3D‐liquid chromatography as a complex mixture characterization tool for knowledge‐based downstream process development

Knowledge‐based development of chromatographic separation processes requires efficient techniques to determine the physicochemical properties of the product and the impurities to be removed. These characterization techniques are usually divided into approaches that determine molecular properties, such as charge, hydrophobicity and size, or molecular interactions with auxiliary materials, commonly in the form of adsorption isotherms. In this study we demonstrate the application of a three‐dimensional liquid chromatography approach to a clarified cell homogenate containing a therapeutic enzyme. Each separation dimension determines a molecular property relevant to the chromatographic behavior of each component. Matching of the peaks across the different separation dimensions and against a high‐resolution reference chromatogram allows to assign the determined parameters to pseudo‐components, allowing to determine the most promising technique for the removal of each impurity. More detailed process design using mechanistic models requires isotherm parameters. For this purpose, the second dimension consists of multiple linear gradient separations on columns in a high‐throughput screening compatible format, that allow regression of isotherm parameters with an average standard error of 8%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1283–1291, 2016     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: II. RELATIONSHIP BETWEEN GROWTH RATE, CARBOHYDRATE CONCENTRATION AND 14C-PARTITIONING WITHIN TUBERS

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. The growth rates of individualtubers were closely reflected by their ¹⁴C-content 20 h after¹⁴CO₂ had been applied to the aerial parts of the shoot for4 h. The ¹⁴C-content of the tuber (sink strength) was significantlycorrelated to the ¹⁴C-concentration of the tuber tissue (¹⁴Cg^–1 fr. wt.=sink activity). The sink activity, which differedbetween individual tubers by up to a factor of 10, was alsoclosely related to the conversion rates of ¹⁴C into the starchand the remainder as well as to the ¹⁴C-content in the ethanolsoluble fraction. This indicates the simultaneous use of photosynthatefor growth and storage in the growing tubers. No preferenceof photosynthate utilization for either of these processes couldbe detected in relation to the sink activity of the tubers.Tubers with high sink activity imported ¹⁴C-labelled photosynthateat higher rates although their tissue contained higher concentrationsof reducing sugars and sucrose than the tissue of tubers withlow sink activity. Despite the close relationship between sinkactivity and the rate of starch synthesis (¹⁴C-conversion intostarch), no significant correlation was found between sink activityand the actual starch concentration of the tissue. The applicationof zeatin riboside directly onto individual tubers increasedtheir growth rates in comparison to non-treated tubers of thesame plant. The results indicate the importance of both growthand storage processes for the regulation of sink activity inyoung potato tubers. Key words: Potato tuber, ¹⁴C-photosynthate partitioning, zeatin riboside application     

ZnCl2 catalyzed new coumarinyl-chalcones as cytotoxic agents

A new series of coumarin-yl-chalcone derivatives (3a-m) had been designed and synthesized through different reactions such as aromatic addition, cyclization and Claisen-Schmidt reactions in good yields (54–78%). 5-acetyl-4-(2-hydroxyphenyl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (1) has been synthesized by multi-component one pot reaction of salicylaldehyde, methyl acetoacetate and urea, which was further reacted with malonic acid employing ZnCl₂ catalyst to yield 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (2). The title compounds (3a-m) were synthesised by reacting 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H)-one (2) with different aromatic aldehydes in the presence of potassium hydroxide. In silico studies, a preliminary screening method for predicting the anti-cancer activity was performed for the synthesized compounds (3a-m) against Src, Alb tyrosine kinase and homology model protein (PDB ID: 4csv). The derivatives 3h and 3m showed moderate binding energies. The in vitro cytotoxic activity was evaluated for the compounds 3h and 3m by using human cancer cell-line morphology and MTT assay against three human cell-lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast). The results indicate that the derivatives 3h and 3m display significant anti-cancer activity, however it was found to be less cytotoxic when compared to the standard used i.e. Imatinib.     

A molecular phylogeny of the cosmopolitan hyperdiverse genus Hydraena Kugelann (Coleoptera,Hydraenidae)

With almost 900 described species, Hydraena Kugelann (Hydraenidae) is one of the largest genera among Coleoptera. The subgeneric classification of Hydraena has been controversial, with 11 subgeneric names having so far been attributed to it. Some of these, Haenydra Rey and Spanglerina Perkins, have been treated as valid genera, as subgenera or as species groups. The most recent complete treatment of the genus, based on a cladistic analysis of morphological characters, recognized two major lineages, and only these were classified as subgenera: Hydraenopsis (mainly Gondwanan distribution), and Hydraena s.str. (mainly Laurasian). Here, we reconstruct the phylogeny of Hydraena using 212 species plus several outgroups and approximately 4 kb of sequence data from two nuclear (SSU and LSU) and four mitochondrial genes (cox1, rrnL, trnL and nad1). Data were aligned with two different strategies of multiple alignment (implemented in mafft and prank ), and the phylogenies reconstructed using maximum likelihood and Bayesian methods. We estimated approximate ages of the main nodes using a relaxed molecular clock with Bayesian methods, and an a priori evolutionary rate of 0.01 substitutions/site/million years (Ma) plus a calibration point based on a biogeographical split. We found strong support for the monophyly of Hydraena and many of the clades recognized with morphological data. The following clades are considered as subgenera: Phothydraena Kuwert, Spanglerina Perkins, Holcohydraena Kuwert, Hydraenopsis Janssens and Hydraena s.str. The placement of three species groups, two Neotropical (H. multispina group, H. paeminosa group) and one South African/Madagascan (H. monikae group), is uncertain, and they are considered incertae sedis within Hydraena. The origin of the genus was estimated to be in the Lower Eocene, with many species complexes diversifying in the Pleistocene. Dispersal events seem to have played a key role in order to determine the current distribution of the species groups in the southern hemisphere (mainly in Hydraenopsis).     

