Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells

Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria

Wireless sensor networks for in-situ image validation for water and nutrient management

Water and Nitrogen (N) are critical inputs for crop production. Remote sensing data collected from multiple scales, including ground-based, aerial, and satellite, can be used for the formulation of an efficient and cost effective algorithm for the detection of N and water stress. Formulation and validation of such techniques require continuous acquisition of ground based spectral data over the canopy enabling field measurements to coincide exactly with aerial and satellite observations. In this context, a wireless sensor in situ network was developed and this paper describes the results of the first phase of the experiment along with the details of sensor development and instrumentation set up. The sensor network was established based on different spatial sampling strategies and each sensor collected spectral data in seven narrow wavebands (470, 550, 670, 700, 720, 750, 790 nm) critical for monitoring crop growth. Spectral measurements recorded at required intervals (up to 30 seconds) were relayed through a multi-hop wireless network to a base computer at the field site. These data were then accessed by the remote sensing centre computing system through broad band internet. Comparison of the data from the WSN and an industry standard ground based hyperspectral radiometer indicated that there were no significant differences in the spectral measurements for all the wavebands except for 790nm. Combining sensor and wireless technologies provides a robust means of aerial and satellite data calibration and an enhanced understanding of issues of variations in the scale for the effective water and nutrient management in wheat.<br /

Design space exploration and optimization of path oblivious RAM in secure processors

Keeping user data private is a huge problem both in cloud computing and computation outsourcing. One paradigm to achieve data privacy is to use tamper-resistant processors, inside which users' private data is decrypted and computed upon. These processors need to interact with untrusted external memory. Even if we encrypt all data that leaves the trusted processor, however, the address sequence that goes off-chip may still leak information. To prevent this address leakage, the security community has proposed ORAM (Oblivious RAM). ORAM has mainly been explored in server/file settings which assume a vastly different computation model than secure processors. Not surprisingly, naïvely applying ORAM to a secure processor setting incurs large performance overheads. In this paper, a recent proposal called Path ORAM is studied. We demonstrate techniques to make Path ORAM practical in a secure processor setting. We introduce background eviction schemes to prevent Path ORAM failure and allow for a performance-driven design space exploration. We propose a concept called super blocks to further improve Path ORAM's performance, and also show an efficient integrity verification scheme for Path ORAM. With our optimizations, Path ORAM overhead drops by 41.8%, and SPEC benchmark execution time improves by 52.4% in relation to a baseline configuration. Our work can be used to improve the security level of previous secure processors.National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374)American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipUnited States. Defense Advanced Research Projects Agency (Clean-slate design of Resilient, Adaptive, Secure Hosts Contract N66001-10-2-4089

Development And Morphology Of Stelar Components In The Stems Of Some Members Of The Leguminosae And Rosaceae

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141585/1/ajb209994.pd

Sequential application of hyperspectral indices for delineation of stripe rust infection and nitrogen deficiency in wheat

© 2015, Springer Science+Business Media New York. Nitrogen (N) fertilization is crucial for the growth and development of wheat crops, and yet increased use of N can also result in increased stripe rust severity. Stripe rust infection and N deficiency both cause changes in foliar physiological activity and reduction in plant pigments that result in chlorosis. Furthermore, stripe rust produce pustules on the leaf surface which similar to chlorotic regions have a yellow color. Quantifying the severity of each factor is critical for adopting appropriate management practices. Eleven widely-used vegetation indices, based on mathematic combinations of narrow-band optical reflectance measurements in the visible/near infrared wavelength range were evaluated for their ability to discriminate and quantify stripe rust severity and N deficiency in a rust-susceptible wheat variety (H45) under varying conditions of nitrogen status. The physiological reflectance index (PhRI) and leaf and canopy chlorophyll index (LCCI) provided the strongest correlation with levels of rust infection and N-deficiency, respectively. When PhRI and LCCI were used in a sequence, both N deficiency and rust infection levels were correctly classified in 82.5 and 55 % of the plots at Zadoks growth stage 47 and 75, respectively. In misclassified plots, an overestimation of N deficiency was accompanied by an underestimation of the rust infection level or vice versa. In 18 % of the plots, there was a tendency to underestimate the severity of stripe rust infection even though the N-deficiency level was correctly predicted. The contrasting responses of the PhRI and LCCI to stripe rust infection and N deficiency, respectively, and the relative insensitivity of these indices to the other parameter makes their use in combination suitable for quantifying levels of stripe rust infection and N deficiency in wheat crops under field conditions

Discovery of a Novel Class of Orally Active Trypanocidal N-Myristoyltransferase Inhibitors

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.</p

Peroxiredoxin II Regulates Effector and Secondary Memory CD8+ T cell Responses

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H2O2on an antigen-specific CD8+ T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8+ T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8+ T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8+ T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8+ T cell pool. During chronic viral infection, increased antigen-specific CD8+ T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8+ T cell expansion, secondary memory generation, and immunopathology

The <i>N</i>-myristoylome of <i>Trypanosoma cruzi</i>

Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%)

Custom Integrated Circuits

Contains reports on nine research projects.Analog Devices, Inc.International Business Machines CorporationJoint Services Electronics Program Contract DAAL03-89-C-0001U.S. Air Force - Office of Scientific Research Contract AFOSR 86-0164BDuPont CorporationNational Science Foundation Grant MIP 88-14612U.S. Navy - Office of Naval Research Contract N00014-87-K-0825American Telephone and TelegraphDigital Equipment CorporationNational Science Foundation Grant MIP 88-5876