Square-to-sine wave filter

Square-to-sine wave filter network tested for regulation and stability under fixed load

Turbolysis: A low-cost, small footprint alternative to commercial bead beaters for cell lysis.

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p < 0.005). Thus, turboLysis offers good cell lytic performance in a small form factor at a low cost

RPGR-associated retinal degeneration in human X-linked RP and a murine model

PURPOSE. We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model. METHODS. XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5–72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset. RESULTS. Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration. CONCLUSIONS. RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy

Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.Martin Donnelley, Kaye S. Morgan, Karen K. W. Siu, Nigel R. Farrow, Charlene S. Stahr, Richard C. Boucher, Andreas Fouras & David W. Parson

Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study.

BACKGROUND: The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and containment strategies. METHODS: In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years. Transmission dynamics, infectiousness, and drug susceptibility were analysed in a subset of patients from the Western Cape using whole-genome sequencing (WGS; n=149), a cough aerosol sampling system (CASS; n=26), and phenotypic testing for 18 drugs (n=179). FINDINGS: Between Oct 1, 2008, and Oct 31, 2012, we enrolled and followed up 273 patients for a median of 20·3 months (IQR 9·6-27·8). 203 (74%) had programmatically incurable tuberculosis and unfavourable outcomes (treatment failure, relapse, default, or death despite treatment with a regimen based on capreomycin, aminosalicylic acid, or both). 172 (63%) patients were discharged home, of whom 104 (60%) had an unfavourable outcome. 54 (31%) home-discharged patients had failed treatment, with a median time to death after discharge of 9·9 months (IQR 4·2-17·4). 35 (20%) home-discharged cases were smear-positive at discharge. Using CASS, six (23%) of 26 home-discharged cases with data available expectorated infectious culture-positive cough aerosols in the respirable range (<5 μm), and most reported inter-person contact with suboptimal protective mask usage. WGS identified 17 (19%) of the 90 patients (with available sequence data) that were discharged home before the diagnosis of 20 downstream cases of extensively drug-resistant tuberculosis with almost identical sequencing profiles suggestive of community-based transmission (five or fewer single nucleotide polymorphisms different and with identical resistance-encoding mutations for 14 drugs). 11 (55%) of these downstream cases had HIV co-infection and ten (50%) had died by the end of the study. 22 (56%) of 39 isolates in patients discharged home after treatment failure were resistant to eight or more drugs. However, five (16%) of 31 isolates were susceptible to rifabutin and more than 90% were likely to be sensitive to linezolid, bedaquiline, and delamanid. INTERPRETATION: More than half of the patients with programmatically incurable tuberculosis were discharged into the community where they remained for an average of 16 months, were at risk of expectorating infectious cough aerosols, and posed a threat of transmission of extensively drug-resistant tuberculosis. Urgent action, including appropriate containment strategies, is needed to address this situation. Access to delamanid, bedaquiline, linezolid, and rifabutin, when appropriate, must be accelerated along with comprehensive drug susceptibility testing. FUNDING: UK Medical Research Council, South African Medical Research Council, South African National Research Foundation, European & Developing Countries Clinical Trials Partnership, Oppenheimer Foundation, Newton Fund, Biotechnology and Biological Sciences Research Council, King Abdullah University of Science & Technology

An empirical investigation of the conditional risk-return trade-off in South Africa.

One of the fundamental tenets of finance is the relationship between risk and return. This research report contributes to the debate by testing the conditional risk-return relationship of shares on the Johannesburg Stock Exchange (JSE) for the period 2001 to 2011. More specifically, the extent to which beta, standard deviation, semi-deviation and value-at-risk (VaR) are individually able to explain total share return, taking into account the conditional framework of up and down markets and sub-periods, is investigated. Portfolios based on these risk measures have been tracked and regressed. The robustness of the relationships are tested by using value and equal weighted portfolios. The study indicates that standard deviation was able to explain the risk-return relationship across all scenarios (overall, up/down markets and sub-periods), while beta proved to be an ineffective measure of risk under all scenarios. The testing of downside risk measures revealed that semi-deviation produced weak results under all scenarios, while value-at-risk proved to be an effective measure of risk both during poor market conditions and on an overall basis

Maeotian Gray Clay Cups of the 4th – 3rd Centuries BC

The article is devoted to the typology and chronology of gray clay cups from the Maeotian burial grounds on the right bank of the Kuban river. Type 1 refers to biconical cups with a high, narrow neck widening upwards (30 items). A little over half of the studied vessels come from assemblages with a wide chronological range dating back to the 4th and early 3rd centuries BC, where they are accompanied by swords of the Sindian-Maeotian type or sets of pottery characteristic of this period. The remaining burials with cups can be dated more precisely by the finds of container amphorae from different centers (Heraclea, Thasos, Ikos, Mende, Sinope, etc.). The wide chronological framework of the existence of type 1 cups among the Maeotians can be determined within the first half of the 4th century BC, but their narrow chronology is limited to the second quarter of this century. Two versions of the origin of cups of this shape are put forward: the first one is from imported red clay vessels; the second one is from hand-made cups of the 6th–5th centuries BC. Type 2 is truncated conical cups widening upwards (13 items). There are not enough strong chronological references for a narrow dating of this type of cups. Furthermore, apart from the cups, imported vessels were found in the three assemblages including amphorae of Rhodes, a black glazed plate and a fish plate. As the analysis of the assemblages shows, type 2 cups existed among the Maeotians for a rather limited period from the late 3rd century BC up until the beginning of the next century

A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis

BACKGROUND: Cystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco®, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediated CFTR gene therapy formulation through preclinical and clinical development. OBJECTIVE: To determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF. DESIGN: This was a randomised, double-blind, placebo-controlled Phase IIb trial of the CFTR gene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers. SETTINGS: Data were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database. PARTICIPANTS: Patients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination of CFTR mutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised). INTERVENTIONS: Subjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year. MAIN OUTCOME MEASURES: The primary end point was the relative change in percentage predicted FEV1 over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference. RESULTS: There was a significant (p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (p = 0.031) and CT gas trapping (p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age or CFTR mutation class. Subjects with a more severe baseline FEV1 had a FEV1 TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups. CONCLUSIONS: Monthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo. LIMITATIONS: Although encouraging, the improvement in FEV1 was modest and was not accompanied by detectable improvement in patients’ quality of life. FUTURE WORK: Future work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration. TRIAL REGISTRATION: ClinicalTrials.gov NCT01621867

A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis