Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

Improved method using muramic acid to estimate biomass of bacteria in sediments

Summary A method, which depends on the measurement of muramic acid content to estimate bacterial biomass, has been improved in sensitivity by two orders of magnitude. It is now applicable to any aquatic sediment, whereas previously it was mainly useful in the analysis of gut contents of deposit-feeding animals. Reduced NAD, a product of the oxidation of d-lactate derived from muramic acid, is assayed using bacterial luciferase. The amount of muramic acid in a number of terrestrial and marine bacteria was measured, and found to be lower than that obtained with the previous, less specific, assay procedure. The muramic acid content of a blue-green alga has been measured, thus allowing blue-green algae to be taken into account when estimating bacterial biomass. Experimental evidence is presented which shows that muramic acid in cell wall fragments of bacteria is rapidly degraded by microorganisms in a marine sediment.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Altered neuropathic pain behaviour in a rat model of depression is associated with changes in inflammatory gene expression in the amygdala

The association between chronic pain and depression is widely recognized, the comorbidity of which leads to a heavier disease burden, increased disability and poor treatment response. This study examined nociceptive responding to mechanical and thermal stimuli prior to and following L5‐L6 spinal nerve ligation (SNL), a model of neuropathic pain, in the olfactory bulbectomized (OB) rat model of depression. Associated changes in the expression of genes encoding for markers of glial activation and cytokines were subsequently examined in the amygdala, a key brain region for the modulation of emotion and pain. The OB rats exhibited mechanical and cold allodynia, but not heat hyperalgesia, when compared with sham‐operated counterparts. Spinal nerve ligation induced characteristic mechanical and cold allodynia in the ipsilateral hindpaw of both sham and OB rats. The OB rats exhibited a reduced latency and number of responses to an innocuous cold stimulus following SNL, an effect positively correlated with interleukin (IL)‐6 and IL‐10 mRNA expression in the amygdala, respectively. Spinal nerve ligation reduced IL‐6 and increased IL‐10 expression in the amygdala of sham rats. The expression of CD11b (cluster of differentiation molecule 11b) and GFAP (glial fibrillary acidic protein), indicative of microglial and astrocyte activation, and IL‐1β in the amygdala was enhanced in OB animals when compared with sham counterparts, an effect not observed following SNL. This study shows that neuropathic pain‐related responding to an innocuous cold stimulus is altered in an animal model of depression, effects accompanied by changes in the expression of neuroinflammatory genes in the amygdala .     

Notch signaling mediates melanoma–endothelial cell communication and melanoma cell migration

Stromal and cellular components within the tumor microenvironment significantly influence molecular signals mediating tumor growth and progression. We recently performed a screen to evaluate critical mediators of melanoma–endothelial communication and identified several molecular pathways associated with these cellular networks, including Notch3. Here, we evaluate the nature of melanoma–endothelial communication mediated by Notch3 and its functional significance. We find that Notch3 is specifically upregulated in melanoma–endothelial cell cocultures and is functionally associated with increased Notch signaling in melanoma cells. Furthermore, induced Notch3 signaling in melanoma cell lines leads to enhanced tumor cell migration without associated increases in tumor cell growth. Additionally, Notch3 expression is specifically associated with malignant patient samples and is not evident in benign nevi. We conclude that Notch3 mediates melanoma–endothelial cell communication and tumor cell migration and may serve as a meaningful therapeutic target for this aggressive malignancy.     

Potential Pitfalls in MALDI-TOF MS Analysis of Abiotically Synthesized RNA Oligonucleotides

Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in “RNA world” research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3′-5′ polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process.     

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2^LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.     

Adsorption of α-amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study

Food Biophysics - Engineered biocatalyst and its desired products using nanotechnology has intensified the research in food industries. Zinc oxide (ZnO) nanosheet is designed and prepared; the...     

Consolidated Pretreatment and Hydrolysis of Plant Biomass Expressing Cell Wall Degrading Enzymes

Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.