Faeces - Urine separation via settling and displacement: Prototype tests for a novel non-sewered sanitation system.

The development of novel, non-sewered sanitation systems like the Nano Membrane Toilet requires thorough investigation of processes that may seem well-understood. For example, unlike the settling of primary sludge, the separation of solids from liquids in a small-volume container at the scale of a household toilet has not been studied before. In two sets of experiments, the settling of real faeces and toilet paper in settling columns and the settling of synthetic faeces in a conical tank are investigated to understand the factors affecting the liquid quality for downstream treatment processes. Toilet paper is found to be a major inhibitor to settling of solids. While a lower overflow point results in better phase separation through displacement of liquid, a higher overflow point and frequent removal of solids may be more advantageous for the liquid quality.

Field testing of a prototype mechanical dry toilet flush.

A prototype of a non-fluid based mechanical toilet flush was tested in a semi-public, institutional setting and in selected peri-urban households in eThekwini municipality, Republic of South Africa. The mechanism's functionality and users' perception of the flush were assessed. User perception varied depending on background: Users accustomed to porcelain water flush toilets were open to, yet reserved about the idea of using a waterless flush in their homes. Those who commonly use Urine Diversion Dehydration Toilets were far more receptive. The user-centred field trials were complemented by a controlled laboratory experiment, using synthetic urine, -faeces, and -menstrual blood, to systematically assess the efficiency of three swipe materials to clean the rotating bowl of the flush. A silicone rubber with oil-bleed-effect was found to be the best performing material for the swipe. Lubrication of the bowl prior to use further reduced fouling. A mechanical waterless flush that does not require consumables, like plastic wrappers, is a novelty and could - implemented in existing dry toilet systems - improve acceptance and thus the success of waterless sanitation.

Planning and communicating prototype tests for the Nano Membrane Toilet: A critical review and proposed strategy.

Urban sanitation in growing cities of the Global South presents particular challenges. This led to the Bill & Melinda Gates Foundation's Reinvent The Toilet Challenge, which sparked the development of various non-sewered sanitation technologies like the Nano Membrane Toilet. Complex disruptive technologies like this entail an extensive product development process, including various types of prototype tests. While there is an abundance of literature discussing how to build prototypes, and the optimal number of tests, there has been little focus on how to plan and conduct tests, especially in a development endeavour of this complexity. Four approaches to testing are reviewed, and their strengths and weaknesses compared. A visualised testing strategy is proposed that encompasses the entire product development process and can be used to plan and communicate prototype tests for the Nano Membrane Toilet to ultimately achieve compliance with international standards.

Analysis of spinal and muscle pathology in transgenic mice overexpressing wild-type and ALS-linked mutant MATR3.

Mutations in MATR3 have been associated with amyotrophic lateral sclerosis (ALS) as well as a form of distal myopathy termed vocal cord pharyngeal distal myopathy (VCPDM). To begin to understand how mutations in MATR3 may cause disease, here we provide initial characterization of transgenic (Tg) mice expressing human wild-type (WT) MATR3 (MATR3WT) and ALS-mutant F115C MATR3 (MATR3F115C) proteins under the control of the mouse prion promoter (MoPrP). For each construct, we established multiple independent lines of mice that stably transmitted the transgene. Unexpectedly, for all stably-transmitting lines examined, MATR3 transgenic mRNA expression was more robust in muscle, with minimal expression in spinal cord. The levels of transgenic mRNA in muscle did not differ between mice from our lead MATR3F115C line and lead MATR3WT line, but mice from the lead MATR3F115C line had significantly higher levels of MATR3 protein in muscle over the lead MATR3WT line. Mice from the three independent, established lines of MATR3F115C mice developed weakness in both fore- and hind-limbs as early as < 1 months of age; whereas, MATR3WT mice aged to > 20 months were not overtly distinguishable from non-transgenic (NT) littermates based on basic motor phenotype. Muscle of both MATR3WT and MATR3F115C mice showed vacuoles by 2 months of age which worsened by ~ 10 months, but vacuolation was noticeably more severe in MATR3F115C mice. Overall, our results indicate that increasing the levels of MATR3 in muscle can cause pathologic changes associated with myopathy, with MATR3F115C expression causing overt muscle atrophy and a profound motor phenotype. The findings suggest that analysis of muscle pathology in individuals harboring ALS-linked MATR3 mutations should be routinely considered.