CO2 responsiveness of plants: a possible link to phloem loading

Of the many responses of plants to elevated CO₂, accumulation of total non-structural carbohydrates (TNC in % dry weight) in leaves is one of the most consistent. Insufficient sink activity or transport capacity may explain this obvious disparity between CO₂ assimilation and carbohydrate dissipation and structural investment. If transport capacity contributes to the problem, phloem loading may be the crucial step. It has been hypothesized that symplastic phloem loading is less efficient than apoplastic phloem loading, and hence plant species using the symplastic pathway and growing under high light and good water supply should accumulate more TNC at any given CO₂ level, but particularly under elevated CO₂. We tested this hypothesis by carrying out CO₂ enrichment experiments with 28 plant species known to belong to groups of contrasting phloem-loading type. Under current ambient CO₂ symplastic loaders were found to accumulate 36% TNC compared with only 19% in apoplastic loaders (P=0.0016). CO₂ enrichment to 600 μmol mol^?1 increased TNC in both groups by the same absolute amount, bringing the mean TNC level to 41% in symplastic loaders (compared to 25% in apoplastic loaders), which may be close to TNC saturation (coupled with chlornplast malfunction). Eight tree species, ranked as symplastic loaders by their minor vein companion cell configuration, showed TNC responses more similar to those of apoplastic herbaceous loaders. Similar results are obtained when TNC is expressed on a unit leaf area basis, since mean specific leaf areas of groups were not significantly different. We conclude that phloem loading has a surprisingly strong effect on leaf tissue composition, and thus may translate into alterations of food webs and ecosystem functioning, particularly under high CO₂.     

Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein

The Rhs family comprises a set of composite elements found in the chromosomes of many natural Escherichia coli strains. Five Rhs elements occur in strain K-12. The most prominent Rhs component is a giant core open reading frame (core ORF) whose features are suggestive of a cell surface ligand-binding protein. This hypothetical protein contains a peptide motff, xxGxxxRYxYDxxGRL(I or T)xxxx, that is repeated 28 times. A similar repeated motif is found in a Bacillus subtilis wall-associated protein. The Rhs core ORFs consist of two distinct parts: a large N-terminal core that is conserved in all Rhs elements, and a smaller C-terminus that Is highly variable. Distinctive G+C contents of Rhs components indicate that the elements have a recent origin outside the E. coli species, and that they are composites assembled from segments with very different evolutionary histories. The Rhs cores fail into three sub-families that are mutually more than 20% divergent Downstream of the core ORF is a second, much shorter ORF. Like the adjacent core extension, these are highly variable. In most examples, the hypothetical product of this ORF has a candidate signal sequence for transport across the cytoplasmic membrane. Another Rhs component, the 1.3 kb H-rpt, has features typical of insertion sequences. Structures homologous to H-rpt have been detected in other bacterial genera, such as Vibrio and Salmonella, where they are associated with loci that determine O-antigen variation.     

Defunct brain stem cardiovascular regulation underlies cardiovascular collapse associated with methamphetamine intoxication

Background  

Intoxication from the psychostimulant methamphetamine (METH) because of cardiovascular collapse is a common cause of death within the abuse population. For obvious reasons, the heart has been taken as the primary target for this METH-induced toxicity. The demonstration that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse induced by the pesticide mevinphos implicates another potential underlying mechanism. The present study evaluated the hypothesis that METH effects acute cardiovascular depression by dampening the functional integrity of baroreflex via an action on brain stem nuclei that are associated with this homeostatic mechanism.     

Carbohydrate cytochemistry on hypodermic lymphatic endothelium of the green lizard,Lacerta hispanica

Summary A cytochemical study on the endothelium of the hypodermic lymphatic capillaries of the green lizard,Lacerta hispanica, has been carried out. The dialysed iron method produced a homogeneous precipitate on the surface of the endothelial cells and on the inside of the endocytic vesicles. The periodic acid-thiocarbohydrazide-silver proteinate, low pH phosphotungstic acid and high iron diamine techniques gave negative results. The carbohydrates in the capillaries thus seem to be glycosaminoglycans with carboxyl groups. The possible role of these glycosaminoglycans in the formation of the endocytic vesicles is discussed.     

Culture media for mouse oocyte maturation affect subsequent embryonic development

These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten's medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten's medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%).(ABSTRACT TRUNCATED AT 250 WORDS)