Passive dispersal of the grape rust mite Calepitrimerus vitis Nalepa 1905 (Acari, Eriophyoidea) in vineyards

Modes of passive dispersal of the grape rust mite Calepitrimerus vitis (Eriophyoidea) were investigated in a vineyard of South-Western Germany. More than 200 Eriophyoidea per month were trapped in a wind chamber during summer (32,1 % C. vitis) suggesting long-distance dispersal by air currents. Rain washed part of the adult C. vitis population from the foliage. SE micrographs suggest that quiescent nymphs are affixed to the leaf by a substance of unknown nature. However, the role of rain in C. vitis colonisation of uninfested vineyards is still unclear, as is the role of phoretic transport by arthropods. For the first time, evidence of rust mite dispersal by human activity is presented. A large number of C. vitis was found adhering to clothes and hands of workers carrying out customary cultural practices in the vineyard. Other arthropods, including Typhlodromus pyri, the main predator of C. vitis, were also passively transported by wind, rain and human activit

Photo-cross-linking and identification of nuclear proteins that bind to DNA containing a site-specific adduct of cis-[Pt(NH₃)(N-6-aminohexyl)-4-benzophenonamide)CI₂]

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.In title on title page, "cis" appears as italic. Vita.Includes bibliographical references.cis-Diamminedichloroplatinum(II), or cisplatin, is the most successful cancer drug ever discovered. It has been used for over 30 years in the treatment of several types of malignancies, with the highest success rates in testicular cancer patients. Despite the triumphs of this drug, the exact mechanisms of cell-killing by cisplatin are not fully understood. A more complete understanding of the ability of this drug to target and destroy cancer cells will lead to advances in overcoming the intrinsic and acquired resistance that many cancer patients encounter with cisplatin. Cisplatin can form several distinct DNA adducts, which are toxic to cells if not repaired. By identifying proteins that bind to platinum-modified DNA, the work in this thesis focuses on the early stages of DNA adduct processing. The first chapter of this thesis gives a review of the identification of proteins with an affinity for platinum-modified DNA in the literature. Different techniques are discussed, and the proteins identified by these methodologies are discussed. The methodologies previously used to identify proteins with an affinity for platinum-modified DNA have led to important advances in our knowledge of the cellular processing of these adducts. The development of an assay that can survey the entire nuclear milieu, allowing for multiprotein complexes to stay intact and proteins to compete for binding to the damaged DNA has been difficult. These challenges were addressed by using a photo-active cisplatin analogue, cis-[Pt(NH3)(N-(6-aminohexyl)-4-benzophenonamide)C12] (PtBP6). Site-specifically modified DNA probes were synthesized and used to identify proteins photo-cross-linked by the benzophenonemodified platinum complex. A 25-bp probe containing a 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand adduct of PtBP6 were synthesized and used in photo-cross-linking experiments. The results demonstrated that the 1,2-d(GpG) adduct binds more strongly to HMG-domain proteins, and the 1,3-d(GpTpG) adduct exclusively binds a nucleotide excision repair protein RPA1.(cont.) A probe containing a mismatched 1,2-d(GpG) adduct was also synthesized, and this DNA photo-cross-linked more strongly to the mismatch repair protein, Msh2. These results demonstrate that PtBP6-modified DNA mimics the behavior of cisplatin-modified DNA published in the literature. These experiments also identify PARP-1 as a protein with an affinity for platinum-modified DNA. This protein will bind to other types of DNA damage, but had not been previously implicated in platinum-DNA damage binding. Photo-cross-linking experiments were carried out using nuclear extracts from cancer cells with varying sensitivity to cisplatin. The results indicate that expression levels of PARP-1 may effect the cell's ability to survive cisplatin exposure. The proteins photo-cross-linked by different nuclear extracts were consistent, however, indicating that DNA damage recognition is not the mechanism for cisplatin resistance in these cell lines. In order to ensure that the proteins identified using the 25-bp probe were not binding to the blunt ends of the DNA and being photo-cross-linked, a 90 base dumbbell probe was synthesized. In this probe, the DNA contains no blunt ends but two loops, which are 19 bases from the platinum adduct. Photo-cross-linking experiments using this probe confirmed the binding of each of the proteins identified using the 25-bp probe. The photo-cross-linking of DDBI, RFC1 and RFC2 demonstrates that a longer platinummodified DNA probe is a better substrate for recognition by nucleotide excision repair proteins. This work also indicates that both mismatch repair recognition complexes Msh2/Msh3 and Msh2/Msh6 bind to platinum-damaged DNA. The affinity of chromatin remodeling proteins for platinum-damaged DNA was also established in this work. The characterization of PARP-1 as a protein with an affinity for platinumdamaged DNA was first discovered using photo-cross-linking experiments with PtBP6. Prior to this discovery, the ability of PARP inhibitors to sensitize cells to cisplatin had already been investigated.(cont.) Conflicting results are reported in the literature about the efficacy of these inhibitors as potentiators of cisplatin toxicity. In order to probe the mechanism of the interaction, assays were carried out using PARP inhibitors synthesized by Alison Ondrus of the Movassaghi lab. These experiments indicated that the activity of PARP proteins following exposure to platinum-modified DNA may be cell-line dependent. We were unable to detect a significant increase in PARP activity following exposure to cisplatin, however, in any cell line tested. These results suggest that more mechanistic studies into the effect of the activity of PARP proteins on cancer cells exposed to cisplatin are warranted. Initial photo-cross-linking experiments using a duplex containing a benzophenone moiety on the DNA indicated that this type of probe could be used to identify proteins with an affinity for various types of DNA lesions. Also, photo-cross-linking using a 157bp probe, site-specifically modified with PtBP6, indicated that there are some obstacles to overcome when performing these types of experiments with longer DNA probes. Initial NMR spectroscopic studies of a PtBP6-modified d(GpG) were carried out to characterize the orientational isomers of this type of DNA lesion.by Evan Ross Guggenheim.Ph.D

