Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure

The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.     

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.     

Low intensity,long-term outpatient rehabilitation in COPD: a randomised controlled trial

Background

Most pulmonary rehabilitation programmes currently involve 2–3 sessions per week as recommended by international guidelines. We aimed to investigate whether relevant improvements in physical capabilities and quality of life in patients with chronic obstructive pulmonary disease (COPD) could be achieved by a long-term, low intensity, once weekly rehabilitation programme using limited resources.

Methods

100 patients with moderate to severe COPD were randomised to a continuous outpatient interdisciplinary rehabilitation programme or standard care. Physiotherapy-led supervised outpatient training sessions were performed once weekly in addition to educational elements. Outcome measures at baseline and after 26 weeks were 6-minute-walk-test, cycle ergometry, and health-related quality of life.

Results

37 patients in the training group and 44 patients in the control group completed the study. After 26 weeks there were clinically significant differences between the groups for 6 minute-walk-distance (+59 m, 95% CI 28–89 m), maximum work load (+7.4 Watt, 95% CI 0.5-13.4 Watt) and St. George’s Respiratory Questionnaire score (−5 points, 95% CI −10 to −1 points). Total staff costs of the programme per participant were ≤ €625.

Conclusion

Clinically meaningful improvements in physical capabilities and health-related quality of life may be achieved using long-term pulmonary rehabilitation programmes of lower intensity than currently recommended. Trial registration: clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01195402","term_id":"NCT01195402"}}NCT01195402.     

Aberrant cytokine pattern of the nasal mucosa in granulomatosis with polyangiitis

Introduction

In granulomatosis with polyangiitis (GPA), a complex autoimmune small-vessel vasculitis frequently associated with chronic necrotizing inflammation of the nasal mucosa, elevated nasal Staphylococcus (S.) aureus carrier rates are a risk factor for relapse. As cytokines are primarily involved in the regulation of defense against potentially pathogenic microorganisms, the aim of this study was to compare healthy individuals and GPA patients with respect to their baseline cytokine expression of nasal epithelial cells (NEC), which form the first barrier against such triggers. The ability of S. aureus to influence the nasal microenvironment''s cytokine secretion was assessed by exemplary stimulation experiments.

Methods

Baseline expression of 19 cytokines of primary NEC of GPA patients and normal controls (NC) was quantified by a multiplex cytokine assay. Stimulation experiments were performed with supernatants of S. aureus and expression of interleukin-8 was determined by ELISA.

Results

In GPA, an altered pattern of baseline cytokine expression with significantly up-regulated G-CSF and reduced interleukin (IL)-8 concentrations was observed. Both NEC of GPA patients and NC responded to stimulation with S. aureus, but GPA patients displayed a significantly lower IL-8 secretion and a diminished dynamic range of response towards the stimulus.

Conclusions

The data presented underline the hypothesis of a disturbed epithelial nasal barrier function in GPA. The dysregulated baseline expression of G-CSF and IL-8 and the reduced response to microbial stimulation may facilitate changes in the composition of the nasal flora and favour an imbalanced inflammatory response, which might be relevant for the disease course.     

GGA1-mediated endocytic traffic of LR11/SorLA alters APP intracellular distribution and amyloid-β production

Proteolytic processing of the amyloid-β precursor protein (APP) and generation of amyloid-β peptide (Aβ) are key events in Alzheimer's disease (AD) pathogenesis. Cell biological and genetic evidence has implicated the low-density lipoprotein and sorting receptor LR11/SorLA in AD through mechanisms related to APP and Aβ production. Defining the cellular pathway(s) by which LR11 modulates Aβ production is critical to understanding how changes in LR11 expression affect the development of Aβ pathology in AD progression. We report that the LR11 ectodomain is required for LR11-mediated reduction of Aβ and that mutagenesis of the LR11 Golgi-localizing, γ-adaptin ear homology domain, ADP-ribosylation factor (GGA)-binding motif affects the endosomal distribution of LR11, as well as LR11's effects on APP traffic and Aβ production. Targeted small interfering RNA (siRNA) knockdown studies of GGA1, GGA2, and GGA3 indicate a surprising degree of specificity toward GGA1, suggesting that GGA1 is a candidate regulator of LR11 traffic. Additional siRNA knockdown experiments reveal that GGA1 is necessary for both LR11 and β-site APP-cleaving enzyme-1 (BACE1) modulation of APP processing to Aβ. Mutagenesis of BACE1 serine 498 to alanine enhances BACE1 targeting to LR11-positive compartments and nullifies LR11-mediated reduction of Aβ. On basis of these results, we propose that GGA1 facilitates LR11 endocytic traffic and that LR11 modulates Aβ levels by promoting APP traffic to the endocytic recycling compartment.     

Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates

Background

Although it has proven to be an important foundation for investigations of carnivoran ecology, biology and evolution, the complete species-level supertree for Carnivora of Bininda-Emonds et al. is showing its age. Additional, largely molecular sequence data are now available for many species and the advancement of computer technology means that many of the limitations of the original analysis can now be avoided. We therefore sought to provide an updated estimate of the phylogenetic relationships within all extant Carnivora, again using supertree analysis to be able to analyze as much of the global phylogenetic database for the group as possible.

Results

In total, 188 source trees were combined, representing 114 trees from the literature together with 74 newly constructed gene trees derived from nearly 45,000 bp of sequence data from GenBank. The greater availability of sequence data means that the new supertree is almost completely resolved and also better reflects current phylogenetic opinion (for example, supporting a monophyletic Mephitidae, Eupleridae and Prionodontidae; placing Nandinia binotata as sister to the remaining Feliformia). Following an initial rapid radiation, diversification rate analyses indicate a downturn in the net speciation rate within the past three million years as well as a possible increase some 18.0 million years ago; numerous diversification rate shifts within the order were also identified.

Conclusions

Together, the two carnivore supertrees remain the only complete phylogenetic estimates for all extant species and the new supertree, like the old one, will form a key tool in helping us to further understand the biology of this charismatic group of carnivores.     

Between Metabolite Relationships: an essential aspect of metabolic change

Not only the levels of individual metabolites, but also the relations between the levels of different metabolites may indicate (experimentally induced) changes in a biological system. Component analysis methods in current 'standard' use for metabolomics, such as Principal Component Analysis (PCA), do not focus on changes in these relations. We therefore propose the concept of 'Between Metabolite Relationships' (BMRs): common changes in the covariance (or correlation) between all metabolites in an organism. Such structural changes may indicate metabolic change brought about by experimental manipulation but which are lost with standard data analysis methods. These BMRs can be analysed by the INdividual Differences SCALing (INDSCAL) method. First the BMR quantification is described and subsequently the INDSCAL method. Finally, two studies illustrate the power and the applicability of BMRs in metabolomics. The first study is about the induced plant response of cabbage to herbivory, of which BMRs are a considerable part. In the second study-a human nutritional intervention study of green tea extract-standard data analysis tools did not reveal any metabolic change, although the BMRs were considerably affected. The presented results show that BMRs can be easily implemented in a wide variety of metabolomic studies. They provide a new source of information to describe biological systems in a way that fits flawlessly into the next generation of systems biology questions, dealing with personalized responses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0316-1) contains supplementary material, which is available to authorized users.