Negative Control Experiments Research Articles

Methodology for fast testing of carbon-based nanostructured 3D electrodes in vanadium redox flow battery

Progress in material chemistry is manifested daily by the variety of prepared functional materials, often with nanodimensional structuring. The electrodes for vanadium redox flow batteries have been shown to benefit from incorporating nanostructured materials such as carbon nanotubes. However, the methods of such incorporation are far from optimal, relying mainly on physical deposition or insertion into a binder. Here, we describe a technique for integrating carbon-based rod-like nanomaterials into a vanadium redox flow battery and a methodology for fast nanomaterial performance testing. The technique is based on creating a fixed nanomaterial bed sandwiched between two graphite felt electrodes, forming a 3D flow-through electrode in the battery. Performing various positive and negative control experiments, we show the beneficial effect of a nanostructured bed on the primary battery characteristics obtained from short-term electrochemical experiments. We characterize carbon nanotubes exhibiting promising electrochemical behavior in vanadium electrolytes, as observed in our previous study. The load curves obtained from charge-discharge steps at various current densities and electrolyte flow rates revealed considerable differences in the performance of the tested materials, with few-walled carbon nanotubes reaching unsurpassable characteristics. At room temperature, with 50 %-SOC-working solutions and the highest tested linear velocity of 14.6 cm/min, the evaluated power density for this material reached values above 500 mW/cm2. For comparison, thermally treated graphite felt, used as a benchmark material, provided a power density of around 300 mW/cm2 under identical conditions. Although developed for vanadium redox flow batteries, the method enables testing tube-like and rod-like (nano-)materials for flow electrochemical systems.

Multidimensional Outcome Parameters in a Cress Seedling-CuCl2 Crystallization Assay to Corroborate Specific Effects of Stannum metallicum 30x Compared to Lactose 30x.

Previously we developed a test system which yielded highly significant evidence for specific effects of a Stannum metallicum 30x preparation in a multi-center replication trial. This test system is based on cress seed germination in homeopathic or control samples, CuCl2 crystallization of the cress extract, and subsequent digital textural image analysis of the resulting crystallization patterns. The current study aimed to investigate whether three novel outcome parameters could further corroborate and possibly characterize the specific effects of Stannum metallicum 30x. To this end, (1) cress seedling length, (2) a second texture analysis parameter, entropy and (3) the local connected fractal dimension (LCFD) of crystallization patterns as a measure of complexity were considered. The stability of the experimental setup was monitored throughout the entire investigation with systematic negative control (SNC) experiments. Cress length and entropy revealed a time-modulated potency treatment effect, in the absence of a significant main treatment effect. This indicated that the effect of the potency treatment varied significantly across the different experimental days. LCFD yielded a highly significant potency treatment effect. In addition, a significant interaction of treatment with experimental day seems to indicate a modulation of this effect. No significant effects were observed in any of the evaluations of the SNC experiments, indicative of a stable experimental setup and a reliable and specific treatment effect. Neither significant nor strong correlations were found between the four parameters, indicating that they reflect different effects of Stannum metallicum 30x on the organism treated. This quadruple characterization of the biological effects of Stannum metallicum 30x provides an unprecedented opportunity for basic homeopathy research into, among others, the presumed specificity of homeopathic preparations.

Need for external replication trials in homeopathic basic research: presenting research on three duckweed test systems as an example

