Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of the complex dielectric permittivity of cells suspended in a physiological medium. It provides knowledge on the polarization–relaxation response of cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of cells and therefore, can reveal rare insights on cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmatic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. Comparing the permittivity spectra of tagged and native cell lines reveals clear differences between the analyzed suspensions. In addition, differences on the matching dielectric properties of cells were obtained. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.     

Systemic and brain macrophage infections in relation to the development of simian immunodeficiency virus encephalitis

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.     

Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.     

Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and very intricate and involves a plethora of genes in the pathogen and in the host. In the pathogen, different genes are activated in response to the changing environment to enable it to survive, adapt, evade host defenses, propagate, and damage the host. To understand the disease process, it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. Here, we have adapted a recombinase-based in vivo expression technology (RIVET) to study the genes activated in X. campestris pv. vesicatoria during its interaction with one of its hosts, tomato. This is the first study that demonstrates the feasibility of this approach for identifying in vivo induced genes in a plant pathogen. RIVET revealed 61 unique X. campestris pv. vesicatoria genes or operons that delineate a picture of the different processes involved in the pathogen-host interaction. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed several genes that may be important for the interaction of the pathogen with its host, including a citH homologue gene, encoding a citrate transporter, which was shown to be required for wild-type levels of virulence.     

Adipocyte Stiffness Increases with Accumulation of Lipid Droplets

Adipogenesis and increase in fat tissue mass are mechanosensitive processes and hence should be influenced by the mechanical properties of adipocytes. We evaluated subcellular effective stiffnesses of adipocytes using atomic force microscopy (AFM) and interferometric phase microscopy (IPM), and we verified the empirical results using finite element (FE) simulations. In the AFM studies, we found that the mean ratio of stiffnesses of the lipid droplets (LDs) over the nucleus was 0.83 ± 0.14, from which we further evaluated the ratios of LDs over cytoplasm stiffness, as being in the range of 2.5 to 8.3. These stiffness ratios, indicating that LDs are stiffer than cytoplasm, were verified by means of FE modeling, which simulated the AFM experiments, and provided good agreement between empirical and model-predicted structural behavior. In the IPM studies, we found that LDs mechanically distort their intracellular environment, which again indicated that LDs are mechanically stiffer than the surrounding cytoplasm. Combining these empirical and simulation data together, we provide in this study evidence that adipocytes stiffen with differentiation as a result of accumulation of LDs. Our results are relevant to research of adipose-related diseases, particularly overweight and obesity, from a mechanobiology and cellular mechanics perspectives.     

The Earliest Lead Object in the Levant

In the deepest section of a large complex cave in the northern Negev desert, Israel, a bi-conical lead object was found logged onto a wooden shaft. Associated material remains and radiocarbon dating of the shaft place the object within the Late Chalcolithic period, at the late 5^th millennium BCE. Based on chemical and lead isotope analysis, we show that this unique object was made of almost pure metallic lead, likely smelted from lead ores originating in the Taurus range in Anatolia. Either the finished object, or the raw material, was brought to the southern Levant, adding another major component to the already-rich Late Chalcolithic metallurgical corpus known to-date. The paper also discusses possible uses of the object, suggesting that it may have been used as a spindle whorl, at least towards its deposition.     

Hemodynamic Response Imaging: A Potential Tool for the Assessment of Angiogenesis in Brain Tumors

Blood oxygenation level dependence (BOLD) imaging under either hypercapnia or hyperoxia has been used to study neuronal activation and for assessment of various brain pathologies. We evaluated the benefit of a combined protocol of BOLD imaging during both hyperoxic and hypercapnic challenges (termed hemodynamic response imaging (HRI)). Nineteen healthy controls and seven patients with primary brain tumors were included: six with glioblastoma (two newly diagnosed and four with recurrent tumors) and one with atypical-meningioma. Maps of percent signal intensity changes (ΔS) during hyperoxia (carbogen; 95%O2+5%CO2) and hypercapnia (95%air+5%CO2) challenges and vascular reactivity mismatch maps (VRM; voxels that responded to carbogen with reduced/absent response to CO2) were calculated. VRM values were measured in white matter (WM) and gray matter (GM) areas of healthy subjects and used as threshold values in patients. Significantly higher response to carbogen was detected in healthy subjects, compared to hypercapnia, with a GM/WM ratio of 3.8 during both challenges. In patients with newly diagnosed/treatment-naive tumors (n = 3), increased response to carbogen was detected with substantially increased VRM response (compared to threshold values) within and around the tumors. In patients with recurrent tumors, reduced/absent response during both challenges was demonstrated. An additional finding in 2 of 4 patients with recurrent glioblastoma was a negative response during carbogen, distant from tumor location, which may indicate steal effect. In conclusion, the HRI method enables the assessment of blood vessel functionality and reactivity. Reference values from healthy subjects are presented and preliminary results demonstrate the potential of this method to complement perfusion imaging for the detection and follow up of angiogenesis in patients with brain tumors.     

Relevance of intra-hospital patient movements for the spread of healthcare-associated infections within hospitals - a mathematical modeling study

The aim of this study is to analyze patient movement patterns between hospital departments to derive the underlying intra-hospital movement network, and to assess if movement patterns differ between patients at high or low risk of colonization. For that purpose, we analyzed patient electronic medical record data from five hospitals to extract information on risk stratification and patient intra-hospital movements. Movement patterns were visualized as networks, and network centrality measures were calculated. Next, using an agent-based model where agents represent patients and intra-hospital patient movements were explicitly modeled, we simulated the spread of multidrug resistant enterobacteriacae (MDR-E) inside a hospital. Risk stratification of patients according to certain ICD-10 codes revealed that length of stay, patient age, and mean number of movements per admission were higher in the high-risk groups. Movement networks in all hospitals displayed a high variability among departments concerning their network centrality and connectedness with a few highly connected departments and many weakly connected peripheral departments. Simulating the spread of a pathogen in one hospital network showed positive correlation between department prevalence and network centrality measures. This study highlights the importance of intra-hospital patient movements and their possible impact on pathogen spread. Targeting interventions to departments of higher (weighted) degree may help to control the spread of MDR-E. Moreover, when the colonization status of patients coming from different departments is unknown, a ranking system based on department centralities may be used to design more effective interventions that mitigate pathogen spread.