SUMMARY Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis (Mtb) in macrophages. However, mechanisms by which Mtb adapts to acidic environments are poorly understood. In this study, we analyzed the physiological functions of OmpATb, a surface-accessible protein of Mtb. OmpATb did not complement the permeability defects of an M. smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of Mtb indicating that OmpATb is not a general porin. Chemical analysis of low pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of Mtb. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which Mtb neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that Mtb has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of Mtb to acidic environments in vitro but not in mice.

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SummaryProteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

Nanostructures with long-term stability at the surface of gold electrodes are generated by reconstituting the porin MspA from Mycobacterium smegmatis into a specially designed monolayer of long-chain lipid surfactant on gold. Tailored surface coverage of gold electrodes with long-chain surfactants is achieved by electrochemically assisted deposition of organic thiosulfates (Bunte salts). The subsequent reconstitution of the octameric-pore MspA is guided by its extraordinary self-assembling properties. Importantly, electrochemical reduction of copper(II) yields copper nanoparticles within the MspA nanopores. Electrochemical impedance spectroscopy, reflection electron microscopy, and atomic force microscopy (AFM) show that: 1) the MspA pores within the self-assembled monolayer (SAM) are monodisperse and electrochemically active, 2) MspA reconstitutes in SAMs and with a 10-nm thickness, 3) AFM is a suitable method to detect pores within SAMs, and 4) the electrochemical reduction of Cu2+ to Cu0 under overpotential conditions starts within the MspA pores.

The unique stability and self-assembling properties of porin MspA, a channel protein isolated from Mycobacterium smegmatis, were established using three very different host systems:(1) A very simple and straightforward nanostructuring process was achieved by depositing buffer droplets containing MspA onto highly ordered pyrolytic graphite (HOPG) from the gas phase, followed by thermal curing and high vacuum treatment. Three lateral nanostructures were obtained depending on the protein mass deposited, the temperature during deposition and the subsequent curing process. We report here a detailed statistical analysis of the MspA pore sizes on HOPG.

Magnetic nanoparticles have continuously gained importance for the purpose of magnetically-aided drug-delivery, magnetofection, and hyperthermia. We have summarized significant experimental approaches, as well as their advantages and disadvantages with respect to future clinical translation. This field is alive and well and promises meaningful contributions to the development of novel cancer therapies.

A novel procedure for nanostructuring on carbon surfaces by depositing buffer droplets containing a unique protein (MspA porin from Mycobacterium smegmatis) and a PMMA prepolymer, followed by thermal curing and high vacuum treatment, was developed. The formation of protein/PMMA nanostructures on carbon surfaces depended on the amount of protein/prepolymer deposited and the temperature during and after the deposition process. The nanostructures were analyzed quantitativly using computer-supported high-resolution transmission electron micrography (TEM).

The photoinitiated polymerization of methyl methacrylate (MMA) using 2,3-diphenylbutadiene (DPB) as cross-linking agent and benzoin (BN), benzoin methyl ether (BME), and irgacure (IGC) as photoinitiators in the presence of poly(N-isopropylacrylamide) (PNIPAM) as a water-soluble, hydrophobic template polymer in H2O/DMF led to the formation of P(MMA/DPB) polymer latex particles. These particles were analyzed by using TEM in combination with the program IMAGE (NIH/USA). After purification by precipitation using THF/H2O, globular latex particles were found. Using BME as photoinitiator, “real nanoparticles” possessing a smaller diameter than 10 nm were formed among bigger particles (d > 20 nm). The CHN analysis of the P(MMA/DPB) polymers indicated that a smaller fraction of PNIPAM (4−10%) was incorporated into the latex particles.