One of the main aims in homeopathic basic research is to find reliable test systems that are externally replicable. In face of the replication crisis in research in general and the specificities that are necessary to include in homeopathic basic research, it is of great importance to encourage high-quality replication trials. One way to achieve that is to build up a register or a data base for test systems in homeopathic basic research that are already developed to the point that external replication trials are possible. Such a register offers the opportunity to include not just the published information but also helpful insight such as material lists and time frames for easier planning and conduction of replication trials. Additionally, further information on critical environmental parameter, unpublished negative results, etc. could be included.
 The general frame work of such a register/ data base is demonstrated with the example of three different duckweed test systems developed in our working group since 2001. The growth rate of duckweed (Lemna gibba L., clone number 9352) was investigated in randomised and blinded settings, using either untreated plants or plants pre-treated with a stressor (arsenic solution or calcium deficiency in the growth medium). Within the original trials, potentised substances in the range of 14x-33x were compared to water control groups (succussed and unsuccussed water). The stability of the test systems was investigated in parallel systematic negative control (SNC) experiments. All original trials revealed a significant influence of some potencies on the growth rate of duckweed and stable test systems as shown with SNC experiments. Four internal replication trials for two of the three test systems were conducted. The replication trials showed effects of homeopathic preparations with some differences in effect size and effect direction compared to original studies.

The CUT&RUN suspect list of problematic regions of the genome

BackgroundCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions.ResultsWe compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified.ConclusionsWe propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

Code-free machine learning for classification of central nervous system histopathology images.

Machine learning (ML), an application of artificial intelligence, is currently transforming the analysis of biomedical data and specifically of biomedical images including histopathology. The promises of this technology contrast, however, with its currently limited application in routine clinical practice. This discrepancy is in part due to the extent of informatics expertise typically required for implementation of ML. Therefore, we assessed the suitability of 2 publicly accessible code-free ML platforms (Microsoft Custom Vision and Google AutoML), for classification of histopathological images of diagnostic central nervous system tissue samples. When trained with typically 100 to more than 1000 images, both systems were able to perform nontrivial classifications (glioma vs brain metastasis; astrocytoma vs astrocytosis, prediction of 1p/19q co-deletion in IDH-mutant tumors) based on hematoxylin and eosin-stained images with high accuracy (from ∼80% to nearly 100%). External validation of the predicted accuracy and negative control experiments were found to be crucial for verification of the accuracy predicted by the algorithms. Furthermore, we propose a possible diagnostic workflow for pathologists to implement classification of histopathological images based on code-free machine platforms.

Convergent cardiorespiratory neurons represent a significant portion of cardiac and respiratory neurons in the vagal ganglia.

Significant cardiorespiratory coordination is required to maintain physiological function in health and disease. Sensory neuronal “cross-talk” between the heart and the lungs is required for synchronous regulation of normal cardiopulmonary function and is most likely mediated by the convergence of sensory neural pathways present in the autonomic ganglia. Using neurotracer approaches with appropriate negative control experiments in a mouse model, presence of cardiorespiratory neurons in the vagal (nodose) ganglia are demonstrated. Furthermore, we found that convergent neurons represent nearly 50% of all cardiac neurons and approximately 35% of all respiratory neurons. The current findings demonstrate a pre-existing neuronal substrate linking cardiorespiratory neurotransmission in the vagal ganglia, and a potentially important link for cardiopulmonary cross-sensitization, which may play an important role in the observed manifestations of cardiopulmonary diseases.

Corrigendum] MicroRNA‑335 is downregulated in bladder cancer and inhibits cell growth, migration and invasion via targeting ROCK1

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that, in Fig.3 on p. 4382, the 'Invasion' assay data for the negative control (NC) experiments for the T24 and EJ cell lines appeared to contain an overlap of data, such that they may have been derived from the same original source even though the data were purportedly intended to show the results from differently peformed experiments. The authors have re‑examined their original data, and realize that this figure was inadvertently assembled incorrectly. The revised version of Fig.3, showing alternative data from one of the repeated experiments, is shown below. Note that this error did not significantly affect either the results or the conclusions reported in this paper, and all the authors agree to this corrigendum. Furthermore, the authors thank the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this corrigendum, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 13: 4379-4385, 2016; DOI: 10.3892/mmr.2016.5055].

Simulated distributions from negative experiments highlight the importance of the body mass index distribution in explaining depression-body mass index genetic risk score interactions.

BackgroundDepression and obesity are complex global health problems. Recent studies suggest that a genetic predisposition to obesity might be accentuated in people with depression, but these analyses are prone to bias. Here, we tested the hypothesis that depression accentuates genetic susceptibility to obesity and applied negative control experiments to test whether any observed interactions were real or driven by confounding and statistical biases.MethodsWe used data from up to 378 000 Europeans in UK Biobank, a 73 variant body mass index (BMI) genetic risk score, two depression measures [depression symptoms (DS), major depression (MD)] and an antidepressant usage variable available. We tested whether (i) depression and (ii) antidepressant treatment accentuated genetic susceptibility to obesity. Finally, we performed negative control experiments by sampling individuals at random so that they had BMI distributions identical to depression cases and controls.ResultsDepression was associated with an accentuation of an individual’s genetic risk of obesity with evidence of interactions for both DS and MD (Pinteraction = 7 × 10–4 and 7 × 10–5 respectively). Antidepressant usage within DS cases accentuated genetic obesity risk (Pinteraction = 9 × 10–4), but not for MD (Pinteraction = 0.13). Negative control experiments suggested that the observed interactions for MD (empirical-P = 0.067) may be driven by statistical biases or confounding factors but were not possible with the larger DS groups. Antidepressant usage interaction also appears to be driven by statistical artefacts (empirical-P = 0.510 using MD and 0.162 using DS).ConclusionWe have highlighted the importance of running negative experiments to confirm putative interactions in gene–environment studies. We provide some tentative evidence that depression accentuates an individual’s genetic susceptibility to higher BMI but demonstrated that the BMI distributions within cases and controls might drive these interactions.

Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions - a replication study

Background: Inhibition of human basophil activation by highly diluted histamine was reported to be a reliable experimental model to examine biological effects of high dilutions. However, independent replications did not always yield concordant results.
 Aims: We aimed at performing an independent replication of a former study [1] using rigorously controlled experimental conditions to minimise confounding factors.
 Materials and Methods: In 20 independent experiments, human basophils were treated with highly diluted histamine (15cH, 16cH, corresponding to 10-30-10-32 M) prior to activation by fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Controls were treated with analogously diluted water (15cH, 16cH). The dilutions were prepared freshly for each experiment in deionised water by successive steps of centesimal dilution and agitation (10 s vortex at high speed). Highly diluted samples were blinded and randomised. All samples were set in triplicates. Activated basophils were determined by flow cytometry using anti-CD203c. 20 independent systematic negative control (SNC) experiments were carried out to investigate possible systematic errors. 
 Results: No difference in basophil activation was observed between the highly diluted histamine samples and the highly diluted water controls. There was no evidence for a blood donor specificity of the results. The SNC experiments demonstrated the stability of the test system. Experimental variability within and between experiments was slightly reduced for the highly diluted histamine samples.
 Discussion: This study was designed as an independent reproduction of a former study [1]. Though we strictly adopted the experimental procedure described in [1], our results do not confirm the large inhibitory effects observed for histamine 15cH and 16cH. This lack of reproducibility might be due to minor differences in the experimental design, such as blinding and randomising of the samples, which we chose to perform in order to reduce the possibility of artifacts but was omitted in the former study.
 Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research.
 Keywords: Human basophils; histamine; high dilutions; flow cytometry
 Reference:
 [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.

Reproducibility of effects of the homeopathic dilutions 14x - 30x of gibberellic acid on growth of Lemna gibba L.

Background: Reproducibility of investigations in homeopathy is still challenging. Duckweed (Lemna gibba L.), a monocotyledonous waterplant which mostly reproduces vegetatively and therefore builds genetically identical clones, may be a suitable test system for standardised trials. 
 Aims: This study investigated if formerly observed effects of gibberellic acid 14x – 30x on growth of Lemna gibba were reproducible. 
 Methododology: Duckweed was grown in dilutions of gibberellic acid (14x–30x) as well as once succussed (c1) and unsuccussed (c0) water control. Area-related growth rate for day 0–7 was determined by a computerised image analysis system. Three series including five independent blinded and randomised experiments each were carried out in the same way as in the original study. Only time and conductor of experiments were modified. System stability was controled by three series of systematic negative control (SNC) experiments with the same set-up, but distilled and autoclaved water was used as the only test substance. According to the series with gibberellic acid, each serie of SNC experiments included five experiments. Full two-way ANOVA (α = 5%) was used for statistical analysis. Independent variables were treatment and experiment number, dependent variable was r(area) for day 0–7. Data of each experiment was normalised to its mean value to allow a better comparison between experiments. Only if the global ANOVA F-test was significant (p < 0.05) we compared the investigated groups with Fisher`s LSD test (protected Fisher`s LSD).
 Results: No specific effects of agitated dilutions of gibberellic acid were found in the first two replication series (p=0.263 and p=0.062). In the third serie with gibbous Lemna gibba L. we observed a significant effect (p=0.009) of the homeopathic treatment, however growth was increased in contrast to decreasing in the former study. Variability in experiments with gibberellic acid 14x – 30x was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. 
 Conclusions: When designing new studies to investigate reproducibility, different physiological states of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

Comparative study of two bioassays with weakened duckweed and yeast treated with homeopathic preparations

In homeopathic basic research, the question as to the most adequate test systems and apt methodology is still open.
 This investigation examined the hypothesis that more complex organisms show stronger reactions to homeopathic remedies than less complex ones.
 We compared two Arsenic (As5+) stressed bioassays with duckweed (Lemna gibba, a multi-cellular autotrophic organism) and yeast (Saccharomyces cerevisiae, a single-cellular heterotrophic organism) regarding their response to homeopathic preparations [1].
 For duckweed, growth rates of leaf area and leaf number were evaluated. For yeast, growth kinetics were determined by measuring slope, yield and Et50 (point in time when yield was half maximum) of the sigmoid growth curve. The experiments with duckweed and yeast were performed in parallel (same day, same location and identical homeopathic preparations).
 After screening 17 substances, three homeopathic preparations (Arsenicum album, nosode, gibberellic acid) were chosen for repeated experimental series [2]. Five independent experiments were conducted for each remedy with both organisms in parallel. Potency levels used were in the range of 17x–33x for duckweed and 17x–30x for yeast. To control for test system stability, systematic negative control experiments were conducted over the complete experimentation period. All experiments were blinded and randomized.
 The systematic negative control experiments did not yield any significant effects. Application of potentized Arsenicum album in the duckweed bioassay yielded the largest effects compared to water controls without remedies for the parameters leaf area and leaf number (p

