Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxgenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.De två första författarna delar förstaförfattarskapet</p

The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence

Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell

Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia pseudotuberculosis Infection

Pathogenic Yersinia spp. depend on the activity of a potent virulence plasmid-encoded ysc/yop type 3 secretion system (T3SS) to colonize hosts and cause disease. It was recently shown that Yersinia pseudotuberculosis upregulates the virulence plasmid copy number (PCN) during infection and that the resulting elevated gene dose of plasmid-encoded T3SS genes is essential for virulence. When and how this novel regulatory mechanism is deployed and regulates the replication of the virulence plasmid during infection is unknown. In the present study, we applied droplet digital PCR (ddPCR) to investigate the dynamics of Y. pseudotuberculosis virulence PCN variations and growth rates in infected mouse organs. We demonstrated that both PCN and growth varied in different tissues and over time throughout the course of infection, indicating that the bacteria adapted to discrete microenvironments during infection. The PCN was highest in Peyer's patches and cecum during the clonal invasive phase of the infection, while the highest growth rates were found in the draining mesenteric lymph nodes. In deeper, systemic organs, the PCN was lower and more modest growth rates were recorded. Our study indicates that increased gene dosage of the plasmid-encoded T3SS genes is most important early in the infection during invasion of the host. The described ddPCR approach will greatly simplify analyses of PCN, growth dynamics, and bacterial loads in infected tissues and will be readily applicable to other infection models

Proposed model for T3SS-dependent protein translocation by a binary AB toxin like mechanism.

<p>T3SS translocators (t) and effectors (e) are secreted by the T3SS across the bacterial envelope (IM and OM) to the surface of the cell before host cell contact (1). Target cell sensing results in release of the surface localized T3SS substrates (2). The translocators (t) assemble into a pore in the target cell plasma membrane (PM) and mediate the translocation of the effectors (e) into the target cell cytoplasm (3).</p

Effects of a home-based physical rehabilitation program on tibial bone structure, density and strength after hip fracture : a secondary analysis of a randomized controlled trial

Weight-bearing physical activity may decrease or prevent bone deterioration after hip fracture. This study investigated theeffects of a home-based physical rehabilitation program on tibial bone traits in older hip fracture patients. A population-basedclinical sample of men and women operated for hip fracture (mean age 80 years, 78% women) was randomly assigned into anintervention (n¼40) and a standard care control group (n¼41) on average 10 weeks postfracture. The intervention groupparticipated in a 12-month home-based rehabilitation intervention, including evaluation and modification of environmentalhazards, guidance for safe walking, nonpharmacological pain management, motivational physical activity counseling, and aprogressive, weight-bearing home exercise program comprising strengthening exercises for the lower legs, balance training,functional exercises, and stretching. All participants received standard care. Distal tibia (5% proximal to the distal end plate)compressive bone strength index (BSI; g2/cm4), total volumetric BMD (vBMDTOT;mg/cm3), and total area (CSATOT;mm2), as wellas midtibia (55%) strength–strain index (SSI; mm3), cortical vBMD (vBMDCO;mg/cm3), and ratio of cortical to total area (CSACO/CSATOT)were assessed in both legs by pQCT at baseline and at 3, 6, and 12 months. The intervention had no effect (group time)on either the distal or midtibial bone traits. At the distal site, BSI of both legs, vBMDTOTof the fractured side, and CSATOTof thenonfractured side decreased significantly over time in both groups 0.7% to 3.1% (12 months,p<0.05). At the midshaft site,CSACO/CSATOTand SSI of both legs, and vBMDCOof the fractured leg, decreased significantly over time in both groups 1.1% to1.9% (12 months,p<0.05). Trabecular and cortical bone traits of the tibia on the fractured and the nonfractured sidedeteriorated throughout follow-up. The home-based physical rehabilitation intervention aimed at promoting mobility recoverywas unable to prevent bone deterioration in older people after hip fracture.peerReviewe

The Amino-Terminal part of the Needle-Tip Translocator LcrV of Yersinia pseudotuberculosis is Required for Early Targeting of YopH and In Vivo Virulence

Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal lcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell