MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

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Effects of grazing intensity and grazing exclusion on litter decomposition in the temperate steppe of Nei Mongol,China

Aims Grazing intensity and grazing exclusion affect ecosystem carbon cycling by changing the plant community and soil micro-environment in grassland ecosystems. The aims of this study were: 1) to determine the effects of grazing intensity and grazing exclusion on litter decomposition in the temperate grasslands of Nei Mongol; 2) to compare the difference between above-ground and below-ground litter decomposition; 3) to identify the effects of precipitation on litter production and decomposition. Methods We measured litter production, quality, decomposition rates and soil nutrient contents during the growing season in 2011 and 2012 in four plots, i.e. light grazing, heavy grazing, light grazing exclusion and heavy grazing exclusion. Quadrate surveys and litter bags were used to measure litter production and decomposition rates. All data were analyzed with ANOVA and Pearson’s correlation procedures in SPSS. Important findings Litter production and decomposition rates differed greatly among four plots. During the two years of our study, above-ground litter production and decomposition in heavy-grazing plots were faster than those in light-grazing plots. In the dry year, below-ground litter production and decomposition in light-grazing plots were faster than those in heavy-grazing plots, which is opposite to the findings in the wet year. Short-term grazing exclusion could promote litter production, and the exclusion of light-grazing could increase litter decomposition and nutrient cycling. In contrast, heavy-grazing exclusion decreased litter decomposition. Thus, grazing exclusion is beneficial to the restoration of the light-grazing grasslands, and more human management measures are needed during the restoration of heavy-grazing grasslands. Precipitation increased litter production and decomposition, and below-ground litter was more vulnerable to the inter-annual change of precipitation than above-ground litter. Compared to the light-grazing grasslands, heavy-grazing grasslands had higher sensitivity to precipitation. The above-ground litter decomposition was strongly positively correlated with the litter N content (R² = 0.489, p < 0.01) and strongly negatively correlated with the soil total N content (R² = 0.450, p < 0.01), but it was not significantly correlated with C:N and lignin:N. Below-ground litter decomposition was negatively correlated with the litter C (R² = 0.263, p < 0.01), C:N (R² = 0.349, p < 0.01) and cellulose content (R² = 0.460, p < 0.01). Our results will provide a theoretical basis for ecosystem restoration and the research of carbon cycling.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Restriction mapping of the rRNA genes from Artemia larvae

A restriction endonuclease analysis of the genes coding for the ribosomal RNA from Artemia larvae has shown that these genes consist of a repeat unit of 16.2 kilobase pairs (10.7 Mdaltons) and that the repeat unit seems to be homogeneous in size.     

Role of Activity of Gastrointestinal Microflora in Absorption of Calcium and Magnesium in Rats Fed β1-4 Linked Galactooligosaccharides

Rats fed a diet containing β1-4 linked galactooligosaccharides (GOS) (5 g/100 g of diet) absorbed calcium and magnesium more efficiently than those fed the control diet. However, the increment obtained through GOS-feeding was reduced by neomycin sulfate 0.67 g/100 g of diet). Since the decrease in cecal pH in rats fed GOS was suppressed by neomycin-feeding, bacterial action in the digestive tract was considered to be reduced by neomycin-feeding. Our findings suggest that the action of intestinal bacteria is necessary for the effects of GOS.     

Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.