Evaluation of a field deployable, high-throughput RT-LAMP device as an early warning system for COVID-19 through SARS-CoV-2 measurements in wastewater.

Quantification of SARS-CoV-2 RNA copies in wastewater can be used to estimate COVID-19 prevalence in communities. While such results are important for mitigating disease spread, SARS-CoV-2 measurements require sophisticated equipment and trained personnel, for which a centralized laboratory is necessary. This significantly impacts the time to result, defeating its purpose as an early warning detection tool. The objective of this study was to evaluate a field portable device (called MINI) for detecting SARS-CoV-2 viral loads in wastewater using real-time reverse transcriptase loop-mediated isothermal amplification (real-time RT-LAMP). The device was tested using wastewater samples collected from buildings (with 430 to 1430 inhabitants) that had known COVID-19-positive cases. Results show comparable performance of RT-LAMP against reverse transcriptase polymerase chain reaction (RT-qPCR) when detecting SARS-CoV-2 copies in wastewater. Both RT-LAMP and RT-qPCR detected SARS-CoV-2 in wastewater from buildings with at least three positive individuals within a 6-day time frame prior to diagnosis. The large 96-well throughput provided by MINI provided scalability to multi-building detection. The portability of the MINI device enabled decentralized on-site detection, significantly reducing the time to result. The overall findings support the use of RT-LAMP within the MINI configuration as an early detection system for COVID-19 infection using wastewater collected at the building scale.

Microplastic accumulation and biomagnification in a coastal marine reserve situated in a sparsely populated area.

Toxic chemicals within and adsorbed to microplastics (0.05-5 mm) have the potential to biomagnify in food webs. However, microplastic concentrations in highly productive, coastal habitats are not well understood. Therefore, we quantified the presence of microplastics in a benthic community and surrounding environment of a remote marine reserve on the open coast of California, USA. Concentrations of microplastic particles in seawater were 36.59 plastics/L and in sediments were 0.227 ±â€¯0.135 plastics/g. Densities of microplastics on the surfaces of two morphologically distinct species of macroalgae were 2.34 ±â€¯2.19 plastics/g (Pelvetiopsis limitata) and 8.65 ±â€¯6.44 plastics/g (Endocladia muricata). Densities were highest in the herbivorous snail, Tegula funebralis, at 9.91 ±â€¯6.31 plastics/g, potentially due to bioaccumulation. This study highlights the need for further investigations of the prevalence and potential harm of microplastics in benthic communities at remote locations as well as human population centers.

Cartilage T1ρ and T2 relaxation times: longitudinal reproducibility and variations using different coils, MR systems and sites.

OBJECTIVE: To evaluate the longitudinal reproducibility and variations of cartilage T1ρ and T2 measurements using different coils, MR systems and sites. METHODS: Single-Site study: Phantom data were collected monthly for up to 29 months on four GE 3T MR systems. Data from phantoms and human subjects were collected on two MR systems using the same model of coil; and were collected on one MR system using two models of coils. Multi-site study: Three participating sites used the same model of MR systems and coils, and identical imaging protocols. Phantom data were collected monthly. Human subjects were scanned and rescanned on the same day at each site. Two traveling human subjects were scanned at all three sites. RESULTS: Single-Site Study: The phantom longitudinal RMS-CVs ranged from 1.8% to 2.7% for T1ρ and 1.8-2.8% for T2. Significant differences were found in T1ρ and T2 values using different MR systems and coils. Multi-Site Study: The phantom longitudinal RMS-CVs ranged from 1.3% to 2.6% for T1ρ and 1.2-2.7% for T2. Across three sites (n = 16), the in vivo scan-rescan RMS-CV was 3.1% and 4.0% for T1ρ and T2, respectively. Phantom T1ρ and T2 values were significantly different between three sites but highly correlated (R > 0.99). No significant difference was found in T1ρ and T2 values of traveling controls, with cross-site RMS-CV as 4.9% and 4.4% for T1ρ and T2, respectively. CONCLUSION: With careful quality control and cross-calibration, quantitative MRI can be readily applied in multi-site studies and clinical trials for evaluating cartilage degeneration.

It's not what you hear, it's the way you think about it: appraisals as determinants of affect and behaviour in voice hearers.

BACKGROUND: Previous studies have suggested that beliefs about voices mediate the relationship between actual voice experience and behavioural and affective response. METHOD: We investigated beliefs about voice power (omnipotence), voice intent (malevolence/benevolence) and emotional and behavioural response (resistance/engagement) using the Beliefs About Voices Questionnaire - Revised (BAVQ-R) in 46 voice hearers. Distress was assessed using a wide range of measures: voice-related distress, depression, anxiety, self-esteem and suicidal ideation. Voice topography was assessed using measures of voice severity, frequency and intensity. We predicted that beliefs about voices would show a stronger association with distress than voice topography. RESULTS: Omnipotence had the strongest associations with all measures of distress included in the study whereas malevolence was related to resistance, and benevolence to engagement. As predicted, voice severity, frequency and intensity were not related to distress once beliefs were accounted for. CONCLUSIONS: These results concur with previous findings that beliefs about voice power are key determinants of distress in voice hearers, and should be targeted specifically in psychological interventions.

Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease.

Mitochondrial diseases are frequently caused by heteroplasmic mitochondrial DNA (mtDNA) mutations. As these mutations express themselves only at high relative ratios, any approach able to manipulate mtDNA heteroplasmy can potentially be curative. In this study, we developed a system to manipulate mtDNA heteroplasmy in all skeletal muscles from neonate mice. We selected muscle because it is one of the most clinically affected tissues in mitochondrial disorders. A mitochondria-targeted restriction endonuclease (mito-ApaLI) expressed from AAV9 particles was delivered either by intraperitoneal or intravenous injection in neonate mice harboring two mtDNA haplotypes, only one of which was susceptible to ApaLI digestion. A single injection was able to elicit a predictable and marked change in mtDNA heteroplasmy in all striated muscles analyzed, including heart. No health problems or reduction in mtDNA levels were observed in treated mice, suggesting that this approach could have clinical applications for mitochondrial myopathies.

Plant genotype and nitrogen loading influence seagrass productivity, biochemistry, and plant-herbivore interactions.

Genetic variation within and among key species can have significant ecological consequences at the population, community, and ecosystem levels. In order to understand ecological properties of systems based on habitat-forming clonal plants, it is crucial to clarify which traits vary among plant genotypes and how they influence ecological processes, and to assess their relative contribution to ecosystem functioning in comparison to other factors. Here we used a mesocosm experiment to examine the relative influence of genotypic identity and extreme levels of nitrogen loading on traits that affect ecological processes (at the population, community, and ecosystem levels) for Zostera marina, a widespread marine angiosperm that forms monospecific meadows throughout coastal areas in the Northern Hemisphere. We found effects of both genotype and nitrogen addition on many plant characteristics (e.g., aboveground and belowground biomass), and these were generally strong and similar in magnitude, whereas interactive effects were rare. Genotypes also strongly differed in susceptibility to herbivorous isopods, with isopod preference among genotypes generally matching their performance in terms of growth and survival. Chemical rather than structural differences among genotypes drove these differences in seagrass palatability. Nitrogen addition uniformly decreased plant palatability but did not greatly alter the relative preferences of herbivores among genotypes, indicating that genotype effects are strong. Our results highlight that differences in key traits among genotypes of habitat-forming species can have important consequences for the communities and ecosystems that depend on them and that such effects are not overwhelmed by known environmental stressors.

What are the most effective intervention techniques for changing physical activity self-efficacy and physical activity behaviour--and are they the same?

There is convincing evidence that targeting self-efficacy is an effective means of increasing physical activity. However, evidence concerning which are the most effective techniques for changing self-efficacy and thereby physical activity is lacking. The present review aims to estimate the association between specific intervention techniques used in physical activity interventions and change obtained in both self-efficacy and physical activity behaviour. A systematic search yielded 27 physical activity intervention studies for 'healthy' adults that reported self-efficacy and physical activity data. A small, yet significant (P < 0.01) effect of the interventions was found on change in self-efficacy and physical activity (d = 0.16 and 0.21, respectively). When a technique was associated with a change in effect sizes for self-efficacy, it also tended to be associated with a change (r(s) = 0.690, P < 0.001) in effect size for physical activity. Moderator analyses found that 'action planning', 'provide instruction' and 'reinforcing effort towards behaviour' were associated with significantly higher levels of both self-efficacy and physical activity. 'Relapse prevention' and 'setting graded tasks' were associated with significantly lower self-efficacy and physical activity levels. This meta-analysis provides evidence for which psychological techniques are most effective for changing self-efficacy and physical activity.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease.

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Automated analysis of postoperative behaviour: assessment of HomeCageScan as a novel method to rapidly identify pain and analgesic effects in mice.

This study evaluated whether the automated behaviour recognition software 'HomeCageScan' (HCS) could detect behaviour changes and any positive analgesic effects in two mouse strains undergoing vasectomy (C3H/HeNCrl and C57BL/6). Another objective was to test the effectiveness of HCS in differentiating between the effects of each treatment relative to conventional manual analysis. Each control (unoperated) group consisted of four mice of each strain. They were either untreated mice, mice given meloxicam alone (10 mg/kg) or mice given either saline or meloxicam (10 mg/kg) 30 min prior to isoflurane anaesthesia. The vasectomized mice received either saline or meloxicam at 5, 10 or 20 mg/kg, again, 30 min prior to isoflurane anaesthesia. Filming began one hour following surgery. Each mouse was filmed for 6 min for the manual analysis and then for a further 20 min for analysis with HCS. In a time-matched test, HCS and the manual analysis produced activity data that generated identical conclusions regarding treatment effects and strain differences. Both HCS and the manual analysis found the C57BL/6 controls were overall more active, but not following vasectomy, when both types of analysis detected markedly reduced activity. Low-dose meloxicam (5 mg/kg) had a positive effect on postoperative mobility in the C3H/HeNCrl mice; however, increasing the dose rate progressively reduced this. These effects were also detected with the manual analysis. Overall, HCS provided a sufficiently accurate and rapid method of analysing mouse behaviour encouraging more prolonged assessments in the future. This capability and the possibility of training the software to recognize a greater range of behaviours, including pain-specific indicators, should be of considerable value for assessing postoperative behaviour in both mice and rats. This would allow analgesic requirements to be investigated in a greater range of rodent models than is currently feasible with conventional analysis methods.

Anxiety in recovery from severe burn injury: an experimental comparison.

Adjustment to burn injury is a slow process that often results in high distress and anxiety. This study aimed to investigate the impact on anxiety of viewing various types of photographic stimuli (including burn injuries), comparing a burn-injured and control sample. An experimental design was used comparing those recovering from a burn injury with age- and gender-matched controls. Thirty participants from a burns support group and their matched controls were asked to complete a trait anxiety measure (STAI Y-2). Participants then completed the state anxiety measure (STAI Y-1) after viewing different types of photographic stimuli (burn injuries, uninjured body parts, neutral) under controlled experimental conditions. Results demonstrated significant differences in anxiety levels between groups with respect to trait anxiety and state anxiety after each experimental condition. Age of injury was also found to be a significant influence over trait anxiety. A significant effect of experimental condition was found, as was a significant interaction between group and condition. Those with burn injuries were more anxious than controls - a difference that was further exaggerated when participants viewed photographs of burned or uninjured body parts in an experimental setting.

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model.

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

Insulin-like growth factor 1 (IGF-1) and its receptor mRNA levels in breast cancer and adjacent non-neoplastic tissue.

UNLABELLED: Previous studies investigating the insulin-like growth factor 1 receptor (IGF-1R) expression in breast cancer tissue and adjacent non-neoplastic breast tissue (ANCT) have produced conflicting results. The IGF-1 and IGF-1R expression in pairs of breast cancer tissue and ANCT were investigated using RT-PCR and immunohistochemistry. The results of both methods were compared. MATERIALS AND METHODS: IGF-1 and IGF-1R mRNA from 31 specimen pairs were estimated using RT-PCR. Immunohistochemistry for IGF-1R was carried out on 20 specimen pairs and the strength of staining was scored. RESULTS: The mean relative IGF-1 mRNA level was lower in the cancerous tissue (mean 0.450 +/- 0.206) than in the ANCT (mean 0.632 +/- 0.384) (paired t-test, p = 0.001). There was no measurable difference in relative IGF-1R mRNA levels in the cancerous tissue (mean 0.146 +/- 0.08) and the ANCT (mean 0.14608 +/- 0.108) (paired t-test, p = 0.807). Using immunohistochemistry, there was no statistical difference (paired t-test, p = 0.910) in IGF-1R staining scores between cancer (mean 1.93) and ANCT (mean 1.90). The comparison between the two methodologies showed no correlation (Pearson's Correlation Coefficient = -0.393). DISCUSSION: It can be concluded that IGF-1 expression is lower in cancerous tissue, thus supporting a paracrine relationship between cancerous tissue and ANCT, which may be useful in the prevention, diagnosis and treatment of breast cancer. There was no difference in the expression of the IGF-1 receptor in both types of tissue, as proven by RT-PCR and immunohistochemistty. Conflicting results in previous studies may be due to the different methods used to measure IGF-1R expression.

Immunological phenotyping of fibroblast cultures from patients with a mitochondrial respiratory chain deficit.

