In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR.

A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases - as seen with the B.1.1.7 variant - can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.

Life cycle environmental impacts of chemical recycling via pyrolysis of mixed plastic waste in comparison with mechanical recycling and energy recovery.

A large portion of plastic produced each year is used to make single-use packaging and other short-lived consumer products that are discarded quickly, creating significant amounts of waste. It is important that such waste be managed appropriately in line with circular-economy principles. One option for managing plastic waste is chemical recycling via pyrolysis, which can convert it back into chemical feedstock that can then be used to manufacture virgin-quality polymers. However, given that this is an emerging technology not yet used widely in practice, it is not clear if pyrolysis of waste plastics is sustainable on a life cycle basis and how it compares to other plastics waste management options as well as to the production of virgin plastics. Therefore, this study uses life cycle assessment (LCA) to compare the environmental impacts of chemical recycling of mixed plastic waste (MPW) via pyrolysis with the established waste management alternatives: mechanical recycling and energy recovery. Three LCA studies have been carried out under three perspectives: waste, product and a combination of the two. To ensure robust comparisons, the impacts have been estimated using two impact assessment methods: Environmental footprint and ReCiPe. The results suggest that chemical recycling via pyrolysis has a 50% lower climate change impact and life cycle energy use than the energy recovery option. The climate change impact and energy use of pyrolysis and mechanical recycling of MPW are similar if the quality of the recyclate is taken into account. Furthermore, MPW recycled by pyrolysis has a significantly lower climate change impact (-0.45 vs 1.89 t CO2 eq./t plastic) than the equivalent made from virgin fossil resources. However, pyrolysis has significantly higher other impacts than mechanical recycling, energy recovery and production of virgin plastics. Sensitivity analyses show that some assumptions have notable effects on the results, including the assumed geographical region and its energy mix, carbon conversion efficiency of pyrolysis and recyclate quality. These results will be of interest to the chemical, plastics and waste industries, as well as to policy makers.

Rapid detection of SARS-CoV-2 by pulse-controlled amplification (PCA).

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/µl This new principle might pave the way to rapid and sensitive point of care testing.

Fast Rotating Relativistic Stars: Spectra and Stability without Approximation.

We study oscillations and instabilities of relativistic stars using perturbation theory in general relativity and take into account the contribution of a dynamic spacetime. We present the oscillation spectrum as well as the critical values for the onset of the secular CFS instability of neutron stars, and propose universal relations for gravitational wave asteroseismology, which may help constrain the neutron star radius and/or the nuclear equation of state. The results are relevant for all stages during a neutron star's life but especially to nascent or remnant objects following a binary merger.

Isolation and Characterization of Blueberry Polyphenolic Components and Their Effects on Gut Barrier Dysfunction.

Highbush blueberries contain anthocyanins and proanthocyanidins that have antimicrobial and anti-inflammatory bioactivities. We isolated and characterized three polyphenolic fractions, a total polyphenol fraction (TPF), an anthocyanin-enriched fraction (AEF), and a proanthocyanidin-enriched fraction (PEF), from freeze-dried blueberry powder and evaluated their effects on an in vitro model of gut barrier dysfunction. High-performance liquid chromatography chromatograms illustrate successful fractionation of the blueberry powder into TPF, AEF, and PEF. AEF contained 21 anthocyanins, and PEF contained proanthocyanidin oligomers of (epi)catechin with primarily B-type interflavan bonds. The model uses a strain of Escherichia coli to disrupt a Caco-2 cell monolayer on Transwell inserts. Barrier function was measured by transepithelial electrical resistance (TEER), a marker of membrane permeability. All fractions were able to restore TEER values after an E. coli challenge when compared to the control, while AEF was able to attenuate the E. coli-induced decrease in TEER in a dose-dependent manner.

A methodology for integrating the biomass balance approach into life cycle assessment with an application in the chemicals sector.

Driven by the need to reduce greenhouse gas emissions and dependence on fossil resources, the chemical and other industries are gradually starting to develop bio-based products. For the introduction of bio-feedstocks in existing production pathways in a cost-effective way, a simplified approach based on mass balance has been proposed. This concept is known as the biomass balance (BMB) approach and the resulting products are called BMB products. They do not necessarily contain biomass material but can contribute to sustainable sourcing and production of bio-based products in the supply chain without any performance loss in comparison to the same products derived from fossil resources. The aim of the study is to show how the BMB approach can be used in life cycle assessment (LCA) while following the requirements set out in the ISO 14040 and 14044 standards. To demonstrate that, the proposed BMB approach has been used to estimate life cycle environmental impacts of a polymer product, which can be produced using fossil or bio-feedstocks. For the polymer derived from bio-feedstocks, bio-naphtha and biogas are considered as replacement to naphtha and its impacts are compared with the fossil-based alternative. The paper demonstrates that the BMB approach provides a quick and pragmatic method for establishing the biomass content in chemical and related products while incentivising the industry to continue increasing the proportion of bio-based products in their product portfolio. It also shows that the environmental performance of BMB products is highly dependent on the particular bio-feedstock used, the way it is sourced and on key modelling assumptions, including the assumptions on biogenic carbon uptake in the bio-feedstocks.

Aß seeding potency peaks in the early stages of cerebral ß-amyloidosis.

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the ß-amyloid peptide (Aß) is an important factor in AD pathogenesis, we asked whether Aß seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aß seeding activity in two mouse models of cerebral Aß amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aß deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aß, the resulting specific seeding activity sharply peaked at the initial phase of Aß deposition, which in turn is characterized by a temporary several-fold increase in the Aß42/Aß40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aß42 than Aß40 to seed activity. Our findings indicate that the Aß seeding potency is greatest early in the pathogenic cascade and diminishes as Aß increasingly accumulates in brain. The present results provide experimental support for directing anti-Aß therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Daytime sleep has no effect on the time course of motor sequence and visuomotor adaptation learning.

Sleep has previously been claimed to be essential for the continued learning processes of declarative information as well as procedural learning. This study was conducted to examine the importance of sleep, especially the effects of midday naps, on motor sequence and visuomotor adaptation learning. Thirty-five (27 females) healthy, young adults aged between 18 and 30years of age participated in the current study. Addressing potential differences in explicit sequence and motor adaptation learning participants were asked to learn both, a nine-element explicit sequence and a motor adaptation task, in a crossover fashion on two consecutive days. Both tasks were performed with their non-dominant left hand. Prior to learning, each participant was randomized to one of three interventions; (1) power nap: 10-20min sleep, (2) long nap: 50-80min sleep or (3) a 45-min wake-condition. Performance of the motor learning task took place prior to and after a midday rest period, as well as after a night of sleep. Both sleep conditions were dominated by Stage N2 sleep with embedded sleep spindles, which have been described to be associated with enhancement of motor performance. Significant performance changes were observed in both tasks across all interventions (sleep and wake) confirming that learning took place. In the present setup, the magnitude of motor learning was not sleep-dependent in young adults - no differences between the intervention groups (short nap, long nap, no nap) could be found. The effect of the following night of sleep was not influenced by the previous midday rest or sleep period. This finding may be related to the selectiveness of the human brain enhancing especially memory being thought of as important in the future. Previous findings on motor learning enhancing effects of sleep, especially of daytime sleep, are challenged.

Technical Note: Simple, scalable, and sensitive protocol for retrieving Bacillus anthracis (and other live bacteria) from heroin.

We describe a culture-based method suitable for isolating Bacillus anthracis and other live bacteria from heroin. This protocol was developed as a consequence of the bioforensic need to retrieve bacteria from batches of the drug associated with cases of injectional anthrax among heroin-consumers in Europe. This uncommon manifestation of infection with the notorious pathogen B. anthracis has resulted in 26 deaths between the years 2000 to 2013. Thus far, no life disease agent has been isolated from heroin during forensic investigations surrounding these incidences. Because of the conjectured very small number of disease-causing endospores in the contaminated drug it is likely that too few target sequences are available for molecular genetic analysis. Therefore, a direct culture-based approach was chosen here. Endospores of attenuated B. anthracis artificially spiked into heroin were successfully retrieved at 84-98% recovery rates using a wash solution consisting of 0.5% Tween 20 in water. Using this approach, 82 samples of un-cut heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2000 and 2014 were tested and found to be surprisingly poor in retrievable bacteria. Notably, while no B. anthracis was isolated from the drug batches, other bacteria were successfully cultured. The resulting methodical protocol is therefore suitable for analyzing un-cut heroin which can be anticipated to comprise the original microbiota from the drug's original source without interference from contaminations introduced by cutting.

Stigmatisation, Avoidance Behaviour and Difficulties in Coping are Common Among Adult Patients with Vitiligo.

Vitiligo is a non-contagious skin disorder with loss of pigmentation, often impairing patients' well-being. This study used Dermatology Life Quality Index (DLQI), Adjustment to Chronic Skin Disorders Questionnaire (ACS), Beck Depression Inventory (BDI) and additional questions to explore quality of life (QoL), coping, depression and stigmatisation and included 96 patients with vitiligo and 23 controls. Stigmatisation was common: 87/96 patients (90%) reported questions/approaches, 23/96 (24%) experienced nasty comments. Sixty-four out of 96 (66.7%) had avoided situations because of vitiligo or concealed their white spots. Sixty patients (62.5%) implied psychological stress as influential on disease's course. Patients scored higher in all questionnaires than controls (DLQI = 4.9/1.6, ACS-social anxiety/avoidance = 36.9/22.1, ACS-helplessness = 27.3/16.0, ACS-anxious-depressive mood = 19.4/15.6), except BDI (6.8/7.3). QoL of 65 patients (67.7%) was hardly impaired, 70 (72.9%) were not depressed. Treatment with pro-pseudocatalase PC-KUS reduced social anxiety/avoidance, anxious-depressive mood and depression. Patients without low-key stigmatisation scored highest in DLQI and social anxiety/avoidance. Avoidance and concealing behavior correlated with all questionnaires' scores.

Disease-related behavioral patterns and experiences affect quality of life in children and adolescents with vitiligo.

BACKGROUND: Vitiligo is an acquired, non-contagious depigmentation disorder involving a patchy loss of skin color. It often leads to stigmatization, embarrassment, and reduced quality of life (QoL) in adult patients. Little is known about children's reactions. OBJECTIVES: This study aimed to explore disease-related QoL and experiences in a multinational group of children and adolescents. METHODS: Quality of life, disease-related experiences and behavior, and sociodemographic data were examined in 24 boys and 50 girls (age range: 7-17 years) using the Children's Dermatology Life Quality Index (CDLQI) and additional questions. Eighteen children without skin disorders served as age-, sex- and skin color-matched controls. RESULTS: The mean disease duration was 3.5 years. The most common sites of onset were the trunk, legs, and head and neck. Overall, 35.1% of the 74 subjects reported a positive family history, 91.9% had visited a doctor, and 75.7% had received treatment. Two-thirds (66.2%) were distressed by their vitiligo, and 93.2% had experienced low-key stigmatization, 44.6% nasty comments, and 21.7% bullying. A total of 24.4% had concealed their disease, and 29.7% had avoided situations because of vitiligo. Frequency of stigmatization influenced avoidant behavior. Parents, particularly mothers, and friends were important sources of support. Patients and controls had similar numbers of friends and leisure time activities. The mean CDLQI score of the group was low (2.8). Higher CDLQI scores were related to stigmatization, hiding of white spots, facial depigmentation, avoidance of situations, and a vitiligo-negative family history. CONCLUSIONS: Disease-related stigmatization, negative experiences, and avoidant behavior affect QoL. Therefore, the CDLQI should be combined with other instruments to screen for disease burden. These results call for the careful evaluation of young patients with vitiligo.

Cumulative life course impairment in vitiligo.

Vitiligo is an acquired, idiopathic skin disease characterized by the mostly progressive loss of the inherited skin color leading to white patches and in some cases to total depigmentation. The course of this ancient disease is still unknown. The worldwide prevalence range is 0.5-1%. The disease burden includes stigmatization, depression, impaired quality of life, lack of self-confidence, embarrassment and self-consciousness. To the best of our knowledge, the extent to which this chronic disease may exert an influence upon the life course of affected individuals has, to date, not been investigated. The material presented herein is the result of an accurate analysis of published literature. Moreover, we included our own data collected in two studies. To apply the concept of cumulative life course impairment in vitiligo, we looked at possible trigger factors, role of patient's age and the age at disease onset, disease duration and stigmatization. Stigmatization had the strongest impact. It is common in patients with an early disease onset, often leading to other disturbances. Our data revealed that older patients or those with a disease onset later in life adjust better to this chronic skin disorder and that they are less socially avoidant. However, long disease duration can also lead to impaired quality of life and obsession, while this group seems to be less depressed or embarrassed. Results from our own work with peer groups of these patients strongly support a positive long-lasting effect of treatment on quality of life of children, adolescents and adults. To which extent vitiligo may contribute to a cumulative life course impairment remains to be shown.

