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We report a new 1-6 self-immolative, traceless crosslinker derived from the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyrogallol orthoester derivative (HMPO) was stable for 24 hours at pH values of 7.4 and 6.6 and in plasma, releasing molecules bound to the hydroxymethyl moiety under acid-dependent stimuli at pH 5.5. The linker was non-toxic and was used for the conjugation of Doxorubicin (Doxo) or Combretastatin A4 with Cetuximab. The ADCs formed showed their pH responsivity reducing cell viability of A431 and A549 cancer cells better than Cetuximab alone.

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.

It is recognized that prostaglandin E2 (PGE2) is one key lipid mediator involved in chronic inflammation, and it is directly implicated in tumor development by regulating cancer cell growth and migration, apoptosis, epithelial–mesenchymal transition, angiogenesis, and immune escape. In addition, the expression of the enzymes involved in PGE2 synthesis, cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES1), positively correlates with tumor progression and aggressiveness, clearly indicating the crucial role of the entire pathway in cancer. Moreover, several lines of evidence suggest that the COX2/mPGES1/PGE2 inflammatory axis is involved in the modulation of epidermal growth factor receptor (EGFR) signaling to reinforce the oncogenic drive of EGFR activation. Similarly, EGFR activation promotes the induction of COX2/mPGES1 expression and PGE2 production. In this review, we describe the interplay between COX2/mPGES1/PGE2 and EGFR in cancer, and new therapeutic strategies that target this signaling pathway, to outline the importance of the modulation of the inflammatory process in cancer fighting.

Cerebral cavernous malformation (CCM) is a rare disease of genetic origin characterized by dilated and leaky capillaries occurring mainly in the central nervous system. CCM can arise sporadically or may be inherited as an autosomal dominant condition with incomplete penetrance and variable clinical expressivity. The sporadic form accounts for up to 80% of cases, whereas the familial form accounts for at least 20% of cases. Genetic studies have identified three genes associated with CCMs: KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3). Recently, great advances in understanding the pathophysiology of CCM disease have been obtained thanks to the use of animal and cellular models displaying all or some of the pathological characteristics that are observed in the human disease. Despite interspecies differences and the difficulty in creating animal models that completely recapitulate the human CCM disease onset and progression, these models have been helpful in identifying new molecular mechanisms underlying CCM development and in testing novel pharmacological therapies.

The genus Vaccinium L. (Ericaceae) includes more than 450 species, which mainly grow in cooler areas of the northern hemisphere. Vaccinium species have been used in traditional medicine of different cultures and the berries are widely consumed as food. Indeed, Vaccinium supplement-based herbal medicine and functional food, mainly from V. myrtillus and V. macrocarpon, are used in Europe and North America. Biological studies support traditional uses since, for many Vaccinium components, important biological functions have been described, including antioxidant, antitumor, anti-inflammatory, antidiabetic and endothelium protective activities. Vaccinium components, such as polyphenols, anthocyanins and flavonoids, are widely recognized as modulators of cellular pathways involved in pathological conditions, thus indicating that Vaccinium may be an important source of bioactive molecules. This review aims to better describe the bioactivity of Vaccinium species, focusing on anti-inflammatory and endothelial protective cellular pathways, modulated by their components, to better understand their importance for public health.

Phaseolus vulgaris L. (common bean) is a leguminous species that is an important dietary component due to its high content of proteins, unsaturated fatty acids, minerals, dietary fibers and vitamins. Due to the high content of polyphenols, several biological activities have been described for bean extracts, making it possible to include P. vulgaris among food with beneficial effects for human health. Moreover, more than 40,000 varieties of beans have been recognised with different nutraceutical properties, pointing out the importance of food biodiversity. In this work, we describe for the first time the chemical composition and biological activity of a newly recognized Italian variety of P. vulgaris grown in a restricted area of the Tuscany region and named &ldquo, Fagiola di Venanzio&rdquo, Fagiola di Venanzio water extract is rich in proteins, sugars and polyphenols and displays antioxidant, anti-inflammatory and antiproliferative activities in in vitro assays on colon cancer cellular models. Our data indicate that this variety of P. vulgaris appears to be a promising source of bioactive compounds and encourage more in-depth studies to better elucidate the implications of its consumption for public health.

