Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H₂O₂ as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.     

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.     

Acute effects of superimposed electromyostimulation during cycling on myokines and markers of muscle damage

Objectives:

The purpose of the present study was to evaluate the effects of superimposed electromyostimulation (E) during cycling on myokines and markers of muscle damage, as E might be a useful tool to induce a high local stimulus to skeletal muscle during endurance training without performing high external workloads.

Methods:

13 subjects participated in three experimental trials each lasting 60 min in a randomized order. 1) Cycling (C), 2) Cycling with superimposed E (C+E) and 3) E. Interleukin-6 (IL-6), brain-derived neurotrophic factor (BDNF), creatine kinase (CK) and myoglobin were determined before (pre) and 0’, 30’, 60’, 240’ and 24h after each intervention.

Results:

Only C+E caused significant increases in levels of CK and myoglobin. BDNF and IL-6 significantly increased after C and C+E, however increases for IL-6 were significantly higher after C+E compared to C.

Conclusion:

The present study showed that superimposed E during cycling might be a useful tool to induce a high local stimulus to skeletal muscle even when performing low to moderate external workloads. This effect might be due the activation of additional muscle fibers and mild eccentric work due to the concomitant activation of agonist and antagonist. However the higher load to skeletal muscle has to be taken into account.     

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice

Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gα_q/11, Gα_12/13 and Gα_i family without interaction with G protein‐coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen‐activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular‐regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein‐dependent signalling in cardiac cells.     

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

Effects of multidisciplinary integrated care on quality of care in residential care facilities for elderly people: a cluster randomized trial

Background:

Sophisticated approaches are needed to improve the quality of care for elderly people living in residential care facilities. We determined the effects of multidisciplinary integrated care on the quality of care and quality of life for elderly people in residential care facilities.

Methods:

We performed a cluster randomized controlled trial involving 10 residential care facilities in the Netherlands that included 340 participating residents with physical or cognitive disabilities. Five of the facilities applied multidisciplinary integrated care, and five provided usual care. The intervention, inspired by the disease management model, consisted of a geriatric assessment of functional health every three months. The assessment included use of the Long-term Care Facility version of the Resident Assessment Instrument by trained nurse-assistants to guide the design of an individualized care plan; discussion of outcomes and care priorities with the family physician, the resident and his or her family; and monthly multidisciplinary meetings with the nurse-assistant, family physician, psychologist and geriatrician to discuss residents with complex needs. The primary outcome was the sum score of 32 risk-adjusted quality-of-care indicators.

Results:

Compared with the facilities that provided usual care, the intervention facilities had a significantly higher sum score of the 32 quality-of-care indicators (mean difference − 6.7, p = 0.009; a medium effect size of 0.72). They also had significantly higher scores for 11 of the 32 indicators of good care in the areas of communication, delirium, behaviour, continence, pain and use of antipsychotic agents.

Interpretation:

Multidisciplinary integrated care resulted in improved quality of care for elderly people in residential care facilities compared with usual care.

Trial registration:

www.controlled-trials.com trial register no. ISRCTN11076857.The quality of care provided in residential care facilities is under pressure worldwide.¹ Facilities are frequently understaffed, and the complexity of care needed by residents increases while expertise of staff does not necessarily keep pace.^2,3 Although most care organizations want to innovate and improve quality of care, many lack expertise or financial resources needed to do so.^4,5 Family physicians are responsible for medical care in residential care facilities in the Netherlands. However, they do not regard themselves as suited for systematic management of chronic diseases and disabilities associated with frail health.⁶About 10% of elderly people aged 75 or older in the Netherlands live in residential care facilities.^7,8 These facilities were established to offer sheltered living for elderly people who are disabled but still relatively healthy. Because of the growing elderly population, the characteristics of elderly people living in residential care facilities have become more comparable to those of people in nursing homes, who need complex care. Residential care facilities in the Netherlands are comparable to residential care facilities in Canada, are publicly funded and are subject to government inspection and approval. Over 70% of the residents need professional care, such as assistance with activities of daily living, nursing care (e.g., medication, wound care) and housekeeping. They have multiple chronic diseases and associated disabilities.^9–12Effective interventions for chronic illnesses generally rely on a multidisciplinary team approach. The elements of this approach include structured geriatric assessment, protocol-based regulation of medications, support for self-reliance and intensive follow-up. The closely related disease management model comprises coordination of care, steering of the care process and patient empowerment.¹³ This model is strongly recommended by Bodenheimer and colleagues to improve the health and quality of life of chronically ill patients.¹⁴ However, no studies have as yet been undertaken to evaluate the effects of disease management on functional health and quality of care for elderly people in residential care facilities who have physical or cognitive disabilities.We developed an approach to multidisciplinary integrated care inspired by the disease management model. The objective of our study was to determine the effects of multidisciplinary integrated care on quality of care and quality of life for elderly people in residential care facilities.     

Biochemical markers of ongoing joint damage in rheumatoid arthritis - current and future applications, limitations and opportunities

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease associated with potentially debilitating joint inflammation, as well as altered skeletal bone metabolism and co-morbid conditions. Early diagnosis and aggressive treatment to control disease activity offers the highest likelihood of preserving function and preventing disability. Joint inflammation is characterized by synovitis, osteitis, and/or peri-articular osteopenia, often accompanied by development of subchondral bone erosions, as well as progressive joint space narrowing. Biochemical markers of joint cartilage and bone degradation may enable timely detection and assessment of ongoing joint damage, and their use in facilitating treatment strategies is under investigation. Early detection of joint damage may be assisted by the characterization of biochemical markers that identify patients whose joint damage is progressing rapidly and who are thus most in need of aggressive treatment, and that, alone or in combination, identify those individuals who are likely to respond best to a potential treatment, both in terms of limiting joint damage and relieving symptoms. The aims of this review are to describe currently available biochemical markers of joint metabolism in relation to the pathobiology of joint damage and systemic bone loss in RA; to assess the limitations of, and need for additional, novel biochemical markers in RA and other rheumatic diseases, and the strategies used for assay development; and to examine the feasibility of advancement of personalized health care using biochemical markers to select therapeutic agents to which a patient is most likely to respond.