IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.     

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3⁺ T-cells than photo-damaged skin, however, the abundance of CD3⁻CD56⁺ Natural Killer (NK) cells, CD11c⁺HLA-DR⁺ conventional Dendritic Cells (cDC), BDCA-2⁺HLA-DR⁺ plasmacytoid DC (pDC), FoxP3⁺ Regulatory T-cells (T-reg), Vα24⁺Vβ11⁺ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4⁺ T-cells were associated with SCC, yet CD8⁺ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8⁺ T-cells than IEC. We propose that differences in CD8⁺ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4⁺ T-cells to CD8⁺ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.     

EPR and ENDOR study of crystalline cytosine x HCl doped with 5-methylcytosine. Radiation-induced radical formation and hole transfer

Radical formation and hole transfer were investigated in crystals of cytosine.HCl (C.HCl) doped with 0-1.1 mol-% 5-methylcytosine x HCl (5MC x HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3alphaH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3alphaH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3alphaH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2'deoxycytidine hydrochloride (CdR x HCl), 5'dCMP and 3'CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC x HCl doped into crystals of C.HCl give rise to a considerable yield of 3alphaH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3alphaH radicals in the crystalline cytosine derivatives CR, CdR.HCl, 5'dCMP and 3'CMP and suggests that the 3alphaH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C x HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3alphaH radical is more stable than the primary cytosine radicals. The relative amounts of the 3alphaH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3alphaH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3alphaH radical being less exposed to recombination than other cytosine radicals.     

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7-7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

The Point Count Transect Method for Estimates of Biodiversity on Coral Reefs: Improving the Sampling of Rare Species

Understanding patterns in species richness and diversity over environmental gradients (such as altitude and depth) is an enduring component of ecology. As most biological communities feature few common and many rare species, quantifying the presence and abundance of rare species is a crucial requirement for analysis of these patterns. Coral reefs present specific challenges for data collection, with limitations on time and site accessibility making efficiency crucial. Many commonly used methods, such as line intercept transects (LIT), are poorly suited to questions requiring the detection of rare events or species. Here, an alternative method for surveying reef-building corals is presented; the point count transect (PCT). The PCT consists of a count of coral colonies at a series of sample stations, located at regular intervals along a transect. In contrast the LIT records the proportion of each species occurring under a transect tape of a given length. The same site was surveyed using PCT and LIT to compare species richness estimates between the methods. The total number of species increased faster per individual sampled and unit of time invested using PCT. Furthermore, 41 of the 44 additional species recorded by the PCT occurred ≤ 3 times, demonstrating the increased capacity of PCT to detect rare species. PCT provides a more accurate estimate of local-scale species richness than the LIT, and is an efficient alternative method for surveying reef corals to address questions associated with alpha-diversity, and rare or incidental events.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

The diversity and phylogeny of the commercially important algal class Eustigmatophyceae,including the new clade Goniochloridales

The Eustigmatophyceae is a class of yellow-green algae allied with the Chrysophyceae and other chlorophyll c possessing stramenopile (heterokont) algae. Some members of the class, especially the marine species of the genus Nannochloropsis, are under intense investigation for their potential for production of biofuels and beneficial fatty acids. The class has generally been thought to comprise a small number of genera and species, and these organisms were considered rare or infrequently encountered. In this study, we examined the phylogeny and diversity of this class by analysis of nuclear 18S rDNA sequence data. Our analysis included sequences from all the named members of the Eustigmatophyceae held in culture collections as well as a number of strains identified in culture collections as Xanthophyceae, new strains with features characteristic of the Eustigmatophyceae, and published data for uncultured DNA clones. The results of these analyses show that the Eustigmatophyceae is far more diverse than generally recognized. Two major lineages are supported in the class, the previously recognized order Eustigmatales and the new clade, Goniochloridales. Additional new lineages were also resolved within each of these major lineages; however, the results of our analyses were considered insufficient for naming these subordinate clades. Several of these lineages comprised only unnamed strains or uncultured DNA clones. Overall, our results indicate that the Eustigmatophyceae is a highly diverse class, with many new species, genera, and families awaiting taxonomic treatment.     

Progression,Symptoms and Psychosocial Concerns among Those Severely Affected by Multiple Sclerosis: A Mixed-Methods Cross-Sectional Study of Black Caribbean and White British People

Objective

Multiple sclerosis is now more common among minority ethnic groups in the UK but little is known about their experiences, especially in advanced stages. We examine disease progression, symptoms and psychosocial concerns among Black Caribbean (BC) and White British (WB) people severely affected by MS.

Design

Mixed methods study of 43 BC and 43 WB people with MS (PwMS) with an Expanded Disability Status Scale (EDSS) ≥6 involving data from in clinical records, face-to-face structured interviews and a nested-qualitative component. Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) were calculated. To control for selection bias, propensity scores were derived for each patient and adjusted for in the comparative statistical analysis; qualitative data were analysed using the framework approach.

Results

Median EDSS for both groups was (6.5; range: 6.0–9.0). Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) based on neurological assessment of current EDSS scores identified BC PwMS were more likely to have aggressive disease (PI F = 4.04, p = 0.048, MSSS F = 10.30, p<0.001). Patients’ reports of the time required to reach levels of functional decline equivalent to different EDSS levels varied by group; EDSS 4: BC 2.7 years v/s WB 10.2 years (U = 258.50, p = 0.013), EDSS 6∶6.1 years BC v/s WB 12.7 years (U = 535.500, p = 0.011), EDSS 8: BC 8.7 years v/s WB 10.2 years. Both groups reported high symptom burden. BC PwMS were more cognitively impaired than WB PwMS (F = 9.65, p = 0.003). Thematic analysis of qualitative interviews provides correspondence with quantitative findings; more BC than WB PwMS referred to feelings of extreme frustration and unresolved loss/confusion associated with their rapidly advancing disease. The interviews also reveal the centrality, meanings and impact of common MS-related symptoms.

Conclusions

Delays in diagnosis should be avoided and more frequent reviews may be justified by healthcare services. Culturally acceptable interventions to better support people who perceive MS as an assault on identity should be developed to help them achieve normalisation and enhance self-identity.