Application of cycling gradient capillary electrophoresis to detection of APC, K-ras, and DCC point mutations in patients with sporadic colorectal tumors

A previously introduced technique of cycling gradient capillary electrophoresis (CGCE) was applied to monitoring of molecular changes during adenoma-carcinoma transition in progression of sporadic colorectal cancer. The purpose of this work was optimization of separation parameters for selected mutation regions in tumor suppressor genes involved in the early stages of colorectal carcinogenesis, followed by scanning for these mutations in clinical tissue samples from patients with adenomatous polyps and early carcinomas. A total of 47 colorectal tumors in various stages of progression were examined. Main emphasis was given to evaluation of mutation detection sensitivity and specificity required for effective early disease detection. A total of 7 different somatic mutations was identified among 32 K-ras mutant samples, 1 inherited mutation and 5 somatic mutations were identified among 15 adenomatous polyposis coli (APC) mutated samples. None of the two previously reported "deleted in colorectal carcinomas" (DCC) mutations was found in any of the clinical samples. In addition to simple optimization of running conditions, CGCE has demonstrated sensitivity and selectivity allowing detecting small mutant fractions as well as combination of multiple mutants within a single target sequence.     

Rheological properties of doughs with buckwheat and quinoa additives

Rheological properties of doughs prepared from wheat flour with buckwheat and quinoa flour addition (2.5 mass %, 5.0 mass %, 7.5 mass %, and 10 mass %) were investigated using a farinograph and compared with those of standard dough (without addition of pseudocereals). The following characteristics were determined: water absorption capacity, water consumption, dough growth time, level of dough softening, dough stability, mechanical resistance, and dough elasticity. Dough stability showed a linear decrease with the increasing content of pseudocereals. Doughs containing quinoa flour were more stable than those with buckwheat flour addition. Dough growth time was reduced with increasing amounts of buckwheat flour but it was not affected in the case of quinoa flour addition. From the comparison of the studied characteristics it can be concluded that an addition of lower amounts of quinoa (up to 5.0 mass %) to wheat flour will not significantly impair rheological properties of the dough but provides for enhanced nutritional value of the prepared bakery products.     

Saccharide-functionalized alkanethiols for fouling-resistant self-assembled monolayers: synthesis, monolayer properties, and antifouling behavior

We describe the synthesis of a series of mono-, di-, and trisaccharide-functionalized alkanethiols as well as the formation of fouling-resistant self-assembled monolayers (SAMs) from these. The SAMs were characterized using ellipsometry, wetting measurements, and infrared reflection-absorption spectroscopy (IRAS). We show that the structure of the carbohydrate moiety affects the packing density and that this also alters the alkane chain organization. Upon increasing the size of the sugar moieties (from mono- to di- and trisaccharides), the structural qualities of the monolayers deteriorated with increasing disorder, and for the trisaccharide, slow reorganization dynamics in response to changes in the environmental polarity were observed. The antifouling properties of these SAMs were investigated through protein adsorption experiments from buffer solutions as well as settlement (attachment) tests using two common marine fouling species, zoospores of the green macroalga Ulva linza and cypris larvae of the barnacle Balanus amphitrite. The SAMs showed overall good resistance to fouling by both the proteins and the tested marine organisms. To improve the packing density of the SAMs with bulky headgroups, we employed mixed SAMs where the saccharide-thiols are diluted with a filler molecule having a small 2-hydroxyethyl headgroup. This method also provides a means by which the steric availability of sugar moieties can be varied, which is of interest for specific interaction studies with surface-bound sugars. The results of the surface dilution study and the low nonspecific adsorption onto the SAMs both indicate the feasibility of this approach.     

Noncanonical hydrogen bonding in nucleic acids. Benchmark evaluation of key base-phosphate interactions in folded RNA molecules using quantum-chemical calculations and molecular dynamics simulations

RNA molecules are stabilized by a wide range of noncanonical interactions that are not present in DNA. Among them, the recently classified base-phosphate (BPh) interactions belong to the most important ones. Twelve percent of nucleotides in the ribosomal crystal structures are involved in BPh interactions. BPh interactions are highly conserved and provide major constraints on RNA sequence evolution. Here we provide assessment of the energetics of BPh interactions using MP2 computations extrapolated to the complete basis set of atomic orbitals and corrected for higher-order electron correlation effects. The reference computations are compared with DFT-D and DFT-D3 approaches, the SAPT method, and the molecular mechanics force field. The computations, besides providing the basic benchmark for the BPh interactions, allow some refinements of the original classification, including identification of some potential doubly bonded BPh patterns. The reference computations are followed by analysis of some larger RNA fragments that consider the context of the BPh interactions. The computations demonstrate the complexity of interaction patterns utilizing the BPh interactions in real RNA structures. The BPh interactions are often involved in intricate interaction networks. We studied BPh interactions of protonated adenine that can contribute to catalysis of hairpin ribozyme, the key BPh interaction in the S-turn motif of the sarcin-ricin loop, which may predetermine the S-turn topology and complex BPh patterns from the glmS riboswitch. Finally, the structural stability of BPh interactions in explicit solvent molecular dynamics simulations is assessed. The simulations well preserve key BPh interactions and allow dissection of structurally/functionally important water-meditated BPh bridges, which could not be considered in earlier bioinformatics classification of BPh interactions.     

A Helquat-like Compound as a Potent Inhibitor of Flaviviral and Coronaviral Polymerases

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.     

Nanostructured films of amphiphilic fluorinated block copolymers for fouling release application

New amphiphilic block copolymers S nSz m consisting of blocks with varied degrees of polymerization, n and m, of polystyrene, S, and polystyrene carrying an amphiphilic polyoxyethylene-polytetrafluoroethylene chain side-group, Sz, were prepared by controlled atom transfer radical polymerization (ATRP). The block copolymers, either alone or in a blend with commercial SEBS (10 wt% SEBS), were spin-coated in thinner films (200-400 nm) on glass and spray-coated in thicker films ( approximately 500 nm) on a SEBS underlayer (150-200 microm). Angle-resolved X-ray photoelectron spectroscopy (XPS) measurements proved that at any photoemission angle, varphi, the atomic ratio F/C was larger than that expected from the known stoichiometry. Consistent with the enrichment of the outer film surface (3-10 nm) in F content, the measured contact angles, theta, with water (theta w > or = 107 degrees ) and n-hexadecane (theta h > or = 64 degrees ) pointed to the simultaneous hydrophobic and lipophobic character of the films. The film surface tension gamma S calculated from the theta values was in the range 13-15 mN/m. However, the XPS measurements on the "wet" films after immersion in water demonstrated that the film surface underwent reconstruction owing to its amphiphilic nature, thereby giving rise to a more chemically heterogeneous structure. The atomic force microscopy (AFM) images (tapping mode/AC mode) revealed well-defined morphological features of the nanostructured films. Depending on the chemical composition of the block copolymers, spherical (ca. 20 nm diameter) and lying cylindrical (24-29 nm periodicity) nanodomains of the S discrete phase were segregated from the Sz continuous matrix (root-mean-square, rms, roughness approximately 1 nm). After immersion in water, the underwater AFM patterns evidenced a transformation to a mixed surface structure, in which the nanoscale heterogeneity and topography (rms = 1-6 nm) were increased. The coatings were subjected to laboratory bioassays to explore their intrinsic ability to resist the settlement and reduce the adhesion strength of two marine algae, viz., the macroalga (seaweed) Ulva linza and the unicellular diatom Navicula perminuta. The amphiphilic nature of the copolymer coatings resulted in distinctly different performances against these two organisms. Ulva adhered less strongly to the coatings richer in the amphiphilic polystyrene component, percentage removal being maximal at intermediate weight contents. In contrast, Navicula cells adhered less strongly to coatings with a lower weight percentage of the amphiphilic side chains. The results are discussed in terms of the changes in surface structure caused by immersion and the effects such changes may have on the adhesion of the test organisms.     

Rapid and simple method for determination of hexabromocyclododecanes and other LC–MS–MS-amenable brominated flame retardants in fish

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C₁₈ and primary–secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS–MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115 % with relative standard deviations (RSDs) <13 % for all analytes. Limits of quantification (LOQs) were in the range 0.1–1 μg kg^?1 wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8–2.5 μg kg^?1 wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6–43.5 and 2.3–7.5 μg kg^?1 wet weight, respectively.