The structure, function and regulation of mycobacterial porin-encoding genes

Two years ago Senaratne et al., (1998) published research into a gene, ompATb, which encodes an outer membrane protein in the genus Mycobacterium. It was shown that the protein, OmpATb, possesses a pore-forming ability in liposomes and can be incorporated into lipid bilayers in which it exhibits voltage-gated pore characteristics. This study examines the distribution of this gene, and the protein product OmpATb, amongst different mycobacterial species using a PCR based method, and demonstrates immunoblotting using an antibody previously raised against the protein. Regulation of the porin genes in Escherichia coli by a two-component sensor-regulator pair is discussed and parallels drawn to a putative method of regulation in the mycobacteria. Regulation of the mycobacterial porin is demonstrated by real-time RT-PCR of Mycobacterium bovis BCG RNA and the addition of osmotic stress to the bacterial culture is shown to have a dramatic effect on the levels of ompATb transcript. Two genes, Rv0902c and Rv0903c, are proposed to have a mode of action in controlling the expression of the porin gene in Mycobacterium tuberculosis. These were identified by homology to their Escherichia coli counterparts by searching the published genome of Mycobacterium tuberculosis (Cole et al., 1998). The regulatory component, Rv0903c, is cloned into an expression vector and the recombinant Rv0903 protein overexpressed in Escherichia coli. This protein product is purified using immobilised metal affinity chromatography and is used in gel retardation assays to show that two regions around the ompATb gene bind the protein. Phosphorylation of the protein in vitro is also shown to enhance the binding affinity. The porin-encoding gene is expressed in a non-native organism, Mycobacterium smegmatis, both from its native promoter and from the mycobacterial hsp60 promoter, the effect of overexpression of this gene and the protein product is investigated. The protein is also cloned into Mycobacterium smegmatis in the presence and the absence of the putative regulatory machinery. An attempt to characterise the promoter region of the ompATb gene is made using the construction of lacZ reporter vectors. These are used for assays of β-galactosidase activity in Mycobacterium smegmatis. It is shown that the ompATb promoter functions in this organism and that the length of upstream sequence included affects the promoter activity. A suicide vector for knockout of the ompATb gene is constructed and transformed into Mycobacterium tuberculosis H37Rv in an attempt to disrupt the function of the gene and a ΔompATb strain of Mycobacterium tuberculosis 1424 is characterised by Southern blot and PCR based methods. This strain is investigated by DNA and cDNA microarray analysis and by growth in media providing a variety of environmental stresses. Gene disruption by homologous recombination is also attempted on the regulatory gene Rv0903c, employing a different technique for suicide vector construction that uses 3 counterselectable markers. This technique has been previously applied with some success in the mycobacteria (Parish and Stoker, 2000; Parish and Stoker, 2000). A number of colonies exhibiting the correct phenotype for gene knockouts are examined using a PCR based method

The role of bile acids and farnesoid X receptor in ileal Crohn's disease

M.D.The aetiopathogenesis of Crohn’s disease (CD) is characterised by epithelial barrier dysfunction and immune dysregulation in a genetically susceptible host. Exposure to a western diet, a dysbiosis and bile acid (BA) dysmetabolism have also been implicated. The BA receptor, farnesoid-x receptor (FXR), is central to the crosstalk between the host and its microbiota. FXR acts to maintain the epithelial barrier and has an immuno-modulatory function although the mechanism is not fully understood. The first aim of this study was to investigate the effect of FXR agonism using invitro models of cytokine-induced epithelial barrier dysfunction. Functional and morphological studies of Caco-2 cell monolayers demonstrated that FXR acts to maintain epithelial cell morphology, probably via a mechanism involving the regulation of myosin light chain kinase expression. FXR agonism was also able to abolish the IL-8 response of HT29 cells to stimulation with TNF. The second aim of this study was to investigate the link between a westernlifestyle diet, impaired BA signalling and ileal inflammation in a mouse model of obesity. Animals fed a western-lifestyle diet expressed more ileal inflammatory cytokine. There was a trend to suggest that supplementation with an FXR agonist reduced inflammatory cytokine expression. Finally, this study aimed to both measure FXR activity in patients with ileal CD, but also to optimise an ex-vivo tissue culture model to assay the effect of FXR agonism on ileal mucosal cytokine production. FXR activity was significantly reduced in patients with ileal CD as compared with controls. An ex-vivo tissue culture model was optimised, but there was no evidence that FXR agonism could attenuate the response of primary tissue to stimulation with inflammatory cytokine. xiv In summary, the data presented support the hypothesis that disrupted bile acid signalling, via FXR, may predispose to the development of ileal CD by impairing gut epithelial immune homeostasis

