A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides

Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.     

ON THE IDENTITY OF KARLODINIUM VENEFICUM AND DESCRIPTION OF KARLODINIUM ARMIGER SP. NOV. (DINOPHYCEAE), BASED ON LIGHT AND ELECTRON MICROSCOPY,NUCLEAR‐ENCODED LSU RDNA,AND PIGMENT COMPOSITION1

An undescribed species of the dinoflagellate genus Karlodinium J. Larsen (viz. K. armiger sp. nov.) is described from Alfacs Bay (Spain), using light and electron microscopy, pigment composition, and partial large subunit (LSU) rDNA sequence. The new species differs from the type species of Karlodinium (K. micrum (Leadbeater et Dodge) J. Larsen) by lacking rows of amphiesmal plugs, a feature presently considered to be a characteristic of Karlodinium. In K. armiger, an outer membrane is underlain by a complex system of cisternae and vacuoles. The pigment profile of K. armiger revealed the presence of chlorophylls a and c, with fucoxanthin as the major carotenoid. Phylogenetic analysis confirmed K. armiger to be related to other species of Karlodinium; thus forming a monophyletic genus, which, in the LSU tree, occupies a sister group position to Takayama de Salas, Bolch, Botes et Hallegraeff. The culture used by Ballantine to describe Gymnodinium veneficum Ballantine (Plymouth 103) was examined by light and electron microscopy and by partial LSU rDNA. Ultrastructurally, it proved identical to K. micrum (cultures Plymouth 207 and K. Tangen KT‐77D, the latter also known as K‐0522), and in LSU sequence, differed in only 0.3% of 1438 bp. We consider the two taxa to belong to the same species. This necessitates a change of name for the most widely found species, K. micrum, to K. veneficum. The three genera Karlodinium, Takayama, and Karenia constitute a separate evolutionary lineage, for which the new family Kareniaceae fam. nov. is suggested.     

Peak Oxygen Uptake after Cardiac Rehabilitation: A Randomized Controlled Trial of a 12-Month Maintenance Program versus Usual Care

Background

Exercise capacity is a strong predictor of survival in patients with coronary artery disease (CAD). Exercise capacity improves after cardiac rehabilitation exercise training, but previous studies have demonstrated a decline in peak oxygen uptake after ending a formal rehabilitation program. There is a lack of knowledge on how long-term exercise adherence can be achieved in CAD patients. We therefore assessed if a 12-month maintenance program following cardiac rehabilitation would lead to increased adherence to exercise and increased exercise capacity compared to usual care.

Materials and Methods

Two-centre, open, parallel randomized controlled trial with 12 months follow-up comparing usual care to a maintenance program. The maintenance program consisted of one monthly supervised high intensity interval training session, a written exercise program and exercise diary, and a maximum exercise test every third month during follow-up. Forty-nine patients (15 women) on optimal medical treatment were included following discharge from cardiac rehabilitation. The primary endpoint was change in peak oxygen uptake at follow-up; secondary endpoints were physical activity level, quality of life and blood markers of cardiovascular risk.

Results

There was no change in peak oxygen uptake from baseline to follow-up in either group (intervention group 27.9 (±4.7) to 28.8 (±5.6) mL·kg (-1) min (−1), control group 32.0 (±6.2) to 32.8 (±5.8) mL·kg (−1) min (−1), with no between-group difference, p = 0.22). Quality of life and blood biomarkers remained essentially unchanged, and both self-reported and measured physical activity levels were similar between groups after 12 months.

Conclusions

A maintenance exercise program for 12 months did not improve adherence to exercise or peak oxygen uptake in CAD patients after discharge from cardiac rehabilitation compared to usual care. This suggests that infrequent supervised high intensity interval training sessions are inadequate to improve peak oxygen uptake in this patient group.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01246570","term_id":"NCT01246570"}}NCT01246570     

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.     

