Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K _i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.     

RKIP Inhibition in Cervical Cancer Is Associated with Higher Tumor Aggressive Behavior and Resistance to Cisplatin Therapy

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.     

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

Low-Anxiety Rat Phenotypes Can Be Further Reduced through Genetic Intervention

Background

A previous study using an intercross between the inbred rat strains Lewis (LEW) and Spontaneously Hypertensive Rats (SHR) identified a locus on chromosome 4, named Anxrr16, influencing an experimental index of anxiety and showing a transgressive effect, with alleles from the LEW strain (more anxious) decreasing rather than increasing anxiety.

Objective

To confirm the location and isolate the effect of a rat genome region named Anxrr16 through a planned genomic recombination strategy, where the target locus in SHR rats was replaced with LEW genetic material.

Methods

A new congenic strain, named SHR.LEW-Anxrr16 (SLA16), was developed from a cross between LEW (donor) and SHR (receptor) rats and then evaluated in several anxiety-related tests. The activity and attention levels of the new strain were also evaluated, since hyperactivity was observed during its construction and because SHR is a model of attention deficit hyperactivity disorder.

Results

Significant effects of Anxrr16 were found for open field central locomotion, as well as for other indices of anxiety from the light/dark box, triple test and T-maze. In all cases, the low-anxiety levels of SHR rats were further reduced by the insertion of LEW alleles. Differences in locomotor activity were found only in unfamiliar (hence stressful) environments and no genetic effects were observed in indices of attention.

Conclusion

The SLA16 strain can help in the identification of the molecular pathways involved in experimental anxiety and it demonstrates how apparently extreme phenotypes sometimes hide major opposite-acting genes.     

Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

Background

Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.

Methodology

In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results.

Principal findings

We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients.

Conclusions

Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.     

Jatropha curcas L. (Euphorbiaceae) exhibits a mixed mating system, high correlated mating and apomixis

The hierarchical mating system among and within fruits of Jatropha curcas was investigated in a base population using five microsatellite loci, employing mixed mating and correlated mating models. Open-pollinated fruits were collected from 15 randomly selected seed trees, sampling seven fruits per tree for a total of 21 seeds from each tree. We detected multilocus genotypes identical to the mother tree in 13 % of offspring, implying the occurrence of apomixis in J. curcas. The presumed apomictic individuals were excluded from the analysis of the remaining results. Evidence of substantial selfing was provided by the average multilocus outcrossing rate (t _m?=?0.683), showing that the species exhibits a mixed mating system. The outcrossing rate showed a large variation among seed trees, ranging from 0.21 to 1.0, indicating that the species is not self-incompatible. Significant differences were detected between the multilocus and the single locus outcrossing rates (t _m???t _s?=?0.347) that suggested mating among related individuals, possibly because of the presence of individuals from the same progeny (sibs) in the base population. The multilocus paternity correlation was extremely high for the population (r _p(m)?=?0.999), indicating that the progenies were manly composed of full-sibs. As a consequence of selfing and a high paternity correlation, the co-ancestry coefficient within the progeny was higher (Θ?=?0.369) than expected for panmictic populations. Our results indicated that J. curcas produces seeds asexually by apomixis and sexually by a mixed mating system, combining selfing and outcrossing.     

Viability of phenanthrene biodegradation by an isolated bacterial consortium: optimization and scale-up

In the present work, biodegradation of phenanthrene by a bacterial consortium (LB2), isolated from lab-polluted soils has been investigated. The 16S rRNA gene-based molecular analysis revealed that the bacterial consortium LB2 consisted of two strains showing a very high homology with Staphylococcus warneri and Bacillus pumilus. The optimization of phenanthrene degradation by the consortium LB2, using a central composite face-centered design was carried out taking into account three important parameters such as temperature, pH, and phenanthrene concentration. Near complete phenanthrene degradation was reached by consortium LB2 at the optimal conditions (pH of 7.5 and 37.5 °C) in less than 48 h. Moreover, the efficiency of phenanthrene biodegradation was assessed by using logistic and Luedeking and Piret-type models. Finally, the process was implemented at bench-scale bioreactor and the main degradation routes were identified based on GC-MS data.