Development of Micro-Sized Algan Deep Ultraviolet Light Emitting Diodes and Monolithic Photonic Integrated Circuits

III-Nitride materials-based visible emission LEDs have emerged as a disruptive technology in the fields of lighting,i) communications,ii,iii,iv) and displays.v,vi) Shorter wavelength LEDs in the DUV spectral region (210nm – 360nm) with ultra-wide bandgap (UWBG) AlxGa1-xN active layers are now poised to displace toxic Mercury-based light sources.vii) Over the past decade AlGaN LEDs operating in the deep ultra-violet (DUV) spectral region (200 nm \u3c λemission \u3c 300 nm) have been deployed in novel applications including autonomous drone-based sterilization and sanitization systems, viii) point-of-use water purification systems, ix) photo-therapeutics,x) gas sensors,xi) and non-line-of-sight (NLOS) communications.xii) Similarly, DUV light detectors using ultra-wide bandgap (UWBG) AlxGa1-xN hetero-junctions have also been reported by several research groups.xiii,xiv,xv) Currently, several DOD early threat warning systems employ such solarblind DUV photodiodes.xvi) These detectors have also garnered attention for environmental safety applications in flame and radiation monitoring systems.xvii) Furthermore, ultra-wide Bandgap (UWBG) AlGaN materials-based devices are robust to the harsh conditions of outer space.xviii) Hence, the integration of UWBG (\u3e 4 eV) AlGaN-based DUV optoelectronics and electronic devices is of great interest for future miniaturized systemon-chip (SOC) applications.xix) Unlike visible emission materials platforms, at the onset of this work, there were no reports of high brightness DUV emitters, DUV Photonic Integrated Circuits (PIC), nor deeply-scaled DUV micro-LEDs. In this work, we designed and characterized the world’s first monolithically integrated DUV PICs comprised of AlGaN MQW-based light emitters and detectors and an AlGaN waveguide, demonstrated sub-20 µm sized DUV micro-LEDs (also referred to as micropixel LEDs) with record kW/cm2 class brightness, and integrated these ultra-bright emitters into a modularly interconnected array architecture with excellent power and area scalability. This work is divided into two sections with the first focused on providing proof-of-concept and experimental characterizations of DUV PICs. The second revolves around the development and characterization of deeply-scaled sub-20 µm diameter DUV emitters with aim to improve the electrical, optical, and thermal performance in addition to opening the door to future applications where traditional large-area DUV LEDs are unsuited such as high speed data transfer, direct write lithography, and high-resolution UV displays. In our pioneering work on DUV PICs, we first qualified MQW AlGaN LED epitaxial layers for use as both an emitter and detector. We next established the suitability of the n-AlGaN contact layer for waveguiding DUV radiation. Using a symmetric array of micro-LEDs with a pixel size of 30 µm, we determined the directional dependence of DUV radiation within the AlGaN waveguide layer. Then, using a neighboring emitter and detector, we etched a trench between the devices down to the sapphire substrate and measured the photocurrent after each etching iteration to determine the distribution of guided light among the epilayers. We next fabricated DUV PICs with different emitterdetector spacings and extracted the optical losses for both planar and ridge waveguides.xx,xxi) In those studies, successful detection of DUV emission at waveguide channel lengths up to 3 mm was realized for the first time. With continued development, such next-generation AlGaN materials-based PICs will have profound impacts in the fields of DUV-based gas and bio-chemical sensing as well as covert and quantum communications. To realize high brightness DUV emitters with theorized high modulation bandwidths, AlGaN-based micro-LEDs (micropixel LEDs) were designed and fabricated with sub-20 µm mesa diameters, slanted sidewalls, and a monolithically integrated heatspreader. Despite the reduced emission area for these AlGaN-based micro-LEDs, the brightness (W/cm2 ) is remarkably enhanced due to their efficient light generation at kA/cm2 -level current densities enabled by a superior uniformity of current injection and removal of the self-generated heat from the device active region.xxii) At these levels of injection current density, the dynamic carrier lifetimes, which chiefly dictate the maximum modulation bandwidth in the case of visible emission micro-LEDs, is significantly reduced.xxiii,xxiv) Further tailoring of our micro-sized devices for applications requiring a high dose of UVC radiation, we developed a novel device layout architecture for large arrays via a hierarchically interconnected micropixel geometry which we showed to decrease the series resistance and thermal impedance of the devices while increasing the EQE, maximum LOP, and peak brightness compared to a single macro-LED with an equal emission area.xxv,xxvi,xxvii) The first-generation 5 µm micropixel of this work,xxii) with vertical sidewalls, had a record-setting brightness of 291 W/cm2 at a current density of 10.2 kA/cm2 under continuous wave (CW) operation. Second generation micro-LEDs with slanted sidewalls and an optimized fabrication procedure yielded a CW brightness of up to 600 W/cm2 at 15 kA/cm2 and a pulsed-mode brightness as high as 10.2 kW/cm2 at a current density of 50 kA/cm2 without flip-chip packaging nor encapsulation.xxviii) This kW/cm2 - class performance is an order of magnitude brighter than some of the most luminescent blue LEDs found in the literature, the current benchmark.xxix,xxx,xxxi) To date, these are the smallest and brightest DUV LEDs globally. This technology, when further matured, will be particularly useful in the areas of DUV direct-write lithography, time-resolved fluorescence, optically pumped polymerbased lasers, charge control systems like envisaged in the evolved Laser Interferometer Space Antenna (eLISA) mission, and optical communications. Future development of selective area growth (SAG) techniques to marry electronic control and read-out devices with DUV micro-LED-based PIC technology is expected to lead to demonstrations of highbandwidth multi-functional UWBG AlGaN-based SOCs

Nahm Transform and Moduli Spaces of CPn Models on the Torus

There is a Nahm transform for two-dimensional gauge fields which establishes a one-to-one correspondence between the orbit space of U(N) gauge fields with topological charge k defined on a torus and that of U(k) gauge fields with charge N on the dual torus. The main result of this paper is to show that a similar duality transform cannot exist for CPn instantons. This fact establishes a significative difference between 4-D gauge theories and CPn models. The result follows from the global analysis of the moduli space of instantons based on a complete and explicit parametrization of all self-dual solutions on the two-dimensional torus. The boundary of the space of regular instantons is shown to coincide with the space of singular instantons. This identification provides a new approach to analyzing the role of overlapping instantons in the infrared sector of CPn sigma models.Comment: 28 pages, 5 eps-figure

A cross-sectional study to evaluate second line virological failure and elevated bilirubin as a surrogate for adherence to atazanavir/ritonavir in two urban HIV clinics in Lilongwe, Malawi

BACKGROUND: Malawi's national antiretroviral therapy program provides atazanavir/ritonavir-based second line regimens which cause concentration-dependent rise in indirect bilirubin. We sought to determine if elevated bilirubin, as a surrogate of atazanavir/ritonavir adherence, can aid in the evaluation of second line virological failure in Malawi. METHODS: We conducted a cross-sectional study of HIV-infected patients ≥15 years who were on boosted protease inhibitor-based second line antiretroviral therapy for at least 6 months in two urban HIV clinics in Lilongwe, Malawi. Antiretroviral therapy history and adherence data were extracted from the electronic medical records and blood was drawn for viral load, complete blood count, total bilirubin, and CD4 cell count at a clinic visit. Factors associated with virological failure were assessed using multivariate logistic regression model. RESULTS: Out of 376 patients on second line antiretroviral therapy evaluated, 372 (98.9%) were on atazanavir/ritonavir-based therapy and 142 (37.8%) were male. Mean age was 40.9 years (SD ± 10.1), mean duration on second line antiretroviral therapy was 41.9 months (SD ± 27.6) and 256 patients (68.1%) had elevated bilirubin >1.3 mg/dL. Overall, 35 (9.3%) patients had viral load >1000 copies/ml (virological failure). Among the virologically failing vs. non-failing patients, bilirubin was elevated in 34.3% vs. 72.0% respectively (p < 0.001), although adherence by pill count was similar (62.9% vs. 60.7%, p = 0.804). The odds of virological failure were higher for adults aged 25-40 years (adjusted odds ratio (aOR) 2.5, p = 0.048), those with CD4 cell count <100 (aOR 17.5, p < 0.001), and those with normal bilirubin levels (aOR 5.4, p < 0.001); but were lower for the overweight/obese patients (aOR 0.3, p = 0.026). Poor pill count adherence (aOR 0.7, p = 0.4) and male gender (aOR 1.2, p = 0.698) were not associated with second line virological failure. CONCLUSIONS: Among patients