CONFORMATIONAL CHANGES OF SINGLE DNA MOLECULES INDUCED BY H-NS AND HU PROTEINS IN A NANOSPACE

Ph.DDOCTOR OF PHILOSOPH

Fluorescentna in situ hibridizacija za dokazivanje virusa klasične svinjske kuge i praćenje njegove lokalizacije u zaraženim tkivima.

Classical swine fever (CSF) may be diagnosed by various conventional techniques but nucleic acid based methods have gained popularity because of their high sensitivity and specificity. For detection of viral RNA in infected tissues, a fluorescent in-situ hybridization (FISH) based method was developed using a 308 bp biotinylated DNA probe targeting E2/NS2 gene of CSFV. In-situ RNA/DNA hybrids were detected with fluorescently labeled Streptavidin conjugate and viewed under a fluorescent microscope. Viral nucleic acids could be demonstrated in the lymphoid tissues such as the spleen and lymph nodes collected from CSFV infected animals. Infected macrophages and reticuloendothelial cells revealed bright intracytoplasmic fluorescence. In conclusion, the fluorescent in situ hybridization technique used in the study provided an effective means of detection and localization of CSFV nucleic acids.Klasična svinjska kuga može se dijagnosticirati različitim uobičajenim metodama, ali se, zbog visoke osjetljivosti i specifičnosti, najčešće rabe metode zasnovane na dokazivanju specifične nukleinske kiseline. Za dokaz virusne RNA u zaraženom tkivu u ovom je radu razvijena fluorescentna in situ hibridizacija (FISH) uporabom biotinom obilježene DNA probe od 308 bp za ciljni gen E2/NS2 virusa klasične svinjske kuge. RNA/DNA hibridne molekule bile su dokazane uporabom konjugata obilježenog streptavidinom pretragom fluorescentnim mikroskopom. Virusna nukleinska kiselina bila je dokazana u limfoidnim tkivima odnosno u slezeni i limfnim čvorovima životinja zaraženih virusom. Fluorescencija je jasno bila izražena u citoplazmi zaraženih makrofaga i retikuloendotelijalnih stanica. Može se zaključiti da fluorescentna in situ hibridizacija opisana u ovom radu pruža učinkovit alat za dokaz nukleinske kiseline virusa klasične svinjske kuge i njegove lokalizacije u zaraženim tkivima

Multi-Intent Detection in User Provided Annotations for Programming by Examples Systems

In mapping enterprise applications, data mapping remains a fundamental part of integration development, but its time consuming. An increasing number of applications lack naming standards, and nested field structures further add complexity for the integration developers. Once the mapping is done, data transformation is the next challenge for the users since each application expects data to be in a certain format. Also, while building integration flow, developers need to understand the format of the source and target data field and come up with transformation program that can change data from source to target format. The problem of automatic generation of a transformation program through program synthesis paradigm from some specifications has been studied since the early days of Artificial Intelligence (AI). Programming by Example (PBE) is one such kind of technique that targets automatic inferencing of a computer program to accomplish a format or string conversion task from user-provided input and output samples. To learn the correct intent, a diverse set of samples from the user is required. However, there is a possibility that the user fails to provide a diverse set of samples. This can lead to multiple intents or ambiguity in the input and output samples. Hence, PBE systems can get confused in generating the correct intent program. In this paper, we propose a deep neural network based ambiguity prediction model, which analyzes the input-output strings and maps them to a different set of properties responsible for multiple intent. Users can analyze these properties and accordingly can provide new samples or modify existing samples which can help in building a better PBE system for mapping enterprise applications

VLSI BASED ROBUST ROUTER ARCHITECTURE

The focus of this Paper is the actual implementation of Network Router and verifies the functionality of the Five port router using the latest verification methodologies and Hardware Verification Languages. In the proposed design the FSM is designed with reduced number of states. Due to reduction of states the amount of time to produce the response became less obviously the frequency is improved. At the same time the memory required to design of this Router chip is also reduced. In the existed design number of LUTS are 724. In the existed design the total memory usage is 297148 kilobytes and the maximum frequency is 76.374MHz, whereas in the proposed design the number of LUTS are 240.In the proposed design, the total memory usage is 249164 kilobytes and the maximum frequency is 81.162MHz

Dc-Bus Voltage Control With A Three-Phase Bidirectional Inverter For Dc Distribution Systems