Thermodynamic analysis of a gamma type Stirling engine in an energy recovery system.

The demand for better hygiene has increased the need for developing more effective sanitation systems and facilities for the safe disposal of human urine and faeces. Non-Sewered Sanitary systems are considered to be one of the promising alternative solutions to the existing flush toilet system. An example of these systems is the Nano Membrane Toilet (NMT) system being developed at Cranfield University, which targets the safe disposal of human waste while generating power and recovering water. The NMT will generate energy from the conversion of human waste with the use of a micro-combustor; the heat produced will power a Stirling engine connected to a linear alternator to generate electricity. This study presents a numerical investigation of the thermodynamic analysis and operational characteristics of a quasi steady state model of the gamma type Stirling engine integrated into a combustor in the back end of the NMT system. The effects of the working gas, at different temperatures, on the Stirling engine performance are also presented. The results show that with the heater temperature of 390 °C from the heat supply via conduction at 820 W from the flue gas, the Stirling engine generates a daily power output of 27 Wh/h at a frequency of 23.85 Hz.

Probabilistic performance assessment of complex energy process systems - The case of a self-sustained sanitation system.

A probabilistic modelling approach was developed and applied to investigate the energy and environmental performance of an innovative sanitation system, the "Nano-membrane Toilet" (NMT). The system treats human excreta via an advanced energy and water recovery island with the aim of addressing current and future sanitation demands. Due to the complex design and inherent characteristics of the system's input material, there are a number of stochastic variables which may significantly affect the system's performance. The non-intrusive probabilistic approach adopted in this study combines a finite number of deterministic thermodynamic process simulations with an artificial neural network (ANN) approximation model and Monte Carlo simulations (MCS) to assess the effect of system uncertainties on the predicted performance of the NMT system. The joint probability distributions of the process performance indicators suggest a Stirling Engine (SE) power output in the range of 61.5-73 W with a high confidence interval (CI) of 95%. In addition, there is high probability (with 95% CI) that the NMT system can achieve positive net power output between 15.8 and 35 W. A sensitivity study reveals the system power performance is mostly affected by SE heater temperature. Investigation into the environmental performance of the NMT design, including water recovery and CO2/NOx emissions, suggests significant environmental benefits compared to conventional systems. Results of the probabilistic analysis can better inform future improvements on the system design and operational strategy and this probabilistic assessment framework can also be applied to similar complex engineering systems.

Conceptual environmental impact assessment of a novel self-sustained sanitation system incorporating a quantitative microbial risk assessment approach.

In many developing countries, including South Africa, water scarcity has resulted in poor sanitation practices. The majority of the sanitation infrastructures in those regions fail to meet basic hygienic standards. This along with the lack of proper sewage/wastewater infrastructure creates significant environmental and public health concerns. A self-sustained, waterless "Nano Membrane Toilet" (NMT) design was proposed as a result of the "Reinvent the Toilet Challenge" funded by the Bill and Melinda Gates Foundation. A "cradle-to-grave" life cycle assessment (LCA) approach was adopted to study the use of NMT in comparison with conventional pour flush toilet (PFT) and urine-diverting dry toilet (UDDT). All three scenarios were applied in the context of South Africa. In addition, a Quantitative Microbial Risk Assessment (QMRA) was used to reflect the impact of the pathogen risk on human health. LCA study showed that UDDT had the best environmental performance, followed by NMT and PFT systems for all impact categories investigated including human health, resource and ecosystem. This was mainly due to the environmental credits associated with the use of urine and compost as fertilizers. However, with the incorporation of the pathogen impact into the human health impact category, the NMT had a significant better performance than the PFT and UDDT systems, which exhibited an impact category value 4E + 04 and 4E + 03 times higher, respectively. Sensitivity analysis identified that the use of ash as fertilizer, electricity generation and the reduction of NOx emissions were the key areas that influenced significantly the environmental performance of the NMT system.

