Hypoxia in tumors contributes to chemotherapy resistance, worsened by acidosis driven by carbonic anhydrases (hCA IX and XII). Targeting these enzymes can mitigate acidosis, thus enhancing tumor sensitivity to cytotoxic drugs. Herein, novel 4-(pyrazolyl)benzenesulfonamide ureas (SH7a−t) were developed and evaluated for their inhibitory activity against hCA IX and XII. They showed promising results (hCA IX: KI =15.9−67.6 nM, hCA XII: KI = 16.7−65.7 nM). Particularly, SH7s demonstrated outstanding activity (KIs = 15.9 nM for hCA IX and 55.2 nM for hCA XII) and minimal off-target kinase inhibition over a panel of 258 kinases. In NCI anticancer screening, SH7s exhibited broad-spectrum activity with an effective growth inhibition full panel GI50 (MG-MID) value of 3.5 μM and a subpanel GI50 (MG-MID) range of 2.4−6.3 μM. Furthermore, SH7s enhanced the efficacy of Taxol and 5-fluorouracil in cotreatment regimens under hypoxic conditions in HCT-116 colorectal cancer cells, indicating its potential as a promising anticancer agent.

This is a detailed and user-friendly protocol for the cultivation and successful crossing of Lotus japonicus (L. japonicus) e.g. for the generation of higher order mutants, based on methods previously reported (Grant et al., 1962; Handberg and Stougaards, 1992; Jiang and Gresshoff, 1997; Pajuelo and Stougaard, 2005).

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.

In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.

The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and quantification of both neutral and acidic sugars.

The Arabidopsis thaliana seed coat produces large amounts of cell wall polysaccharides, which swell out of the epidermal cells upon hydration of the mature dry seeds. While most mucilage polymers immediately diffuse in the surrounding solution, the remaining fraction tightly adheres to the seed, forming a dense gel-like capsule (Macquet et al., 2007). Recent evidence suggests that the adherence of mucilage is mediated by complex interactions between several cell wall components (Griffiths et al., 2014; Voiniciuc et al., 2015a). Therefore, it is important to evaluate how different cell wall mutants impact this mucilage property. This protocol facilitates the analysis of monosaccharides in sequentially extracted mucilage fractions, and quantifies the detachment of each component from seeds.

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.

Many tumor cells exhibit a disturbed intracellular redox state resulting in higher levels of reactive oxygen species (ROS). As these contribute to tumor initiation and sustenance, catalytic redox agents combining significant activity with substrate specificity promise high activity and selectivity against oxidatively stressed malignant cells. We describe here the design and synthesis of novel organochalcogen based redox sensor/effector catalysts. Their selective anticancer activity at submicromolar and low micromolar concentrations was established here in a range of tumor entities in various biological systems including cell lines, primary tumor cell cultures, and animal models. In the B-cell derived chronic lymphocytic leukemia (CLL), for instance, such compounds preferentially induce apoptosis in the cancer cells while peripheral blood mononuclear cells (PBMC) from healthy donors and the subset of normal B-cells remain largely unaffected. In support of the concept of sensor/effector based ROS amplification, we are able to demonstrate that underlying this selective activity against CLL cells are pre-existing elevated ROS levels in the leukemic cells compared to their nonmalignant counterparts. Furthermore, the catalysts act in concert with certain chemotherapeutic drugs in several carcinoma cell lines to decrease cell proliferation while showing no such interactions in normal cells. Overall, the high efficacy and selectivity of (redox) catalytic sensor/effector compounds warrant further, extensive testing toward transfer into the clinical arena.

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer’s disease. The hallmark of this principle is the prevention of the formation of Aβ3,11(pE)-40,42, as these Aβ-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Aβ-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Aβ3,11(pE)-40,42 formation.

Tyrosine phosphorylation represents a unique signaling process that controls metabolic pathways, cell activation, growth and differentiation, membrane transport, apoptosis, neural, and other functions. We present here the three-dimensional structure of Fyn tyrosine kinase, a Src-family enzyme involved in T-cell receptor signal transduction. The structure of Fyn was modeled for homology using the Sybyl-Composer suite of programs for modeling. Procheck and Prosa II programs showed the high quality of the obtained three-dimensional model. Rosmarinic acid, a secondary metabolite of herbal plants, was discovered as a new Fyn kinase inhibitor using immunochemical and in silico methods. Two possible binding modes of rosmarinic acid were evaluated here, i.e., near to or in the ATP-binding site of kinase domain of Fyn. Enzyme kinetic experiments revealed that Fyn is inhibited by a linear-mixed noncompetitive mechanism of inhibition by rosmarinic acid. This indicates that rosmarinic acid binds to the second “non-ATP” binding site of the Fyn tyrosine kinase.