H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Plasma endocannabinoid behaviour in total knee and hip arthroplasty.

Endocannabinoids are a class of lipid mediators involved in a wide range of physiological pathways including pain perception, and immunological defences. In particular, the involvement of endocannabinoids in bone metabolism and bone resorption has recently been studied. Moreover, one study on total knee arthroplasty describes the probable role of endocannabinoids in pain perception after surgery. The aim of the present study was to evaluate variations of endocannabinoid concentrations in patients undergoing total hip or total knee arthroplasty before and after surgery. Sera from 23 patients were collected at three different times: before surgery and at two different times during rehabilitation, and endocannabinoids were quantified by HPLC-MS/MS analysis. Mean values of endocannabinoids in presurgical serum samples were: 6.11±0.5 ng/ml for N-palmitoylethanolamide, 1.39±0.08ng/ml for N-stearoylethanolamide, 4.84±0.04 ng/ml for N-oleoylethanolamide, 0.44±0.03ng/ml for N-arachidonoylethanolamide, 0.84±0.05ng/ml for N-linoleoylethanolamide, 0.17±0.01ng/ml for N-α-linolenoylethanolamide. Statistical analysis showed a significant decrease of all the endocannabinoids after surgery, while there were no remarkable differences between total hip and total knee arthroplasties or between genders. Moreover, the results show no significant correlation between endocannabinoid concentrations and C-reactive protein and Erythrocyte sedimentation rate. The present study shows for the first time a specific and univocal behaviour of six endocannabinoids and N-acylethanolamides in orthopaedic surgery, suggesting the endocannabinoid system as a possible pharmacological target for presurgical therapeutics.

Effects of bioactive fatty acid amide derivatives in zebrafish scale model of bone metabolism and disease.

The endocannabinoid system (which includes fatty acid derivatives, receptors, and metabolizing enzymes) is involved in a variety of physiological processes, including bone metabolism in which it regulates the function of osteoblasts and osteoclasts, as well as differentiation of their precursors. The zebrafish (Danio rerio) provides a useful animal model for bone research since zebrafish bones develop rapidly and are anatomically similar to mammalian bones. Putative orthologues and paralogs of endocannabinoid genes have recently been identified in zebrafish, demonstrating the presence of cannabinoid type 1 (CB1) and type 2 (CB2) receptors with affinity to endocannabinoid ligands. To identify therapeutic molecules potentially useful in bone-related diseases, we evaluated the in vivo effects of exposure to long-chain fatty acid amides in adult zebrafish. Using a well-established zebrafish scale model, we found that anandamide and N-linoleoylethanolamine are able to stimulate bone formation by increasing alkaline phosphatase activity in physiological conditions. In addition, they prevent the alteration of bone markers in a prednisolone-induced osteoporosis model in adult zebrafish scales, whereas their esterified forms do not. These data suggest that long-chain fatty acid amides are involved in regulating bone metabolism in zebrafish scales and that the CB2 receptor is a key mediator in this process.

Mechanism of traumatic knee injuries and MRI findings.

Bone bruises are focal abnormalities in subchondral bone marrow due to trabecular microfractures as a result of traumatic force. These trauma-induced lesions are better detected with magnetic resonance (MR) imaging using water-sensitive sequences. Moreover, the pattern of bone bruise is distinctive and allows us to understand the dynamics of trauma and to predict associated soft injuries. This article discusses the mechanism of traumatic injury and MR findings.

Financial impact of radiological reports on medical-legal evaluation of compensation for meniscal lesions.

To evaluate any discrepancy between radiological reports for clinical purposes and for medicolegal purposes and to quantify its economic impact on repayments made by private insurance companies for meniscal injuries of the knee. The medical records obtained pertaining to 108 knee injury patients (mean age 43.3 years) assessed over a period of 12 months were analysed. Clinical medical reports, aimed at assessing the lesion, and medicolegal reports, drawn up with a view to quantifying compensation, were compared. Unlike reports for clinical purposes in reports for medicolegal purposes, in the evaluation of meniscal lesions, in addition to morphological features of lesions, chronological, topographical, severity and exclusion criteria were applied. To estimate the economic impact resulting from the biological damage, we consulted an actuarial table based on the 9-point minor incapacity classification system. Meniscal lesions not compatible with a traumatic event and therefore not eligible for an insurance payout were found in 56 patients. Of these, 37 failed exclusion criteria, while 19 failed to meet chronological criteria. This difference resulted in a reduction in compensation made by private insurance companies with savings estimated with a saving between euro 203,715.41 and euro 622,315.39. The use of a clinical report for medicolegal purposes can be a source of valuation error, as chronological and/or dynamic information regarding the trauma mechanism may be lacking. Therefore, the use of a full radiological appraisal allows a better damage's assessment and an adequate compensation for injuries.

