Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallopeptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked `CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWV X GP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel `raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWV X GP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.

Chromosome numbers for eight Trifolium species were determined. These represented first counts for T. philisticum Zoh. and T. strepens Crantz. and confirmation for T. amabile H.B.K., T. badium Schreb., T. megalanthum Steud., T. virginicum Small., T. aureum Poll., and T. physodes Stev. ex M.B. Six of the eight species had 2n = 16 chromosomes. Trifolium megalanthum, T. badium, T. aureum and T. strepens were found to have 2n = 32, 2n = 14, 2n = 16, and 2n = 16 chromosomes, respectively. T. strepens and T. aureum have the same chromosome number and morphology, and we accept them as the same species under the name of T. aureum. Karyotypic analysis of T. physodes revealed two basic types of chromosomes with nearly median and nearly submedian centromeres. Chromosome mean percentage lengths for T. physodes varied from 16.24 to 10.18 and the arm ratios varied from 1.14 to 1.68.

Breeding for pest resistance has become a major objective of most clover breeding programs. Periodic plant introductions are a valuable source of breeders' germplasm. Introductions are received from breeding programs in other countries and from plant collection expeditions. In the United States, the annual clovers are evaluated at the Southern Regional Plant Introduction Center, Griffin, Georgia; and perennials, at the Northeast Regional Plant Introduction Center, Geneva, New York. Germplasm releases provide an important source of genetic variability. Genotypic diversity is evident from the development of numerous ecotypes in the important agronomic clover species, particularly crimson clover in the USA and subterranean clover in Australia. Variability in phenotype expressed in such ecotypes includes tolerance to acid soils, earliness of flowering, seedling vigor and seedling establishment, season of greatest vegetative growth, proportion of hard seed needed for reseeding (crimson clover), and resistance to endemic pests.

The objective of this investigation was to ascertain the variation in and the effectiveness of phenotypic recurrent selection for multiple‐parted heads in red clover ( Trifolium pratense L.). Additional objectives were to examine the effect of changes in this character on seed yield and other morphological and physiological characters. Six generations of selection were conducted on an annual basis under field conditions involving 1040 to 2548 plants and selection intensity of 2.0 to 6.8%. The effectiveness of selection was evaluated using spaced plants established from remnants eeds. Number of head parts in largest heads increased linearly from 1 to 7.4 with a regression (b) coefficient of 0.92 head parts/generation. The check cultivar Kenstar from which the original selections were made, had 93% single‐parted heads whereas Generation 6 had only 37% single‐parted heads and 9% of the heads with 5 or more parts. Number of seeds per plant, persistence, and vigor (forage yield) declined over generations, probably due, in part, to inbreeding depression and partly due to other factors. Extreme selections for multiple‐parted heads exhibited weak and lodged, fasciated stems containing many growing points. Recurrent selection was quite effective, but changes associated with increasing multiple‐parted heads were deleterious and mayb e difficult to overcomeS. election is a powerful tool that can generate genotypes not previously seen, and conceivably could reduce the need of plant exploration for desirable genes, at least in cross‐pollinated species.

Supplementary Figure 1 from Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Supplementary from Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells

Mitochondria are important organelles for providing the ATP and carbon skeletons required to sustain cell growth. While these organelles also participate in other key metabolic functions across species, they have a specialized role in plants of optimizing photosynthesis through participating in photorespiration. It is therefore critical to map the protein composition of mitochondria in plants to gain a better understanding of their regulation and define the uniqueness of their metabolic networks. To date, <30% of the predicted number of mitochondrial proteins has been verified experimentally by proteomics and/or GFP localization studies. In this mini-review, we will provide an overview of the advances in mitochondrial proteomics in the model plant Arabidopsis thaliana over the past 5 years. The ultimate goal of mapping the mitochondrial proteome in Arabidopsis is to discover novel mitochondrial components that are critical during development in plants as well as genes involved in developmental abnormalities, such as those implicated in mitochondrial-linked cytoplasmic male sterility.

Members of the cysteine- and glycine-rich protein family (CRP1, CRP2, and CRP3) contain two zinc-binding LIM domains, LIM1 (amino-terminal) and LIM2 (carboxyl-terminal), and are implicated in diverse cellular processes linked to differentiation, growth control, and pathogenesis. Here we report the solution structure of full-length recombinant quail CRP2 as determined by multi-dimensional triple-resonance NMR spectroscopy. The structural analysis revealed that the global fold of the two LIM domains in the context of the full-length protein is identical to the recently determined solution structures of the isolated individual LIM domains of quail CRP2. There is no preference in relative spatial orientation of the two domains. This supports the view that the two LIM domains are independent structural and presumably functional modules of CRP proteins. This is also reflected by the dynamic properties of CRP2 probed by 15N relaxation values (T1, T2, and nuclear Overhauser effect). A model-free analysis revealed local variations in mobility along the backbone of the two LIM domains in the native protein, similar to those observed for the isolated domains. Interestingly, fast and slow motions observed in the 58-amino acid linker region between the two LIM domains endow extensive motional freedom to CRP2. The dynamic analysis indicates independent backbone mobility of the two LIM domains and rules out correlated LIM domain motion in full-length CRP2. The finding that the LIM domains in a protein encompassing multiple LIM motifs are structurally and dynamically independent from each other supports the notion that these proteins may function as adaptor molecules arranging two or more protein constituents into a macromolecular complex.

The authors received funding from the German Research Foundation (SFB/TRR 57) and the Interdisciplinary Center for Clinical Research (IZKF) Aachen. Potential conflict of interest: Nothing to report. Author names in bold designate shared co‐first authorship. To the Editor: There are numerous experimental and clinical studies available showing that lipocalin 2 (LCN2), also known as neutrophil gelatinase‐associated lipocalin (NGAL), is up‐regulated during inflammation and in response to cellular stress, promoting protective effects during acute and chronic injury.1 In a recent report, Xu et al. demonstrated that hepatocyte‐derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration.2 The presented data corroborate our previous findings that injury‐induced up‐regulation of hepatic LCN2 has a significant hepatoprotective effect in acute liver injury and that hepatocytes are the major source for hepatic LCN2.3 Furthermore, these data suggest that LCN2 might act as an intrinsic “help‐me” sensor that, upon injury, develops an activity necessary to recruit inflammatory cells. In line with this assumption, we observed that lcn2‐deficient mice showed a significant lower recruitment of neutrophils and leukocytes, compared to wild‐type (WT) animals, when fed with a methionine‐choline–deficient (MCD) diet that induces hepatic inflammation and injury (Fig. 1). The staining and activity measurement of myeloperoxidase (MPO), representing an enzyme that is most abundantly expressed in neutrophilic granulocytes, further confirmed the assumption that LCN2 is an essential factor promoting hepatic neutrophil recruitment (Fig. 2). In line with this, after MCD feeding, hepatic MPO expression analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR) was higher in WT animals than in mice lacking LCN2 (not shown). Moreover, the important role of LCN2 for neutrophil recruitment was also documented in mice that were injected with lipopolysaccharide (LPS). When MPO‐positive neutrophil infiltrates were comparatively analyzed in WT and Lcn2 null mice that received LPS for 6 hours, we found a statistically significant higher number of respective cells (P = 0.0139 when counting each 10 representative high power fields) in WT animals (not shown).Figure 1: Leukocyte/neutrophil recruitment in hepatic inflammation. WT or Lcn2‐deficient mice were fed with a standard chow or MCD diet for 4 weeks. (A) Liver samples were stained with Oil Red, hematoxylin and eosin (H & E), or antibodies directed against the leukocyte common antigen CD45 or neutrophil marker MPO. (B) RNA was prepared and subjected to qRT‐PCR analysis for CD45 expression. (C) Protein extracts from respective livers were subjected to western blotting analysis for analysis of CD45 expression. Abbreviations: GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA.Figure 2: Quantitative analysis of neutrophil recruitment. (A) MPO was stained in liver cryosections obtained from WT or Lcn2‐deficient mice fed with either a control or an MCD diet for 4 weeks (magnification, ×40). As a negative control, livers were stained with a nonimmune immunoglobulin. As a positive control, tonsillitis tissue was stained with the antibody directed against MPO. (B) Cells positive for MPO were counted in 10 representative high power fields each using the public domain Java image processing and analysis program (ImageJ, version IJ 1.46r). Statistically significant differences are indicated. (C) MPO activity was quantified in liver tissue samples using the MPO activity quantification kit (ab105136; Abcam, Cambridge, MA), according to the manufacturer's instructions. Statistically significant differences are indicated. (D) Liver cryosections taken from WT or Lcn2‐deficient animals that received a single injection of LPS or a standard saline solution (NSS) for 6 hours were stained for LCN2 and MPO. Magnification, ×200. Abbreviation: DAPI, 4’,6‐diamidino‐2‐phenylindole.It is most likely that this biological activity of LCN2 is organ independent given that LCN2 was already reported to promote production of neutrophil‐attracting chemokines and neutrophil recruitment during mycobacterial pulmonary infection by inducing alveolar macrophage‐produced granulocyte colony‐stimulating factor, a key regulator of neutrophil production and keratinocyte chemoattractant (KC/CXCL1) that is involved in chemotaxis and cell activation of neutrophils through chemokine receptor CXCR2.5 Of note, another recent report has demonstrated that inflammatory cell‐derived LCN2 at the sites of inflammation plays important roles in central sensitization and the subsequent nociceptive behavior in the complete Freund's adjuvant‐induced chronic inflammatory pain mouse model.6 All these attributes render this lipocalin as a reliable biomarker for hepatic inflammation and as a novel drug target for lesions associated with overshooting inflammatory reactions in liver and other organs.

Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mitochondrial biochemical pathways, and consider the impact of N-terminal pre-sequence cleavage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels.

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.

Background: Toxic metal accumulation in the body that may be caused by inherited disorders, chronic intake, or acute intoxication. Vice versa, inadequate daily supply with a specific trace element results in shortcomings and manifests in clinical symptoms. In diagnostics, the determination of exact metal concentrations or the measurement of proteins correlating with the concentration of a specific metal is widespread. In addition, several histopathological stains are available, allowing identification of specific metal deposits in tissue. The introduction of novel metal imaging techniques has extended the repertoire of analytical possibilities. In particular, laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) has undergone an intensive progress in recent years. It has multi-element capability, granting the analysis of a large variety of biological materials with high spatial resolution. It further can identify and quantitate a large number of metals and metalloids at extremely low concentrations. Methods and findings: We here optimized and extended previous studies in which we used LA-ICP-MS for trace metal imaging in livers and brains of wild type and Atp7b deficient mice that represents an experimental model of human Wilson's disease. We show that the observed alterations in metal content and distribution are closely linked to the progression of the disease. While the accumulation of copper in the brain can be assigned to special regions, the overload with copper is initially distributed uniformly, while the regional distribution of individual metals is significant modulated in necrotic and tumorigenic tissue areas. Conclusion: These illustrations show that LA-ICP-MS is a highly powerful and innovative analytical technique that will have tremendous impact on biomedical research and diagnostics of metal disorders. Abbreviations: Atp7b: gene encoding a copper-transporting P-type ATPase; EDX: Energy-Dispersive X-ray spectroscopy; LA-ICP-MS: Laser Ablation Inductively Coupled Plasma Mass Spectrometry; LM: Light Microscopic; SIMS: Secondary Ion Mass Spectrometry; SXRF: Synchrotron X-ray Fluorescence.

A number of previous studies have documented the gross response of mitochondrial respiration to salinity treatment, but it is unclear how NaCl directly affects the kinetics of plant phosphorylating and non-phosphorylating electron transport pathways. This study investigates the direct effects of NaCl upon different respiratory pathways in wheat, by measuring rates of isolated mitochondrial oxygen consumption across different substrate oxidation pathways in saline media. We also profile the abundance of respiratory proteins by using targeted selected reaction monitoring (SRM) mass spectrometry of mitochondria isolated from control and salt-treated wheat plants. We show that all pathways of electron transport were inhibited by NaCl concentrations above 400 mM; however electron transfer chains showed divergent responses to NaCl concentrations between 0 and 200 mM. Stimulation of oxygen consumption was measured in response to NaCl in scenarios where exogenous NADH was provided as substrate and electron flow was coupled to the generation of a proton gradient across the inner membrane. Protein abundance measurements show that several enzymes with activities less affected by NaCl are induced by salinity, whereas enzymes with activities inhibited by NaCl are depleted. These data deepen our understanding of how plant respiration responds to NaCl, offering new mechanistic explanations for the divergent salinity responses of whole-plant respiratory rate in the literature.