Impact of different SARS-CoV-2 assays on laboratory turnaround time.

Introduction. Clinical microbiology laboratories have had to cope with an increase in the volume of tests due to the emergence of the SARS-CoV-2 virus. Short turnaround times (TATs) are important for case tracing and to help clinicians in patient management. In such a context, high-throughput systems are essential to process the bulk of the tests. Rapid tests are also required to ensure shorter TATs for urgent situations. In our laboratory, SARS-CoV-2 assays were initially implemented on our custom platform using a previously published method. The commercial cobas 6800 (Roche diagnostics) assay and the GeneXpert Xpress (Cepheid) SARS-CoV-2 assay were implemented on 24 March and 8 April 2020, respectively, as soon as available.Hypothesis/Gap Statement. Despite the abundant literature on SARS-CoV-2 assays, the articles focus mainly on the diagnostic performances. This is to our knowledge the first article that specifically studies the TAT of different assays.Aim. We aimed to describe the impact of various SARS-CoV-2 assays on the TAT at the beginning of the outbreak.Methodology. In this study, we retrospectively analysed the TAT of all SARS-CoV-2 assays performed in our centre between 24 February and 9 June, 2020.Results. We retrieved 33 900 analyses, with a median TAT of 6.25 h. TATs were highest (6.9 h) when only our custom platform was used (24 February to 24 March, 2020). They were reduced to 6.1 h when the cobas system was introduced (24 March to 8 April, 2020). The implementation of the GeneXpert further reduced the median TAT to 4.8 h (8 April to 9 June, 2020). The GeneXpert system had the shortest median TAT (1.9 h), followed by the cobas (5.5 h) and by our custom platform (6.9 h).Conclusion. This work shows that the combination of high-throughput systems and rapid tests allows the efficient processing of a large number of tests with a short TAT. In addition, the use of a custom platform allowed the quick implementation of an in-house test when commercial assays were not yet available

Resistance training volume, energy balance and weight management: Rationale and design of a 9 month trial

The increased prevalence of obesity and the lack of treatment success both argue for the design and evaluation of strategies to prevent the development of overweight and obesity. To date, the role of resistance training (RT) in this regard is largely unexplored. RT may be effective for weight management as a result of increased fat-free mass (FFM), which may result in increased resting metabolic rate and increased physical activity energy expenditure. However, the literature relative to the efficacy of RT protocols recommended for healthy adults to alter the aforementioned parameters is inconsistent or inadequately evaluated. We will conduct a 9 month randomized controlled efficacy trial to compare changes in body composition (fat mass, FFM, % body fat) and energy balance in response to 2 volumes of RT (1 vs. 3 sets vs. non-exercise control) both at the completion of training (9 months) and 1 year later (body composition). This investigation will be conducted in a sample of healthy, normal and overweight, sedentary, young adult men and women; a group at high risk for development of overweight and obesity. Our results will provide information relative to the minimum volume of RT that may be associated with body weight/fat gain which may inform the development of guidelines for RT to prevent weight gain or to alter body composition

Water and sewage disposal for farm homes

March, 1939

DISPOSITION AND TOXICITY OF A MIXED BACKBONE ANTISENSE OLIGONUCLEOTIDE, TARGETED AGAINST HUMAN CYTOMEGALOVIRUS, AFTER INTRAVITREAL INJECTION OF ESCALATING SINGLE DOSES IN THE RABBIT

This paper is available online at http://www.dmd.org ABSTRACT: The ocular disposition and toxicity of GEM132, a mixed backbone phosphorothioate oligonucleotide developed for the treatment of cytomegalovirus-induced retinitis, were studied in rabbits for 6 months following single intravitreal injection of 5, 20, or 100 g/eye (toxicity) and 3.7, 15.7, or 78.5 g/eye (disposition). Intraocular pressure, electroretinograms, and ophthalmoscopy were evaluated in the toxicity arm as well as gross and microscopic pathology at the termination of the study. Vitreous humor, retina, and the remaining ocular tissues were collected from all animals in the disposition arm. No toxicities were observed in the low-dose group. Intraocular pressure was transiently mildly increased in the mid-and high-dose groups; macroscopic findings were mild and infrequent. Changes in electroretinograms and histopathological findings attributed to GEM132 were observed by 4 weeks postdose in the high-dose group. Area under the curve values in all ocular tissues sampled were proportional to dose, suggesting GEM132 disposition exhibited first-order kinetics. Vitreous humor concentrations decreased in a multiphasic manner, consistent with rapid distribution. Polyacrylamide gel electrophoresis analysis of retinal extracts indicated that, at 4 weeks postdose, 90% of the radioactivity was associated with parent compound. At 8 weeks postdose, this had decreased to 70%, and subsequently to 50% at 21 weeks postdose. In retina, GEM132 reached concentrations >5 times IC 90 by 1 week postdose, with maximum concentrations 4 to 8 weeks postdose. Retinal concentrations of intact GEM132 then declined at a very slow rate. Microautoradiography suggested that radioactivity was distributed throughout the retinal layers, the largest amount being located in the middle layers. The advancement of antisense oligonucleotides to human clinical trials as anticancer Human cytomegalovirus (HCMV), a common opportunistic infection in immunocompromised persons, is a major cause of life-threatening disease. CMV-induced retinitis, if left untreated, will cause severe, irreversible damage to the retina, resulting in progressive loss of vision in the involved eye(s). Currently available therapies for CMV-induced retinitis include ganciclovir, foscamet, cidofovir, and the recently approved PS antisense oligonucleotide, fomivirsen (ISIS 2922). GEM132 is a 20-mer mixed backbone oligonucleotide (MBO) with 2Ј-O-methylribonucleosides at the two 3Ј-and four 5Ј-terminal nucleotides. It is antisense, complementary to the intron-exon boundary of the UL35 and UL37 premRNA transcripts of HCMV, and has shown antiviral activity in vitro with an IC 90 of about 0.1 M against standard laboratory strains of the virus Although studies have been published on the pharmacokinetics of PS oligonucleotides after intravitreal dosing Materials and Methods Synthesis and Purification of GEM132. GEM132 is a 20-base phosphorothioate MBO (5Ј-UGGGGCTTACCTTGCGAACA-3Ј), 6605-Da molecular 1255 mass (free acid), in which two deoxynucleosides at the 5Ј end and four deoxynucleosides at the 3Ј end (underlined) are substituted with 2Ј-O-methylribonucleosides. GEM132 was chemically synthesized using deoxynucleoside phosphoramidites (Milligen, Milford, MA) and 2Ј-O-methylribonucleoside phosphoramidites (Glen Research, Sterling, VA) on an automated synthesizer as previously described Experimental Design. Male Dutch Belted rabbits were given a single intravitreal injection (50 l) of GEM132 in both eyes. Control animals received saline only. For the disposition study, [ 14 C]GEM132 was administered; eyes and selected tissues were harvested at various times postdosing (n ϭ 3 eyes/time point). The oligonucleotide was dissolved in 0.9% saline, and the doses administered were 3.7 g/eye (0.07 Ci), 15.7 g/eye (0.3 Ci), and 