Molecular Characterization, Tissue Distribution, Subcellular Localization and Actin-Sequestering Function of a Thymosin Protein from Silkworm

We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development

Large-Molecule Decomposition Products of Electrolytes and Additives Revealed by On-Electrode Chromatography and MALDI

The decomposition of electrolyte and additive molecules has a critical impact on battery performance. In this work, large-molecule decomposition products in solid-electrolyte interphase (SEI) are identified with clear structure assignment by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The MALDI analysis is facilitated by on-electrode chromatography that serves to fractionate different molecular species on the electrode surfaces prior to MS measurements. These methods exemplify a practical and readily adoptable strategy to characterize the organic elements in SEIs

Large-Molecule Decomposition Products of Electrolytes and Additives Revealed by On-Electrode Chromatography and MALDI

The decomposition of electrolyte and additive molecules has a critical impact on battery performance. In this work, large-molecule decomposition products in solid-electrolyte interphase (SEI) are identified with clear structure assignment by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The MALDI analysis is facilitated by on-electrode chromatography that serves to fractionate different molecular species on the electrode surfaces prior to MS measurements. These methods exemplify a practical and readily adoptable strategy to characterize the organic elements in SEIs

Optimizing an Antioxidant TEMPO Copolymer for Reactive Oxygen Species Scavenging and Anti-Inflammatory Effects in Vivo.

Oxidative stress is broadly implicated in chronic, inflammatory diseases because it causes protein and lipid damage, cell death, and stimulation of inflammatory signaling. Supplementation of innate antioxidant mechanisms with drugs such as the superoxide dismutase (SOD) mimetic compound 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is a promising strategy for reducing oxidative stress-driven pathologies. TEMPO is inexpensive to produce and has strong antioxidant activity, but it is limited as a drug due to rapid clearance from the body. It is also challenging to encapsulate into micellar nanoparticles or polymer microparticles, because it is a small, water soluble molecule that does not efficiently load into hydrophobic carrier systems. In this work, we pursued a polymeric form of TEMPO [poly(TEMPO)] to increase its molecular weight with the goal of improving in vivo bioavailability. High density of TEMPO on the poly(TEMPO) backbone limited water solubility and bioactivity of the product, a challenge that was overcome by tuning the density of TEMPO in the polymer by copolymerization with the hydrophilic monomer dimethylacrylamide (DMA). Using this strategy, we formed a series of poly(DMA-co-TEMPO) random copolymers. An optimal composition of 40 mol % TEMPO/60 mol % DMA was identified for water solubility and O2•- scavenging in vitro. In an air pouch model of acute local inflammation, the optimized copolymer outperformed both the free drug and a 100% poly(TEMPO) formulation in O2•- scavenging, retention, and reduction of TNFα levels. Additionally, the optimized copolymer reduced ROS levels after systemic injection in a footpad model of inflammation. These results demonstrate the benefit of polymerizing TEMPO for in vivo efficacy and could lead to a useful antioxidant polymer formulation for next-generation anti-inflammatory treatments

Optimizing an Antioxidant TEMPO Copolymer for Reactive Oxygen Species Scavenging and Anti-Inflammatory Effects in Vivo

Oxidative stress is broadly implicated in chronic, inflammatory diseases because it causes protein and lipid damage, cell death, and stimulation of inflammatory signaling. Supplementation of innate antioxidant mechanisms with drugs such as the superoxide dismutase (SOD) mimetic compound 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) is a promising strategy for reducing oxidative stress-driven pathologies. TEMPO is inexpensive to produce and has strong antioxidant activity, but it is limited as a drug due to rapid clearance from the body. It is also challenging to encapsulate into micellar nanoparticles or polymer microparticles, because it is a small, water soluble molecule that does not efficiently load into hydrophobic carrier systems. In this work, we pursued a polymeric form of TEMPO [poly(TEMPO)] to increase its molecular weight with the goal of improving in vivo bioavailability. High density of TEMPO on the poly(TEMPO) backbone limited water solubility and bioactivity of the product, a challenge that was overcome by tuning the density of TEMPO in the polymer by copolymerization with the hydrophilic monomer dimethylacrylamide (DMA). Using this strategy, we formed a series of poly(DMA-co-TEMPO) random copolymers. An optimal composition of 40 mol % TEMPO/60 mol % DMA was identified for water solubility and O(2)(•−) scavenging in vitro. In an air pouch model of acute local inflammation, the optimized copolymer outperformed both the free drug and a 100% poly(TEMPO) formulation in O(2)(•−) scavenging, retention, and reduction of TNFα levels. Additionally, the optimized copolymer reduced ROS levels after systemic injection in a footpad model of inflammation. These results demonstrate the benefit of polymerizing TEMPO for in vivo efficacy and could lead to a useful antioxidant polymer formulation for next-generation anti-inflammatory treatments

Transcription and expression level of BmTHY in different development stages of <i>Bombyx mori</i>.

<p>(a) Analysis of BmTHY expression was performed by RT-PCR. Relative BmTHY expression was determined in relation to the corresponding BmTHY expression level in the silkworm moth: ΔΔC_T (stage) = ΔC_T (stage)−ΔC_T (egg); (b) Western blotting analysis of the expression levels of of BmTHY in different development stages. 1,egg;2,pupa; 3,larva;4,moth.</p

Determine of polyclonal antibody titer by ELISA.

<p>ELISA was used to determine the following ratio for antibodies against rBmTHY: positive serum extinction value/negative serum extinction value (P/N)≥2.1 was positive; 1.5≤P/N<2.1 was suspicious expression; P/N<1.5 was negative. From this, the titer of the antibodies was greater than 1∶25600 at a concentration of 10 µg/mL.</p

Western blotting analysis of the His-tag-BmTHY fusion protein expression.

<p>Samples were resolved by 12% SDS PAGE under reducing conditions (M,1,2,3 SDS-PAGE; 1′, 2′,3′ Western blotting). M: protein molecular weight marker (low); 1: purified fusion protein expressed in <i>E.coli</i> Rosetta; 2: supernatant of <i>E.coli</i> Rosetta/pET-28a-BmTHY induced by IPTG after supersonic treatment; 3: <i>E.coli</i> Rosetta/pET-28a. Arrow indicates the fragment of the His-tag fusion BmTHY.</p

Expression and purification of the His-tag-BmTHY fusion protein.

<p>Samples were resolved by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. A: Expression of fusion protein in Rosetta (DE3); M: protein molecular weight marker (low); 1: Rossetta (pET-28a-BmTHY) without induction; 2: Rossetta (pET-28a-BmTHY) after induction; B: Purification of the His-tag fusion protein in Rosetta (DE3); M: protein molecular weight marker (low); 1: supernatant of <i>E.coli</i> Rosetta/pET-28a-BmTHY induced by IPTG after supersonic treatment; 2: purified fusion protein expressed in <i>E.coli</i> Rosetta.</p