Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Survival of Plants During Short-Term BOA-OH Exposure: ROS Related Gene Expression and Detoxification Reactions Are Accompanied With Fast Membrane Lipid Repair in Root Tips

For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10–30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30–60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

Discover, Sample and Refine: Exploring Chemistry with Enhanced Sampling Techniques

Over the last few decades enhanced sampling methods have made great strides. Here, we exploit this progress and propose a modular workflow for blind reaction discovery and characterization of reaction paths. Central to our strategy is the use of the recently developed explore variant of the on-the-fly probability enhanced sampling method. Like metadynamics, this method is based on the identification of appropriate collective variables. Our first step is the discovery of new chemical reactions and it is performed biasing a one dimensional collective variable derived from spectral graph theory. Once new reaction pathways are detected, we construct ad-hoc tailored neural-network based collective variables to improve sampling of specific reactions and finally we refine the results using free energy perturbation theory. Our workflow has been successfully applied to both intramolecular and intermolecular reactions. Without any chemical hypothesis, we discovered several possible products, computed the free energy surface at semiempirical level, and finally refined it with a more accurate Hamiltonian. Our workflow requires minimal user input, and thanks to its modularity and flexibility, can extend the scope of ab initio molecular dynamics for the exploration and characterization of reaction space.

Detection of invasive Trichosporon asahii in patient blood by a fungal PCR array

Rare invasive fungal infections are increasingly emerging in hosts with predisposing factors such as immunodeficiency. Their timely diagnosis remains difficult, as their clinical picture may initially mimic infections with more common fungal species and species identification may be difficult with routine methods or may require time-consuming subcultures. This often results in ineffective drug administration and fatal outcomes. We report on a patient in their early twenties with mixed cellularity classical Hodgkin lymphoma with a disseminated Trichosporon asahii (T. asahii) infection. Even though pathogen detection and identification was possible via the standard procedure consisting of culture followed by matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry, the patient passed away in the course of multi organ failure. Herein, we report on a retrospectively applied experimental diagnostic fungal PCR-analysis used on an EDTA blood sample and consisting of two pan-fungal reactions and seven branch-specific reactions. Regarding invasive T. asahii infection, this PCR array could considerably shorten time to diagnosis and switch to a targeted therapy with triazoles.

SPECIFIC DETECTION OF SARS-COV-2 VARIANTS B.1.1.7 (ALPHA) AND B.1.617.2 (DELTA) USING A ONE-STEP QUANTITATIVE PCR ASSAY

In this report, we describe the development of an RT-qPCR assay, termed Alpha Delta assay, which can detect SARS-COV-2 (SC-2) and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in N gene, and the Delta reaction targets the spike gene 156-158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha Delta assay that targets the 119-120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15-25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha Delta assay and the Orf8-119del assay were successfully used to classify clinical samples that were subsequently analyzed by whole genome sequencing. Lastly, the capability of the Alpha Delta assay and Orf8-119del assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health.

Longitudinal estimation of stress-related states through bio-sensor data

PurposeThe authors aim to develop a conceptual framework for longitudinal estimation of stress-related states in the wild (IW), based on the machine learning (ML) algorithms that use physiological and non-physiological bio-sensor data.Design/methodology/approachThe authors propose a conceptual framework for longitudinal estimation of stress-related states consisting of four blocks: (1) identification; (2) validation; (3) measurement and (4) visualization. The authors implement each step of the proposed conceptual framework, using the example of Gaussian mixture model (GMM) and K-means algorithm. These ML algorithms are trained on the data of 18 workers from the public administration sector who wore biometric devices for about two months.FindingsThe authors confirm the convergent validity of a proposed conceptual framework IW. Empirical data analysis suggests that two-cluster models achieve five-fold cross-validation accuracy exceeding 70% in identifying stress. Coefficient of accuracy decreases for three-cluster models achieving around 45%. The authors conclude that identification models may serve to derive longitudinal stress-related measures.Research limitations/implicationsProposed conceptual framework may guide researchers in creating validated stress-related indicators. At the same time, physiological sensing of stress through identification models is limited because of subject-specific reactions to stressors.Practical implicationsLongitudinal indicators on stress allow estimation of long-term impact coming from external environment on stress-related states. Such stress-related indicators can become an integral part of mobile/web/computer applications supporting stress management programs.Social implicationsTimely identification of excessive stress may improve individual well-being and prevent development stress-related diseases.Originality/valueThe study develops a novel conceptual framework for longitudinal estimation of stress-related states using physiological and non-physiological bio-sensor data, given that scientific knowledge on validated longitudinal indicators of stress is in emergent state.