receiving atazanavir/ritonavir-based second line antiretroviral therapy, bilirubin levels better predicted virological failure than pill count adherence. Therefore, strategic use of bilirubin and viral load testing to target adherence counseling and support may be cost-effective in monitoring second line antiretroviral therapy adherence and virological failure. Drug resistance testing targeted for patients with virological failure despite elevated bilirubin levels would facilitate timely switch to third line antiretroviral regimens whenever available

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P

Genetic diversity of Koala retrovirus (KoRV) env gene subtypes: Insights into northern and southern koala populations

Koala retrovirus (KoRV) is a recently endogenised retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the “northern” and “southern” koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV’s ongoing entry into the koala genome

Multimodality imaging with CT, MR and FDG-PET for radiotherapy target volume delineation in oropharyngeal squamous cell carcinoma

Background: This study aimed to quantify the variation in oropharyngeal squamous cell carcinoma gross tumour volume (GTV) delineation between CT, MR and FDG PET-CT imaging. Methods: A prospective, single centre, pilot study was undertaken where 11 patients with locally advanced oropharyngeal cancers (2 tonsil, 9 base of tongue primaries) underwent pre-treatment, contrast enhanced, FDG PET-CT and MR imaging, all performed in a radiotherapy treatment mask. CT, MR and CT-MR GTVs were contoured by 5 clinicians (2 radiologists and 3 radiation oncologists). A semi-automated segmentation algorithm was used to contour PET GTVs. Volume and positional analyses were undertaken, accounting for inter-observer variation, using linear mixed effects models and contour comparison metrics respectively. Results: Significant differences in mean GTV volume were found between CT (11.9 cm³) and CT-MR (14.1 cm³), p < 0.006, CT-MR and PET (9.5 cm³), p < 0.0009, and MR (12.7 cm³) and PET, p < 0.016. Substantial differences in GTV position were found between all modalities with the exception of CT-MR and MR GTVs. A mean of 64 %, 74 % and 77 % of the PET GTVs were included within the CT, MR and CT-MR GTVs respectively. A mean of 57 % of the MR GTVs were included within the CT GTV; conversely a mean of 63 % of the CT GTVs were included within the MR GTV. CT inter-observer variability was found to be significantly higher in terms of position and/or volume than both MR and CT-MR (p < 0.05). Significant differences in GTV volume were found between GTV volumes delineated by radiologists (9.7 cm³) and oncologists (14.6 cm³) for all modalities (p = 0.001). Conclusions: The use of different imaging modalities produced significantly different GTVs, with no single imaging technique encompassing all potential GTV regions. The use of MR reduced inter-observer variability. These data suggest delineation based on multimodality imaging has the potential to improve accuracy of GTV definition. Trial registration: ISRCTN Registry: ISRCTN34165059. Registered 2nd February 2015

