Primary Succession on a Hawaiian Dryland Chronosequence

We used measurements from airborne imaging spectroscopy and LiDAR to quantify the biophysical structure and composition of vegetation on a dryland substrate age gradient in Hawaii. Both vertical stature and species composition changed during primary succession, and reveal a progressive increase in vertical stature on younger substrates followed by a collapse on Pleistocene-aged flows. Tall-stature Metrosideros polymorpha woodlands dominated on the youngest substrates (hundreds of years), and were replaced by the tall-stature endemic tree species Myoporum sandwicense and Sophora chrysophylla on intermediate-aged flows (thousands of years). The oldest substrates (tens of thousands of years) were dominated by the short-stature native shrub Dodonaea viscosa and endemic grass Eragrostis atropioides. We excavated 18 macroscopic charcoal fragments from Pleistocene-aged substrates. Mean radiocarbon age was 2,002 years and ranged from < 200 to 7,730. Genus identities from four fragments indicate that Osteomeles spp. or M. polymorpha once occupied the Pleistocene-aged substrates, but neither of these species is found there today. These findings indicate the existence of fires before humans are known to have occupied the Hawaiian archipelago, and demonstrate that a collapse in vertical stature is prevalent on the oldest substrates. This work contributes to our understanding of prehistoric fires in shaping the trajectory of primary succession in Hawaiian drylands.     

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice

Background

Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.

Methods

Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.

Results

RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.

Conclusions

MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0176-8) contains supplementary material, which is available to authorized users.     

Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda R ed operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements.     

Spectroscopic characterization and molecular weight distribution of dissolved organic matter in sediment porewaters from Lake Erhai, Southwest China

Dissolved organic matter (DOM) in sediment porewaters from Lake Erhai, Southwest China was investigated using dissolved organic carbon (DOC) concentration, UV absorbance, fluorescence and molecular weight distribution. DOC exhibited a high concentration at the sediment–water interface with a rapid decrease to the oxic–anoxic interface at approximately 7 cm, and then increased with depth. Similar trends were also found for the UV absorption coefficients at 254 and 280 nm in the porewaters. DNA in the sediment was also measured, which confirmed the high abundance of aerobic bacteria in the upper layer of the sediment. Both humic-like (peaks A and C) and protein-like (peaks B and D) fluorescence were observed in the porewater DOM, and their fluorescence intensities exhibited a similar porewater profile as DOC concentration. A strong correlation was found between the peak fluorescence intensity ratio r(A, C) and r(D, B). Both the fluorescence index and UV absorption coefficient at 254 nm suggested a dramatic increase in aromaticity of porewater DOM across the oxic–anoxic interface. Porewater DOM exhibited a multimodal distribution of molecular weight with a relatively low polydispersity. The results of this study offer significant insight into the nature and properties of DOM in freshwater ecosystems.     

Fine mapping of the recessive genic male-sterile gene (Bnms1) in Brassica napus L.

A recessive genic male sterility (RGMS) system, S45 AB, has been developed from spontaneous mutation in Brassica napus canola variety Oro, and is being used for hybrid cultivar development in China. The male sterility of S45 was controlled by two duplicated recessive genes, named as Bnms1 and Bnms2. In this study, a NIL (near-isogenic line) population from the sib-mating of S45 AB was developed and used for the fine mapping of the Bnms1 gene, in which the recessive allele was homozygous at the second locus. AFLP technology combined with BSA (bulked segregant analysis) was used. From a survey of 2,560 primer combinations (+3/+3 selective bases), seven AFLP markers linked closely to the target gene were identified, of which four were successfully converted to sequence characterized amplified region (SCAR) markers. For further analysis, a population of 1,974 individuals was used to map the Bnms1 gene. On the fine map, Bnms1 gene was flanked by two SCAR markers, SC1 and SC7, with genetic distance of 0.1 cM and 0.3 cM, respectively. SC1 was subsequently mapped on linkage group N7 using doubled-haploid mapping populations derived from the crosses Tapidor × Ningyou7 and DH 821 × DHBao 604, available at IMSORB, UK, and our laboratory, respectively. Linkage of an SSR marker, Na12A02, with the Bnms1 gene further confirmed its location on linkage group N7. Na12A02, 2.6 cM away from Bnms1, was a co-dominant marker. These molecular markers developed from this research will facilitate the marker-assisted selection of male sterile lines and the fine map lays a solid foundation for map-based cloning of the Bnms1 gene.     

Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

Solution structure of mu-conotoxin PIIIA,a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.     

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

狼毒种子萌发特性与种群更新机制的研究

研究了采集于植株上的和收集于土壤种子库的狼毒(Stellera chamaejasme)种子在不同温度、光照和5种预处理(即破裂种皮、去除种皮、98％H2SO4浸种5min、0．2％KNO3浸种24h、10℃低温保存1周)条件下的萌发力。结果表明,狼毒种子萌发率较低,25℃恒温、黑暗条件下萌发率为13％,较适宜的萌发温度为30℃恒温或10～30℃变温,破裂种皮和去除种皮萌发率显著提高,25℃恒温、光暗交替条件下萌发率分别为49％和47％,浓硫酸浸种5min处理萌发率可达到32％,KON3浸种和10℃低温保存两个处理对促进狼毒种子萌发效果不明显,狼毒种子萌发对光照条件不敏感,种子硬实性是导致狼毒种子萌发率较低的主要原因,取自土壤种子库内的狼毒种子萌发率高于当年采集的种子,在自然条件下,并非每年都有狼毒种子萌发长成幼苗,种群更新时机是随机的或周期性的。