This study was undertaken to determine whether exposure of operating room personnel to inhalation anesthetics, including nitrous oxide, isoflurane, and sevoflurane was associated with any hepatotoxic or nephrotoxic changes. Fifty-two operating room personnel and 52 non-exposed subjects were studied. A questionnaire pertaining to demographic characteristics and medical history of participants was completed. Fasting blood samples were taken from all subjects to measure the functional parameters of kidneys and liver. Biological monitoring was also performed to detect the urinary concentration of IAs. Urinary concentrations of nitrous oxide, isoflurane, and sevoflurane were found to be 175.8 ± 77.52, 4.95 ± 3.43, and 15.0 3± 16.06 ppm, respectively. The mean levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, Alpha-glutathione-S-transferase, as well as the serum levels of kidney injury molecule-1, creatinine and calcium were significantly higher in the exposed group. Statistically significant associations were observed between exposure to inhalation anesthetics and the mean levels of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase, serum creatinine, kidney injury molecule-1, and calcium. Under the exposure scenario described in the present study, occupational exposure to inhalation anesthetics was associated with subtle, subclinical, pre-pathologic changes in the parameters of liver and kidneys. Additionally, Alpha-glutathione-S-transferase and kidney injury molecule-1 were found to be sensitive markers for early detection of subclinical changes in the parameters of kidney and liver function in subjects who are exposed to inhalation anesthetics. BMI body mass indexIA inhalation anestheticsAlpha-GST alpha-glutathione-S-transferaseALP alkaline phosphataseALT alanine aminotransferaseASP aspartate aminotransferaseAST aspartate aminotransferaseBUN blood urea nitrogenGGT gamma-glutamyltransferaseKFT kidney function testsKIM-1 kidney injury molecule-1LFT liver function testsORP operating room personnnelWAG waste anesthetic gasesAnesthetic gases or inhalation anesthetics (IAs) are widely used for the induction and maintenance of anesthesia during surgery . NitrouIt is estimated that more than 200,000 health care workers in the United States are potentially exposed to WAGs in their workplace and are at the risk of developing adverse health effects due to long-term exposure to these agents . Acute While in some studies exposure to WAGs has been shown to be associated with hepatotoxic and nephrotoxic outcomes  for these discrepancies are not known, they could be explained, at least in part, by differences in the type and concentrations of anesthetic gases, length of exposure, co-exposure to other chemicals, type of studies, sample size, statistical analyses, and shortcomings, such as lack of control for potential confounders and selection of inappropriate control groups.The present study was undertaken to examine, more thoroughly, whether exposure to WAGs, including nitrous oxide, isoflurane, and sevoflurane, by a group of ORP, under their usual working condition, is associated with any significant changes in the conventional and/or more sensitive parameters of kidney and liver function.In this historical cohort study, hepatotoxic and nephrotoxic effects of chronic exposure to WAGs, including nitrous oxide, isoflurane, and sevoflurane were assessed in a group of ORP at a large public hospital in Shiraz, south of Iran. The exposed group composed of 52 ORP  with a history of at least one-year exposure to the WAGs. Similarly, 52 unexposed employees from administrative staff were randomly selected and served as the referent group.A questionnaire pertaining to demographic characteristics and medical history, including age, sex, level of education, smoking habits, alcohol consumption, duration of employment (exposure), mental and physical diseases, history of exposure to hepatotoxic and/or nephrotoxic chemicals, and previous or current history of kidney and liver diseases was completed for all subjects. Individuals with a history of renal and liver disease, history of taking hepatotoxic and nephrotoxic drugs, and those with previous or current exposure  to other known hepatotoxic and nephrotoxic agents were excluded from the study.Subjects signed an informed consent before participating in the study. The protocol of the study was approved by the ethics committee of Shiraz University of Medical Sciences (IR.SUMS.RE C.1396.S388) and the study was conducted in accordance with the Helsinki declaration of 1964 as revised in 2013.Fasting blood samples (5 ml) were taken from the antecubital vein of the studied subjects at their workplace. Clot blood samples were centrifuged at 3500 rpm for 5 minutes to obtain the sera and stored at -80 °C until analysis. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), albumin, total protein, total bilirubin, direct bilirubin, blood urea nitrogen (BUN), creatinine, calcium, and phosphorus were determined by colorimetric methods using commercially available diagnostic kits  by an auto analyzer . The Alpha-glutathione S-transferase  activity was determined spectrophotometrically according to the method published by Mannervik and Guthenberg (1981). All substrates for the activity measurements and kinetic studies were purchased from Sigma-Aldrich. Serum levels of kidney injury molecule-1 (KIM-1) were measured using the ELISA kit , according to the manufacturer's instructions. Serum Potassium level was measured by flame photometry method.Concentrations of nitrous oxide, isoflurane and sevoflurane were measured in 30 urinary samples taken from anesthesiologists, surgeons, surgical technicians, and nurses. Urine samples were collected at the end of the morning operating shift (after at least three hours of exposure). About 10 ml of a urine sample was transferred with a disposable Luer lock syringe to a 20 ml screw cap headspace vial pre-sealed with a PTFE/ rubber septum. Samples were kept on ice packs in a cold box and immediately transported to the laboratory where they were analyzed. Analysis of the urine samples was performed in accordance with a previously published method  coupled with an Agilent 7697A headspace auto-sampler. The separation was performed in an Hp-5ms capillary column (30 m × 0.25 mm × 0.25 µm) . The injection block temperature was set at 250 °C. Initial oven temperature was 40 °C for 4 min, followed by an increase of 40 °C /min to 140 °C. The injection volume was 1 µl (split 1:2). Helium was used as the carrier gas at a flow rate of 1.2 mL/min. MS interface temperature was set at 250 °C. Headspace loop, vials equilibrium, and transfer line temperature were set at 60, 42, and 70 °C, respectively. The running time was 10 min. A solvent delay time was 0.5 min. Quantification in MS was performed in selected ion monitoring (SIM) mode at a mass-to-charge ratio (m/z) of 30 for nitrous oxideAll statistical analyses were performed using version 20.0 of statistical package for social sciences (SPSS) software. Descriptive results are presented as arithmetic mean ± SD. Independent sample t-test and Chi-square were used for comparing the means of quantitative and qualitative variables, respectively. Multiple linear regression analysis was used to adjust the effects of confounding variables, such as age, sex, and body mass index (BMI) on the relationships between exposure to WAGs and the parameters of kidney function tests (KFT) and liver function tests (LFT).Demographic characteristics of the studied subjects are shown in Table 1The results of the KFT and LFT of the studied subjects are shown in Table 2The associations between exposure to the WAGs and the parameters of liver and kidney function tests are shown in Tables 3In this historical cohort study, possible hepatotoxic and nephrotoxic effects of chronic exposure to nitrous oxide, isoflurane and sevoflurane were studied in a group of ORP. No significant differences were noted between the exposed and referent subjects as far as their demographic characteristics were concerned. Additionally, none of the studied subjects had a history of exposure to hepatotoxic and/or nephrotoxic substances.The American Conference of Governmental Industrial Hygienists (ACGIH) has proposed the TLV values of 50 and 5 ppm for nitrous oxide and isoflurane respectively ACGIH, 2019). Also, 019. AlsoIn this study, urinary concentrations of nitrous oxide, isoflurane, and sevoflurane in the ORP were found to be 175.8 ± 77.53, 4.95 ± 3.43, and 15.03 ± 16.06 ppm, respectively. The IAs were not detectable in the urine of the non-exposed group. These concentrations, quantitatively, correspond with the findings of Kargar et al. for anesthetic gases in the air. They have shown that environmental exposures of ORP to these agents were higher than national recommended exposure limits . The mean concentration of nitrous oxide in urine was about 7 and 5 fold higher than the limits set by Imbriani et al. 1995) and Acc995 and AThe levels of exposure to WAGs depend on several parameters, including absence or presence of proper ventilation and scavenging systems in operating rooms, type of surgery, the extent of leaks from anesthesia face masks during the administration of the anesthetic gases to the patients, leakage from cylinders, whether there is a regular check for detecting gas leakage from anesthetic machines and length of daily shifts. Also, inappropriate work practices such as starting anesthetic gas flow before applying a mask on the patient's face or closing the anesthetic gas flow after removing a face mask and poorly fitted face masks play a major role in this scenario.The finding of the present study revealed that the mean values of all parameters of liver function test for both groups were within the normal range. However, serum AST, ALT, and GGT were significantly higher in the exposed group Table 2 than in The mean serum alpha-GST activity was significantly higher in the exposed group than in the referent individuals. Interestingly, after adjusting for important confounders, statistically significant positive associations were found between exposures to the WAGs and serum activities of ALT, AST, GGT and alpha-GST Table 3. These iHalogenated anesthetic agents have been reported to cause liver damage. Reported injury has ranged from mild injury to fulminant hepatic failure. Mild injury is characterized with a slight increase in serum activity of aminotransferases while fulminant hepatic failure is associated with the significant increase in the activity of liver enzymes and bilirubin level which lead to severe liver necrosis . Our findings are in line with the proposition that long-term exposure to IAs  is associated with subtle, sub-clinical, pre-pathologic changes in both conventional and more sensitive parameters of liver function. Additionally, it is in agreement with the findings of some experimental and epidemiological studies. For example, Casale et al. 2014) reporte reporte1These controversial findings suggest that other factors, including duration and severity of exposure, the type of anesthetic gases, age, gender, co-exposure to other chemicals may affect the hepatotoxicity of these agents. Overall, the side effects of anesthetic agents, including nitrous oxide, isoflurane and sevoflurane are dependent on the magnitude and frequency of exposure  reporteThe majority of previous studies have assessed the relationship between WAGs and kidney injuries in anesthetized patients and limited studies have investigated such a relationship in subjects occupationally exposed to these chemicals. For instance, Cohen et al. reported a high frequency of kidney disease as a result of chronic occupational exposure to anesthetic gases among operating room staff, especially female . CaciarSerum creatinine and BUN are two useful indicators for evaluating kidney function. However, their uses as sensitive and specific biomarkers for early detection of kidney injuries have numerous limitations. First, many non-renal factors such as age, sex, race, body weight, diet, and drug consumption can influence serum creatinine level. Second, the production rate of urea is not stable and increases with the consumption of protein-rich diets or under situations such as bleeding, muscle trauma, and/or steroid administration .Therefore, it may be argued that the elevated creatinine levels observed in this study may not necessarily be attributed to the exposure to the anesthetic gases. While true, the following lines of circumstantial evidence indicate that the observed effects are very likely to be related to exposure to the anesthetic gases. First, KIM-1, as a sensitive and specific biomarker for early detection of renal injury, is not virtually detectable in subjects with healthy kidneys and within hours after kidney injury, its level markedly increased in both animals and humans . TherefFinally, as shown in Table 3Subjects were free from any preexisting medical conditions, particularly, liver and renal diseases and did not take any hepatotoxic and/or nephrotoxic drugs. Also, they had no history of exposure to hepatotoxic and nephrotoxic agents.The major organs affected by anesthetic agents (isoflurane and sevoflurane) and their metabolites are the liver and kidneys because they are metabolized through the metabolic pathway involving cytochrome P-450 2E1 in the liver and kidneys  and Iran National Science Foundation (Grant number: 96005391).The authors declared that they have no conflict of interest.


We analyzed the scientific outputs of global atrial fibrillation (AF) researches, developed a model to qualitatively and quantitatively evaluate the AF research productions from 2004 to 2018.The data was retrieved from the Web of Science Core Collection (WOSCC) on June 15, 2019. Bibliometrics tools—CiteSpace V  and VOSviewer  --were used for bibliometric analyzing published outputs and finding research hotspots.Journal of Cardiovascular Electrophysiology published the greatest number of articles, and the leading country was the United States. The leading institution was the Mayo Clinic, and the most productive researchers were: LIP GYH, Natale A, Chen SA, DI Biase L, and Kuck KH. The keywords analysis showed that catheter ablation, risk, heart failure, stroke, and management were research hotspots.There were a total of 21,839 research articles on AF, and the annual publication rate increased over time from 2004 to 2018. The Bibliometric analysis of the literature shows that research on AF continues to be a hot spot, and the clinical treatment of AF is an important research frontier. However, further research and collaboration are needed worldwide. Our findings aim to provide valuable information for the editors of journals that publish research on arrhythmia, and to help researchers identify new perspectives for future researches. And there will be about 5.2 million in the United States, which will increase to 12.1 million by 2030. The increasing prevalence of AF has made it a global concern.Atrial fibrillation (AF) occurs when the atriums do not coordinate with the conduction of impulses, which results in irregular fibrillation of atrial muscle fibers and loss of effective mechanical contractions. According to a recent research, there are 20.9 million male and 12.6 million female patients with AF (excluding asymptomatic patients). The incidence of stroke in patients with AF is much higher than in those without AF –7 and sudden cardiac death associated with AF contributes to the high mortality.,9 Due to its widespread prevalence and potentially serious complications, AF puts a huge economic burden on the public health and medical systems in both developed and developing countries. It has been estimated that AF accounted for about 1% of the UK National Health Service's (NHS) budget, and $16 to 26 billion has been spent on AF health care in the United States per year.–12The progression of AF is often associated with high mortality and disability risk.–15 Our bibliometrics study provides an overview of these outputs and identifies the influential countries, institutions, authors, publications, and milestone theories. We hope our results could contribute to the development of AF research and give some suggestions to the policy makers, researchers, and funding agencies that work on AF researches and treatments.Over the past 15 years, a large number of new discoveries on AF have been made and published by researchers, indicating continuous progress in this field. However, these researches have not been analyzed and mapped systematically, with only a few bibliometric studies focused on cardiovascular disease. 22.1For this bibliometric analysis, we selected the Web of Science Core Collection (WOSCC) of Thomson Reuters as our database. The search parameters were: ; time span: 2004 to 2018; category refined: Cardiac Cardiovascular Systems; literature type: Article; index: sci-expanded. No language restrictions. All records  and references in our search results were exported in plain text format. Retrieval work was performed in 1 day  to avoid variations due to daily updates to the database. The data is all secondary data and does not contain any personal information, therefore informed consent was not required.2.2 and VOSviewer   were used for bibliometric analysis of the data we retrieved. Excel 2019 was used to display the number of published articles and the journal distribution trends in each year. CiteSpace V was used for the co-authorship network of Countries/Institutions/Authors, dual-map analysis and co-occurrence of keywords. To understand the research frontiers, burst detection was applied to investigate the growth rate of citations of keywords using CiteSpace. The co-authorship/co-occurrence maps consists of nodes representing elements such as countries, institutions, authors, and keywords, as well as links between nodes representing co-author relationships and co-occurrence relationships. The color of the links between the nodes represents the year of the first common reference. Centrality is an indicator used to measure the importance of an element. If the centrality is more than 0.1, the element is considered relatively important and represented by a purple ring. The VOSviewer was used for co-citation analysis of References/Journals/Authors to construct maps of relevant knowledge. In the co-citation maps, different nodes representing elements such as cited references, cited journals and cited authors, and the size of nodes reflect the number or frequency of citations. The links between nodes represent co-citation relationships.,20 The colors of nodes and lines indicate different clusters or years. The impact factor of each journal was derived from the 2019 journal citation report (JCR) .Excel 2019, CiteSpace V , CiteSpace parameters were used as follows: time slice (2004–2018), year of each slice (1), term source , node type (1 at a time), selection criteria (50), pruning (Pathfinder), and visualization . The fractional counting method in VOSviewer was used and the chosen threshold was set to select publications. Finally, the top 100 cited references/journals/authors were selected.33.1Based on the selection criteria, 21,852 publications of AF in WOSCC from 2004 to 2018 were indexed. After duplicates (12) and retraction (1) removed, there were a total of 21,839 publications left . The other articles were published in following languages: 389 (1.781%) in Russian, 135 (0.618%) in German, 123 (0.563%) in Spanish, 96 (0.440%) in Portuguese, 81 (0.371%) in French, 64 (0.293%) in Polish, 37 (0.169%) in Italian, and 22 (0.101%) in Turkish.Journal of Cardiovascular Electrophysiology had the largest number of published articles , followed by Euro Pace , Heart Rhythm , International Journal of Cardiology , and American Journal of Cardiology .The total 21,839 articles were published in 193 journals  ranked first, followed by J Am Coll Cardiol , Eur Heart J, New Engl J Med, and Am J Cardiol.In total, 15,805 journals were cited in 21,839 articles Fig. , Circula92,877 cited authors were cited in 21,839 articles  was labeled as “catheter ablation”, followed by the second largest cluster (#1) labeled as “cardiac surgery”, and the third largest cluster (#2) was labeled as “atrial fibrillation”. These clusters were also shown in a timeline view , Circulation (IF2019 = 23.054), and European Heart Journal (IF2019 = 23.239). About 13.33% (2/15) had an impact factor between 5 and 10, including the Euro pace (IF2019 = 6.100) and Heart Rhythm (IF2019 = 5.225). 40% (6/15) had an impact factor between 3 and 5, 26.67% (4/15) had an impact factor lower than 3. In summary, publications in the AF field were mainly published on middle-high IF journals and it was still a challenge to publish papers about AF in high IF journals .Analysis of the top 15 most popular journals shows 20% (3/15) of the journals had an impact factor greater than 10. These journals were We speculate that scholars on AF choose journals where their papers are most likely to be accepted. At the same time, they searched publications in cutting-edge research journals. Articles use new scientific research technologies, and provided significant evidence for clinical practices were most easily accepted.4.2Co-operations between countries played a vital role in the development of AF researches. The top 10 countries  and linear ablation surgical treatment of AF;2.the key members of the academic group 2 were Natale A, Di Biase L, and Hindricks G. They mainly engaged in the separation of the pulmonary vein cavity;3.the main members of the academic group 3 were Lip GYH, Soliman EZ, and Alonso A, who mainly engaged in the epidemiology of AF;4.the main members of academic group 4 included Chen SA, Chang SL, and Lin YJ, who mainly engaged in research on AF electrophysiology.4.3 The second most frequently co-cited reference was the 2010 ESC guidelines for the managements of AF. The guidelines proposed a new scoring system--CHA2DS2VASC score, which changed the score from 1 to 2 for age ≥75 years on the basis of CHADS2 score, and added 3 risk factors including vascular disease, people age 65 to 74 years, and gender  with a maximum score of 9. The third most frequently co-cited reference was published by Lip et al in 2010. In this paper, the authors proposed a simple and novel model for improving stroke risk stratification in patients with AF, and the real value of this model was demonstrated by the Birmingham 2009 risk trial in AF populations of different race/ethnicity. The paper with the highest centrality value was published in 2004 by Nadenanee K et al. This study discovered and verified that areas with complex fractionated electrograms (CFAEs) represent a defined electrophysiologic substrate and were ideal target sites for ablations to eliminate AF and restore normal sinus rhythm. The paper with the second highest centrality value was published in 2002 by Van Gelder et al. This study illustrated that rate control was not inferior to rhythm control for the prevention of death and morbidity from cardiovascular causes. Therefore, it may be an appropriate therapy for patients with a recurrence of persistent AF after electrical cardioversion.Through the analysis of the ranking of citation frequency and centrality, we found 5 “classic texts”. These texts had laid a theoretical and practical foundation for related research of AF, so we can call them landmark studies. The most frequent co-cited reference was published in 2009 by Connolly et al. This was the first large-scale study to compare the effects of 2 doses of dabigatron and warfarin on stroke risk in patients with AF and to identify effective strategies for preventing stroke and systemic embolism with AF.4.4 Based on the keywords co-occurrence map obtained before, the top 20 keywords were selected to get the top 3 research hotspots and list accordingly.Keywords reflected the core theme and main content of papers, therefore they can provide a reasonable description of research hotspots.4.4.1 race, and sex. In addition, some modifiable factors which could increase risk of AF include congestive heart failure (HF),–36 hypertension(HTN),,38 diabetes mellitus (DM), obesity,,41 alcohol consumption,–44 and obstructive sleep apnea (OSA). Behavioral interventions for AF must be based on clearly defined risk factors in order to be effective. Moreover, risk management also has important influence: stroke prevention in AF must balance the patient's risk of thromboembolism with the risk of bleeding. CHA2DS2-VASc was the most authoritative risk stratification tool recommended by the guidelines to assess the risk of stroke and bleeding.,46At present, relatively clear risk factors for AF are age, 4.4.2 When AF was triggered, it was difficult to suppress if it was not treated in time (that AF begets AF). In the later stages of AF, atrial structural remodeling and atrial electrophysiological remodeling occurs. These atrial histological changes could lead to slower atrial conduction speed, local heterogeneous conduction, and local blockage. All of these are the biological substrates for AF. In addition, the autonomic nervous system may have a role in heterogeneous atrial electrical conduction, and may trigger atrial arrhythmias, including AF.At present, scholars have not got a consensus on the specific mechanism of AF. The triggers and progression of AF appear to be a result of multiple mechanisms. In 2003, Sanders. proposed the theory of triggers of AF, a milestone in the history of AF, which included the spontaneous depolarization of atrial cells. These transient ectopic tachycardia initially reduced the atrial electrical instability, leading to atrial fibrillation.4.4.3 As mentioned above, pulmonary veins, which trigger AF, are the most common anatomic source of ectopic atrial beats. The advantages of catheter PVI over antiarrhythmic drugs alone in maintaining the sinus rhythm have been demonstrated in several clinical trials.–55 These trials reported success rates ranging from 66% to 86% at 12 months, indicating that CA was the most successful way for patients with paroxysmal AF. Favorable success rates driving the inclusion of CA in the guidelines. However, it should be emphasized that CA had a low ablation success rate in persistent AF and long-term AF (> at 12 months), and it was associated with frequent recurrent surgical treatment. Therefore, it was not usually for these patients. In addition, the evidence for whether CA can reduce the risk of stroke, heart failure, and death is still insufficient.Over the past decades, catheter ablation (CA) for AF has become one of the most common heart procedures in the United States due to its safety and efficacy.5In our paper, CiteSpace and VOSviewer were used to do literature measurement on a large amount of WOSCC data. The graphics produced from this analysis show that this area of research has been growing markedly over the last 15 years and the graphs illustrate this in a relatively intuitive way. Our aim was to assess past research activities in the field of atrial fibrillation, to track their evolution and to predict new trends in specific subjects or sub-areas. Understanding these aspects is another way to help us understanding the clinical study of atrial fibrillation as well as the disease itself. The data also preliminarily revealed cooperation between authors and institutions, and may provide information for future research.6As far as we know, this is the first bibliometric analysis of research specifically for AF. In order to fully understand the current research status of AF, 3 bibliometric tools were used to identify the countries, institutions, authors, and hotspots in this field. The data analyzed was large enough to reflect the current status of AF research. However, this study also has some limitations. First, the data were retrieved from WOSCC and only contained research papers . Second, since some authors used the same abbreviations of their names and some keywords could be included as multiple expressions, some accuracy may have been lost despite our efforts to standardize and correct them.Conceptualization: Shuqing Shi, Jingjing Shi.Data curation: Shuqing Shi, Qiulei Jia.Formal analysis: Shuqing Shi, Jingjing Shi.Investigation: Shuqing Shi, Qiulei Jia.Methodology: Shuai Shi, Guozhen Yuan.Project administration: Yuanhui Hu.Resources: Shuqing Shi, Jingjing Shi.Software: Shuqing Shi, Dandan Wang.Writing – original draft: Shuqing Shi.Writing – review & editing: Shuai Shi, Yi Wei.


Achromobacter spp. are ubiquitous in nature and are increasingly being recognized as emerging nosocomial pathogens. Nevertheless, to date, only 30 complete genome sequences of Achromobacter phages are available in GenBank, and nearly all of those phages were isolated on Achromobacter xylosoxidans. Here, we report the isolation and characterization of bacteriophage vB_AchrS_AchV4. To the best of our knowledge, vB_AchrS_AchV4 is the first virus isolated from Achromobacter spanius. Both vB_AchrS_AchV4 and its host, Achromobacter spanius RL_4, were isolated in Lithuania. VB_AchrS_AchV4 is a siphovirus, since it has an isometric head (64 ± 3.2 nm in diameter) and a non-contractile flexible tail (232 ± 5.4). The genome of vB_AchrS_AchV4 is a linear dsDNA molecule of 59,489 bp with a G+C content of 62.8%. It contains no tRNA genes, yet it includes 82 protein-coding genes, of which 27 have no homologues in phages. Using bioinformatics approaches, 36 vB_AchrS_AchV4 genes were given a putative function. A further four were annotated based on the results of LC–MS/MS. Comparative analyses revealed that vB_AchrS_AchV4 is a singleton siphovirus with no close relatives among known tailed phages. In summary, this work not only describes a novel and unique phage, but also advances our knowledge of genetic diversity and evolution of Achromobacter bacteriophages. Achromobacter spp. are ubiquitous, lactose nonfermenting, Gram-negative bacilli that are frequently isolated from a wide range of environmental habitats  isTo investigate whether DNA metabolism and lysis genes follow the same evolutionary trajectory as the structural module, phylogenetic analysis was also performed on DNA polymerase and endolysin sequences .From the phylogenetic incongruence observed, it is evident that each AchV4 genome module has its own evolutionary history. The latter result is in keeping with a number of studies suggesting that siphophage genomes are mosaics of genes from various sources, including other phages and their hosts ,59. IndeThe remote relationship of AchV4 with bacteriophages Phobos, PspYZU01, YMC16/01/N133_KPN_BP, and phiAxp-2 was also demonstrated by the proteome-based tree generated using ViPTree web-service . NotablyG scores , 23 (28%), 24 (29%), and 28 (34%) of its proteins with phiAxp-2, Phobos, PspYZU01, and YMC16/01/N133_KPN_BP, respectively .Achromobacter phage genome sequences available in the NCBI database  was used to estimate all-vs.-all distances between these phages. Based on ANI and AAI values  and two myoviruses (Mano and Motura).There are currently 29 annotated database . To defiI values , the genobtained . As seenAchromobacter phages analyzed into 19 species, 9 genera, and 3 families.Of note, a very similar clustering was observed  on the tAchromobacter-infecting viruses sequenced to date, 15 belong to the family Siphoviridae, AchV4 included. Although all of these viruses have a canonical virion structure common to phages with siphoviral morphologies, comparative genomics analysis indicated that the genome structure of AchV4 significantly differs from that of other Achromobacter siphoviruses  the rearrangements within a structural module, (ii) the location of lysis and DNA metabolism genes, (iii) the number and location of putative genes of unknown function, and (iv) the presence of the lysogeny module. Although two Achromobacter siphoviruses, JWX and JWF, have been previously reported to be capable of lysogeny, as seen in At a glance, the AchV4 virion assembly genes, which comprise the most conserved module in all siphoviruses, are positioned in the same order and orientation as those of phiAxp-2. Upon closer inspection, however, it is evident that the structural module of these two phages differs profoundly over the tail assembly region, and with respect to a number of putative genes of mostly unknown function interspersed among structural genes. Among the many differences observed between the genome of AchV4 and those of other Achromobacter phages.Taken together, the results of the comparative genomics not only demonstrate the uniqueness of AchV4, whose closest relatives have not yet been identified and characterized, but also highlight the diversity of Achromobacter spp. do not typically cause disease in humans, some members of this genus  are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis [Achromobacter, a total of 20 species and 24 genogroups have been recognized thus far; however, little is known about their predators in nature—bacteriophages.Achromobacteria are ubiquitous Gram-negative bacilli that belong to the phylum Proteobacteria, class Betaproteobacteria. The organisms have a global distribution, and may be found in water (both fresh and marine), soil, municipal and hospital water supplies as well as human clinical specimens ,2. They fibrosis ,18. As dAchromobacter viruses sequenced to date have been isolated on Achromobacter xylosoxidans suggests that the phage community has been predominantly focused on clinically relevant hosts, which in turn suggests that Achromobacter phage biodiversity is greatly under-sampled.According to the literature, bacteriophages are the most abundant organisms in the biosphere that typically outnumber bacterial populations by at least 10-fold ,64. In tAchromobacter spanius-infecting bacteriophage AchV4. To the best of our knowledge, AchV4 is the first virus reported to be active on bacteria of this particular species. Comparative genome analysis indicated that AchV4 is a unique virus that shows no nucleotide sequence similarity to any of the other bacteriophages sequenced thus far. AchV4 has been predicted to encode 82 proteins, yet no tRNAs. The latter, in combination with a high G+C content , suggests that the phage is well adapted to the translation machinery of the host, and possibly attests their long-term “relationships”. On the other hand, it has been previously suggested that the virulent phages contain more tRNAs and have a higher A+T content than temperate ones [Achromobacter spanius isolate RL_4 cells under standard laboratory conditions. However, bioinformatics analysis allowed the identification of the lysogeny cassette in the genome of AchV4, suggesting that this phage, under different conditions and/or in the case of a different host, may be capable of lysogeny. Since in the lysogenic state temperate phages share the same mutational biases as the host [Klebsiella phage YMC16/01/N133_KPN_BP  than it does with any known Achromobacter phage or, in fact, with any other phage that infects Betaproteobacteria.In this study, we report the isolation and characterization of ate ones . Bacterithe host , lysogenthe host ,67. Yet,Achromobacter phages available in GenBank, to obtain a better view of their relationships with AchV4 and to explore Achromobacter phage diversity. Our results indicate that AchV4 is distantly related to one out of 30 sequenced Achromobacter viruses, Achromobacter xylosoxidans bacteriophage phiAxp-2. Although AchV4 and phiAxp-2 share 26 proteins (mostly those involved in virion morphogenesis), both phages are singletons of the family Siphoviridae, and their closest relatives are yet to be isolated/sequenced. For such a small group , the diversity of Achromobacter viruses is remarkable. These 30 phages differ in their virion morphology, genome length, DNA sequence, G+C content, and, as illustrated by the comparison of 15 Achromobacter siphophages, both in their genome organization and in gene content. The 30 Achromobacter phages considered here represent fourteen distinct lineages that group into four genetically unrelated clusters and five singletons: three siphoviruses  and two myoviruses (Mano and Motura). Within the clusters, phages shared substantial DNA sequence similarity, whereas no similarity was observed between viruses from different clusters. Such great genome sequence diversity is rather unexpected, given that all of these viruses infect Achromobacter  and supposedly should be in genetic communication with each other. However, as discussed above, the number of sequenced Achromobacter phages is too small and by no means reflects the nature or complexity of the population of viruses that infect this particular host. Hence, more phage genome sequences are needed, from a diverse array of species, to obtain a more complete understanding of Achromobacter viruses, especially considering the prevalence and importance of their host, Achromobacter spp.We also extended our analysis to the sequenced complete genomes of Achromobacter spanius. The data presented here indicate that AchV4 is a singleton siphovirus that belongs to no known genus of tailed phages. Moreover, we show that this particular group of viruses is highly diverse, and emphasize the need for more representative Achromobacter phage sequencing data.In this work, we characterized a novel bacteriophage, AchV4, the first virus reported to be active on


Aromatase inhibitors are the first-choice drugs for the treatment of hormone sensitive breast cancer. However, in addition to the scarcity of studies, there are controversies about their effects on vaginal epithelial cell proliferation in rats, especially those in persistent estrus.To investigate vaginal epithelial cell proliferation by Ki-67 antigen expression, persistent estrus was induced in 42 randomly selected rats. These rats were randomly divided into 2 groups: group I , which received 0.1 mL of propylene glycol (vehicle) daily, and group II , which received 0.5 mg/kg or 0.125 mg/day of anastrozole diluted with 0.1 mL of propylene glycol.p<0.003).Light microscopy showed a higher concentration of cells with brown Ki-67 stained nuclei in the control compared to the experimental group. The mean percentage of Ki-67 stained nuclei per 500 cells in the vaginal epithelium was 68.64±2.64 and 30.46±2.00 [mean±standard error of the mean (SEM)] in the control and experimental groups, respectively (This study showed that anastrozole, at the dose and treatment duration selected, significantly decreased cell proliferation in the vaginal mucosa of the rats in persistent estrus. Vaginal atrophy and hormone-sensitive breast cancer are common conditions in postmenopausal women, for which tamoxifen is the standard endocrine therapy; however, although tamoxifen may have trophic effects on the vaginal epithelium, severe adverse effects such as endometrial cancer lead to the search of alternative endocrine therapies The use of anastrozole for long periods does not show any effect on the levels of steroid hormones, such as cortisol, aldosterone, androstenedione, and 16-hydroxyprogesterone, confirming anastrozole's high selectivity in inhibiting aromatase without interfering with other adrenal steroidogenesis pathways The persistent estrus female rat is a model that is under constant estrogenic stimulation, mimicking polycystic ovarian syndrome. Therefore this model is interesting and useful to study the effects of hormonal drugs th day, the rats from both groups were euthanized by an intraperitoneal injection containing an excessive dose of anesthetics, 300 mg/kg ketamine and 15 mg/kg midazolam. The rats were then immobilized on a cork board to remove the vagina, and the tissue was fixed in buffered formalin (pH approximately 7.5). After 24h fixation, the vaginal epithelium was subjected to immunohistochemical analysis.This study was approved by the Animal Experimentation Ethics Committee of the Federal University of Piauí (UFPI) and conducted according to the ethical principles established by the Brazilian College of Animal Experimentation (COBEA). We used 42 Wistar-Hannover rats, weighing approximately 250g each, from the Veterinary Sciences Laboratory of the Federal University of Piauí. Persistent estrus was induced in the animals through a subcutaneous injection of 1.25 mg of testosterone propionate on the second day of life. During the study, all animals were housed in plastic cages with metal tops (grills) at an ambient temperature ranging from 20°C to 24°C. Fluorescent lamps provided 12-h light/12-h dark cycles. While their food portions were rationed, the rats had free access to filtered water. At 90 days of life, androgenized rats were selected for research. Persistent estrus rats presented with occlusion of the distal third of the vagina and keratinization of the vaginal epithelium (the main characteristic of persistent estrus); the presence of polycystic ovaries was noted at the time of autopsy Histological sectioning to produce the histological slides was performed using the Minota-type microtome adjusted to 5-μm thickness. The preparation of the histological slides and respective immunohistochemistry, were performed simultaneously under the same conditions. Immunohistochemical evaluation of the Ki-67 marker was performed using a detection system combined with an antigen retrieval method. For this, the sections were treated with 3% hydrogen peroxide diluted in buffered solution for 5 min to block the endogenous peroxide. Following antigen recovery, tissue samples were incubated with anti-Ki-67 rat primary monoclonal antibody (clone MIB-5/1:100) for 16h overnight in a refrigerator at approximately 4°C. The samples were then washed with buffered saline and incubated for 45 min with the New Link Polymer detection system. To read these reactions, all histological slides were treated for 5 min with a 3-3 solution of benzidine diamine tetrahydrochloride at a concentration of 1 mg/mL of Tris buffered saline and hydrogen peroxide solution, then contrasted with Harris Hematoxylin for 5 min, followed by dehydration in ethyl alcohol and xylol baths. The cells were considered positive for immunohistochemical expression of the Ki-67 antigen when their nuclei were stained with a brown color.® Image Analysis and Processing Software .Cell counting was performed manually by two blind observers in relation to the groups studied, and the positive and negative cells were labeled separately to avoid counting the same cells more than once. Cell counting was performed at a research laboratory located at the Gynecological Coordination of the Getulio Vargas Hospital/Federal University of Piaui, where a computerized system consisting of a Light Microscope Eclipse E-400  was used, coupled with a color camcorder . Images were captured on a Pentium IV microcomputer with an 80-Gigabyte hard drive, 3.0-GHz processor, 1024 RAM, and graphics card using Windows XP. Cell counting for Ki-67 positive and negative cells was performed at 400× magnification. At least 500 cells in the vaginal epithelium were counted in each slide in random fields, starting in the area of higher nuclei concentration with Ki-67 expression, using Imagelabp<0.05.The data were analyzed using the non-parametric Mann–Whitney test. The data were tabulated using the IBM SPSS Statistics V21 program, with the significance level set at p<0.003)  . Figure Studies on the effects of drugs used to treat hormone-sensitive breast cancer in human tissue are limited for ethical reasons; hence experiments using animal models are reasonable alternatives. This led us to study the effects of anastrozole, an inhibitor of the synthesis of estrogens, in a rat experimental model where the rats are in persistent estrus as the vaginal epithelium is under constant estrogen stimulation. In the present study, rats in persistent estrus (control) showed a higher concentration of Ki-67-stained nuclei in the epithelium compared to the group treated with anastrozole. The mean number of Ki-67-stained nuclei was significantly reduced in the group treated with anastrozole than in the control group.The persistent estrus female rat is an animal model that mimics polycystic ovarian syndrome. The female rat in persistent estrus an intensely proliferated vaginal epithelium with vaginal cornification, a characteristic that defines this experimental model In this study, anastrozole was administered by oral gavage, which, although more laborious, is similar to the route commonly used by human females. In postmenopausal women, anastrozole is generally administered at a dose of 1 mg/day for breast cancer treatment Some studies have evaluated cell proliferation in the mammary, uterine, and vaginal epithelia of rats based on the expression of the Ki-67 antigen. Gerdes et al. The few studies that do exist on the effects of aromatase inhibitors in the uterine and vaginal epithelia of rats present controversial results. Kubatka et al. According to the aforementioned studies, the drugs were administered in the animal feed, and as reported, letrozole caused uterine and vaginal atrophy. These authors also did not identify any vaginal alterations after administering exemestane and anastrozole in the animal feed. On the other hand, Barros-Oliveira et al. showed significantly decreased expression of cell proliferation based on Ki-67 expression in the mammary epithelium of rats in persistent estrus receiving 0.125 mg daily of anastrozole by oral gavage for 28 days Thus, we conclude that anastrozole administered to female rats in persistent estrus at a dose of 0.5 mg/kg/day, that is 0.125 mg/day, by oral gavage for 28 days, significantly decreased Ki-67 antigen expression in the vaginal epithelium, showing an anti-proliferative effect of anastrozole on the vaginal mucosa. However, further studies are necessary and important to confirm these scientific findings.Chagas DC, Barros-Oliveira MC, Lopes-Costa PV and da Silva BB conceived and designed the study, and wrote the final version of the manuscript. Pereira RO, Melo MA, dos Santos AR, Costa-Silva DR, Borges CS, Viana JL, Facina G, da Silva BB were responsible for conducting the study, statistically analyzing the data, and manuscript drafting and review. All of the authors reviewed the manuscript.


The causal link between chronic hepatitis C and glycometabolic alterations has been confirmed by much biochemical, clinical, and epidemiological research work, but what is still controversial is the long-term clinical impact of sustained virologic response (SVR) achieved by direct-acting antiviral agents (DAAs) on patients with type 2 diabetes mellitus (DM). The aim of this paper is to summarize the biochemical and clinical consequences to DM of DAA-based therapy for hepatitis C virus (HCV) infection. An electronic search of Embase, PubMed, MEDLINE, Ovid, and the Cochrane Database of Systematic Reviews was conducted for publications assessing whether clearance of HCV achieved by interferon (IFN)-free antiviral therapy determines significant changes in glycometabolic control and clinical outcomes of diabetic patients. A beneficial effect of SVR obtained by DAA therapy on DM prevention and the short-term outcome of glycometabolic alterations are acknowledged by most of the studies. Whether this effect is maintained over the long term with a significant clinical impact on diabetic and liver disease is still a matter of debate. Hepatitis C virus (HCV) infection has been associated with the development of extrahepatic manifestations , includiAccording to a recent meta-analysis , in patiWhat is still controversial is the role played by SVR in HCV-positive patients with glucose intolerance  or established DM. In fact, the mechanism by which viral eradication would determine an improvement of the biochemical parameters of HCV-positive patients with DM or IFG has not yet been clarified. In addition to the uncertainty about how viral eradication would act on glycemic dysmetabolism, there is a lack of prospective studies with appropriate follow-up on the outcomes of diabetic patients achieving SVR. With the advent of DAAs and consequent high SVR rates, several observational studies ,25,26,27In order to address these issues, we performed a systematic review of pertinent studies published up to 30 June 2019.An electronic search of Embase, PubMed, MEDLINE, Ovid, and the Cochrane Database of Systematic Reviews was conducted for publications, with no language and study type restrictions, up to 30 June 2019. The keywords used were: chronic hepatitis C, diabetes mellitus, diabetes, type 2 diabetes mellitus, insulin resistance, cardiovascular diseases, renal insufficiency, retinopathy, microangiopathy, hepatitis, chronic hepatitis C, hepatitis C virus, HCV, meta-analysis, systematic review, review, direct-acting antiviral agents, cirrhosis, hepatocellular carcinoma (HCC). All the articles obtained were evaluated and accepted if they met one or more of the following criteria: (1) data on the incidence of IR and/or DM in the follow-up of patients treated with DAAs; (2) sufficient data to establish the glycometabolic response in HCV-positive diabetic patients after DAA therapy; (3) data on the incidence of DM-related events in the follow-up of diabetic patients treated with DAAs; and (4) data on the incidence of liver-related events in the follow-up of diabetic patients treated with DAAs. We excluded studies that involved patients without IR and/or DM and conference abstracts.Data were independently extracted by two researchers (D.G.R. and M.S.) and discrepancies were resolved by the third reviewer (G.M.S.). The following information was collected from each study: first author, year of publication, country of origin, study and design type , control group (untreated patients or nonresponders), total sample size, gender, age (mean and range), glycemic and HbA1c levels pre/post DAA therapy and during post-SVR follow-up, length of post-SVR follow-up, number and percentage of patients with reduced antidiabetic therapy, and number and type of DM-related and liver-related events in diabetic patients during post-SVR follow-up.(1)Does SVR achieved by DAAs significantly prevent the onset of IR and DM?(2)Does HCV clearance with DAAs lead to significant improvement of glycometabolic control in patients with DM? If so, is this control maintained over the long term?(3)Does SVR-related glycometabolic improvement induce de-escalation/withdrawal of antidiabetic therapy?(4)Do DAA-induced glycemic changes determine a significant clinical impact on the outcome of DM and its complications?(5)What is the clinical impact of DAA-induced SVR on the incidence of HCC in diabetic patients?Five issues regarding glycometabolic control in diabetic patients with CHC who were successfully treated with DAAs were identified, and the following pertinent questions were posed:In the pre-DAA era, several reviews and meta-analyses ,34,35 shp = 0.001). Contradictory results were reported by an American [Adinolfi and colleagues  prospectAmerican  and a CaAmerican  study: IAmerican : 23 of 2p < 0.001) and those treated with PEG-IFN/RBV , p < 0.001). The difference between the two types of treatment can be explained by the different efficacy rates of viral eradication obtained with IFN compared to IFN-free treatment. Moreover, these rates were not reported by the study, which merely describes an overall SVR rate of 78.82%. In multivariate analysis, the reduction in the risk of developing DM in patients treated with DAAs was quantified as 47% (relative risk (RR) 0.53, 95% CI 0.46–0.63, p < 0.0001) and the unfavorable predictive factors were ethnic origin (black or Hispanic), male sex, high BMI, and advanced liver fibrosis. Surprisingly, the results of this study contrast with those of a study [p = 0.86). Of interest, an American study [Regarding the impact of DAAs on the incidence of DM , recent  a study  performean study  conductean study . It is tp = 0.048). However, this decrease was found to be particularly marked in patients with SVR obtained before transplantation , which adds another element in favor of the proposal to administer antiviral therapy before transplantation rather than after.Other studies on less selected and therefore more representative series showed a significant reduction of the onset of de novo DM in HCV-positive patients with SVR. In the study of Li and colleagues , 5127 noIn conclusion, studies that evaluated the effect of DAA-induced SVR on the incidence of IR and DM in sustained responders seem to confirm what was already observed in the IFN era. However, it should be noted that comparing the effects of IFN treatment with those of DAAs is not relevant since they represent two completely different mechanisms of viral eradication. Last but not least, although the majority of the