Application of the BIOLOG microplate system to monitor the physiological response of heat-stressed bacteria

The BIOLOG microplate system was originally evaluated by Miller and Rhoden (1991) for its ability to correctly identify clinical and environmental isolates. The system determines identity based on the exchange of electrons produced during utilization of a carbon substrate and subsequent respiration, leading to the reduction of a tetrazolium-based color change. Enteric bacteria ( Escherichia coli, Enterobacter sakazakii, and Salmonella enterica serovar Typhimurium) were exposed to heat stress (52°C) and samples were removed as a function of time (0, 15, 30 and 90 min). Stressed cells were monitored with traditional viable cell counts and inoculated into BIOLOG GN plates. The stress response of enteric bacteria results in both physical changes, as reflected by their D-values and sublethal injury, and metabolic changes, as reflected by differential utilization of carbon substrates. Results indicate that the BIOLOG system may be used as a tool to monitor the physiological change of heat-stressed enteric bacteria. The BIOLOG system allows for three types of analysis of the metabolic changes taking place in heat-stressed enteric bacteria. The first two applications of the BIOLOG system demonstrated a generalized approach using AWCD and guild groupings as a tool to monitor physiological changes of heat-stressed enteric bacteria. The use of AWCD and guild groupings proved to be relatively ineffective in monitoring physiological changes in heat-stressed enteric bacteria. The third application that may be used demonstrate possible specific substrate utilization differences in heat-stressed enteric bacteria. Notable changes in specific substrate utilization were observed in thermally-stressed enteric bacteria with the use of the BIOLOG system

Quantification of the Energetic and Microcirculatory Heterogeneity in the Renal System

Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of kidney function and is typically diagnosed by an increase in blood-urea-nitrogen and serum creatinine, a decrease in the glomerular filtration rate (GFR), and a decrease in urine output. AKI can be brought on by a myriad of events: physical damage to the kidney, cardiac arrest, blood loss, toxicologic effects from pharmacological drug use, and in most cases seen, sepsis. These events introduce global and or local ischemic insult to the kidney, causing a decrease in renal functionality. Originally, global renal hypoperfusion was thought to be the culprit causing AKI. However, evidence is showing that AKI can occur in the absence of this, proved by the normal or even increased blood flow seen in sepsis-induced AKI. In fact, studies are finding similar results that show microcirculatory dysfunction, inflammation, and tubular oxidative stress are the driving physiological factors for sepsis-induced AKI. The development and use of intravital video microscopy (IVVM) allows \textit{in vivo} studies of biological systems to be conducted. The excitation and emission of Flourophores are used to visualize specific structures and interactions within a system, and provide the means for analysis. Visualization of renal system structure and dynamics have be captured using IVVM, specifically ATP generation activity seen in the tubular epithelial cells and the microvascular dysfunction of blood flow associated with sepsis-induced AKI. The work proposed here focuses on using these images to quantify and explain the heterogeneity seen in the microhemodynamics of the cortical peritubular capillaries as well as mitochondrial energetics of the renal system. The information learned regarding oxygen delivery and energy consumption can be used to further understand the physio/pathophysio-logical interactions of the renal system in states of health and AKI

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors

<p>Abstract</p> <p>Background</p> <p>The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as <it>ATM </it>(Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity.</p> <p>Results</p> <p>Using a replication-free assay in <it>Xenopus </it>extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells.</p> <p>Conclusions</p> <p>These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.</p

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish

Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans

Publisher Correction: Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

The original version of this article contained an error in the name of the author Ramachandran S. Vasan, which was incorrectly given as Vasan S. Ramachandran. This has now been corrected in both the PDF and HTML versions of the article

Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second

To study the behavior of freely moving model organisms such as zebrafish (Danio rerio) and fruit flies (Drosophila) across multiple spatial scales, it would be ideal to use a light microscope that can resolve 3D information over a wide field of view (FOV) at high speed and high spatial resolution. However, it is challenging to design an optical instrument to achieve all of these properties simultaneously. Existing techniques for large-FOV microscopic imaging and for 3D image measurement typically require many sequential image snapshots, thus compromising speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over a 135-cm^2 area, achieving up to 230 frames per second at throughputs exceeding 5 gigapixels (GPs) per second. 3D-RAPID features a 3D reconstruction algorithm that, for each synchronized temporal snapshot, simultaneously fuses all 54 images seamlessly into a globally-consistent composite that includes a coregistered 3D height map. The self-supervised 3D reconstruction algorithm itself trains a spatiotemporally-compressed convolutional neural network (CNN) that maps raw photometric images to 3D topography, using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. As a result, our end-to-end 3D reconstruction algorithm is robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. The scalable hardware and software design of 3D-RAPID addresses a longstanding problem in the field of behavioral imaging, enabling parallelized 3D observation of large collections of freely moving organisms at high spatiotemporal throughputs, which we demonstrate in ants (Pogonomyrmex barbatus), fruit flies, and zebrafish larvae

Oxygen stable isotope ratios from British oak tree-rings provide a strong and consistent record of past changes in summer rainfall

United Kingdom (UK) summers dominated by anti-cyclonic circulation patterns are characterised by clear skies, warm temperatures, low precipitation totals, low air humidity and more enriched oxygen isotope ratios (δ18O) in precipitation. Such conditions usually result in relatively more positive (enriched) oxygen isotope ratios in tree leaf sugars and ultimately in the tree-ring cellulose formed in that year, the converse being true in cooler, wet summers dominated by westerly air flow and cyclonic conditions. There should therefore be a strong link between tree-ring δ18O and the amount of summer precipitation. Stable oxygen isotope ratios from the latewood cellulose of 40 oak trees sampled at eight locations across Great Britain produce a mean δ18O chronology that correlates strongly and significantly with summer indices of total shear vorticity, surface air pressure, and the amount of summer precipitation across the England and Wales region of the United Kingdom. The isotope-based rainfall signal is stronger and much more stable over time than reconstructions based upon oak ring widths. Using recently developed methods that are precise, efficient and highly cost-effective it is possible to measure both carbon (δ13C) and oxygen (δ18O) isotope ratios simultaneously from the same tree-ring cellulose. In our study region, these two measurements from multiple trees can be used to reconstruct summer temperature (δ13C) and summer precipitation (δ18O) with sufficient independence to allow the evolution of these climate parameters to be reconstructed with high levels of confidence. The existence of long, well-replicated oak tree-ring chronologies across the British Isles mean that it should now be possible to reconstruct both summer temperature and precipitation over many centuries and potentially millennia