A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein

The investigation of hormone-receptor interaction normally needs isolation and extensive purification of the receptor protein or a particular receptor-containing fraction. To bypass these time- and resource-consuming procedures, we have established a live cell-based assay using transgenic bacteria expressing single eukaryotic receptors. Here we describe some biochemical features of the Arabidopsis cytokinin receptor CRE1/AHK4 expressed in Escherichia coli. The data show that the main characteristics of the ligand-receptor interaction, including binding affinity and ligand specificity, can be determined using intact bacteria expressing a functional receptor.     

Small and overlooked: Phylogeny of the genus Trigonodactylus (Squamata: Gekkonidae), with the first record of Trigonodactylus arabicus from Jordan

Geckos of the genus Trigonodactylus are widely distributed in the sand deserts of the Arabian Peninsula. Three species of this genus are currently recognized, with a fourth one, Stenodactylus pulcher, which placement within Trigonodactylus has been tentatively suggested, but not yet confirmed. We present a phylogenetic analysis of the genus Trigonodactylus with new specimens collected in central Saudi Arabia and southern Jordan. New genetic data has been generated from three mitochondrial markers to investigate the phylogenetic relationships of all species of the genus and to assess the putative generic assignment of S. pulcher. Our results confirm that S. pulcher indeed belongs within Trigonodactylus, branching as a sister lineage to all other species of the genus. The new samples cluster within Trigonodactylus arabicus, thus confirming the genetic homogeneity of the species across its large and seemingly inhospitable range. The new specimen collected in southern Jordan represents the first record for the country and a considerable range extension to the northwest from all previously reported localities. Our findings and discovery of a new species for Jordan highlight the need of more field surveys to be carried out in the underexplored parts of Jordan and northern Saudi Arabia, as these places still hold a potential for new discoveries and are crucial for understating the biogeography of the Arabian herpetofauna.     

Binpairs: Utilization of Illumina Paired-End Information for Improving Efficiency of Taxonomic Binning of Metagenomic Sequences

Motivation

Paired-end sequencing protocols, offered by next generation sequencing (NGS) platforms like Illumia, generate a pair of reads for every DNA fragment in a sample. Although this protocol has been utilized for several metagenomics studies, most taxonomic binning approaches classify each of the reads (forming a pair), independently. The present work explores some simple but effective strategies of utilizing pairing-information of Illumina short reads for improving the accuracy of taxonomic binning of metagenomic datasets. The strategies proposed can be used in conjunction with all genres of existing binning methods.

Results

Validation results suggest that employment of these “Binpairs” strategies can provide significant improvements in the binning outcome. The quality of the taxonomic assignments thus obtained are often comparable to those that can only be achieved with relatively longer reads obtained using other NGS platforms (such as Roche).

Availability

An implementation of the proposed strategies of utilizing pairing information is freely available for academic users at https://metagenomics.atc.tcs.com/binning/binpairs.     

Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic 15N chemical shielding anisotropy tensors

Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic ¹⁵N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic ¹⁵N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated ¹⁵N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic ¹⁵N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.     

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia

Background

White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown.

Methodology/Principal Findings

We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval.

Conclusions/Significance

G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.     

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Characterizing Thalamocortical Disturbances in Cervical Spondylotic Myelopathy: Revealed by Functional Connectivity under Two Slow Frequency Bands

Background and Purpose

Recent advanced MRI studies on cervical spondylotic myelopathy (CSM) revealed alterations of sensorimotor cortex, but the disturbances of large-scale thalamocortical systems remains elusive. The purpose of this study was to characterizing the CSM-related thalamocortical disturbances, which were associated with spinal cord structural injury, and clinical measures.

Methods

A total of 17 patients with degenerative CSM and well-matched control subjects participated. Thalamocortical disturbances were quantified using thalamus seed-based functional connectivity in two distinct low frequencies bands (slow-5 and slow-4), with different neural manifestations. The clinical measures were evaluated by Japanese Orthopaedic Association (JOA) score system and Neck Disability Index (NDI) questionnaires.

Results

Decreased functional connectivity was found in the thalamo-motor, -somatosensory, and -temporal circuits in the slow-5 band, indicating impairment of thalamo-cortical circuit degeneration or axon/synaptic impairment. By contrast, increased functional connectivity between thalami and the bilateral primary motor (M1), primary and secondary somatosensory (S1/S2), premotor cortex (PMC), and right temporal cortex was detected in the slow-4 band, and were associated with higher fractional anisotropy values in the cervical cord, corresponding to mild spinal cord structural injury.

Conclusions

These thalamocortical disturbances revealed by two slow frequency bands inform basic understanding and vital clues about the sensorimotor dysfunction in CSM. Further work is needed to evaluate its contribution in central functional reorganization during spinal cord degeneration.     

S3EPY: a Sparky extension for determination of small scalar couplings from spin-state-selective excitation NMR experiments

S3EPY is a Python extension to the program Sparky written to facilitate the assessment of coupling constants from in-phase/antiphase and spin-state-selective excitation (S³E) experiments. It enables the routine use of small scalar couplings by automating the coupling evaluation procedure. S3EPY provides an integrated graphical user interface to programs which outputs graphs and the table of determined couplings.