REHBaR: a Publication Guideline for Homeopathic Basic Research

Background: Specific efficacy of homeopathically prepared substances in high dilution levels is controversial due to the comparably low number of randomized controlled clinical trials and due to the low concentrations of the substances potentised. No generally accepted theoretical model is currently available to explain specific effects of such preparations. In order to unequivocally demonstrate any such effects, experimental research has to meet very high standards. 
 Aims: Objective was to compile guidelines for homeopathic basic research regarding experimental design, implementation, execution, evaluation and publication. 
 Methods: A Delphi Process was conducted, involving European researchers who published experimental work within the last five years. The Delphi process included a total of five rounds, three rounds of adjusting and phrasing plus two consensus conferences [1]. Eligible criteria were collected from existing publications concerned with the quality of homeopathic basic research. In advance a short questionnaire was sent to a selection of research institutes in Germany active in non-homeopathic basic research. 
 Results: Regarding experimental design, the most important points to consider are: randomized and coded (blinded) allocation of the treatments, several independent experiments (including independent production lots), potentised or succussed controls, positive controls (to control reactivity of the system) and systematic negative control experiments to document test system stability and adequacy of the statistical evaluation [1–4]. A detailed publication guideline for authors was developed. REHBaR (Reporting experiments in Homeopathic Basic Research) provides a checklist of 23 items, supplemented with detailed examples [4]. Background, objectives and possible hypotheses should be given in the part ‘introduction’. Special emphasis is put on the ‘materials and methods’ section, where a detailed description of chosen controls, object of investigation, experimental setup, replication, parameters, intervention, allocation, blinding, and statistical methods is required. The section ‘results’ should present sufficient details on analyzed data, descriptive as well as inferential. Authors should discuss their results and give an interpretation in the context of current evidence.
 Conclusions: Guidelines how to prepare detailed and informative publications are very common in clinical research [5]. To the best to our knowledge REHBaR is the first guideline to be applied by authors when preparing their manuscripts and to be used by scientific journals in the reviewing process in the field of homeopathic basic research. Furthermore the REHBaR guideline can be helpful for planning and conducting experiments as it includes fundamental qualitative standards. To which extent REHBAR can be used also as an instrument to evaluate the quality of a publication will be discussed.

Use of plant bioassays in homeopathic basic research - a systematic review

Background:
 
 Experimental research on the effects of treatments with homeopathic preparations on plants was last reviewed in 1990.
 
 
 
 Aims:
 
 The objective was to compile a systematic literature review on plant bioassays in homeopathic basic research using predefined criteria.
 
 
 
 Methods:
 
 Literature search was carried out on publications that reported experiments with homeopathic preparations on whole plants, seeds, plant parts or cells from 1920 to 2010, in healthy, abiotically or biotically stressed conditions. Outcomes had to be measured by established state-of-the-art procedures and statistically evaluated. Using a Manuscript Information Score (MIS) those publications were identified that provided sufficient information for proper interpretation (MIS > 5). Further evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations and on the use of systematic negative control experiments to ensure proper system performance.
 
 
 
 Results:
 
 A total of 157 publications with plants were identified [1–3]. The 157 publications described a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS > 5 allowing proper interpretation. 29 studies were identified with adequate controls to identify specific effects of homeopathic preparations, reporting significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro’s number. Studies that tested series of consecutive potency levels reported a non-linear and discontinuous relation between effect and potency level. There were many individual studies with diverse methods and very few replication trials. 10 studies reported use of systematic negative control experiments, yielding evidence for the stability of the experimental set-up.
 
 
 
 Conclusions:
 Plant models appear to be a useful approach to investigate basic research questions on homeopathic preparations, but more independent replication trials and systematic research are needed. Systematic negative control experiments should be implemented on a routine basis to exclude false-positive and false-negative results.

Novel electrochemiluminescent assay for the aptamer-based detection of testosterone

This work presents a proof-of-concept assay for the detection and quantification of small molecules based on aptamer recognition and electrochemiluminescence (ECL) readout. The testosterone-binding (TESS.1) aptamer was used to demonstrate the novel methodology. Upon binding of the target, the TESS.1 aptamer is released from its complementary capture probe – previously immobilized at the surface of the electrode – producing a decrease in the ECL signal after a washing step removing the released (labeled) TESS.1 aptamer. The analytical capability of the ECL assay towards testosterone detection was investigated displaying a linear range from 0.39 to 1.56 μM with a limit of detection of 0.29 μM. The selectivity of the proposed assay was assessed by performing two different negative control experiments; i) detection of testosterone with a randomized ssDNA sequence and ii) detection of two other steroids, i.e. deoxycholic acid and hydrocortisone with the TESS.1 aptamer. In parallel, complementary analytical techniques were employed to confirm the suggested mechanism: i) native nano-electrospray ionization mass spectrometry (native nESI-MS) was used to determine the stoichiometry of the binding, and to characterize aptamer-target interactions; and, ii) isothermal titration calorimetry (ITC) was carried out to elucidate the dissociation constant (Kd) of the complex of testosterone and the TESS.1 aptamer. The combination of these techniques provided a complete understanding of the aptamer performance, the binding mechanism, affinity and selectivity. Furthermore, this important characterization carried out in parallel validates the real functionality of the aptamer (TESS.1) ensuring its use towards selective testosterone binding in further biosensors. This research will pave the way for the development of new aptamer-based assays coupled with ECL sensing for the detection of relevant small molecules.

