Correlations between the alpha-Gal antigen, antibody response and calcification of cardiac valve bioprostheses: experimental evidence obtained using an alpha-Gal knockout mouse animal model

IntroductionPreformed antibodies against αGal in the human and the presence of αGal antigens on the tissue constituting the commercial bioprosthetic heart valves (BHVs, mainly bovine or porcine pericardium), lead to opsonization of the implanted BHV, leading to deterioration and calcification. Murine subcutaneous implantation of BHVs leaflets has been widely used for testing the efficacy of anti-calcification treatments. Unfortunately, commercial BHVs leaflets implanted into a murine model will not be able to elicit an αGal immune response because such antigen is expressed in the recipient and therefore immunologically tolerated.MethodsThis study evaluates the calcium deposition on commercial BHV using a new humanized murine αGal knockout (KO) animal model. Furtherly, the anti-calcification efficacy of a polyphenol-based treatment was deeply investigated. By using CRISPR/Cas9 approach an αGal KO mouse was created and adopted for the evaluation of the calcific propensity of original and polyphenols treated BHV by subcutaneous implantation. The calcium quantification was carried out by plasma analysis; the immune response evaluation was performed by histology and immunological assays. Anti-αGal antibodies level in KO mice increases at least double after 2 months of implantation of original commercial BHV compared to WT mice, conversely, the polyphenols-based treatment seems to effectively mask the antigen to the KO mice’s immune system.ResultsCommercial leaflets explanted after 1 month from KO mice showed a four-time increased calcium deposition than what was observed on that explanted from WT. Polyphenol treatment prevents calcium deposition by over 99% in both KO and WT animals. The implantation of commercial BHV leaflets significantly stimulates the KO mouse immune system resulting in massive production of anti-Gal antibodies and the exacerbation of the αGal-related calcific effect if compared with the WT mouse. DiscussionThe polyphenol-based treatment applied in this investigation showed an unexpected ability to inhibit the recognition of BHV xenoantigens by circulating antibodies almost completely preventing calcific depositions compared to the untreated counterpart

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Attività antiproliferativa di derivati furocumarinici attivati da blue light in linee cellulari di carcinoma prostatico e vescicale

