EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker‐free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone‐MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113‐7D) and a diploid industrial strain (Ethanol Red) for production of 3‐hydroxypropionic acid, where we tested three different acetyl‐CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone‐MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.     

Characterization of three newly established rat sarcoma cell clones

Establishment of new animal models using selected cell lines with different behaviour is very important for cancer investigations. In this study, we describe three morphologically distinct rat sarcoma clones??C4, C7 and D6??isolated from the R5-28 cell line. Cells of all clones expressed vimentin, fibronectin, laminin, collagen IV and matrix metalloproteinases 2 and 9. However, desmin, cytokeratins 8 and 18, ZO-1 and desmoplakins I and II were not detected. Significant proliferative capacity was documented by proliferating cell nuclear antigen expression and BrdU positivity. Karyotype of the C4, C7 and D6 cells greatly differed from diploid chromosome number of normal rat somatic cells. High expression of three cytokines??monocyte chemoattractant protein 1, tissue inhibitor of metalloproteinases 1 and vascular endothelial growth factor??was observed in all three clones. However, they varied in concentration of chemokines associated with neutrophil migration and activation??cytokine induced neutrophil chemoattractant 2 and lipopolysaccharide induced CXC chemokine. The C4 clone showed spontaneous tumour regression in vivo that was associated with significant changes in lymphocyte subpopulations.     

Structure and magnetic properties of a trinuclear nickel(II) complex with benzenetricarboxylate bridge

Novel trinuclear Ni(II) complex [Ni₃(pmdien)₃(btc)(H₂O)₃](ClO₄)₃ · 4H₂O, 1 where pmdien = N,N,N′,N′,N″-pentamethyldiethylenetriamine, H₃btc = 1,3,5-benzenetricarboxylic (trimesic) acid, has been prepared and structurally characterized. Three nickel atoms are bridged by btc trianion and their coordination sphere is completed by three N atoms of pmdien and O atom of the water molecule. The three nickel(II) magnetic centers are equivalent and their coordination spheres are completed to deformed octahedrons. Magnetic susceptibility was measured over the temperature range 1.8–300 K and zJ′ = ?0.19 cm^?1, D = 3.79 cm^?1, g = 2.18 parameters were calculated.     

Clinical and Histopathological Characterization of Cutaneous Melanomas in the Melanoblastoma‐Bearing Libechov Minipig Model

Spontaneous animal tumors appear to be highly suitable models to study human oncology and cancer therapy. The aim of this study was to characterize the clinical and histological features of hereditary melanocytic lesions found in the French herd of melanoblastoma‐bearing Libechov minipigs (MeLiM) and their Duroc crossbreeds. Clinically, we discriminated between three types of melanocytic skin lesions, which offer a lesion continuum from lentigo to metastatic melanomas. More than 70% of these lesions appear on piglets before they are 3 months old and preferentially on homogeneous black coat piglets. The incidence of melanoma reaches 50% in MeLiM. Most of the highly invasive melanomas regressed spontaneously in the first year of the piglet's life and the regression was followed by hair, skin and iris depigmentation. A histopathological study was conducted according to the human melanoma classification. Except for lentigo maligna, we observed the three main types of human melanoma in swine [superficial spreading melanoma (SSM), nodular or unclassified melanoma] with an excess of SSM (59–67%). The histological events leading to total spontaneous regression are chronologically described. The genetic predisposition, the high incidence of melanoma, the clinical and histopathological features similar to the human disease and the high rate of spontaneous regression offer an opportunity to use this model for studying genetic events controlling melanoma development and regression and the biological mechanisms involved in oncogenesis and anti‐cancerous self‐defense.     

Clinical and histopathological characterization of cutaneous melanomas in the melanoblastoma-bearing Libechov minipig model

