The Crystal Structure of the Complexes of Concanavalin A with 4′-Nitrophenyl-α-d-mannopyranoside and 4′-Nitrophenyl-α-d-glucopyranoside

Concanavalin A (Con A) is the best-known plant lectin and has importantin vitrobiological activities arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complexes of Con A with 4′-nitro-phenyl-α-d-mannopyranoside (α-PNM) and 4′-nitrophenyl-α-d-glucopyranoside (α-PNG) have been crystallized in space group P2₁2₁2 with cell dimensionsa= 135.19 Å,b= 155.38 Å,c= 71.25 Å anda= 134.66 Å,b= 155.67 Å, andc= 71.42 Å, respectively. X-ray diffraction intensities to 2.75 Å for the α-PNM and to 3.0 Å resolution for the α-PNG complex have been collected. The structures of the complexes were solved by molecular replacement and refined by simulated annealing methods to crystallographic R-factor values of 0.185/0.186 and free-R-factor values of 0.260/0.274, respectively. In both structures, the asymmetric unit contains four molecules arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site near the surface of each monomer. The nonsugar (aglycon) portion of the compounds used helps to identify the exact orientation of the saccharide in the sugar-binding pocket and is involved in major interactions between tetramers. The hydrogen bonding network in the region of the binding site has been analyzed, and only minor differences with the previously reported Con A–methyl-α-d-mannopyranoside complex structure have been observed. Structural differences that may contribute to the slight preference of the lectin for mannosides over glucosides are discussed. Calculations indicate a negative electrostatic surface potential for the saccharide binding site of Con A, which may be important for its biological activity. It is also shown in detail how a particular class of hydrophobic ligands interact with one of the three so-called characteristic hydrophobic sites of the lectins.     

Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.     

Inside the brain of a neuron

For many decades, neurons were considered to be the elementary computational units of the brain and were assumed to summate incoming signals and elicit action potentials only in response to suprathreshold stimuli. Although modelling studies predicted that single neurons constitute a much more powerful computational entity, able to perform an array of nonlinear calculations, this possibility was not explored experimentally until the discovery of active mechanisms in the dendrites of most neuron types. Here, we review several modelling studies that have addressed information processing in single neurons, starting with those characterizing the arithmetic of different dendritic components, to those tackling neuronal integration at the cell body and, finally, those analysing the computational abilities of the axon. We present modelling predictions along with supporting experimental data in an effort to highlight the significant contribution of modelling work to enhancing our understanding of single-neuron arithmetic.     

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells)

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional ‘types’ have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.     

Host species identity in annual Brassicaceae has a limited effect on the assembly of root-endophytic fungal communities

ABSTRACT

Background

Establishing the contribution of host plant genotype to the assembly of root-associated microbial communities can be challenging, since multiple ecological factors may confound the effects purely due to the host, and plant species often do not occur sympatrically.     

High postprandial triglyceridemia in patients with type 2 diabetes and microalbuminuria

Microalbuminuria (MA) is an independent risk factor for atherosclerosis in patients with type 2 diabetes mellitus (T2DM). Postprandial lipemia is also associated with excess cardiovascular risk. However, the association between MA and postprandial lipemia in diabetes has not been investigated. A total of 64 patients with T2DM, 30 with and 34 without MA, were examined. Plasma total triglycerides (TGs), triglycerides contained in chylomicrons (CM-TG), and TGs in CM-deficient plasma were measured at baseline and every 2 h for 6 h after a mixed meal. Postheparin LPL and HL activities were also determined. Plasma levels of apolipoprotein A-V (apoA-V), apoC-II, and apoC-III were measured in the fasting state and 2 h postprandially. Patients with MA had higher postprandial total TG levels than those without MA (P < 0.001); this increase been attributed mainly to CM-TG. LPL activity and fasting concentrations of the measured apolipoproteins were not different between the studied groups, whereas HL activity was higher in the patients with MA. ApoC-II and apoC-III levels did not change postprandially in either study group, whereas apoA-V increased more in the patients with MA. These data demonstrate for the first time that MA is characterized by increased postprandial lipemia in patients with T2DM and may explain in part the excess cardiovascular risk in these patients.     

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry’s disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.     

Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

Background—Aim

Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.

Methods

Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.

Results

Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.

Conclusions

Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.     

Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7

Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.