Reproducibility of effects of homeopathically potentised Argentum nitricum on the growth of Lemna gibba L. in a randomized and blinded bioassay

Background: Reproducibility of basic research investigations in homeopathy is still a challenging issue. A former study reported a significant interaction between date of experiment and treatment effect of Argentum nitricum 14x–30x on the growth rate of duckweed (Lemna gibba L.).
 In a reproduction trial with duckweed (Lemna gibba L.) and potencies of gibberellic acid duckweed appeared only then sensitive to treatment with homeopathic potencies of gibberellic acid potencies when the plants were in the growth state of gibbosity.
 Aims: This study investigated if formerly observed effects of Argentum nitricum 14x – 30x on growth of Lemna gibba were reproducible and if sensitivity to homeopathic treatment was influences by the growth state of duckweed.
 Methods: Duckweed was grown in potencies of Argentum nitricum (14x–30x) and one time succussed and unsuccussed water control (c0 and c1). Area-related growth rate for day 0–7 was determined by a computerised image analysis system. The original study including six independent blinded and randomised experiments each were repeated one time completely and a second time with 3 experiments. Every repetition experiment was carried out in the same way as in the original study, only time and conductor of experiments were modified. System stability was controled by systematic negative control (SNC) experiments with the same set-up, but water as the only test substance. All water used in potency and SNC experiments was distilled and autoclaved before.
 Statistical analysis was evaluated with full two-way ANOVA (α = 5%), using treatment and experiment number as independent variables and r(area) for day 0–7 as dependent variable. Data of each experiment was normalised to its mean value to allow a better comparison between experiments. Only if the global ANOVA F-test was significant (p < 0.05) investigated groups were compared with Fisher`s LSD test (protected Fisher`s LSD).
 Results: The original study observed a significant interaction between treatment and experiment number compared to cpool (c0+c1).Caused to the fact that in the first repetition series a significant treatment difference between c0 and c1 was found, additionally a significant interaction between experiment number and c0 and c1, it was not possible to pool c0 and c1. All data (original study, as well as repetition series 1 and 2) was evaluated comparing potencies to one time succussed control c1. This new statistical evaluation confirmed the significant interaction between treatment and experiment number for data of the original study (p = 0.0055). In the first repetition series a significant treatment effect (p = 0.016) was observed, in the second series no effect.Variability in experiments with Argentum nitricum 14x – 30x was higher than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. An a posteriori subgroup analysis suggests that using a specific growth state of Lemna gibba (gibbosity) seems to be crucial for successful reproduction of the significant interaction of the original study.
 Conclusions: With the original study design chosen (disregarding gibbosity of Lemna gibba), results of the original study could not be reproduced. Bioassays for use in homeopathic basic research must carefully consider different physiological states of the test organisms. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

Electrochemical impedance biosensor for detection of saxitoxin in aqueous solution.

Saxitoxin is a cyanotoxin which is very harmful to human health; the concentration limit in drinking water is only 3μg/L. Therefore, a simple, fast, sensitive, low-cost, and specific method for its detection, quantification, and monitoring in water bodies is needed to avoid adverse effects on animal and human health. In this work, we developed an electrochemical impedimetric biosensor using a specific aptamer as recognition element for saxitoxin detection. This method allies the superior sensing characteristics of aptamers with the nondestructive, label-free, and easy working principles of the electrochemical impedance technique. The device presented sensitivity for detecting saxitoxin concentrations above 0.3μg/L, with high selectivity in negative control experiments, demonstrating a promising alternative for water toxin detection.

Noninvasively visualize the expression of LAPTM4B protein using a novel 18F-labeled peptide PET probe in hepatocellular carcinoma

ObjectiveLysosomal protein transmembrane 4 beta (LAPTM4B) is selectively expressed in hepatocellular carcinoma (HCC) cells and thus a potential biomarker for diagnosing HCC. In this study, we designed a novel 18F-labeled PET probe to non-invasively visualize LAPTM4B expression in mouse model of HCC tumor. MethodsA PET targeting tracer named [18F]FP-LAP2H was radio-synthesized using a LAPTM4B targeting peptide, LAP2H, coupled with 4-nitrophenyl-2-[18F]fluoropropionate ([18F]NFP). Radio-stability, cell uptake, micro PET/CT imaging and ex vivo biodistribution were performed for determining its stability, cell binding specificity, and tumor targeting in vivo. Results[18F]FP-LAP2H was successfully synthesized with radiochemical yields of 6–14% (decay-corrected yield) and molar activity of 10–44 GBq/μmol. The tracer showed stable (~90%) in phosphate-buffered saline, pH 7.4, and in human serum (~80%) for 2 h. In vitro cell uptake studies indicated the radioactivity accumulation in HCC cells was LAPTM4B protein-specific. Micro PET/CT demonstrated that implanted LAPTM4B positive HepG2 and BEL7402 tumors could be clearly visualized. The ex vivo biodistribution studies demonstrated that the tumor/liver ratio were 1.80 ± 0.65 and 2.09 ± 0.68 in implanted HepG2 and BEL7402 tumors respectively. Negative control and blocking experiments revealed that the radioactivity uptake in the HCC tumor was LAPTM4B protein-specific. Conclusions[18F]FP-LAP2H appears to be a potential PET tracer for imaging LAPTM4B-positive HCC tumor. Further endeavors need to do to improve tumor/liver ratio.

Effects of Homeopathic Preparations of Mercurius corrosivus on the Growth Rate of Moderately Mercury-Stressed Duckweed Lemna gibba L.

Background A bioassay with severely mercury-stressed duckweed ( Lemna gibba L.) had revealed growth-inhibiting effects of homeopathically potentised mercury(II) chloride ( Mercurius corrosivus, Merc-c. ). We hypothesised that effects of potentised preparations are dependent on the stress level of the organisms used in the bioassay. The aim of the present investigation was to examine the response of duckweed to potentised Merc-c. at a lower stress level. Methods Duckweed was moderately stressed with 2.5 mg/L mercury(II) chloride for 48 hours. Afterwards plants grew in either Merc-c. (seven different potency levels, 24x–30x) or water controls (unsuccussed or succussed water) for 7 days. Growth rates of the frond (leaf) area were determined using a computerised image-analysis system for day 0–3 and 3–7. Three independent experiments with potentised Merc-c. and three systematic negative control experiments were performed. All experiments were randomised and blinded. Results Unsuccussed and succussed water did not significantly differ in their effects on duckweed growth rate. The systematic negative control experiments did not yield any significant effects, thus providing evidence for the stability of the experimental system. Data from the two control groups and the seven treatment groups ( Merc-c. 24x–30x) were each pooled to increase statistical power. Duckweed growth rates for day 3–7 were enhanced ( p < 0.05) after application of Merc-c. compared with the controls. Growth rates for day 0–3 were not influenced by the homeopathic preparations. Conclusions Moderately mercury-stressed Lemna gibba L. yielded evidence of growth-enhancing specific effects of Merc-c. 24x–30x in the second observation period (day 3–7). This observation is complementary to previous experiments with severely mercury-stressed duckweed, in which a decrease in growth was observed in the first observation period (day 0–3). We hypothesise that the differing results are associated with the level of stress intensity (moderate vs. severe).

