Polyphosphate - an ancient energy source and active metabolic regulator

There are a several molecules on Earth that effectively store energy within their covalent bonds, and one of these energy-rich molecules is polyphosphate. In microbial cells, polyphosphate granules are synthesised for both energy and phosphate storage and are degraded to produce nucleotide triphosphate or phosphate. Energy released from these energetic carriers is used by the cell for production of all vital molecules such as amino acids, nucleobases, sugars and lipids. Polyphosphate chains directly regulate some processes in the cell and are used as phosphate donors in gene regulation. These two processes, energetic metabolism and regulation, are orchestrated by polyphosphate kinases. Polyphosphate kinases (PPKs) can currently be categorized into three groups (PPK1, PPK2 and PPK3) according their functionality; they can also be divided into three groups according their homology (EcPPK1, PaPPK2 and ScVTC). This review discusses historical information, similarities and differences, biochemical characteristics, roles in stress response regulation and possible applications in the biotechnology industry of these enzymes. At the end of the review, a hypothesis is discussed in view of synthetic biology applications that states polyphosphate and calcium-rich organelles have endosymbiotic origins from ancient protocells that metabolized polyphosphate

Cellulose: from biocompatible to bioactive material

International audienceSince the papyri, cellulose has played a significant role in human culture, especially as paper. Nowadays, this ancient product has found new scientific applications in the expanding sector of paper-based technology. Among paper-based devices, paper-based biosensors raise a special interest. The high selectivity of biomolecules for target analytes makes these sensors efficient. Moreover, simple paper-based detection devices do not require hardware or specific technical skill. They are inexpensive, rapid, user-friendly and therefore highly promising for providing resource-limited settings with point-of-care diagnostics. The immobilization of biomolecules onto cellulose is a key step in the development of these sensing devices. Following an overview of cellulose structural features and physicochemical properties, this article reviews current techniques for the immobilization of biomolecules on paper membranes. These procedures are categorized into physical, biological and chemical approaches. There is no universal method for biomolecule immobilization. Thus, for a given paper-based biochip, each strategy can be considered

Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal

Mapping of Chemical Heterogeneity at Surfaces with High-Throughput Wide-Field Second Harmonic Microscopy

Many industrial processes are based on electrochemical reactions using engineered electrocatalysts. However, the current lack of theoretical knowledge obscures efforts to maximize catalysts' efficiency, and the optimization process is mostly empirical. To fully understand the structure/chemical function relationship on complex surfaces, new experimental tools are needed that can evaluate electrochemical processes on nm to tens of ÎŒm length scales. These tools have to be surface-specific and ideally label-free in order not to influence the reactions. In this thesis, we apply high-throughput wide-field second harmonic (SH) microscopy together with in-situ electrochemical techniques to i) study the extent of surface heterogeneity, ii) surface changes during electrochemical cycling, and iii) to identify and characterize surface active sites. We first investigated surface heterogeneity at the glass/electrolyte interface with the novel SH microscope developed in our laboratory, as this interface has been widely discussed in the literature with inconsistent results. The spatial resolution of our SH microscope, ~400 nm, together with the temporal resolution of 250 ms, allowed us to spatially resolve the surface pKa,s for the silica deprotonation reaction with values ranging from 2.3 to 10.7. The average value, 6.7, coincides well with the reported values from experimental studies lacking spatial resolution. Furthermore, we demonstrated the ability of SH microscopy to image the orientation of bulk water molecules under an externally-applied electric field in a confined 1 ÎŒm-sized glass pore. We further applied SH imaging coupled in-situ with cyclic voltammetry to characterize the surface of gold electrodes during electrochemical cycling. Analyzing the voltage dependence of the SH signal and utilizing a novel correlation coefficient procedure for wide-field SH imaging, we have identified two types of surface areas on the polycrystalline gold. The first type remains stable during electrochemical cycling, while the second type undergoes surface reconstruction. We assign the second type of gold surface areas to domains of higher roughness, where anion adsorption occurs at lower potentials than expected. Lastly, we focused on studying the oxygen evolution reaction (OER) on polycrystalline gold working electrodes by coupling SH imaging with electrochemical methods. We observe a large spatial heterogeneity of the OER, and we quantify that only 2 V vs. the reverse hydrogen electrode (RHE), which is the onset of the OER. This type is stable under potential cycling, presumably extending multiple layers deep into the gold electrode. The second, anomalous type, is observed at potentials < 2 V vs. RHE. This type is removed by potential cycling, suggesting it involves a structural motif only 1-2 gold layers deep. The findings in this thesis highlight the importance of surface heterogeneity on a sub-ÎŒm level, showing that the surface structure influences the chemical properties and reactivity. This heterogeneity needs to be considered and utilized in the industrial context to establish more efficient electrochemical processes with lower energy requirements

Tunneled Data Extraction into Separate Flows

The goal of this work is to design and implement an application for extraction of tunneled data into seperate flows. The app will be removing all layers of encapsulation, that the file contains.The use of the app lays in better analysis and diagnostics of network communication. Thanks to removing the tunnels from the network flow, it will only contain data we can use. In the theoretical part, the work focuses on network architecture TCP/IP, the tunneling protocols and ways of capturing communication on the network. The practical part describes the way of retrieving test data, it also contains a design of the target application, as well as implementation of this design and testing of the final application

Preparation of micro and nanostructures using matrix laser lithography

katedra fyzikální elektronik

Tunneled Data Extraction into Separate Flows

Cílem této práce je navrhnout a implementovat aplikaci pro extrakci tunelovaných dat do samostatných toků. Aplikace bude odstraňovat všechny vrstvy zapouzdření, které se ve vstupním souboru nachází. Využití nástroje spočívá v lepší analýze a diagnostice síťové komunikace. Díky odstranění tunelů ze síťového toku už bude tok obsahovat pouze data, se kterými se má dále pracovat. Práce se v teoretické části také zabývá síťovou architekturou TCP/IP, tunelovacími protokoly a způsoby, jakými lze na síti zachytávat komunikace. V praktické části je popsán způsob, jakým byly získávány testovací data, návrh cílové aplikace, implementace tohoto návrhu a testování výsledné aplikace.The goal of this work is to design and implement an application for extraction of tunneled data into seperate flows. The app will be removing all layers of encapsulation, that the file contains.The use of the app lays in better analysis and diagnostics of network communication. Thanks to removing the tunnels from the network flow, it will only contain data we can use. In the theoretical part, the work focuses on network architecture TCP/IP, the tunneling protocols and ways of capturing communication on the network. The practical part describes the way of retrieving test data, it also contains a design of the target application, as well as implementation of this design and testing of the final application.