The yeast two-hybrid system was originally designed to detect protein-protein interactions using yeast transcriptional activators. Since its original description, this technique has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. Nowadays, the yeast two-hybrid screen remains one of the leading molecular tools to study protein-protein interactions in native intracellular conditions. In these years, the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches.Several groups studying molecular mechanisms underlying the Cerebral Cavernous Malformation disease have successfully used the yeast two-hybrid system or related methods to isolate, identify, and characterize molecular interactions involved in the onset and progression of the pathology.Here we describe general principles, strengths, and limits of the yeast two-hybrid technology, and the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay by using a GAL4-based system.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified three genes associated to CCMs: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), which account for about 50%, 20%, and 10% of the cases, respectively. The great advances in the knowledge of the physiopathological functions of CCM genes, such as their involvement in the angiogenic process, have allowed to propose distinct putative therapeutic compounds, which showed to be effective at least in limiting some pathological phenotypes in cellular and animal models of the disease. However, despite numerous efforts, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking.Here we describe simply and low-cost assays as in vitro endothelial cell proliferation and migration assays that can be used to better understand the role of CCM genes on endothelial cell functions and to screen potential new compounds for CCM therapy.

Cerebral cavernous malformation (CCM) is a rare cerebrovascular disorder of genetic origin consisting of closely clustered, abnormally dilated and leaky capillaries (CCM lesions), which occur predominantly in the central nervous system. CCM lesions can be single or multiple and may result in severe clinical symptoms, including focal neurological deficits, seizures, and intracerebral hemorrhage. Early human genetic studies demonstrated that CCM disease is linked to three chromosomal loci and can be inherited as autosomal dominant condition with incomplete penetrance and highly variable expressivity, eventually leading to the identification of three disease genes, CCM1/KRIT1, CCM2, and CCM3/PDCD10, which encode for structurally unrelated intracellular proteins that lack catalytic domains. Biochemical, molecular, and cellular studies then showed that these proteins are involved in endothelial cell-cell junction and blood-brain barrier stability maintenance through the regulation of major cellular structures and mechanisms, including endothelial cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, autophagy, and endothelial-to-mesenchymal transition, suggesting that they act as pleiotropic regulators of cellular homeostasis, and opening novel therapeutic perspectives. Indeed, accumulated evidence in cellular and animal models has eventually revealed that the emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses.In this introductory review, we present a general overview of 20 years of amazing progress in the identification of genetic culprits and molecular mechanisms underlying CCM disease pathogenesis, and the development of targeted therapeutic strategies.

Krev interaction trapped protein 1 (KRIT1) is a scaffold protein known to form functional complexes with distinct proteins, including Malcavernin, PDCD10, Rap1 and others. It appears involved in several cellular signaling pathways and exerts a protective role against inflammation and oxidative stress. KRIT1 has been studied as a regulator of endothelial cell functions and represents a determinant in the pathogenesis of Cerebral Cavernous Malformation (CCM), a cerebrovascular disease characterized by the formation of clusters of abnormally dilated and leaky blood capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage. Although KRIT1 is ubiquitously expressed, few studies have described its involvement in pathologies other than CCM including cancer. Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic propensity. Despite the numerous efforts made to define the signaling pathways activated during melanoma progression, the molecular mechanisms at the basis of melanoma growth, phenotype plasticity and resistance to therapies are still under investigation.

Cerebral cavernous malformation (CCM) is a rare cerebrovascular disorder of genetic origin consisting of closely clustered, abnormally dilated and leaky capillaries (CCM lesions), which occur predominantly in the central nervous system. CCM lesions can be single or multiple and may result in severe clinical symptoms, including focal neurological deficits, seizures, and intracerebral hemorrhage. Early human genetic studies demonstrated that CCM disease is linked to three chromosomal loci and can be inherited as autosomal dominant condition with incomplete penetrance and highly variable expressivity, eventually leading to the identification of three disease genes, CCM1/KRIT1, CCM2, and CCM3/PDCD10, which encode for structurally unrelated intracellular proteins that lack catalytic domains. Biochemical, molecular, and cellular studies then showed that these proteins are involved in endothelial cell-cell junction and blood-brain barrier stability maintenance through the regulation of major cellular structures and mechanisms, including endothelial cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, autophagy, and endothelial-to-mesenchymal transition, suggesting that they act as pleiotropic regulators of cellular homeostasis, and opening novel therapeutic perspectives. Indeed, accumulated evidence in cellular and animal models has eventually revealed that the emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses.In this introductory review, we present a general overview of 20 years of amazing progress in the identification of genetic culprits and molecular mechanisms underlying CCM disease pathogenesis, and the development of targeted therapeutic strategies.

