Vertebrate development requires ARVCF and p120 catenins and their interplay with RhoA and Rac

Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context.     

Woody plant encroachment impacts on soil carbon and microbial processes: results from a hierarchical Bayesian analysis of soil incubation data

Quantitative methods were used to examine soil properties and their spatial heterogeneity in a 0-year fenced mobile dune (MD0), an 11-year fenced mobile dune (MD11) and a 20-year fenced mobile dune (MD20) in Horqin Sandy Land, Northern China. The objective of the study was to assess the effect of vegetation restoration on heterogeneity of soil properties in sand dunes and to provide a concept model to describe the relationship between vegetation succession and spatial heterogeneity variation of soil properties in the dunes. The results showed that the average values of vegetation cover, species number and diversity, soil organic carbon (C), total nitrogen (N), and electrical conductivity (EC) increased with the increase in fenced age of mobile dunes, while soil water content (0–20 cm) showed the reverse trend. Geostatistical analysis revealed that the spatial heterogeneity of soil organic C, total N, EC, very fine sand content, and soil water content (0–20 cm) increased from MD0 to MD11 with succession from sand pioneer plant to shrub species then decreased from MD11 to MD20 due to continuous development of herbaceous plants. Canonical correspondence analysis (CCA) showed that there was a relatively high correspondence between vegetation and soil factors, suggesting that the major gradients relating soil organic C, total N, EC, pH, slope, very fine sand content, and soil water content are the main factors for the distribution of dune plants and account for 68.1% of the species-environment relationship among the three sites. In addition, the distribution of the sand pioneer plant was positively related to the relative height of the sampling site and soil water content, and that of most herbaceous plants were determined by soil organic C, total N, EC, pH, and very fine sand content in mobile dunes. The conceptual model of relationship between vegetation succession and spatial heterogeneity of soil properties in mobile dunes suggests spatial patterns of soil properties are most strongly related to plant-induced heterogeneity in dune ecosystems prone to wind erosion, and conversely, the magnitude and degree of spatial heterogeneity in soil properties can influence the plant distribution pattern and vegetation succession of mobile dunes.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Retrovirus-like Gag Protein Arc1 Binds RNA and Traffics across Synaptic Boutons

1. Download high-res image (178KB)
2. Download full-size image

     

Clinical utility of tumor genomic profiling in patients with high plasma circulating tumor DNA burden or metabolically active tumors

Background

This retrospective study was undertaken to determine if the plasma circulating tumor DNA (ctDNA) level and tumor biological features in patients with advanced solid tumors affected the detection of genomic alterations (GAs) by a plasma ctDNA assay.

Method

Cell-free DNA (cfDNA) extracted from frozen plasma (N?=?35) or fresh whole blood (N?=?90) samples were subjected to a 62-gene hybrid capture-based next-generation sequencing assay FoundationACT. Concordance was analyzed for 51 matched FoundationACT and FoundationOne (tissue) cases. The maximum somatic allele frequency (MSAF) was used to estimate the amount of tumor fraction of cfDNA in each sample. The detection of GAs was correlated with the amount of cfDNA, MSAF, total tumor anatomic burden (dimensional sum), and total tumor metabolic burden (SUVmax sum) of the largest ten tumor lesions on PET/CT scans.

Results

FoundationACT detected GAs in 69 of 81 (85%) cases with MSAF >?0. Forty-two of 51 (82%) cases had ≥?1 concordance GAs matched with FoundationOne, and 22 (52%) matched to the National Comprehensive Cancer Network (NCCN)-recommended molecular targets. FoundationACT also detected 8 unique molecular targets, which changed the therapy in 7 (88%) patients who did not have tumor rebiopsy or sufficient tumor DNA for genomic profiling assay. In all samples (N?=?81), GAs were detected in plasma cfDNA from cancer patients with high MSAF quantity (P?=?0.0006) or high tumor metabolic burden (P?=?0.0006) regardless of cfDNA quantity (P?=?0.2362).

Conclusion

This study supports the utility of using plasma-based genomic assays in cancer patients with high plasma MSAF level or high tumor metabolic burden.

     

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.     

Insertional Editing of Mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G·C base pair. Uridine insertions have been identified in the T loop of tRNA^Lys from Didymium and tRNA^Glu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTΨC (Ψ = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.     

Functional epitope on human neutrophil flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.     

Anion binding to a protein-protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein-protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from approximately -1 to -2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge-charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.     

Effects of hydroxylamine and N-Methyl-N′-Nitro-N-nitrosoguanidine in Mimulus cardinalis (Scrophulariaceae): Survival curves and dominant mutants

Summary The lethal and mutagenic effects of hydroxylamine (HA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were investigated in the higher plant Mimulus cardinalis. MNNG was found to be more toxic than HA. The shapes of the survival curves obtained at different concentrations of HA and MNNG are interpreted on the basis of decreased biological activity of the solution to increased age of solution. Based on the appearance of chlorophyll-deficient mutants, MNNG is mutagenic in Mimulus. No albinos were detected in HA treated plants. A total of 67 putative mutants were isolated in the mutation spectra of HA and MNNG treated plants. The frequency of mutants induced by HA and MNNG are different. MNNG is mutagenic at 1/10 the concentration of HA in inducing putative mutations in M ₁ plants.A portion of this work will be submitted by the senior author to the Faculty of Miami University in partial fulfillment of the Doctor of Philosophy degree.