Report on Vincristine-Producing Endophytic Fungus Nigrospora zimmermanii from Leaves of Catharanthus roseus.

Vincristine is an anti-cancer compound and one of the most crucial vinca alkaloids produced by the medicinal plant Catharanthus roseus (L.) G. Don. (Apocynaceae). This plant is home to hundreds of endophytic microbes, which produce a variety of bioactive secondary metabolites that are known for their medicinal properties. In this study, we focused on isolating an endophytic fungus that could increase the yield of vincristine under laboratory conditions as an alternative to plant-mediated extraction of vincristine. The endophytic fungus Nigrospora zimmermanii (Apiosporaceae) was isolated from Catharanthus roseus and it was found to be producing the anticancer compound vincristine. It was identified using high-performance thin-layer chromatography by matching the Rf value and spectral data with the vincristine standard and mass spectrometry data and the reference molecule from the PubChem database. The generation study of this microbe showed that the production of vincristine in the parent fungus was at its maximum, i.e., 5.344 µg/mL, while it was slightly reduced in subsequent generations. A colonization study was also performed and it showed that the fungus N. zimmermanii was able to re-infect the plant Catharanthus roseus after 20 days of inoculation. The colonization study showed that N. zimmernanii could infect the plant after isolation. This method is an efficient and easy way to obtain a high yield of vincristine, as compared to plant-mediated production.

Real time optical detection of gold in living cells through genetically-encoded probe.

To study the efflux of gold (Au) in living cells, a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor has been developed. The gold-sensing domain GolB from Salmonella typhimurium has been fused to the N- and C-termini of the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus respectively. In living cells, this probe is highly selective and sensitive to gold and it can withstand changes in variable pH ranges. GolSeN-25, the most efficient sensor variant, binds gold with an affinity (K d) of 0.3 × 10-6 M, covering gold concentrations of nM to µM, and can be used for non-invasive real-time in vivo gold measurement in living cells. A simple and sensitive FRET probe was designed for the detection of gold with high selectivity and can be applied to the analysis of real samples.

FRET-Based Genetically Encoded Sensor to Monitor Silver Ions.

Silver is commonly used in wound dressing, photography, health care products, laboratories, pharmacy, biomedical devices, and several industrial purposes. Silver (Ag+) ions are more toxic pollutants widely scattered in the open environment by natural processes and dispersed in soil, air, and water bodies. Ag+ binds with metallothionein, macroglobulins, and albumins, which may lead to the alteration of various enzymatic metabolic pathways. To analyze the uptake and metabolism of silver ions in vitro as well as in cells, a range of high-affinity fluorescence-based nanosensors has been constructed using a periplasmic protein CusF, a part of the CusCFBA efflux complex, which is involved in providing resistance against copper and silver ions in Escherichia coli. This nanosensor was constructed by combining of two fluorescent proteins (donor and acceptor) at the N- and C-terminus of the silver-binding protein (CusF), respectively. SenSil (WT) with a binding constant (K d) of 5.171 µM was more efficient than its mutant variants (H36D and F71W). This nanosensor allows monitoring the level of silver ions in real time in prokaryotes and eukaryotes without any disruption of cells or tissues.

Enhanced sensitivity and detection range of FRET-based vitamin B12 nanosensor.

Vitamin B12 (cobalamin) is a cobalt-containing compound that acts as an essential co-factor for various enzymes involved in the metabolic processes of the living cells. The constructed FRET Sensor for Vitamin Anemia Linked (SenVitAL) displayed marginal FRET efficiency. Here, we report the development of a molecular SenVitAL containing enhanced cyan fluorescent protein (ECFP) and venus as FRET pair to improve the FRET efficiency for optical imaging and screening of already developed sensor by our group. The sensor is the improved version of previously reported SenVitAL and consists of ECFP/venus as FRET pair instead of the originally used pair CFP/YFP. To increase the physiological range of vitamin B12 measurement, affinity mutants were created. Compared to the wild type, SenVitAL-5 with W44Q mutation has higher affinity and displayed large dynamic detection range (0.10-480 µM) in response to vitamin B12 binding. For cell-based monitoring and dynamic measurement of vitamin B12 flux rates, SenVitAL-5 was successfully expressed in cytosol of yeast and mammalian cells. Changes in the emission intensities of the two fluorophores were detected using confocal microscopy in both cell types in response to vitamin B12. With the addition of 50 µM extracellular vitamin B12 to the cells, the emission intensity of venus increased and that of ECFP decreased over the time. Furthermore, the results show that the variant SenVitAL-5 measures the vitamin B12 in a concentration-dependent manner, showing the resulting increase in the FRET ratio and thus confirming its utility as an ideal fluorescent indicator for the detection of vitamin B12 in eukaryotic systems in real time.