Recent studies on acute mountain sickness (AMS) have used fixed-location and fixed-time measurements of environmental and physiological variable to determine the influence of AMS-associated factors in the human body. This study aims to measure, in real time, environmental conditions and physiological variables of participants in high-altitude regions to develop an AMS risk evaluation model to forecast prospective development of AMS so its onset can be prevented.2 coefficients of determination between the LLS and the measured altitude, ambient temperature, atmospheric pressure, relative humidity, climbing speed, heart rate, blood oxygen saturation (SpO2), heart rate variability (HRV), were 0.1, 0.23, 0, 0.24, 0, 0.24, 0.27, and 0.35 respectively; incorporating all aforementioned variables, the R2 coefficient is 0.62. The bagged trees classifier achieved favorable classification results, yielding a model sensitivity, specificity, accuracy, and area under receiver operating characteristic curve of 0.999, 0.994, 0.998, and 1, respectively.Thirty-two participants were recruited, namely 25 men and 7 women, and they hiked from Cuifeng Mountain Forest Park parking lot  to Wuling . Regression and classification machine learning analyses were performed on physiological and environmental data, and Lake Louise Acute Mountain Sickness Scores (LLS) to establish an algorithm for AMS risk analysis. The individual RThe experiment results indicate the use of machine learning multivariate analysis have higher AMS prediction accuracies than analyses utilizing single varieties. The developed AMS evaluation model can serve as a reference for the future development of wearable devices capable of providing timely warnings of AMS risks to hikers. Acute mountain sickness (AMS) is a maladaptation syndrome that occurs at high altitudes , 2. The 2), and HRV) [2, and HRV to diagnose mountain sickness [As of this writing, the mechanism and diagnosis of AMS remain uncertain. The 2018 Lake Louise Acute Mountain Sickness (LLS) is currently used to assess the severity of AMS. The score is based on headache, gastrointestinal symptoms, fatigue and/or weakness, and dizziness or lightheadedness. The maximum total score is 12 points, with 3–5, 6–9, and 10–12 points indicating mild, moderate, and severe levels of AMS, respectively –17. Releand HRV) . Recent sickness .2 and HRV) that are collected daily. [The majority of studies on AMS perform linear analyses to build AMS prediction models with environmental and biological data , and heart rate variability (HRV) was performed using MATLAB R2020a to obtain the coefficient of determination (R2) of each variable and all variables. The coefficients of determination for altitude, ambient temperature, atmospheric pressure, relative humidity, climbing speed, heart rate, SpO2, HRV, and all variables were 0.1, 0.23, 1.2 × 10–6(≒0), 0.24, 1.5 × 10–5(≒0), 0.24, 0.27, 0.35, and 0.62, respectively. Single variable analysis revealed that HRV had the highest coefficient of determination, namely 0.35. The coefficient of determination for all eight variables was 0.62. The correlation analysis results are presented in Table We used linear regression analysis of the LLSs and the recorded altitude, ambient temperature, atmospheric pressure, relative humidity, climbing speed, heart rate, blood oxygen saturation  for LLS. And the binary classification method using bagged trees classifiers to obtain a high degree of accuracy modeling, a variety of factors in the machine learning to build models, can be conducive to the development of the AMS risk assessment model.In the results show that using MATLAB R2020a line regression analysis and machine learning model to diagnose mild AMS. We use measured environmental and physiological factors to Coefficient of determination . In the experiment, the mean SpO2 was approximately 83.5 . HRV are conducive to evaluating autonomic nervous system activity , 14, 20 2. In high-altitude environments with low pressure and low oxygen, the autonomic nervous system response of patients with AMS decreases. Decrease in HRV demonstrates to more suggestive of AMS symptoms than decrease in oxygen levels. During the on-site experiment, some participants experienced an increased heart rate and expressed discomfort.In the measured results, the participants’ HRV significantly decreased in the mountain regions, thereby suggesting a correlation between the HRV-measured decrease in autonomic nervous system activity and anoxia severity, as indicated by SpORelevant studies on the factors that influence AMS have revealed a low correlation between heart rate and LLSs. During the hiking process, the participants’ physiological indicators changed with hiking duration and altitude changes; in particular, participants experienced an increase in heart rate relative to their usual state.2 and HRV exhibited a negative correlation with LLSs. This result is consistent with that of most studies on mountain sickness [The on-site experiment results indicate that in the hiking process, the variations of environmental factors resulted in changes in the participants’ physiological factors. Additionally, a positive correlation was revealed between heart rate and LLSs, whereas SpOsickness , 13, 20.However, the coefficient of determination between atmospheric pressure and climbing speed with LLSs in this study was 0. Some studies have shown that the above two factors are related to AMS , 21. ThiFrom the results, the 25 machine learning algorithms were employed to analyze the collected environmental and physiological factors, and demonstrated to yield sensitivity, specificity, and accuracy better than those of previous studies , 28, thuThe experiment location was at an altitude over 2500 m, thereby meeting the threshold for acute mountain sickness (AMS). However, because no participant exceeded six hours of hiking, the recorded data are considered short-term measurements. Additionally, the data may have been influenced by underlying factors, including the amount of exercise the participant had performed in the previous day and their rest and sleep duration. Therefore, this study had inadequate data when compared with studies that collected data for three consecutive days , 28, 29.After participants put on the wearable devices and hiked from the Cuifeng Mountain Forest Park parking lot to Wuling, the ECG pads may have loosened due to participant activity, resulting in data inaccuracies. During data processing, we eliminated data collected during incidences when recording equipment was loosened to increase data accuracy. However, this also reduced the number of data collected and may have resulted in error values. In future studies, we plan to use more secure physiological monitoring equipment to prevent the occurrence of errors.Participants were predominantly male and were relatively old in age. In the future, we will recruit more woman participants ranging between 18 and 40 years old and increase the data collection instances along the planned hiking path to increase the AMS diagnosis accuracy.The highest LLS recorded in this study was 7, thereby indicating moderate AMS (LLS range: 6–9 points). During the measurement, the participant expressed immense discomfort and an inability to continue hiking. In accordance with institutional review board regulations, participants had the right to stop hiking. Therefore, this study did not collect data on participants experiencing severe AMS (LSS range: 10–12). This study focused on diagnosing whether AMS occurred. In the future, we will more thoroughly research all levels of AMS severity.2 and HRV are more prone to experiencing AMS [When traveling to mountain regions, the low-pressure and low-oxygen environment may result in the weakening of the human heart self-regulating function. This increases the possibility of AMS or other high-altitude conditions. The literature on AMS suggests that among hikers exposed to simulated atmospheric pressure or low-pressure and low-oxygen conditions, those with lower SpOcing AMS . The anaWe employed machine learning algorithms and various environmental and physiological factors for AMS diagnosis. Our model achieved a sensitivity and AUC of 0.99, indicating that the use of multivariate factors to train multivariate analysis-based machine learning models exhibit high developmental value in LLS evaluation. This machine learning algorithm can be coupled with equipment capable of the timely detection of multiple factor data to increase the accuracy for AMS predictions. Additionally, this application may prevent hikers from overlooking AMS symptoms and remind them to take preventive measures , thereby preventing the risk of severe AMS.2 and HRV with AMS observed in this study was consistent with that of previous studies [This study is the first to employ timely recorded environmental and physiological factors and multivariate analysis for AMS diagnosis. In terms of using LLS to diagnose AMS symptom severity, correlation analysis results revealed that the diagnosis effect of a single variable and LLS was unsatisfactory. However, the use of multiple variables and LLS exhibited satisfactory diagnosis effectiveness. The degree of correlation of SpO studies . Additio2), and heart rate variability (HRV) in real-time. The Lake Louise Acute Mountain Sickness score (LLS) was adopted to evaluate AMS severity. The flowchart  , 2, 6, tThe experimental protocol was approved by the Tri-Service General Hospital Human Ethics Committee under registration number IRB: B202005136, and the informed consent of the experiment participants was obtained before this study. Adults between 25 and 55 years old capable of engaging in hiking activities and living below an altitude of 500 m were recruited as participants. Additionally, participants with diabetes, neuropathy, cardiovascular and pulmonary diseases, or other symptoms that could influence heart rate variability (HRV) were excluded. Before testing, participants were required to administered the 2017 Physical Activity Readiness Questionnaire (2017PAR-Q +)  to confiThis study initially recruited 34 participants, however, only 32 fulfilled the inclusion criteria; two participants with self-reported cardiovascular disease and diabetes respectively were excluded. The final sample set were comprised of 25 men and 7 women. The mean participant age was 36.5 ± 8.1 years, and the mean body mass index (BMI) was 24.3 ± 2.36. The mean home altitude of the participants was 65.4 ± 90.4 m. No participant had consumed drugs  30 days before the experiment. The most common acute mountain sickness (AMS) symptoms were weakness and dizziness, which were observed in 13 (40.6%) of the participants. The baseline demographics of the participants are listed in Table 2), and ECG. The monitor was connected to a Smart Link I application and a USB interface to transmit the data to a personal computer for data observation and analysis. Time domain analysis was performed on the collected ECG data, and the standard deviation of the R–R intervals was calculated to obtain the HRV data [This study used the MD-670P Plus portable pulse oximeter and electrocardiogram (ECG) monitor  to measure heart rate, blood oxygen saturation  was employed to measure the environmental factors of a participant’s position, including altitude, ambient temperature, atmospheric pressure, relative humidity, and climbing speed. Throughout the experiment, the participants were required to wear the pulse oximeter and ECG monitor and the SensorTag and hike along Provincial Highway 14. The participants embarked from the Cuifeng Mountain Forest Park parking lot in Nantou , passed the Yuanfeng Lookout , the Kunyuan parking lot , Wuling , and arrived at Shimen Mountain Class 3 Triangulation Point No. 6389 . The total hiking distance was 16.3 km, and the experiment duration was 8 h. The map of the experimental route is presented in Fig. 2, and ECG records of the portable pulse oximeter and ECG monitor. During the hiking activity, participants who experienced discomfort were immediately required to complete the online digital LLS. Additionally, professionals were assigned to the Cuifeng Checkpost, Yuanfeng, Kunyang parking, Wuling, and Shimenshan Trail to verbally administer the LLS to passing participants, thereby preventing the participants’ subjective factors from influencing the acute mountain sickness assessment. The recording equipment automatically measured and recorded the data without requiring manual operation by the participants.The participants’ physiological variables were collected from the heart rate, SpO2, and HRV values of participants in mountainous areas were 115.84, 83.5, and 39.17, respectively; and were slightly lower than in their usual state. The measured physiological variables of the participants engaging in hiking activities were roughly equal to those recorded in previous studies [In the experimental process, data were not recorded during incidences when recording equipment (ECG pads and pulse oximeter) were loosened; a total of 8410 data points were collected and used for analysis. Table  studies –17.TableThe MATLAB R2020a machine learning and deep learning tool   was empl2) of each individual factor and all factors was calculated to determine factor correlations with the LLSs.First, the linear regression model of regression learner applications was adopted to analyze the measured environmental and physiological factors. The coefficient of determination , Discriminant Analysis , Logistic Regression Classifiers (Logistic Regression), Naive Bayes Classifiers , Support Vector Machines 、Nearest Neighbor Classifiers , Ensemble Classifiers .Finally, the characteristics of the developed AMS prediction model were evaluated based on sensitivity, specificity, accuracy, and area under the receiver operating characteristic curve (AUC). To prevent training and or testing bias, the 8410 data points were randomly divided into 90: 10 training and testing sets respectively. The aforementioned 25 machine learning algorithms were used to build the model, and were validated with the tenfold cross validation to prevent reliance on sample characteristics; and also to stabilize the final model.After a classification algorithm has trained on data, we want to examine the performance of the algorithm on a specific test dataset. We assessed the predictive performance of our model using a range of common performance metrics: sensitivity, specificity, accuracy, and area under the curve (AUC), all of which range from 0 to 1 . From TaThe three conventional evaluation indicators  were calculated as follows.Sensitivity = TP/(TP + FN).Specificity = TN/(FP + TN).Accuracy = (TP + TN)/(TP + FN + FP + TN).We can adjust different thresholds to get the true positive rate versus false positive rate , by plotting a Receiver Operating Characteristic curve (ROC) to get the area under the curve (AUC) size, we can use the ROC curve to find the classifier that maximizes the classification accuracy, or to evaluate the performance of the classifier in high sensitivity and high specificity regions. We adopted tenfold cross-validation to obtain the mean values and standard deviations of these evaluation indicators to fairly compare their performance. In detail, the dataset is divided into ten approximately equal-sized sub-datasets, and the positive samples are divided equally in each sub-dataset.


Objectives: Regulatory clinical Phase I studies are aimed at establishing the human safety of an active pharmaceutical agent to be later marketed as a drug. Since homeopathic medicines are prepared by a potentizing method using alcohol, past a certain dilution, their toxicity/infectivity is assumed to be unlikely. We aimed to develop a bridge study between homeopathic pathogenetic trials and clinical trials. The primary purpose was to evaluate the safety of a nosode, developed from clinical samples of a COVID-19 patient. The secondary objectives were to explore whether a nosode developed for a specific clinical purpose, such as use during an epidemic, may elicit laboratory signals worthy of further exploration. Methods: An open-label study was designed to evaluate the safety and immune response of the Coronavirus nosode BiosimCovex, given orally on three consecutive days to ten healthy volunteers. Clinical examinations, laboratory safety and immune parameters were established. Interferon–gamma, Interleukin-6, and CD 4 were measured. (CTRI registration number: CTRI/2020/05/025496). Results: No serious/fatal adverse events were reported. Laboratory tests to measure safety were unchanged. Three subjects showed elevated Interleukin-6 (IL-6) on day 17 in comparison to the baseline, and ten subjects showed elevated IL-6 on day 34. A significant difference between IL-6 observations, calculated by repeated measures ANOVA, was found to be highly significant. On day 60, the IL-6 values of nine subjects were found to return to normal. Corresponding CD4 cell elevation was observed on day 60, when compared to day 34. Conclusions: HPT may potentially extend into physiological changes with regards to immune response and should encourage future studies. Clinical Phase I trials for the regulatory approval of drugs are generally small and are conducted to primarily examine the safety of new medicines in healthy subjects before proceeding further into clinical trials that modify disease. The treatment of illnesses caused by SARS-CoV-2 is always potentially limited by the toxicity of medications . The conIn the homeopathic system of medicine, the drugs are potentized via dilution and succussion, rendering the source material open to potential nanoparticles . It is aThere are nearly 1500 drug substances recorded in various homeopathic pharmacopeias  ,7 that aAs an example, ‘The Drug and Cosmetic Act’ (India) states that, to be included in the pharmacopeia, the drug must be along-used homeopathic drug whose therapeutic efficacy is recorded in data and established by long clinical use . The ActControversy remains surrounding nosode use as a potential disease prophylactic during epidemics. Loeb et al.  showed tThe aim of this study is to bridge the homeopathic pathogenetic trial  techniquTo evaluate the safety of BiosimCovex in healthy volunteers, this study evaluated potential immune response signals and recorded subjective symptoms elicited in the trial, historically and commonly used in homeopathic pathogenetic trials.A Coronavirus nosode, initially coded as CVN01 and subsequently labeled as BiosimCovex, was prepared from a clinical sample  of an orThe clinical sample selected to prepare the nosode had the lower cycle threshold (CT) and was identified/characterized by gene sequencing. The CT value was approximately 17 and corresponded to 4–9 log10, amounting to the count of more than 20 billion. Based on the CT value of the sample, the corresponding viral copies, as per the published literature, were sufficient to start the homeopathy potencies . PotenciAn open-label, non-randomized study was designed to examine the response of BiosimCovex administered orally to healthy volunteers. This study aimed to examine safety in terms of clinical effects and blood parameters, and to evaluate certain immune responses at the baseline and at days 17, 34, and 60.This study was conducted at a single center in Mumbai. The first volunteer was enrolled on 10 June 2020, and the last volunteer’s visit was completed on 12 August 2020. The study protocol, amendments, and informed consent forms (ICF) were reviewed and approved by the Ethics Committee constituted by Homeopathy India Private Limited. Written informed consent was obtained from each volunteer before the performance of any study-specific procedures. This study was conducted in accordance with the guidelines of the Good Clinical Practice and the ethical principles outlined in the Declaration of Helsinki 2008.This is a category ‘nosode’ preliminary HPT, with ten individuals proposed by the scientific advisory board as appropriate for a Phase I trial. Ten healthy volunteers , with no known major untreated diseases, with normal routine laboratory parameters during screening, were enrolled in the trial . The basAge: 18–65 years, both males and females, healthy individuals with no major untreated diseases and normal routine laboratory parameters during screening. Participants who were informed of the nature of the study and were willing to give written informed consent were enrolled in the study.Any subject with a known disease or condition which might compromise the general health or comorbid conditions, subjects who had been confirmed to have COVID-19 and had been isolated for its treatment, subjects who had recently suffered and recovered from COVID-19, persons with a known history of allergies and food hypersensitivity, women during pregnancy, puerperium and breast-feeding, subjects who had participated in another clinical trial during the last 6 months and with other conditions, which, in the opinion of the investigators, could make the patient unsuitable for enrolment or could interfere in their adherence to of the study protocol, were excluded from the study.Subjects were administered with six doses of nosode BiosimCovex 30C (pill size 30)  as six pills, twice daily, for three consecutive days. Pre and post examinations, such as physical examinations, vital signs, and laboratory investigations were taken at baseline, on days 17, 34, and 60. The participants were monitored thoroughly. Based on the data in the source documents and laboratory values, the safety results were reported. Samples were sent to the testing laboratory within 30 min of collection at ambient conditions to avoid any degradation. The central laboratory carefully processed the samples upon arrival.The trial protocol was reviewed by the Scientific Advisory Board and approved by an Ethics Committee, named ‘Institutional Ethics Committee’, letter dated 27 May 2020. The trial was registered at the Clinical Trial Registry of India (CTRI) with the trial registration number: CTRI/2020/05/025496.The primary endpoint was the determination of the safety of the BiosimCovex, and the secondary endpoint was the evaluation of the changes in the immune response parameters.As the observations made after a specific time interval demonstrate the pre- and post-treatment effects, and the data is continuous in nature, the t test for Paired Two Sample for Means was explored. Statistical analyses were performed using Software SPSS . For a continuous dependent variable IL-6, where the sample was collected at multiple visits, a significant difference between observations in each visit was calculated by repeated measures ANOVA.Safety assessments included the monitoring of subjects for any unexpected symptoms, adverse events, serious adverse events, as assessed by predefined questionnaires, clinical and laboratory investigation results, blood pressure, physical examination findings, and general well-being, as assessed by questionnaire.Complete Blood Count (CBC), C-Reactive Protein (CRP), Liver Function Test (LFT), performed on the fully automated analyzer (XL-200), and Serum ferritin level, measured by CMIA, and the chemiluminescent assay, were measured at baseline and day 17. The RT-PCR tests for the laboratory verification of COVID-19 were not permitted by the government for healthy persons (due to the shortage of kits in the country during the study period). Instead, the status of the COVID-19 IgG/IgM against the SARS-CoV-2 Spike Protein, measured by enzyme-linked immunosorbent assay (ELISA) method , were investigated at baseline, days 17, 34, and 60, along with the CBC and ferritin, to confirm that the subjects were not infected.An interferon–gamma (IFN-γ) assay was conducted by the Chemiluminescent assay (CLIA) method, using a value less than 0.438 IU/ML as negative, and above that as positive; this was measured at the baseline and day 17. In addition, Interleukin-6, using a value greater than 7.00 pg/mL as positive and an electro chemiluminescent immunoassay  method, was measured at the baseline and days 17, 34 and 60. After initiating the study, and observing elevations of Interleukin-6, we amended the protocol to also investigate the CD4 panel, measured at days 34 and 60.There were no serious or fatal adverse events during the study. The laboratory basic biochemistry and liver function tests were not affected by BiosimCovex 30C . The P-vAs this manuscript is focused on safety and potential immune response signals, symptoms that are usually cataloged during a homeopathic pathogenetic trial were cataloged and are published elsewhere.p value 0).  compared to the baseline. A significant difference between IL-6 observations, calculated by repeated measures ANOVA, was found highly significant .  The subjalue 0). . IndividIFN-γ was measured at the baseline and day 17, [Baseline Mean (SD) 0.016 (0.025) and day 17 Mean (SD) 0.13 (0.12)] and COVID-19 IgG and IgM measured at the baseline (0.157/0.264), days 17 (0.352/0.207), 34 (0.220/0.231), or 60 (0.24/0.25), did not show any activity indicating active exposure to SARS-CoV-2 virus during the dates measured.In conventional homeopathy, one conducts human pathogenetic trials (known as drug provings), without carrying out any animal toxicity studies or safety studies, found in precedents with earlier commonly-used nosodes such as Psorinum , MedorrhThe current trial of BiosimCovex in 30C potency studied in ten human subjects did not elicit safety signals. It remains debatable within the homeopathic community whether the conventional homeopathic pathogenetic trials should require acute, subacute, or chronic toxicity in animal models, for examining the safety of nosodes; this is due to its potentized process which kills the original germs, if diluted, succussed and immersed in 90% alcohol  during tThis human safety study was combined with the evaluation of the potential effects of the nosode on the immune response. If the nosode was to be examined particularly for its prophylactic potential during the pandemic, it is reasonable to expect an immune response during the trial, and therefore this was evaluated. Being a pleiotropic cytokine, Interleukin-6 (IL-6) is known to induce an antigen-specific immune response and inflammatory reaction, activating the host defense mechanisms performing diverse functions. Studies of genetically modified animal models suggest that IL-6 has a role in both the innate and adaptive immune responses that protect the host from a variety of infections .In this study, the mild elevation of IL-6 in all the subjects may be linked to an immune response, as there were no corresponding clinical signs and symptoms of inflammatory or pathological response. In addition, there was no increase in the C-Reactive Protein, despite an increase in IL-6, suggestive of non-inflammatory and non-pathological responses, thereby suggesting an immune response .IL-6 levels, increasing by several tens or even hundreds of pg/mL, are observed in chronic diseases, depending on severity. Values of more than 1000 pg/mL can occur during septic shock or a cytokine storm and in severe cases . The excThe roles of IL-6 and IFN-γ are both important and well described, and both are elevated greatly in COVID-19 patients . Importap value ≤ 0.05). The current cytokine fluctuation was observable on days 17 and 30, but not on day 60. IL-6 levels were reduced to baseline in 9 of the 10 subjects by day 60. Interestingly, the timing of elevated IL-6 found in these subjects corresponds to the commonly observed timing of symptom production during a conventional homeopathic pathogenetic trial [In our studies, unlike extreme changes, the IL-6 levels increased to an average of five folds , but not as effective as vaccinations (100%) . If therAs epidemic and pandemic emergencies present with time constraints, immediate prophylactic and therapeutic solutions may not wait for the lengthy process of new nosode drug discovery. It is essential to develop faster, effective, yet safer methods of therapy; these may include the development of nosodes from clinical samples or inactivated microbial components, conducting safety studies, and evaluating specific immunological tests to assess potential clinical usefulness. Should this strategy prove even partially effective, it may fill the gap that occurs from the start of an epidemic until vaccine development, production and distribution are underway. Further, with genomic variations, mutations, and adoptions , it is iThe limitations include Type 1 errors due to the small sample size in the trial and a lack of a placebo-controlled arm. The lack of a placebo arm points us to the potential association of the immune response, but not to causation. Another limitation is potentially the too few immune markers being examined. Unfortunately, we added the CD4 count after we observed a rise in IL-6, and, therefore, did not capture CD4 and Lymphocyte count baseline levels. The study result is indicative of the immune response; however, a confirmatory study should be repeated in the future in a larger sample size, with controls, and testing a wider number of immune markers.No safety concerns were observed in the small study population by consuming BiosimCovex. In this noncontrolled open-label study, while antibodies to COVID-19 remained negative, a transient immune response may have been observed in elevated IL-6 in all the subjects, with a corresponding increase in the CD4 count on day 60 in comparison to day 34. A larger placebo-controlled bridge study may be possible to explore the induction of biochemical changes that may occur by ingestion of the nosode.


Pseudomonas fluorescens, Trichoderma viride, Pleurotus ostreatus, Mucor racemosus, Burkholderia sp., Cupriavidus sp., Pseudonocardia sp., and a community of anaerobic microorganisms. Many aldrin/dieldrin resistance genes have been identified from insects and microorganisms, such as Rdl, bph, HCo-LGC-38, A302SS2-RDL, CSRDL1A, CSRDL2S, HaRdl-1, and HaRdl-2. Aldrin degradation includes three pathways: the oxidation pathway, the reduction pathway, and the hydroxylation pathway, with dieldrin as a major metabolite. Degradation of dieldrin includes four pathways: oxidation, reduction, hydroxylation, and hydrolysis, with 9-hydroxydieldrin and dihydroxydieldrin as major products. Many studies have investigated the toxicity and degradation of aldrin/dieldrin. However, few reviews have focused on the microbial degradation and biochemical mechanisms of aldrin/dieldrin. In this review paper, the microbial degradation and degradation mechanisms of aldrin/dieldrin are summarized in order to provide a theoretical and practical basis for the bioremediation of aldrin/dieldrin-polluted environment.As members of the organochlorine group of insecticides, aldrin and dieldrin are effective at protecting agriculture from insect pests. However, because of excessive use and a long half-life, they have contributed to the major pollution of the water/soil environments. Aldrin and dieldrin have been reported to be highly toxic to humans and other non-target organisms, and so their use has gradually been banned worldwide. Various methods have been tried to remove them from the environment, including xenon lamps, combustion, ion conversion, and microbial degradation. Microbial degradation is considered the most promising treatment method because of its advantages of economy, environmental protection, and convenience. To date, a few aldrin/dieldrin-degrading microorganisms have been isolated and identified, including  Due to the largescale exploitation of natural resources and the destruction of the natural environment by human beings, the pressure on the biosphere is increasing . The mai12H8Cl6 and its half-life in soil is estimated to be 1.5–5.2 years  and dieldrin (CAS 60-57-1) are synthetic organochlorine cyclodiene pesticides used to control subterranean insect pests such as nargles root maggots, mole cricket grubs and weevils, in agriculture. Aldrin’s  molecular formula is C.2 years ,b. Dield 5 years . In the  5 years ,b. They  5 years . Organoc 5 years .Although reports of resistance to the cyclodiene pesticides aldrin and dieldrin account for more than 60% of reported cases of resistance, the overall incidence of related cases of resistance are declining . TherefoAldrin and dieldrin have been used for more than 80 years, and a lot of research has been conducted to investigate their toxicity and degradation . HoweverAldrin and dieldrin belong to the same group of pesticides, most of which have the same toxicology . They havia volatilization, washing, dust, etc., which in turn causes wider pollution (–1)  . A healtion (–1) . The estion (–1) . An inveion (–1) . Estimation (–1) .–1⋅dw (–1 (–1) were found in the sediments of southern and southern river ecosystems in Nigeria (–1) exceeding the EU environmental quality standards (–1 (–1) and dieldrin (71.19 ng⋅L–1) were found in stream water in Mumbai, India, exceeding their respective standard levels but below the range set by the America Environmental Protection Agency (EPA) at 0.13 65.1 ng⋅L–1  embryos  to ferrous iron (Fe2+) within 72 h. The effective removal time of many organochlorine pesticides (OCPs) is greatly reduced by the catalysis of ferrous iron (Fe2+). When the iron dosage was increased from 2.5 × 103 mg⋅L–1 to 1.5 × 104 mg⋅L–1, the removal rate of dieldrin was increased by 23.3% , which can degrade 49 and 38% of dieldrin, respectively. Enterobacter sp. LY402 is able to degrade 40.4% of dieldrin (5.0 mg⋅L–1) in 168 h  have been especially well studied. In previous studies, WRF was able to oxidize a wider variety of refractory compounds, such as lindane, lignin, dichlorodiphenyltrichloroethane (DDT) . TherefoPhanerochaete chrysosporium and Trametes versicolor, which can completely degrade 5–30 mg⋅L–1 of dieldrin within 25 days. The wood-rotting fungal strain YK543, isolated from rotten wood, can degrade approximate 39.1% of dieldrin within 30 days  of dieldrin within 14 days  medium within 14 days  and 65.4% of aldrin (100 mg⋅L–1) within 2 weeks (–1) within 3 months, and 70% of aldrin (7 mg⋅L–1) within 160 days   . AccordiThe degradation pathways of aldrin and dieldrin found in previous research are shown in Degradation of dieldrin includes four pathways: oxidation, reduction, hydroxylation, and hydrolysis . In the Based on this degradation pathway, it is inferred that oxidative degradation and reductive dechlorination are important degradation mechanisms of aldrin and dieldrin . HoweverRdl) was first identified in mutant Drosophila, where the single amino acid Ala at position 302 was replaced by Ser/Gly  and the alternative splicing is complex.The gene identified as having a high level of resistance to dieldrin , which are likely derived from recent gene duplicates . Although CSRDL2S expression is twice as high as CSRDL1A at all growth stages, they have similar expression patterns (Bombyx mori) has the largest known insect GABA gene family in the world, including three Rdls . The three Rdls subunits may be generated by two repeated events, among which Rdl1 gene has an RNA editing site, and Rdl1 and Rdl3 can perform alternative splicing (When the gene at the  survive . In gene species . Howeverplicates . Chilo spatterns . G-aminosplicing .HCo-LGC-38) that is moderately sensitive to fipronil and dieldrin  population, thus developing resistance to fipronil (Rdl was replaced by a serine in the cat flea population, leading to resistance to fipronil (Rdl has limited cross-resistance to fipronil and is not detected in field populations of the American housefly (Worryingly,  avoided . Lindanel losses . Alaninefipronil . Alaninefipronil . Howeverhousefly . These aAldrin and dieldrin are widely used for their insecticidal activity, but are restricted because of the toxic effect on the environment. Both insecticides have been used for a long time, and their toxic effects on non-target organisms cannot be ignored as aldrin and dieldrin accumulate in the environment and in food chains. Residual aldrin and dieldrin are toxic to a variety of animals, including humans, dogs, fish, shrimp, and other non-target organisms. Therefore, it is necessary to develop efficient, economical, and environmentally friendly technologies for the degradation of aldrin and dieldrin. Microorganisms have the potential to detoxify exogenous compounds easily through several metabolic pathways, and detoxification by indigenous microorganisms is recognized as the most promising remediation approach. A microbial consortium has reported as having superior performance over a pure culture. The reason for the improved performance is the reduction of the metabolic burden. However, the published literature on the microbial remediation of aldrin/dieldrin-contaminated sites remains inadequate. In addition, the development of biodegradable genes/enzymes and degradation microorganisms must be an ongoing project in the future. In recent years, the biodegradation of pesticides based on omics technology has provided a new direction for the bioremediation of environmental pollution by pesticides. Omics-based evaluations could explore in more depth the mechanisms involved in the bioremediation of these xenobiotics.SC conceived of the presented idea. SP and ZL contributed to the writing and prepared the figures and tables. JL, YZ, SM, PB, and SC participated in revising the manuscript. All authors approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We previously found that cordycepin inhibits the growth and metastasis formation of MDA-MB-231 cells through the Hedgehog pathway but has not validated this in vivo. In this study, we confirmed cordycepin’s anti-triple-negative breast cancer (TNBC) effect in nude mice and documented its mechanism. We found that cordycepin reduced the volume and weight of MDA-MB-231 xenografts and affected the expression of proliferation-, apoptosis-, epithelial–mesenchymal transition-, and matrix metalloproteinase-related proteins without side effects. RNA sequencing screening, pathway enrichment, and the protein network interaction analysis revealed enriched pathways and targets mainly concentrated on the Hedgehog pathway and its core components of SHH and GLI2. This indicates that the Hedgehog pathway plays a central role in the cordycepin-mediated regulation of growth and metastasis formation in TNBC. The database analysis of the Hedgehog pathway markers  revealed that the Hedgehog pathway is activated in breast cancer tissues, and its high expression is not conducive to a patient’s survival. Finally, we verified that cordycepin effectively inhibited the Hedgehog pathway in TNBC through Western blotting and immunohistochemistry. This study found that cordycepin could regulate the growth and metastasis formation of TNBC through the Hedgehog pathway in vivo, which provides new insights for targeting and treating breast cancer. Cordyceps militaris is a nutrient-rich edible fungus containing cordycepin; cordyceps polysaccharide; pentostatin; ergosterol; and other active