Development and Evaluation of a Fusion Polyprotein Based on HspX and Other Antigen Sequences for the Serodiagnosis of Tuberculosis

BackgroundThe lack of suitable diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. Serological tests, based on multiple target antigens, represent an attractive option for diagnosis of this disease due to their rapidity, convenience, and low cost.MethodsMeasures to reduce non-specific reactions and thereby improve the specificity of serological tests were investigated, including blocking antibodies against common bacteria in serum samples and synthesizing polypeptides covering non-conserved dominant B-cell epitopes of antigens. In addition, a fusion polyprotein containing HspX and eight other antigen sequences was constructed and expressed to increase overall sensitivity of the tests.ResultsInclusion of Escherichia coli lysate partially increased the specificity of the serological tests, while synthesis and inclusion of peptides containing non-conserved sequences of TB antigens as well as dominant B-cell epitopes reduced non-specific reactions without a decrease in sensitivity of the tests. A polyprotein fusing HspX and eight other antigen sequences was constructed and displayed 60.2% sensitivity, which was higher than that of HspX and the other individual antigen segments. Moreover, the specificity of the polyprotein was 93.8%, which was not significantly decreased when compared with HspX and the other individual antigen segments.ConclusionsThe roles of the fusion polyprotein in the humoral immune response against TB infection were demonstrated and provide a potential novel approach for the development of TB diagnostics.

Shortageflation 3.0. Gospodarka wojenna – państwowy socjalizm – kryzys pandemiczny

The crisis caused by the pandemic has induced governments and central banks to undertake non-orthodox actions aimed at the protection of people’s living standards and the maintenance of production and service activities of enterprises. The policy of the aggressive rise in money supply has resulted in a considerable increase in budget deficits and foreign debts. In this context, it is important to seek an answer to the question how this can accelerate inflationary processes and to formulate proper suggestions addressed to economic policy. In fact, inflation now is higher than the official price indices because it is partly dampened. The rise in the general price level does not reflect fully the actual intensity of inflation. We have to do with a price and resource inflation, called shortageflation. Methodologically, it is interesting to compare this contemporary phenomenon (3.0) with inflation suppression in a war economy (1.0) and in the economies of real socialism (2.0). Such comparisons show some similarities of these processes but also significant differences due to the specific reactions of households and enterprises. The author discusses five channels of liquidating the excessive money resources and indicates the ways most advisable from the point of view of the sustainable development in the post-pandemic future. Especially important is to stimulate the transformation of a part of the inflationary money surplus into the market demand for goods and services which increases the utilization of the existing production potential and leads to investments which create new production capacities as well as the conversion of compulsory savings into voluntary savings.

OBSERVING PEOPLE'S REACTIONS AND RESPONSES TO URBAN ROAD TRAFFIC NOISE (RTN) IN JORDAN

The subjective aspect of assessing Road Traffic Noise (RTN) in urban agglomerations is crucial, as it takes into consideration the sensitivity and specific reactions of residents to Road Traffic noise (RTN) in their living environments. This paper aims to present a detailed sociological study initiated to measure the level of public awareness regarding RTN and estimate the impact of RTN disturbance on RTN disturbance their daily activities. For this purpose, an attitudinal survey was conducted using a predesigned questionnaire with limited noise measurements. The questioners were distributed to the neighboring residents of one of the most congested arterial roads in Amman, Jordan’s capital. The questioner firstly focused on evaluating the awareness of respondents of the problem magnitude and its environmental and health impacts, and secondly, assessing the effects of RTN as perceived by the respondents on different daily activities. The analysis of the collected data found that around 50% of respondents believe that RTN impacts the environment, and 60% believe that RTN affects human health, while 38% consider changing their place of residence and moving to a quieter place. The main results of the study showed that RTN causes annoyance to people while performing their daily activities, especially while studying and resting. Further main reported impacts included causing fatigue (64%), anxiety (75%), nervousness (87%), focus reduction (89%), and discomfort (90%).

Influence of vehicular emissions on metropolitan trees using chlorophyll contents

The study assessed carbon sequestration potential of the identified trees {Gmelina, Mango, Neem, Masquerade, Step and Cassia} along the five selected roadsides in Abeokuta metropolitan. Leaves of the identified trees were plucked, digested in triplicate and analysed for chlorophyll (Ch) A, B and A+B contents using UV-visible spectrophotometer. Obtained data were subjected to descriptive and inferential statistics while correlation analysis for relationship between the analysed leaf Ch contents. Results indicated that leaf chlorophyll contents varied. The ranking of tree to metropolitan vehicular emission along roadsides was Neem < Gmelina < Mango < Masquerade tree. Correlation matrix analysis indicated that production of the Ch contents across the roadsides tree species was not alike and might be due to their specific reactions to vehicular emission. In conclusion, the order of Ch contents might be the tree species potential to withstand vehicular pollutants.