Visualizing plant development and gene expression in three dimensions using optical projection tomography

A deeper understanding of the mechanisms that underlie plant growth and development requires quantitative data on three-dimensional (3D) morphology and gene activity at a variety of stages and scales. To address this, we have explored the use of optical projection tomography (OPT) as a method for capturing 3D data from plant specimens. We show that OPT can be conveniently applied to a wide variety of plant material at a range of scales, including seedlings, leaves, flowers, roots, seeds, embryos, and meristems. At the highest resolution, large individual cells can be seen in the context of the surrounding plant structure. For naturally semitransparent structures, such as roots, live 3D imaging using OPT is also possible. 3D domains of gene expression can be visualized using either marker genes, such as beta-glucuronidase, or more directly by whole-mount in situ hybridization. We also describe tools and software that allow the 3D data to be readily quantified and visualized interactively in different ways.     

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.     

Phenacetin acts as a weak genotoxic compound preferentially in the kidney of DNA repair deficient Xpa mice

Cell cycle checkpoints and DNA repair capacity are critical for the maintenance of genome integrity. We hypothesized that, in comparison to healthy controls, esophageal cancer patients might have a higher frequency of deficiencies in cell cycle checkpoints and/or DNA repair system. Using flow cytometry and comet assay, we assessed the γ-radiation-induced S phase and G2-M phase accumulation, and benzo(a)pyrene-diol-epoxide (BPDE)- and γ-radiation-induced DNA damage, in peripheral blood lymphocytes of 99 newly diagnosed esophageal cancer patients and 112 age-, gender-, and ethnicity-matched healthy controls. The mean γ-radiation-induced cell accumulation at G2-M phase was significantly lower in esophageal cancer patients than the control subjects (case versus control: 5.27% ± 5.11% versus. 7.06% ± 5.04%, P = 0.013). The less G2-M phase cell accumulation resulted in a significant increased risk for esophageal cancer with an odds ratio of 2.08 (95% confidence interval 1.15–3.77). After normalization to baseline S fraction, the radiation-induced increment in the 4N/2N ratio was also significantly lower in esophageal cancer patients than in controls (case versus control: 0.76% versus 1.04%, P = 0.0039). The less increment in the radiation-induced 4N/2N ratio was associated with 2.24(95% confidence interval 1.22–4.11)-fold increase of esophageal cancer risk. We also compared the mutagen-induced DNA damage level among individuals with different S or G2-M phase cell accumulation. We found that the less G2-M phase accumulation was associated with both high BPDE induced and γ-radiation-induced DNA damage in the healthy controls (P for trend = 0.023 and 0.015, respectively). Similar pattern was observed for S phase accumulation (P for trend = 0.033 and 0.022, respectively). However, such association was not seen in esophageal cancer patients. This study provides the first molecular epidemiologic evidence linking increased esophageal cancer risk with defects in cell-cycle checkpoints and DNA repair capacity.     

Circulating oxidized LDL: determinants and association with brachial flow-mediated dilation

Circulating oxidized LDL (oxLDL) levels are strongly correlated to LDL-cholesterol (LDL-c) and apolipoprotein-B100 (apoB100), making it difficult to disentangle their independent contributions to cardiovascular risk. We explored the determinants of oxLDL and the relation between oxLDL and flow-mediated dilation (FMD) of the brachial artery to investigate whether the oxLDL/LDL-c and oxLDL/apoB100 ratios are more informative than the separate variables. FMD of the brachial artery and plasma concentrations of oxLDL, LDL-cholesterol, and apoB100 were measured in 624 men and women (age range 50 to 87 years), participating in a population-based cohort study. OxLDL was strongly correlated with apoB100 (r = 0.82, P < 0.001) and LDL-c (r = 0.67, P < 0.001). Other major independent determinants of oxLDL were sex, HDL-cholesterol, and LDL particle size. LDL-c and apoB100 concentrations were not significantly associated with FMD. After adjustment for age, sex, glucose tolerance status, and Framingham risk score, the oxLDL/apoB100 ratio was negatively related to FMD (P = 0.017). This association was weaker for the oxLDL/ LDL-c ratio (P = 0.062) and absent for oxLDL level (P = 0.27). In contrast to oxLDL, the oxLDL/apoB100 ratio, and to a lesser extent the oxLDL/LDL-c ratio, are related to a functional measure of atherosclerosis. Therefore correction of oxLDL for LDL particle number may improve the clinical usefulness of oxLDL measurement.     

Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca²⁺ handling, as indicated by analysis of Ca²⁺ transients in intact myofibers and expression of Ca²⁺-handling proteins, but can be explained by the reduced myofilament Ca²⁺ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca²⁺ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.     

Cryptic Variation between Species and the Basis of Hybrid Performance

Crosses between closely related species give two contrasting results. One result is that species hybrids may be inferior to their parents, for example, being less fertile [1]. The other is that F1 hybrids may display superior performance (heterosis), for example with increased vigour [2]. Although various hypotheses have been proposed to account for these two aspects of hybridisation, their biological basis is still poorly understood [3]. To gain further insights into this issue, we analysed the role that variation in gene expression may play. We took a conserved trait, flower asymmetry in Antirrhinum, and determined the extent to which the underlying regulatory genes varied in expression among closely related species. We show that expression of both genes analysed, CYC and RAD, varies significantly between species because of cis-acting differences. By making a quantitative genotype-phenotype map, using a range of mutant alleles, we demonstrate that the species lie on a plateau in gene expression-morphology space, so that the variation has no detectable phenotypic effect. However, phenotypic differences can be revealed by shifting genotypes off the plateau through genetic crosses. Our results can be readily explained if genomes are free to evolve within an effectively neutral zone in gene expression space. The consequences of this drift will be negligible for individual loci, but when multiple loci across the genome are considered, we show that the variation may have significant effects on phenotype and fitness, causing a significant drift load. By considering these consequences for various gene-expression–fitness landscapes, we conclude that F1 hybrids might be expected to show increased performance with regard to conserved traits, such as basic physiology, but reduced performance with regard to others. Thus, our study provides a new way of explaining how various aspects of hybrid performance may arise through natural variation in gene activity.     

Is serum HMGB1 a biomarker in ANCA-associated vasculitis?

Background

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease.

Methods

AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses.

Results

HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 ± 1.80 ng/ml vs. 2.39 ± 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 ± 1.83 ng/ml vs. MPA: 3.11 ± 1.91 ng/ml vs. RLV: 1.92 ± 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 ± 1.48 ng/ml vs. 3.52 ± 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (ρ = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients).

Conclusions

Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. In contrast to SLE, HMGB1 is not a useful biomarker in AAV.     

Multiple Inflammatory Biomarker Detection in a Prospective Cohort Study: A Cross-Validation between Well-Established Single-Biomarker Techniques and an Electrochemiluminescense-Based Multi-Array Platform

Background

In terms of time, effort and quality, multiplex technology is an attractive alternative for well-established single-biomarker measurements in clinical studies. However, limited data comparing these methods are available.

Methods

We measured, in a large ongoing cohort study (n = 574), by means of both a 4-plex multi-array biomarker assay developed by MesoScaleDiscovery (MSD) and single-biomarker techniques (ELISA or immunoturbidimetric assay), the following biomarkers of low-grade inflammation: C-reactive protein (CRP), serum amyloid A (SAA), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1). These measures were realigned by weighted Deming regression and compared across a wide spectrum of subjects’ cardiovascular risk factors by ANOVA.

Results

Despite that both methods ranked individuals’ levels of biomarkers very similarly (Pearson’s r all≥0.755) absolute concentrations of all biomarkers differed significantly between methods. Equations retrieved by the Deming regression enabled proper realignment of the data to overcome these differences, such that intra-class correlation coefficients were then 0.996 (CRP), 0.711 (SAA), 0.895 (sICAM-1) and 0.858 (sVCAM-1). Additionally, individual biomarkers differed across categories of glucose metabolism, weight, metabolic syndrome and smoking status to a similar extent by either method.

Conclusions

Multiple low-grade inflammatory biomarker data obtained by the 4-plex multi-array platform of MSD or by well-established single-biomarker methods are comparable after proper realignment of differences in absolute concentrations, and are equally associated with cardiovascular risk factors, regardless of such differences. Given its greater efficiency, the MSD platform is a potential tool for the quantification of multiple biomarkers of low-grade inflammation in large ongoing and future clinical studies.     

Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi''s sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.     

Non-proteolytic functions of calpain-3 in sarcoplasmic reticulum in skeletal muscles

Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using “knock-in” (referred to as Capn3^CS/CS) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672-2683]. However, compared to Capn3-null (Capn3^−/−) mice, Capn3^CS/CS mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3^CS/CS mice with Capn3^−/− mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3^CS/CS mice showed that the nonenzymatic role of calpain-3 is required for proper Ca²⁺ efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca²⁺ efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A.