components with pharmacological effects, such as antibacterial, antiviral, antitumor, immunity regulation, lipid-lowering, antiplatelet agglutination, and memory improvement activities [Cordyceps sinensis, but its factory production and relatively low price make it the best substitute [tivities ,2,3,4. Dtivities . Its actbstitute .Cordyceps militaris is the adenosine analog cordycepin (3-deoxyadenosine) [Cordyceps militaris contains more cordycepin than Cordyceps sinensis. Most of the cordycepin used in the current study was extracted from a Cordyceps militaris fermentation broth. Cordycepin has antitumor, antivirus, and immune regulation effects (among others) and is used to treat leukemia [One of the earliest and arguably most important natural products isolated from enosine) . Cordyceleukemia . Cordyceleukemia . AlthougBreast cancer is one of the most common malignant tumors in women worldwide, ranking among the top three gynecological tumors in the world in terms of both morbidity and mortality. Furthermore, triple-negative breast cancer (TNBC) is more prone to metastasis and difficult to treat than other breast cancers due to the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 on its surface . CurrentOur group previously demonstrated, through in vitro experiments, that cordycepin induced apoptosis and regulated the apoptotic factors in breast cancer cells; affected the expression of motility-, migration, invasion-, and epithelial–mesenchymal transition (EMT)-related proteins; inhibited the expression of the Hedgehog pathway components and GLI transcription activity; and knocking out GLI blocked the effects of cordycepin on apoptosis, EMT, and the Notch pathways, suggesting that the Hedgehog pathway and its related targets play an important role in breast cancer inhibition . Thus, wWe first measured the effects of cordycepin on the growth of MDA-MB-231 xenografts. This study used 20 nude mice to establish a human breast cancer MDA-MB-231 xenograft model, and 20 of them developed tumors (the tumor formation rate was 100%). The skin surface of the mice at the tumor formation site was pink, and the xenograft was raised on the skin surface with an irregular shape, clear boundary, and rough texture a,b. CordThe occurrence and development of tumors are closely related to the activation of oncogenes, inactivation of tumor suppressor genes, and abnormal expression of apoptosis genes and cell proliferation genes; breast cancer is no exception . To confMatrix metalloproteins and EMT are important factors of tumor cell metastasis and invasion . Therefop < 0.05 and log2 (FC) < 1) and then performed the KEGG and GO function analyses. In line with the fast growth and easy metastasis formation of tumors, we found terms related to growth and metastasis among the significantly enriched GO. We analyzed the growth and metastasis differential genes by an Enrichr online enrichment analysis, and the results showed (p < 0.05)  c. TherefWe drew the protein–protein interaction network map of 78 genes and found that the core genes SHH and GLI2 of the Hedgehog signaling pathway had high degree values a. High dp < 0.05, To clarify the clinical significance of the Hedgehog pathway in the treatment of TNBC, we used the Oncomine database and Kaplan–Meier analysis and assessed the expression of key factors  of the Hedgehog pathway in normal breast tissue and breast cancer tissue, along with their relationship to the prognosis of breast cancer patients. First, we analyzed the expression of SHH, PTCH1, SMO, GLI1, and GLI2 in normal breast and breast cancer tissues through the Oncomine database. We found that normal breast tissue and breast cancer tissue expressed significantly different levels of SHH, PTCH1, SMO, GLI1, and GLI2 , GEO (Gene Expression Omnibus), and the published literature; it is a gene-on-a-chip database that analyzes their differential expression in cancer and normal tissues ,33. The www.wssydw.com, accessed on 17 March 2017). We bought doxorubicin from Sigma . We purchased RPMI 1640 medium and fetal bovine serum from Gibco . We obtained NaCl, KCl, KH2SO4, absolute ethanol, xylene, H2O2, and neutral gum from Sinopharm Chemical Reagent Co., Ltd . We purchased Trypsin K from Roche . We obtained phosphate-buffered solution (PBS); hematoxylin dye; EDTA (pH 9.0) antigen retrieval solution; bovine serum albumin (BSA); hematoxylin differentiation solution; hematoxylin blue liquid; neutral gum; citrate buffer; primary antibodies , caspase-3, E-cadherin, N-cadherin, snail, ZEBI, mmp2, mmp9, cyclin D, and Bax); secondary antibody (HRP-conjugated goat anti-rabbit IgG (H+L)); and histochemistry kit DAB chromogenic reagent from servicebio . We purchased Trizol from Life Technologies. Finally, we bought chloroform, phenol, isopropanol, ethanol, and agarose from Guangzhou Chemical Reagent Factory  ).We purchased MDA-MB-231 cells from iCell Bioscience Inc. . We obtained 20 BALB/C nude mice , feed, litter, and cages from Wu’s laboratory animals  (8 cells/mL with a serum-free cell culture medium and mixed them with BD Matrigel (1:1) on ice for later use. We reared the nude mice in a sterile environment for one week, then injected the mixture of cells and BD Matrigel subcutaneously using a 1-mL syringe. We monitored the tumor formation daily. When the tumors on the tumor-bearing area on the back of the mice reached the size of a mung bean, we randomly divided the 20 mice into a control group and an administration group. Based on our previous in vitro experiments and data from the literature, the control group received sterile water in the stomach daily, and the administration group received 20 mg/kg of cordycepin daily.We diluted the MDA-MB-231 cells to 1 × 102)/2, where D is the long diameter of the tumor, and d is the short diameter of the tumor). We used the average tumor volume of each group to draw a tumor growth curve. Seven weeks after the administration, we photographed the mice, sacrificed them, collected the tumor tissue, and weighed the xenografts.We observed the growth of the nude mice daily, recorded their weight weekly, and noted any mental sluggishness signs and weight loss. After administration, we checked the tumor volume with Vernier calipers every other week , the samples were centrifuged for 10 min, 12,000× g at 4 °C, and the upper aqueous phase was taken. Samples were added to an equal volume of chloroform, mixed well, and centrifuged for 10 min, 12,000× g at 4 °C. RNA was precipitated by an equal volume of Isopropanol and kept for 1 h at −20 °C, then centrifuged for 10 min, 12,000× g at 4 °C. Precipitation was washed by 1 mL 75% ethanol and centrifuged for 5 min, 800× g at 4 °C, and the upper aqueous phase was taken. Ethanol was sucked up and vacuum-dried for 2 to 3 min. Add 20–50 μL RNAse-free water to the sample, put it in a bath at room temperature for 10 min, then centrifuge instantly. RNA samples were stored at −80 °C.RNA was extracted from liver tissue using the conventional method of Trizol . RNA conWe prepared lysates from the xenografts using a homogenizer and lysis solution. We obtained a protein standard curve using BSA and Coomassie brilliant blue solution in a microplate reader. We diluted the lysate to reach the concentration range of the standard curve and then determined its concentration. We prepared a separation gel with a lower layer containing 10% ammonium persulfate and 10 mL of 10% polyacrylamide and an upper layer of concentrated glue containing 6 mL of 5% polyacrylamide and filled with neutral balsam. After the gel solidified, we added the samples, then placed it in the electrophoresis tank and added the electrophoresis buffer to start running the gel.After transferring the membrane, we took it out and placed it in a petri dish. We washed the membranes three times with tris-buffered saline with Tween 20 (TBST). Next, we prepared 5% skimmed milk powder with TBST to seal the membrane; added the diluted primary antibody ; and incubated it overnight at 4 °C. We then washed the membranes three times with TBST and then incubated them with the secondary antibody (HRP-conjugated goat anti-rabbit IgG (H+L)) for 2 h. Finally, we used a BCIP/NBT color development kit in a dark place for about 10 min to reveal the color. We recorded the results with a gel imaging system.2O2, incubated in a wet box for 10 min, and then washed off the H2O2 on the slices with PBS. After that, we immersed the slides in citrate buffer and placed them in a stainless-steel pot for 20 min to achieve antigen retrieval. We then added 10% goat serum dropwise and sealed the slides in a humidified box at room temperature for 30–60 min. Then, we incubated the tissue sections with the primary antibodies overnight at 4 °C. After washing the sections twice with PBS to remove the primary antibodies, we added the secondary antibody (biotin-labeled goat anti-mouse/rabbit) and incubated it at 37 °C for 30 min. We next dyed and stained the sections with SABC, DAB, and hematoxylin. After staining, we put the slices in 1% hydrochloric acid alcohol for a few seconds, then in 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol for 5 min each. Finally, we mounted the sections using neutral balsam, inspected them, and took photographs.We fixed liver tissue specimens in formalin and embedded them in paraffin. We incubated the sections for 60 min at room temperature, added xylene, incubated 10 min, and then added xylene again and incubated 10 min to melt the wax. Then, we rehydrated the slides by immersing them in ethanol absolute, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and pure water for 5 min each. After washing three times with PBS, we added 3% Hwww.oncomine.org, (accessed on 15 October 2020) ) database can collect and analyze microarray data of gene expression profiles in tumor samples, providing real-time transcriptome data. Currently, Oncomine includes 65 gene expression datasets, more than 4700 microarrays, and 48,000,000 gene expression results, primarily for the differential expression analysis of most cancer types, the corresponding normal tissues, and various cancer subtypes. It can be used to analyze the differential expression analyses of multiple studies and draw relatively comprehensive conclusions. Inputting gene names, setting fold changes, and sequencing of the p-value can yield differential expression data of this gene in various tumors. Thus, selecting breast cancer can yield the expression levels of target genes in tumor tissues and normal tissues.We used the Oncomine database to analyze the mRNA expression levels of growth and metastasis differential genes screened in the early stage in the clinical database. The Oncomine ), clicked on the breast cancer related database, and entered the target gene. Next, we divided these cohorts into two groups according to their median gene expression levels, and assessed the relationship between the genes expression levels and the survival of breast cancer patients. We performed this for each of the genes that interested us. We logged in to the kmplot website .We processed data with the SPSS 23.0 statistical software package and compared the groups by one-way ANOVA, with We established a nude mouse xenograft model to confirm that cordycepin significantly inhibited the expression level of the factors related to growth, apoptosis, metastasis formation, and other processes in TNBC. An analysis of the RNA sequencing data revealed that the Hedgehog pathway was the most enriched pathway in breast cancer tissues. By analyzing the markers of the Hedgehog pathway and assessing their expression changes in xenografts, we confirmed that the Hedgehog signaling pathway plays an important role in the anti-breast cancer effect of cordycepin. In summary, cordycepin can regulate growth and metastasis formation in TNBC by inhibiting the Hedgehog pathway.


Uroplatus include peculiar endemic species to Madagascar. Even though they have been the subject of several morphological and molecular studies, karyological analyses have been performed only on U. phantasticus, leaving the chromosomal diversity of the genus completely unexplored. In this study, we performed a preliminary molecular analysis and a comparative cytogenetic study providing the first karyotype description of eight species of Uroplatus and an assessment of their karyological variability. We found chromosome diversity in the species studied in terms of total chromosome number (2n = 34–38), localization of loci of Nucleolar Organizer Regions (NORs) , heterochromatin composition and occurrence of heteromorphic sex chromosome pairs. Adding our newly generated data to those available from the literature, we show that in the genus Uroplatus, as well as in a larger group of phylogenetically related gecko genera, chromosome diversification mainly occurred toward a reduction in the chromosome number by means of chromosome fusions and translocation of NOR-bearing chromosomes. We also hypothesize that the diversification of sex chromosome systems occurred independently in different genera.The geckos of the genus Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4′,6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34–38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) . Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.We provide here the first karyotype description of eight  Blaesodactylus Boettger, 1893, Ebenavia , Geckolepis Grandidier, 1867, Gehyra , Hemidactylus Oken, 1817, Lygodactylus Gray, 1864, Matoatoa Nussbaum, Raxworthy & Pronk, 1998, Paragehyra Angel, 1929, Paroedura Günther, 1879, Phelsuma Gray, 1825 and Uroplatus Duméril, 1806), with a total of more than 100 species currently described [Madagascar is one of the world’s “hottest” biodiversity hotspots and an ideal region to better understand complex evolutionary dynamics ,2,3. TheHowever, even if recent research started to better define the phylogeny and the taxonomy of many different groups, only a small fraction of species has been studied with cytogenetic methods, despite an increasing evidence that their species diversity is reflected at the karyotype level ,10,11,12Uroplatus, which have been the subject of several morphological and molecular studies  has a known karyotype, leaving the chromosome diversity of the genus completely unexplored. Overall, the karyotypes of geckos exhibit a wide variability in terms of the total number of chromosomes, number of uni-armed and bi-armed chromosomes, localization of different chromosome markers and presence or absence of differentiated sex chromosomes [U. phantasticus, the karyotype is composed of 2n = 36, all acrocentric chromosomes, Nucleolar Organizer Regions (NORs) on the second pair and absence of differentiated sex chromosomes [This applies also to the geckos of the genus ee e.g., ,21,22,23omosomes ,8,9,24. Uroplatus currently includes 21, mostly nocturnal, forest-dwelling species, which are overall widespread in Madagascar and surrounding islands (such as Nosy Be), with the exception of the arid southern spiny forest and regions 2400 m asl [The genus 00 m asl . The gen00 m asl ,23).Uroplatus samples from distinct Malagasy areas. We provide the first karyotype description of eight species of the genus and a characterization of their chromosomal diversity. Then, superimposing our newly generated karyological data on available phylogenies [In this paper we performed a preliminary molecular taxonomic analysis and a comparative cytogenetic study with standard karyotyping, Ag-NOR staining and sequential C-banding on different logenies ,25 and clogenies ,8,24,26,Paroedura, Lygodactylus and Christinus Wells & Wellington, 1983.We also provide a first record of a putative heteromorphic sex chromosome system in the genus and hypothesize that sex chromosome diversification occurred multiple times, independently in the phylogenetically related genera Uroplatus. The samples were collected during fieldwork in 1999–2004 by various collaborators and no animal was sampled during the realization of this study. Taxonomic attribution, field number, sex, and origin of all the samples analysed in this study are provided in We examined 13 samples of 8 different species of the genus After capture, animals were injected with a 0.5 mg/mL colchicine solution (0.1 mL/10 g body weight). Tissue samples  were incubated for 30 min in hypotonic solution , fixed and conserved in Carnoy’s solution . The fixed material was preserved at 4 °C and transferred to the laboratory of University of Naples Federico II where it was processed as described below.Uroplatus geckos and the number of available sequences in public repositories [DNA was extracted from tissue samples following Sambrook et al. . A fragmsitories ,21,22,23PCR was conducted in 25 μL using the following parameters: initial denaturation at 94 °C for 5 min, followed by 36 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s and a final extension for 7 min at 72 °C. Amplicons were sequenced on an automated sequencer ABI 377  using BigDye Terminator 3.1 .Chromatograms were manually checked and edited using Chromas Lite 2.6.6  and BioEdit 7.2.6.1 . All newUroplatus , we are confident in the taxonomic attribution provided in U. prope guentheri . Considering the pairwise distance threshold usually used for species identification in squamates for the 12S (3–4%) see e.g., [Uroplatus, but more focused morphological and molecular analyses employing a combination of mitochondrial and nuclear markers should be performed to better assess the taxonomic placement of these samples. This result is not surprising considering the significant number of newly described Uroplatus species in the last years and the molecular identification of different undescribed lineages ..UroplatuMetaphase plates were obtained from tissues sampled during previous fieldwork (see above) using the air-drying method as described in Mezzasalma et al. .3), +4′,6-diamidino-2-phenylindole (DAPI). following Mezzasalma et al. [Chromosomes were stained with conventional colorations (5% Giemsa solution at pH 7), silver staining (Ag-NOR) , C-bandia et al. .Karyotype reconstruction was performed after scoring at least five plates per sample and chromosomes were classified following Levan et al. .Our chromosome analysis showed the occurrence of karyological variability among the studied samples in terms of chromosome number, number and chromosome location of loci of NORs, pattern of heterochromatin and the occurrence of a putative heteromorphic sex chromosome pair.U. prope guentheri) to 2n = 38 of (in U. ebenaui and U. fiera). A karyotype of 2n = 36 was the most common condition in the samples studied and shown by five different species . The karyotypes of all the analysed specimens were composed of all acrocentric chromosomes, gradually decreasing in length. The only exception was represented by the studied female of U. alluaudi, whose karyotype showed a heteromorphic pair (10th pair) including an acrocentric chromosome which was distinctively shorter than a metacentric chromosome. This pair, also in consideration of C-banding results (see below), can be considered as a putative heteromorphic sex chromosome pair with female heterogamety (ZZ/ZW) (Chromosome number varied from 2n = 34 (in  (ZZ/ZW) .U. alluaudi, U. guentheri, and U. pietschmanni), loci of NORs were localised in a telomeric position on the chromosomes of the 2nd pair. In two species (U. fimbriatus and U. henkeli), loci of NORs were in a peritelomeric position on the 6th chromosome pair. In U. finiavana NORs were on the chromosomes of the 10th and 16th pair, while in U. ebenaui NORs were localised on the chromosomes of the 16th pair. Loci NORs were peculiar in U. fiera, residing on pericentromeric regions of the chromosomes of the 2nd pair and on one of the chromosomes of the 16th pair  or positive to either CMA3 and DAPI (in U. pietschmanni)  . In U. aand DAPI . Interesand DAPI . BecauseUroplatus and represents the first step in describing the karyological variability of the genus, as well as a new contribution to reconstruct chromosomal evolutionary dynamics in a larger clade of leaf-toed geckos.Our cytogenetic analysis provided the first karyotype description of eight Malagasy gecko species of Uroplatus mostly encompasses the total chromosome number (from 2n = 34 to 38), a different localization of loci of NORs and the raising of putative heteromorphic sex chromosomes. Chromosome morphology resulted almost invariably acrocentric in the genus with the exception of a large metacentric chromosome found in U. alluaudi, here considered as the W sex chromosome (see below).Overall, we found that the chromosomal diversity in U. ebenaui  should be considered as a primitive state in Uroplatus. From karyotypes with a similar structure, the chromosomal diversification in the genus probably proceeded toward a progressive reduction in the total chromosome number    to middle-sized (6th pair in U. fimbriatus and U. henkeli) and large chromosomes  , is quite rare in reptiles, but similar configurations have been documented in Lacertidae, Opluridae, Leiocephalidae and Helodermatidae  , Lygodactylus [Matoatoa [Paroedura and Christinus [Lygodactylus, Matoatoa, Paroedura and Christinus) and/or the translocation of small NOR-bearing chromosomes on larger chromosomes   see .We highlight that this group of geckos provides an example of an early stage of the transition between “symmetrical” (mostly composed by acrocentric chromosomes and without a clear distinction between macro- and microchromosomes) and “asymmetrical” karyotypes (with a high number of biarmed chromosomes and a clear distinction between macro- and microchromosomes) , which iUroplatus of a putative heteromorphic sex chromosome system . However, only a single female was studied in this work and more karyological data on males and females of U. alluaudi should be gathered in order to confirm this observation. Nevertheless, we highlight that the occurrence of a sex chromosome system is the most robust explanation of the heteromorphic pair found in the female specimen here studied. Notably, the largely heterochromatic W chromosome found in U. alluaudi is much bigger than the Z, a condition rarely observed in squamates, e.g., in Clelia clelia  and Phisalixella variabilis  [Another interesting outcome of our cytogenetic analysis is the first record in r, 1896) ,50.Uroplatus species studied so far, also seems to support the relatively recent origin of the heteromorphic pair in U. ebenaui.Bigger dimensions of the heteromorphic chromosome (Y/W) usually indicate its relatively recent diversification by means of heterochromatin addition and amplification, which is usually followed by the degeneration of the Y/W chromosome, down to the size of a microchromosome ,51,52,53Paroedura (different species with ZW and Z1Z2W chromosomes), C. marmoratus (ZW as the 4th pair) and L. tuberosus (ZW as the 1st pair) [In phylogenetically related geckos, heteromorphic sex chromosome systems are not a common feature and are known mainly in st pair) ,8,24,26.Reptiles are a well-known model organism in the study of sex chromosome diversification and include species with temperature dependent sex determination (TSD) and genetic sex determination (GSD) with either male or female heterogamety  [Paroedura) [U. alluaudi) (this study).In the gecko clade considered here, sex chromosome diversification seems to have followed different pathways in different genera. Diversification by progressive addition of heterochromatin probably occurred in berosus) , largelyroedura) ,24,57 orC. marmoratus, whose euchromatic, submetacentric W started its diversification from the Z by means of an inversion [P. gracilis from Fiherenena  probably originated from an autosome-sex chromosome fusion [The alternative model has been proposed for nversion , while te fusion .Paroedura, most species and genera , localization of loci of NORs , heterochromatin composition and the occurrence of a putative heteromorphic sex chromosome pair.We provide here the first karyotype description of eight gecko species of the genus Uroplatus. Progressive chromosome rearrangements eventually led to karyotypes with a lower chromosome number (2n = 34–36) and NORs on medium or large chromosomes.Considering the occurrence of chromosome characters which are considered plesiomorphic in squamates, we hypothesise a karyotype of 2n = 38 with NORs on one of the smallest pairs as the primitive condition in Uroplatus species studied here resemble those of phylogenetically related leaf-toed geckos, including Paroedura, Ebenavia, Phelsuma and Matoatoa and the Australian genus Christinus. We show that the whole group is characterized by a tendency toward a reduction in the chromosome number (from 2n = 42 to 2n = 34), the formation of metacentric chromosomes and/or the translocation of NORs on middle-sized or large chromosomes.Overall, the karyotypes of the Uroplatus , with a largely heterochromatic W chromosome which is much bigger than the Z. We discuss similarities and differences of sex chromosome diversification in phylogenetically related taxa , hypothesizing that the rise of non-homologous sex chromosomes occurred independently in different genera.We also found a first case of a putative heteromorphic sex chromosome pair in


The hepatotoxicity of many drugs, including antibiotics, is poorly analyzed in human in vitro models. (2) A standardized assay with a human hepatoma cell line was used to test the hepatotoxicity of various concentrations  of antibiotics. In an ICU, the most frequently prescribed antibiotics, ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, meropenem, rifampicin, tigecycline, and vancomycin, were incubated with HepG2/C3A cells for 6 days. Cell viability , albumin synthesis, and cytochrome 1A2 activity were determined in cells. (3) In vitro, vancomycin, rifampicin, and tigecycline showed moderate hepatotoxic potential. The antibiotics ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, and meropenem were associated with mild hepatotoxic reactions in test cells incubated with the testes Cmax concentration. Rifampicin and cefuroxime showed significantly negative effects on the viability of test cells. (4) Further in vitro studies and global pharmacovigilance reports should be conducted to reveal underlying mechanism of the hepatotoxic action of vancomycin, rifampicin, tigecycline, and cefuroxime, as well as the clinical relevance of these findings. Infections are a common problem in the intensive care unit (ICU), mainly due to the severity of patient illness and the frequent use of invasive procedures. Recent data suggest that 51% of ICU patients are infected, and 71% receive antibiotics , with brAdverse drug-related events (ADEs) are more frequent and severe among critically ill patients as a result of their weakened immune systems. There is often the need for prophylactic calculated administration of antibiotics as a result of the patient’s critical condition; thus, symptoms indicative of infection may be treated immediately without a full diagnosis.p < 0.001) [Generally, evaluation of the incidence of antibiotic-associated ADEs in hospitalized patients is unavailable. Shehab et al. conducted a retrospective analysis of ADEs among patients admitted to emergency departments at the Johns Hopkins Hospital . The aim of the study was to estimate and compare the number and frequency of emergency department visits for drug-related side effects associated with systemic antibiotics in the US by event type, individual drug, and drug class. The results of the study show that nearly 20% of patients treated with antibiotics for at least 24 h experience adverse side effects . Potenti< 0.001) . It is e< 0.001) . WhereasAntibiotics are the single largest class of agents that cause drug-induced liver injury (DILI) . DILI caAntibiotic-induced hepatotoxicity is usually abnormal, momentary, unpredictable (depending on the pharmacology of the antibiotic), and mainly dose-independent. Rifampicin is an antibiotic belonging to the class of antituberculosis medications; it has been associated with major side effect, such as DILI . AntibioThe frequency of severe antibiotic-related hepatotoxicity is low relative to the quantities prescribed annually. Population-based estimates suggest that <5 cases per 100,000 antibiotic prescriptions are associated with hepatotoxicity, which remains a major reason for withdrawal of approved drugs .Nevertheless, it is important to understand that most antibiotics can cause idiosyncratic hepatotoxic reactions. Diagnosis of DILI remains a challenge; therefore, the development of reliable hepatotoxic biomarkers is in full progress. Several methods have been developed to facilitate the assessment of hepatotoxicity causality. These liver-specific causality assessment tools fall into three categories: (1) probabilistic approaches, (2) expert judgments, and (3) algorithms or scales ,14. Clinhttps://livertox.nih.gov (accessed on 22 August 2022)), a clinical research database that provides an overview of medications, includes a description of the pattern and history of liver damage, as well as case studies supported with lab data and a comprehensive list of references. However, the standard drug compendia and published reports on the potential hepatotoxicity of drugs vary widely. Therefore, it is recommended to routinely consult LiverTox to stay up-to-date on the latest DILI reports [LiverTox ( reports .The purpose of this study is to examine often-used antibiotics in intensive care unit (ICU) settings for their hepatotoxic potential. An established and standardized hepatocyte biosensor was successfully employed for antibiotic assessment in immortalized human hepatocyte HepG2/C3A. These cells are characterized by their functional similarities to the human liver, as well as their physiological response to toxic insults and metabolic markers ,26,27,282 humidified incubator. The passage number did not exceed 10, and cells were regularly subcultured every 2–3 days.Human hepatocellular carcinoma cells  were cultured as adherent monolayers in Dulbecco’s Modified Eagle Medium  supplemented with 2 mM L-glutamine, 10% FBS , and 1% antibiotic solution  and placed in a 37 °C, 5% CO5 cells/mL in 24-well tissue culture plates [Depending on the specific established microtiter plate hepatotoxicity assay, HepG2/C3A cells were seeded at a density of 5 × 10e plates ,27,28. Ce plates , cefepime plates ,31 cefure plates ,33, levoe plates , linezole plates ,36, meroe plates , rifampie plates , tigecyce plates , and vane plates  for 2 × w/v); Sigma, Seelze, Germany). Lactate dehydrogenase (LDH) was released into the culture medium following the loss of membrane integrity as a result of cell death. After harvesting the supernatants, LDH activity was measured photometrically by detecting the change in absorbance at 340 nm  according to the optimized standard method of the Deutsche Gesellschaft für Klinische Chemie (DGKC) [A simple in vitro output measure of hepatotoxicity is cell death assessment. Cell death was calculated by lactate dehydrogenase and trypan blue exclusion assays ((0.4% (e (DGKC) . On the The XTT assay -2H-Tetrazolium-5-Carboxanilide) 3--2,5-diphenyltetrazolium) is a colorimetric tetrazolium reduction assay  used to quantify functional cell impairment. It is based on the measurement of mitochondrial enzyme activity in viable cells that