A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity

Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 – 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP₆), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB.     

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.     

Internal Jugular Vein Cross-Sectional Area Enlargement Is Associated with Aging in Healthy Individuals

Background

Internal jugular vein (IJV) narrowing has been implicated in central nervous system pathologies, however normal physiological age- and gender-related IJV variance in healthy individuals (HIs) has not been adequately assessed.

Objectives

We assessed the relationship between IJV cross-sectional area (CSA) and aging.

Materials and Methods

This study involved 193 HIs (63 males and 130 females) who received 2-dimensional magnetic resonance venography at 3T. The minimum CSA of the IJVs at cervical levels C2/C3, C4, C5/C6, and C7/T1 was obtained using a semi-automated contouring-thresholding technique. Subjects were grouped by decade. Pearson and partial correlation (controlled for cardiovascular risk factors, including hypertension, heart disease, smoking and body mass index) and analysis of variance analyses were used, with paired t-tests comparing side differences.

Results

Mean right IJV CSA ranges were: in males, 41.6 mm² (C2/C3) to 82.0 mm² (C7/T1); in females, 38.0 mm² (C2/C3) to 62.3 mm² (C7/T1), while the equivalent left side ranges were: in males, 28.0 mm² (C2/C3) to 52.2 mm² (C7/T1); in females, 27.2 mm² (C2/C3) to 47.8 mm² (C7/T1). The CSA of the right IJVs was significantly larger (p<0.001) than the left at all cervical levels. Controlling for cardiovascular risk factors, the correlation between age and IJV CSA was more robust in males than in the females for all cervical levels.

Conclusions

In HIs age, gender, hand side and cervical location all affect IJV CSA. These findings suggest that any definition of IJV stenosis needs to account for these factors.     

Song and brain development in canaries raised under different conditions of acoustic and social isolation over two years

Early isolation experiments indicate that male songbirds learn their songs during an early sensitive period, although later work has shown that some open-ended learners modify songs in later years. Recent isolation experiments suggest that in some species song has a stronger genetic basis than previously thought. This study raised domestic canaries under different combinations of acoustic and social isolation and followed song development into the second year. Males raised alone in acoustic isolation developed songs with normal syllables, but larger repertoires and also produced syllables with lower repetition rates when compared to controls. The smallest repertoire occurred in males raised in a peer group. Isolate males had a smaller song control nucleus HVC than controls, but there was no effect on nucleus RA or on brain weight in general. In the second year, after introduction into a large normal colony, isolate and peer group males adjusted their syllable repertoire to normal size. In particular, the isolates reduced their repertoire even though the size of HVC showed a significant increase in volume. However, songs of isolate and peer group males still differ in repetition rate and number of single syllables in the common aviary. In contrast, control males showed low syllable turnover and no significant change in repertoire size. Nor did they show any significant change in the volumes of song control nuclei. It seems that complete isolation affects only some aspects of song and brain development, and later socialization corrects some but not all of these in the second year.     

Suction blister fluid as potential body fluid for biomarker proteins

Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.     

Deep-water fisheries along the British Isles continental slopes: status,ecosystem effects and future perspectives

In this paper, we revisit the state of deep-water fisheries to the west of the British Isles and aim to provide an overview on the key drivers behind community changes along continental margins. The deep-water fisheries to the west of the British Isles that extend from the shelf-slope break down to the lower slope and along banks and seamounts of the Rockall Basin, mainly target blue ling Molva dypterygia, roundnose grenadier Coryphaenoides rupestris, orange roughy Hoplostethus atlanticus, with by-catches of black scabbardfish Aphanopus carbo and tusk Brosme brosme. These fishing grounds experienced a long period of exhaustive exploitation until the early 2000s, but subsequently the implementation of management strategies has helped to relieve excessive fishing pressure. It is widely accepted that a better understanding of the long-term implications of disturbance is needed to understand patterns in deep-water communities and what sustainable use and exploitation of resources might look like in this context.     

Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (R_e) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (R_e > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.     

A rich and bountiful harvest: Key discoveries in plant cell biology

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke’s discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Seven senior plant cell biologists reflect on foundational contributions to a variety of topics, including pollen tube signaling, cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology.     

Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants. In contrast, M. loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS). Here we show by hybridization analysis that most M. loti strains contain the VirB/D4 T4SS and not the T3SS. Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures. A rhcJ T3SS mutant of strain MAFF303099 also nodulated L. leucocephala, unlike the wild type. On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type. Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins. Both Msi059 and Msi061 were translocated through the A. tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT). Taken together, these results suggest that the VirB/D4 T4SS of M. loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia. The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M. loti and A. tumefaciens T4SS.