Simple Summary Inflammation is assessed as a hallmark of cancer and it is now widely recognized that there exists a direct causal link between inflammation and tumors. Among the inflammatory mediators, prostaglandin E2 (PGE2), the major product of cyclooxygenases (COXs), plays a pivotal role in tumor progression. Numerous pieces of evidence suggest that drugs, such as aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit PGE2 production, may exert a protective effect against tumor initiation and may play a role during tumor progression. In fact, a number of studies suggest that PGE2 increases tumor growth and invasion, reduces apoptosis, increases metastasis and angiogenesis, and suppresses antitumor immunity. In this review, we describe the current knowledge on the pro-tumoral activity of PGE2 focusing on its role in cancer progression and in the regulation of the tumor microenvironment. Abstract The involvement of inflammation in cancer progression has been the subject of research for many years. Inflammatory milieu and immune response are associated with cancer progression and recurrence. In different types of tumors, growth and metastatic phenotype characterized by the epithelial mesenchymal transition (EMT) process, stemness, and angiogenesis, are increasingly associated with intrinsic or extrinsic inflammation. Among the inflammatory mediators, prostaglandin E2 (PGE2) supports epithelial tumor aggressiveness by several mechanisms, including growth promotion, escape from apoptosis, transactivation of tyrosine kinase growth factor receptors, and induction of angiogenesis. Moreover, PGE2 is an important player in the tumor microenvironment, where it suppresses antitumor immunity and regulates tumor immune evasion, leading to increased tumoral progression. In this review, we describe the current knowledge on the pro-tumoral activity of PGE2 focusing on its role in cancer progression and in the regulation of the tumor microenvironment.

The yeast two-hybrid system was originally designed to detect protein-protein interactions using yeast transcriptional activators. Since its original description, this technique has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. Nowadays, the yeast two-hybrid screen remains one of the leading molecular tools to study protein-protein interactions in native intracellular conditions. In these years, the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches.Several groups studying molecular mechanisms underlying the Cerebral Cavernous Malformation disease have successfully used the yeast two-hybrid system or related methods to isolate, identify, and characterize molecular interactions involved in the onset and progression of the pathology.Here we describe general principles, strengths, and limits of the yeast two-hybrid technology, and the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay by using a GAL4-based system.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified three genes associated to CCMs: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3), which account for about 50%, 20%, and 10% of the cases, respectively. The great advances in the knowledge of the physiopathological functions of CCM genes, such as their involvement in the angiogenic process, have allowed to propose distinct putative therapeutic compounds, which showed to be effective at least in limiting some pathological phenotypes in cellular and animal models of the disease. However, despite numerous efforts, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking.Here we describe simply and low-cost assays as in vitro endothelial cell proliferation and migration assays that can be used to better understand the role of CCM genes on endothelial cell functions and to screen potential new compounds for CCM therapy.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function. In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells. Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.

Avenanthramides (Avns), polyphenols found exclusively in oats, are emerging as promising therapeutic candidates for the treatment of several human diseases, including colon cancer. By engineering a Saccharomyces cerevisiae strain, we previously produced two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, YAvnI) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, YAvnII), which are endowed with a structural similarity to bioactive oat avenanthramides and stronger antioxidant properties. In this study, we evaluated the ability of these yeast-derived recombinant avenanthramides to inhibit major hallmarks of colon cancer cells, including sustained proliferation, migration and epithelial-mesenchymal transition (EMT). Using the human colon adenocarcinoma cell line HT29, we compared the impact of YAvns and natural Avns, including Avn-A and Avn-C, on colon cancer cells by performing MTT, clonogenic, adhesion, migration, and anchorage-independent growth assays, and analyzing the expression of EMT markers. We found that both YAvns and Avns were able to inhibit colon cancer cell growth by increasing the expression of p21, p27 and p53 proteins. However, YAvns resulted more effective than natural compounds in inhibiting cancer cell migration and reverting major molecular features of the EMT process, including the down-regulation of E-cadherin mRNA and protein levels.