Cryo-Preparation and Planar Magnetron Sputtering for Low Temperature Scanning Electron Microscopy

Cryo-preparation is a reliable technique for the structural investigation of food products in low temperature scanning electron microscopy (SEM). Artifacts, such as, the segregation of water/non-water ingredients, occur during the freezing process by the crystallization of ice; they can be helpful for correct interpretation of visualized details, e.g., the detection of water containing compartments. The size of the segregation structures depends on water concentration and specimen thickness. The condensation of water vapor (ice contamination) is influenced by the specimen temperature and the partial pressure of the water inside the vacuum system. Furthermore, the evaporation (sublimation, etching) of specimen water can be regulated by monitoring the specimen temperature. Sublimation under SEM observation, i.e., in situ etching at low acceleration voltage, allows the progress of etching to be observed continuously, prior to the coating of the specimen inside a dedicated cryo-preparation system attached to the SEM. Coating of specimens provides superior structural resolution compared with the observation of uncoated samples. A coating layer of platinum ( ~ 1-2 nm thick), deposited on a cold substrate by planar magnetron sputtering, is almost homogenous and has a density close to that of the solid metal. Its use allows bulk biological specimens to be observed in low temperature SEM with a structural resolution up to the visualization of transmembrane proteins

Poly(ADP-ribose) polymerase-1 activity facilitates the dissociation of nuclear proteins from platinum-modified DNA

The affinity of the poly(ADP-ribose) polymerase-1 (PARP-1) for platinum-damaged DNA was first discovered during photo-cross-linking experiments using the photoactive compound Pt-BP6 [J. Am. Chem. Soc. 2004, 126, 6536–6537], an analogue of the anticancer drug cis-diamminedichloroplatinum(II), cisplatin. Although PARP inhibitors sensitize cancer cells to cisplatin, there are conflicting reports in the literature about their efficacy. In order to improve our understanding of the mechanism by which PARP inhibition might potentiate the cell-killing ability of cisplatin, and to shed light on the source of the discrepancy among different laboratories, we have in the present study probed the influence of three PARP inhibitors in four types of cancer cells, cervical (HeLa), testicular (NTera2), pancreatic (BxPC3), and osteosarcoma (U2OS), on the results of Pt-BP6 photo-cross-linking experiments and cytotoxicity assays. We find that the activity of PARP proteins following exposure to platinum-modified DNA results in the dissociation of DNA-bound proteins. PARP inhibitors were able to sensitize some, but not all, of the cell lines to cisplatin. This cell line-dependence and the potential consequences of PARP-initiated protein removal from platinum–DNA lesions are discussed. Control experiments revealed that NTera2 cells are especially sensitive to PARP inhibition