The use of hydrogen to separate and recycle neodymium–iron–boron-type magnets from electronic waste

AbstractThe rare earth metals have been identified by the European Union and the United States as being at greatest supply risk of all the materials for clean energy technologies. Of particular concern are neodymium and dysprosium, both of which are employed in neodymium–iron–boron based magnets. Recycling of magnets based on these materials and contained within obsolete electronic equipment, could provide an additional and secure supply. In the present work, hydrogen has been employed as a processing agent to decrepitate sintered neodymium–iron–boron based magnets contained within hard disk drives into a demagnetised, hydrogenated powder. This powder was then extracted mechanically from the devices with an extraction efficiency of 90 ± 5% and processed further using a combination of sieves and ball bearings, to produce a powder containing <330 parts per million of nickel contamination. It is then possible for the extracted powder to be re-processed in a number of ways, namely, directly by blending and re-sintering to form fully dense magnets, by Hydrogenation, Disproportionation, Desorption, Recombination processing to produce an anisotropic coercive powder suitable for bonded magnets, by re-melting; or by chemical extraction of the rare earth elements from the alloy. For example, it was shown that, by the re-sintering route, it was possible to recover >90% of the magnetic properties of the starting material with significantly less energy than that employed in primary magnet production. The particular route used will depend upon the magnetic properties required, the level of contamination of the extracted material and the compositional variation of the feedstock. The various possibilities have been summarised in a flow diagram

Anemia in people on second line antiretroviral treatment in Lilongwe, Malawi: a cross-sectional study

Abstract Background Anemia is common among people living with HIV infection and is frequently associated with poor quality of life and poor prognosis. It has been well described in antiretroviral naïve individuals and those on non-nucleoside reverse transcriptase inhibitor-based first line antiretroviral therapy (ART) regimens. However there is limited information on anemia for ART experienced individuals on protease inhibitor-based second line ART regimens in resource limited settings. Our objective was to describe the prevalence and risk factors of anemia in this ART experienced population in Malawi. Methods We conducted a cross-sectional study using routine facility data at two HIV clinics in Lilongwe, Malawi. The analysis included individuals receiving protease inhibitor-based second line ART. Clinical and laboratory data were collected at routine clinic visits. We used descriptive statistics, two-sample t-tests and multivariate logistic regression for data analysis. Results Three hundred seventy-seven records were included in this analysis (37% male, median age 41 years, median CD4 count 415 cells/μL). The prevalence of anemia was 125/377 (33.2%) − mild, moderate and severe anemia was 17.5%, 13.8%, and 1.9% respectively. Female participants had a higher prevalence than male participants (43.6% vs. 15.7%, p < 0.001). In multivariate logistic regression, female sex (adjusted odds ratio (aOR) 5.3; 95% CI 2.9–9.5) and a CD4 count <200 cell/ul (aOR 3.1; 95%CI 1.6–6.0) were associated with increased risk of having anemia while a BMI ≥30 kg/m2 (aOR 0.8; 95% CI 0.6–1.0) and being on ART for more than 10 years (aOR 0.4; 95% CI 0.2–0.9) were associated with reduced risk of anemia. Being on a zidovudine- containing ART regimen was not associated with anemia. Conclusion Anemia is common in people on second line ART in Lilongwe, Malawi. Screening for anemia in this population would be a useful strategy; especially for female patients, those who are underweight and have a low CD4 cell counts