Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles

We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.     

SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer

SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.     

Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.     

Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms

Background aimsMesenchymal stromal cells (MSC) can be isolated from the perivascular connective tissue of umbilical cords, called Wharton's jelly. These human umbilical cord perivascular cells (HUCPVC) might provide therapeutic benefits when treating skeletal or cutaneous malformations in neonatal patients.MethodsHUCPVC were isolated, and their proliferation rate, marker expression and multilineage differentiation potential determined. HUCPVC or their conditioned medium (HUCPVC-CM) was injected into the excisional wound of a mouse splinted-wound model. The effects of the treatment on wound closure were examined by morphohistochemical and gene expression analyses.ResultsHUCPVC expressed typical MSC markers and could differentiate into osteoblastic and adipogenic lineages. HUCPVC transplanted into the mouse wound accelerated wound closure. Immunohistologic analysis showed that the HUCPVC accelerated wound healing by enhancing collagen deposition and angiogenesis via paracrine mechanisms. Furthermore, treatment with HUCPVC-CM alone significantly enhanced wound closure. HUCPVC-CM increased the number of anti-inflammatory M2 macrophages expressing resistin-like molecule (RELM)-α/CD11b and promoted neovessel maturation. Quantitative polymerase chain reaction (PCR) analysis showed that HUCPVC-CM increased the expression of tissue-repairing cytokines interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound.ConclusionsOur results show that HUCPVC promotes wound healing via multifaceted paracrine mechanisms. Together with their ability to differentiate into the osteogenic linage, HUCPVC may provide significant therapeutic benefits for treating wounds in neonatal patients.     

A highly efficient construction of GM1 epitope tetrasaccharide and its conjugation with KLH

GM1 epitope tetrasaccharide was synthesized by a condensation of sialyl-alpha(2-3)-gal acceptor and gal-beta(1-3)-GalN donor in a highly efficient manner. After introduction of mercaptohexanol to the tetrasaccharide, it was coupled to maleimide-activated KLH carrier protein to give the desired GM1 epitope-KLH conjugate.     

Design and synthesis of versatile ganglioside probes for carbohydrate microarrays

A series of ganglioside GM1-, GM2-, and GM3-type probes, in which the ceramide portion is replaced with a glucose residue, were systematically synthesized based on a convergent synthetic method.     

Short-term cold storage of blowfly Lucilia sericata embryos

The developmental rate under low temperatures and cold tolerance were investigated in embryos of the blowfly Lucilia sericata. The larvae of this species are now widely used in maggot debridement therapy. Embryonic development was dependent on temperature, with a lower developmental threshold of 9.0 °C. The duration of the egg stage at a rearing temperature of 25 °C was 14 h, and a low temperature of 12.5 °C successfully prolonged this period to 66 h. Embryonic stages differed markedly in their cold tolerance; young embryos were less tolerant to cold than old ones. Late embryonic stages are suitable for cold storage at 5 °C and the storage for 72 h did not decrease the hatching rate by more than 50%. In the mass‐rearing process required for maggot debridement therapy, either of these two simple protocols would be beneficial.     

Circatidal activity rhythm in the mangrove cricket Apteronemobius asahinai

Mangrove forests are influenced by tidal flooding and ebbing for a period of approximately 12.4 hours (tidal cycle). Mangrove crickets (Apteronemobius asahinai) forage on mangrove forest floors only during low tide. Under constant darkness, most crickets showed a clear bimodal daily pattern in their locomotor activity for at least 24 days; the active phases of approximately 10 hours alternated with inactive phases of approximately 2 hours, which coincided with the time of high tide in the field. The free-running period was 12.56+/-0.13 hours (mean+/-s.d. n=11). This endogenous rhythm was not entrained by the subsequent 24 hours light-dark cycle, although it was suppressed in the photophase; the active phase in the scotophase continued from the active phase in the previous constant darkness, with no phase shift. The endogenous rhythm was assumed to be a circatidal rhythm. On the other hand, the activity under constant darkness subsequent to a light-dark cycle was more intense in the active phase continuing from the scotophase than from the photophase of the preceding light-dark cycle; this indicates the presence of circadian components. These results suggest that two clock systems are involved in controlling locomotor activity in mangrove crickets.     

Total synthesis of 6-O-sulfo-sialylparagloboside: a widely useful glycoprobe for biochemical research

The total synthesis of 6-O-sulfo-sialylparagloboside is described. A suitably protected beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-GlcpOSE derivative was glycosylated with an alpha-D-Neup5Ac-(2-->3)-D-Galp derived imidate to give the corresponding protected alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-d-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-GlcpOSE pentasaccharide derivative. Proper manipulation of the protecting groups of the pentasaccharide afforded the corresponding glycosyl imidate, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol. Selective reduction of the azido group, N-acylation with octadecanoic acid, 6-O-sulfation of the GlcpNAc residue, and complete removal of the protecting groups gave the desired 6-O-sulfo-sialylparagloboside.