Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (～1/100 of the exciting wavelength).     

Pancreatic anticancer activity of a novel geranylgeranylated coumarin derivative

A series of hydroxycoumarin derivatives has been synthesized and evaluated against human pancreatic PANC-1 cancer cells under nutrient-deprived conditions. Several compounds exhibited 100% preferential cytotoxicity at low micromolar concentrations under nutrition starvation, and showed no cytotoxicity under nutrient-rich conditions. In this study, a novel geranylgeranylated ether coumarin derivative 9 was found to exhibit the highest cytotoxic activity of 6.25 μM within 24h. The preferential anti-tumor activity exhibited by compound 9 against PANC-1 under low oxygen and nutrient environment illustrates its great potential as a promising lead structure for the development of novel agents to combat pancreatic cancer.     

Marine actinomycetes as a source of novel secondary metabolites

A set of 600 actinomycetes strains which were isolated from marine sediments from various sites in the Pacific and Atlantic Oceans were screened for the production of bioactive secondary metabolites. Marine streptomycete strains were found to be producers of well known chemically diverse antibiotics isolated from terrestrial streptomycetes, as in the case of marine Micromonospora strains. New marine members of the rare genus Verrucosispora seem to be a promising source for novel bioactive secondary metabolites as shown in the case of the abyssomicin producing strain AB-18-032.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

Natural strategies for the spatial optimization of metabolism in synthetic biology

Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

Quantifying the effects of switchgrass (Panicum virgatum) on deep organic C stocks using natural abundance 14C in three marginal soils

Perennial bioenergy crops have been shown to increase soil organic carbon (SOC) stocks, potentially offsetting anthropogenic C emissions. The effects of perennial bioenergy crops on SOC are typically assessed at shallow depths (<30 cm), but the deep root systems of these crops may also have substantial effects on SOC stocks at greater depths. We hypothesized that deep (>30 cm) SOC stocks would be greater under bioenergy crops relative to stocks under shallow‐rooted conventional crop cover. To test this, we sampled soils to between 1‐ and 3‐m depth at three sites in Oklahoma with 10‐ to 20‐year‐old switchgrass (Panicum virgatum) stands, and collected paired samples from nearby fields cultivated with shallow rooted annual crops. We measured root biomass, total organic C, ¹⁴C, ¹³C, and other soil properties in three replicate soil cores in each field and used a mixing model to estimate the proportion of recently fixed C under switchgrass based on ¹⁴C. The subsoil C stock under switchgrass (defined over 500–1500 kg/m² equivalent soil mass, approximately 30–100 cm depth) exceeded the subsoil stock in neighboring fields by 1.5 kg C/m² at a sandy loam site, 0.6 kg C/m² at a site with loam soils, and showed no significant difference at a third site with clay soils. Using the mixing model, we estimated that additional SOC introduced after switchgrass cultivation comprised 31% of the subsoil C stock at the sandy loam site, 22% at the loam site, and 0% at the clay site. These results suggest that switchgrass can contribute significantly to subsoil organic C—but also indicated that this effect varies across sites. Our analysis shows that agricultural strategies that emphasize deep‐rooted grass cultivars can increase soil C relative to conventional crops while expanding energy biomass production on marginal lands.