Discrimination of human cytotoxic lymphocytes from regulatory and B-lymphocytes by orthogonal light scattering

Light scattering properties of human lymphocyte subpopulations selected by immunofluorescence were studied with a flow cytometer. Regulatory and B-lymphocytes showed a low orthogonal light scatter signal, whereas cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15 revealed a large orthogonal light scatter signal. Two populations in light scatter histograms could be observed with monoclonal antibodies directed against determinants present on both regulatory and cytotoxic lymphocytes. By analysis of the lymphocytes of 16 individuals we found a linear relation between the number of cells with a large orthogonal light scattering and the number of cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15. These observations demonstrate physical differences between cytotoxic lymphocytes and regulatory and B lymphocytes. Moreover, the results suggest a method to estimate the amount of cytotoxic lymphocytes without using monoclonal antibodies

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Landscape Values and Aesthetic Preferences Across the Front Range

Boulder’s Open Space and Mountain Parks (OSMP) lands are managed to provide a diverse set of benefits valued by Boulder’s residents as well as tourists. Not all OSMP lands provide the same set of benefits however. Understanding how the values associated with OSMP lands vary across the region can provide managers with insights into how best to allocate resources so that they yield the maximum public benefit. In addition to an understanding of the values visitors associate with OSMP lands, management can benefit from knowledge of how different features of the landscape impact user experiences, both positively and negatively

Aesthetic Characteristics of the Front Range: An Analysis of Viewsheds Provided by Boulder OSMP Lands

The city of Boulder’s Open Space and Mountain Parks (OSMP) lands offer residents and visitors a variety of unique recreational, scenic, and cultural experiences that are often captured and shared publicly via social media. Given the diversity of OSMP lands, visitor experiences likely differ based on the aesthetic and biophysical features that can be viewed from these landscapes. For instance, the peaks of the iconic Flatirons provide visitors with different scenic views than the low-lying grasslands in the southeastern area of the city. Furthermore, visitor use and enjoyment of OSMP lands could be directly related to the landscape features that are visible from these different locations. Understanding how visible landscape features vary across OSMP lands can help managers target their planning efforts to improve the quality of outdoor recreation experiences, and potentially identify new locations for outdoor recreation infrastructure (e.g., trails, pavilions, etc.) that offer the ability to see the regions most desirable landscape features. This study: (1) identifies points in the landscape where users are often inspired to take photographs; (2) maps the landscapes most often viewed by visitors; (3) summarizes the types of landscape features viewed from OSMP lands; and (4) determines how these landscape features vary across LCAs

Editorial: Actinobacteria, a Source of Biocatalytic Tools

Actinobacteria (Actinomycetes) represent one of the largest and most diverse phyla among the Bacteria. The characteristics and phylogeny of actinobacteria have been well-described throughout the years (Anteneh and Franco; Embley et al., 1994; Stackebrandt et al., 1997a,b; Stach and Bull, 2005; Stackebrandt and Schumann, 2006; Ventura et al., 2007; Gao and Gupta, 2012; Goodfellow, 2012a,b; Schrempf, 2013; Lawson, 2018; Lewin et al., 2016). Still actinobacteria are hotspots for discovery of new biomolecules and enzyme activities, fueling an active field of research. The remarkable diversity is displayed by various lifestyles, distinct morphologies, a wide spectrum of physiological and metabolic activities, as well as genetics

Mixed-Integer MPC Strategies for Fueling and Density Control in Fusion Tokamaks

Model predictive control (MPC) is promising for fueling and core density feedback control in nuclear fusion tokamaks, where the primary actuators, frozen hydrogen fuel pellets fired into the plasma, are discrete. Previous density feedback control approaches have only approximated pellet injection as a continuous input due to the complexity that it introduces. In this letter, we model plasma density and pellet injection as a hybrid system and propose two MPC strategies for density control: mixed-integer (MI) MPC using a conventional mixed-integer programming (MIP) solver and MPC utilizing our novel modification of the penalty term homotopy (PTH) algorithm. By relaxing the integer requirements, the PTH algorithm transforms the MIP problem into a series of continuous optimization problems, reducing computational complexity. Our novel modification to the PTH algorithm ensures that it can handle path constraints, making it viable for constrained hybrid MPC in general. Both strategies perform well with regards to reference tracking without violating path constraints and satisfy the computation time limit for real-time control of the pellet injection system. However, the computation time of the PTH-based MPC strategy consistently outpaces the conventional MI-MPC strategy

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ¿ABC, which lacks helices aA, aB and aC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered

Abnormal distribution of CD8 subpopulation in B-chronic lymphocytic leukemia identified by flow cytometry

We studied the occurrence of T-cell subpopulations for patients with B-cell chronic lymphocytic leukemia. The CD8+ population was divided into CD8+ suppressor (CD8a+) and CD8+ cytotoxic (CD8b+) lymphocytes using difference in orthogonal light scattering.\ud \ud Average CD4+/CD8+ratios determined for all patients were decreased. For individual patients this sometimes was not true. In contrast CD4+/CD8a+ ratios were markedly increased in all individual patients. The CD8+ lymphocytes appeared to consist mainly of CD8b+lymphocytes. Moreover the CD8b+/CD8+ ratio correlated with clinical stage: untreated patients (stage 0 of Rai) have smaller CD8b+/CD8+ ratios than patients with advanced stages of Rai