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3–0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies., Graphical abstract Schematic models representing adaptive redox responses associated with KRIT1 loss-of-function. KRIT1 loss-of-function causes a persistent activation of the redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS* and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp70 and AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. Inter-individual differences in Nrf2-mediated adaptive defense mechanisms might influence susceptibility to CCM disease onset and progression. *The generic ROS term refers to O2•−and H2O2as well as to putative secondary oxidative products that might be implicated without certainty.fx1, Highlights • KRIT1 loss causes a chronic adaptive redox response based on the JNK-Nrf2-Glo1 axis. • Phospho-JNK, Nrf2 and Glo1 are upregulated in endothelial cells lining human CCMs. • Defective autophagy contributes to the sustained upregulation of the Nrf2-Glo1 axis. • Nrf2-Glo1 upregulation causes a drop of AP-modified Hsp70 and Hsp27 proteins. • Sustained Nrf2-Glo1 activation sensitizes cells to oxidative stress and apoptosis.

Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease of genetic origin affecting 0.3-0.5% of the population and still awaiting therapies other than neurosurgery. It is characterized by abnormally enlarged and leaky capillaries, which predispose to seizures, neurological deficits and intracerebral hemorrhage (ICH), and may occur sporadically or is dominantly inherited with incomplete penetrance and variable expressivity. Three disease genes have been identified, KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), whose loss-of-function mutations are major pathogenic determinants, accounting for the main phenotypic hallmarks of CCM disease, including destabilization of endothelial cell-cell junctions and increased vascular permeability [1]. However, accumulating evidence in animal models clearly demonstrate that homozygous loss of CCM genes is not fully sufficient to cause CCM lesion formation and disease progression, suggesting the necessary contribution of additional determinants, including microenvironmental stress events [1]. Indeed, the clinical behavior in individual patients, including development of numerous and large lesions, and risk of ICH, remains highly unpredictable, while novel pharmacological strategies are particularly needed to limit disease progression and severity in susceptible individuals [1]. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins. Previously, we found that CCM proteins, including KRIT1, play an important role in maintaining intracellular redox homeostasis through the modulation of master regulators of ROS production/detoxification and cell responses to oxidative stress, thereby limiting altered redox signaling and oxidative damage, and preserving cellular resistance to oxidative stress [1-3]. Consistently, recently we demonstrated that KRIT1 loss-of-function causes upregulation of NADPH oxidase-mediated redox signaling, leading to enhanced endothelial cell sensitivity to oxidative stress and inflammation, and decreased microvessel barrier function, further suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis [4]. Moreover, preliminary results indicate that these events involve also carbonyl compounds generated through the lipid peroxidation process, and a sustained upregulation of Nrf2, suggesting a complex but unifying pathogenic mechanism that reconciles both the pleiotropic functions of CCM proteins and the distinct therapeutic approaches proposed so far. In addition, we identified genetic modifiers influencing disease severity, including polymorphisms in genes related to inter-individual variability in susceptibility to oxidative stress [5]. Taken together, our findings point to a major role for altered redox signaling in CCM pathogenesis and indicate that inter-individual variability in cell responses to oxidative stress may impact disease onset, progression and severity, suggesting novel preventive and therapeutic approaches.

Saccharomyces cerevisiae has been proven to be a valuable tool for the expression of plant metabolic pathways. By engineering a S. cerevisiae strain with two plant genes (4cl-2 from tobacco and hct from globe artichoke) we previously set up a system for the production of two novel phenolic compounds, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (Yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (Yeast avenanthramide II, Yav II). These compounds have a structural similarity with a class of bioactive oat compounds called avenanthramides. By developing a fermentation process for the engineered S. cerevisiae strain, we obtained a high-yield production of Yav I and Yav II. To examine the biological relevance of these compounds, we tested their potential antioxidant and antiproliferative properties upon treatment of widely used cell models, including immortalized mouse embryonic fibroblast cell lines and HeLa cancer cells. The outcomes of our experiments showed that both Yav I and Yav II enter the cell and trigger a significant up-regulation of master regulators of cell antioxidant responses, including the major antioxidant protein SOD2 and its transcriptional regulator FoxO1 as well as the down-regulation of Cyclin D1. Intriguingly, these effects were also demonstrated in cellular models of the human genetic disease Cerebral Cavernous Malformation, suggesting that the novel phenolic compounds Yav I and Yav II are endowed with bioactive properties relevant to biomedical applications. Taken together, our data demonstrate the feasibility of biotechnological production of yeast avenanthramides and underline a biologically relevant antioxidant activity of these molecules.

