Reaction intensification for biocatalytic production of polyphenolic natural product di-C-β-glucosides

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4–0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

Diversity of Siphonaria Sowerby I, 1823 (Gastropoda,Siphonariidae) in the Seychelles Bank and beyond

Molecular phylogenetic studies have shown that the characters of the reduced shell of the false limpets of the genus Siphonaria Sowerby I, 1823 are highly variable and often insufficient for species delimitation. The taxonomy and distribution of Siphonaria in the Indian Ocean are poorly known. We sampled Siphonaria in the Seychelles Bank to check the occurrence of recorded species using DNA sequences and to study the paths through which Siphonaria species have colonised the Seychelles Bank by reconstructing their phylogenetic relationships. Analyses of a dataset comprising 16 S rRNA gene sequences of 33 specimens from the Seychelles Bank and 300 additional Siphonaria sequences from other regions from GenBank with various methods for species delimitation resulted in 19–102 primary species hypotheses. Assemble Species by Automatic Partitioning provided a conservative estimate of the species number (42) in which several indisputable species were lumped. The results of Automatic Barcode Gap Discovery depended strongly on the assumed prior maximum intraspecific divergence, whereas the tree-based methods Generalised Mixed Yule Coalescent and Poisson Tree Processes resulted in high overestimates. The specimens from the Seychelles Bank represent three clades, belonging to the Siphonaria ‘atra’ group, the Siphonaria ‘normalis’ group and a possibly undescribed species recorded previously only from Hainan. At least two of the three species recorded from the Seychelles Bank came from the east, i.e., from the Coral Triangle in the Indo-Australian Archipelago, the region with the highest marine biodiversity worldwide. A major transport mechanism across the Indian Ocean was probably the South Equatorial Current.     

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.     

Morphological parameters affecting false lumen thrombosis following type B aortic dissection: a systematic study based on simulations of idealized models

Type B aortic dissection (TBAD) carries a high risk of complications, particularly with a partially thrombosed or patent false lumen (FL). Therefore, uncovering the risk factors leading to FL thrombosis is crucial to identify high-risk patients. Although studies have shown that morphological parameters of the dissected aorta are related to FL thrombosis, often conflicting results have been reported. We show that recent models of thrombus evolution in combination with sensitivity analysis methods can provide valuable insights into how combinations of morphological parameters affect the prospect of FL thrombosis. Based on clinical data, an idealized geometry of a TBAD is generated and parameterized. After implementing the thrombus model in computational fluid dynamics simulations, a global sensitivity analysis for selected morphological parameters is performed. We then introduce dimensionless morphological parameters to scale the results to individual patients. The sensitivity analysis demonstrates that the most sensitive parameters influencing FL thrombosis are the FL diameter and the size and location of intimal tears. A higher risk of partial thrombosis is observed when the FL diameter is larger than the true lumen diameter. Reducing the ratio of the distal to proximal tear size increases the risk of FL patency. In summary, these parameters play a dominant role in classifying morphologies into patent, partially thrombosed, and fully thrombosed FL. In this study, we point out the predictive role of morphological parameters for FL thrombosis in TBAD and show that the results are in good agreement with available clinical studies.

     

Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.     

POPULATION GENETIC STRUCTURE IN DIPLOID BLUEBERRY VACCINIUM SECTION CYANOCOCCUS (ERICACEAE)

Vaccinium section Cyanococcus comprises diploid, tetraploid, and hexaploid species (x = 12) all of which are highly self-sterile. In order to assess the distribution of genetic variation within and among the diploids, population genetic analyses of allozyme data were conducted on three species: V. elliottii, V. myrtilloides, and V. tenellum. Populations were located throughout the range of these taxa in eastern North America. Data were collected at 20 loci, 12 of which are mendelian based on formal genetic analyses. Consistent with expectations for out-crossing taxa, these species exhibited high levels of variation within populations. Eighteen loci were polymorphic with the number and frequency of alleles varying among taxa. Mean values for the number of alleles per polymorphic locus, proportion of polymorphic loci, and expected heterozygosity were 2.8, 48.2, and 0.148, respectively. High infraspecific genetic identities exceeding 0.9 indicated that these taxa are homogeneous. All populations were in Hardy-Weinberg equilibrium with slight heterozygote excess observed in V. myrtilloides and V. tenellum. Although total genetic diversity was lower than that observed for other predominantly out-crossing species, it was apportioned similarly. The majority could be attributed to differences among individuals within populations. Conspecific populations were relatively undifferentiated with genetic differentiation similar to other self-incompatible species.     

Modulation of the heart rate by thyroid hormone in the newborn rat

Confirming the literature data the authors describe that the heart rate is smaller in the newborn rats than in adult ones and increases until the adult values during the first two weeks of life. On the other hand, the blood thyroid hormone exhibits the same pattern, showing an early postnatal increment. As, according the Adolph's data (1967), the heart rate enhancement is not due to the catecholamines, the authors suppose that such enhancement might conceivably depend on thyroid hormone increment.     

Effects of Removal of Successful Males in a Fallow Deer Lek

Removal of 5 of the 8 most successful males in a fallow deer lek between breeding seasons led to an increase in fighting rate of males and a decrease in mating rate of both sexes. The increase in fighting was likely due to disruption of the social hierarchy. The lower mating rate of females was not due to disruption of copulations by territorial males, nor did it appear to result from disturbance from fights. We suggest that difficulties in mate choice related to an unstable male hierarchy resulted in females either increasing their length of stay in the lek, visiting the lek more than once before mating, or leaving the lek without mating.