78.5 g/eye (1.5 Ci). For the toxicity study, unlabeled GEM132 was administered at doses of 5, 20, or 100 g/eye. Electroretinograms (ERGs). Electroretinograms were obtained from all animals in the study at regular intervals. Light stimuli included scotopic blue, red, and white single flash and 30 Hz white flicker. The animals were anesthetized with ketamine (50 mg/kg)/xylazine (5 mg/kg) and dark-adapted at least 30 min before recording of the ERG. A mydriatic agent was applied to the eyes to dilate the pupils before testing. Sample Processing and Storage Conditions. At each time point, the left eye from the even-numbered animals and both eyes from the odd-numbered animals were dissected, and retina, vitreous humor, and remaining ocular tissue were removed and weighed. The weighed vitreous humor (0.724 Ϯ 0.031 g, mean Ϯ S.D.) and retina (0.0283 Ϯ 0.0003 g) samples were homogenized in 2 ml of 0.9% saline in preweighed glass tubes with a Teflon probe. After homogenization, aliquots of retina were solubilized in 35% tetraethylammonium hydroxide. Ten milliliters of Hionic Fluor scintillation fluid (Canberra Packard, Ontario, Canada) was added to vitreous humor and solubilized retina, and radioactivity was measured by liquid scintillation spectroscopy. Remaining ocular tissue samples (1.47 Ϯ 0.18 g) were minced and digested in toto in 35% tetraethylammonium hydroxide. Duplicate aliquots of the digest were added to 10 ml of scintillation fluid, and radioactivity was determined. Samples having a radioactivity level of less than or equal to double background were considered below the limit of quantitation and considered zero. Autoradiography and Microautoradiography Conditions. At each time point, the right eye of each even-numbered animal was removed and fixed in a 2.5% gluteraldehyde solution, stored, refrigerated, and transferred within 24 h to 10% neutral buffered Formalin where they remained for at least 24 h before trimming. Eyes were then trimmed, dehydrated, infiltrated, and imbedded in paraffin within 8 days after necropsy. Six-micrometer-thick sections were prepared. The following procedures were conducted under reduced safety light conditions: sections were subjected to deparaffination, dipped in diluted and warmed Kodak NTB-2 nuclear track emulsion, followed by a 30-min rinse in hot distilled water and controlled drying. After drying, the emulsion-coated slides were stored in lightproof plastic boxes at 4 -8°C. All slides were developed after 15 days of exposure using a solution of Kodak D-19 developer, washed, fixed in Kodak Rapid Fixer, and washed. Slides were stained with H&E, mounted, and evaluated for the localization and relative concentration of radioactivity (visualized as small, reduced silver grains lying on cellular surfaces). A four-grade (1, slight; 2, mild; 3, moderate; and 4, severe) grading scheme was used to semiquantify the amount of radioactivity. The amount of radioactivity present in underlying sclera was used to establish a threshold between a negative and a slight deposition of radioactivity in the retina. Polyacrylamide Gel Electrophoresis (PAGE) and Phosphor Image Analysis. Retinal homogenates were subjected to protein digestion by incubation with proteinase K enzyme (0.25 ml of a 20 mg/ml solution) containing 20 mM Tris EDTA for 3 h at 60°C. Samples were extracted twice with Tris ETDA-saturated phenol-chloroform solution (1:1, v/v) and once with chloroform to remove proteinase K, digested protein, and lipids from nucleic acids. After extraction, an aliquot of the organic and aqueous phase from each sample was removed, and total radioactivity was determined by liquid scintillation spectroscopy. This allowed for the determination of extraction recovery. The average recoveries (ϮS.D.) were 79.7 Ϯ 13.2% (quality control standards), 74.3 Ϯ 19.1% (day 7), 75.7 Ϯ 6.4% (day 14), 