Structural Insights of Non-canonical U*U Pair and Hoogsteen Interaction Probed with Se Atom

Unlike DNA, in addition to the 20 -OH group, uracil nucleobase and its modifications play essential roles in structure and function diversities of non- coding RNAs. Non-canonical U.U base pair is ubiquitous in non-coding RNAs, which are highly diversified. However, it is not completely clear how uracil plays the diversifing roles. To investigate and compare the uracil in U-A and U.U base pairs, we have decided to probe them with a selenium atom by synthesizing the novel 4-Se-uridine (SeU) phosphoramidite and Se-nucleobase-modified RNAs (SeU-RNAs), where the exo-4-oxygen of uracil is replaced by selenium. Our crystal structure studies of U-A and U.U pairs reveal that the native and Se-derivatized structures are virtually identical, and both U-A and U.U pairs can accommodate large Se atoms. Our thermostability and crystal structure studies indicate that the weakened H-bonding in U-A pair may be compensated by the base stacking, and that the stacking of the trans- Hoogsteen U.U pairs may stabilize RNA duplex and its junction. Our result confirms that the hydrogen bond (O4.. .H-C5) of the Hoogsteen pair is weak. Using the Se atom probe, our Se- functionalization studies reveal more insights into the U.U interaction and U-participation in structure and function diversification of nucleic acids

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

<p>Abstract</p> <p>Background</p> <p>Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.</p> <p>Results</p> <p>We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.</p> <p>Conclusion</p> <p>We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.</p

Design of Mixing Station for Alternative Gaseous Fuels

Úkolem diplomové práce je navrhnout potrubní rozvod plynů do směšovací stanice a následně i navrhnout samotnou směšovací stanici. V textové části je nejprve vytvořena rešerše týkající se využívání technických a topných plynů v praxi. Na tuto část postupně navazují specifikace plynů, které se budou dopravovat do směšovací stanice. Ve výpočtové části se práce prostřednictvím výpočtových vztahů zabývá složením směsného plynu, fyzikálními vlastnostmi jednotlivých plynů i samotného plynu směsného, přičemž jsou vypočteny a navrženy potrubní tratě jednotlivých plynů směřujících do směšovací stanice. Konstrukční část práce se zabývá návrhem směšovací stanice a směšovací komory, ve které se jednotlivé plyny mísí. Grafická část práce obsahuje schémata jednotlivých potrubních tratí a směšovací stanice, 3D model směšovací komory a výkres směšovací komory.The task of the diploma thesis is to design the pipeline distribution of gases to the mixing station and then design the mixing station itself. In the text part is first created a search regarding the use of technical and heating gases in practice. This part is followed by the gas specifications that will be transported to the mixing station. In the computational part, the work with the help of calculation relations deals with the composition of the mixed gas, the physical properties of the individual gases and the mixing gas itself, and the pipelines of the individual gases leading to the mixing station are calculated and designed. The construction part deals with the design of the mixing station and the mixing chamber, in which the individual gases are mixed. The graphical part of the thesis contains diagrams of individual pipeline lines and mixing stations, a 3D model of the mixing chamber and a drawing of the mixing chamber.361 - Katedra energetikyvýborn

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library

Hitting the target: fragment screening with acoustic in situ co-crystallization of proteins plus fragment libraries on pin-mounted data-collection micromeshes

A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin

The Gravity Collective: A Search for the Electromagnetic Counterpart to the Neutron Star-Black Hole Merger GW190814

We present optical follow-up imaging obtained with the Katzman Automatic Imaging Telescope, Las Cumbres Observatory Global Telescope Network, Nickel Telescope, Swope Telescope, and Thacher Telescope of the LIGO/Virgo gravitational wave (GW) signal from the neutron star-black hole (NSBH) merger GW190814. We searched the GW190814 localization region (19 deg$^{2}$ for the 90th percentile best localization), covering a total of 51 deg$^{2}$ and 94.6% of the two-dimensional localization region. Analyzing the properties of 189 transients that we consider as candidate counterparts to the NSBH merger, including their localizations, discovery times from merger, optical spectra, likely host-galaxy redshifts, and photometric evolution, we conclude that none of these objects are likely to be associated with GW190814. Based on this finding, we consider the likely optical properties of an electromagnetic counterpart to GW190814, including possible kilonovae and short gamma-ray burst afterglows. Using the joint limits from our follow-up imaging, we conclude that a counterpart with an $r$-band decline rate of 0.68 mag day$^{-1}$, similar to the kilonova AT 2017gfo, could peak at an absolute magnitude of at most $-17.8$ mag (50% confidence). Our data are not constraining for ''red'' kilonovae and rule out ''blue'' kilonovae with $M>0.5 M_{\odot}$ (30% confidence). We strongly rule out all known types of short gamma-ray burst afterglows with viewing angles $<$17$^{\circ}$ assuming an initial jet opening angle of $\sim$$5.2^{\circ}$ and explosion energies and circumburst densities similar to afterglows explored in the literature. Finally, we explore the possibility that GW190814 merged in the disk of an active galactic nucleus, of which we find four in the localization region, but we do not find any candidate counterparts among these sources.Comment: 86 pages, 9 figure

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia