Evaluation of the genotoxic and mutagenic potentials of a living nosode compounded with Influenza A virus

Background: The influenza virus has been responsible for contagious respiratory diseases with high mortality rates [1]. Some drugs have been used to treat human influenza. However, these drugs cause many common side effects and induce the appearance of resistant viral strains [2]. The impact caused by the influenza virus has motivated the development of new approaches for the prevention and control of influenza [3]. Therefore, a new homeopathic medicine was developed using, as a starting point, the infectious influenza virus [4]. This belongs to a group called living nosodes [5]. However, its mutagenic and genotoxic potentials, especially when used in low dilutions, has not yet been evaluated and it is important because this biotherapic is prepared from living microorganisms. Different methods can be used to detect mutagenic and genotoxicic effects. Aims: This study aims to evaluate the genotoxic and mutagenic potentials of influenza A living nosode at different homeopathic potencies. Methodology: 1 ml of purified viral suspension was diluted in 9 ml of sterile distilled water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 sucussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x [6]. By the same technique, water vehicle was prepared until 30x potency to be used as control. All samples were prepared in sterile and under aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: The Inductest showed no decrease in the survival fraction of the bacteria used, and no increase in the formation of lysogenic induction, in any tested potency. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: We can conclude that this living nosode compounded with Influenza A virus is not able to induce DNA damage in prokaryotic cells. This result permits us to conclude that patients who use this medicine have no side effects related to mutagenesis and genotoxicity.

Evaluation of the genotoxic and mutagenic potentials of phytotherapic and homeopathic solutions of Euphorbia tirucalli Lineu (Aveloz)

Introduction: Euphorbia tirucalli Lineu, commonly known as Aveloz, is a very common plant found in tropical regions [1]. The ingestion or contact with its latex causes symptoms such as vomiting and diarrhea, pallor, skin irritation, hepatotoxicity as well as carcinogenesis [2]. Moreover, the Aveloz latex is also responsible for a few important activities against some infectious and neoplastic diseases. Aveloz latex phytochemical composition may vary according to seasonal aspects and geographic location [3], and it is used either orally or topically in traditional medicine. Popularly known as an antitumoral agent (breast, prostate, lung, kidney), it is used not only in Brazil, but in several other countries. According to the literature, the latex could have a dual behaviour, activating or inhibiting tumoral events [3-6]. However, there are few reports discussing these mechanisms. Besides, the mutagenic and genotoxic potentials of phytochemical and homeopathic Aveloz have not yet been described. Several experimental methods have been used to evaluate the mutagenic and genotoxic effects, such as Inductest, the Ames test and the chromotest. Objective: This study aims to evaluate the genotoxic and mutagenic potentials of Aveloz latex and Aveloz phytotherapic and homeopathic solutions. Methodology: In this study, Aveloz 5 and 30cH are prepared according to Brazilian Homeopathic Pharmacopoeia [7], from Aveloz latex collected in the Center for Natural Products Research (NPPN) at the Universidade Federal do Rio de Janeiro [8]. The Aveloz phytochemical solution was prepared following the doses used in folk medicine: 2 drops diluted in 250ml of water and 2 drops diluted in 25 ml of water. All test solutions were submitted to the following methodologies: (a) Inductest: assesses the ability of physical or chemical agents to promote lysogenic induction as a response to DNA damage in lysogenic bacteria; (b) The Ames test: uses indicator strains of Salmonella typhimurium, which are sensitive to substances that can induce different types of mutation; (c) Chromotest: evaluates the genotoxicity of chemicals through the induction of the bacterial SOS system. Results: In the Inductest there was no decrease in bacterial survival fraction and no increase in lysogenic cycle. As measured by The Ames test and the chromotest no mutagenic or genotoxic potentials were detected. Discussion: The homeopathic and the phytochemical Aveloz solutions were unable to produce lysogenic induction or mutagenesis in bacterial cells and they were also unable to produce genotoxic effects, as measured by chromotest. Conclusion: Our results showed that no mutagenic or genotoxic damages were induced by all Aveloz preparations studied, thus we are led to believe that patients using Aveloz as a complementary therapy present no side effects in relation to mutagenesis and genotoxicity.

Evaluation of the genotoxic and mutagenic potentials of homeopathic Candida albicans solutions

Background: Candida spp is naturally found in humans’ flora of skin, gastrointestinal and genitourinary tracts and, in general, up to 75% of the population does not have any symptom [1]. However, oral candidiasis is very common among HIV patients and patients undergoing chemotherapy. The treatment of oral candidiasis is necessary once the disease causes discomfort and dysphagia, resulting in poor nutrition, slow recovery, and prolonged hospital stay [2,3]. Preliminary results obtained by our group with a new biotherapic prepared from Candida albicans (Candida 30x) showed a great potential to reduce the candida yeast adhesion rate when the epithelial cells were pre-treated. This study is currently being developed with the evaluation of mutagenic and genotoxic potentials of several homeopathic solutions. Aims: The goal of this study was to assess the genotoxic and mutagenic potentials of different homeopathic potencies of C. albicans. Methodology: One part of C. albicans yeast obtained from Brazilian patient’s blood [4] was diluted in 9 parts of sterile water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution (1x). Then, 1 ml of this solution was diluted in 9 ml of solvent, submitted to 100 succussions, obtaining 2x potency. This procedure was successively repeated to obtain 30x potency, according to Brazilian Homeopathic Pharmacopoeia [5]. By the same technique, water vehicle was prepared until 30x to be used as control. All samples were prepared in sterile and aseptic conditions, using laminar flow cabinet, class II and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x of C. albicans and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: In the Inductest no decrease in survival fraction of bacteria and no increase in the formation of lysogenic induction were detected independently of the homeopathic potency employed. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: Afterwards, we can conclude that these samples are not able to induce DNA damage in the cells tested. So, the use of this medicine does not present any side effects related to mutagenesis and genotoxicity.

Cell Survival Computation via the Generalized Stochastic Microdosimetric Model (GSM2); Part I: The Theoretical Framework

The current article presents the first application of the Generalized Stochastic Microdosimetric Model (GSM2) for computing explicitly a cell survival curve. GSM2 is a general probabilistic model that predicts the kinetic evolution of DNA damages taking full advantage of a microdosimetric description of a radiation energy deposition. We show that, despite the high generality and flexibility of GSM2, an explicit form for the survival fraction curve predicted by the GSM2 is achievable. We illustrate how several correction terms typically added a posteriori in existing radiobiological models to improve the prediction accuracy, are naturally included into GSM2. Among the most relevant features of the survival curve derived from GSM2 and presented in this article, is the linear-quadratic behavior at low doses and a purely linear trend for high doses. The study also identifies and discusses the connections between GSM2 and existing cell survival models, such as the Microdosimetric Kinetic Model (MKM) and the Multi-hit model. Several approximations to predict cell survival in different irradiation regimes are also introduced to include intercellular non-Poissonian behaviors.

Existence of log-phase Escherichia coli persisters and lasting memory of a starvation pulse

The vast majority of a bacterial population is killed when treated with a lethal concentration of antibiotics. The time scale of this killing is often comparable with the bacterial generation time before the addition of antibiotics. Yet, a small subpopulation typically survives for an extended period. However, the long-term killing dynamics of bacterial cells has not been fully quantified even in well-controlled laboratory conditions. We constructed a week-long killing assay and followed the survival fraction of Escherichia coli K12 exposed to a high concentration of ciprofloxacin. We found that long-term survivors were formed during exponential growth, with some cells surviving at least 7 d. The long-term dynamics contained at least three time scales, which greatly enhances predictions of the population survival time compared with the biphasic extrapolation from the short-term behavior. Furthermore, we observed a long memory effect of a brief starvation pulse, which was dependent on the (p)ppGpp synthase relA. Specifically, 1 h of carbon starvation before antibiotics exposure increased the surviving fraction by nearly 100-fold even after 4 d of ciprofloxacin treatment.

Long-read sequencing reveals increased occurrence of genomic variants and adenosine methylation in Bacillus pumilus SAFR-032 after long-duration flight exposure onboard the International Space Station

Bacillus pumilus SAFR-032, an endospore-forming bacterial strain, was investigated to determine its methylation pattern (methylome) change, compared to ground control, after direct exposure to space conditions onboard the International Space Station (ISS) for 1.5 years. The resulting ISS-flown and non-flown strains were sequenced using the Nanopore MinION and an in-house method and pipeline to identify methylated positions in the genome. Our analysis indicated genomic variants and m6A methylation increased in the ISS-flown SAFR-032. To complement the broader omics investigation and explore phenotypic changes, ISS-flown and non-flown strains were compared in a series of laboratory-based chamber experiments using an X-ray irradiation source (doses applied at 250, 500, 750, 1000 and 1250 Gy); results show a potentially higher survival fraction of ISS-flown DS2 at the two highest exposures. Taken together, results from this study document lasting changes to the genome by methylation, potentially triggered by conditions in spaceflight, with functional consequences for the resistance of bacteria to stressors expected on long-duration missions beyond low Earth orbit.

The impact of heating, ventilation, and air conditioning design features on the transmission of viruses, including the 2019 novel coronavirus: a systematic review of ultraviolet radiation

Respiratory viruses are capable of transmitting via an aerosol route. Emerging evidence suggests that SARS-CoV-2 which causes COVID-19 can be spread through airborne transmission, particularly in indoor environments with poor ventilation. Heating, ventilation, and air conditioning (HVAC) systems can play a role in mitigating airborne virus transmission. We conducted a systematic review of the scientific literature examining the effectiveness of HVAC design features in reducing virus transmission; here we report results for ultraviolet (UV) radiation. Following international standards for systematic reviews, we conducted a comprehensive search and synthesized findings from 32 relevant studies published between 1936 and 2020. Research demonstrates that: viruses and bacteriophages are inactivated by UV radiation; increasing UV dose is associated with decreasing survival fraction of viruses and bacteriophages; increasing relative humidity is associated with decreasing susceptibility to UV radiation; UV dose and corresponding survival fraction are affected by airflow pattern, air changes per hour, and UV device location; and UV radiation is associated with decreased transmission in both animal and human studies. This comprehensive synthesis of the scientific evidence examining the impact of UV radiation on virus transmission can be used to guide implementation of systems to mitigate airborne spread and identify priorities for future research.