In the present study different actinomycete strains were collected and isolated from Algerian Sahara soil with the aim to select novel enzymes with promising features for biotechnological applications. The Ms1 strain was selected, amongst the others, for its capability to produce melanin in different solid media. Ms1 chromosomal DNA was sequenced and the strain assigned to Streptomyces cyaneofuscatus sp. A tyrosinase (MW∼30kD) encoding sequence was identified and the corresponding enzyme was isolated and biochemically characterized. The tyrosinase showed the highest activity and stability at neutral and alkaline pH and it was able to oxidize l-DOPA at T=55°C and pH 7. The enzyme showed variable stability in presence of various water-miscible organic solvents, while it was inactivated by reducing agents. The tyrosinase activity was unaffected by NaCl and enhanced by different cations. Furthermore, the enzyme showed a higher specificity for diphenols than monophenols showing a higher diphenolase than monophenolase activity. Finally, tyrosinase was stabilized by immobilization on nylon nanofiber membranes with a payload of 82% when 1% glutaraldeyde was used. Taken all together, these results show that the enzyme displays interesting properties for biotechnological purposes.

Loss-of-function mutations in the KRIT1 gene (CCM1) have been associated with the pathogenesis of cerebral cavernous malformations (CCM), a major cerebrovascular disease. However, KRIT1 functions and CCM pathogenetic mechanisms remain incompletely understood. Indeed, recent experiments in animal models have clearly demonstrated that the homozygous loss of KRIT1 is not sufficient to induce CCM lesions, suggesting that additional factors are necessary to cause CCM disease. Previously, we found that KRIT1 is involved in the maintenance of the intracellular reactive oxygen species (ROS) homeostasis to prevent ROS-induced cellular dysfunctions, including a reduced ability to maintain a quiescent state. Here, we show that KRIT1 loss of function leads to enhanced expression and phosphorylation of the redox-sensitive transcription factor c-Jun, as well as induction of its downstream target COX-2, in both cellular models and human CCM tissues. Furthermore, we demonstrate that c-Jun upregulation can be reversed by either KRIT1 re-expression or ROS scavenging, whereas KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. Taken together with the reported role of c-Jun in vascular dysfunctions triggered by oxidative stress, our findings shed new light on the molecular mechanisms underlying KRIT1 function and CCM pathogenesis., Graphical abstract, Highlights • KRIT1 loss of function leads to the upregulation of c-Jun. • c-Jun upregulation occurs in human cerebral cavernous malformation (CCM) lesions. • KRIT1 loss-dependent c-Jun upregulation can be reversed by ROS scavenging. • KRIT1 overexpression prevents forced upregulation of c-Jun induced by oxidative stimuli. • A novel mechanism for CCM pathogenesis is suggested.

The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis. However, the molecular mechanisms underlying the fine-tuned functional communication between cadherins and integrins are still elusive. This paper focuses on recent findings towards the involvement of reactive oxygen species (ROS) in the regulation of cell adhesion and signal transduction functions of integrins and cadherins, pointing to ROS as emerging strong candidates for modulating the molecular crosstalk between cell-matrix and cell-cell adhesion receptors.

The members of the RasGTPase superfamily are involved in various signaling networks responsible for fundamental cellular processes. Their activity is determined by their guanine nucleotide-bound state. Recent evidence indicates that some of these proteins may be regulated by redox agents. Reactive oxygen species (ROSs) and reactive nitrogen species (RNSs) have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that the intracellular production of ROS is tightly regulated and that these redox agents serve as signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some Ras GTPases appear to regulate ROS production and that oxidants function as effector molecules for the small GTPases, thus contributing to their overall biological function. Thus, redox agents may act both as upstream regulators and as downstream effectors of Ras GTPases. Here we discuss current understanding concerning mechanisms and physiopathological implications of the interplay between GTPases and redox agents.

An actinobacteria strain was isolated from Algerian Sahara soil and assigned to Streptomyces cyaneofuscatus Pridham et al. 1958 species. This strain was selected for its ability to produce melanin exopigments in liquid and solid media. Melanin synthesis was associated with tyrosinase activity and the enzyme from this strain was isolated and biochemically characterized. Synthetic melanin was then enzymatically produced using the S. cyaneofuscatus Pridham et al. 1958 tyrosinase. As this enzyme showed a higher diphenolase activity, a synthetic melanin from the enzymic oxidation of 3,4-dihydroxyphenylalanine (dopa) was obtained by the use of a Trametes versicolor (L.) Lloyd laccase for comparison. The natural and synthetic pigments were physico-chemically characterized by the use of ultraviolet (UV)-Visible, and Fourier transform infrared (FT-IR) and multifrequency electron paramagnetic resonance (EPR) spectroscopies. All the melanin samples displayed a stable free radical when analyzed by X-band EPR spectroscopy. Once the samples were recorded at Q-band EPR, a copolymer derived from a mixture of different constituents was evident in the natural melanin. All radical species were analyzed and discussed. The use of water-soluble melanin naturally produced by S. cyaneofuscatus Pridham et al. 1958 represents a new biotechnological alternative to commercial insoluble pigments.

Cerebral Cavernous Malformation (CCM) is a neurovascular disease characterized by abnormal vascular structures associated with defects on vascular lumen formation and vascular fragility, mostly localized in the brain. Lack of KRIT1 protein associated to CCM drives the stable and quiescent endothelium toward an active state characterized by loss of barrier function, increased vascular permeability, loss of adherens junction integrity, increased β-catenin nuclear localization and vascular endothelial growth factor (VEGF) expression and angiogenesis. Multiple pathways are responsible for changes in endothelial phenotype and we contributed to demonstrate that the dysregulation of reactive oxygen species (ROS) production following loss of KRIT1 expression play a key role in increasing vascular permeability and is linked to NADPH oxidase (NOX) up-regulation. By using wt and KRIT1-/- mouse embryonic fibroblasts (MEFs) we showed that loss of KRIT1 induced NOX4 overexpression that is associated with alteration of angiogenic growth factors expression and with the angiogenic switch of endothelial cells. Our data contribute to demonstrate the pivotal role of oxidative stress in the progression of CCM disease, indicating NOX4 and ROS as potential targets for pharmaceutical approaches.

Background Familial Cerebral Cavernous Malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions. CCM lesions manifest across a range of different phenotypes, including wide differences in lesion number, size and susceptibility to intracerebral hemorrhage (ICH). Oxidative stress plays an important role in cerebrovascular disease pathogenesis, raising the possibility that inter-individual variability in genes related to oxidative stress may contribute to the phenotypic differences observed in CCM1 disease. Here, we investigated whether candidate oxidative stress-related cytochrome P450 (CYP) and matrix metalloproteinase (MMP) genetic markers grouped by superfamilies, families or genes, or analyzed individually influence the severity of CCM1 disease. Methods Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging (SWI) were performed to determine total and large (≥5 mm in diameter) lesion counts as well as ICH in 188 Hispanic CCM1 patients harboring the founder KRIT1/CCM1 ‘common Hispanic mutation’ (CCM1–CHM). Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 1,122 genetic markers (both single nucleotide polymorphisms (SNPs) and insertion/deletions) grouped by CYP and MMP superfamily, family or gene for association with total or large lesion count and ICH adjusted for age at enrollment and gender. Genetic markers bearing the associations were then analyzed individually. Results The CYP superfamily showed a trend toward association with total lesion count (P=0.057) and large lesion count (P=0.088) in contrast to the MMP superfamily. The CYP4 and CYP8 families were associated with either large lesion count or total lesion count (P=0.014), and two other families (CYP46 and the MMP Stromelysins) were associated with ICH (P=0.011 and 0.007, respectively). CYP4F12 rs11085971, CYP8A1 rs5628, CYP46A1 rs10151332, and MMP3 rs117153070 single SNPs, mainly bearing the above-mentioned associations, were also individually associated with CCM1 disease severity. Conclusions Overall, our candidate oxidative stress-related genetic markers set approach outlined CYP and MMP families and identified suggestive SNPs that may impact the severity of CCM1 disease, including the development of numerous and large CCM lesions and ICH. These novel genetic risk factors of prognostic value could serve as early objective predictors of disease outcome and might ultimately provide better options for disease prevention and treatment., Graphical abstract, Highlights • Inter-individual variability in oxidative stress-related genes may impact CCM1 disease severity. • CYP4 and CYP8 families were associated with either large lesion or total lesion count, while CYP46 and MMP Stromelysin families were associated with ICH. • Single SNPs in CYP4F8, CYP4F11, CYP4F12, CYP8A1, CYP19A1, CYP27A1, CYP27B1, CYP46A1 and CYP51A1 genes showed significant associations with at least one disease severity phenotype. • A single SNP in the MMP3 gene was strongly associated with ICH. • CYP and MMP SNPs associated with CCM1 disease severity could serve as early objective predictors of disease susceptibility and outcome.

