Studying the Morphology of Lyophilized Protein Solids Using X-ray Micro-CT: Effect of Post-freeze Annealing and Controlled Nucleation

The objective of this study was to determine how different techniques used during the freezing step of lyophilization affect morphology of the dried protein solids. Aqueous solutions containing recombinant human albumin, trehalose, and sodium phosphate buffer were dried after their freezing by shelf-ramp cooling, immersion in liquid nitrogen, or controlled ice nucleation. Some shelf-frozen solutions were heat treated (annealed) before the vacuum drying. We used three-dimensional (3D) X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) to study the morphology of solids. The X-ray micro-CT images of the lyophilized microporous solids showed traces of varied size and structure ice crystals that were comparable to corresponding SEM images. A post-freeze heat treatment and a controlled nucleation both induced larger ice crystal ghosts in the solids. The variations in the structure of walls surrounding ice crystals, formed by the different freezing procedures, should affect the water vapor transition during the primary and secondary drying. Some solids also showed higher-density layer in the upper surface. Overall, the simple sample preparation procedures and the ample morphological information make the X-ray micro-CT appropriate for analyzing lyophilized pharmaceuticals.     

Reply to: “Comment on root orientation can affect detection accuracy of ground-penetrating radar”

Introduction

We showed that root orientation affected a parameter of ground penetrating radar (GPR), amplitude area (A) (Tanikawa et al. Plant Soil 373:317–327, 2013). The aims of this reply to Wu et al. (2014) are (i) to correct the two inaccuracies in Tanikawa et al. (2013) and (ii) to improve our method of estimating A(90°) using A(x) of root angle x.

Methods

Measured A values of Tanikawa et al. (2013) were analyzed with the modified equations.

Results

The first inaccuracy was the use of incorrect units for the coefficient b (the phase shift) in the sinusoidal waveform of A(x). The units should have been radians instead of degrees. The second inaccuracy was the mis-derivation of A(x) into A(x?+?90°). In the modified method, A(90°) was estimated by A(x) from two orthogonally intersecting transect lines and a transect line at a diagonal to them.

Conclusions

The two inaccuracies did not affect the previous main conclusions that the parameter T was suitable for estimating root diameter and that grid transects are likely to identify clear hyperbolas reflecting roots in radar profiles (Tanikawa et al. 2013). By the improved method, we could accurately estimate root diameter by scanning using three transect lines intersecting at angles of x, x?+?45°, and x?+?90°.     

Attentional Control and Interpretation of Facial Expression after Oxytocin Administration to Typically Developed Male Adults

Deficits in attentional-inhibitory control have been reported to correlate to anger, hostility, and aggressive behavior; therefore, inhibitory control appears to play an important role in prosocial behavior. Moreover, recent studies have demonstrated that oxytocin (OT) exerts a prosocial effect (e.g., decreasing negative behaviors, such as aggression) on humans. However, it is unknown whether the positively valenced effect of OT on sociality is associated with enhanced attentional-inhibitory control. In the present study, we hypothesized that OT enhances attentional-inhibitory control and that the positively valenced effect of OT on social cognition is associated with enhanced attentional-inhibitory control. In a single-blind, placebo-controlled crossover trial, we tested this hypothesis using 20 healthy male volunteers. We considered a decrease in the hostility detection ratio, which reflects the positively valenced interpretation of other individuals’ facial expressions, to be an index of the positively valenced effects of OT (we reused the results of our previously published study). As a measure of attentional-inhibitory control, we employed a modified version of the flanker task (i.e., a shorter conflict duration indicated higher inhibitory control). These results failed to demonstrate any significant behavioral effects of OT (i.e., neither a positively valenced effect on facial cognition nor an effect on attentional-inhibitory control). However, the enhancement of attentional-inhibitory control after OT administration significantly correlated to the positively valenced effects on the interpretation of uncertain facial cognition (i.e., neutral and ambiguous facial expressions).     

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp. nov., an Agarolytic Marine Bacterium,Forms a New Clade in Vibrionaceae

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7^T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146^T (97.8% similarity) and V. agarivorans CECT 5085^T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7^T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085^T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7^T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085^T, V. hangzhouensis JCM 15146^T V. maritimus LMG 25439^T, and V. variabilis LMG 25438^T). In silico DDH data also supported the genomic relationship. The strains C7^T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1^T, V. brasiliensis LMG 20546^T, V. orientalis ATCC 33934^T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058^T and V. astriarenae C7^T are members of the newest clade of Vibrionaceae named Agarivorans.     