reduce the tetrazolium derivative XTT. After 1 h of incubation, the optical density (OD) was measured with an ELISA reader  at a test wavelength of 450 nm and a reference wavelength of 650 nm .Metabolic activity in the form of ethoxyresorufin-O-deethylase activity  1A2) was quantified using a methodology previously established in our laboratory based on a method described by Donato et al. ,43. The The concentration of human albumin in the supernatants was determined nephelometrically from 0.2 mL cell culture medium or plasma  .p < 0.05.Non-normally distributed experimental data were analyzed and presented as median and 25th–75th percentiles with GraphPad Prism 5 . Kruskal–Wallis and Mann–Whitney non-parametric tests were used for pairwise comparisons of groups. Statistical significance was set at The effects on cell proliferation and vitality varied significantly among the tested antibiotics. Compared to the negative control, vitality was significantly reduced after incubation with the Cmax concentrations of levofloxacin and linezolid in medium and plasma, whereas vancomycin was only decreased in medium A,B. The Whereas there was no effect observed at Cmax, cell vitality was significantly decreased at higher concentrations of rifampicin and cefuroxime, especially in medium C,D. For A significant reduction in cell count compared to the negative control was detected after incubation with cefuroxime, levofloxacin, linezolid, rifampicin, and tigecycline at Cmax in medium; for tigecycline, a reduction was observed in medium, as well as in plasma C,D.As the concentration was increased, a further decrease was observed for rifampicin and cefuroxime, principally in medium A,B. The A significant increase in lactate dehydrogenase (LDH) at Cmax was detected after incubation with ampicillin, cefepime, cefuroxime, meropenem, rifampicin, tigecycline, and vancomycin. In particular LDH values increased by more than 50% compared to the negative controls (91 U/L) after treatment with ampicillin, meropenem, and rifampicin at Cmax in medium A. The teThe activity of mitochondrial dehydrogenases (XTT-test) of HepG2/C3A cells was significantly decreased at the Cmax of levofloxacin  1.065), rifampicin (0.984), tigecycline (1.054), and vancomycin (1.079) compared with the negative control in medium (1.301), whereas it was significantly increased for ampicillin (1.523), cefepime (1.486), cefuroxime (1.55), meropenem (1.327), and linezolid (1.394) A. A negaCells incubated in plasma showed increased activity of mitochondrial dehydrogenases after treatment with ampicillin (1.430), cefuroxime (1.674), linezolid (1.273), and rifampicin (1.382) at the Cmax concentration B. In conIn the hepatocyte cultures, the control group showed an EROD activity level of around 8.7 pmol/L in medium and 5.3 pmol/L in plasma . A signiFollowing exposure to higher concentrations of drugs (5× Cmax and 10× Cmax), only ampicillin-treated cells showed an increase in EROD activity in medium of more than 70%  and in plasma of about 65% . However, the various EROD levels of ampicillin-treated cells were still decreased compared to the medium control (8.7 pmol/L). Compared to the plasma control, the EROD levels were slightly higher . The other antibiotics did not result in a significant decrease.We observed a significant increase in albumin synthesis after incubation at Cmax with ampicillin, cefepime, linezolid, and meropenem . In contIn addition, in plasma, cefuroxime, meropenem, tigecycline, and vancomycin influenced negative microalbumin synthesis at the Cmax concentrations. Treatment with ampicillin, cefepime, cefuroxime, levofloxacin, linezolid, and rifampicin increased the microalbumin synthesis of the hepatocytes  are listed in The majority of classes of antibiotics have hepatotoxic potential and are considered a leading cause of DILI ,45. WithPrediction of drug-induced hepatotoxicity in humans from in vitro data remains a significant challenge . First, The discovery of antibiotics as a cure for bacterial infections represents a considerable advance for medicine. However, in recent decades, it has also become clear that there is no ideal antibiotic with exclusive effect on the disease-causing microorganism and that every antibiotic therapy is associated with certain risks. Collectively, many classes of antibiotics are a major cause of drug-induced liver injury (DILI) . Toxic ehttp://livertox.nih.gov) were classified into five categories based on the number of published cases .For the study of drug metabolism, there is no single approach that provides all the necessary information for complete understanding; therefore, it is necessary to verify whether the conclusions derived from in vitro experiments actually correspond to the events occurring in vivo. In this discussion, we attempt to harmonize the approaches with the information from various studies to enable a better evaluation of the results. An important element in assessing causality in drug-induced liver injury is whether the implicated agent is known to cause hepatotoxicity. In a review by Einar S. Björnsson and Jay H. Hoofnagle (2015), various drugs with potential hepatotoxicity were categorized . In the According to some statistics, antibiotics are the second most hepatotoxic class of drugs . AccordiAccording to the in vitro results of the antibiotics tested in this study, rifampicin, tigecycline, and vancomycin have the highest hepatotoxic potential; furthermore, rifampicin and cefuroxime at higher concentration led to a destruction of cell integrity.Only rifampicin and cefuroxime led to a dose-dependent decrease in vitality and cell-count (proliferation) in medium and plasma. Additionally, incubation of cefuroxime at higher concentrations with the hepatoma cell line resulted in a significant increase in lactate dehydrogenase (LDH) in the cell culture supernatant. Cefuroxime is a second-generation cephalosporin. It is very rarely known to cause DILI ,65. OnlyThe activity of mitochondrial dehydrogenases (XTT-test) of HepG2/C3A cells, as a basic functional test, was significantly decreased after incubation with therapeutic concentrations (Cmax) of levofloxacin, rifampicin, tigecycline, and vancomycin. A negative dose-dependent effect on mitochondrial activity was only observed for cefepime at higher concentrations (but with an increase at Cmax). Vancomycin therapy has been widely associated with hypersensitivity reactions that may be associated with mild liver injury but rarely with severe or life-threatening liver injury. These include Stevens–Johnson syndrome, toxic epidermal necrolysis, and the characteristic syndrome of drug reaction, eosinophilia, as well as systemic symptoms . Liver fThe cytochrome (P450) 1A2 enzyme (CYP 1A2) is most important for the metabolism of foreign substances. In our in vitro study, we observed a significant increase in CYP 1A2 activity after incubation with levofloxacin and linezolid at therapeutic concentrations (Cmax), whereas ampicillin, cefepime, cefuroxime, meropenem, rifampicin, tigecycline, and vancomycin led to a significant decrease. At higher concentrations of the tested drugs (5× Cmax and 10× Cmax), only ampicillin-treated cells showed a slight increase in CYP 1A2 activity in the range of the cell-culture control. The decrease in CYP 1A2 activity after incubation with levofloxacin and linezolid in vitro is surprising, as neither substance is known as a CYP 1A2 inducer or substrate ,91,92. IThe production of proteins, such as albumin, is an important function of the liver. In our in vitro model, we found that incubation with cefuroxime, tigecycline, and vancomycin at Cmax led to a significant decrease in albumin synthesis in medium. Furthermore, a dose-dependent decrease in albumin was determined after incubation with all antibiotics in medium; however, after incubation with the 10× Cmax concentration of tigecycline, a complete breakdown of albumin synthesis was observed. These are new findings, and the mechanisms are currently unclear. Treatment with tigecycline can lead to inhibition of mitochondrial respiration and β-oxidation . Under iThe used “broad-spectrum” hepatotoxicity test was previously validated in some clinical and experimental studies ,26,27,28The antibiotics meropenem, linezolid, and ampicillin should be also discussed. Meropenem often causes only mild, transient elevations in aminotransferase and rarely causes clinically apparent cholestatic liver damage . The cauIt is difficult to identify the action of single hepatotoxic drugs because treatment is often administered in combination with many other drugs, and most antibiotics can cause hepatotoxic reactions. In our experiments, we tested antibiotics that are frequently use in intensive care units. Rifampicin, tigecycline, and vancomycin are the most commonly used drugs with high hepatotoxic potential; therefore, they should be used with caution in patients with pre-existing liver disease/insufficiency (summarized in Further in vitro studies and global pharmacovigilance reports should be conducted to reveal the underlying mechanism of the hepatotoxic action of vancomycin, rifampicin, tigecycline, and cefuroxime, as well as the clinical relevance of these findings. Unfortunately, recognition of the toxicity of certain drugs is limited by inadequate reporting and the relatively rare overall frequency of hepatotoxicity. Diligent reporting in DILI cases registries ,22,110 cCareful drug monitoring is particularly important in patients with a history of hepatotoxic reactions to antibiotics, the elderly, and patients with impaired liver function. Due to the increase in old and sick patients in intensive care units, as well as the increasing tendency of predamaged livers, the risk–benefit ratio of antibiotic therapy should be maximized.