Purpose. Phospholipase activity, one of Helicobacter pylori pathogenicity factors, has not been investigated enough, so far, although it may induce a remarkable damage to the gastric mucosa. In the present work, we have compared the whole phospholipase activity of H. pylori strains isolated from patients with gastric carcinoma with that of strains isolated from dyspeptic patients without gastric carcinoma. Methods. We measured the phospholipase activity of one distinct H. pylori colony isolated from each of 10 patients with gastric carcinoma and 10 controls, dyspeptic patients without endoscopic and histological signs of gastric carcinoma. We also determined the phospholipase activity of 20 additional strains isolated from different areas of neoplastic and non-neoplastic tissue of two patients with gastric carcinoma, the cagA and vacA positive G27 and 328 wild strains and their respective vacA and cagA negative isogenic mutants. The whole phospholipase activity of strains was determined by measuring the release of 14C-labeled palmitic acid from the radioactive l -3-phosphatidylcholine, 1,2-di[1-14C]palmiloyl substrate; results were expressed in pmol of palmitic acid per mg of protein. Results. H. pylori strains isolated from patients with gastric carcinoma had levels of phospholipase activity significantly higher than those of strains isolated from controls (99.37 [S.D. 40.45] versus 34.46 [S.D. 16.46], P Conclusions. The infection by H. pylori strains that produce high levels of phospholipase may increase the risk of developing gastric carcinoma. We hypothesise that indirect products of phospholipase activity, such as prostaglandins, leukotrienes and lysophospholipids, may mediate carcinogenesis.

Plasma protein adsorption patterns on surfaces may give vital information to evaluate biocompatibility of biomaterials designed for direct blood-contacting applications or tissue integration. Adsorption of human serum proteins on four different types of biomaterials (glass, aminosilanized glass, hyaluronan and sulfated hyaluronan) was analyzed by two-dimensional electrophoresis. Desorption of proteins from the surfaces was first classically achieved by sodium dodecyl sulfate (SDS) elution. We introduced a second elution step (by use of isoelectric focusing (IEF) sample buffer consisting of urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, and dithioerythritol) which allows more stringent elution conditions and is a tool to evaluate the protein adsorption strength to biomaterials. Moreover, the two-step elution may discriminate between irreversible and reversible adsorption of plasma proteins for biomaterials, thus helping to elucidate the structure of protein multilayers which form a complex system at the surfaces. The IEF sample buffer proved not to alter the biomaterial structure and integrity. Hydrophobic bonds resulted to be the main strength driving protein adsorption onto our biomaterials. Apolipoproteins were the most important proteins interacting with the surfaces suggesting that high-density lipoprotein (HDL) particles could play a role in biocompatibility due to their beneficial effects on endothelial cells.

Background. The cytotoxin-associated protein CagA is a Helicobacter pylori immunodominant antigen whose gene resides in the cag pathogenicity island. Our purpose was to determine if the disruption or deletion of cagA gene could have an effect on the expression of other proteins at the proteome level. We analyzed two H. pylori strains, 328 and G27 wild-type, bearing the cag pathogenicity island, and their respective isogenic cagA− mutants. Methods. The proteomes of two H. pylori strains (328 and its isogenic mutant SPM328_ΔcagA) were resolved by two-dimensional electrophoresis and the digitalized images obtained were analysed both quantitatively and qualitatively. Peculiar spots of each strain were identified by mass spectrometry or by Western blotting. Results. The comparison between the proteome expression of an H. pylori cagA+ strain and an isogenic mutant strain where the cagA gene was disrupted showed that, as well as the lack of expression of CagA, both flagellin A and flagellin B expressions were significantly decreased. The cagA− isogenic mutant was nonmotile. G27_ΔcagA, in which CagA was inactivated by gene deletion, was nonmotile as well respecting to motile G27 wild-type strain. Moreover, reintroduction of cagA in G27_ΔcagA restored motility. Conclusions. Our results suggest that CagA could quantitatively influence flaA and flaB transcription or their subsequent translation and/or correct folding.

This paper describes the project "Biotech a Scuola" ("Biotech at School"), financed by the Italian Ministry of Education within the SeT program (Special Project for Scientific-Technological Education). The project involved the University of Siena, five senior and junior secondary schools in the Siena area, and a private company. Twenty-three teachers from diverse fields and 318 students from 15 classes were involved. The aim of the project was to improve scientific-technological teaching by providing schools with the support and materials necessary to understand some fundamental aspects of biotechnology. With this project we propose a model of close cooperation among various educational sectors with the goal of teaching junior and senior high school students some of the theory and practice of modern biotechnology.

