Fluorometric Beam Profiling of UV MALDI Lasers

The photon distribution (beam profile) of the laser as projected onto the sample is an important variable in matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Measurement of the beam profile is, therefore, an important factor within MALDI-MS. In this study a simple, low-cost fluorometric laser beam profiling technique is presented and applied in conjunction with MALDI-MS experiments. A comparison of the beam profile information afforded by a commercial system and the fluorometric method is carried out to determine the variation of beam profile for an Nd:YVO₄ laser operated between 1 and 25 kHz. The beam profile information can be used, in conjunction with corresponding ion yields, to inform MALDI-MS experiments. The fluorometric beam profiling technique is used to obtain information about the beam dimensions as incident upon the MALDI-MS sample plate in-source. These values are compared with equivalent information obtained from ablation of thin film α-cyano-4-hydroxycinnamic acid (CHCA). In this study, area estimation by ablation provided a value 1.6 times smaller than that obtained by the fluorometric method, demonstrating the need for caution when measuring beam profile and, therefore, fluence, in MALDI-MS.

Computer‐aided Discovery of Potential Inhibitors for Transthyretin‐related Amyloidosis

Transthyretin (TTR) is a homotetrameric plasma protein associated with human amyloid diseases. Although Tafamidis has been recently approved for the treatment of TTR familial amyloid polyneuropathy (FAP), there is still a need for more effective drugs in the treatment of TTR amyloidosis diseases. In this study, a computer‐aided approach combining molecular docking, virtual screening and molecular dynamics (MD) simulations was employed to identify potent TTR amyloidosis inhibitors from National Cancer Institute (NCI), Maybridge and Asdi databases. A receptor‐specific scoring function was also developed using comparative binding energy (COMBINE) method to accurately predict the inhibitory activities for the selected compounds during virtual screening. The developed receptor‐specific scoring function demonstrated good predictive ability by yielding strong correlation coefficients between experimental activities and estimated activities for 32 training set and 9 test set compounds, respectively. Moreover, it was successfully applied to rank the candidate compounds from structure‐based virtual screening. Finally, three compounds (NSC220163, MFCD00276817 and SPB06319) were identified as potential leads, which exhibited higher predicted inhibitory activities and higher binding affinities in comparison to the Tafamidis. Our results further suggest that halogen bonding interaction plays a crucial role in stabilizing the TTR‐inhibitor complex. These results indicate that our computational approach could effectively discover more potent TTR amyloidosis inhibitors, which can be further validate by in vitro and in vivo biological tests.     

Discovery and SAR Studies of Potent Modulators of BMI-1 Expression

In our efforts to identify molecules that selectively reduce the expression of BMI1, a stem cell gene, we discovered and characterized the first-in-class series of small molecules that modulate the expression of BMI1 protein in cancer cells. Structure–activity and structure–property relationships associated with this series were investigated through medicinal chemistry efforts. These studies revealed important structural features required for achieving anti-tumor activity and acceptable pharmacokinetic properties within this series. The 4-CF₃−Ph at the left-side of the molecule, a proper placement of the N-atom on the six-membered heterocycle in the middle, combined with a properly substituted C(2)-methyl benzimidazole on the right-hand side were required to achieve potency, microsomal stability, and exposure upon oral dosing. A compound (PTC-02) with acceptable pharmacological properties and efficacious in vivo in several tumor animal models was identified.     

A microfluidic "baby machine" for cell synchronization

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.     

Pharmacophore Mode lingand Virtual Screening to Design the Potential Influenza Virus Endonuclease Inhibitors

Influenza virus endonuclease is an attractive target for antiviral therapy in the treatment of influenza infection. The purpos e of this study is to design a novel antiviral agent with improved biological activities against the influenza virus endonuclease. In this study, chemical feature‐based 3D pharmacophore models were developed from 41 known influenza virus endonuclease inhibitors. The best quantitative pharmacohore model (Hypo 1), which consists of two hydrogen‐bond acceptors and two hydrophobic features, yields the highest correlation coefficient (R = 0.886). Hypo 1 was further validated by the cross validation method and the test set compounds. The application of this model for predicting the activities of 11 known influenza virus endonuclease inhibitors in the test set shows great success. The correlation coefficient of 0.942 and a cross validation of 95;% confidence level prove that this model is reliable in identifying structurally diverse compounds for influenza virus endonuclease inhibition. The most active compound (compound 1) from the training set was docked into the active site of the influenza virus endonuclease as an additional verification that the pharmacophore model is accurate. The docked conformation showed important hydrogen bond interactions between the compound and two amino acids, Lys 134 and Lys 137. After validation, this model was used to screen the NCI chemical database to identify new influenza virus endonuclease inhibitors. Our study shows that the to pranking compound out of the 10 newly identified compounds using fit value ranking has an estimated activity of 0.049 μM. These newly identified lead compounds can be further experimentally validated using in vitro techniques.     

