peptide GE11 for the targeting of the epidermal growth factor receptor, expressed on many cells and, in particular, on many types of cancer cells. We show how a combination of spectroscopic measurements and molecular dynamics simulations allows the comprehension of the targeting activity of peptides linked to the surface of gold nanostructures and how the targeting is tuned by the presence of polyethylene glycol chains.

Associations of clock genes polymorphisms with soft tissue sarcoma susceptibility and prognosis.

BACKGROUND: Dysfunction of the circadian clock and polymorphisms of some circadian genes have been linked to cancer development and progression. We investigated the relationship between circadian genes germline variation and susceptibility or prognosis of patients with soft tissue sarcoma. PATIENTS AND METHODS: We considered the 14 single nucleotide polymorphisms (SNPs) of 6 core circadian genes that have a minor allele frequency > 5% and that are known to be associated with cancer risk or prognosis. Genotyping was performed by q-PCR. Peripheral blood and clinic-pathological data were available for 162 patients with liposarcoma or leiomyosarcoma and 610 healthy donors. Associations between the selected clock genes polymorphisms and sarcoma susceptibility or prognosis were tested assuming 3 models of inheritance: additive, recessive and dominant. Subgroup analysis based on sarcoma histotype was performed under the additive genetic model. Multivariate logistic regression and multivariate Cox proportional hazard regression analyses were utilized to assess the association between SNPs with patient susceptibility and survival, respectively. Pathway variation analysis was conducted employing the Adaptive Rank Truncated Product method. RESULTS: Six out of the 14 analyzed SNPs were statistically significantly associated with susceptibility or prognosis of soft tissue sarcoma (P < 0.05). The present analysis suggested that carriers of the minor allele of the CLOCK polymorphism rs1801260 (C) or of PER2 rs934945 (T) had a reduced predisposition to sarcoma (26% and 35% respectively with the additive model) and liposarcoma (33% and 41% respectively). The minor allele (A) of NPAS2 rs895520 was associated with an increased predisposition to sarcoma of 33% and leiomyosarcoma of 44%. RORA rs339972 C allele was associated with a decreased predisposition to develop sarcoma assuming an additive model (29%) and leiomyosarcoma (36%). PER1 rs3027178 was associated with a reduced predisposition only in liposarcoma subgroup (32%). rs7602358 located upstream PER2 was significantly associated with liposarcoma survival (HR: 1.98; 95% CI 1.02-3.85; P = 0.04). Germline genetic variation in the circadian pathway was associated with the risk of developing soft tissue sarcoma (P = 0.035). CONCLUSIONS: Genetic variation of circadian genes appears to play a role in the determinism of patient susceptibility and prognosis. These findings prompt further studies to fully dissect the molecular mechanisms.

Biofabrication of a novel leukocyte-fibrin-platelet membrane as a cells and growth factors delivery platform for tissue engineering applications.

Autologous platelet-rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte-fibrin-platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin-10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface-protein level (CD44high /CD73+ /CD34+ /CD117+ /CD31+ ). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.

Genetic susceptibility to bone and soft tissue sarcomas: a field synopsis and meta-analysis.

BACKGROUND: The genetic architecture of bone and soft tissue sarcomas susceptibility is yet to be elucidated. We aimed to comprehensively collect and meta-analyze the current knowledge on genetic susceptibility in these rare tumors. METHODS: We conducted a systematic review and meta-analysis of the evidence on the association between DNA variation and risk of developing sarcomas through searching PubMed, The Cochrane Library, Scopus and Web of Science databases. To evaluate result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Integrative analysis of genetic and eQTL (expression quantitative trait locus) data was coupled with network and pathway analysis to explore the hypothesis that specific cell functions are involved in sarcoma predisposition. RESULTS: We retrieved 90 eligible studies comprising 47,796 subjects (cases: 14,358, 30%) and investigating 1,126 polymorphisms involving 320 distinct genes. Meta-analysis identified 55 single nucleotide polymorphisms (SNPs) significantly associated with disease risk with a high (N=9), moderate (N=38) and low (N=8) level of evidence, findings being classified as noteworthy basically only when the level of evidence was high. The estimated joint population attributable risk for three independent SNPs (rs11599754 of ZNF365/EGR2, rs231775 of CTLA4, and rs454006 of PRKCG) was 37.2%. We also identified 53 SNPs significantly associated with sarcoma risk based on single studies.Pathway analysis enabled us to propose that sarcoma predisposition might be linked especially to germline variation of genes whose products are involved in the function of the DNA repair machinery. CONCLUSIONS: We built the first knowledgebase on the evidence linking DNA variation to sarcomas susceptibility, which can be used to generate mechanistic hypotheses and inform future studies in this field of oncology.

Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide.

Plasmonic nanostructures show important properties for biotechnological applications, but they have to be guided on the target for exploiting their potentialities. Antibodies are the natural molecules for targeting. However, their possible adverse immunogenic activity and their cost have suggested finding other valid substitutes. Small molecules like peptides can be an alternative source of targeting agents, even if, as single molecules, their binding affinity is usually not very good. GE11 is a small dodecapeptide with specific binding to the epidermal growth factor receptor (EGFR) and low immunogenicity. The present work shows that thousands of polyethylene glycol (PEG) chains modified with lysines and functionalized with GE11 on clusters of naked gold nanoparticles, obtained by laser ablation in water, achieves a better targeting activity than that recorded with nanoparticles decorated with the specific anti-EGFR antibody Cetuximab (C225). The insertion of the cationic spacer between the polymeric part of the ligand and the targeting peptide allows for a proper presentation of GE11 on the surface of the nanosystems. Surface enhanced resonance Raman scattering signals of the plasmonic gold nanoparticles are used for quantifying the targeting activity. Molecular dynamic calculations suggest that subtle differences in the exposition of the peptide on the PEG sea are important for the targeting activity.