Influenza infections impose a high burden of morbidity and mortality among older adults, at great cost to individuals and society. Enhanced influenza vaccines, which contain either an immune adjuvant or higher than normal doses of influenza virus antigens, are recommended for older adults. We used a health economics model to evaluate the cost effectiveness in Spain of a recently licensed recombinant quadrivalent influenza vaccine (QIVr), which contains three times the standard dose of influenza virus hemagglutinin but no neuraminidase, compared with an MF59-adjuvanted quadrivalent influenza vaccine (aQIV). Based on current tender prices in Spain and a conservative assumption that QIVr is 10.7% relatively more effective than aQIV, the incremental cost-effectiveness ratio (ICER) for QIVr was EUR 101,612.41 per quality-adjusted life year (QALY) gained. To meet the Spanish willingness-to-pay threshold of EUR 25,000 per QALY gained, QIVr would need to be 34.1% relatively more effective than aQIV. In a probabilistic sensitivity analysis conducted to confirm the robustness of the analysis, 99.7% of simulations for QIVr were higher than the willingness-to-pay curve. These findings suggest that QIVr is not currently a cost-effective influenza vaccine option relative to aQIV for older persons living in Spain. Seasonal influenza carries a high burden of disease on individuals and society. Of ~1 billion infections occurring worldwide each year, 3–5 million are severe and up to 650,000 result in death—67% of which occur among persons aged 65 years and older . The mor® adjuvant, an oil-in-water emulsion of squalene oil that produces a stronger, broader, and longer immune responses relative to standard influenza vaccines in older adults ..3].In addition, the model used the inputs shown in As shown in All costs were in 2021 euros and did not include administration or transportation costs, which are expected to be the same across vaccines. Tender prices for vaccines included in the model were EUR 13 for aQIV and EUR 25 for QIVr . ResourcLost productivity due to influenza infection was calculated using the discounted human capital approach and based on working days lost multiplied by the probability of being employed, or 1.2% for persons aged 65–69 years and 0.3% for those aged ≥70 years ,42. ProdAs mentioned, randomized, head-to-head clinical trials comparing the efficacy of aQIV and QIVr have not been conducted, and no observational studies have yet compared aQIV and QIVr. The only relevant comparison is a single observational study by Izurieta et al. comparing QIVr with an aTIV, as well as other formulations, in older adults aged ≥65 years during the 2019–2020 season. In this study, rVE was calculated as (1—relative risk [RR]) × 100%, and the rVE for the prevention of influenza-related inpatient stays was 10.7%  . This waTwo analysis scenarios were conducted to test the impact of the model assumptions on the incremental cost-effectiveness ratios (ICERs). One-way deterministic sensitivity analysis (DSA) was used to evaluate the impact of input uncertainty.In the first analysis scenario, the rVE of 10.7% for QIVr vs. aQIV was used based on the QIVr vs. aTIV comparison reported in Izurieta et al. This value was used because it is the highest rVE available for a recombinant vs. and adjuvanted influenza vaccine . BecauseIn the first scenario based on a 10.7% rVE favoring QIVr over aQIV, QIVr was associated with incremental costs of EUR 71,054,601.74 and 699.27 QALY gained, for an ICER of EUR 101,612.41 per QALY gained. In the second scenario, to achieve a cost-effective ICER of EUR 25,000 per QALY gained, QIVr would need to be associated with an rVE of 34.12%, incremental costs of EUR 55,747,194.30, and QALY gained of 2229.88 .A DSA was conducted with the tornado plot summarizing the most influential parameters for the ICER presented in In the PSA conducted to confirm the robustness of the cost-effectiveness analysis, 99.7% of simulations were higher than the willingness-to-pay curve .Our results show that QIVr at its current price and the assumed increased effectiveness over aQIV is not cost effective for vaccination against influenza in older adults aged ≥65 years in Spain. At an ICER of EUR 101,612.41/QALY gained and at an rVE of 10.7% favoring QIVr (as considered in the model), the current tender price of EUR 25 per QIVr dose (vs. EUR 13 for aQIV ) is too The rVE of 10.7% used in this analysis was based on a comparison of QIVr with an adjuvanted trivalent formulation, which was missing antigens for one of the B viruses contained within QIVr. No published studies have compared the efficacy or effectiveness of QIVr and aQIV, so it is unknown how inclusion of the fourth virus strain would affect the rVE value between these vaccines. QIVr contains three times the dose of hemagglutinin (45 μg per antigen) as standard dose vaccines, including aQIV (15 μg per antigen) and, thus, may be considered a “high-dose” influenza vaccine. The results of a meta-analysis of real world evidence comparisons between aTIV and HD-TIV (60 μg per antigen) showed no significant difference between the adjuvanted and high-dose formulations, with a point estimate of 3.2% , slightly favoring aTIV . Based oOur group recently conducted another cost-effectiveness analysis comparing aQIV and HD-QIV in Spain, and our results in that study support the findings of the present analysis. In the previous study, we demonstrated cost savings of EUR 63.6 million if aQIV rather than HD-QIV were used to vaccinate a population of ~5 million older adults because aQIV is less expensive than HD-QIV and presents a minor advantage in effectiveness, with 5405 fewer symptomatic cases, 760 fewer primary care visits, 171 fewer emergency room visits, 442 fewer hospitalizations, and 26 fewer deaths, with a corresponding increase of 206 QALYs each year. In addition, aQIV fell below the Spanish cost-effectiveness threshold in this study . The finOur analysis is limited by the dearth of available data comparing adjuvanted with recombinant influenza vaccines. To date, only a single observational study from a single season has been published, which compared QIVr to aTIV rather than aQIV . In addiThe model inputs used in the study were primarily drawn from Spanish data on outcomes such as influenza incidence, primary care and ED visits, hospitalizations, resource use, costs, vaccine coverage, and mortality. Although some utility data are from Spain, data from the UK, Belgium, and Germany were also used, as in previous Spanish influenza models .Adults ≥65 years of age are most vulnerable to the impact of influenza, with the highest rates of infection and poor outcomes. Enhanced vaccines were developed to overcome the immunosenescence—an age-related process that leads to dysregulation of the innate and adaptive immune systems and less effective humoral and cellular immune responses against pathogens or to vaccines  support


PKD2 gene of an autosomal dominant polycystic kidney disease (ADPKD) patient with multiple liver cysts. A 57-year-old woman presented to our university hospital with abdominal fullness, decreasing appetite, and dyspnea for three months. A percutaneous drainage of hepatic cysts was performed with no significant symptomatic relief. A computed tomography (CT) scan revealed a hepatic cyst in the lateral portion of the liver with appreciable compression of the stomach. Prior to this admission, the patient had undergone three drainage procedures with serial CT-based follow-up of the cysts over the past 37 years. With a presumptive diagnosis of extrarenal manifestation of ADPKD, we performed both a hepatic cystectomy and a hepatectomy. Because the patient reported a family history of hepatic cysts, we conducted a postoperative genetic analysis. A novel missense mutation, p.Ile424Ser, was detected in the PKD2 gene. Mutations in either the PKD1 or PKD2 genes account for most cases of ADPKD. To the extent of our knowledge, this point mutation has not been reported in the general population. Our in-silico analysis suggests a hereditary likely pathogenic mutation.We report a novel missense mutation, p.Ile424Ser, in the  PKD1 (polycystin 1) at position 16p13.3 and PKD2 (polycystin 2) at 4q22.1, account for 80% and 15% of ADPKD cases, respectively [Studies have shown that mutations in one of two genes, PKD2 gene of an autosomal dominant polycystic kidney disease (ADPKD) patient with multiple liver cysts. To the extent of our knowledge, this point mutation has not been reported in the general population. Our in-silico analysis suggests a hereditary likely pathogenic mutation.We report a novel missense mutation, p.Ile424Ser, in the A 57-year-old female, with an established history of multiple liver cysts, presented to the Internal Medicine Department of our university hospital with a three-month history of epigastric discomfort and reduced appetite. The patient’s hepatic cysts were incidentally found on a computed tomography (CT) scan at 20 years of age. There existed a family history of bilateral renal cysts and multiple liver cysts in her maternal grandfather and mother and liver cancer in her father. The patient previously underwent three rounds of percutaneous drainage of hepatic cysts at ages 48, 53, and 54 years. A percutaneous cyst drainage, CT-guided, was performed this time as well, by the treating physician at the Interventional Radiology Department in collaboration with the Internal Medicine Department; however, it led to minimal symptomatic relief. Subsequently, the patient was referred to our surgical department for further evaluation and treatment of the hepatic cysts.2; urinalysis was not particular; and systolic blood pressure was 110 mmHg.A physical examination showed mild icterus and palpable liver with associated mild abdominal tenderness. The patient was found to be mildly anemic with a hemoglobin level of 10.5 g/dL. Moreover, she presented with hypokalemia with a potassium level of 2.5 mEq/L and elevated levels of biliary enzymes, including γGTP (117 U/L) and alkaline phosphatase (564 U/L). The level of follicle-stimulating hormone was 83.3 mIU/mL, and that of estradiol was 8 pg/mL. The alpha-fetoprotein level was slightly elevated to 16.5 ng/mL (≤10.0). The indocyanine green plasma disappearance rate  was 0.202, which was within the normal range. The level of serum creatine was 0.8 mg/dL, eGFR 80 mL/min/1.73 mAbdominal magnetic resonance imaging (MRI) T2 horizontal section and magnetic resonance cholangiopancreatography revealed no dilation of the intrahepatic bile duct, but cystic lesions of various sizes were scattered across both the lobes. The cysts protruding from the right lobe, S5, were slightly heterogenous and septate with low signal intensity, indicating intracystic bleeding. Some cysts showed a decrease in size after drainage, but the extrahepatic cysts exhibited no change. These extrahepatic laterally located cysts measured up to 90 mm in diameter and were found to be compressing the stomach, causing a posterior deviation. In addition, numerous bilateral renal cysts were visualized . Thus, wIntraoperative findings were found to be consistent with the MRI findings. A cyst protruding from the left lobe of the liver compressed the stomach, causing a posterior deviation. Hence, a left hepatic lobectomy, a caudate lobectomy, an S5 cystectomy, and a cholecystectomy were performed. Intraoperative cytological analysis was negative for malignancy. In addition, we performed fenestration of the cysts located on the liver surface and incineration of the luminal surface .The patient suffered no complications during the one-year postoperative follow-up period. No change in cyst size or extent with CT was noted upon initiation of nonhormonal management of perimenopausal symptoms.https://www.ncbi.nlm.nih.gov/snp/ (accessed on 13 March 2022) and genomAD; https://gnomad.broadinstitute.org/ (accessed on 13 March 2022), revealing a novel missense variant NM_000297.4:c.1271T>G p.Ile424Ser in the PKD2 gene , while the presentation in the case of a PKD2 mutation is less severe and less likely to lead to ESRF. The most common extrarenal lesion in ADPKD occurs in the form of polycystic liver disease, which is classified via Gigot classification based on the number, size, and distribution of cysts observed on CT to evaluate the severity of the disease. Accordingly, sclerotherapy, coronary embolization, cyst opening, hepatectomy, or liver transplantation is performed to treat the lesions. Symptoms of multiple hepatic cysts are mainly compression symptoms, including gastrointestinal disturbances such as abdominal distention, upper abdominal pain, and decreased food intake; respiratory disturbances due to compression of the lungs and heart, pain, and changes in vital signs because of rupture; and anemia due to bleeding. In addition, if there appears to be an irregular thickening of the cyst wall or a contrast effect upon imaging, mucinous cystic neoplasm or intraductal papillary neoplasm of the bile duct cannot be ruled out, and malignant transformation or precancerous lesions may be detected. Therefore, the patient is subject to surgical resection to eliminate malignant transformation and precancerous lesions. ADPKD was reported by Bear et al. in 1984, Ravine et al. in 1994, and Pei et al. in 2009, and evidence-based guidelines for the treatment of polycystic kidney disease were published in Japan in 2017 ,6,7,8. TPKD2 gene, which was thought to be one of the causative genes of the disease. This missense variant has not been registered in the general population previously, and our in-silico analysis strongly suggested that it was a pathogenic mutation.In our case, the patient was classified as Gigot type II , and surgical resection was effective . Since hPKD1  and PKD2 genes , respectively, which form heterodimers in the cilia of cells  and PC2 , encoded by the of cells . A reducHowever, mechanisms underlying the formation of multiple hepatic cysts remain to be clarified. These mechanisms may be specific to hepatic cysts derived from the bile duct epithelium. Hence, as the gene mutation has been identified in the present study, future research should aim at providing further evidence for the pathogenicity of this mutation and revealing possible mechanisms of hepatic cysts development. In addition to the findings in regular imaging analysis, the frequency of occurrence of multiple hepatic cysts in patients with ADPKD ranges from 20% (for patients in their twenties) to 75% (for patients in their fifties) . MultiplPKD2 missense mutation p.Ile424Ser in a patient with ADPKD mainly presenting with polycystic liver disease, and our in-silico analysis strongly indicated that this mutation is a likely pathogenic mutation. Hepatic cystectomy and hepatectomy were performed to control the disease. We believe our findings indicate the pathogenic nature of the PKD2 mutation and provide a clue to the molecular mechanism of hepatic cyst formation.We found a novel