Scientific guidelines for preclinical research on potentised preparations manufactured according to current pharmacopoeias-the PrePoP guidelines.

OBJECTIVE: Pharmacopoeias regulate the manufacture of potentised pharmaceutical preparations used in different branches of complementary and integrative medicine. The physicochemical properties and biological activity of these preparations are often investigated in preclinical research, yet no guidelines for experimental research currently exist in this area. The present PrePoP guidelines aim to provide recommendations to promote high-quality, statistically sound, and reproducible preclinical research on potentised preparations. METHODS: Input was gathered from researchers nominated by the relevant scientific societies using a simplified Delphi consensus approach covering the most relevant aspects of basic research methodology in the field including appropriate controls, sample preparation and handling, and statistics. After three rounds of feedback, a consensus was finally reached on the most important aspects and considerations for conducting high-quality research on potentised preparations. RESULTS: We present a series of recommendations on a range of topics including experimental controls, system stability, blinding and randomisation, environmental influences, and procedures for the preparation of potentised samples and controls, and we address some specific challenges of this research field. CONCLUSION: This expert consensus process resulted in a robust set of methodological guidelines for research on potentised preparations and provides a valuable framework that will inform and improve the quality of subsequent research in this emerging field. PLEASE CITE THIS ARTICLE AS: Tournier AL, Bonamin LV, Buchheim-Schmidt S, Cartwright S, Dombrowsky C, Doesburg P, Holandino C, Kokornaczyk MO, van de Kraats EB, López-Carvallo JA, Nandy P, Mazón-Suástegui JM, Mirzajani F, Poitevin B, Scherr C, Thieves K, Würtenberger S, Baumgartner S. Scientific guidelines for preclinical research on potentised preparations manufactured according to current pharmacopoeias-the PrePoP guidelines. J Integr Med. 2024; Epub ahead of print.

Chronobiology of Viscum album L.: a time series of daily metabolomic fingerprints spanning 27 years.

Introduction: European mistletoe (Viscum album L.) has been gaining increasing interest in the field of oncology as a clinically relevant adjunctive treatment in many forms of cancer. In the field of phytopharmacology, harvesting time is pivotal. In the last century, a form of metabolomic fingerprinting based on pattern formation was proposed as a way to determine optimal harvesting times to ensure high quality of mistletoe as raw material for pharmaceutical use. In order to further evaluate the information obtained with this metabolomic fingerprinting method, we analysed a large time series of previously undigitised daily mistletoe chromatograms dating back to the 1950s. Methods: These chromatograms were scanned and evaluated using computerized image analysis, resulting in 12 descriptors for each individual chromatogram. We performed a statistical analysis of the data obtained, investigating statistical distributions, cross-correlations and time self-correlations. Results: The analysed dataset spanning about 27 years, contains 19,037 evaluable chromatograms in daily resolution. Based on the distribution and cross-correlation analyses, the 12 descriptors could be clustered into six independent groups describing different aspects of the chromatograms. One descriptor was found to mirror the annual rhythm being well correlated with temperature and a phase shift of 10 days. The time self-correlation analysis showed that most other descriptors had a characteristic self-correlation of ∼50 days, which points to further infradian rhythms (i.e., more than 24 h). Discussion: To our knowledge, this dataset is the largest of its type. The combination of this form of metabolomic fingerprinting with the proposed computer analysis seems to be a promising tool to characterise biological variations of mistletoe. Additional research is underway to further analyse the different rhythms present in this dataset.

Environmental Homeopathy: Homeopathic Potencies Regulate the Toxicity and Growth of Raphidiopsis raciborskii (cyanobacteria) and can be Tracked Physico-Chemically. Part 1: Biological Results.

INTRODUCTION: Cyanobacteria are microorganisms found in many parts of the world and several genera, such as Raphidiopsis raciborskii, are producers of cyanotoxins. Homeopathic potencies have been found to modulate toxicity in different biological models, and the present study endeavors to discover whether this might also be the case with cyanobacteria. OBJECTIVES: Our objective was to investigate the possible effects of homeopathic potencies on the resilience of Artemia franciscana (brine shrimp) embryos to saxitoxin (STX; cyanotoxin) and on controlling the growth of R. raciborskii in vitro. METHOD: A. franciscana cysts were cultivated in seawater in 96-well plates to evaluate the hatching rate and vitality, plus the gene expression of heat shock proteins (HSPs), after being challenged with R. raciborskii extract containing 2.5 µg/L of STX and treated with different homeopathic potencies. Untreated wells were used as controls ("base-line"). Potencies were chosen from a screening process based on seven selected homeopathic preparations according to the similitude of STX symptoms (Sulphur, Zincum metallicum, Nitric acidum, Plumbum metallicum, Mercurius solubilis, Phosphoric acidum, Isotherapic from R. raciborskii extract; all at 6cH, 30cH and 200cH). Cultures of R. raciborskii maintained in an artificial seawater medium were equally treated with screened homeopathic potencies selected from the same list but specifically for their growth control as a function of time. RESULTS: A 15% lower rate of hatching of A. franciscana cysts was observed after treatment with Nitric acidum 6cH in comparison with baseline (p = 0.05). A complete toxicity reversal was seen after treatment with Isotherapic 200cH, with a 23-fold increase of Hsp 26 gene expression (p = 0.023) and a 24-fold increase of p26 gene expression (p ≤ 0.001) in relation to baseline. Nitric acidum 200cH and Mercurius solubilis 30cH limited the exponential growth of cyanobacteria up to 95% and 85% respectively (p ≤ 0.003) in relation to baseline. Succussed water presented only a transitory 50% inhibition effect. CONCLUSION: Isotherapic 200cH improved A. franciscana bioresilience to STX; Nitric acidum 200cH and Mercurius solubilis 30cH showed the optimal performance on limiting R. raciborskii growth. The results point to the potential of homeopathic potencies to mitigate environmental problems related to water quality.

Environmental Homeopathy: Homeopathic Potencies Regulate the Growth and Toxicity of Raphidiopsis raciborskii (cyanobacteria) and Can be Tracked Physico-Chemically. Part 2: Physico-chemical Results.

INTRODUCTION: The control of cyanobacterial toxicity and growth by homeopathic potencies was described in Part 1 of this two-part report. Here, a parallel approach characterized the physico-chemical features of the potencies used and the liquid media treated with them, correlating these results with their respective biological effects. OBJECTIVES: Our objective was to establish if physico-chemical parameters can track homeopathic potencies in seawater or artificial seawater medium (ASM)-1 and to discover whether these parameters correlate with previously described biological effects. METHOD: Artemia franciscana (brine shrimp) cysts were cultivated in seawater challenged with Raphidiopsis raciborskii extract and treated with different homeopathic potencies chosen from a screening process. Cultures of R. raciborskii maintained in ASM-1 were also treated with previously screened homeopathic potencies, and their growth was monitored as a function of time. The physico-chemical properties of the treated media (seawater or ASM-1) were evaluated by their interaction with solvatochromic dyes and changes in pH, conductivity and temperature. RESULTS: Coumarin 7 was found to be a marker for Nitric acidum 6cH and Isotherapic (R. raciborskii extract) 200cH in seawater (analysis of variance [ANOVA], p = 0.0015). Nile red was found to be a marker for Nitric acidum 200cH and Mercurius solubilis 30cH in ASM-1 (ANOVA, p ≤ 0.001). An increase in pH of ASM-1 and endothermic effects were observed after these treatments (two-way ANOVA, p = 0.0001). Seawater and ASM-1 to which potencies had been added were also subjected to a constant unidirectional 2,400 Gauss static magnetic field and found to have enhanced effects on the solvatochromic dyes tested. CONCLUSION: Homeopathic potencies were specifically traceable in aqueous media using solvatochromic dyes, especially when the samples were subjected to a magnetic field. Results from monitoring other physical parameters, such as pH and temperature, were less specific in relation to potency tracking. However, potency-induced endothermic effects might provide valuable thermodynamic data relating to the nature of potencies.

HRI London 2023: The Homeopathy Research Community Reunites after a 4-Year Hiatus, Sparking Fresh Collaborations between Researchers 'Old and New'.

The Homeopathy Research Institute's (HRI) 5th international research conference took place in the heart of London, from 16th to 18th June 2023. With 230 attendees from 27 countries, HRI's conferences remain truly international. HRI London 2023 will be remembered for its inspiring blend of 'old and new', with a programme notable for the maturity of the evidence presented by experienced names drawing on decades of work, as well as the enthusiasm and skill of up-and-coming researchers who took the floor to present their new findings. In this report, we present scientific highlights from the event.

Highly Diluted Glyphosate Mitigates Its Effects on Artemia salina: Physicochemical Implications.

Glyphosate is an herbicide widely used in agriculture but can present chronic toxicity in low concentrations. Artemia salina is a common bio-indicator of ecotoxicity; it was used herein as a model to evaluate the effect of highly diluted-succussed glyphosate (potentized glyphosate) in glyphosate-based herbicide (GBH) exposed living systems. Artemia salina cysts were kept in artificial seawater with 0.02% glyphosate (corresponding to 10% lethal concentration or LC10) under constant oxygenation, luminosity, and controlled temperature, to promote hatching in 48 h. Cysts were treated with 1% (v/v) potentized glyphosate in different dilution levels (Gly 6 cH, 30 cH, 200 cH) prepared the day before according to homeopathic techniques, using GBH from the same batch. Controls were unchallenged cysts, and cysts treated with succussed water or potentized vehicle. After 48 h, the number of born nauplii per 100 µL, nauplii vitality, and morphology were evaluated. The remaining seawater was used for physicochemical analyses using solvatochromic dyes. In a second set of experiments, Gly 6 cH treated cysts were observed under different degrees of salinity (50 to 100% seawater) and GBH concentrations (zero to LC 50); hatching and nauplii activity were recorded and analyzed using the ImageJ 1.52, plug-in Trackmate. The treatments were performed blind, and the codes were revealed after statistical analysis. Gly 6 cH increased nauplii vitality (p = 0.01) and improved the healthy/defective nauplii ratio (p = 0.005) but delayed hatching (p = 0.02). Overall, these results suggest Gly 6cH treatment promotes the emergence of the more GBH-resistant phenotype in the nauplii population. Also, Gly 6cH delays hatching, another useful survival mechanism in the presence of stress. Hatching arrest was most marked in 80% seawater when exposed to glyphosate at LC10. Water samples treated with Gly 6 cH showed specific interactions with solvatochromic dyes, mainly Coumarin 7, such that it appears to be a potential physicochemical marker for Gly 6 cH. In short, Gly 6 cH treatment appears to protect the Artemia salina population exposed to GBH at low concentrations.

HRI Online 2022: Leading International Experts Illustrate the Positive Impact of Increased Collaboration in Homeopathy Research.

The Homeopathy Research Institute (HRI) welcomed more than 450 people from 35 countries to their first online event on June 25, 2022. The one-day programme featured an excellent line-up of international speakers and provided a unique interactive platform in keeping with the theme of the event - Key Collaborations in Homeopathy Research. Scientists from a range of different research fields gave an exceptional insight into the current status of homeopathy research. Here we give an overview of the most significant findings in both clinical and basic research presented during HRI Online 2022.

HRI London 2019: 10 Years of Excellence in Homeopathy Research.

The Homeopathy Research Institute's (HRI's) 4th International Research Conference took place in the heart of London from 14 to 16 June 2019. With 352 attendees from 38 countries, this was HRI's largest and most international conference to date. HRI London 2019 will be remembered for the highest quality scientific programme so far, combined with a vibrant, positive atmosphere throughout, making it the perfect way to celebrate HRI's 10th Anniversary. Here we summarise the science presented during the intense 2.5-day programme.

