Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

BACKGROUND: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism. RESULTS: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca(2+), features typical of the subtilisins. CONCLUSIONS: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates

The role of Comprehension in Requirements and Implications for Use Case Descriptions

Within requirements engineering it is generally accepted that in writing specifications (or indeed any requirements phase document), one attempts to produce an artefact which will be simple to comprehend for the user. That is, whether the document is intended for customers to validate requirements, or engineers to understand what the design must deliver, comprehension is an important goal for the author. Indeed, advice on producing ‘readable’ or ‘understandable’ documents is often included in courses on requirements engineering. However, few researchers, particularly within the software engineering domain, have attempted either to define or to understand the nature of comprehension and it’s implications for guidance on the production of quality requirements. Therefore, this paper examines thoroughly the nature of textual comprehension, drawing heavily from research in discourse process, and suggests some implications for requirements (and other) software documentation. In essence, we find that the guidance on writing requirements, often prevalent within software engineering, may be based upon assumptions which are an oversimplification of the nature of comprehension. Hence, the paper examines guidelines which have been proposed, in this case for use case descriptions, and the extent to which they agree with discourse process theory; before suggesting refinements to the guidelines which attempt to utilise lessons learned from our richer understanding of the underlying discourse process theory. For example, we suggest subtly different sets of writing guidelines for the different tasks of requirements, specification and design

In vitro culture with gemcitabine augments death receptor and NKG2D ligand expression on tumour cells

Much effort has been made to try to understand the relationship between chemotherapeutic treatment of cancer and the immune system. Whereas much of that focus has been on the direct effect of chemotherapy drugs on immune cells and the release of antigens and danger signals by malignant cells killed by chemotherapy, the effect of chemotherapy on cells surviving treatment has often been overlooked. In the present study, tumour cell lines: A549 (lung), HCT116 (colon) and MCF-7 (breast), were treated with various concentrations of the chemotherapeutic drugs cyclophosphamide, gemcitabine (GEM) and oxaliplatin (OXP) for 24 hours in vitro. In line with other reports, GEM and OXP upregulated expression of the death receptor CD95 (fas) on live cells even at sub-cytotoxic concentrations. Further investigation revealed that the increase in CD95 in response to GEM sensitised the cells to fas ligand treatment, was associated with increased phosphorylation of stress activated protein kinase/c-Jun N-terminal kinase and that other death receptors and activatory immune receptors were co-ordinately upregulated with CD95 in certain cell lines. The upregulation of death receptors and NKG2D ligands together on cells after chemotherapy suggest that although the cells have survived preliminary treatment with chemotherapy they may now be more susceptible to immune cell-mediated challenge. This re-enforces the idea that chemotherapy-immunotherapy combinations may be useful clinically and has implications for the make-up and scheduling of such treatments

Are there gender differences in the geography of alcohol-related mortality in Scotland? An ecological study

<b>Background</b> There is growing concern about alcohol-related harm, particularly within Scotland which has some of the highest rates of alcohol-related death in western Europe. There are large gender differences in alcohol-related mortality rates in Scotland and in other countries, but the reasons for these differences are not clearly understood. In this paper, we aimed to address calls in the literature for further research on gender differences in the causes, contexts and consequences of alcohol-related harm. Our primary research question was whether the kind of social environment which tends to produce higher or lower rates of alcohol-related mortality is the same for both men and women across Scotland. <b>Methods</b> Cross-sectional, ecological design. A comparison was made between spatial variation in men's and women's age-standardised alcohol-related mortality rates in Scotland using maps, Moran's Index, linear regression and spatial analyses of residuals. Directly standardised mortality rates were derived from individual level records of death registration, 2000–2005 (n = 8685). <b>Results</b> As expected, men's alcohol-related mortality rate substantially exceeded women's and there was substantial spatial variation in these rates for both men and women within Scotland. However, there was little spatial variation in the relationship between men's and women's alcohol-mortality rates (r2 = 0.73); areas with relatively high rates of alcohol-related mortality for men tended also to have relatively high rates for women. In a small number of areas (8 out of 144) the relationship between men's and women's alcohol-related mortality rates was significantly different. <b>Conclusion</b> In as far as geographic location captures exposure to social and economic environment, our results suggest that the relationship between social and economic environment and alcohol-related harm is very similar for men and women. The existence of a small number of areas in which men's and women's alcohol-related mortality had an different relationship suggests that some places may have unusual drinking cultures. These might prove useful for further investigations into the factors which influence drinking behaviour in men and women

Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy

3D functional imaging of neuronal activity in entire organisms at single cell level and physiologically relevant time scales faces major obstacles due to trade-offs between the size of the imaged volumes, and spatial and temporal resolution. Here, using light-field microscopy in combination with 3D deconvolution, we demonstrate intrinsically simultaneous volumetric functional imaging of neuronal population activity at single neuron resolution for an entire organism, the nematode Caenorhabditis elegans. The simplicity of our technique and possibility of the integration into epi-fluoresence microscopes makes it an attractive tool for high-speed volumetric calcium imaging.Comment: 25 pages, 7 figures, incl. supplementary informatio

Meditation-induced near-death experiences: a 3-year longitudinal study

Near-death experiences (NDEs) are life transformational events that are increasingly being subjected to empirical research. However, to date, no study has investigated the phenomenon of a meditation-induced near-death experience (MI-NDE) that is referred to in ancient Buddhist texts. Given that some advanced Buddhist meditators can induce NDEs at a pre-planned point in time, the MI-NDE may make NDEs more empirically accessible and thus advance understanding into the psychology of death-related processes. The present study recruited 12 advanced Buddhist meditators and compared the MI-NDE against two other meditation practices (i.e. that acted as control conditions) in the same participant group. Changes in the content and profundity of the MI-NDE were assessed longitudinally over a 3-year period. Findings demonstrated that compared to the control conditions, the MI-NDE prompted significantly greater pre-post increases in NDE profundity, mystical experiences and non-attachment. Furthermore, participants demonstrated significant increases in NDE profundity across the 3-year study period. Findings from an embedded qualitative analysis (using grounded theory) demonstrated that participants (i) were consciously aware of experiencing NDEs, (ii) retained volitional control over the content and duration of NDEs and (iii) elicited a rich array of non-worldly encounters and spiritual experiences. In addition to providing corroborating evidence in terms of the content of a “regular” (i.e. non-meditation-induced) NDE, novel NDE features identified in the present study indicate that there exist unexplored and/or poorly understood dimensions to NDEs. Furthermore, the study indicates that it would be feasible - including ethically feasible - for future research to recruit advanced meditators in order to assess real-time changes in neurological activity during NDEs

HIV non-B subtype distribution: emerging trends and risk factors for imported and local infections newly diagnosed in South Australia

Monitoring HIV subtype distribution is important for understanding transmission dynamics. Subtype B has historically been dominant in Australia, but in recent years new clades have appeared. Since 2000, clade data have been collected as part of HIV surveillance in South Australia. The aim of this study was to evaluate the prevalence of and risk factors for HIV-1 non-B subtypes. The study population was composed of newly diagnosed, genotyped HIV subjects in South Australia between 2000 and 2010. We analyzed time trends and subtype patterns in this cohort; notification data were aggregated into three time periods (2000–2003, 2004–2006, and 2007–2010). Main outcome measures were number of new non-B infections by year, exposure route, and other demographic characteristics. There were 513 new HIV diagnoses; 425 had information on subtype. The majority (262/425) were in men who have sex with men (MSM), predominantly subtype B and acquired in Australia. Infections acquired in Australia decreased from 77% (2000–2003) to 64% (2007–2010) ( p = 0.007) and correspondingly the proportion of subtype B declined from 85% to 68% ( p = 0.002). Non-B infections were predominantly (83%) heterosexual contacts, mostly acquired overseas (74%). The majority (68%) of non-B patients were born outside of Australia. There was a non-significant increase from 1.6% to 4.2% in the proportion of locally transmitted non-B cases (p = 0.3). Three non-B subtypes and two circulating recombinant forms (CRFs) were identified: CRF_AE (n = 41), C (n = 36), CRF_AG (n = 13), A (n = 9), and D (n = 2). There has been a substantial increase over the past decade in diagnosed non-B infections, primarily through cases acquired overseas

A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry

<p>Abstract</p> <p>Background</p> <p>Relatively recently, the software KB™ Basecaller has replaced <it>phred </it>for identifying the bases from raw sequence data in DNA sequencing employing dideoxy chemistry. We have measured quantitatively the consequences of that change.</p> <p>Results</p> <p>The high quality sequence segment of reads derived from the KB™ Basecaller were, on average, 30-to-50 bases longer than reads derived from <it>phred</it>. However, microbe identification appeared to have been unaffected by the change in software.</p> <p>Conclusions</p> <p>We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to <it>phred</it>. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.</p