Prostate and bladder cancer are currently the most common tumors in the male population and those with the highest mortality rates. The latter is related to the high incidence of relapses resulting from a lack of efficacy of available treatments, often characterized by invasiveness and toxicity. A new approach for the management of these cancers could be represented by photochemotherapy where the drugs are directly activated on the diseased area, thus reducing toxicity to neighboring healthy cells. The antiproliferative activity of the compounds, psoralenes and angelicins, following activation with UVA, light is well known and largely due to their ability to interact with DNA forming monoadducts (MAs) and cross-links (XLs). However, the major disadvantage of the use of furocumarins in combination with UVA light is represented by the risk of mutagenicity linked both to their ability to covalently bind the nucleic acid and to the toxicity of the radiation. The possibility of photoactivate this type of molecule with blue light (BL) could represent an innovation in the field of photochemotherapy because it would improve the most common operational limits and side effects connected with the use of UVA. BL, unlike UVA, has a deeper tissue penetration, thus allowing the potential treatment of more invasive tumours, and at the same time has a lower mutagenicity. Moreover, from a technical point of view, the optical fibers used in photodynamic therapy (PDT) have a better performance to deliver BL than UVA both from the point of view of light diffusion and the amount of transmitted radiation. Thiswork has demonstrated that 8-MOP and TMA are able to be photoactivated by BL, despite their low coefficients of molar extinction, and exert an antiproliferative effect on prostate (DU145) and bladder (T24) cancer cell lines. Experiments on isolated DNA have confirmed the ability of these molecules to form MAs and XLs, produce strand breaks and photooxidation of nucleic acid bases when activated by UVA. However, photoactivation by BL induced, both a quantitative decrease and a variation in the type of lesions in the macromolecule. This decrease was far greater in the case of TMA, which, unlike 8-MOP, was unable to form XLs and photooxide DNA after activation with BL. 8-MOP and TMA plus BL had good antiproliferative activity resulting from the induction of the apoptotic process and the formation of ROS. In addition, these molecules modulated the activation status of p38 and ERK1/2 with both types of irradiation. However, TMA had a higher activity than 8-MOP being active at lower doses, and was not genotoxic, as shown by the evaluation of the phosphorylation status of histone H2AX in both DU145 and T24 cells, when irradiated with UVA or BL. TMA/BL also modulated Wnt's canonical signal pathway in a negative way; in fact, it increased the phosphorylated forms of β-catenin and GSK3β (to tyrosine 216) and decreases the nuclear levels of β-catenin. The inhibition of this pathway lead to a decrease in the expression of some of its target genes, such as cyclin D1, c-Myc and CD44v6, as demonstrated by RT-PCR. In addition to decreased expression at transcript level, there was also a reduction in protein expression of CD44 in all samples treated with TMA. The latter was due to the modulation of the phosphorylation state at tyrosine 216 of GSK3β, as suggested by the partial recovery of the expression of nuclear β-catenin and the reduction of its phosphorilated form after treatment with LiCl. The collected data also suggest that the activation status of GSK3β may be mediated by ERK1/2, as TMA/BL induced a decrease in its phosphorylated form. In conclusion, TMA photoactivated by BL may represent an interesting option for photochemotherapy of non-invasive prostate and bladder carcinomas, because this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects.Il carcinoma protatico e quello vescicale sono le patologie tumorali a maggiore incidenza con i più alti tassi di mortalità. Quest’ultima è connessa all’elevata frequenza di ricadute derivante da una scarsa efficacia dei trattamenti disponibili, spesso caratterizzati da invasività e tossicità. Un nuovo approccio potrebbe essere rappresentato dalla fotochemioterapia nella quale i farmaci vengono attivati per svolgere la loro funzione direttamente sul sito interessato, riducendo quindi la tossicità alle cellule sane limitrofe. L’attività antiproliferativa dei composti usati, psoraleni ed angelicine in associazione con luce UVA, è ben nota ed in gran parte dovuta alla loro capacità di interagire con il DNA formando monoaddotti (MA) e legami crociati (XL). Tuttavia, il maggior svantaggio è rappresentato dal rischio di mutagenicità legato sia alla loro capacità di legare covalentemente l’acido nucleico che alla tossicità della stessa radiazione utilizzata. La possibilità di fotoattivare questo tipo di molecole con blue light (BL) potrebbe rappresentare un’innovazione nel campo della fotochemioterapia poichè BL, diversamente da UVA, possiede una più profonda penetrazione tessutale, permettendo quindi il potenziale trattamento di tumori più invasivi, e al contempo presenta una minore mutagenicità. In questa tesi è stato dimostrato che 8-MOP e TMA, nonostante i loro bassi coefficienti di estinzione molare, sono in grado di essere fotoattivate da BL ed esercitano un effetto antiproliferativo su linee cellulari di tumore prostatico (DU145) e vescicale (T24). Esperimenti su DNA isolato hanno confermato la capacità di queste molecole di formare MA e XL, produrre tagli ai filamenti e fotoossidare le basi dell’acido nucleico quando attivate da UVA; la fotoattivazione mediante BL induceva, invece, sia una diminuzione quantitativa che una variazione nella tipologia delle lesioni alla macromolecola. Questa diminuzione risultava di gran lunga maggiore nel caso di TMA che, al contrario di 8-MOP, in seguito ad attivazione con BL non era in grado di formare XL e di fotoossidare il DNA. L’ attività antiproliferativa di 8-MOP e TMA risultava dall’induzione del processo apoptotico e dalla formazione di ROS. Inoltre, veniva modulato lo stato di attivazione di p38 ed ERK1/2 con entrambe le tipologie di irradiazione. Tuttavia, i dati raccolti hanno evidenziato che TMA possedeva una maggiore efficacia rispetto a 8-MOP essendo attiva a dosi minori. Inoltre, non era genotossica, come mostrato dalla valutazione dello stato di fosforilazione dell’istone H2AX sia nelle cellule DU145 che nelle T24, quando irradiata con UVA o BL. TMA/BL, inoltre, modulava in modo negativo la via di segnale canonica di Wnt; aumentava le forme fosforilate di β-catenina e di GSK3β (tirosina 216) e diminuiva i livelli nucleari di β-catenina. L’inibizione di questa via si traduceva nella diminuzione dell’espressione di ciclina D1, c-Myc e CD44 nella sua variante v6, come evidenziato dalla RT-PCR; per quest’ultima si registrava anche una riduzione dell’espressione proteica. L’inibizione della via Wnt era dovuta alla modulazione dello stato di fosforilazione di GSK3β, come suggerito dal parziale recupero dell’espressione di β-catenina nucleare ed alla riduzione della sua forma fosforilata in seguito a trattamento con LiCl. I dati raccolti hanno suggerito, inoltre, che lo stato di attivazione di GSK3β potesse essere mediato da ERK1/2, in quanto TMA/BL induceva una diminuzione della sua forma fosforilata. In conclusione, TMA fotoattivata da BL può rappresentare un’interessante opzione per la fotochemioterapia dei carcinomi prostatico e vescicale non invasivo, in quanto tale trattamento è in grado di inibire vie chiave per la crescita e la progressione tumorale in assenza di effetti genotossici