HRI Malta 2017-Cutting Edge Research in Homeopathy: HRI's Third International Research Conference in Malta.

The third international conference on "Cutting Edge Research in Homeopathy" organised by the Homeopathy Research Institute (HRI) was held on the inspiring and historic island of Malta from 9th to 11th of June, 2017. One hundred and two abstracts underwent peer review by the HRI Scientific Advisory Committee and external experts to produce the programme of 36 oral presentations and 37 posters, presented by researchers from 19 countries. The 2.5-day programme covered a diverse range of topics, including quantitative and qualitative clinical research, basic research, veterinary research, and provings. These intensive plenary and parallel sessions were interspersed with multiple opportunities for delegates to discuss and exchange ideas, in particular through interactive panel discussions and a pre-conference workshop. The continuing commitment of the homeopathy research community to generate high-quality studies in this rapidly evolving field was clear. In this conference report, we present highlights from this memorable event.

Cutting Edge Research in Homeopathy: HRI's second international research conference in Rome.

Rome, 3rd-5th June 2015, was the setting for the Homeopathy Research Institute's (HRI) second conference with the theme 'Cutting Edge Research in Homeopathy'. Attended by over 250 delegates from 39 countries, this event provided an intense two and a half day programme of presentations and a forum for the sharing of ideas and the creation of international scientific collaborations. With 35 oral presentations from leaders in the field, the scientific calibre of the programme was high and the content diverse. This report summarises the key themes underpinning the cutting edge data presented by the speakers, including six key-note presentations, covering advancements in both basic and clinical research. Given the clear commitment of the global homeopathic community to high quality research, the resounding success of both Barcelona 2013 and Rome 2015 HRI conferences, and the dedicated support of colleagues, the HRI moves confidently forward towards the next biennial conference.

EpiTools: An Open-Source Image Analysis Toolkit for Quantifying Epithelial Growth Dynamics.

Epithelia grow and undergo extensive rearrangements to achieve their final size and shape. Imaging the dynamics of tissue growth and morphogenesis is now possible with advances in time-lapse microscopy, but a true understanding of their complexities is limited by automated image analysis tools to extract quantitative data. To overcome such limitations, we have designed a new open-source image analysis toolkit called EpiTools. It provides user-friendly graphical user interfaces for accurately segmenting and tracking the contours of cell membrane signals obtained from 4D confocal imaging. It is designed for a broad audience, especially biologists with no computer-science background. Quantitative data extraction is integrated into a larger bioimaging platform, Icy, to increase the visibility and usability of our tools. We demonstrate the usefulness of EpiTools by analyzing Drosophila wing imaginal disc growth, revealing previously overlooked properties of this dynamic tissue, such as the patterns of cellular rearrangements.

Do Homeopathic Pathogenetic Trials generate recognisable and reproducible symptom pictures?: Results from a pilot pathogenetic trial of Ozone 30c.

BACKGROUND: Homeopathic Pathogenetic Trials (HPTs) are a pillar of homeopathy, a key source of the symptoms characteristic of a particular homeopathic medicine. Homeopaths choose homeopathic medicines by comparing these remedy pictures with the symptoms the patient is presenting. Thus, recognition of these symptom sets underpins the clinical practice of homeopathy. OBJECTIVE: To test whether HPTs generate consistent and recognisable sets of symptoms in consecutive trials. DESIGN: Practising homeopaths, blinded to the homeopathic medicine under investigation, were given the set of symptoms generated during an unpublished HPT and asked to identify the homeopathic medicine used. HOMEOPATHIC TRIAL SUBSTANCE: Ozone, prepared by homeopathic method to the ultramolecular dilution of 30c (10(-60) dilution), was chosen at random from twenty potential medicines. RESULTS: Seven practising homeopaths were asked to make three guesses as to the identity of the remedy. Initially from the full list of possible remedies (N = 2372). Two of the seven homeopaths guessed the identity of the remedy correctly (p < 0.0001). Subsequently, when their choice of possible medicines was restricted to a list of 20, the same two homeopaths selected the correct medicine, however none of the other practising homeopaths did so (p = 0.2). DISCUSSION: The selection of the correct homeopathic medicine from the unrestricted list (N = 2372 medicines) by two homeopaths is noteworthy given that the homeopathic medicine used during the HPT was diluted well beyond Avogadro's number and would not be expected to produce any detectable or recognisable symptomatology. Possible reasons why the remaining five homeopaths did not guess correctly are discussed. CONCLUSION: The results show that practising homeopaths may be able to correctly identify a homeopathic medicine from the set of symptoms generated during an HPT. This suggests that such symptom pictures generated by taking an ultramolecular homeopathic medicine are recognisable and specific to the substance taken. Since identification of the remedy was based on past HPT information held in the materia medica, this demonstrates that HPT-generated symptom pictures are reproducible, thus validating the HPT methodology. These promising preliminary findings warrant replication; possible improvements to the trial design to be incorporated in future studies were identified.

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth.

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal-distal (P-D) axis by the Dachsous-Fat-Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P-D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.

Matrix geometry determines optimal cancer cell migration strategy and modulates response to interventions.

The molecular requirements and morphology of migrating cells can vary depending on matrix geometry; therefore, predicting the optimal migration strategy or the effect of experimental perturbation is difficult. We present a model of cell motility that encompasses actin-polymerization-based protrusions, actomyosin contractility, variable actin-plasma membrane linkage leading to membrane blebbing, cell-extracellular-matrix adhesion and varying extracellular matrix geometries. This is used to explore the theoretical requirements for rapid migration in different matrix geometries. Confined matrix geometries cause profound shifts in the relationship of adhesion and contractility to cell velocity; indeed, cell-matrix adhesion is dispensable for migration in discontinuous confined environments. The model is challenged to predict the effect of different combinations of kinase inhibitors and integrin depletion in vivo, and in confined matrices based on in vitro two-dimensional measurements. Intravital imaging is used to verify bleb-driven migration at tumour margins, and the predicted response to single and combinatorial manipulations.

Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila.

Tissues can grow in a particular direction by controlling the orientation of cell divisions. This phenomenon is evident in the developing Drosophila wing epithelium, where the tissue becomes elongated along the proximal-distal axis. We show that orientation of cell divisions in the wing requires planar polarization of an atypical myosin, Dachs. Our evidence suggests that Dachs constricts cell-cell junctions to alter the geometry of cell shapes at the apical surface, and that cell shape then determines the orientation of the mitotic spindle. Using a computational model of a growing epithelium, we show that polarized cell tension is sufficient to orient cell shapes, cell divisions, and tissue growth. Planar polarization of Dachs is ultimately oriented by long-range gradients emanating from compartment boundaries, and is therefore a mechanism linking these gradients with the control of tissue shape.

Alternating evolutionary pressure in a genetic algorithm facilitates protein model selection.

BACKGROUND: Automatic protein modelling pipelines are becoming ever more accurate; this has come hand in hand with an increasingly complicated interplay between all components involved. Nevertheless, there are still potential improvements to be made in template selection, refinement and protein model selection. RESULTS: In the context of an automatic modelling pipeline, we analysed each step separately, revealing several non-intuitive trends and explored a new strategy for protein conformation sampling using Genetic Algorithms (GA). We apply the concept of alternating evolutionary pressure (AEP), i.e. intermediate rounds within the GA runs where unrestrained, linear growth of the model populations is allowed. CONCLUSION: This approach improves the overall performance of the GA by allowing models to overcome local energy barriers. AEP enabled the selection of the best models in 40% of all targets; compared to 25% for a normal GA.