Quantum monitoring of cellular metabolic activities in single mitochondria

Free radicals play a vital role in all kinds of biological processes including immune responses. However, free radicals have short lifetimes and are highly reactive, making them difficult to measure using current methods. Here, we demonstrate that relaxometry measurement, or T1, inherited from the field of diamond magnetometry can be used to detect free radicals in living cells with subcellular resolution. This quantum sensing technique is based on defects in diamond, which convert a magnetic signal into an optical signal, allowing nanoscale magnetic resonance measurements. We functionalized fluorescent nanodiamonds (FNDs) to target single mitochondria within macrophage cells to detect the metabolic activity. In addition, we performed measurements on single isolated mitochondria. We were able to detect free radicals generated by individual mitochondria in either living cells or isolated mitochondria after stimulation or inhibition

Imaging the distribution of an antibody-drug conjugate constituent targeting mesothelin with Zr-89 and IRDye 800CW in mice bearing human pancreatic tumor xenografts

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibodydrug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (Zr-89) labeled anti-mesothelin antibody (Zr-89-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of Zr-89-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of Zr-89-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent Zr-89-AMA tumor uptake. Tumor uptake of Zr-89-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. Zr-89-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. Zr-89-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting

Recombination efficiency of different recombineering strains.

<p>90-nt homologous oligos incorporating a stop codon into the <i>lacZ</i> gene were electroporated into <i>E. coli</i> cells to analyze the recombination efficiency of the pRED integrated EcSIM strains as compared to EcNR2. Recombinants were selected on X-gal/IPTG plates. The recombination efficiency was calculated by estimating the fraction of white colonies in the total number of colonies.</p

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters

Quantum monitoring of cellular metabolic activities in single mitochondria

Free radicals play a vital role in all kinds of biological processes including immune responses. However, free radicals have short lifetimes and are highly reactive, making them difficult to measure using current methods. Here, we demonstrate that relaxometry measurement, or T1, inherited from the field of diamond magnetometry can be used to detect free radicals in living cells with subcellular resolution. This quantum sensing technique is based on defects in diamond, which convert a magnetic signal into an optical signal, allowing nanoscale magnetic resonance measurements. We functionalized fluorescent nanodiamonds (FNDs) to target single mitochondria within macrophage cells to detect the metabolic activity. In addition, we performed measurements on single isolated mitochondria. We were able to detect free radicals generated by individual mitochondria in either living cells or isolated mitochondria after stimulation or inhibition