Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

Background

Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).

Results

Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.

Conclusion

Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.     

Protein kinase D as a potential new target for cancer therapy

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.     

The Exo70 Subunit of the Exocyst Is an Effector for Both Cdc42 and Rho3 Function in Polarized Exocytosis

The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation of exocytosis by Rho GTPases.     

Macro-arthropod succession in grassland growing on bauxite residue

Ecosystem functions such as nutrient cycling are crucial components of revegetation programmes for bauxite residues and other tailings. Whilst vegetation establishment on bauxite residue is well studied, little is known about the development of the soil-biota. Macro-arthropod assemblages have been used to monitor and evaluate restoration success on a variety of mine spoils, but there is no information on its presence in bauxite residues.In order to understand the colonization and community development of macro-arthropods in revegetated residue, we compared species from two revegetated residues with an unamended site using pitfall and pan trap techniques. Whilst relatively few immobile species were present in the unamended site, high diversity was recorded for the revegetated sites. A number of species representative of predatory and carnivorous trophic structure were identified for the two vegetated sites. Additionally, many species identified are indicative of later stages of succession.These findings indicate that bauxite residues can be successfully restored to a functioning grassland type ecosystem. Monitoring of key arthropod species is recommended as part of the restoration evaluation process.     

Mini-Tn7 vectors as genetic tools for single copy gene cloning in Acinetobacter baumannii

Acinetobacter baumannii is an emerging pathogen that causes serious infections with high mortality rates in immunocompromised individuals. Genetic manipulations in this medically-relevant pathogen are limited by the paucity of molecular tools. In this study, we show the application of the mini-Tn7-based single copy insertion system in A. baumannii. Mini-Tn7 elements are known to integrate at a naturally evolved, therefore presumably neutral location (intergenic region) downstream of the glmS gene (glucosamine-fructose-6-phosphate aminotransferase) in Gram-negative bacteria. We identified the site of insertion of mini-Tn7 in A. baumannii and demonstrated application of this useful cloning tool by inserting the gfp gene into the chromosome. Our work shows that mini-Tn7 elements are useful tools for genetic studies in this important pathogen.     

Inhibition of angiogenesis- and inflammation-inducing factors in human colon cancer cells <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in ovo</Emphasis> by free and nanoparticle-encapsulated redox dye,DCPIP

Background  

The redox dye, DCPIP, has recently shown to exhibit anti-melanoma activity in vitro and in vivo. On the other hand, there is increasing evidence that synthetic nanoparticles can serve as highly efficient carriers of drugs and vaccines for treatment of various diseases. These nanoparticles have shown to serve as potent tools that can increase the bioavailability of the drug/vaccine by facilitating absorption or conferring sustained and improved release. Here, we describe results on the effects of free- and nanoparticle-enclosed DCPIP as anti-angiogenesis and anti-inflammation agents in a human colon cancer HCT116 cell line in vitro, and in induced angiogenesis in ovo.     

Ionizing radiation and hematopoietic malignancies: Altering the adaptive landscape

Somatic evolution, which underlies tumor progression, is driven by two essential components: (1) diversification of phenotypes through heritable mutations and epigenetic changes and (2) selection for mutant clones which possess higher fitness. Exposure to ionizing radiation (IR) is highly associated with increased risk of carcinogenesis. This link is traditionally attributed to causation of oncogenic mutations through the mutagenic effects of irradiation. On the other hand, potential effects of irradiation on altering fitness and increasing selection for mutant clones are frequently ignored. Recent studies bring the effects of irradiation on fitness and selection into focus, demonstrating that IR exposure results in stable reductions in the fitness of hematopoietic stem and progenitor cell populations. These reductions of fitness are associated with alteration of the adaptive landscape, increasing the selective advantages conferred by certain oncogenic mutations. Therefore, the link between irradiation and carcinogenesis might be more complex than traditionally appreciated: while mutagenic effects of irradiation should increase the probability of occurrence of oncogenic mutations, IR can also work as a tumor promoter, increasing the selective expansion of clones bearing mutations which become advantageous in the irradiation-altered environment, such as activated mutations in Notch1 or disrupting mutations in p53.Key words: Notch, p53, fitness, irradiation, hematopoietic, evolution     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe³⁺ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe³⁺] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe³⁺ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.     

Chronic cardiac resynchronization therapy and reverse ventricular remodeling in a model of nonischemic cardiomyopathy

While cardiac resynchronization therapy (CRT) has been shown to reduce morbidity and mortality in heart failure (HF) patients, the fundamental mechanisms for the efficacy of CRT are poorly understood. The lack of understanding of these basic mechanisms represents a significant barrier to our understanding of the pathogenesis of HF and potential recovery mechanisms. Our purpose was to determine cellular mechanisms for the observed improvement in chronic HF after CRT. We used a canine model of chronic nonischemic cardiomyopathy. After 15 months, dogs were randomized to continued RV tachypacing (untreated HF) or CRT for an additional 9 months. Six minute walk tests, echocardiograms, and electrocardiograms were done to assess the functional response to therapy. Left ventricular (LV) midmyocardial myocytes were isolated to study electrophysiology and intracellular calcium regulation. Compared to untreated HF, CRT improved HF-induced increases in LV volumes, diameters and mass (p<0.05). CRT reversed HF-induced prolongations in LV myocyte repolarization (p<0.05) and normalized HF-induced depolarization (p<0.03) of the resting membrane potential. CRT improved HF-induced reductions in calcium (p<0.05). CRT did not attenuate the HF-induced increases in LV interstitial fibrosis. Using a translational approach in a chronic HF model, CRT significantly improved LV structure; this was accompanied by improved LV myocyte electrophysiology and calcium regulation. The beneficial effects of CRT may be attributable, in part, to improved LV myocyte function.