Structure and function of the Ts2631 endolysin of <i>Thermus scotoductus</i> phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding

Abstract To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents

Biomimetic Silica Particles with Self-Loading BMP-2 Knuckle Epitope Peptide and Its Delivery for Bone Regeneration

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis

The Ardón L6 ordinary chondrite : a long-hidden Spanish meteorite fall

We report and describe an L6 ordinary chondrite fall that occurred in Ardón, León province, Spain (longitude 5.5605°W, latitude 42.4364°N) on July 9th, 1931. The 5.5 g single stone was kept hidden for 83 yr by Rosa Gonzólez Perez, at the time an 11 yr old who had observed the fall and had recovered the meteorite. According to various newspaper reports, the event was widely observed in Northern Spain. Ardón is a very well-preserved, fresh, strongly metamorphosed (petrologic type 6), and weakly shocked (S3) ordinary chondrite with well-equilibrated and recrystallized minerals. The mineral compositions (olivine Fa23.v±o.3, low-Ca pyroxene Fs2o.4±o.2WoL5±o.2, plagioclase An10.3i0.5Ab84.3iL2), magnetic susceptibility (log v = 4.95 i 0.05 x 10-9 m3 kg-1), bulk density (3.49 i 0.05 g cm-3), grain density (3.58 i 0.05 g cm-3), and porosity (2.5 vol%) are typical for L6 chondrites. Short-lived radionuclides confirm that the meteorite constitutes a recent fall. The 21Ne and 38Ar cosmic ray exposure ages are both about 20-30 Ma, similar to values for many other L chondrites. The cosmogenic 22Ne/21Ne ratio indicates that preatmospheric Ardón was a relatively large body. The fact that the meteorite was hidden in private hands for 83 yr makes one wonder if other meteorite falls may have experienced the same fate, thus possibly explaining the anomalously low number of falls reported in continental Spain in the 20th century

Efficient active transport of gene nanocarriers to the cell nucleus

The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of nature's most efficient DNA viruses and nonviral polyethylenimine/DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation