Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease

Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this phenomenon as endogenous MceA bactericidal activity to differentiate it from the action of extracellularly secreted MccE492. The toxicity of endogenous MceA is enhanced by an efficient targeting to the inner membrane. However, a periplasmic intermediate state is not required for MceA toxicity. Indeed, endogenous MceA remains fully active when it is fused to thioredoxin-1, a fast-folding protein that promotes retention of the C terminus of MceA in the cytoplasm. The C-terminal domain of MccE492 is required only for delivery from the extracellular environment to the periplasm, and it is not required for inner membrane damage. A common component is absolutely essential for the bactericidal activity of both endogenous MceA and extracellular MccE492. Indeed, toxicity is strictly dependent on the presence of ManYZ, an inner membrane protein complex involved in mannose uptake. Based on these findings, we propose a new model for cell entry, inner membrane insertion, and toxic activity of MccE492.     

Biaxial cell stimulation: A mechanical validation

To analyse mechanotransduction resulting from tensile loading under defined conditions, various devices for in vitro cell stimulation have been developed. This work aimed to determine the strain distribution on the membrane of a commercially available device and its consistency with rising cycle numbers, as well as the amount of strain transferred to adherent cells.The strains and their behaviour within the stimulation device were determined using digital image correlation (DIC). The strain transferred to cells was measured on eGFP-transfected bone marrow-derived cells imaged with a fluorescence microscope. The analysis was performed by determining the coordinates of prominent positions on the cells, calculating vectors between the coordinates and their length changes with increasing applied tensile strain.The stimulation device was found to apply homogeneous (mean of standard deviations approx. 2% of mean strain) and reproducible strains in the central well area. However, on average, only half of the applied strain was transferred to the bone marrow-derived cells. Furthermore, the strain measured within the device increased significantly with an increasing number of cycles while the membrane's Young's modulus decreased, indicating permanent changes in the material during extended use. Thus, strain magnitudes do not match the system readout and results require careful interpretation, especially at high cycle numbers.     

DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone

The environmental contaminant 3-nitrobenzanthrone (3-nitro-7H-benz[d, e]anthracen-7-one) was recently shown to be a very strong bacterial mutagen, suggesting a new class of mutagenic compounds present in airborne particulate matter and diesel exhaust. Using the 32P-postlabeling assay, we investigated the capacity for 3-nitrobenzanthrone to form DNA adducts in vitro. Calf thymus DNA was incubated with 3-nitrobenzanthrone and either xanthine oxidase, a mammalian nitroreductase or rat liver S9 or zinc. Under these conditions 3-nitrobenzanthrone formed a total of seven adducts detectable by 32P-postlabeling. Using enrichment by butanol extraction the highest level of DNA adduct formation was found with activation by zinc (RAL: 88.4+/-32 per 108 nucleotides) followed by activation with xanthine oxidase (RAL: 75.5+/-12) and activation by rat liver S9 (RAL: 48.6+/-8). Three of the seven adduct spots were detected in all activation systems, however different amounts of individual spots were obtained in the different in vitro systems. The adduct pattern observed for the enzymatic incubations consisted of three major spots and was essentially identical. Chemical reduction of 3-nitrobenzanthrone by zinc resulted in five adduct spots whose formation was found to be concentration dependent. All adducts of 3-nitrobenzanthrone observed in this study migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of airborne particulate matter. When butanol enrichment was compared with nuclease P1 enrichment one adduct was clearly sensitive to the 3'-monophosphatase activity of nuclease P1. Our results demonstrate that 3-nitrobenzanthrone binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vitro all of which are reduction products. These adducts may contribute to the known genotoxicity and carcinogenicity of extracts from airborne particulates.     

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.     

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.     

Automated lab-scale production of PVA/PEG-enzyme immobilisates

Entrapment of biocatalysts by cryogelation is a gentle method to extend the scope of biocatalysis. To foster the use of this versatile method we devised an automated injector for the production of PVA/PEG beads. The device consists of a thermostated reservoir connected to a programmable injector nozzle and an agitated receiving bath for the droplets. This lab-scale production unit yields up to 1500 beads with immobilized enzyme per minute with a narrow size distribution and good roundness.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.