In this paper a new energy management system has been proposed for three-phase bidirectional inverter with dc-bus voltage control. The advantage of this bidirectional inverter is that it can operate in both grid connection and rectification modes with power factor correction. The proposed control system take into account dc-bus capacitance and control dc-bus voltage to track a linear relationship between the dc-bus voltage and inverter inductor current. The inverter tunes the dc-bus voltage every line cycle, which can reduce the frequency of operation-mode change and current distortion. With the increase in the demand of renewable energy sources, placed important role in distributed systems. In this paper battery storage system has been proposed with DG system. Battery storage system will give additional support to the DG system and also helps in controlling the DC bus voltage. The simulation results have been presented using MATLAB/SIMULINK software. The simulation results validated for the rectification mode with battery storage. This system will improve the performance of the DG system

Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists.

BACKGROUND: There is emerging evidence that Wnt pathway activity may increase during the progression from colorectal adenoma to carcinoma and that this increase is potentially an important step towards the invasive stage. Here, we investigated whether epigenetic silencing of Wnt antagonists is the biological driver for this increased Wnt activity in human tissues and how these methylation changes correlate with MSI (Microsatelite Instability) and CIMP (CpG Island Methylator Phenotype) statuses as well as known mutations in genes driving colorectal neoplasia. METHODS: We conducted a systematic analysis by pyrosequencing, to determine the promoter methylation of CpG islands associated with 17 Wnt signaling component genes. Methylation levels were correlated with MSI and CIMP statuses and known mutations within the APC, BRAF and KRAS genes in 264 matched samples representing the progression from normal to pre-invasive adenoma to colorectal carcinoma. RESULTS: We discovered widespread hypermethylation of the Wnt antagonists SFRP1, SFRP2, SFRP5, DKK2, WIF1 and SOX17 in the transition from normal to adenoma with only the Wnt antagonists SFRP1, SFRP2, DKK2 and WIF1 showing further significant increase in methylation from adenoma to carcinoma. We show this to be accompanied by loss of expression of these Wnt antagonists, and by an increase in nuclear Wnt pathway activity. Mixed effects models revealed that mutations in APC, BRAF and KRAS occur at the transition from normal to adenoma stages whilst the hypermethylation of the Wnt antagonists continued to accumulate during the transitions from adenoma to carcinoma stages. CONCLUSION: Our study provides strong evidence for a correlation between progressive hypermethylation and silencing of several Wnt antagonists with stepping-up in Wnt pathway activity beyond the APC loss associated tumour-initiating Wnt signalling levels.A.L.S. was supported by the Fundacao para a Ciencia e Tecnologia (Portugal); A.I. by a Clinician Scientist Fellowship from Cancer Research UK (grant no C10112/A11388); M.B. by the Medical Research Council (U105192713) and by Cancer Research UK (grant no C7379/A8709).This is the final published version. It first appeared at http://www.biomedcentral.com/1471-2407/14/891

Calcium-Mediated Protein Folding and Stabilisation of Salmonella Biofilm-Associated Protein a

Biofilm-associated proteins (BAPs) are important for early biofilm formation (adhesion) by bacteria and are also found in mature biofilms. BapA from Salmonella is a ~386 kDa surface protein, comprised of 27 tandem repeats predicted to be bacterial Ig-like (BIg) domains. Such tandem repeats are conserved for BAPs across different bacterial species, but the function of these domains is not completely understood. In this work, we report the first study of the mechanical stability of the BapA protein. Using magnetic tweezers, we show that the folding of BapA BIg domains requires calcium- binding and the folded domains have differential mechanical stabilities. Importantly, we identify that >100 nM concentration of calcium is needed for folding of the BIg domains, and the stability of the folded BIg domains is regulated by calcium over a wide concentration range from sub-micromolar (?M) to millimolar (mM). Only at mM calcium concentrations, as found in the extracellular environment, do the BIg domains have the saturated mechanical stability. BapA has been suggested to be involved in Salmonella invasion, and it is likely a crucial mechanical component of biofilms. Therefore, our results provide new insights into the potential roles of BapA as a structural maintenance component of Salmonella biofilm and also Salmonella invasion

Amplified stretch of bottlebrush-coated DNA in nanofluidic channels

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.Singapore-MIT Alliance for Research and TechnologyNational Science Foundation (U.S.

Effect of H-NS on the elongation and compaction of single DNA molecules in a nanospace

10.1039/c3sm51214bSoft Matter9409593-960