Sustainability of cattle farms in Italy

This paper aimed to analyze the sustainability of Italian cattle farms, in terms of nutrient surplus and land use, greenhouse gasses emission and animal welfare. Intensive livestock farms are concentrated in Northern Italy, especially in Po valley, in which the average livestock density is 1.7 livestock unit/ha of agricultural area. A high percentage of agricultural areas are Nitrates Vulnerable Zones, and the farmers were asked to adapt the farm management to the limits established by the Nitrates Directive. Also the mitigation of greenhouse gasses emitted by ruminants, in particular enteric methane, has become an important issue for livestock sustainability and an area of research in animal science. Indeed, agriculture is responsible for about 50% of global production of methane from human activities and the largest amount is produced by rumen fermentation in cattle. Furthermore, the importance of animal welfare in intensive livestock farms is well recognized by EU citizens. For Italian cattle farms, the improvement of animal welfare is an emerging issue especially for beef and calves. Strategies aimed at improve the sustainability of Italian cattle farms are discussed, including farm management, research approaches and regional planning and policies

PBL (PSORALENS + BLUE LIGHT): BLUE LIGHT ACTIVATES 8-MOP AND TMA TRIGGERING PROSTATE (DU145) AND VESICAL (T24) TUMOR CELL APOPTOSIS AND DEATH

BACKGROUND. Psoralens and angelicins (furocoumarins) are natural and synthetic compounds with high antiproliferative potency under UVA irradiation mainly used for the treatment of skin diseases (PUVA therapy) or immunological disorders in extracorporeal photopheresis (ECP). To improve their activity against psoriasis or vitiligo and avoid severe side effects mainly related to the formation of interstrand crosslinks (XLs) with DNA pyrimidine bases, a variety of derivatives, hopefully monofunctional, have been synthesized. Although angelicins, due to their angular geometry, do not generally form XLs, some of them, i.e. (TMA), can crosslink folded DNA upon UVA. Furthermore, furocoumarins produce ROS that impair cellular functions through lipid peroxidation, oxidation of guanine and strand breaks in nucleic acids, oxidation of proteins and inactivation of enzymes. To photoactivate 8- MOP and 4,6,4'-trimetylangelicin (TMA) towards human prostate (DU145 PCa) and bladder (T24) cancer cell lines, a new approach based on less toxic and more penetrating visible radiation (BL, 420 nm) is proposed. RESULTS. TMA and 8-MOP showed high antiproliferative activity towards both cancer cell lines, through induction of apoptosis. Besides ROS generation (less efficient under BL than UVA), the proapoptotic effect seemed related to the activation of p38 and inhibition of p44/42 phosphorylation. Moreover, no phosphorylation of the histone H2AX, nuclear \u3b2 -catenin and GSK3\u3b2 occurred. Moreover, Cyclin D1, c-Myc and CD44v6 expression were reduced through inhibition of the Wnt pathway. Overall, DU145 cells appeared more sensitive to PBL than T24, showing a specificity of the test compounds towards different tumor cell lines. The strong photocytotoxicity of TMA and 8-MOP can be related to the kind and number of DNA lesions. Under BL, no mutagenic crosslinks, no photocleavage nor photooxidative lesions were detected on isolated DNA by TMA phototreatment, but only MAs can form. However, generation of XLs still remained for 8-MOP under BL but in a lower amount than under UVA. CONCLUSIONS. Overall, our results indicate that 8-MOP, and particularly TMA, can be efficiently activated by BL and may be considered good candidates for targeted PBL of prostate and bladder cancers and possibly for other solid tumors

