MP13, a generalized transducing bacteriophage for Bacillus megaterium.

The first generalized transducing bacteriophage reported for Bacillus megaterium has been characterized. Optimum conditions for lysate production and transduction procedures were established so that transducing frequencies of 8 x 10(-6) and higher are now possible. The phage, MP13, has a head diameter of 97 nm and a contractile tail (202 by 17 nm) and adsorbs to the periphery of the cell. MP13 was inactivated rapidly at 60 degrees C, but not at 55 degrees C, and was sensitive to toluene, ether, and chloroform. When centrifuged in a neutral CsCl gradient, two bands were observed, a major band of 1.490 g cm-3 and a minor band of 1.482 g cm-3 buoyant density. The major band contained only infective particles, whereas the minor band contained both infective and transducing particles. Phage DNA was resistant to several restriction endonucleases, but yielded 9 fragments with MboI, more than 34 with HindIII, and 7 with BstEII. The molecular weights for the fragments from MboI-BstEII double digests total 97 x 10(9).     

Axon guidance at the midline: from mutants to mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.     

Brain structure variation in great apes, with attention to the mountain gorilla (Gorilla beringei beringei)

This report presents data regarding the brain structure of mountain gorillas (Gorilla beringei beringei) in comparison with other great apes. Magnetic resonance (MR) images of three mountain gorilla brains were obtained with a 3T scanner, and the volume of major neuroanatomical structures (neocortical gray matter, hippocampus, thalamus, striatum, and cerebellum) was measured. These data were included with our existing database that includes 23 chimpanzees, three western lowland gorillas, and six orangutans. We defined a multidimensional space by calculating the principal components (PCs) from the correlation matrix of brain structure fractions in the well-represented sample of chimpanzees. We then plotted data from all of the taxa in this space to examine phyletic variation in neural organization. Most of the variance in mountain gorillas, as well as other great apes, was contained within the chimpanzee range along the first two PCs, which accounted for 61.73% of the total variance. Thus, the majority of interspecific variation in brain structure observed among these ape taxa was no greater than the within-species variation seen in chimpanzees. The loadings on PCs indicated that the brain structure of great apes differs among taxa mostly in the relative sizes of the striatum, cerebellum, and hippocampus. These findings suggest possible functional differences among taxa in terms of neural adaptations for ecological and locomotor capacities. Importantly, these results fill a critical gap in current knowledge regarding great ape neuroanatomical diversity.     

Inhibitor-associated transposition events in<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

In up to 100% of all bacteria grown in the presence of initially inhibitory concentrations of five diverse inhibitors, an extra copy of the resident insertion element IS31831 was found in specific chromosomal regions, the sites of which apparently depended on the inhibitor used. Thus, in nine out of nine independently isolated cyanide-associated transpositions, the acquired copy was located within an ORF encoding a protein related to the hypothetical but conserved protein YeiH of Escherichia coli. A putative Sox box upstream of the yeiH gene implicates superoxide as a potential regulator of the gene, a possibility further supported by the finding that superoxide dismutase (SodA) is overexpressed in cells cultured in cyanide-containing medium. Neither the cyanide-associated nor any of the other transposition mutations appeared to confer any discernible phenotypic advantage upon cells grown in the presence or absence of the inhibitors, as revealed most stringently by mixed-cell experiments. An alternative, albeit heterodox, explanation for the emergence of the mutants postulates a very high rate of transpositional activity in the presence of inhibitors. The initial emergence of the mutants was found to depend crucially upon the cell density. Thus, when growth medium was supplemented with 50 mM fluoropyruvate and inoculated to a density of 2×10⁷ cfu/ml, single colonies with heterogeneous restriction fragment length polymorphisms (RFLPs) were routinely isolated at a frequency of 6 to 16% after 1–2 days of incubation. After 3 days, 10–36% of the colonies showed RFLPs, but the type was now dominated by the fluoropyruvate-specific RFLP, which, at higher resolution, invariably proved to be heterogeneous. This heterogeneity proved that these specific mutants were of multiple origin, indicating that clonal enrichment was irrelevant to their emergence. It is suggested that the presence of the inhibitor induces the development of hyper-transpositional activity, which is regulated by a soluble bacterial product.Communicated by W. Arber     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

Imbalance of dendritic cell co-stimulation in COPD

Background

Dendritic cells (DCs) control immunity and play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the expression of function-associated surface molecules on circulating DCs in COPD is unknown.

Methods

Four-colour flow cytometry was used to compare blood DC surface molecules of 54 patients with COPD (median age: 59 years; median FEV₁: 38% predicted, median CAT score: 24) with two age-matched control groups with normal lung function: 21 current smokers and 21 never-smokers.

Results

Concentrations of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) and the mDC/pDC ratio did not differ between the groups. The increased expression of BDCA-1, BDCA-3, CD86 and CCR5 on mDCs in patients with COPD did not significantly differ from smokers with normal lung function. In contrast, COPD was specifically characterised by a decreased expression of the anti-inflammatory co-stimulatory molecule PD-L1 on pDCs and an increased expression of the pro-inflammatory co-stimulatory molecule OX40 ligand (OX40L) on mDCs. These changes were not confined to patients with elevated systemic inflammation markers (leukocytes, c-reactive protein, interleukin-6, fibrinogen). The ratio of OX40L to PD-L1 expression (OX40L/PD-L1 ratio), a quantitative measure of imbalanced DC co-stimulation, correlated with the severity of pulmonary emphysema in patients with COPD.

Conclusion

An imbalance of DC co-stimulation might contribute to the pathogenesis of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0174-x) contains supplementary material, which is available to authorized users.     

Quantitative Measurement of Melanoma Spread in Sentinel Lymph Nodes and Survival

Background

Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and Findings

We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3–123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61–2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02–2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions

Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically. Please see later in the article for the Editors'' Summary     

Cysteine proteinase SpeB from Streptococcus pyogenes - a potent modifier of immunologically important host and bacterial proteins

Group A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates that S. pyogenes possess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein from S. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.     

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.