The products of hexavalent chromium [Cr(VI)] reduction by glutathione (GSH) alone or in the presence of equimolar quantities of aspartate (Asp) and/or glutamate (Glu) and a chromium-containing material extracted from bovine liver were studied by ultraviolet-visible spectrum (UV-vis) studies, electrospray mass spectrometry (ES-MS), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). Reduction of chromate by GSH was followed by UV-vis and NMR, revealing the formation of a paramagnetic complex in which GSH acts as a ligand. ES-MS and EPR measurements provided unequivocal evidence of a dimeric Cr(V)(2)GSH(2) species in which the two metal ions are bridged by the Gamma-Glu carboxylate. The analysis of the (1)H and (13)C shifts experienced by GSH protons and the values of paramagnetic contributions to proton spin-lattice relaxation rates provided a set of constraints for structural determination. The same experiments were repeated in the presence of an equimolar concentration of Asp, revealing the formation of a dimeric Cr(V) paramagnetic complex in which the two metals are now bridged by Asp. Nuclear magnetic resonance dispersion profiles show that water is not displaced by Asp and that the correlation time of this complex is slowed by the increased complexity. When Glu is also included in the solution in equimolar concentration to GSH and Asp, data are consistent with the formation of many mono- and dinuclear species, with the three ligands competing with each other. Finally, the spectroscopic investigation of the chromium-containing material extracted from bovine liver revealed the presence of a complicate mixture of Cr(IV) or Cr(V) complexes, among which some Cr(V)-GSH species are present alone or with other ligands in the metal coordination sphere.

We have discovered, by immunocytochemistry and immunoelectronmicroscopy, that humanin (HN) is expressed in human ejaculated sperm and testis. In sperm, the HN immunolabeling pattern depends on sperm morphology; in particular, HN is mainly localized in the midpiece of sperm in semen samples with normal morphology and in cytoplasmic residues and entire tail in those with abnormal morphology. We also found HN in the cytoplasm and nucleus of spermatocytes and spermatids and in experimentally uncoiled chromatin of mature ejaculated sperm. Because it has been established that HN has antiapoptotic properties, it is reasonably hypothesized that HN may play an important role in preventing apoptosis in human sperm and testis. Thus, the examination of the HN localization in normal and abnormal sperm could be proposed as an auxiliary test to better define sperm quality.

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.

The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes. Fuelled by the original identification in 1982 of mutationally activated and transforming human Ras genes in human cancer cell lines, a variety of powerful experimental techniques have been intensively focused on discovering and studying structure, biochemistry, and biology of Ras and Ras-related small GTPases, leading to fundamental research breakthroughs into identification and structural and functional characterization of a huge number of Ras superfamily members, as well as of their multiple regulators and effectors. In this review we provide a general overview of the major milestones that eventually allowed to unlock the secret treasure chest of this large and important superfamily of proteins.

Redox agents have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that mammalian cells can rapidly respond to ligand stimulation with a change in intracellular ROS thus indicating that the production of intracellular redox agents is tightly regulated and that they serve as intracellular signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some members of the Ras GTPase superfamily appear to regulate the production of redox agents and that oxidants can function as effector molecules for the small GTPases, thus contributing to their overall biological function. In addition, many of the Ras superfamily small GTPases have been shown to be redox sensitive, thanks to the presence of redox-sensitive sequences in their primary structure. The action of redox agents on these redox-sensitive GTPases is similar to that of guanine nucleotide exchange factors in that they perturb GTPase nucleotide-binding interactions that result in the enhancement of the guanine nucleotide exchange of small GTPases. Thus, Ras GTPases may act both as upstream regulators and downstream effectors of redox agents. Here we overview current understanding concerning the interplay between Ras GTPases and redox agents, also taking into account pathological implications of misregulation of this cross talk and highlighting the potentiality of these cellular pathways as new therapeutical targets for different pathologies.

Since its original description, the yeast two-hybrid system has been extensively used to identify protein-protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. In these years the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be assayed by hybrid-based approaches. Several groups studying molecular mechanisms that underlie signal transduction pathways regulated by Ras GTPases have successfully used the yeast two-hybrid system or related methods to isolate and identify new binding partners of Ras proteins. Here we describe the basic protocol for a yeast two-hybrid library screening and for a small-scale yeast two-hybrid assay.