Imbalance of Mg Homeostasis as a Potential Biomarker in Colon Cancer

Background: Increasing evidences support a correlation between magnesium (Mg) homeostasis and colorectal cancer (CRC). Nevertheless, the role of Mg and its transporters as diagnostic markers in CRC is still a matter of debate. In this study we combined X-ray Fluorescence Microscopy and databases information to investigate the possible correlation between Mg imbalance and CRC. Methods: CRC tissue samples and their non-tumoural counterpart from four patients were collected and analysed for total Mg level and distribution by X-Ray Fluorescence Microscopy. We also reviewed the scientific literature and the main tissue expression databases to collect data on Mg transporters expression in CRC. Results: We found a significantly higher content of total Mg in CRC samples when compared to non-tumoural tissues. Mg distribution was also impaired in CRC. Conversely, we evidenced an uncertain correlation between Mg transporters expression and colon malignancies. Discussion: Although further studies are necessary to determine the correlation between different cancer types and stages, this is the first report proposing the measurement of Mg tissue localisation as a marker in CRC. This study represents thus a proof-of-concept that paves the way for the design of a larger prospective investigation of Mg in CRC

現地観測に基づく河川流の乱流特性に関する研究

博士（工学）神戸大

Caratterizzazione della O-fosfoetanolamina fosfoliasi umana, una beta-liasi piridossalfosfato dipendente non convenzionale

Abstract Subgroup-II aminotransferases (AT-II) are a structural subclass of PLP-dependent enzymes, specialized in transforming for compounds containing an amino group not adjacent to a carboxylate. As the name suggests, the vast majority of these enzymes are in fact transaminases, endowed with some structural peculiarities that help explain their specificity for terminal amines. In this thesis work, we analyzed the specificity of one of the few AT-II enzymes that is not a transaminase, namely O-phosphoethanolamine phospho-lyase lyase (PEA-PL), in terms of both reaction and substrate. This enzyme is quite peculiar for several reasons. First, it is the only known AT-II enzyme (together with its closely related paralog, O-phospho-5-hydroxy-L-lysine phospho-lyase) whose primary function is to catalyze an elimination reaction. Second, because its substrate is, quite unusually, an amine containing a phosphate group. Third, because the enzyme, despite being phylogenetically very close to pyruvate-dependent AT-II transaminases (in particular, to the promiscuous AGXT2) was reported to lack any transaminase activity. The obtained results show that this human PEA-PL is at least 500-fold more active as a lyase than as an aminotransferase and that the lyase reaction is very selective for O-phosphoethanolamine, strongly discriminating against closely related compounds. Dissecting the factors that contribute to such narrow substrate specificity we found that PEA-PL is in fact able to recognize only amines and not amino acids, while the phosphate was shown to be a key determinant of substrate specificity. The results of this first part of the work allowed us to conclude that both the substrate- and reaction specificity of PEA-PL are remarkably strict for a PLP-dependent enzyme. To rationalize these findings, and to better understand their mechanistic basis, we undertook a wide kinetic study encompassing (i) the relative ability of various anionic compounds in inhibiting the PEA-PL reaction; (ii) the pH dependence of the enzyme’s steady-state catalytic parameters and of the inhibitors effectiveness; (iii) a spectroscopic study of the interaction of the enzyme with substrates and inhibitors and (iv) a pre-steady state analysis of the PEA-PL reaction, to gain more information on the formation of catalytic intermediates and generally on the reaction mechanism of the enzyme. The results of these studies confirm that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies showed that upon reaction with PEA the main absorption band of PLP (lambdamax=412 nm) rapidly blue-shifts to ~400 nm. Further experiments suggested that the newly formed and rather stable 400-nm species most likely represents a Michaelis (non-covalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity-efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines (structurally resembling PEA) that occur in the brain. Having established the high substrate specificity of PEA-PL, we also decided to explore its practical usefulness, by designing an enzymatic assay for the selective detection of PEA in liquid samples. This assay would be most useful for the clinical laboratory: as PEA levels are increased in in some biological fluids in the course of metabolic, genetic and psychiatric diseases, a method for the rapid and quantitative detection of this analyte would be of relevance to the diagnosis and monitoring of such diseases. The fluorometric assay we begun to develop achieved the desired sensitivity, as it could satisfactorily quantitate micromolar amounts of PEA in a buffer-water system

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

The present review focuses on a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, belonging to the broader fold-type I structural group and whose archetypes can be considered ornithine δ-transaminase and γ-aminobutyrate transaminase. These proteins were originally christened “subgroup-II aminotransferases” (AT-II) but are very often referred to as “class-III aminotransferases”. As names suggest, the subgroup includes mainly transaminases, with just a few interesting exceptions. However, at variance with most other PLP-dependent enzymes, catalysts in this subfamily seem specialized at utilizing substrates whose amino function is not adjacent to a carboxylate group. AT-II enzymes are widespread in biology and play mostly catabolic roles. Furthermore, today several transaminases in this group are being used as bioorganic tools for the asymmetric synthesis of chiral amines. We present an overview of the biochemical and structural features of these enzymes, illustrating how they are distinctive and how they compare with those of the other fold-type I enzymes

Potential Effects of Corneal Cross-Linking upon the Limbus

Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies done in vitro or ex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it

Kinetic characterization of the human O-phosphoethanolamine phospho-lyase reveals unconventional features of this specialized pyridoxal phosphate-dependent lyase

Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies show that upon reaction with PEA the main absorption band of PLP (λmax = 412 nm) rapidly blue-shifts to ~ 400 nm. Further experiments suggest that the newly formed and rather stable 400-nm species most probably represents a Michaelis (noncovalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity–efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines, structurally resembling PEA, that occur in the brain

GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the β-catenin and TGFβ pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium

: Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction

CRISPR/Cas9-mediated specific knock-down of dominant mutations in Rhodopsin gene

Rhodopsin (RHO) mutations represent a common cause of blindness, accounting for 25% of autosomal dominant Retinitis Pigmentosa (RP) and 8-10% of all RP. Although gene therapy has been successfully applied to retinal degeneration caused by recessive mutations, therapeutic intervention for dominant mutations are still lagging behind. In this study, we explored the efficacy of newly described CRISPR/Cas9 variants with altered PAM specificity and nearly completely reduced off-target effects, to specifically inactivate two highly frequent dominant mutations, P23H and P347S, mapped in the N-terminal and the C-terminal region of the RHO gene, respectively. We designed gRNAs on the mutations to compare allele-specific targeting of the high fidelity SpCas9 (SpCas9-HF1), the respective VQR variant (SpCas9-VQR-HF1) or the SaCas9, and we tested gRNAs in vitro on HeLa clones stably expressing P23H, P347S or wild-type RHO. Analysis of insertions or deletions (Indels) in the genomic DNA specifically in the RHO gene, by Cel-I assay and sequencing, identified the most efficient and mutation-specific system able to induce Indels in the P23H or P347S RHO mutated allele, with almost undetectable editing of the wild-type allele. We are going to package the selected CRISPR/Cas9-gRNA in AAV2/8 particles to test this approach in P23H or P347S RHO transgenic mice, to evaluate retina functionality and vision recovery upon CRISPR/Cas9-mediated editing. Our results will provide clear evidences about the employment of CRISPR/Cas9 system to selectively target dominant mutations and the preclinical application of this strategy for patients affected by RP due to mutations in the RHO gene