Genetic variation of clock genes and cancer risk: a field synopsis and meta-analysis.

BACKGROUND: The number of studies on the association between clock genes' polymorphisms and cancer susceptibility has increased over the last years but the results are often conflicting and no comprehensive overview and quantitative summary of the evidence in this field is available. RESULTS: Literature search identified 27 eligible studies comprising 96756 subjects (cases: 38231) and investigating 687 polymorphisms involving 14 clock genes. Overall, 1025 primary and subgroup meta-analyses on 366 gene variants were performed. Study distribution by tumor was as follows: breast cancer (n=15), prostate cancer (n=3), pancreatic cancer (n=2), non-Hodgkin's lymphoma (n=2), glioma (n=1), chronic lymphocytic leukemia (n=1), colorectal cancer (n=1), non-small cell lung cancer (n=1) and ovarian cancer (n=1).We identified 10 single nucleotide polymorphisms (SNPs) significantly associated with cancer risk: NPAS2 rs10165970 (mixed and breast cancer shiftworkers), rs895520 (mixed), rs17024869 (breast) and rs7581886 (breast); CLOCK rs3749474 (breast) and rs11943456 (breast); RORA rs7164773 (breast and breast cancer postmenopausal), rs10519097 (breast); RORB rs7867494 (breast cancer postmenopausal), PER3 rs1012477 (breast cancer subgroups) and assessed the level of quality evidence to be intermediate. We also identified polymorphisms with lower quality statistically significant associations (n=30). CONCLUSIONS: Our work supports the hypothesis that genetic variation of clock genes might affect cancer risk. These findings also highlight the need for more efforts in this research field in order to fully establish the contribution of clock gene variants to the risk of developing cancer. METHODS: We conducted a systematic review and meta-analysis of the evidence on the association between clock genes' germline variants and the risk of developing cancer. To assess result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Subgroup meta-analysis was also performed based on participant features and tumor type. The breast cancer subgroup was further stratified by work conditions, estrogen receptor/progesterone receptor status and menopausal status, conditions associated with the risk of breast cancer in different studies.

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue-engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein-loading ability and biodegradability. Copyright © 2015 John Wiley & Sons, Ltd.

Gene and MicroRNA Expression Are Predictive of Tumor Response in Rectal Adenocarcinoma Patients Treated With Preoperative Chemoradiotherapy.

Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT. Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one-color microarray technique that compares signatures between responders (R) and non-responders (NR), as measured based on tumor regression grade. Two groups composed of 38 "exploration cohort" and 21 "validation cohort" LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti-correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR-630 appeared only with the NR patients and was anti-correlated with a single transcript: RAB5B. After PAM, the following eight transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7, and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity, and 82% specificity. All three parameters reached 100% when both cohorts were considered together. In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. J. Cell. Physiol. 232: 426-435, 2017. © 2016 Wiley Periodicals, Inc.

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs.

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of ß-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote ß-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated ß-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen ß-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total ß-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.

Differential activation of sporamin expression in response to abiotic mechanical wounding and biotic herbivore attack in the sweet potato.

BACKGROUND: Plants respond differently to mechanical wounding and herbivore attack, using distinct pathways for defense. The versatile sweet potato sporamin possesses multiple biological functions in response to stress. However, the regulation of sporamin gene expression that is activated upon mechanical damage or herbivore attack has not been well studied. RESULTS: Biochemical analysis revealed that different patterns of Reactive oxygen species (ROS) and antioxidant mechanism exist between mechanical wounding (MW) and herbivore attack (HA) in the sweet potato leaf. Using LC-ESI-MS (Liquid chromatography electrospray ionization mass spectrometry analysis), only the endogenous JA (jasmonic acid) level was found to increase dramatically after MW in a time-dependent manner, whereas both endogenous JA and SA (salicylic acid) increase in parallel after HA. Through yeast one-hybrid screening, two transcription factors IbNAC1 (no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), and cup-shaped cotyledon (CUC)) and IbWRKY1 were isolated, which interact with the sporamin promoter fragment of SWRE (sporamin wounding-responsive element) regulatory sequences. Exogenous application of MeJA (methyl jasmonate), SA and DIECA (diethyldithiocarbamic acid, JAs biosynthesis inhibitor) on sweet potato leaves was employed, and the results revealed that IbNAC1 mediated the expression of sporamin through a JA-dependent signaling pathway upon MW, whereas both IbNAC1 and IbWRKY1 coordinately regulated sporamin expression through JA- and SA-dependent pathways upon HA. Transcriptome analysis identified MYC2/4 and JAZ2/TIFY10A (jasmonate ZIM/tify-domain), the repressor and activator of JA and SA signaling among others, as the genes that play an intermediate role in the JA and SA pathways, and these results were further validated by qRT-PCR (quantitative real-time polymerase chain reaction). CONCLUSION: This work has improved our understanding of the differential regulatory mechanism of sporamin expression. Our study illustrates that sweet potato sporamin expression is differentially induced upon abiotic MW and biotic HA that involves IbNAC1 and IbWRKY1 and is dependent on the JA and SA signaling pathways. Thus, we established a model to address the plant-wounding response upon physical and biotic damage.