Data representativeness in LCA: A framework for the systematic assessment of data quality relative to technology characteristics

A shortcoming in current data quality assessment schemes is that the data quality information is not used systematically to identify the critical data in a life cycle inventory (LCI) model. In addition, existing criteria employed to evaluate representativeness lack relevance to the specific context of a study. A novel framework is proposed herein for the evaluation of the representativeness of LCI data, including an analysis of the importance of the data and a modification of quality criteria based on unit process characteristics. Temporal characteristics are analyzed by identifying the technology shift, because data generated before this time are considered outdated. Geographical and technological characteristics are analyzed by defining a “related area” and a “related technology,” which is done by identifying a number of relevant geographical and technical factors, and then comparing the collected data with these factors. The framework was illustrated in a case study on household waste incineration in Denmark. The results demonstrated the applicability of the method in practice, and they provided data quality criteria unique to waste incineration unit processes, for example, different time intervals to evaluate temporal representativeness. However, the proposed method is time demanding, and thus sector‐level characteristic analyses are feasible instead of the user having to do the analyses.     

Voltage and Calcium Dynamics Both Underlie Cellular Alternans in Cardiac Myocytes

Cardiac alternans, a putative trigger event for cardiac reentry, is a beat-to-beat alternation in membrane potential and calcium transient. Alternans was originally attributed to instabilities in transmembrane ion channel dynamics (i.e., the voltage mechanism). As of this writing, the predominant view is that instabilities in subcellular calcium handling are the main underlying mechanism. That being said, because the voltage and calcium systems are bidirectionally coupled, theoretical studies have suggested that both mechanisms can contribute. To date, to our knowledge, no experimental evidence of such a dual role within the same cell has been reported. Here, a combined electrophysiological and calcium imaging approach was developed and used to illuminate the contributions of voltage and calcium dynamics to alternans. An experimentally feasible protocol, quantification of subcellular calcium alternans and restitution slope during cycle-length ramping alternans control, was designed and validated. This approach allows simultaneous illumination of the contributions of voltage and calcium-driven instability to total cellular instability as a function of cycle-length. Application of this protocol in in vitro guinea-pig left-ventricular myocytes demonstrated that both voltage- and calcium-driven instabilities underlie alternans, and that the relative contributions of the two systems change as a function of pacing rate.     

DISPERSAL and BYCATCH MORTALITY IN GRAY, HALICHOERUS GRYPUS, AND HARBOR, PHOCA VITULINA, SEALS TAGGED AT THE NORWEGIAN COAST

Between 1975 and 1998, 3,571 gray and 630 harbor seal pups were tagged along the Norwegian coast, and 259 (7%) gray and 80 (13%) harbor seal tags were returned. Incidental mortality, mainly in bottom-set nets, accounted for the majority of deaths (79% in gray and 48% in harbor seals, respectively). Seals were most vulnerable to incidental mortality in fishing gear during the first three months after birth, but high incidental mortality prevailed during the first 8–10 mo. Gray seals dispersed more widely (mean distance: 120 km) than harbor seals (mean distance: 69 km). Both species dispersed most widely during the two first months after tagging. The maximum distance moved was 739 km for gray and 463 km for harbor seals. Strong fidelity for their place of birth was observed in adult gray seals during breeding season. No significant difference in incidental mortality was detected between the areas of tagging. However, for 37 harbor seals tagged in a 724 km nature reserve no returns were reported.     

Specific prediction of mortality by oxidative stress-induced damage to RNA vs. DNA in humans

Modifications of nucleic acids (DNA and RNA) from oxidative stress is a potential driver of aging per se and of mortality in age-associated medical disorders such as type 2 diabetes (T2D). In a human cohort, we found a strong prediction of all-cause mortality by a marker of systemic oxidation of RNA in patients with T2D (n = 2672) and in nondiabetic control subjects (n = 4079). The finding persisted after the adjustment of established modifiers of oxidative stress (including BMI, smoking, and glycated hemoglobin). In contrast, systemic levels of DNA damage from oxidation, which traditionally has been causally linked to both T2D and aging, failed to predict mortality. Strikingly, these findings were subsequently replicated in an independent general population study (n = 3649). The data demonstrate a specific importance of RNA damage from oxidation in T2D and general aging.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide