Tenecteplase is a modified recombinant tissue-plasminogen activator, which is effective and safe in the treatment of acute ischemic stroke. However, the therapeutic dose of tenecteplase has been controversial. The purpose of this study is to systematically investigate the efficacy and safety of different doses of tenecteplase thrombolytic therapy for acute ischemic stroke.Computer retrieval of English databases  and Chinese databases  is conducted for a randomized controlled clinical study on thrombolytic treatment of acute ischemic stroke with different doses of tenecteplase from the establishment of the database to October 2020. Two researchers independently conduct data extraction and literature quality evaluation on the quality of the included studies, and meta-analysis is conducted on the included literatures using RevMan5.3 software.In this study, National Institute of Health Stroke Scale (NIHSS) score, Modified Rankin Scale (mRS) score scale, symptomatic intracranial hemorrhage (SICH) incidence, All-cause mortality, and so on are used to evaluate the efficacy and safety of tenecteplase thrombolytic therapy in acute ischemic stroke with different doses.This study will provide reliable evidence-based evidence for the clinical application of different doses of tenecteplase in thrombolytic therapy for acute ischemic stroke.DOI 10.17605/OSF.IO/2MPCW. The lifetime risk of stroke for patients aged 55 to 75 years is 20% in women and 15% in men. Moreover, about 10% of patients with AIS die within 1 year, and 20% to 25% of patients suffer from severe disability.Stroke is the second leading cause of death after malignant tumors. Every year, about 22 million people around the world suffer from stroke, mainly including 3 categories: ischemic (87%), hemorrhagic (10%), and subarachnoid hemorrhage (3%). and timely intravenous thrombolysis could make blocked blood vessels recanalize, thereby saving ischemic pentheus and finally reducing the mortality and disability rate of ischemic stroke. Recombinant tissue-plasminogen activator (RT-PA) and intravenous thrombolysis within a time window are currently internationally recognized as effective treatment methods for acute ischemic stroke. Alteplase, the active ingredient in the recombinant tissue-type plasminogen activator (RT-PA), can activate the conversion of plasminogen into plasmin to achieve a good thrombolytic effect, while tenecteplase is a biologically engineered variant of alteplase, which has a longer t1/2 and stronger anti-plasminogen activator inhibitor effect. In addition, tenecteplase is cheaper and convenient to administer. At present, the clinical efficacy of intravenous thrombolysis with different doses of tenecteplase has not been clearly defined. A non-randomized dose-increasing safety study has shown that tenecteplase of 0.1 to 0.4 mg/kg is safe for ischemic stroke, but there is no systematic evaluation of which dose is more beneficial for the treatment of acute ischemic stroke.The key point of treatment of AIS is to achieve blood vessel recanalization and restore brain tissue irrigation effectively in early stage,–10 on the doses of tenecteplase, ranging from 0.1 to 0.4 mg/kg, but the results are uneven. Therefore, this plans to systematically evaluate the effectiveness and safety of intravenous thrombolysis of AIS with different doses of tenecteplase, so as to provide a reliable reference basis for clinical application.At present, there have been a number of randomized controlled trials22.1 It has been registered on open science framework (OSF) on November 14, 2020. (Registration number: DOI 10.17605/OSF.IO/2MPCW).This study of systematic review and meta-analysis has been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P).2.2Since this is a protocol with no patient recruitment and personal information collection, the approval of the ethics committee is not required.2.32.3.1We will collect a randomized controlled study of tenecteplase at different doses in the treatment of AIS in Chinese and English only.2.3.2Patients who are clinically diagnosed as AIS and confirmed by brain computed tomography (CT) and magnetic resonance imaging (MRI), with no limitation on nationality, race, age, sex, and course of disease.2.3.3The observation group receive intravenous thrombolysis with different doses of tenecteplase . The control group receive intravenous thrombolytic therapy with alteplase 0.9 mg/kg.2.3.4(1)Primary outcome: National Institute of Health Stroke Scale (NIHSS) . Modified Rankin Scale (mRS) rating scale ;(2)). All-cause mortality.Secondary outcomes: symptomatic intracranial hemorrhage (SICH) risk, (incidence of SICH as defined by the European Cooperative Acute Stroke Study (ECASS)II standard2.4(1)Studies with incomplete data and repeated publication;(2)Articles which are published as abstracts and whose author are unable to offer data after being contacted;(3)Studies with no relevant outcome indicators or obvious error in the research data;(4)The risk assessment of bias is the literature with high risk of bias.2.5The computer searches CNKI, WanFang, Viper, Chinese Biomedical Literature Database, and Chinese search terms are “Ti Nai Pu Mei (TNK-tPA)” or “Zhong Feng (Stroke)” or “Ji Xing Que Xue Xing Nao Zu Zhong (Acute ischemic stroke)” or “Ji Xing Nao Geng Si ,” and “Ji Xing Nao Geng Se .” Retrieval in English databases including PubMed, EMBASE, Web of Science, the Cochrane Library, Search terms in English are tenecteplase, TNK-tPA, acute ischemic stroke, AIS. The retrieval time is from the establishment of the database to October 2020, and all domestic and foreign literatures on different doses of tenaiprase for AIS treatment are collected. Take PubMed as an example, and the retrieval strategy is shown in Table 2.6Data are extracted independently by 2 researchers and cross-checked. If there are different opinions, they would be discussed or solved with the assistance of a third researcher. The extracted contents include the first author, year of publication, country, study start and end time, number of cases, intervention measures, treatment time window, and outcome indicators. The literature selection process is shown in Fig. 2.7 According to the performance of the included literature in the above evaluation items, the 2 researchers will give low risk, unclear and high risk judgments one by one, and cross-check after completion respectively. In case of any disagreement, discussion will be carried out. If no agreement can be reached, discussion will be made by the researchers in the third party.The Cochrane Risk Bias Assessment Tool is used to assess the risk bias of the included studies.2.82.8.1P ≥ .10 and I2 ≤ 50%, heterogeneity is low. Fixed-effect model is used for meta-analysis. When P < .1, I2 > 50% indicates inter-study heterogeneity, and the source of heterogeneity should be analyzed. Clinical heterogeneity is treated by subgroup analysis. If no significant clinical or methodological heterogeneity can be found, statistical heterogeneity would be considered, and the random-effect model would be used for analysis. If the clinical heterogeneity is too obvious and the subgroup analysis cannot be performed, the meta-analysis would not be performed, but descriptive analysis is only performed.RevMan5.2 software is used for Meta-analysis. Odds ratio (RR) is used as the combined statistic for counting data, and standardized mean difference (SMD) is used as the combined statistic for measurement data. Point estimate and 95% confidence interval (95% CI) are given for each effect. Heterogeneity among study results is analyzed by chi-squared test. When 2.8.2If there is missing data in the article, contact the author via email for additional information. If the author cannot be contacted, or the author has lost relevant data, descriptive analysis will be conducted instead of meta-analysis.2.8.3Subgroup analysis is carried out according to the dose and treatment of tenecteplase.2.8.4In order to determine the stability of outcome indicators, sensitivity analysis is used to analyze each outcome indicator.2.8.5Funnel plots will be used to assess publication bias if no fewer than 10 studies are included in an outcome measure. In addition, Egger and Begg test were used for the evaluation of potential publication bias.2.8.6 The evaluation includes bias risk, interconnectedness, inconsistency, inaccuracy, and publication bias. The quality of evidence will be rated as high, medium, low, or very low.We grade the outcome indicators through the Grading of Recommendation Assessment, Development and Evaluation (GRADE).3,16 There are many pathological mechanisms of ischemic stroke, among which acute arterial thrombosis leads to vascular occlusion is a common cause, and venous thrombolysis is the most important measure to restore blood flow.With the aging of the population, acute ischemic stroke has become one of the main diseases leading to death and disability, and the trend is increasing year by year, seriously affecting the physical health and quality of life of residents.–19: Convenience: compared with the traditional alteplase (RT-PA) usage , tenecteplase has a longer half-life and only needs a single injection within 10 seconds; Efficacy: Compared with RT-PA, it has higher specificity to fibrin and higher recanalization rate; compared with common RT-PA, tenecteplase has higher specificity for fibrin at thrombus site, and its activity can be inhibited by endogenous inhibitors; compared with alteplase, it is cheaper to reduce the financial concerns of patients before use, thus increasing the proportion of patients with thrombolysis. Although it has been proved that tenecteplase is as effective as alteplase, the issue of dosage has been controversial. Studies have shown that 0.40 mg/kg tenecteplase is no more advantageous than 0.25 mg/kg tenecteplase in patients with large vessel occlusie ischemic stroke, indicating that dose size is not positively correlated with benefit, so it is necessary and urgent to explore the most appropriate dose.The emergence of tenecteplase as the third generation of thrombolytic drugs enriches and improves the clinical selection of thrombolytic drugs, with the following advantages The efficacy has been verified clinically after extensive clinical use, so it is reliable to evaluate the efficacy and safety of tenecteplase in different doses as a control. This systematic evaluation will evaluate the reliability of the efficacy of tenecteplase in the treatment of acute ischemic stroke at different doses based on available evidence, providing an evidence-based basis for the selection of clinicians.According to the trial of the National Institute of Neurological Disorders and Stroke (NINDS) in the United States, the recommended dose of IV-TPA is 0.9 mg/kg (maximum 90 mg).However, this systematic review has some limitations. At present, there are few randomized controlled studies on tenecteplase doses, which may lead to publication bias due to the small number of included studies. At the same time, due to the limitation of language ability, we only search literature in English and Chinese, and may ignore research or reports in other languages.Data curation: Guanfeng Chen, Chunfeng Bai.Funding acquisition: Sen Shao.Literature retrieval: Guanfeng Chen and Chunfeng Bai.Software: Zhou Zhu, Junheng Li.Supervision: Sen Shao.Writing – original draft: Guanfeng Chen, Chunfeng Bai.Writing – review & editing: Guanfeng Chen, Sen Shao.


Streptococcus anginosus group (SAG). In the population of 463 patients, the male-to-female ratio was 1.95:1, and the patient age ranged from 6 months to 103 years. There were 49 children (10.58%), 311 young and middle-aged adults (67.17%), and 103 elderly adults (22.25%). Approximately 45.4% had underlying conditions, which were mostly malignant tumors and diabetes. Of the 463 specimens, 254 were S. anginosus (54.86%), 173 were S. constellatus (37.37%), and 36 were S. intermedius (7.77%). According to the age distribution, the incidence peaked in the 35–54 year age group. Different sites of infection had statistically significant differences regarding the constituent ratios of these three species. Different age groups also exhibited statistically significant differences in constituent ratios of the pathogenic organisms, as well as organ infections. In our population, 269 were clinically cured, 184 reported satisfactory improvement, and 10 died. SAG, as an opportunistic pathogen, can induce pyogenic infections in patients of all ages and shows no significant gender predilection in any age group. The three pathogenic organisms had differences with respect to patient age and infections of body sites.This study aimed to investigate the clinical characteristics, distribution of different strains and risk factors of patients infected with  Streptococcus anginosus group (SAG) is a group of gram-positive streptococci normally colonizing the upper respiratory, digestive and reproductive tracts and consists of three distinct species, S. anginosus, S. constellatus and S. intermedius. SAG is not recognized as a causative pathogen. However, with the presence of certain incentive factors, colonized SAG directly induces noninvasive infections and causes invasive infections after entering normal sterile sites in the body, including the blood and serosal cavity, which eventually affect the tissues and organs of various systems of the body. The number of patients infected with SAG is increasing1. In fact, there is a lack of knowledge of these bacteria as opportunistic pathogens that may cause invasive infections2. Infections caused by SAG should be considered in the clinical diagnosis and treatment of related infections. On this basis, a retrospective analysis of patients diagnosed with infections with SAG admitted by the general teaching hospitals of two regional medical centers between Jan. 2014 and Nov. 2019 was conducted, and the findings are reported below.S. anginosus, S. constellatus and S. intermedius) were collected from the bacterial laboratory database of the Affiliated Hospital of Jining Medical University Hospital and Jining No. 1 People’s Hospital, and blood, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid, seroperitoneum, specimens by surgical drainage, mediastinum or soft tissue aspirate specimens were collected from these patients. A total of 463 subjects, including 306 males and 157 females, were enrolled in the present study after the clinical history of the patients was reviewed.Patients with cultures positive for SAG  plates and cultured at 35 °C for 24 h until tiny pinpoint, circular, convex, alpha- or beta-hemolytic colonies with entire edges appeared. According to the Gram stain procedure, these bacteria were gram-positive cocci that were nonmotile, non-spore-forming, catalase-negative and arranged in pairs and in chains of varying lengths.SAG consists of gram-positive, catalase-negative cocci and nonmotile facultative anaerobes that have typically small colonies (≤0.5 mm diameter) and demonstrate variable hemolysis patterns . The identification of this bacterium in our laboratory is implemented by GP card (gram-positive bacteria identification card) of VITEK2 Compact, a fully automatic bacterial identification susceptibility instrument, and the flight mass spectrometer. Specific biochemical identification: the physiological and biochemical characteristics of SAG showed that urinase and sorbitol were negative; acetoin (VP) and arginine were positive. The species identification of SAG could be identified by the biochemical examination items including β-D fucosidase,β-D acetyl semi-glycosidase, neuraminidase and hyaluronidase. See the following table for details Table .Table 1KMedical records of these patients were obtained from the medical record department, and the following data were collected: (1) general information such as sex and age, etc.; (2) basic conditions at presentation, including underlying diseases , use of glucocorticoids and immunosuppressants 3 months before admission, and the presence of implants and surgical history in the last year. (3) laboratory test results of patients, including routine blood tests, C-reactive protein (CRP), serum albumin level (Alb) and bacterial culture; (4) type, dosage and date of antibiotics used; (5) infections, including the site, treatment and the presence of other bacterial infections; and (6) prognosis, i.e., the outcome (death or discharge after improvement) of the patients.Data were analyzed using SPSS 22 statistical software. Measurement data that fit a normal distribution are expressed by “x ± s”, while enumeration data are represented by the number of cases and constituent ratio. Univariate analysis was performed using the Q test (for measurement data). Chi-squared tests were performed to compare multiple constituent ratios; p ≤ 0.05 was considered statistically significant.This study was approved by the Human Research Ethics Committee of the Affiliated Hospital of Jining Medical University. All patients were approached in accordance with approved ethical guidelines. They agreed to participate in this study and signed informed consent forms. We state that all methods in the study were performed in accordance with the relevant guidelines and regulations developed by the aforementioned ethics committee.2DM), 23 (10.95%) with central nervous system diseases , 20 (9.52%) with chronic kidney failure, 12 (5.71%)with chronic respiratory disease, 6 (2.86%) with viral hepatitis, and 10 (4.77%)with connective tissue disease with oral hormone and immunosuppressive agents were found.210 of the 463 patients had major underlying diseases. Among them, 63 (30%) with solid tumors, 6 (2.86%) with hematological malignancies, 70 (33.33%) with type 2 diabetes mellitus , 173 for S. constellatus (37.37%), and 36 for S. intermedius (7.77%). Clinical diagnoses suggested infections in different body sites, which could be categorized as follows: cranial infection (5), oral and maxillofacial infection (43), ear-nose-throat (ENT) and cervical infection (61), chest infection (78), abdominal infection (134), pelvic infection (13), perianal infection (48), extremity and trunk (skin and soft tissue) infection (55), and occult infection (26). The abdomen was the most common site of infection, followed by the chest, ENT and neck. There were statistically significant differences regarding the constituent ratios of the SAG organisms  in different body sites of infection. By comparing the constituent ratios, it was noted that S. constellatus was more likely to cause infection of the thorax than of other body sites; S. anginosus had a closer association with cranial infection than infections of other body sites; S. intermedius infection was mostly found in the brain  was more common in patients aged between 35 and 54 than in other age groups; S. anginosus infection (68.75%) had a higher prevalence in those between the ages of 18 and 34 years old. For patients at the age of 65 and above, S. intermedius infection (11.65%) was a greater threat . According to the age structure, the three age groups were further divided into children (0–17 years old), young and middle-aged adults I (18–34 years old), II (35–54 years old), III (55–64 years old), and senior adults (≥65 years old). In terms of patient age and onset of infection, patients aged 35–54 were exposed to the highest risk of infection (30.24%), followed by those aged 55–64 (23.76%). Different age groups also exhibited statistically significant differences in constituent ratios of the pathogenic organisms. According to the constituent ratios, we found that the constituent ratios of the three pathogenic organisms were different in the various age groups. In terms of organ involvement, most cases were associated with infection of the appendix , followed by pulmonary infection 76, 16.41%). Differences with respect to patient age and the constituent ratios of associated organs of infection showed statistical significance. From the constituent ratios, it was found that infection of the appendix (27.18%) mostly occurred in patients at the age of 18 and below. For the group between 18 and 34 years of age, a higher proportion of anal infection (37.21%) was observed in comparison to that in other age groups. Oral and maxillofacial infection (53.85%) was the most commonly seen condition in those aged between 35 and 54. The prevalence of pulmonary infection was higher in patients aged 55 and above  than in other age groups (Table 1%. DiffeWhite blood cell (WBC) count, CRP level, and serum albumin level were measured. Elevated WBC count and CRP level were observed in most patients, and no significant differences were observed between the 3 groups. The serum albumin level dropped to varying degrees, and the albumin level was significantly different between the elderly group and the other groups (P < 0.01) Table .Table 5TBased on drug sensitivity test results, diagnosed patients were given anti-infection treatment and invasive treatment , nutritional support, or appropriate treatment for underlying diseases. In our population, 269 were clinically cured, 184 reported satisfactory improvement, and 10 died.1. However, relevant data are not available on China’s part. To date, only limited clinical studies have presented detailed analyses of SAG7. However, these studies have investigated SAG organisms in children and adults separately. We retrospectively studied the clinical data of patients of all age groups infected with this pathogen to analyze the clinical characteristics of diseases due to SAG.The anginosus group streptococci were first described by Guthof in 1956 after being isolated from dental abscesses. Since then, there have been reports on varied types of abscesses and relatively small studies on this subject. The incidence rate has reportedly increased continuously in the past two decades7. By comparing the numbers of patients in all age groups, it was noted that adult patients at the age of 35–54 accounted for the largest proportion of our study population, which agrees with a previously reported study6. There were statistically significant differences regarding the constituent ratios of the three pathogenic organisms in the age groups. Based on the constituent ratios, the three pathogenic organisms showed an unequal distribution in every age group. S. constellatus had a higher proportion in patients aged between 35 and 54 than in other age groups; similarly, S. anginosus had the highest proportion in the group that was 18-34 years old compared to other age groups. The proportion of S. intermedius in patients aged 65 and above was higher than that in the other age groups.From the patients aged 6 months to 103 years in the present study, it was found that SAG bacterial infections could occur in individuals despite their ages and show a clear male predominance. The male-to-female ratios in the different age groups had no significant differences, which is similar to the results reported by several previously published studies11. Malignancy and diabetes were predominant among the underlying conditions, which is similar to previous studies10. However, the similar proportions of malignancy and diabetes might relate to the distribution of the underlying conditions. Among the patients with diabetes, 11 had diabetic foot disease, and 7 had subcutaneous abscesses, subcutaneous wounds, and nonhealing decubiti. Of the 13 patients with malignant tumors in their heads and necks, 9 had postlaryngectomy stomas, 2 had tongue cancer resection, 1 had hypopharynx cancer resection, and 11 received radiotherapy. All these factors seem to be closely associated with immunodeficiency, direct damage to the local mucous membrane, and disruption of the microbial balance in the oral cavity following radiotherapy13. In addition, the infection cases in this study mostly occurred after 2016, which is probably associated with the use of hormonal agents and immunosuppressants, active interventions, sample harvesting, and improvements in bacterial cultivation.In this study, 93.30% of the 463 patients had community-acquired infections; 210 (45.36%) had serious underlying conditions, including chest surgery, malignancy, nervous system disease, drinking problems, and diabetes, which were believed to make these patients susceptible to SAGS. anginosus, which agrees with previously published studies15. However, the constituent ratios of the pathogenic organisms in different body sites exhibited statistically significant differences. S. constellatus was more likely to produce chest infection; S. anginosus was closely associated with perianal abscess; and S. intermedius was a major cause of cranial infection. However, further evidence is needed to support the point of view that these species are strongly associated with infections of different body sites6. Further analysis of the infected organs revealed that most cases were associated with purulent appendicitis, followed by pulmonary infection. The constituent ratios of organ involvement also differed among the age groups. In the patients under 18, most cases involved appendicitis; for those between 18 and 34, anal abscess was the most common infection; and oral and maxillofacial infection was found in most patients aged 35 to 54. The prevalence of pulmonary infection was higher in patients aged 55 and above  than in other age groups.SAG bacterial infections often induce pyogenic infections such as superficial or deep soft-tissue infections and involve multiple organs. In this study, common superficial soft-tissue infections included infections of wounds, operative incisions, and pressure sores; deep soft-tissue infections included oral and maxillofacial infections, cellulitis, and necrotizing fasciitis. Chest infections could have produced pulmonary abscesses, pneumonia, pleural effusions, pyopneumothorax, and pleural fistulas. The conditions of patients with infection of the appendix progressed rapidly to abscesses and perforation and possibly resulted in peritonitis; perianal abscesses largely led to complex conditions. In these cases, the abdomen and chest were mainly involved, and most infections were caused by Laboratory test results showed that in these patients, the white blood cell count increased, the CRP level generally increased, and the serum albumin level dropped to varying degrees. Although such parameters have not been discussed in the preceding studies, they may provide us with a new direction from which to look closely at relevant cases. Comparing the white blood cell counts and the CRP levels in different age groups, there was no significant difference. However, senior adults had a lower level of albumin, and the difference showed statistical significance.Although SAG is not the most common cause of appendicitis, perianal abscess, parapneumonic effusion, and tonsillar abscess, it may complicate a patient’s condition with abscesses and lead to interventions that include invasive procedures such as surgery, puncture, and drainage. The main treatment options for these 463 patients included anti-infection treatment, invasive procedures and symptomatic supportive care. A total of 121 patients were given combined anti-infection treatment, and 341 were administered anti-infection monotherapy . Most patients had a favorable prognosis. Ten patients died, with a total mortality of 2.16%.This study has a few limitations. First, this was a retrospective study limited by its non-multicenter design and relatively small sample size, and thus, the results might not be generalizable to other populations. Second, despite the observation of clinical practices, this study adopted sampling criteria and body-site categories that differed from those in previous studies.As an opportunistic pathogen, SAG was found to produce pyogenic infections in patients of all ages and exhibited a clear male predominance in all age groups where there was no significant difference regarding the male-to-female ratio. The three pathogenic organisms showed differences with respect to patient age and infections of body sites. These patients required puncture drainage, surgical debridement and anti-infection treatment, and most patients had a favorable outcome.


Individual cells and epithelia control the chemical exchange with the surrounding environment by the fine-tuned expression, localization, and function of an array of transmembrane proteins that dictate the selective permeability of the lipid bilayer to small molecules, as actual gatekeepers to the interface with the extracellular space. Among the variety of channels, transporters, and pumps that localize to cell membrane, organic cation transporters (OCTs) are considered to be extremely relevant in the transport across the plasma membrane of the majority of the endogenous substances and drugs that are positively charged near or at physiological pH. In humans, the following six organic cation transporters have been characterized in regards to their respective substrates, all belonging to the solute carrier 22 (SLC22) family: the organic cation transporters 1, 2, and 3 (OCT1–3); the organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2); and the organic cation transporter 6 (OCT6). OCTs are highly expressed on the plasma membrane of polarized epithelia, thus, playing a key role in intestinal absorption and renal reabsorption of nutrients , in the elimination of waste products , and in the kinetic profile and therapeutic index of several drugs . As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article critically presents the physio-pathological, pharmacological, and toxicological roles of OCTs in the tissues in which they are primarily expressed. SLC22A1, -2, and -3. The organic cation/carnitine transporter novel 1 and 2 (OCTN1 and N2) and the organic cation transporter 6 (OCT6) are encoded by the SLC22A4, -5, and -16, respectively -N-methylpyridinium (ASP+). In most cases, OCTs are Na+-independent electrogenic transporters, whose activity is driven by the membrane potential across the plasma membrane. Thus, according to the electrochemical gradient of the substrate, OCTs can act as either influx or efflux systems. An exception is represented by OCTN2, which displays a bifunctional mode of transport. OCTN2 transports carnitine and its precursor γ-butyrobetaine in a Na+-dependent manner, and other organic cations in a Na+-independent manner [OCTs are known as polyspecific transporters because they recognize and transport a broad range of molecules, typically positively charged or zwitterions at physiological pH, such as the organic amines choline and carnitine, the neurotransmitters dopamine and serotonin, the microbiota products trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and the vitamin B1 thiamine ,16. OCTst manner . OCTs art manner . OCT1 ant manner . Likewist manner ,22,23.Expression and localization studies in different species have revealed that OCT1, OCT2, and OCT6 displayed relatively narrow patterns of expression limited to individual organs or tissues. In humans, OCT1 is primarily expressed on the basolateral membrane of enterocytes and hepatocytes (intestines and liver) ,25, and As part of the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations, this article provides a critical overview of the physiological, pharmacological, and toxicological impact and function of organic cation transporters in the key organ systems in which they are expressed. Sources for this review were obtained through extensive literature searches of publications browsing PubMed. Only papers published in the English language were considered.SLC22A1 gene is under the control of the hepatic nuclear transcription factor HNF-4a. When it binds to the promoter region of the SLC22A1 gene, HNF-4a activates the transcription of the OCT1 mRNA. The HNF-4a-mediated transcriptional activation of the SLC22A1 gene is inhibited by the bile acid chenodeoxycholic acid, which is the most potent endogenous ligand of the nuclear receptor farnesoid X receptor (FXR) [++/calmodulin [There is a good deal of evidence that OCT1 and OCT3 are expressed in rodent, as well as human liver, whereas OCTN1 and OCTN2 may be expressed in rodent but not in the human hepatocytes. OCT1 represents the most studied hepatic OCT . In thisor (FXR) . Indeed,or (FXR) ,56,57,58or (FXR) ,60,61. Almodulin . Recentllmodulin .-/- mouse, the transgenic liver appears to become prone to steatosis, indicating a role of OCT1 in hepatic lipid and energy metabolism [SLC22A1 gene encoding for OCT1. In the SLC22A1 gene, many nonsynonymous single nucleotide polymorphisms (SNPs) have been identified, some affecting expression or transport activity and others altering substrate selectivity [SLC22A1 gene have been associated with altered pharmacokinetics and pharmacodynamics of several drugs including opioids, the β2 agonist fenoterol, and metformin [Thus far, OCT1 has been primarily characterized from a pharmacological perspective, and its physiological role has only been partially defined. Recently, it has been shown that the total Oct1 knockout mouse, viable with no apparent deficiencies or phenotype, displayed an increased ratio of AMP to ATP, which activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride levels in the liver . This phtabolism . The phaectivity . Geneticetformin ,70,71,72etformin ,69.4) treatment was significantly higher in Oct3-/- than wild-type mice, because of an overproduction of TGFβ by stellate cells [-/- mice. Similar to OCT1, the hepatic expression of OCT3 is significantly affected by cholestasis in both humans and rodents; however, the mechanism of transcription repression might be different [OCT3 colocalizes with OCT1 in the sinusoidal hepatocyte membrane . The rolte cells . As adrete cells ,76, it iifferent .SLC22A5 gene, is a high-affinity, Na+-dependent, electrogenic carnitine carrier [+-dependent manner, γ-butyrobetaine, the carnitine precursor (Km~13 µM) [m of ~5 µM, consistent with a high-affinity system [OCTN2, encoded by the  carrier . Carniti carrier . About 2 carrier . Carniti carrier . An impo carrier . Shortly carrier . Notablym~13 µM) . The roly system ,53. Carny system ,85,86,87y system  has beeny system . Taken tMouse Octn1 was found to be expressed in non-parenchymal mouse liver cells, with reports showing functional expression in stellate cells. Upregulation of Octn1 and activation of stellate cells, after treatment with the liver toxin dimethylnitrosamine, were seen to lead to increased liver levels of the natural, nutrient-derived, OCTN1 substrate, antioxidant ergothioneine, which resulted in protection from inflammation, oxidative stress, and more severe liver fibrosis . Althoug-/- are normal, suggesting that the expression of Oct1 alone is sufficient to sustain normal renal function. Because of this functional redundancy, our understanding of the potential role of OCT2 primarily relies on studies that employ mice lacking both Oct1 and Oct2 (Oct1/2-/-), which display an impaired tubular secretion of organic cations [OCT2 and OCTN2, and to a lesser extent, OCT3 and OCTN1 are expressed in the human kidney. OCT2 and OCT3 are considered to be expressed on the basolateral side of proximal tubule cells ,93, whil cations .OCT2 has been well characterized for its relevance in creatinine tubular secretion, although creatinine has been shown to be a substrate of all the above listed transporters, at least in vitro . Creatin-/- mice are partially resistant to cisplatin-induced nephrotoxicity [Actual kidney injury mediated by substrates of OCT2 most notably includes anticancer platinum agents, of which cisplatin is the most studied . Cisplattoxicity ,118,119.toxicity ,117 and toxicity ,121, wittoxicity . Anothertoxicity . In rodetoxicity , and thetoxicity  was depetoxicity , and thetoxicity .Genetic polymorphisms in OCT2 and OCTN1 have been identified to affect metformin renal excretion, leading to significantly increased peak concentrations and larger serum areas under the curve. On the one hand, patients carrying the OCT2 p.Ala270Ser (rs316019) variant or the OCTN1 p.Thr306Ile (rs272893) may require, similar to those with renal impairment, metformin-dosing reductions . Therefo+-independent manner, whereas cephaloridine interaction with OCTN2 is Na+-dependent [OCTN2 is physiologically most relevant for the reabsorption of carnitine, where loss or non-functionality of this transporter leads to primary systemic carnitine deficiency through carnitine wasting by renal excretion ,130,131.ependent ,139,140.OCT1, OCT3, OCTN1, and OCTN2 are expressed in the intestines, where OCT1 and -3 are located at the basolateral membrane of enterocytes and OCTN1 and -2 at the brush border membrane ,141. PhyTo briefly summarize the above sections, framed in the context of what is known in terms of pathophysiology and pharmacological relevance, OCT1 plays more significant roles in the liver, while OCT2 and OCTN2 have very important functions in the kidney. The following sections address additional tissues, which are selected based on current research trends. We discuss even less well known or described functions of OCTs in additional (human) tissues, where much of the existing work stems from animal studies. We critically assess current themes in animal studies on OCTs and detail the limited studies on humans for each respective tissue. For a comprehensive summary on the state of knowledge about OCTs in human and rodent models based on tissue expression, we suggest consulting the extensive review by H. Koepsell .A rather quickly growing number of studies have addressed Oct2 and Oct3 in regard to their roles in the central nervous system and blood-brain barrier in rodents ,163,164.As mentioned in the kidney section of this review, aminoglycoside antibiotics and anticancer platinum agents, most notably gentamicin and cisplatin, respectively, are known for their nephrotoxicity and also to induce irreversible hearing loss. While it cannot be disputed that these agents lead to ototoxicity, since evidence for the relevance of OCT2 in these processes has been presented in both rodents and gineau pigs, literature on humans on this topic is not abundant . AdditioInterestingly, in humans, mutations in OCTN1 that seemingly affect the correct trafficking of the protein to the apical membrane of stria vascularis endothelial cells, were identified as causative in the screening of consanguineous Tunisian families with autosomal recessive non-syndromic hearing loss. Although the reasons behind the hearing loss were not clear, it was postulated in the study that altered energy status via reduced carnitine uptake in the stria vascularis in such patients may have led to oxidative stress and consequent cell damage resulting in profound hearing loss . It is i-/- or Ldlr-/- genotype, also with or without high-fat diet [-/- mice, to further study the role of TMAO, as well as the effects of dietary choline and carnitine handling, on cardiovascular health. It seems clear that more studies in both mice and man are required to fully understand the processes involved in the development of atherosclerosis and the contribution of choline, carnitine, and TMAO transport.Trimethylamine N-oxide (TMAO), which is produced from trimethylamine (TMA) stemming from OCT substrates choline and carnitine from protein and lipid nutrients converted by microbiota in the gut, is associated with cardiovascular disease, and thus considered to be a potential novel pro-atherosclerotic molecule ,174. In fat diet ,189. It Response to metformin treatment underlies intestinal (OCTN1 and OCT1), hepatic (OCT1), and renal (OCT2) handling, and also transports into peripheral tissues on the level of the effect on metabolism in both skeletal muscle and adipose tissue. Indeed, the more ubiquitously expressed OCT3 has been implicated, both in mice and man, in the metabolic response to metformin in muscle tissue ,190. In OCT6 was cloned in 2002 and directly identified as a carnitine transporter specifically expressed in the human testis in Sertoli cells and epididymal epithelium ,27, and The recognition of OCTs as low affinity transporters of frequently prescribed drugs, such as several antibiotics and metformin, the flourishing of pharmacogenetics, and the development of rigorous drug–drug interaction studies for marketing approval have decisively contributed to elucidating the impact of the organ-specific and interorgan functions of these transporters and, in conjunction, their high pharmacological impact. However, with the exception of OCTN2, the understanding of the physio-pathological roles played by OCTs in humans is not fully understood. Comprehension of the roles played by OCTs in physiology is hampered by the partially different substrate specificity and tissue expression between rodents and humans and the lack of obvious phenotypes associated with loss of, or gain of, function of any of these transporters. In general, the phenotype of an organism is not the mere product of its genetic constitution but rather the manifestation of the interaction of the genetic background with various environmental influences. In the study of the physio-pathological role of any gene, the best-case scenario is that the phenotype of a genetic variant is apparent under standard environmental conditions. This is the case of the jvs animals lacking Octn2. Alternatively, the phenotype develops only in specific circumstances that the investigator must understand and optimize. For instance, the potential role of OCTs in the elimination of toxins whose chronic exposure is associated with several aging-related diseases such as Parkinson’s and cardiovascular disease, might suggest that the phenotype of Oct-deficient animals does not manifest just because the animals are not examined in the proper environment, or under the correct challenge or insult, or in the right moment of their life. This seems to be the case for OCT1 in steatosis onset  and for


Background and objectives: Exposure to ionizing radiation (IR) has increased immensely over the past years, owing to diagnostic and therapeutic reasons. However, certain radiosensitive individuals show toxic enhanced reaction to IR, and it is necessary to specifically protect them from unwanted exposure. Although predicting radiosensitivity is the way forward in the field of personalised medicine, there is limited information on the potential biomarkers. The aim of this systematic review is to identify evidence from a range of literature in order to present the status quo of our knowledge of IR-induced changes in protein expression in normal tissues, which can be correlated to radiosensitivity. Methods: Studies were searched in NCBI Pubmed and in ISI Web of Science databases and field experts were consulted for relevant studies. Primary peer-reviewed studies in English language within the time-frame of 2011 to 2020 were considered. Human non-tumour tissues and human-derived non-tumour model systems that have been exposed to IR were considered if they reported changes in protein levels, which could be correlated to radiosensitivity. At least two reviewers screened the titles, keywords, and abstracts of the studies against the eligibility criteria at the first phase and full texts of potential studies at the second phase. Similarly, at least two reviewers manually extracted the data and accessed the risk of bias  for the included studies. Finally, the data were synthesised narratively in accordance to synthesis without meta analyses (SWiM) method. Results: In total, 28 studies were included in this review. Most of the records (16) demonstrated increased residual DNA damage in radiosensitive individuals compared to normo-sensitive individuals based on γH2AX and TP53BP1. Overall, 15 studies included proteins other than DNA repair foci, of which five proteins were selected, Vascular endothelial growth factor (VEGF), Caspase 3, p16INK4A , Interleukin-6, and Interleukin-1β, that were connected to radiosensitivity in normal tissue and were reported at least in two independent studies. Conclusions and implication of key findings: A majority of studies used repair foci as a tool to predict radiosensitivity. However, its correlation to outcome parameters such as repair deficient cell lines and patients, as well as an association to moderate and severe clinical radiation reactions, still remain contradictory. When IR-induced proteins reported in at least two studies were considered, a protein network was discovered, which provides a direction for further studies to elucidate the mechanisms of radiosensitivity. Although the identification of only a few of the commonly reported proteins might raise a concern, this could be because (i) our eligibility criteria were strict and (ii) radiosensitivity is influenced by multiple factors. Registration: PROSPERO (CRD42020220064). IARC Global Cancer Observatory reports more than 18 million new cases of cancer in 2018 (arc.fr/) . HoweverRadiosensitivity is a measure for the reactions of cells, tissues, or individuals to ionizing radiation (IR). Subjects with increased reactions are described as radiosensitive, when compared to a majority of other “normal” responding individuals ,5,6. TheIn a small subset of patients the severe reactions can be ascribed to known radiation hypersensitivity syndromes, such as Ataxia–Telangiectasia (A–T), Fanconi anaemia (FA) or Nijmegen Breakage Syndrome (NBS) ,12,13. ASome further genetic associations were suggested by candidate gene approaches as well as by genome-wide association studies in radiotherapy patients. However, only a small proportion of radiosensitive individuals could be identified . Additiostatus quo of our knowledge of IR-induced changes in protein expression in normal tissue that can be correlated to radiosensitivity. We focus on proteins and protein modifications, as, due to posttranscriptional regulatory processes, the alterations in protein levels may describe the actual cell state, inclusive stress responses, more precisely than transcriptome changes [Therefore, the purpose of this paper is to present the  changes . The futThe aim of this systematic review (SR) is to investigate the IR-induced changes, both in vivo and in vitro, in the human proteome that can be correlated to radiosensitivity. The review protocol  was regiin vitro cultures. Changes in expression of proteins after the exposure, which were associated with radiosensitivity, were defined as the outcome of this review. Only primary peer-reviewed published studies in English language were considered. As a study on ionizing radiation protein biomarkers for epidemiological studies was published in 2012 [Studies that comply with elements of Population, Exposure, Comparators, and Outcome (PECO) were eligible for this SR. The full description of PECO parameters was provided in the protocol . In shor in 2012 , studiesStudies were identified using electronic databases and with consultations of field experts. The authors of the studies were not contacted for further studies or questions regarding the paper. https://pubmed.ncbi.nlm.nih.gov/) [https://www.webofknowledge.com/) [NCBI PubMed (ih.gov/)  and ISI ge.com/)  were useA two-phase screening was performed by authors Dietz and Subedi in parallel. In phase I screening, title, abstract, and key words of all of the studies were cross-checked manually with the inclusion and exclusion criteria provided in the protocol . The artThe data collection was performed in Google Sheets by Subedi, Dietz, and Moertl, with one reviewer entering the data and the other person confirming it. The data were finally processed with Microsoft Excel. The form for data extraction is submitted in We extracted information about: the name of the protein; the fold change ratio after IR; bio fluids or cell lines being investigated; the method used to determine the fold change; the quality and quantity of IR; the characteristics of the donor(s) ; eligibility criteria of the patients; the method used to quantify radiosensitivity ; the replicates performed for the experiment and the statistics to accompany the fold changes; the outcome of the change in protein expression; post-translational modification; and conflict of interest. The findings were summarised and the heterogeneity of the data was compared visually in form of tables. This SR was performed to investigate the changes in protein expression in normal tissue after exposure to ionizing radiation. Therefore, the in vivo and in vitro studies were grouped together and no differences were made between the different radiation qualities. The doses are provided in Gray (Gy) and the dose-rates are provided in (Gy/min).The increase or decrease in protein expression after IR ) was used as a measure of effect size of the exposure. The fold changes were not calculated in this manuscript but taken from the respective studies.p-value, or in certain cases by an adjusted p-value resulting from multiple testing, was used as synthesis method for each outcome. For each comparison, the null hypothesis represented by Studies which contained commonly deregulated proteins were pooled together. Studies were given an initial confidence rating of high, moderate, low, or very low based on the presence of features . Following the OHAT method, which is based on Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group guidelines, the studies were up- or downgraded. The factors increasing confidence were magnitude of the effect, dose response, residual confounding, and consistency, whereas the factors decreasing confidence were risk of bias, unexplained inconsistency, indirectness, and imprecision. After database searching and inclusion of manual sources, 2733 studies were identified. The records were screened for title, abstract, and key words, and 100 articles were selected for a full-text review. Finally, 28 articles were included for this SR . In the The 16 studies that used repair foci to determine individual differences in radiosensitivity included 10 cohort studies , Interleukin-6, and Interleukin-1B.In total, the 28 included studies identified 76 proteins, which were correlated with normal tissue radiosensitivity. The results were prioritised so that the proteins identified in more than one study, regardless of the direction of regulation, along with their role in radiation response, were described further. Besides changes in repair foci (γH2AX and TP53BP1 quantities), the proteins were identified in more than one study are Vascular endothelial growth factor (VEGF), Caspase 3, p16H2AX, a variant of the histone protein H2A, is located in the nucleus and its functions include chromatin organisation and DNA damage response. In case of DNA double strand break damage, its phosphorylation by PI3 kinases ATM, ATR, and DNAPKcs signals the damaged site, and recruits downstream DNA repair proteins ,53,54,55In all studies, irradiation was performed with gamma or X-ray radiation at a high dose rate and doses from 0.5 2.0 Gy. Studies were performed in different cell lines  harbouring DNA repair defects, or in primary cells  from cancer patients. From all parameters investigated, such as basal foci level, radiation induced foci and residual foci at later repair time points, elevated levels of residual γH2AX or TP53BP1 foci appear to be robust to identify radiosensitive cells or individuals. in vitro challenging assay and investigating signal development and disappearance [DNA repair deficient individuals demonstrate delayed development of the initial DNA damage or delayed DNA repair, resulting in an increased level of residual damage after 24 hours ,36. Therpearance ,56,58. Ppearance ,36,59, Lpearance ,36, and pearance ,28,29,32pearance ,60. Our pearance ,61.Can we be confident in the exposure assessment?Can we be confident in the outcome assessment?Did the study design or analyses account for important confounding and modifying variables?An aim of our SR is to discover new feasible markers on protein levels that are associated with radiosensitivity, besides repair foci proteins. To provide a rich reflection of evidence for the reader, we included both significantly deregulated and not deregulated proteins in None of the studies were categorised in T1, one study  was categorised into T3, and the rest were categorised as T2. The RoB questions are suited to cohort and human clinical trials compared to model systems. Concealment, randomisation, and blinding in most studies on model systems are not performed because (i) it is usually a single person that performs the studies and (ii) it is not a common practice to conceal the study groups from the researcher. Therefore, most studies received a ‘probably high risk of bias’ assessment in randomisation, concealment, and blinding domains. Although randomisation is performed during the accessing of outcomes, for example, when performing mass spectrometric analyses or measuring γH2AX quantities on coded slides, more often than not, it is not reported to ensure brevity during publication. Based on the results from this SR, we can recommend that studies on model systems should take care of randomisation, concealing of study groups, blinding the accessors, and, most important, reporting them. The proteins that were reported in at least two studies  are explVEGF induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces permeabilization of blood vessels ,64. Furt137Cs, dose rate 2.7 Gy/min) in CCR6+Th17 T cells, which are highly sensitive to IR-induced senescence. This may contribute to IR-induced normal tissue damage and might facilitate tumour recurrence and metastasis after radiotherapy [137Cs) [Nguyen et al. reported an increase in VEGF secretion 48 h after exposure to 2 Gy IR (otherapy . Braicu otherapy . Fekete  [137Cs) . VEGF is a key mediator of neovascularisation and is highly expressed in cancer cells and tumour-associated stromal cells . In a meThe effect of radiation exposure on VEGF seems to be cell type dependent. However, first in vitro and in vivo studies suggest its importance for normal tissue radiosensitivity. Therefore, it is a promising candidate marker to study radiosensitivity in future projects. Caspase 3 is involved in the activation cascade of several caspases responsible for apoptosis by proteolytically cleaving poly(ADP-ribose) polymerase (PARP). Furthermore it cleaves and activates Caspase-6, -7, and -9 .137Cs irradiated T cells  and observed a radiation induced increase in Caspase 3 concentration, where Cao et al. reported a higher increase in radiosensitive CD4+CD25+ Treg cells compared to normo-sensitive CD4+CD25- T cells after overnight incubation post 0.94, 1.875, and 7.5 Gy. Nguyen et al. described a greater Caspase 3 activation (48 h post 2 Gy) in CCR6negTh cells compared to CCR6+Th17 that are rather prone to IR-induced senescence than to apoptosis. When lymphocytes from healthy donors were irradiated with 1, 2, or 4 Gy (60Co), a dose-dependent increase in active Caspase 3 was observed that included high intra-individual variability [Both, Cao et al.  and Nguyiability . Althougp16 acts as a negative regulator of normal cell proliferation by inhibiting CDK 4 and CDK 6 interaction with cyclin D and the phosphorylation of retinoblastoma protein, prohibiting progression from G1 phase to S phase ,78. p16 60Co γ irradiation (dose rate 2.7 Gy/min) [137Cs with a dose rate of 2.7 Gy/min IR and led to IR-induced senescence [Miyake et al. observed that an increase in p16 expression in keratinocytes , was characterised as radio-resistant but not in fibroblasts or induced pluripotent stem cells (iPSCs) 72 h after 2 Gy  Gy/min) . Nguyen nescence . In contnescence ,81,82. SThe pleiotropic cytokine IL-6 comprises a wide variety of biological functions including immunity, tissue regeneration, and metabolism . It is a137 Cs, dose rate 4.8 Gy/min) in radiosensitive Treg cells, but not in T cells showing a normal sensitivity [137 Cs [Cao et al. reported that IL-6 is significantly downregulated in response to 0.94 and 1.87 Gy  irradiation according to Nguyen et al. [Like for IL-6, Cao et al found a significantly downregulated IL-1β in response to 0.94 and 1.87 Gy  T cells . Secretin et al. . Chen etn et al. . RegardiThe studies that contained the previous markers were further evaluated based on a Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach . Each stSignificant interactions for aforementioned proteins, TP53BP1, and γH2AX , were idFirst of all, it is important to understand the proteomic landscape of normal tissues. Different tissues and cell types harbour divergent baseline protein expression . Most ofSecond, radiosensitivity is a complex issue as many risk factors modify the radiation reaction, thus determining each predictor’s overall impact is difficult to characterise. Some of the factors that influence radiosensitivity and complicate the discovery of a ubiquitous applicable biomarker are specified in this section. There are several known hereditary hyper-radiosensitive disorders arising from rare mutations in DNA repair genes of large effect. All belong to XCIND syndromes, named after distinct hypersensitivity to ionizing radiation (X-ray), cancer susceptibility, immunodeficiency, neurological abnormality, and double-strand DNA breakage. Examples of such syndromes are Ataxia telangiectasia, Fanconi anemia, Ligase IV syndrome, Radiosensitive severe combined immunodeficiency disease (RS-SCID), Radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome, or ataxia telangiectasia and Rad3-related protein (ATR)-Seckel syndrome ,97,98,99Age and gender are crucial factors influencing individual differences in radiosensitivity. Children aged 0–5 years are expected to be the most sensitive group concerning radiation-induced leukaemia, as well as skin, breast, thyroid, and brain cancer for both high and low dose radiation exposures ,104,105.The anatomical structure , as well as breathing rates, and individual metabolism of exposed individuals alter radiation doses received by organs and tissues, which leads to inter-individual variations ,115,116.The fact that there is a clear evidence that not all individuals share the same radiation-induced risk of adverse health outcomes is also backed by the reports from the advisory group on ionizing radiation (UK)  and InteThe need to discover biomarkers for disease risk or susceptibility of radiation related risks for individuals or population subgroups is vital and also stressed by MELODI platform . Not onlThe GRADE tool to up- or downgrade studies was not performed on all studies but only on studies that included proteins, other than repair foci, reported in at least two studies.


Wasabia japonica rhizome. Several scientific studies have shown that 6-MITC possesses interesting antimicrobial, anti-inflammatory, antiplatelet and antioxidant properties which therefore suggested us it could have an interesting chemopreventive potential. In a recent publication, we demonstrated, in two different leukemia cell lines, its ability to modulate several mechanisms supporting its antitumor activity. For this reason, we thought useful to continue the research, by investigating the potential antimutagenic activity of 6-MITC and thus better define its profile as a possible chemopreventive agent. 6-MITC antimutagenic effect against two known mutagenic agents: the clastogen Mitomycin C (MMC) and the aneuplodogen Vinblastine (VINB), was analyzed, in terms of micronuclei frequency decrease, after short- and long- time treatment on TK6 human cells, using a new automated protocol of the “In Vitro Mammalian Cell Micronucleous Test” by flow cytometry. The results showed a different behavior of the isothiocyante. In particular, 6-MITC was unable to counteract the MMC genotoxicity, but when it was associated with VINB a statistically significant decrease in the micronuclei frequency was registered. Overall, the results obtained suggest a potential antimutagenic activity of 6-MITC, in particular against the aneuploidogen agents. This ability, to inhibit or counteract the mutations at the cellular level has a great therapeutic value and it represents a mechanism through a chemopreventive agent can express its activity.6-(methylsulfinyl) hexyl isothiocyanate (6-MITC), is the main bioactive compound present in  Wasabia japonica, a plant belonging to the Brassicaceae family, also called “Japanese radish”. A green paste with a particularly spicy taste is made from the rhizome of this plant, that is used in traditional Japanese cuisine and commonly known as Wasabi (6-(Methylsulfinyl) hexil isothiocyanate (6-MITC) is the main bioactive compound present in s Wasabi .Isothiocyanates have long been considered by the scientific community, for the numerous pharmacological properties demonstrated . SeveralThese results suggested us a potential interest 6-MITC as a chemopreventive agent. In a recent publication, we therefore demonstrated, in two different leukemia cell lines (Jurkat and HL-60), its ability to modulate several mechanisms supporting its antitumor activity, such as the cyotodifferentiation and apoptosis induction or the cellular proliferation inhibition .Beside the ability to interact with cellular and molecular targets, crucial in the development of cancer, also the study and the identification of compounds capable of counteracting the genotoxicity, it is recognized of great interest in the field of chemoprevention . In facti.e the clastogen Mitomycin C (MMC) and the aneuploidogen Vinblastine (VINB). For this purpose, we decided to use a new automated protocol of the Micronucleous (MN) Test by flow cytometry (FCM)  .In Vitro Mammalian Cell Micronucleous Test”, . In this work, we select the “s Test”,  because s Test”, .Among several cell lines  validated by the OECD guideline no. 487 that can be used to assess the genotoxicity of a xenobiotic, we selected TK6 cells . Our choDimethyl sulfoxide (DMSO), Ethanol, Ethylenediaminetetraacetic acid (EDTA), Fetal Bovine Serum (FBS), L-Glutamine (L-GLU), Mitomycin C (MMC), Nonidet, Penicillin-Streptomycin solution (PS), Potassium Chloride, Potassium Dihydrogen Phosphate, Roswell Park Memorial Institute (RPMI) 1640 medium, Vinblastine (VINB),Water bpc grade, Sodium Chloride, Sodium Hydrogen Phosphate , Guava Nexin Reagent, Guava ViaCount Reagent , RNase A, SYTOX Green, 7-aminoactinomycinD (7-AAD) .6-MITC was purchased from Abcam, Cambridge, UK. The purity of ITC was >98%. The isothiocyanate was dissolved in DMSO up to 97.39mM stock solution and stored in the dark at −20°C. Increasing concentrations of 6-MITC from 0 to 64µM were tested. DMSO concentration was always in the range 0.05–1% in all the experimental conditions.2 in RPMI-1640 supplemented with 10% FBS, 1% L-GLU and 1% PS. To maintain exponential growth, the cultures were divided every third day in fresh medium and the cell density did not exceed the critical value of 9x105 cells/mL.TK6 human lymphoblast cells were purchased by Sigma-Aldrich  and were grown at 37°C and 5% CO5 of TK6 cells were treated with increasing concentrations of 6-MITC (0 to 64µM) and incubated for 3h followed by 23h of recovery in fresh medium, 26h total, corresponding to two replication cycles, in the presence or absence of MMC (400ng/mL) or VINB (25ng/mL) (co-treatment). We selected these concentrations on the basis of the literature  and incubated for 26h consecutive, corresponding to two replication cycles, in the presence or absence of MMC (200ng/mL) or VINB (6.25ng/mL) (co-treatment). We selected these concentrations on the basis of the literature .In order to detect cytotoxicity and cytostasis the percentage of viable cells was evaluated by the Guava ViaCount Assay. Briefly, cells were stained with Guava ViaCount Reagent  and analyzed by Guava ViaCount software.In particular, to assess the cytotoxicity, the results obtained in the sample treated with different concentrations of 6-MITC were normalized on those obtained in the control cultures.In parallel, the number of cells seeded at time 0 and that measured at the end of the treatment time, was used to check the correct replication in the control cultures and compare it to that measured in the treated cultures using the relative population doubling (RPD).The percentage of apoptotic cells was evaluated by the Guava Nexin Assay. Briefly, the percentage of live, apoptotic and necrotic cells was assessed using the Guava Nexin Reagent (containing 7-AAD and Annexin-V-PE) and analyzed by Guava Nexin software.The results obtained were expressed as apoptotic fold increase of treated cultures compared to control cultures and were used to select MNs test concentrations. In fact, it is necessary that the percentage of apoptotic cells measured in treated cultures is comparable to that present in the control cultures, in order to avoid possible false positives, due to the presence of apoptotic bodies.5 cells were collected and stained with 7-AAD and SYTOX Green. The discrimination between nuclei and MNs was performed on the basis of the different size analyzed by Forward Scatter (FSC), and the different intensity of green fluorescence. For each sample the MNs frequency was measured on 10,000 nuclei derived from viable and proliferating cells on the basis of different red fluorescence. The results were expressed as MNs frequency fold increase in treated cultures compared to that present in the control cultures , followed by Dunnett or Bonferroni as post-test. All the statistical analyses were performed using Prism Software 4.In order to select the concentrations to be used in the following experiments, we, first, evaluated the cytotoxic and cytostatic potential of 6-MITC after 3h treatment followed by 23h of recovery in complete medium at different concentrations . In fact, the OECD guideline no. 487 recommends proceeding to assess the genotoxicity of a xenobiotic, only if the highest concentration tested shows cytotoxicity and cytostasis at most equal to 55 ± 5% .At the same time, using RPD value, the cytostasis was checked. Similarly, to cytotoxicity, all concentrations tested, except the 6-MITC 64µM, respect the threshold , similarly after short- and long- term treatment.A co-treatment of 3h, followed by 23h of recovery in complete medium, was performed and, also in this case, the cytotoxicity, cytostasis and apoptosis were checked, before proceeding with the genotoxicity analysis. As show in An average apoptosis fold increase equal to three time in MMC+6-MITC associations treated cultures respect to the control cultures was observed  (vs 4.2 times)  (vs 4.7 times)  allowed us to identify only two publications. In particular, Kinae and collaborators demonstrated, using the Ames Test, the antimutagenic activity (in terms of gene mutations) of wasabi root, against the 2-amino-3,8-dimethylimidazoquinoxaline, a well-known mutagen/carcinogen compound present in broiled fish and meat (According to our knowledge, no study has addressed the antigenotoxicity of 6-MITC, the main bioactive compound present on and meat .More recently, the study conducted by Shimamura et al. documented, through Micronucleus Test and Alkaline Comet Assay, the inhibitory effect of Japanese horseradish, on the acrylamide formation and genotoxicity .W. japonica antimutagenic activity was attributable to the 6-MITC.These evidences suggest us to verify if the proven Despite Wasabi has long been used in traditional Japanese cuisine, it was initially checked the absence of 6-MITC mutagenicity. For this purpose, the non-cytotoxic and cytostatic doses, after short- and long- term treatment of TK6 cells, were defined. In fact, the OECD guideline no.487 recommends proceeding with the evaluation on genotoxicity, only if the treated population shows a viability and cell proliferation of at least 40% when compared to the control cultures .At the same time, the induction of apoptosis was analyzed, as cell death alternative mechanism and in order to exclude false positive results, due to the possible confounding between apoptotic bodies and MNs by FCM. Overall, based on the results obtained, the concentrations to be used for the evaluation of mutagenicity were selected and, as can be easily predictable, 6-MITC did not show any mutagenic activity both after 3 and 26h treatment.Subsequently, the study focused on the analysis of the isothiocyanate antimutagenic potential, against two known mutagenic agents: the clastogen MMC and the aneuploidogen VINB.2, H2O2, OH*, so the association with antioxidant molecules represent a possible approach to prevent DNA damage (MMC is characterized by a complex mechanism of action, being able to generate monoalkylation or dialkylation products, and to form covalent cross-linking, between the DNA complementary strands. This interaction prevents strands separation, inhibits DNA replication and causes its break . FurtherA damage . Since tA damage , it madeOn the contrary, the isothiocyanate has shown to counteract the mutagenic capacity of the aneuploidogen VINB, which acts at the level of cellular mitosis, by preventing tubulin polymerization and consequently, inhibiting the microtubules aggregation .The statistical analysis evidenced a significant decrease in the MNs frequency equal to about on half after the long treatment with 6 MITC 1µM concentration.Overall, our work suggests to impute to 6-MITC an antimutagenic capacity. Our findings, are preliminary, since they are obtained against only two mutagens, but allow to highlight the possible mechanism underlying this activity.In fact, from our data it seems that the isothiocyanate does not counteract the structural DNA damage, but rather the genomic DNA damage, highlighting the possibility that it acts on the mitotic spindle formation or at the chromosomal segregation time.Alternatively, the co-treatment could suggest a direct extracellular interaction between the isothiacyanate and the mutagenic agent.These hypothesis needs to be confirmed on a greater number of mutagens, but from the present research emerges an additional interesting biological potential of the 6-MITC. Indeed, the ability to inhibit or counteract the mutations at the cellular level has a great therapeutic value and it represents a less investigated mechanisms through which a chemopreventive agent can express its activity . In concThe datasets generated for this study are available on request to the corresponding author.PH and ML designed the project and supervised the experiments. VC performed the experiments and data analysis. VC writing—original draft preparation. VC, PH, and ML writing—review and editing. All authors contributed to the article and approved the submitted version.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.


Although the prognostic value of lncRNA small nucleolar RNA host gene 15 (SNHG15) expression in cancers has been evaluated in many studies, the results remain controversial. This meta-analysis aimed to clarify the role of SNHG15 in the prognosis of different cancer patients.  Eligible studies were selected from PubMed, PMC, EMBASE, Web of Science, and Cochrane Library according to the inclusion and exclusion criteria . The primary outcome was overall survival (OS) and recurrence-free survival (RFS). The secondary outcome was other clinicopathological parameters . The Cancer Genome Atlas (TCGA) dataset was used to verify the analysis results. P < 0.00001). Subgroup analysis showed that the high expression of SNHG15 was associated with a significantly poor OS of patients with digestive cancer , but not lung cancer . The RFS of patients with high expression of SNHG15 was shorter than that of patients with low expression of SNHG15 . In addition, high SNHG15 expression level was significantly correlated with later TNM stage , lymphatic metastasis , and distant metastasis . The TCGA verification results were consistent with those observed in our meta-analysis.  Eleven eligible studies were eventually included, involving 9 different types of cancer and 1,079 patients. The high expression of SNHG15 was indicative of a significantly poor OS of cancer patients (HR = 1.96, 95% CI = 1.55–2.47,  High expression of the long noncoding RNA SNHG15 in cancer tissue samples predicts an unfavorable prognosis for cancer patients. LncRNA SNHG15 can be used as an adverse prognostic biomarker for cancer patients. Malignant tumors are among the leading causes of morbidity and mortality for people of all ages worldwide , 2. DespLong noncoding RNAs (lncRNAs) are transcripts with a length of more than 200 nucleotides and are widely found in nucleus, cytoplasm, and exosome . They usSmall nucleolar RNA host gene 15 (SNHG15) is a newly discovered LncRNA located on chromosome 7p13 . SNHG15 Article search was performed in the databases, including PubMed, PMC, EMBASE, Web of Science, and Cochrane Library. The time limit for searching was from the construction of these databases to December 20, 2019. The terms used for the search were as follows:  AND . References of the literature included were also traced back.The inclusion criteria for research studies were as follows: (1) research conducted on any type of human cancer; (2) measuring the expression of SNHG15 in tumor tissue; (3) investigating the relationship between SNHG15 expression level and prognosis or clinicopathological characteristics; and (4) reporting the hazard ratio (HR) or odds ratio (OR) with 95% confidence interval (CI), or with sufficient data available for calculation.The exclusion criteria were as follows: (1) duplicate articles; (2) editorials, letters, expert opinions, case reports, and reviews.The primary outcome was overall survival (OS) and recurrence-free survival (RFS). The secondary outcome was other clinicopathological characteristics .Two researchers independently (Cheng Zhang and Yang Ke) extracted data from each original publication, and a third researcher (Xin Liu) resolved the differences. The extracted information included name of the first author, year of publication, country, type of cancer, sample size, the detection method for lncRNA, internal control, outcome index, and cutoff value. For studies reporting only Kaplan–Meier curves, Engauge Digitizer (version 12.1) software was used to extract survival data  and the http://gepia.cancer-pku.cn/) was used to analyze the differential expression of SNHG15 between cancer and normal tissues and the correlation of SNHG15 expression with OS of different cancer types.Gene Expression Profiling Interactive Analysis (GEPIA) database , the results were summarized using a random-effects model. Instead, fixed-effects models were used for analysis. Otherwise, a random-effects model was applied. Begg's funnel plot and Egger's test were used to assess publication bias. A sensitivity analysis was applied to assess the stability of the results. P ≤ 0.05 was considered statistically significant.Statistical analysis was performed using RevMan 5.3.3 software and Stata SE 15.1 software. HR for death or recurrence and 95% CI were calculated between the high and low SNHG15 expression group. Patients with HR  ≥ 1 indicated a poor prognosis. Furthermore, the correlation between SNHG15 expression and clinicopathological characteristics was evaluated using OR and 95% CI. Heterogeneity between the included studies was determined by the As shown in All the 11 studies included were from China, with 9 different types of cancer, viz., hepatocellular carcinoma (HCC) , gastricP=0.99, I2 = 0%), a fixed-effects model was chosen to estimate the combined HR and 95% CI. The results showed that the HR for death between the high SNHG15 expression group and the low SNHG15 expression group was 1.96 , we used a fixed-effects model to complete the synthesis of the data. The results of the subgroup analysis showed that the HR and 95% CI for death between the high SNHG15 expression group and the low SNHG15 expression group in digestive, respiratory, and other cancers were 1.91 , 1.83 , and 2.05  , 21, 27,r OS see .I2 = 0%, P=0.56), a fixed-effects model was used. The combined HR for RFS between the high SNHG15 expression group and the low SNHG15 expression group was 2.03 , which was consistent with our meta-analysis results. We also evaluated the correlation between SNHG15 expression and OS in digestive , respiratory (including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)) and in other  malignant tumors in TCGA. The result showed that patients with high SNHG15 expression levels had significantly poorer OS than those with low SNHG15 expression levels in digestive cancers , and stimulate the invasion and metastasis of the colon, breast, and renal cancer cells , 34. In Different cancers display distinct clinical characteristics and biological behaviors. To reduce the heterogeneity between studies, a subgroup study was performed based on pathological classification. The results suggested that the increase of SNHG15 expression in the digestive subgroup  and RCC, TC, BC, and OC subgroup was significantly associated with a shorter OS, which suggested that SNHG15 may be a reliable prognostic biomarker for cancers of the digestive, urinary, thyroid, and reproductive organs. However, there was no significant correlation detected between SNHG15 and NSCLC.Chemotherapy is a main treatment approach for various cancers. Previous studies have found that SNHG15 expression contributes to cisplatin resistance in BC  and temoOur study included a large number of samples, which confers a high statistical power. Therefore, the results of the present study are more stable and accurate than those of previous individual studies. However, some limitations should be noted in this meta-analysis. First, all studies were from China, so we used the TCGA database, which includes cancer cases from multiple regions including African American, American native, and Caucasian, to verify our meta-analysis results. Indeed, the bioinformatics results support our meta-analysis. A large sample size with multiple races is needed to confirm our findings in the future. Second, given the fact that individual patient data were not analyzed, certain data on the relationships between clinical parameters and SNHG15 could not be combined.In conclusion, this is the first meta-analysis reporting that LncRNA SNHG15 can be used as a prognostic biomarker for cancer patients, especially cancers in the digestive, urinary, thyroid, and reproductive systems. In addition, the high expression level of SNHG15 was closely related to the advanced characteristics of cancer, indicating that patients without these advanced characteristics may be prime candidates for chemotherapy. More high-quality and large-sample studies are required to further confirm the prognostic role of SNHG15 in cancer.


The effect of welding speed on microstructure, mechanical properties, and corrosion properties of laser-assisted welded joints of a twinning-induced plasticity (TWIP) steel was investigated by using X-ray diffraction (XRD), scanning electron microscopy (SEM), electron backscattered diffraction (EBSD) analysis, electrochemical test, and micro-area scanning Kelvin probe test (SKP). The results reveal that the welded joints, with a fully austenitic structure, are obtained by laser welding. In addition, the preferred orientation of grains in fusion zone (FZ) increased with the increase of welding speed. Additionally, the coincidence site lattice (CSL) grain boundaries of FZ decreased with increasing welding speed. However, potentiodynamic polarization and SKP results demonstrated that the welding speed of 1.5 m/min renders superior corrosion resistance. It can also be inferred that the corrosion properties of the welded joints are related to the grain size and frequency of CSL grain boundary in FZ. The effect of twinning-induced plasticity (TWIP) has been developed to fabricate advanced high-strength steel, where mechanical deformation is introduced to produce twins. TWIP steel renders a high tensile strength of >600 MPa, an extremely high elongation of ~95% and superior toughness. The combination of high strength, high plasticity, and high strain hardening makes TWIP steel an ideal candidate for automobiles ,3,4,5,6.Laser welding has the characteristics of high energy density, high efficiency, large temperature gradients, high repeatability of the process, and formation of a narrow heat affected zone (HAZ), which can significantly improve welding accuracy and production efficiency in actual processing ,8,9. In As a steel plate for automobiles, TWIP steel serves in various corrosive environments, such as salt spray and dust in humid environments, antifreeze salt in cold environments, etc. Herein, the corrosion property of TWIP steel is worthy of attention. In addition, the effect of grain structure ,20, alloMoreover, as the weakest part of TWIP steel components, the welded joint determines the lifetime of TWIP steel components in harsh working environments ,26. Due The microstructure and corrosion properties for laser-welded TWIP steel, with different welding speeds, have been studied in detail to explore the influence of laser welding speed on corrosion resistance of TWIP steel welded joints. Moreover, micro-area scanning Kelvin probe (SKP) test has been applied to study the corrosion properties of the micro-area. The relationship between the welding process, microstructure, and corrosion property has thus been established.The as-prepared TWIP steel was used for welding experiments. The sample of TWIP steel and welded joints was ground with Silicon carbide(SiC) sandpaper up to 2000 grit, polished with diamond paste, and etched by using a mixture of nitric acid (2%) and ethanol (98%) to observe the optical microstructure . The cheThe chemical composition and metallographic structure are shown in The welding specimens, with the dimensions of 150 mm × 50 mm × 1.5 mm, were processed from the base material of TWIP steel. The length of 50 mm was parallel to the rolling direction, and the welding direction was perpendicular to the rolling direction. The specimens were polished by using an 800 grits sandpaper to obtain a smooth welding interface, which is free of macroscopic defects.The Adopt TruDiode4006 semiconductor laser was designed and produced by TRUMPF. The IRB4600M2004-type ABB six-axis linkage manipulator was assembled for laser welding. In the rolling direction, laser welding was carried out by using Trudiode4006 semiconductor laser, as the laser which was autogenous, was perpendicular to the specimen surface. The laser power was 3000 W, the spot diameter was 1.5 mm, the focal length was 16 mm, the beam parameter product was 30 mm-mrad, the defocus amount was 14 mm, the focus was below the work piece, and argon gas was purged for protection. The laser welding speed was 1.5 m/min and 2.5 m/min. As shown in The phase analysis was carried out at ambient temperature by using micro-area X-ray diffraction (XRD) . The XRD patterns were collected in the 2θ range of 10° to 100° with an incident spot of 0.1 mm. The scanning electron microscopy (SEM) , equipped with electron backscattered diffraction (EBSD), was used to characterize the microstructure. EBSD analysis was carried out with a scan step of 0.5 μm. The grain size distribution and misorientation between grains were obtained in EBSD data.To further explore the corrosion resistance of laser-welded TWIP steel joints, a micro-area SKP test was carried out on the micro-area electrochemical testing device . The test was performed at ambient temperature in air to obtain the distribution of micro-area volta potential on the surface of a welded joint. The open-circuit potential (OCP) and potentiodynamic polarization curves were measured by electrochemical workstation (AMETEK P4000) in 3.5 wt.% NaCl solution at ambient temperature, using platinum as a counter electrode and a standard calomel electrode (SCE) as reference electrode. The potentiodynamic polarization measurements were carried out after immersion for an hour, while the fluctuations of OCP were less than 5 mv in 10 min. The scanning started from −250 mV vs. OCP toward the anodic potential with a scanning rate of 0.1666 mV/s.The micro-morphology of the welded joints is shown in n grain boundaries could be observed in HAZ. The annealing twins were generally produced in Σ3n grain boundaries of face-centered cubic metals with low layer fault energy. Hence, the low-angle Σ3CSL grain boundaries changed to high-angle grain boundaries  along <111> during migration, which could be ascribed to the inconsistency between the lattice of nucleus and substrate [Recent research indicates that the grain boundary characteristics, especially coincidence site lattice (CSL) grain boundaries, have a very important influence on the mechanical and corrosion properties of metals ,31,32,33ubstrate . MeanwhiThe misorientation distribution shows that the proportion of 60° orientation difference in HAZ increased, the proportion of 0° orientation difference decreased, and the proportion of 38.74° orientation difference remained unchanged with increasing welding speed . In geneThe inverse pole figures (IPFs) and average grain size of FZ in welded joints are shown in −4 A·cm−2, which was much higher than the welded joint prepared under the welding speed of 1.5 m/min (2.73 × 10−4 A·cm−2). Therefore, the corrosion resistance of TWIP steel welded joint decreased with the increase of welding speed.Potential polarization is an usual way to characterize the corrosion performance of materials. Through the analysis of the electrode process of metals and the fitting of Tafel zones, the corrosion properties of metals can be quantitatively compared. In this paper, a potential polarization test was carried out on TWIP steel welded joints with different welding speeds. As shown in As the most corroded zone, FZ determined the corrosion resistance of the welded joint ,28. In oThe voltaic potential difference between the probe and the thin water film on the surface of the corroded metal electrode could be measured by SKP. There is a close relationship between the microstructure of the material surface and volta potential ,38,39. Ied metal .(1)EcorHence, the micro-area SKP technology can be used to characterize the distribution of corrosion potential on the metal surface.The volta potential steps between FZ, HAZ, and BM are demonstrated in Considering that the It can be seen from n CSL grain boundaries and surface potential of FZ. The number of Σ3n CSL grain boundaries in FZ for 1.5 m/min sample was higher than that for 2.5 m/min sample. In general, there were the least Σ3n CSL grain boundaries in FZ in the 2.5 m/min sample, and it showed the worst corrosion resistance. One should note that the Σ3n CSL grain boundaries were beneficial to improve the corrosion resistance of the TWIP steel [n grain boundaries exhibited high resistance to grain boundary failure [The comparison of IP steel . This wa failure ,30,31 an failure . TherefoOn the other hand, the grain size of FZ in 1.5 m/min sample was less than that in the 1.5 m/min sample c. DuringThe preferred orientation of grains and the grain size in FZ increased with the increase of welding speed. However, the CSL grain boundary of FZ decreased with increasing welding speed.The surface potential of FZ, HAZ, and BM in each welded joint sequentially increased. The corrosion resistance of the BM in laser-welded joints was the best, followed by HAZ, and that of FZ as the worst.The 1.5 m/min sample rendered better corrosion resistance, due to the higher frequency of CSL grain boundaries and finer grains in FZ.In summary, the influence of laser welding rate on TWIP steel has been investigated. The following conclusions can be drawn from the current results:This study provides ideas for research on the corrosion properties of TWIP steel laser-welded joints under specific conditions, and provides a reference for the TWIP steel laser welding process under chloride ion corrosion environment. Subsequent studies could focus on the corrosion behavior and mechanism of TWIP steel laser welded joints in different corrosive environments.