How Blue Light Activates Furocoumarin Derivatives Triggering Tumor Cell Apoptosis

Furocoumarins comprise natural and synthetic compounds: linear molecules, so called psoralens, and the angular ones, the angelicins. The photobiological effects of furocoumarins plus UVA are mainly related to their capacity to bind DNA and form monoadducts (MAs) and interstrand crosslinks (XLs), mainly with pyrimidine bases. Furthermore, furocoumarins produce ROS that impair cellular functions through lipid peroxidation, oxidation of guanine and strand breaks in nucleic acids, oxidation of proteins and inactivation of enzymes. It is known that the combination of 8-MOP and UVA radiation causes apoptosis of treated leucocytes and may cause preferential apoptosis of activated or abnormal T cells. Moreover, these apoptotic cells may promote immune tolerance, production of antigen-specific regulatory lymphocytes (CD4/8 T, B) and rebalance of immune system. Even though furocoumarins possess high chemotherapeutic potency under UVA and lack toxicity in the dark, genotoxicity, mutagenicity and skin phototoxicity have been observed. 8-MOP was found to photoreact under blue light (BL), leading to less mutagenic lesions in the DNA, that is preferentially MAs over XLs. Furthermore, cells treated with 419 nm light resumed normal growth rates faster than cells which received the same UVA dose

Effectiveness of photoactivatable drugs on prostate cancer stem cells: a pilot study

Currently available treatment modalities in prostate cancer (PCa) target mature and proliferating tumor cells without affecting the tumor-initiating PCa stem cells (SCs) Many patients initially experience a positive treatment response but some develop progressive disease, including tumor recurrence, metastasis, and therapy resistance. Various focal treatments, such as phototherapy, cryotherapy, interstitial thermotherapy and dynamic high-frequency ultrasound (HIFU), are being evaluated for PCa treatment. Among photoactivatable compounds, furocoumarins possess the interesting property to be highly effective when UVA irradiated, whereas are inert in the dark, thus lacking toxicity. Furthermore, it has been suggested a potential role of the bioactive compounds isolated from the medicinal plant Psoralea corylifolia (neobavaisoflavone and psoralidin) in prostate cancer chemoprevention through enhancement of TRAIL-mediated apoptosis. In this context, this work was aimed at verifying whether photoactivation of a linear furocoumarin, 8-methoxy psoralen (8-MOP), and an angular one, trimethyl angelicin (TMA) could target PCaSCs. Other than UVA light, longer wavelengths, such as 420 nm (Vis), were investigated in order to minimize surrounding tissue damage induced by light, reduce the mutagenicity of the DNA lesions induced by furocoumarins (monoadducts over cross-links), increase light penetration into the target tissue, thus allowing the use of more convenient light sources for in vivo application. While 8-MOP was able to form both crosslinks and monoadducts under the two wavelengths, TMA demonstrated to induce only the less mutagenic monoadducts in the DNA under Vis light. Production of singlet oxygen was detected only under UVA light for the two furocumarins, while Vis light was ineffective. Superoxide anion formed as a minor component of the ROS under both lights. Biological assays were carried out on DU145 cells, a prostate cancer cell line and sphere formation was used as a model for cancer cell stemness (see figure). After irradiation with 2 J/cm2 UVA, both 1 \u3bcM 8-MOP and TMA completely abrogated sphere formation. On the contrary, upon activation with 2 J/cm2 of VIS light, TMA was more effective than 8-MOP. Indeed, sphere formation was totally inhibited by 10 \u3bcM TMA, whereas 10 \u3bcM 8-MOP had no effect. Collectively, our data suggest that TMA plus VIS may be a potential focal treatment towards PCaSCs. It will be noteworthy to ascertain whether, other than DNA lesions, other molecular targets, such signaling pathways involved in the induction and maintenance of stemness in cancer cells, could be affected