Linear systems on tropical curves

A tropical curve Γ is a metric graph with possibly unbounded edges, and tropical rational functions are continuous piecewise linear functions with integer slopes. We define the complete linear system |D| of a divisor D on a tropical curve Γ analogously to the classical counterpart. We investigate the structure of |D| as a cell complex and show that linear systems are quotients of tropical modules, finitely generated by vertices of the cell complex. Using a finite set of generators, |D| defines a map from Γ to a tropical projective space, and the image can be modified to a tropical curve of degree equal to deg(D) when |D| is base point free. The tropical convex hull of the image realizes the linear system |D| as a polyhedral complex. We show that curves for which the canonical divisor is not very ample are hyperelliptic. We also show that the Picard group of a ${\mathbb{Q}}$ -tropical curve is a direct limit of critical groups of finite graphs converging to the curve.     

Intramolecular Pd-catalyzed carboetherification and carboamination. Influence of catalyst structure on reaction mechanism and product stereochemistry

The intramolecular Pd-catalyzed carboetherification of alkenes affords 2-indan-1-yltetrahydrofuran products in moderate to good yield with good to excellent levels of diastereoselectivity. The stereochemical outcome of these reactions is dependent on the structure of the Pd catalyst. Use of PCy(3) or P[(4-MeO)C(6)H(4)](3) as the ligand for Pd leads to syn-addition of the arene and the oxygen atom across the double bond, whereas use of (+/-)-BINAP or DPP-benzene affords products that result from anti-addition. The catalyst-induced change in stereochemistry is likely due to a change in reaction mechanism. Evidence is presented that suggests the syn-addition products derive from an unprecedented transannular alkene insertion of an 11-membered Pd(Ar)(OR) complex. In contrast, the anti-addition products appear to arise from Wacker-type anti-oxypalladation. Studies on analogous Pd-catalyzed intramolecular carboamination reactions, which afford 2-indan-1-ylpyrrolidines that result from syn-addition, are also described.     

A Pro-Fluorescent Ubiquitin-Based Probe to Monitor Cysteine-Based E3 Ligase Activity

Protein post-translational modification with ubiquitin (Ub) is a versatile signal regulating almost all aspects of cell biology, and an increasing range of diseases is associated with impaired Ub modification. In this light, the Ub system offers an attractive, yet underexplored route to the development of novel targeted treatments. A promising strategy for small molecule intervention is posed by the final components of the enzymatic ubiquitination cascade, E3 ligases, as they determine the specificity of the protein ubiquitination pathway. Here, we present UbSRhodol, an autoimmolative Ub-based probe, which upon E3 processing liberates the pro-fluorescent dye, amenable to profile the E3 transthiolation activity for recombinant and in cell-extract E3 ligases. UbSRhodol enabled detection of changes in transthiolation efficacy evoked by enzyme key point mutations or conformational changes, and offers an excellent assay reagent amenable to a high-throughput screening setup allowing the identification of small molecules modulating E3 activity.     

Ultra-high-performance supercritical fluid chromatography-mass spectrometry for the analysis of organic contaminants in sediments

A nontarget screening method was developed based on D-optimal designs for ultra-high performance supercritical fluid chromatography with positive and negative electrospray ionization mode mass spectrometry. A mixture of organic contaminants such as pesticides, steroids, surfactants, phenolic and fatty acids, and polycyclic aromatic hydrocarbon derivatives, was used for the optimization. An aprotic mixture of dichloromethane and acetone [3:1] performed overall best as the injection solvent. The highest peak capacities (n) were accomplished at the shallowest gradient (1%B/min), ammonium formate (n = 378 in negative ionization mode), or ammonium acetate (n = 327 in positive ionization mode) in methanol as the modifier. Capillary voltage, make-up solvent flow rate, water, and additive concentration were the most significant factors for improving peak intensity: higher peak intensities were obtained at lower additive concentrations (5mM ammonium formate), and with 5% water in positive ionization mode. Conversely, water had detrimental effects in negative ionization mode. The optimized method was used to quantify organic contaminants in 17 freshwater sediment samples from Copenhagen, Denmark. Out of 50 monitored contaminants, 35 were detected in at least one sample. Further, the method has a potential for target and nontarget screening analysis of organic contaminants in solid matrices.