Biodistribution Investigations of Technetium-Labelllled Murine Bone Marrow-Derived Extracellllular Vesicles by Nanospect/Ct

The in vivo tracing of the biodistribution of extracellular vesicles (EVs) is a pre-requisite in identifying their target cells and understanding their function. Although luorescent labelling of EVs is already used, radiolabelling can provide more details in understanding biodistribution of EVs. In the present paper we report radiolabelling of bone marrow-derived EVs and in vivo tracing of their biodistribution. EVs isolated from the bone marrow supernatant of űő7ŰL/6 mice were labelled with the technetium-99m (99mTc) isotope. Labelling was eficient and labelled EVs were stable during the 2Ő hours follow-up. Detection of labelled EVs after intravenous injection in mice was performed using ex vivo measurements and in vivo imaging. Ex vivo examinations revealed that at Ő hours post-injection, the highest accumulation rate was in the liver, kidney, spleen and femur epiphysis. In vivo imaging using nanoSPEűT/űT conirmed the ex vivo examinations and demonstrated slow elimination of the radioactivity, 2Ő hours post- injection the bone marrow-containing epiphysis and lymph nodes showed the highest retention values; liver, spleen and kidney were also clearly detectable. In summary, labelling of bone marrow-derived EVs with 99mTc coupled with SPEűT/űT detection was a reliable method for quantitative distribution studies of EVs in vivo

Extracellular vesicles mediate radiation-induced systemic bystander signals in the bone marrow and spleen

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EV) in mediating radiation-induced bystander signals but in vivo investigations are still lacking. Here we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), 24 hours later EVs were isolated from the bone marrow and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similarly to direct radiation EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of 8 differentially expressed miRNAs were identified in the EVs originating from both low and high dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. ​ In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms

Structure of a Nudix hydrolase (MutT) in the Mg2+-­bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution

Knockdown of MTDH Sensitizes Endometrial Cancer Cells to Cell Death Induction by Death Receptor Ligand TRAIL and HDAC Inhibitor LBH589 Co-Treatment

Understanding the molecular underpinnings of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH correlates with poor clinical outcome in breast cancer, neuroblastoma, hepatocellular carcinoma and prostate cancer. MTDH is also highly expressed in advanced endometrial cancers, a disease for which new therapies are urgently needed. In this present study, we focused on the therapeutic benefit of MTDH depletion in endometrial cancer cells to restore sensitivity to cell death. Cells were treated with a combination of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), which promotes death of malignant cells of the human reproductive tract, and histone deacetylase (HDAC) inhibitors, which have been shown to increase the sensitivity of cancer cells to TRAIL-induced apoptosis. Our data indicate that depletion of MTDH in endometrial cancer cells resulted in sensitization of cells that were previously resistant in response to combinatorial treatment with TRAIL and the HDAC inhibitor LBH589. MTDH knockdown reduced the proportion of cells in S and increased cell arrest in G2/M in cells treated with LBH589 alone or LBH589 in combination with TRAIL, suggesting that MTDH functions at the cell cycle checkpoint to accomplish resistance. Using microarray technology, we identified 57 downstream target genes of MTDH, including calbindin 1 and galectin-1, which may contribute to MTDH-mediated therapeutic resistance. On the other hand, in MTDH depleted cells, inhibition of PDK1 and AKT phosphorylation along with increased Bim expression and XIAP degradation correlated with enhanced sensitivity to cell death in response to TRAIL and LBH589. These findings indicate that targeting or depleting MTDH is a potentially novel avenue for reversing therapeutic resistance in patients with endometrial cancer

Cefotaxime and desacetyl cefotaxime in human bile

Ten patients were injected with 2 g cefotaxime i. v. The antibacterial activity in the bile was measured by the agar diffusion test and the concentrations of cefotaxime and desacetyl cefotaxime were determined by high performance liquid chromatography. The values found allow the use of cefotaxime in infectious biliary diseases