Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing

Background

Improved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.

Methods

We sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.

Results

Isolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.

Conclusion

Whole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.     

Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis

BackgroundPsoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)₂D₃.MethodsSerum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)₂D₃. Osteoclast differentiation and cytokine secretion were assessed.ResultsPsoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)₂D₃, compared with psoriasis vulgaris and control PBMCs.ConclusionsOur data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)₂D₃ abrogated these effects.     

Surface glycoprotein of Borna disease virus mediates virus spread from cell to cell

Borna disease virus (BDV) is a non‐segmented negative‐stranded RNA virus that maintains a strictly neurotropic and persistent infection in affected end hosts. The primary target cells for BDV infection are brain cells, e.g. neurons and astrocytes. The exact mechanism of how infection is propagated between these cells and especially the role of the viral glycoprotein (GP) for cell–cell transmission, however, are still incompletely understood. Here, we use different cell culture systems, including rat primary astrocytes and mixed cultures of rat brain cells, to show that BDV primarily spreads through cell–cell contacts. We employ a highly stable and efficient peptidomimetic inhibitor to inhibit the furin‐mediated processing of GP and demonstrate that cleaved and fusion‐active GP is strictly necessary for the cell‐to‐cell spread of BDV. Together, our quantitative observations clarify the role of Borna disease virus‐glycoprotein for viral dissemination and highlight the regulation of GP expression as a potential mechanism to limit viral spread and maintain persistence. These findings furthermore indicate that targeting host cell proteases might be a promising approach to inhibit viral GP activation and spread of infection.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Proteomic Profiling of Mouse Helper T Cell Differentiation

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high‐throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.     

The association between sociodemographic,hormonal, tubo-ovarian factors and bacterial count in Chlamydia and Mycoplasma infections with infertility

Aim: To determine if there is an association between the Chlamydia and Mycoplasma infections with socio-demographic and clinical factors, and also with infertility. Methods: We conducted a study on 100 infertile married women and 100 control group, and collected data on the socio-demographic, hormonal and tubo-ovarian factors. The results of the endocervical swabs were analyzed for Mycoplasma and Chlamydia infection, the bacterial counts were also determined. Results: The percentage positivity to infection was significantly more among the infertile group compared to the control group, and also significantly more among the age group <30 years old. The positivity for infection with Chlamydia and/or Mycoplasma was significantly correlated with age, history of irregular menstruation, and history of previous abortion. Further sub-analysis of the infertile group showed that positivity to Chlamydia and/or Mycoplasma infection was significantly correlated to hormonal factors, ovarian factors, irregular menstruation, and previous abortion. Regression analysis showed that hormonal, ovarian factors, and irregular menstruation were the most significant factors in the positivity to Chlamydia and Mycoplasma infection. Bacterial count was significantly correlated with age, history of irregular menstruation, and history of previous abortion. Conclusion: Infection to Chlamydia and Mycoplasma is associated to younger age (?30 years old), and occurs in the infertile women. There is an interplay between infection in younger women, irregular menstruation, hormonal, and tubo-ovarian factors with infertility. Bacterial count was significantly correlated with age, history of irregular menstruation, and history of previous abortion.     

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi‐chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division‐replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.