72.4 Ϯ 10.3% (day 28), 74.1 Ϯ 8.2% (day 56), 87.3 Ϯ 3.6% (day 114), and 87.4 Ϯ 12.0% (day 149). No radioactivity (Ն2ϫ background) was measurable in the organic phase, indicating that all the GEM132-associated radioactivities were retained in the aqueous phase following extraction. Loss of radioactivity was believed to be due to the difficulty in completely separating the two phases; a thin layer of the aqueous phase was always left behind. An 8-l aliquot of the aqueous phase was loaded on a 20% polyacrylamide gel containing 7.5 M urea and subjected to electrophoresis (70 -290 V, 3-14 mA, 60 -153 min). Quality control standards (0.11, 0.60, and 4.1 mg/ml, corresponding to 780, 4,500, and 21,000 dpm) were prepared by adding known amounts of radiolabeled GEM132 to retinal homogenates obtained from control rabbits. Duplicate standards were loaded on each of the two outer channels of the gel. After electrophoresis, Southern blots of the gels were performed on 0.45-nitrocellulose membranes over a period of about 12 h. The nitrocellulose membranes were then removed and allowed to dry at room temperature. Membranes were exposed to the phosphor screen for 25 to 288 h (Molecular Dynamics Storage Phosphor Screen, Sunnyvale, CA) at room temperature, after which the phosphor screen was scanned by a phosphor imager (Molecular Dynamics Phosphor Imager SI) to obtain radioactivity profiles. The lower limit of detection was determined to be an area of Ն15 phosphor image units. Concentrations of intact GEM132 were calculated by multiplying the measured total radioactivity in the retina (total MBO) by the fraction eluting with the same mobility as the standards, as determined by PAGE/phosphor image analysis and correcting for purity (90%). Intergel variability and intragel variability were determined for phosphor image analysis. Intergel variability ranged from 8.6 to 5.3%. For intragel variability, the %CV for four samples processed on the same gel ranged from 5.5 to 11.1%. The limit of quantification for phosphor image analysis, determined for four different exposure times, was as follows: 295 dpm (24 h), 200 dpm (48 and 96 h), and 100 dpm (144 h). Results Ophthalmic Examinations. Signs of ocular irritation attributed to the injection of GEM132 were infrequent, mild, and transient. Mild cellular infiltrate in the anterior vitreous occurred almost exclusively in the high-dose animals from 2 to 16 weeks postdose. Signs of iritis were also mild and infrequent. Retinal lesions were observed almost exclusively in the high-dose animals. Changes in intraocular pressures were mild and transient and considered only potentially related to treatment. Electroretinograms. Representative data for ERGs are shown in Histopathology. Histopathological findings attributed to the administration of GEM132 were seen in the retina, lens, and optic nerve of a few animals in the mid-dose group at 8 and 21 weeks and the high-dose group at 4, 8, and 16 weeks postdose. Slight retinal degen- DVORCHIK AND MARQUIS eration characterized by partial disorganization of the inner and outer plexiform layer and the ganglion cell layer was observed in one high-dose animal at week 4 and in three high-dose animals at week 8. This finding was also observed in one mid-dose animal at weeks 8 and 21 and in one control and two low-dose animals at week 21. Retinal detachment was observed in one high-dose animal at week 8. A basophilic granular material was observed in the retina of one highdose animal at week 4, in three high-dose animals at week 8, and in two high-dose animals at week 16. One mid-dose animal at week 8 also presented with this finding. A slight to mild mononuclear infiltration was seen in the optic nerves of some high-dose animals at weeks 4 and 6 and in one high-dose animal at week 16. This was also observed in some mid-dose animals at week 8

