Mechanisms of interferon‐beta‐induced inhibition of Toxoplasma gondii growth in murine macrophages and embryonic fibroblasts: role of immunity‐related GTPase M1

The apical complex of Toxoplasma gondii enables it to invade virtually all nucleated cells in warm‐blooded animals, including humans, making it a parasite of global importance. Anti‐T. gondii cellular defence mechanisms depend largely on interferon (IFN)‐γ production by immune cells. However, the molecular mechanism of IFN‐β‐mediated defence remains largely unclear. Here, mouse peritoneal macrophages and murine embryonic fibroblasts (MEFs) primed with recombinant IFN‐β and IFN‐γ showed different pathways of activation. Treatment of these cells with IFN‐β or IFN‐γ inhibited T. gondii (type II PLK strain) growth. Priming macrophages with IFN‐β had no effect on inflammatory cytokine expression, inducible nitric oxide synthase or indoleamine 2,3‐dioxygenase, nor did it have an effect on their metabolites, nitric oxide and kynurenine respectively. In contrast, IFN‐γ stimulation was characterized by classical macrophage activation and T. gondii elimination. IFN‐β activation recruited the immunity‐related GTPase M1 (IRGM1) to the parasitophorous vacuole in the macrophages and MEFs. Anti‐toxoplasma activities induced by IFN‐β were significantly reduced after IRGM1 knockdown in murine macrophages and in IRGM1‐deficient MEFs. Thus, this study unravels an alternative pathway of macrophage activation by IFN‐β and provides a mechanistic explanation for the contribution of IRGM1 induced by IFN‐β to the elimination of T. gondii.     

A parallel phase I/II clinical trial design for combination therapies

The use of multiple drugs in a single clinical trial or as a therapeutic strategy has become common, particularly in the treatment of cancer. Because traditional trials are designed to evaluate one agent at a time, the evaluation of therapies in combination requires specialized trial designs. In place of the traditional separate phase I and II trials, we propose using a parallel phase I/II clinical trial to evaluate simultaneously the safety and efficacy of combination dose levels, and select the optimal combination dose. The trial is started with an initial period of dose escalation, then patients are randomly assigned to admissible dose levels. These dose levels are compared with each other. Bayesian posterior probabilities are used in the randomization to adaptively assign more patients to doses with higher efficacy levels. Combination doses with lower efficacy are temporarily closed and those with intolerable toxicity are eliminated from the trial. The trial is stopped if the posterior probability for safety, efficacy, or futility crosses a prespecified boundary. For illustration, we apply the design to a combination chemotherapy trial for leukemia. We use simulation studies to assess the operating characteristics of the parallel phase I/II trial design, and compare it to a conventional design for a standard phase I and phase II trial. The simulations show that the proposed design saves sample size, has better power, and efficiently assigns more patients to doses with higher efficacy levels.     

Magnetic nanofactories: localized synthesis and delivery of quorum-sensing signaling molecule autoinducer-2 to bacterial cell surfaces

Magnetic 'nanofactories', for localized manufacture and signal-guided delivery of small molecules to targeted cell surfaces, are demonstrated. They recruit nearby raw materials for synthesis, employ magnetic mobility for capture and localization of target cells, and deliver molecules to cells triggering their native phenotypic response, but with user-specified control. Our nanofactories, which synthesize and deliver the "universal" bacterial quorum-sensing signal molecule, autoinducer AI-2, to the surface of Escherichia coli, are assembled by first co-precipitating nanoparticles of iron salts and the biopolymer chitosan. E. coli AI-2 synthases, Pfs and LuxS, constructed with enzymatically activatable "pro-tags", are then covalently tethered onto the chitosan. These enzymes synthesize AI-2 from metabolite S-adenosylhomocysteine. Chitosan serves as a molecular scaffold and provides cell capture ability; magnetite provides stimuli responsiveness. These magnetic nanofactories are shown to modulate the natural progression of quorum-sensing activity. New prospects for small molecule delivery, based on localized synthesis, are envisioned.     

Design,synthesis and biological evaluation of bifunctional inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP)

Collagen degradation and proMMP-2 activation are major functions of MT1-MMP to promote cancer cell invasion. Since both processes require MT1-MMP homodimerization on the cell surface, herein we propose that the use of bifunctional inhibitors of this enzyme could represent an innovative approach to efficiently reduce tumor growth. A small series of symmetrical dimers derived from previously described monomeric arylsulfonamide hydroxamates was synthesized and tested in vitro on isolated MMPs. A nanomolar MT1-MMP inhibitor, compound 6, was identified and then submitted to cell-based assays on HT1080 fibrosarcoma cells. Dimer 6 reduced MT1-MMP-dependent proMMP-2 activation, collagen degradation and collagen invasion in a dose-dependent manner with better results even compared to its monomeric analogue 4. This preliminary study suggests that dimeric MT1-MMP inhibitors might be further developed and exploited as an alternative tool to reduce cancer cell invasion.     

Mutational analysis of block and facilitation of HERG current by a class III anti-arrhythmic agent, nifekalant

Chemicals and toxins are useful tools to elucidate the structure-function relationship of various proteins including ion channels. The HERG channel is blocked by many compounds and this may cause life-threatening cardiac arrhythmia. Besides block, some chemicals such as the class III anti-arrhythmic agent nifekalant stimulate HERG at low potentials by shifting its activation curve towards hyperpolarizing voltages. This is called "facilitation". Here, we report mutations and simulations analyzing the association between nifekalant and channel pore residues for block and facilitation. Alanine-scanning mutagenesis was performed in the pore region of HERG. The mutations at the base of the pore helix (T623A), the selectivity filter (V625A) and the S6 helix (G648A, Y652A and F656A) abolished and S624A attenuated both block and facilitation induced by the drug. On the other hand, the mutation of other residues caused either an increase or a decrease in nifekalant-induced facilitation without affecting block. An open-state homology model of the HERG pore suggested that T623, S624, Y652 and F656 faced the central cavity, and were positioned within geometrical range for the drug to be able to interact with all of them at the same time. Of these, S649 was the only polar residue located within possible interaction distance from the drug held in its blocking position. Further mutations and flexible-docking simulations suggest that the size, but not the polarity, of the side chain at S649 is critical for drug induced facilitation.     

SNAP-23 is not essential for constitutive exocytosis in HeLa cells

We applied the small interfering RNA (siRNA) technique and over-expression of a dominant-negative mutant to evaluate the role of SNAP-23, a non-neuronal isoform of SNAP-25, in constitutive exocytosis from HeLa cells. Although the protein level of SNAP-23 was reduced to less than 10% of the control value by siRNA directed against SNAP-23, exocytosis of SEAP (secreted alkaline phosphatase) was normal. Double knockdown of SNAP-23 and syntaxin-4 also failed to inhibit the secretion. Furthermore, over-expression of deltaC8-SNAP-23, a dominant-negative mutant of SNAP-23, did not abrogate SEAP secretion. These results suggest that SNAP-23 is not essential for constitutive exocytosis of SEAP.