Genome-wide association studies for corneal and refractive astigmatism in UK Biobank demonstrate a shared role for myopia susceptibility loci

Previous studies have suggested that naturally occurring genetic variation contributes to the risk of astigmatism. The purpose of this investigation was to identify genetic markers associated with corneal and refractive astigmatism in a large-scale European ancestry cohort (UK Biobank) who underwent keratometry and autorefraction at an assessment centre. Genome-wide association studies for corneal and refractive astigmatism were performed in individuals of European ancestry (N = 86,335 and 88,005 respectively), with the mean corneal astigmatism or refractive astigmatism in fellow eyes analysed as a quantitative trait (dependent variable). Genetic correlation between the two traits was calculated using LD Score regression. Gene-based and gene-set tests were carried out using MAGMA. Single marker-based association tests for corneal astigmatism identified four genome-wide significant loci (P < 5 × 10−8) near the genes ZC3H11B (1q41), LINC00340 (6p22.3), HERC2/OCA2 (15q13.1) and NPLOC4/TSPAN10 (17q25.3). Three of these loci also demonstrated genome-wide significant association with refractive astigmatism: LINC00340, HERC2/OCA2 and NPLOC4/TSPAN10. The genetic correlation between corneal and refractive astigmatism was 0.85 (standard error = 0.068, P = 1.37 × 10−35). Here, we have undertaken the largest genome-wide association studies for corneal and refractive astigmatism to date and identified four novel loci for corneal astigmatism, two of which were also novel loci for refractive astigmatism. These loci have previously demonstrated association with axial length (ZC3H11B), myopia (NPLOC4), spherical equivalent refractive error (LINC00340) and eye colour (HERC2). The shared role of these novel candidate genes for astigmatism lends further support to the shared genetic susceptibility of myopia and astigmatism

Probing the optical readout characteristics of Fabry-Perot ultrasound sensors through realistic modelling

The Fabry-Perot interferometer (FPI) is widely used in photoacoustic imaging (PAI) as an ultrasound (US) sensor due to its high sensitivity to weak US waves. Such high sensitivity is important as it allows for increasing the depth in tissue at which PAI can access, thus strongly influencing its clinical applicability. FPI sensitivity is impacted by many factors including the FPI mirror reflectivity, focussed beam spot size, FPI cavity thickness and aberrations introduced by the optical readout system. Improving FPI sensitivity requires a mathematical model of its optical response which takes all of these factors into account. Previous attempts to construct such a model have been critically limited by unrealistic assumptions. In this work we have developed a general model of FPI optical readout which based upon electromagnetic theory. By making very few assumptions, the model is able to replicate experimental results and allows insight to be gained into the operating principles of the sensor

Increasing the Q-factor of Fabry-Perot etalons using focused Bessel beam illumination

Sensing and filtering applications often require Fabry-Perot (FP) etalons with an Interferometer Transfer Function (ITF) having high visibility, narrow Full Width at Half Maximum (FWHM), and high sensitivity. For the ITF to have these characteristics, the illumination beam must be matched to the modes of the FP cavity. This is challenging when a small illumination element size is needed, as typical focused beams are not matched to the FP cavity modes. Bessel beams are a potential alternative as their structure resembles the FP cavity modes while possessing a focused core. To study the feasibility of using Bessel beam illumination, in this Letter, ITFs of an FP etalon were measured using Bessel and Gaussian illumination beams. A Bessel beam with core size of 28 µm provided an ITF with visibility 3.0 times higher, a FWHM 0.3 times narrower, and a sensitivity 2.2 times higher than a Gaussian beam with waist 32 µm. The results show that Bessel beam illumination can provide ITFs similar to that of collimated beam illumination while also having with a focused core.</p