Matrix Gla protein and osteopontin genetic associations with coronary artery calcification and bone density: the CARDIA study

A role for matrix proteins has previously been proposed in the pathogenesis of arterial calcification in the setting of atherosclerosis, and a link has been suggested between osteoporosis and arterial calcification. Our aim has been to investigate whether matrix Gla protein (MGP) T-138C, osteopontin (SPP1) T-443C, and Asp94Asp single nucleotide polymorphisms are associated with the development of arterial calcification and bone density. The individual effects of the MGP and SPP1 polymorphisms with coronary calcification are weak and not statistically significant. Bone mineral density differences at both the hip and spine do not vary statistically by genotype for any of the polymorphisms studied. Given the significant role of both MGP and SPP1 in arteriosclerosis, further research in higher risk, older populations are needed to determine fully the way in which MGP and SPP1 polymorphisms are associated with disease.     

Design and application of circular RNAs with protein-sponge function

Circular RNAs (circRNAs) are a class of noncoding RNAs, generated from pre-mRNAs by circular splicing of exons and functionally largely uncharacterized. Here we report on the design, expression, and characterization of artificial circRNAs that act as protein sponges, specifically binding and functionally inactivating hnRNP (heterogeneous nuclear ribonucleoprotein) L. HnRNP L regulates alternative splicing, depending on short CA-rich RNA elements. We demonstrate that designer hnRNP L-sponge circRNAs with CA-repeat or CA-rich sequence clusters can efficiently and specifically modulate splicing-regulatory networks in mammalian cells, including alternative splicing patterns and the cellular distribution of a splicing factor. This new strategy can in principle be applied to any RNA-binding protein, opening up new therapeutic strategies in molecular medicine.     

Basolateral sorting signals regulating tissue-specific polarity of heteromeric monocarboxylate transporters in epithelia

Many solute transporters are heterodimers composed of non-glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue-specific functions. To date, the mechanisms regulating the tissue-specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton-coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C-terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin-dependent but clathrin adaptor AP1B-independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue-specific polarity.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Selective detection of crystalline cellulose in plant cell walls with sum-frequency-generation (SFG) vibration spectroscopy

The selective detection of crystalline cellulose in biomass was demonstrated with sum-frequency-generation (SFG) vibration spectroscopy. SFG is a second-order nonlinear optical response from a system where the optical centrosymmetry is broken. In secondary plant cell walls that contain mostly cellulose, hemicellulose, and lignin with varying concentrations, only certain vibration modes in the crystalline cellulose structure can meet the noninversion symmetry requirements. Thus, SFG can be used to detect and analyze crystalline cellulose selectively in lignocellulosic biomass without extraction of noncellulosic species from biomass or deconvolution of amorphous spectra. The selective detection of crystalline cellulose in lignocellulosic biomass is not readily achievable with other techniques such as XRD, solid-state NMR, IR, and Raman analyses. Therefore, the SFG analysis presents a unique opportunity to reveal the cellulose crystalline structure in lignocellulosic biomass.     

Long signal peptides of RGMa and DCBLD2 are dissectible into subdomains according to the NtraC model

Targeting of proteins to the endoplasmic reticulum (ER) usually requires N-terminal signal peptides (SP) of approximately 22 amino acids in length. However, a substantial number of proteins contain exceptionally long SPs of 40 amino acids and more, an example being protein shrew-1/AJAP1. Using shrew-1's SP as example, the NtraC model has been developed by dissecting long SPs into two functionally distinct subdomains ("N" and "C") separated by a β-turn rich transition area ("tra"). Further proteins have been identified by computational analysis complying with the NtraC model. Here we used the SPs of two of these proteins, DCBLD2 and RGMa (including three isoforms), to show that the NtraC model applies to a growing group of SPs. We demonstrate that the full-length SPs of RGMa and DCBLD2 are functional and furthermore that the C-domains are sufficient and essential for ER targeting, whereas the N-domains are dispensable. Thus, the N-domains are available for additional functions.     

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.