Spontaneous animal tumors appear to be highly suitable models to study human oncology and cancer therapy. The aim of this study was to characterize the clinical and histological features of hereditary melanocytic lesions found in the French herd of melanoblastoma-bearing Libechov minipigs (MeLiM) and their Duroc crossbreeds. Clinically, we discriminated between three types of melanocytic skin lesions, which offer a lesion continuum from lentigo to metastatic melanomas. More than 70% of these lesions appear on piglets before they are 3 months old and preferentially on homogeneous black coat piglets. The incidence of melanoma reaches 50% in MeLiM. Most of the highly invasive melanomas regressed spontaneously in the first year of the piglet's life and the regression was followed by hair, skin and iris depigmentation. A histopathological study was conducted according to the human melanoma classification. Except for lentigo maligna, we observed the three main types of human melanoma in swine [superficial spreading melanoma (SSM), nodular or unclassified melanoma] with an excess of SSM (59-67%). The histological events leading to total spontaneous regression are chronologically described. The genetic predisposition, the high incidence of melanoma, the clinical and histopathological features similar to the human disease and the high rate of spontaneous regression offer an opportunity to use this model for studying genetic events controlling melanoma development and regression and the biological mechanisms involved in oncogenesis and anti-cancerous self-defense.     

Role of distinct dimorphic transitions in territory colonizing and formation of yeast colony architecture

Microbial populations in nature often form organized multicellular structures (biofilms, colonies) occupying different surfaces including host tissues and medical devices. How yeast cells within such populations cooperate and how their dimorphic switch to filamentous growth is regulated are therefore important questions. Studying population development, we discovered that Saccharomyces cerevisiae microcolonies early after their origination from one cell successfully occupy the territory via dimorphic transition, which is induced by ammonia and other volatile amines independently on cell ploidy and nutrients. It results in oriented pseudohyphal cell expansion in the direction of ammonia source, which consequently leads to unification of adjacent microcolonies to one more numerous entity. The further population development is accompanied by another dimorphic switch, which is strictly dependent on Flo11p adhesin and is indispensable for proper formation of biofilm-like aerial 3-D colony architecture. In this, Flo11p is required for both elongation of cells organized to radial clusters (formed earlier within the colony) and their subsequent pseudohyphal expansion. Just before this expansion, Flo11p relocalizes from the bud-neck of radial cell clusters also to the tip of elongated cells.     

Synthesis and structure elucidation of novel fused 1,2,4-triazine derivatives as potent inhibitors targeting CYP1A1 activity

Synthesis and structure elucidation of new series of novel fused 1,2,4-triazine derivatives 3a-3f, 4a-4i and 6a-6b and their inhibitory activities are presented. Molecular structures of the synthesized compounds were confirmed by (1)H NMR, (13)C NMR, MS spectra and elemental analyses. X-ray crystallographic analysis was performed on 2-acetyl-8-(N,N-diacetylamino)-6-(4-methoxybenzyl)-3-(4-methoxy-phenyl)-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine 3d and 2-acetyl-8-(N-acetylamino)-6-benzyl-3-(4-chlorophenyl)-3-methyl-7-oxo-2,3-dihydro-7H-[1,2,4]triazolo[4,3-b][1,2,4]triazine 4e to secure their structures. The inhibitory effect of these compounds toward the CPY1A1 activity was screened to determine their potential as promising anticancer drugs. Our data showed that compounds 4e, 5a, 5b and 6b possess the highest inhibitory effects among all tested compounds. Furthermore, analysis of triazolotriazine derivatives docking showed that these compounds bind only at the interface of substrate recognition site 2 (SRS2) and (SRS6) at the outer surface of the protein. Amino-acids ASN214, SER216 and ILE462 participate in the binding of these compounds through H-bonds.     

Stemodin-derived analogues with lipid peroxidation, cyclooxygenase enzymes and human tumour cell proliferation inhibitory activities

A series of analogues, derived from the antiviral and cytotoxic diterpene stemodin, were prepared and evaluated for their lipid peroxidation (LPO), cyclooxygenase enzyme-1 (COX-1) and -2 (COX-2), and tumour cell proliferation inhibitory activities. Oxidation of stemodin produced stemodinone, which was then converted to stemod-12-en-2-one. Reaction of the latter under Petrow conditions (bromine; silver acetate/pyridine) yielded mainly dibrominated abeo-stachanes. Solvolysis of the dibromo compounds gave products of hydrolysis, some with rearranged skeleta. In the lipid peroxidation inhibitory assay three of the compounds exhibited prominent activity. Interestingly, all the analogues showed higher COX-1 enzyme inhibition than COX-2. Although a few of the diterpenes limited the growth of some human tumour cell lines, most compounds induced proliferation of such cells.     

Detection of self-incompatible oilseed rape plants (Brassica napus L.) based on molecular markers for identification of the class I S haplotype

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F₂ oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.