4,6,4′-Trimethylangelicin Photoactivated by Blue Light Might Represent an Interesting Option for Photochemotherapy of Non-Invasive Bladder Carcinoma: An In Vitro Study on T24 Cells

Photodynamic therapy (PDT) is frequently used to treat non-muscle invasive bladder cancer due its low toxicity and high selectivity. Since recurrence often occurs, alternative approaches and/or designs of combined therapies to improve PDT effectiveness are needed. This work aimed to evaluate the cytotoxicity of 4,6,4′-trimethylangelicin (TMA) photoactivated by blue light (BL) on human bladder cancer T24 cells and investigate the mechanisms underlying its biological effects. TMA/BL exerted antiproliferative activity through the induction of apoptosis without genotoxicity, as demonstrated by the expression levels of phospho-H2AX, an indicator of DNA double-stranded breaks. It also modulated the Wnt canonical signal pathway by increasing the phospho-β-catenin and decreasing the nuclear levels of β-catenin. The inhibition of this pathway was due to the modulation of the GSK3β phosphorylation state (Tyr 216) that induces a proteasomal degradation of β-catenin. Indeed, a partial recovery of nuclear β-catenin expression and reduction of its phosphorylated form after treatment with LiCl were detected. As demonstrated by RT-PCR and cytofluorimetric analysis, TMA/BL also decreased the expression of CD44v6, a marker of cancer stem cells. Taken together, our data suggest that TMA photoactivated by BL may represent an interesting option for the photochemotherapy of noninvasive bladder carcinomas, since this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects

4,6,4′-Trimethylangelicin Photoactivated by Blue Light Might Represent an Interesting Option for Photochemotherapy of Non-Invasive Bladder Carcinoma: An In Vitro Study on T24 Cells

Photodynamic therapy (PDT) is frequently used to treat non-muscle invasive bladder cancer due its low toxicity and high selectivity. Since recurrence often occurs, alternative approaches and/or designs of combined therapies to improve PDT effectiveness are needed. This work aimed to evaluate the cytotoxicity of 4,6,4′-trimethylangelicin (TMA) photoactivated by blue light (BL) on human bladder cancer T24 cells and investigate the mechanisms underlying its biological effects. TMA/BL exerted antiproliferative activity through the induction of apoptosis without genotoxicity, as demonstrated by the expression levels of phospho-H2AX, an indicator of DNA double-stranded breaks. It also modulated the Wnt canonical signal pathway by increasing the phospho-β-catenin and decreasing the nuclear levels of β-catenin. The inhibition of this pathway was due to the modulation of the GSK3β phosphorylation state (Tyr 216) that induces a proteasomal degradation of β-catenin. Indeed, a partial recovery of nuclear β-catenin expression and reduction of its phosphorylated form after treatment with LiCl were detected. As demonstrated by RT-PCR and cytofluorimetric analysis, TMA/BL also decreased the expression of CD44v6, a marker of cancer stem cells. Taken together, our data suggest that TMA photoactivated by BL may represent an interesting option for the photochemotherapy of noninvasive bladder carcinomas, since this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects

Antiproliferative activity of 8-methoxypsoralen on DU145 prostate cancer cells under UVA and blue light

The use of photoactivatable 8-methoxypsoralen (8-MOP) as potential focal treatment towards prostate cancer cells is proposed here. Our results, obtained on isolated DNA and DU145 cells, indicate that blue light, besides UVA, is able to activate 8-MOP. When compared to UVA, blue light irradiation led to a modulation of the extent and the types of 8-MOP-DNA damage, specially cross-links, coupled to a still valuable antiproliferative effect. Our data suggest that the proapototic activity of 8-MOP is related not only to DNA damage and reactive oxygen species generation but also to the modulation of cell signalling pathways. In particular, a different activation of p38 and p44/42 mitogen-activated protein kinases was detected depending on the light wavelengths