Improved estimates of 222 nm far-UVC susceptibility for aerosolized human coronavirus via a validated high-fidelity coupled radiation-CFD code

Transmission of SARS-CoV-2 by aerosols has played a significant role in the rapid spread of COVID-19 across the globe. Indoor environments with inadequate ventilation pose a serious infection risk. Whilst vaccines suppress transmission, they are not 100% effective and the risk from variants and new viruses always remains. Consequently, many efforts have focused on ways to disinfect air. One such method involves use of minimally hazardous 222 nm far-UVC light. Whilst a small number of controlled experimental studies have been conducted, determining the efficacy of this approach is difficult because chamber or room geometry, and the air flow within them, influences both far-UVC illumination and aerosol dwell times. Fortunately, computational multiphysics modelling allows the inadequacy of dose-averaged assessment of viral inactivation to be overcome in these complex situations. This article presents the first validation of the WYVERN radiation-CFD code for far-UVC air-disinfection against survival fraction measurements, and the first measurement-informed modelling approach to estimating far-UVC susceptibility of viruses in air. As well as demonstrating the reliability of the code, at circa 70% higher, our findings indicate that aerosolized human coronaviruses are significantly more susceptible to far-UVC than previously thought.

In vitro Study of Radiosensitivity Effects of Galbanic Acid on Ovarian Tumor Cells (OVCAR-3 Cell Line)

Background and aims: Radiotherapy ranks among the most important procedures in ovarian cancer therapy. However, radioresistance is becoming more prevalent and is one of the main causes of poor clinical outcomes. To overcome this problem, radiosensitizers may be used. The present study aimed to evaluate the radiosensitizing properties of galbanic acid (GBA) on ovarian cancer cells in vitro. Materials and methods: OVCAR-3 cells, an ovarian cancer cell line, were treated with increasing concentrations of GBA (5, 10, 20, and 40 μg/mL) for 24, 48, and 72 h to determine its half-maximal inhibitory concentration (IC50). Cell viability was assessed by alamar Blue assay. The cells treated with 10 μg/mL GBA for 24 h were exposed to increasing doses of radiation (1, 2, and 4 Gy) and the survival fraction was investigated by clonogenic assay. Results: Assessment of cell viability indicated that GBA caused toxicity in a dose-dependent manner. Additionally, GBA pretreatment significantly improved the radiosensitivity of the cells, and survival fraction data indicated synergy between GBA and radiation. Conclusion: Taken together, the current findings highlight GBA as a potent radiosensitizing agent; however, further research is required to determine the molecular mechanisms of the observed effect both in vitro and in vivo. It is also suggested that the radiosensitization effect of GBA on other cell types should be studied in the future.

Soft threshold partial least squares predicts the survival fraction of malignant glioma cells against different concentrations of methotrexate’s derivatives

Chemotherapy appeared to be a significant advancement in cancer research, with fewer side effects. Methotrexate (MTX) is a widely used anticancer drug with strong activity but serious side effects. Several MTX derivatives have been reported, with modifications at various sites to reduce side effects and increase efficacy. The current study uses FTIR spectroscopy to predict the survival fraction of human malignant glioma U87 (MG-U87) cell lines against MTX derivatives. Together with Parent MTX several aldehydes viz. Benzaldehyde, Chlorobenzaldehyde, 2-Chlorobenzaldehyde, 3-Nitrobenzaldehyde, 5-Chloro-2-hydroxybenz-aldehyde, 2-Hydroxy-5-Nitrobenzaldehyde, 2-Thiocarboxyaldehyde, Trans-2-pentenal, and Glutaraldehyde are treated with MTX to obtain MTX derivatives. The prediction of survival fraction of malignant glioma cells is carried out by Lasso, Elastic net and Soft PLS at different concentration levels of synthesized derivatives, including 400 μM, 200 μM, 100 μM, 50 μM, 25 μM and 12.5 μM. The cross-validated prediction error is minimised to optimise spectral wavelength selection and model parameters. It appears that the RMSE computed from test data is significantly varying with the change of models (p = 0.012), with the change of concentrations levels (p $$\le 0.001$$ ≤ 0.001 ) and with the change of combination of models and concentration level (p $$\le 0.001$$ ≤ 0.001 ). StPLS outperforms in predicting survival fraction of glioma cells at the concentration level 50 μM, 100 μM and 400 μM respectively with relative RMSE = 0.1,0.14 and 0.55. Lasso outperforms at the concentration level 12.5 μM, and 200 μM respectively with relative RMSE = 0.4 and 0.14. Elastic net outperforms at the concentration level 25 μM with relative RMSE = 0.8. Consistently appeared influential wavelength identifies the influential functional compounds which best predicts the survival fraction. Hence FTIR appears potential candidate for estimating survival fraction of MTX derivatives.