Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.     

Heavy metal transport by the CusCFBA efflux system

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi‐protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu⁺ and Ag⁺ ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.     

Enhanced enterovirus 71 virus‐like particle yield from a new baculovirus design

Enterovirus 71 (EV71) is responsible for the outbreaks of hand‐foot‐and‐mouth disease in the Asia‐Pacific region. To produce the virus‐like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co‐express EV71 P1 polypeptide and 3CD protease using the Bac‐to‐Bac^® vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD? vector system which was deficient in v‐cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD? system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF‐P1‐C3CD, a recombinant baculovirus constructed using the flashBAC GOLD^TM system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five^TM cells with BacF‐P1‐C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 μg purified VLP induced cross‐protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 μg VLP, one liter High Five^TM culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines. Biotechnol. Bioeng. 2015;112: 2005–2015. © 2015 Wiley Periodicals, Inc.

     

Relationship between angiotensin-converting enzyme ID polymorphism and VO(2max) of Chinese males

Several studies have shown that the angiotensin-converting enzyme (ACE) I allele is associated with enhanced physical performance. We investigated whether this phenomenon is observed in a cohort of 67 Chinese men in Singapore. Angiotensin-converting enzyme ID polymorphism was typed with PCR method and maximal oxygen uptake (VO(2max)) of the DD, ID, and II genotypes was compared. Analysis of covariance revealed that VO(2max) was significantly higher (p<0.05) for the DD genotype (57.86 +/- 3.5 ml.kg.(-1)min(-1)) versus the ID (50.58 +/- 1.80 ml.kg.(-1)min(-1)) or II (50.48 +/- 1.58 ml.kg.(-1) min(-1)) genotype. Our findings suggest that the ACE DD genotype in young adult Chinese males is associated with higher levels of VO(2max).     

Solution‐Processed TiO2 Nanoparticles as the Window Layer for CuIn(S,Se)2 Devices

As a wide‐bandgap semiconductor, titanium dioxide (TiO₂) with a porous structure has proven useful in dye‐sensitized solar cells, but its application in low‐cost, high‐efficiency inorganic photovoltaic devices based on materials such as Cu(InGa)Se₂ or Cu₂ZnSnS₄ is limited. Here, a thin film made from solution‐processed TiO₂ nanocrystals is demonstrated as an alternative to intrinsic zinc oxide (i‐ZnO) as the window layer of CuInS_xSe_1?x solar cells. The as‐synthesized, well‐dispersed, 6 nm TiO₂ nanocrystals are assembled into thin films with controllable thicknesses of 40, 80, and 160 nm. The TiO₂ nanocrystal films with thicknesses of 40 and 80 nm exhibit conversion efficiencies (6.2% and 6.33%, respectively) that are comparable to that of a layer of the typical sputtered i‐ZnO (6.42%). The conversion efficiency of the devices with a TiO₂ thickness of 160 nm decreases to 2.2%, owing to the large series resistance. A 9‐hour reaction time leads to aggregated nanoparticles with a much‐lower efficiency (2%) than that of the well‐dispersed TiO₂ nanoparticles prepared using a 15‐hour reaction time. Under optimized conditions, the champion TiO₂ nanocrystal‐film‐based device shows even higher efficiency (9.2%) than a control device employing a typical i‐ZnO film (8.6%).     

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161^Bright mucosal-associated invariant T (MAIT) and CD56^Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.     

Food mechanical properties in three sympatric species of Hapalemur in Ranomafana National Park,Madagascar

We investigated mechanical dietary properties of sympatric bamboo lemurs, Hapalemur g. griseus, H. aureus, and H. (Prolemur) simus, in Ranomafana National Park, Madagascar. Each lemur species relies on bamboo, though previous behavioral observations found that they specialize on different parts of a common resource (Tan: Int J Primatol 20 1999 547–566; Tan: PhD dissertation 2000 State University of New York, Stony Brook). On the basis of these earlier behavioral ecology studies, we hypothesized that specialization on bamboo is related to differences in mechanical properties of specific parts. We quantified mechanical properties of individual plant parts from the diets of the bamboo lemur species using a portable tester. The diets of the Hapalemur spp. exhibited high levels of mechanical heterogeneity. The lemurs, however, could be segregated based on the most challenging (i.e., mechanically demanding) foods. Giant bamboo culm pith was the toughest and stiffest food eaten, and its sole lemur consumer, H. simus, had the most challenging diet. However, the mechanical dietary properties of H. simus and H. aureus overlapped considerably. In the cases where lemur species converged on the same bamboo part, the size of the part eaten increased with body size. Plant parts that were harvested orally but not necessarily masticated were the most demanding, indicating that food preparation may place significant loads on the masticatory apparatus. Finally, we describe how mechanical properties can influence feeding behavior. The elaborate procurement processes of H. simus feeding on culm pith and H. griseus and H. aureus feeding on young leaf bases are related to the toughnesses of protective coverings and the lemurs' exploitation of mechanical vulnerabilities in these plants. Am J Phys Anthropol, 2009. © 2008 Wiley‐Liss, Inc.     

Speciation processes in putative island endemic sister bat species: false impressions from mitochondrial DNA and microsatellite data

Cases of geographically restricted co‐occurring sister taxa are rare and may point to potential divergence with gene flow. The two bat species Murina gracilis and Murina recondita are both endemic to Taiwan and are putative sister species. To test for nonallopatric divergence and gene flow in these taxa, we generated sequences using Sanger and next‐generation sequencing, and combined these with microsatellite data for coalescent‐based analyses. MtDNA phylogenies supported the reciprocally monophyletic sister relationship between M. gracilis and M. recondita; however, clustering of microsatellite genotypes revealed several cases of species admixture suggesting possible introgression. Sequencing of microsatellite flanking regions revealed that admixture signatures stemmed from microsatellite allele homoplasy rather than recent introgressive hybridization, and also uncovered an unexpected sister relationship between M. recondita and the continental species Murina eleryi, to the exclusion of M. gracilis. To dissect the basis of these conflicts between ncDNA and mtDNA, we analysed sequences from 10 anonymous ncDNA loci with *beast and isolation‐with‐migration and found two distinct clades of M. eleryi, one of which was sister to M. recondita. We conclude that Taiwan was colonized by the ancestor of M. gracilis first, followed by the ancestor of M. recondita after a period of allopatric divergence. After colonization, the mitochondrial genome of M. recondita was replaced by that of the resident M. gracilis. This study illustrates how apparent signatures of sympatric divergence can arise from complex histories of allopatric divergence, colonization and hybridization, thus highlighting the need for rigorous analyses to distinguish between such scenarios.     

NELL-1 binds to APR3 affecting human osteoblast proliferation and differentiation

Nel-like protein 1 (NELL-1) is an osteoinductive molecule associated with premature calvarial suture closure. Here we identified apoptosis related protein 3 (APR3), a membrane protein known as a proliferation suppressor, as a binding protein of NELL-1 by biopanning. NELL-1 and APR3 colocalized on the nuclear envelope of human osteoblasts. NELL-1 significantly inhibited proliferation of osteoblasts co-transfected with APR3 through further down-regulation of Cyclin D1. The co-expression of NELL-1 and APR3 enhanced Ocn and Bsp expression and mineralization. RNAi of APR3 significantly reduced the differentiation effect of NELL-1. These findings suggest that the effects of NELL-1 on osteoblastic differentiation and proliferation are partly through binding to APR3.     

Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix

The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.