Conventional approaches to the diagnosis of mitochondrial respiratory chain diseases, using enzyme assays and histochemistry, are laborious and give limited information concerning the genetic basis of a deficiency. We have evaluated the diagnostic value of 12 monoclonal antibodies to subunits of the four respiratory chain enzyme complexes and F(1)F(0)-ATP synthase. Antibodies were used in immunological studies with skin fibroblast cultures derived from patients with diverse mitochondrial diseases, including patients in which the disease was caused by a nuclear genetic defect and patients known to harbor a heteroplasmic mutation in a mitochondrial tRNA gene. Immunoblotting experiments permitted the identification of specific enzyme assembly deficits and immunocytochemical studies provided clues regarding the genetic origin of the disease. The immunological findings were in agreement with the biochemical and genetic data of the patients. Our study demonstrates that characterization of the fibroblast cultures with the monoclonal antibodies provides a convenient technique to complement biochemical assays and histochemistry in the diagnosis of mitochondrial respiratory chain disorders.

A novel mutation in SURF1 causes skipping of exon 8 in a patient with cytochrome c oxidase-deficient leigh syndrome and hypertrichosis.

Leigh syndrome is a rare pediatric neurodegenerative disorder attributed to impaired mitochondrial energy metabolism. Mutations in SURF1 have been described in several patients with Leigh syndrome associated with cytochrome c oxidase deficiency. We report a new 18-bp deletion (821del18), spanning the splice donor junction of exon 8 of SURF1, in an infant presenting with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. cDNA sequencing demonstrated that this deletion results in a messenger lacking exon 8. RT-PCR experiments suggested a rapid degradation of the aberrant mRNA species from the 5'-end.

Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency?

Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain. It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons. Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation. In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase. Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups. Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease. To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques. These studies have allowed us to identify some of the steps of the assembly process.

Methylene blue based protein solder for vascular anastomoses: an in vitro burst pressure study.

BACKGROUND AND OBJECTIVE: Attempts at sutureless anastomoses have used protein-based solders containing chromophores [Oz et al., J Vasc Surg 1990;11:718; Poppas et al., J Urol 1998150:1052] to enhance the strength of laser anastomoses. Reports have described the use of indocyanine green [Oz et al., Surg Forum 1989;316.], fuschin, and fluorescein isothiocyanate as chromophores [Chuck et al. , Lasers Surg Med 1989;9:471; Vance et al., Lasers Med Sci 1988;3:219]. Methylene blue (MB) is a chromophore with absorption peaks in the 600-700 nm region whose use has not been reported in laser-assisted vascular anastomoses. Therefore, we set out to produce and characterise a MB-containing protein solder. The absorption and burst pressure characteristics have been investigated and described as well as a brief review of the chemical and biological properties of MB. STUDY DESIGN/MATERIALS AND METHODS: The MB and porcine serum albumin (PSA)-based solder was produced and used to form end-to-end anastomoses in porcine splenic arteries. The solder was activated using a laser diode emitting at 670 nm. The burst pressures of the anastomoses were tested, and the results analysed as a function of MB concentration and absorption. In addition, the relationship between MB concentration and absorption was examined. RESULTS: A dose-response relationship was found between the measured absorption of the solder and the burst pressure of the anastomoses formed. Burst pressures exceeding physiological levels were found. Changes in MB concentration revealed a marked negative deviation from Beer's law at 670 nm, owing to the monomer-dimer-trimer equilibria. CONCLUSION: PSA with MB solder is able to form high-quality end-to-end anastomoses, with immediate burst pressure profiles similar to those previously described for sutured [Quigley et al., Microsurgery 1985;6:229], lasered [Quigley et al., Microsurgery 1985;6:229], and soldered anastomoses [Small et al., J Clin Laser Med Surg 1997;15:205]. The relationship between burst pressure strength and chromophore absorption is discussed.

Vocal expression of anger and cardiovascular reactivity within dyadic interactions.

This experiment was conducted to examine whether the vocal expression of anger correlated with cardiovascular reactivity within dyadic interactions. Participants selected three social issues on which they had a strong opinion and, with a confederate who opposed their views, debated these opinions in each of the three vocal styles. The three vocal styles were (1) Anger-out during which they described their view in a loud, fast voice, (2) Mood-incongruent during which they described their view in a soft, slow voice, and (3) Anger-in during which they listened to the confederate oppose their view and only responded from a list of neutral phrases given to them. Cardiovascular reactivity measures (heart rate and blood pressure) were taken during the initial baseline and the three expression of anger conditions. Both the anger-out and mood-incongruent vocal styles significantly correlated with systolic blood pressure and heart-rate reactivity measures. The disparity between the results of this experiment and previous ones on anger and cardiovascular response may be related to differences in the method of anger-arousal (memory-evoked versus dyadic interactions). Results are discussed in terms of similarities to active and passive coping and defensiveness.