Retraction Note: Transgenic mice overexpressing the ALS-linked protein Matrin 3 develop a profound muscle phenotype.

The authors are retracting this article. The article describes mice expressing wild-type human MATR3. However, since publication the authors have become aware that all of the lines of mice described express human MATR3 containing the F115C mutation. Transgenic mice expressing wild-type and mutant Matrin were created simultaneously in their laboratory and, at a crucial stage of generating the DNA for embryo injection, as confirmed by an investigation by the University of Florida, the DNA preparations were accidentally mislabelled. All of the founders created were mosaic, requiring extensive breeding to isolate stable lines. Mice mislabelled as expressing wild-type MATR3 were the first to produce lines that stably transmitted the transgene and thus were the first to be characterized. However, as lines of mice that were mislabelled as expressing the mutant (F115C) MATR3 were ultimately established, the data began to suggest that an error had been made. Sequence analysis of amplified tail DNA from mice descended from the lines reported in the article have revealed that they express the F115C variant. The data described are therefore an accurate description of the pathology of mice that express the F115C variant of MATR3, but not of mice expressing wild-type MATR3. The authors are preparing a new manuscript reporting data from both mice expressing the F115C variant of MATR3 and mice expressing wild-type MATR3.

Transgenic mice overexpressing the ALS-linked protein Matrin 3 develop a profound muscle phenotype.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of upper and lower motor neurons. Mutations in the gene encoding the nuclear matrix protein Matrin 3 have been found in familial cases of ALS, as well as autosomal dominant distal myopathy with vocal cord and pharyngeal weakness. We previously found that spinal cord and muscle, organs involved in either ALS or distal myopathy, have relatively lower levels of Matrin 3 compared to the brain and other peripheral organs in the murine system. This suggests that these organs may be vulnerable to any changes in Matrin 3. In order to determine the role of Matrin 3 in these diseases, we created a transgenic mouse model for human wild-type Matrin 3 using the mouse prion promoter (MoPrP) on a FVB background.We identified three founder transgenic lines that produced offspring in which mice developed either hindlimb paresis or paralysis with hindlimb and forelimb muscle atrophy. Muscles of affected mice showed a striking increase in nuclear Matrin 3, as well as the presence of rounded fibers, vacuoles, nuclear chains, and subsarcolemmal nuclei. Immunoblot analysis of the gastrocnemius muscle from phenotypic mice showed increased levels of Matrin 3 products migrating at approximately 120 (doublet), 90, 70, and 55 kDa. While there was no significant change in the levels of Matrin 3 in the spinal cord in the phenotypic mice, the ventral horn contained individual cells with cytoplasmic redistribution of Matrin 3, as well as gliosis. The phenotypes of these mice indicate that dysregulation of Matrin 3 levels is deleterious to neuromuscular function.

A highly intensified ART regimen induces long-term viral suppression and restriction of the viral reservoir in a simian AIDS model.

Stably suppressed viremia during ART is essential for establishing reliable simian models for HIV/AIDS. We tested the efficacy of a multidrug ART (highly intensified ART) in a wide range of viremic conditions (10³-107) viral RNA copies/mL) in SIVmac251-infected rhesus macaques, and its impact on the viral reservoir. Eleven macaques in the pre-AIDS stage of the disease were treated with a multidrug combination (highly intensified ART) consisting of two nucleosidic/nucleotidic reverse transcriptase inhibitors (emtricitabine and tenofovir), an integrase inhibitor (raltegravir), a protease inhibitor (ritonavir-boosted darunavir) and the CCR5 blocker maraviroc. All animals stably displayed viral loads below the limit of detection of the assay (i.e. <40 RNA copies/mL) after starting highly intensified ART. By increasing the sensitivity of the assay to 3 RNA copies/mL, viral load was still below the limit of detection in all subjects tested. Importantly, viral DNA resulted below the assay detection limit (<2 copies of DNA/5*105 cells) in PBMCs and rectal biopsies of all animals at the end of the follow-up, and in lymph node biopsies from the majority of the study subjects. Moreover, highly intensified ART decreased central/transitional memory, effector memory and activated (HLA-DR⁺) effector memory CD4⁺ T-cells in vivo, in line with the role of these subsets as the main cell subpopulations harbouring the virus. Finally, treatment with highly intensified ART at viral load rebound following suspension of a previous anti-reservoir therapy eventually improved the spontaneous containment of viral load following suspension of the second therapeutic cycle, thus leading to a persistent suppression of viremia in the absence of ART. In conclusion, we show, for the first time, complete suppression of viral load by highly intensified ART and a likely associated restriction of the viral reservoir in the macaque AIDS model, making it a useful platform for testing potential cures for AIDS.

Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension.

OBJECTIVES: A small pool of long-lived memory CD4 T cells harboring the retroviral genome is one main obstacle to HIV eradication. We tested the impact of the gold compound, auranofin, on phenotype and viability of CD4 T cells in vitro, and on persistence of lentiviral reservoir cells in vivo. DESIGN: In-vitro and in-vivo study. The pro-differentiating effect of auranofin was investigated in human primary CD4 T cells, and its capacity to deplete the viral DNA (vDNA) reservoir was tested in a pilot study involving six SIVmac251-infected macaques with viral loads stably suppressed by antiretroviral therapy (ART) (tenofovir/emtricitabine/raltegravir). The study was then amplified by intensifying ART using darunavir/r and including controls under intensified ART alone. All therapies were eventually suspended and viro-immunological parameters were monitored over time. METHODS: Cell subpopulations were quantitated by flow cytometry following proper hematological analyses. Viral load and cell-associated vDNA were quantitated by Taqman real-time PCR. RESULTS: In naïve, central memory and transitional memory CD4 T cells, auranofin induced both phenotype changes and cell death which were more pronounced in the memory compartment. In the pilot study in vivo, auranofin transiently decreased the cell-associated vDNA reservoir in peripheral blood. When ART was intensified, a sustained decrease in vDNA was observed only in auranofin-treated monkeys but not in controls treated with intensified ART alone. After therapy suspension, only monkeys that had received auranofin showed a deferred and subsequently blunted viral load rebound. CONCLUSION: These findings represent a first step towards a remission of primate lentiviral infections.

Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy.

BACKGROUND: In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class. RESULTS: In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy. CONCLUSIONS: In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.

Enantiomeric radiochemical synthesis of R and S (1-(6-amino-9H-purin-9-yl)-3-fluoropropan-2-yloxy)methylphosphonic acid (FPMPA).

Therapy for human immunodeficiency virus (HIV)-infected patients requires chronic multidrug administration. The eventual failure of therapy in some patients has brought into question the tissue concentration of the drugs. With an appropriately radiolabeled compound, we could utilize positron emission tomography to provide quantitative time-activity curves for various tissues. We have developed a fluorine-18 labeled analog of Tenofovir, the active metabolite of Tenofovir DF, a commonly prescribed component of multidrug therapy. Because (1-(6-amino-9H-purin-9-yl)-3-fluoropropan-2-yloxy)methylphosphonic acid (FPMPA) has a chiral center, we prepared both enantiomers and confirmed that the S-isomer exhibited significantly higher antiviral activity than the R-isomer. In viral replication inhibition assays in human MT4 cells infected with SHIV(DH12R), S-FPMPA had an IC(50) of 1.85 muM (95% CI; 0.8-5.53), while the R-isomer was inactive. An appropriate chiral precursor was prepared to allow the incorporation of fluorine-18. The [(18)F]FPMPA in racemic, R, or S form was prepared in a 50 min synthesis in 38+/-5% yield (n = 23, corrected for decay). The product was of high radiochemical and enantiomeric purity. The specific activity of the final product was 4.0+/-1.8 Ci/mumol at EOB (end of bombardment). This product may provide information about drug tissue distribution in animal models under chronic drug treatment.

The genome of the kinetoplastid parasite, Leishmania major.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.