The Complexity of Repairing, Adjusting, and Aggregating of Extensions in Abstract Argumentation

We study the computational complexity of problems that arise in abstract argumentation in the context of dynamic argumentation, minimal change, and aggregation. In particular, we consider the following problems where always an argumentation framework F and a small positive integer k are given. - The Repair problem asks whether a given set of arguments can be modified into an extension by at most k elementary changes (i.e., the extension is of distance k from the given set). - The Adjust problem asks whether a given extension can be modified by at most k elementary changes into an extension that contains a specified argument. - The Center problem asks whether, given two extensions of distance k, whether there is a "center" extension that is a distance at most (k-1) from both given extensions. We study these problems in the framework of parameterized complexity, and take the distance k as the parameter. Our results covers several different semantics, including admissible, complete, preferred, semi-stable and stable semantics

Reasons, Coherence, and Group Rationality

Philosophy and Phenomenological Research, EarlyView

Rhesus macaque model of chronic opiate dependence and neuro-AIDS: longitudinal assessment of auditory brainstem responses and visual evoked potentials

Our work characterizes the effects of opiate (morphine) dependence on auditory brainstem and visual evoked responses in a rhesus macaque model of neuro-AIDS utilizing a chronic continuous drug delivery paradigm. The goal of this study was to clarify whether morphine is protective, or if it exacerbates simian immunodeficiency virus (SIV) related systemic and neurological disease. Our model employs a macrophage tropic CD4/CCR5 co-receptor virus, SIVmac239 (R71/E17), which crosses the blood brain barrier shortly after inoculation and closely mimics the natural disease course of human immunodeficiency virus (HIV) infection. The cohort was divided into 3 groups: morphine only, SIV only, and SIV + morphine. Evoked potential (EP) abnormalities in sub-clinically infected macaques were evident as early as eight weeks post-inoculation. Prolongations in EP latencies were observed in SIV-infected macaques across all modalities. Animals with the highest CSF viral loads and clinical disease showed more abnormalities than those with sub-clinical disease, confirming our previous work (Raymond et al, 1998, 1999, 2000). Although some differences were observed in auditory and visual evoked potentials in morphine treated compared to untreated SIV-infected animals, the effects were relatively small and not consistent across evoked potential type. However, morphine treated animals with subclinical disease had a clear tendency toward higher virus loads in peripheral and CNS tissues (Marcario et al., 2008) suggesting that if had been possible to follow all animals to end-stage disease, a clearer pattern of evoked potential abnormality might have emerged

Theranostic SPECT reconstruction for improved resolution: application to radionuclide therapy dosimetry

BACKGROUND: SPECT-derived dose estimates in tissues of diameter less than 3× system resolution are subject to significant losses due to the limited spatial resolution of the gamma camera. Incorporating resolution modelling (RM) into the SPECT reconstruction has been proposed as a possible solution; however, the images produced are prone to noise amplification and Gibbs artefacts. We propose a novel approach to SPECT reconstruction in a theranostic setting, which we term SPECTRE (single photon emission computed theranostic reconstruction); using a diagnostic PET image, with its superior resolution, to guide the SPECT reconstruction of the therapeutic equivalent. This report demonstrates a proof in principle of this approach. METHODS: We have employed the hybrid kernelised expectation maximisation (HKEM) algorithm implemented in STIR, with the aim of producing SPECT images with PET-equivalent resolution. We demonstrate its application in both a dual 68Ga/177Lu IEC phantom study and a clinical example using 64Cu/67Cu. RESULTS: SPECTRE is shown to produce images comparable in accuracy and recovery to PET with minimal introduction of artefacts and amplification of noise. CONCLUSION: The SPECTRE approach to image reconstruction shows improved quantitative accuracy with a reduction in noise amplification. SPECTRE shows great promise as a method of improving SPECT radioactivity concentrations, directly leading to more accurate dosimetry estimates in small structures and target lesions. Further investigation and optimisation of the algorithm parameters is needed before this reconstruction method can be utilised in a clinical setting

A qualitative study of Parent to Parent support for parents of children with special needs. Consortium to evaluate Parent to Parent.

OBJECTIVE: To examine qualitatively the experiences of parents participating in Parent to Parent programs. METHOD: Twenty-four parents of children with special needs, a subset of subjects in a larger quantitative study, participated in a semi-structured telephone interview to explore the impact and meaning of being matched with a trained supporting parent. RESULTS: Qualitative analysis reveals a successful match is contingent upon creation of a reliable ally in the supporting parent, comprised of four main components: (1) perceived sameness, (2) situational comparisons that enable learning and growth, (3) round-the-clock availability of support, and (4) mutuality of support. CONCLUSIONS: Parent to Parent support creates a community of similar others trained to listen and be supportive and provides an opportunity for matched parents to experience equality and mutuality in their relationship. Findings also identify the need for quality control in Parent to Parent programs and the importance of such programs as an adjunct to traditional professional services