Active vs passive novelty-related strategies: Sex differences in exploratory behaviour and monoaminergic systems.

Sex differences are apparent in numerous behavioural characteristics. In order to compare and characterise male and female variability of exploratory behaviour, 365 male and 401 female rats were assessed in a task where a bimodal response distribution had previously been established in males. Female rats had significantly higher exploratory activity, and presented normal distribution of the behaviour, very differently from the bimodal distribution of males. No major effect of litter or oestrous cycle was detected. Several differences between male and female rats were found in monoamine metabolism measured ex vivo. Male rats had lower levels of dopamine (DA) in frontal cortex, and higher levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in raphe area; higher levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in dorsal striatum but lower levels of 5-HT and 5-HIAA in locus coeruleus area, 5-HIAA levels were also lower in hippocampus as compared to females. Males had higher noradrenaline (NA) levels in hippocampus and lower normetanephrine (NMN) levels in striatum, in both brain regions male animals had lower NMN/NA ratio. No sex difference was found in accumbens. The only brain region with an interaction between sex and the expression of exploratory activity was raphe: Here 5-HT levels were lower, and DOPAC levels and DOPAC/DA and 5-HIAA/5-HT ratios higher in low exploring male but not female rats. Conclusively, female rats not only display higher levels of exploration but the population distribution of this behaviour is distinct; this may be related to differences in the monoaminergic systems between female and male animals.

Senolytic Cocktail Dasatinib+Quercetin (D+Q) Does Not Enhance the Efficacy of Senescence-Inducing Chemotherapy in Liver Cancer.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death, which develops in the context of fibrosis and cirrhosis caused by chronic inflammation, in turn due to non-alcoholic fatty liver disease (NAFLD), alcohol consumption and/or hepatitis viral infection. An increased number of senescent cells are associated with age-related tissue degeneration during NAFLD-induced HCC, or during chemotherapeutic treatment. Senolytic agents target selectively senescent cells. A combination of the senolytic drugs dasatinib and quercetin (D+Q) reduced hepatic lipid accumulation and alleviated age-associated physical dysfunction in mice. However, whether D+Q can impact the treatment of HCC, at the end-stage of the NAFLD inflammatory spectrum, is unknown. Here, using two well-established HCC cell lines (HepG2, Huh-7), we demonstrate that the maximal cytostatic doses for D and/or Q (1 + 1 µM) lacked efficacy in removing doxorubicin-induced ß-gal-positive senescent cells. Moreover, D+Q did not affect doxorubicin-dependent induction of flattened morphology, activation of p16, expression of SASP-associated genes or formation of γH2AX foci. We then investigated the antitumor efficacy of doxorubicin, D+Q, or the combination, in xenograft studies conducted with HCC cells inoculated in athymic nude mice. Doxorubicin reduced tumor growth by 30% compared to control mice, while D+Q was ineffective in synergizing with doxorubicin and in clearing doxorubicin-induced HCC senescent cells. Unexpectedly, D+Q alone appeared to have acute pro-tumorigenic effects in control mice. While our data need to be confirmed in animal models that fully recapitulate NAFLD, we demonstrate that these compounds are ineffective, alone or in synergy with senescence-inducing chemotherapy, against experimental HCC.

Purification, characterization and plasma half-life of PEGylated soluble recombinant non-HA-binding CD44.

BACKGROUND AND OBJECTIVES: The aim of this study was to increase the serum half-life of recombinant CD44 hyaluronan (HA) binding domain by PEGylation. We have previously found that recombinant soluble CD44 HA binding domain (CD44HABD) and its non-HA-binding triple mutant CD44HABD(R41AY78SY79S) (CD44-3MUT) inhibits angiogenesis and subcutaneous tumor growth. However, this ~12 kDa recombinant protein displays a high serum clearance rate. METHODS: Here, we report the purification of monomeric CD44-3MUT from urea solubilized inclusion bodies using weak anion exchange chromatography and gel filtration. To increase the serum residence time of CD44-3MUT we PEGylated the resulting protein using 20 kDa methoxy-PEG-propionaldehyde. RESULTS: PEGylation of CD44-3MUT prolonged its in vivo serum half-life about 70-fold from 0.03 to 1.8 hours. Along with extended plasma residence time, PEGylation also increased the systemic exposure. By cell impedance assay we confirmed that PEGylated CD44-3MUT maintained its in vitro function. The results from the impedance assay additionally demonstrate that the CD44-3MUT effect on endothelial cells is mediated by vimentin. CONCLUSIONS: In summary, we have developed a purification protocol for large-scale production of CD44-3MUT and generated a PEGylated form of CD44-3MUT. HA binding domain of CD44(CD44HABD) and its modified non-HA binding form (CD44-3MUT) inhibit angiogenesis and tumor growth in vivo without disturbing HA-binding functions. CD44-3MUT has been PEGylated for use as a new type of anti-angiogenic human drug. PEGylation of CD44-3MUT improved pharmacokinetic properties but retains its functional activity.