Renin–angiotensin system blockers and susceptibility to COVID-19: an international, open science, cohort analysis

SummaryBackgroundAngiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) have been postulated to affect susceptibility to COVID-19. Observational studies so far have lacked rigorous ascertainment adjustment and international generalisability. We aimed to determine whether use of ACEIs or ARBs is associated with an increased susceptibility to COVID-19 in patients with hypertension.MethodsIn this international, open science, cohort analysis, we used electronic health records from Spain (Information Systems for Research in Primary Care [SIDIAP]) and the USA (Columbia University Irving Medical Center data warehouse [CUIMC] and Department of Veterans Affairs Observational Medical Outcomes Partnership [VA-OMOP]) to identify patients aged 18 years or older with at least one prescription for ACEIs and ARBs (target cohort) or calcium channel blockers (CCBs) and thiazide or thiazide-like diuretics (THZs; comparator cohort) between Nov 1, 2019, and Jan 31, 2020. Users were defined separately as receiving either monotherapy with these four drug classes, or monotherapy or combination therapy (combination use) with other antihypertensive medications. We assessed four outcomes: COVID-19 diagnosis; hospital admission with COVID-19; hospital admission with pneumonia; and hospital admission with pneumonia, acute respiratory distress syndrome, acute kidney injury, or sepsis. We built large-scale propensity score methods derived through a data-driven approach and negative control experiments across ten pairwise comparisons, with results meta-analysed to generate 1280 study effects. For each study effect, we did negative control outcome experiments using a possible 123 controls identified through a data-rich algorithm. This process used a set of predefined baseline patient characteristics to provide the most accurate prediction of treatment and balance among patient cohorts across characteristics. The study is registered with the EU Post-Authorisation Studies register, EUPAS35296.FindingsAmong 1 355 349 antihypertensive users (363 785 ACEI or ARB monotherapy users, 248 915 CCB or THZ monotherapy users, 711 799 ACEI or ARB combination users, and 473 076 CCB or THZ combination users) included in analyses, no association was observed between COVID-19 diagnosis and exposure to ACEI or ARB monotherapy versus CCB or THZ monotherapy (calibrated hazard ratio [HR] 0·98, 95% CI 0·84–1·14) or combination use exposure (1·01, 0·90–1·15). ACEIs alone similarly showed no relative risk difference when compared with CCB or THZ monotherapy (HR 0·91, 95% CI 0·68–1·21; with heterogeneity of >40%) or combination use (0·95, 0·83–1·07). Directly comparing ACEIs with ARBs demonstrated a moderately lower risk with ACEIs, which was significant with combination use (HR 0·88, 95% CI 0·79–0·99) and non-significant for monotherapy (0·85, 0·69–1·05). We observed no significant difference between drug classes for risk of hospital admission with COVID-19, hospital admission with pneumonia, or hospital admission with pneumonia, acute respiratory distress syndrome, acute kidney injury, or sepsis across all comparisons.InterpretationNo clinically significant increased risk of COVID-19 diagnosis or hospital admission-related outcomes associated with ACEI or ARB use was observed, suggesting users should not discontinue or change their treatment to decrease their risk of COVID-19.FundingWellcome Trust, UK National Institute for Health Research, US National Institutes of Health, US Department of Veterans Affairs, Janssen Research & Development, IQVIA, South Korean Ministry of Health and Welfare Republic, Australian National Health and Medical Research Council, and European Health Data and Evidence Network.

ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the pfMSP119 antigen

Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-pfMSP119, in Escherichia coli, which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBP-pfMSP119 antigen surface that is recognized by the anti-pfMSP119 antibody G17.12. We identified three epitope-carrying peptides, 386GRNISQHQCVKKQCPQNSGCFRHLDE411, 386GRNISQHQCVKKQCPQNSGCFRHLDEREE414, and 415CKCLLNYKQE424, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-pfMSP119 antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.