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.     

Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II)

Resveratrol (1, 3,5,4'-trihydroxy-trans-stilbene), a polyphenol found in grapes and other food products, is known as an antioxidant and cancer chemopreventive agent. However, 1 was shown to induce genotoxicity through a high frequency of micronucleus and sister chromatid exchange in vitro and DNA-cleaving activity in the presence of Cu(II). The present study was designed to explore the structure-activity relationship of 1 in DNA strand scission and to characterize the substrate specificity for Cu(II) and DNA binding. When pBR322DNA was incubated with 1 or its analogues differing in the number and positions of hydroxyl groups in the presence of Cu(II), the ability of 4-hydroxystilbene analogues to induce DNA strand scission is much stronger than that of 3-hydroxy analogues. The high binding affinity with both Cu(II) and DNA was also observed by 4-hydroxystilbene analogues. The reduction of Cu(II) which is essential for activation of molecular oxygen proceeded by addition of 1 to the solution of the Cu(II)-DNA complex, while such reduction was not observed with the addition of isoresveratrol, in which the 4-hydroxy group of 1 is changed to the 3-position. The results show that the 4-hydroxystilbene structure of 1 is a major determinant of generation of reactive oxygen species that was responsible for DNA strand scission.     

Enzymatic assay of allantoin in serum using allantoinase and allantoate amidohydrolase

A new enzymatic assay for specifically measuring allantoin concentration in serum has been developed. The currently used methods for allantoin analysis are time consuming and nonspecific or depend on the use of expensive equipment. In our method, allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5). The allantoate produced is hydrolyzed to ureidoglycine and ammonia by the action of allantoate amidohydrolase (EC 3.5.3.9). Nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (EC 1.4.1.4) subsequently acts on the ammonia produced, resulting in a change in absorbance at 340nm due to the consumption of reduced nicotinamide adenine dinucleotide phosphate. The amount of allantoin present is related to the change in the absorbance. The standard curve is linear up to at least 1mM allantoin. The procedure is simple, rapid, and accurate. The method has been used to measure serum allantoin levels after oral administration of purine nucleotides to experimental animals, including rats that have uricase catalyzing the conversion of urate to allantoin.     

HpSulf,a heparan sulfate 6-O-endosulfatase,is involved in the regulation of VEGF signaling during sea urchin development

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.     

Abstracts from the Japanese Journal of Limnology

The Japanese Journal of Limnology is another official publication of the Japanese Society of Limnology. The original papers in the journal were peer-reviewed by a few authorized referees, and appeared in Japanese with English abstracts.An erratum to this article can be found at     

Development of an oligo(ethylene glycol)-based SPR immunosensor for TNT detection

This paper describes the development of novel biosensor surfaces supported by robust self-assembled monolayers (SAMs) of aromatic alkanedithiol and oligo(ethylene glycol) (OEG) linker for highly sensitive surface plasmon resonance (SPR) detection of 2,4,6-trinitrotoluene (TNT). Aromatic alkanedithiol SAMs were firstly formed on Au sensor surface and TNT analogues were immobilized on it through OEG chain. Two kinds of OEG containing amine compounds, where H(2)N(C(2)H(4)O)(11)C(2)H(4)NHCOOC(CH(3))(3) served as a linker to react with carboxyl groups of TNT analogues while H(2)N(C(2)H(4)O)(3)C(2)H(4)OH served as a protein non-fouling background, were covalently bound to carboxyl terminal groups of SAMs with a certain ratio. Optimal ratio of them was also examined. Three kinds of TNT analogues, namely TNP-glycine, DNP-glycine, and DNP-acetic acid were used as immobilized ligands. Highly sensitive TNT detection by indirect competitive assay was conducted on the fabricated sensor surfaces; we examined how structural variations of them affect sensitivity in order to choose optimal hapten as well to improve sensitivity. The DNP-acetic acid immobilized surface, which had the lowest affinity to the TNT antibody among the three, showed the best limit of detection (LOD) value (ca. 80ppt (pgml(-1))). On the other hand, the TNP-glycine immobilized surface, which had the highest affinity, showed the worst LOD value (ca. 220ppt). The LOD got lower to ca. 50ppt by the use of the secondary antibody on the DNP-acetic acid immobilized surface. The sensor surfaces are durable for more than 100 times repeated use without any noticeable deterioration by their chemical stability and rather mild regeneration condition.