Nitric oxide (NO) serves as a messenger for cellular signaling. To visualize NO in living cells, we synthesized a turn-on fluorescent probe for use in combination with microscopy. Unlike existing fluorescent sensors, the construct--a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL)--directly and immediately images NO rather than a derivative reactive nitrogen species. Using spectroscopic and mass spectrometric methods, we established that the mechanism of the reaction responsible for the NO-induced fluorescence involves reduction of the complex to Cu(I) with release of the nitrosated ligand, which occurs irreversibly. We detected NO produced by both constitutive and inducible NO synthases (cNOS and iNOS, respectively) in live neurons and macrophages in a concentration- and time-dependent manner by using the Cu(II)-based imaging agent. Both the sensitivity to nanomolar concentrations of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications.

The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25-bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand cross-link were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photo-cross-linked and identified in cervical, testicular, pancreatic and bone cancer cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA-PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photo-cross-linked only by the probe containing a 1,3-d(G*pTpG*) intrastrand cross-link. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photo-cross-linked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photo-cross-linking experiments using this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early step of the mechanism of action of the cisplatin and its close analogue carboplatin.

Inulin is an important reserve polysaccharide in Asteraceae plants, and is also widely used as a sweetener, a source of dietary fibre and prebiotic. Nevertheless, a lack of genomic resources for inulin‐producing plants has hindered extensive studies on inulin metabolism and regulation. Here, we present chromosome‐level reference genomes for four inulin‐producing plants: chicory (Cichorium intybus), endive (Cichorium endivia), great burdock (Arctium lappa) and yacon (Smallanthus sonchifolius), with assembled genome sizes of 1.28, 0.89, 1.73 and 2.72 Gb, respectively. We found that the chicory, endive and great burdock genomes were shaped by whole genome triplication (WGT‐1), and the yacon genome was shaped by WGT‐1 and two subsequent whole genome duplications (WGD‐2 and WGD‐3). A yacon unique whole genome duplication (WGD‐3) occurred 5.6–5.8 million years ago. Our results also showed the genome size difference between chicory and endive is largely due to LTR retrotransposons, and rejected a previous hypothesis that chicory is an ancestor of endive. Furthermore, we identified fructan‐active‐enzyme and transcription‐factor genes, and found there is one copy in chicory, endive and great burdock but two copies in yacon for most of these genes, except for the 1‐FEH II gene which is significantly expanded in chicory. Interestingly, inulin synthesis genes 1‐SST and 1‐FFT are located close to each other, as are the degradation genes 1‐FEH I and 1‐FEH II. Finally, we predicted protein structures for 1‐FFT genes to explore the mechanism determining inulin chain length.