A review of the worldwide prevalence of vitiligo in children/adolescents and adults.

BACKGROUND: Vitiligo is an acquired, idiopathic, and worldwide common depigmentation disorder with an estimated prevalence from 0.1 to 8%. These numbers are based on clinical population studies and field research examining inhabitants of geographically enclosed areas. Our aim was to collect all available data on the prevalence of vitiligo in the general population, paying particular attention to children/adolescent groups and adults. MATERIALS AND METHODS: Screening of available literature and online databases using several key words. RESULTS: We found more than 50 studies that used several methods and subgroups of the general population. The prevalence of vitiligo ranges from 0.06 to 2.28%, whereas this was 0.0-2.16% in children/adolescents populations. CONCLUSIONS: The often cited prevalence of 8% could not be confirmed after excluding clinical patient populations. Accordingly, the worldwide prevalence of vitiligo ranges between 0.5 and 2%.

Significant immediate and long-term improvement in quality of life and disease coping in patients with vitiligo after group climatotherapy at the Dead Sea.

Quality of life in patients with vitiligo is impaired. This study explored the immediate effect of 20 days of climatotherapy at the Dead Sea on quality of life, coping with the disease, general well-being and individual stress levels in a group of 71 patients with vitiligo and 42 matched controls. The long-term effect was assessed after 12 months in 33/71 patients and 12/42 controls. Study instruments were Dermatology Life Quality Index, Beck Depression Inventory and the Adjustment to Chronic Skin Disorders Questionnaire. Stress measurements were based on cortisol and ß-endorphin concentrations in saliva samples. Quality of life was significantly improved at day 20 at the Dead Sea compared with day 1, and this was still significant after 12 months. Moreover, social anxiety/avoidance, anxious-depressive mood and helplessness as measured by the Adjustment to Chronic Skin Disorders Questionnaire were significantly reduced. There was no difference in levels of cortisol and ß-endorphin between patients and controls, indicating that stress per se is not a significant contributor in vitiligo. In conclusion, therapy in patient groups offers an effective tool for long-lasting improvement in quality of life and patients' well-being.

From basic research to the bedside: efficacy of topical treatment with pseudocatalase PC-KUS in 71 children with vitiligo.

BACKGROUND: The epidermal accumulation of hydrogen peroxide (H(2)O(2)) has been documented in vitiligo. AIM: To assess the effect on disease cessation and repigmentation of the reduction/removal of H(2)O(2) using low-dose, narrow-band, ultraviolet-B (UV-B)-activated pseudocatalase PC-KUS in 71 children with vitiligo. METHODS: This uncontrolled and retrospective study included 45 girls and 26 boys (mean age, 10.3 years) who applied topical PC-KUS twice daily to the entire body surface without narrow-band UV-B dose increments. The affected body areas were documented by special photography at the first visit and after 8-12 months. The response was evaluated by two independent physicians as > 75% vs. < 75% total repigmentation of the face/neck, trunk, extremities, and hands/feet. Generalized (n = 61) and segmental (n = 10) vitiligo were evaluated as different entities. The effect of total-body, low-dose, narrow-band UV-B (0.15 mJ/cm(2)) monotherapy once daily without any increments and without application of PC-KUS was tested over 6 months in 10 children with vitiligo vulgaris (mean age, 8.4 years). RESULTS: One hundred per cent cessation was observed in 70 of the 71 children. More than 75% repigmentation was achieved in 66 of 71 patients on the face/neck, 48 of 61 on the trunk, and 40 of 55 on the extremities; however, repigmentation on the hands/feet was disappointing (five of 53). The response was independent of skin color, age of onset, duration of disease, other demographic features, and previous treatments. The follow-up after narrow-band UV-B monotherapy showed no significant repigmentation in all areas. Seven of 10 patients showed progression of their vitiligo. CONCLUSION: A reduction in epidermal H(2)O(2) using low-dose, narrow-band UV-B-activated pseudocatalase PC-KUS is an effective treatment for childhood vitiligo which can be safely performed at home.

Stoichiometric functionalization of gold nanoparticles in solution through a free radical polymerization approach.

A new simple concept for the stoichiometrical functionalization of nanoparticles based on free radical polymerization of vinyl protected nanoparticles is presented. To demonstrate this concept 2-bis(4-vinylphenyl)disulfane was synthesized and used in the synthesis of gold nanoparticles, leading to 4-vinylthiophenol functionalized nanoparticles. Simple free radical polymerization of these particles initiated by 4,4'-azobis-(4-cyanopentanoic acid) delivered nanoparticles with a single carboxyl group. These monofunctionalized gold nanoparticles were utilized for chemical preparation of gold nanoparticle dimers as well as for construction of gold nanoparticle arrays via binding to polyallylamine.

The NAD(P)H oxidase inhibitor apocynin improves endothelial NO/superoxide balance and lowers effectively blood pressure in spontaneously hypertensive rats: comparison to calcium channel blockade.

The vascular NAD(P)H oxidase contributes to endothelial dysfunction and high blood pressure in the spontaneously hypertensive rat by enhancing superoxide production. We investigated the effects of apocynin, a NAD(P)H oxidase inhibitor, on blood pressure and vascular radical and nitric oxide formation in SHR and compared its effects to the calcium channel blocker nifedipine. Apocynin (over four weeks) lowered systolic blood pressure significantly and as effectively as nifedipine. Both apocynin and nifedipine significantly reduced superoxide production. In parallel, vascular nitric oxide production and ecNOS activity was significantly increased by apocynin treatment. Therefore, apocynin may be an effective antihypertensive drug in essential hypertension.

Basic research confirms coexistence of acquired Blaschkolinear Vitiligo and acrofacial Vitiligo.

We report about a female patient with bilateral and unilateral blaschkolinear depigmentation on the extremities and coexistence of acrofacial vitiligo, who initially presented her first signs of depigmentation at the age of 32 years. The patient was otherwise healthy. The correct diagnosis was based on the latest up to date technology utilizing in vivo FT-Raman and Fluorescence spectroscopy, Wood's light examination of the depigmented skin and immunoreactivity of epidermal catalase expression in 3 mm punch biopsies from the linear depigmented area. The results yielded decreased catalase protein expression compared to healthy controls as well as complete absence of melanocytes. FT-Raman spectroscopy identified the presence of hydrogen peroxide (H(2)O(2)) in the mM range and Fluorescence spectroscopy demonstrated H(2)O(2)-mediated oxidation of tryptophan residues in the depigmented area. The results were in agreement with vitiligo. Repigmentation of the linear lesion was initiated after reduction/removal of epidermal H(2)O(2) with pseudocatalase PC-KUS further supporting the correct diagnosis. To the best of our knowledge this is the first case documented with vitiligo following Blaschko lines in coexistence with classical acrofacial vitiligo. This observation raises the question whether besides H(2)O(2)-mediated stress in association with genomic mosaicism could play a role in some cases with vitiligo.

Colloidal assemblies on patterned silane layers.

The site-selective assembly of colloidal polymer particles onto laterally patterned silane layers was studied as a model system for the object assembly process at mesoscale dimensions. The structured silane monolayers on silicon oxide substrates were fabricated by a combination of liquid- and gas-phase deposition of different trialkoxysilanes with a photolithographic patterning technique. By using this method various types of surface functionalizations such as regions with amino functions next to areas of the bare silica surface or positively charged regions of a quaternary ammonium silane surrounded by a hydrophobic octadecylsilane film could be obtained. Furthermore, a triethoxysilane with a photoprotected amino group was synthesized, which allowed direct photopatterning after monolayer preparation, leading to free NH(2) groups at the irradiated regions. The different silane monolayer patterns were used to study the surface assembly behavior of carboxylated methacrylate particles by optical and scanning electron microscopy. In dependence of the assembly conditions (different surface functionalizations, pH, and drying conditions), a selective preference of the particles for a specific surface type versus others was found. Site-specific colloid adsorption could be observed also on the photosensitive silane layers after local deprotection with light. From the photosensitive silane and positively charged ammonium silane, molecularly mixed monolayers were prepared, which allowed particle adsorption and photoactivation within the same monolayer as shown by fluorescence labeling.