Lesions of the rotator cuff footprint: diagnostic performance of MR arthrography compared with arthroscopy.

BACKGROUND: To evaluate the diagnostic performance of magnetic resonance arthrography (MR-A) of the shoulder in the diagnosis of rotator cuff tears involving the humeral insertion of the supraspinatus and infraspinatus tendon (footprint), using arthroscopy as the reference standard. MATERIALS AND METHODS: The study population included 90 consecutive patients with history and clinical diagnosis of instability of the shoulder, rotator cuff tear or posterosuperior glenoid impingement. A total of 108 MR arthrograms were performed, since 18 patients had undergone a bilateral procedure. Arthroscopy, which was performed within 45 days after MR-A, was used as the reference standard. Sensitivity, specificity, accuracy, positive and negative predictive values were then calculated. RESULTS: Magnetic resonance arthrography showed a sensitivity of 92 % and a specificity of 78 % for the overall detection of tears involving the rotator cuff footprint. The diagnostic accuracy was 90 %, and the positive and negative predictive values were 95 and 64 %, respectively. Ten lesions were non-classifiable on surgery, of which eight were non-classifiable on MR-A also. CONCLUSIONS: Magnetic resonance arthrography is extremely accurate for the detection and classification of rotator cuff footprint tears. Most of these lesions are articular-sided (partial articular-sided supraspinatus tendon avulsion lesions) with predominance in younger patients and concealed type of tear (concealed interstitial delamination lesions).

Complete (1)H and (13)C NMR spectral assignment of alpha- and beta-adenosine, 2'-deoxyadenosine and their acetate derivatives.

(1)H and (13)C NMR chemical shifts of alpha- and beta-anomers of adenosine, 2'-deoxyadenosine and their acetate derivatives were completely and definitely assigned using the concerted application of one- and two-dimensional experiments (gCOSY, gNOESY, gHSQC and gHMBC). The influence of the stereochemistry of the purine base on the NMR data of the hydrogen and carbon atoms of the furanose moiety was estimated.

Complete 1H and 13C NMR spectral assignment of 17-hydroxy epimeric sterols with planar A or A and B rings.

Complete 1H and 13C spectral assignments of 17beta- and 17alpha-hydroxy epimers of three biologically active sterols (boldenone, 3-methoxyestradiol and 3-methoxydihydroequilenin) were achieved making use of one- and two-dimensional NMR techniques (1D-HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and COLOC).

Lipolytic activity of porcine pancreas lipase on fatty acid esters of dialkylglycerols: a structural basis for the design of new substrates for the assay of pancreatic lipases activity.

For the design of new synthetic substrates for the assay of pancreatic lipases activity, acyl dialkylglycerols of variable chain length were prepared. Titrimetric assay of these substrates showed the highest lipolytic activity of porcine pancreas lipase (pPL) with butanoyl dibutylglycerol. The activity is lower but comparable to that shown by pPL towards the classical substrate tributyrin. The 4-nitrophenylcarbonate of 1,2-di-O-butylglycerol, has been prepared and proposed as synthetic substrate for a new spectrophotometric assay of pancreatic lipases.

Lipase-catalyzed protection of the hydroxy groups of the nucleosides inosine and 2'-deoxyinosine: a new chemoenzymatic synthesis of the antiviral drug 2',3'-dideoxyinosine.

The selective acylation of the hydroxy groups of the nucleosides inosine 1a and 2'-deoxyinosine 1b has been achieved in the presence of Candida antarctica and Pseudomonas sp. lipases in organic solvents; starting from the 5'-acetyl derivative of 2'-deoxyinosine, compound 5a, an efficient chemoenzymatic synthesis of the antiviral drug 2',3'-dideoxyinosine 1c has been achieved.

Beta-ethoxyacrolein contamination increases malondialdehyde inhibition of milk xanthine oxidase activity.

beta-Ethoxyacrolein (BEA), a side product that forms during the preparation of malondialdehyde (MDA) by acidic hydrolysis of tetraethoxypropane (TEP), has been found to be an inhibitor of milk xanthine oxidase (XO) several times more potent than pure MDA (NaMDA). The incubation of XO with 10 microM BEA abolished 50% of the enzyme activity within 1 min; the inhibited enzyme was totally regenerated by dialysis and filtration through Sephadex. The BEA inhibition mode of the enzyme was mixed-type with the apparent inhibition constants (Ki) of 2.4 x 10(-6) M. An HPLC method for quantitation of BEA in the crude commonly used MDA preparation was set up.

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3,803 nucleotides and has five open reading frames (ORFs). The 5' ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

Structural requirements of aldehydes produced in LPO for the activation of the heat-shock genes in HeLa cells.

The ability of various aldehydes, some of which are produced in lipid peroxidation, to effect heat-shock gene expression and heat-shock proteins synthesis was evaluated in HeLa cells. Only (E)-4-hydroxyalk-2-enals were active both in racemic and homochiral form. Between the reported primary metabolic products of (E)-4-hydroxynon-2-enal, only the glutathione conjugates were active, whereas (E)-4-hydroxynon-2-enoic acid and 2-nonen-1,4-diol were inactive. Also, unnatural (E)-5-hydroxynon-2-enal and (E)-5-hydroxyhex-2-enal were active, whereas (E)-6-hydroxynon-2-enal was inactive. Thus, it was established that the active aldehydic compounds must possess an (E)-2 double bond and an hydroxy group in a position suitable for the formation of a cyclic hemiacetal in a possible adduct of these aldehydes with proteins. An irreversible binding to proteins could be the first step of the mechanism by which these compounds exert their biological activity.

The relationship between gamma-glutamyl transpeptidase and Hg levels in Se/Hg antagonism in mouse liver and kidney.

The gamma-glutamyl transpeptidase (gamma-GT) activity and Hg concentrations were studied in Se/Hg antagonism in mouse liver and kidney after treatment with methyl mercury (MM) (MM group) and MM + sodium selenite (SE) (SM group). In acute treatment (dietary doses: MM = 250 p.p.m.; SE = 90 p.p.m.; length of treatment 11 days), hepatic gamma-GT activity increased in both protected and unprotected animals with respect to controls and reached a peak after 3 days with respect to controls, its value being relatively greater in the MM group. On the contrary, renal gamma-GT decreased with time with respect to controls and was higher in the SM group at 3 and 7 days. Liver and kidney accumulation of Hg increased and decreased respectively with time, and was higher in SM groups in most cases. In chronic treatment (dietary doses: MM = 12.5 p.p.m.; SE = 9 p.p.m.; length of treatment 12 months) hepatic gamma-GT activity in the MM group was higher than in the SM group at 1.5 and 7 months, whereas the renal activity was lower at 7 months and unchanged at 1.5 and 12 months. In comparison with the acute treatment, the trend of Hg accumulation was similar in liver and different in kidney; Hg concentrations of the SM group were always greater than those of the MM group. Glutathione (GSH) in liver and non-protein SH groups (NPSH) in kidney were also measured in acutely treated animals. On the first day GSH was about 50% of the control value in both the MM and SM groups; it subsequently remained constant in the MM group, but increased to a peak at 7 days, without reaching the control value, in the SM group. Unlike the liver, renal NPSH increased in both groups on the first day, and then decreased with time without reaching the control value, SM group values always exceeding those of MM group. The modulation of gamma-GT activity in liver and kidney caused by SE suggests that the enzyme plays a role in Hg accumulation.

Synthesis of 7 alpha,12 alpha-dihydroxy-3 alpha- [2-(beta-D-glucopyranosyl)acetyl]-5 beta-cholan-24-oic acid.

A C-glucoside of cholic acid was synthesized by the introduction of an acetyl group at position 3 alpha and direct one-pot C-glucosidation using 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl chloride.

Identification of [14C]carmoisine metabolites in bacterial suspension of rat faeces.

An in vitro system consisting of a bacterial suspension of human or rat faecal microflora brought about the biological reduction of the red azo dye [14C]carmoisine to 1-naphthyl-amine-4-sulphonic acid (NA) and 2-amino-1-naphthol-4-sulphonic acid (ANA). These metabolites have been unequivocally identified by radio-HPLC, spectroscopic methods, dilution with cold authentic standards and evidence that the specific activity of the diluted compounds remained constant throughout repeated crystallization, acetylation and purification. The results clearly indicated that samples derived from anaerobic incubations have to be processed for analysis in the complete absence of oxygen. In the presence of oxygen, the formation of a complex pattern of compounds in addition to NA was observed as a consequence of the chemical decomposition of ANA.

Synthesis of 14 beta-cholesta-5,7-dien-3 beta-ol.

5,7-Cholestadien-3 beta-ol was transformed into 14 beta-cholesta-5,7-dien-3 beta-ol in six steps. The inversion of the stereochemistry at C-14 was obtained by a selective protection of the delta 5 and the elaboration of the delta 7 double bond.

A convenient isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids.

Direct isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids was accomplished by pyridinium hydrobromide in dimethyl-formamide.