above-mentioned studies indicate a beneficial effect of SVR in reducing the risk of onset of IR/DM, appropriate follow-up is still lacking. Therefore, definite conclusions cannot be drawn on the lasting efficacy of antiviral therapy with DAAs with regard to the incidence of glycometabolic abnormalities.Data coming from further follow-up studies will be crucial to determine beyond any reasonable doubt whether viral eradication significantly reduces the onset of IR and DM, resulting in an important benefit to global public health.p < 0.001) with strong heterogeneity between the studies . Glycemic values were evaluated by three studies [p = 0.03) was observed, with wide heterogeneity among the studies . According to the reported data, the number of published studies that agreed with the glycometabolic amelioration induced by SVR was higher than the number of studies that disagreed with this hypothesis.A recent review with a meta-analysis  demonstr studies ,20,21: AActually, several studies  show sigTwo studies ,19, althThe number of recruited patients is extremely variable, ranging from a minimum of 13 to a maximum of 2435 patients, and methods regarding outcome evaluation vary. The follow-up is not homogeneous: it ranges from a minimum of 12 weeks to a maximum of 60 months. A decrease of HbA1c of at least 0.5% when compared to baseline values is usually considered a significant improvement of the glycemic state , but thip = 0.002) and HbA1c  values at 12 weeks from the end of therapy; no significant glucose  and HbA1c  reduction was observed among group 2 patients. Abdel Alem and colleagues [p = 0.005) and HbA1c  levels at 24 weeks from the end of therapy. Subgroup analysis showed that the decrease in glucose values occurred mainly in patients treated with oral hypoglycemic agents  while decreased HbA1c was present in both insulin-treated and oral therapy patients . Dawood et al. [p < 0.01) in HbA1c was found 1.5 years after the end of therapy with DAAs (8.4% ± 1.9% versus 7.8% ± 1.7%). By defining glycometabolic improvement as a reduction of HbA1c (with or without reduction of antidiabetic therapy) or antidiabetic therapy (even without a significant variation of HbA1c), the authors showed an amelioration rate of 34%. The predictors of glycometabolic improvement were the presence of at least one antidiabetic therapy, the type of antidiabetic therapy (insulin), and higher creatinine levels. Hum and colleagues [p = 0.02). In sustained responders with pretherapy HbA1c levels < 7.2% and in cirrhotic patients, no significant reduction was observed. These conclusions cannot be easily reproducible as the patients were American veterans, predominantly obese (average BMI > 30) males (98%) with genotype 1 (>99%). Limiting factors of the study were the inability to verify any other therapies besides the antidiabetic one and the impossibility of identifying any changes in lifestyle during follow-up, with all factors potentially influencing glycometabolic control. Pavone et al. [p = 0.007) and HbA1c of −1.95% (p = 0.021). Fabrizio and colleagues [p < 0.01), but the study did not report glycometabolic data concerning diabetic patients only. Morales et al. [p < 0.005) without a significant average weight loss. Data regarding HbA1c changes were not reported according to the presence/absence of DM, but the authors showed a larger drop in HbA1c among diabetic compared to nondiabetic patients, although this was not statistically significant. Weidner and colleagues [p = 0.04) and HbA1c (p = 0.0367) were observed in diabetic patients with SVR 6 months after the end of therapy, but this effect was observed only in noncirrhotic patients. Finally, Pashun et al. [In the study by Ciancio et al. , baselinlleagues  found a d et al.  stratifid et al. , a decree et al.  measuredlleagues  confirmelleagues  measureds et al.  retrospelleagues  evaluaten et al.  publishep = 0.03) at 44 weeks from the end of treatment in 91 liver transplant recipients with recurrent HCV infection treated with DAAs. Unfortunately, it is not possible to establish from the reported data the HbA1c reduction in the 26 diabetic patients treated and cured by DAAs, even though the authors report a 40% decrement of DM treatment. These conclusions were not confirmed by Saab and colleagues [p = 0.098). However, two patients discontinued insulin therapy and a decrease in the cumulative insulin dosage  was recorded at the end of treatment in the absence of weight changes.The effect of antiviral therapy with DAAs on diabetic patients has also been studied in the liver transplant setting ,29,30. Bp = 0.268). Furthermore, 31% of patients had to increase their dosage of insulin therapy. Chaudhury and colleagues [p = 0.17). These three studies show important methodological limitations; the number of patients recruited was rather low (range 13–42); in one study [In addition to the studies by Saab et al.  and Teeglleagues  found nolleagues  evaluatene study , it is nOnce it is established that most of the current evidence agrees that SVR induces a significant improvement in glycometabolic control in a substantial proportion of patients, the next question is whether this effect is maintained over the long term. Data addressing this question are scant, heterogeneous, and often contradictory. Most studies ,27,31,41In conclusion, an improvement of glycometabolic control at the end of therapy or in the immediate post-therapy months in diabetic patients, or only in some subgroups, as reported by Hum and colleagues  and by WFew studies have analyzed this aspect  and oftep = 0.03). In detail, for 8 of the 19 patients (42.1%) taking oral hypoglycemic therapy and 13 of the 46 (28.2%) on insulin therapy, administration of the drug was decreased or suspended. It should be noted that these therapeutic variations occurred in the presence of an average weight gain of 2.85 ± 4.33 kg among sustained responders. Of the 101 patients with SVR, 9 (8.9%) had to have their therapeutic dosage increased compared to 1 nonresponder/untreated patient . According to Dawood et al. [p = 0.04). This reduction was not accompanied by an increase in the proportion of patients treated with metformin, which, on the contrary, was also reduced by 2.2%.In the study by Ciancio et al. , antidiad et al. , dosage d et al. : 23% of d et al.  showed tRegarding patients with OLT , of the p = 0.009).In the study by Morales et al. , 2 of 16In conclusion, most of the studies reporting improvements in glycometabolic control showed reduction/suspension of antidiabetic therapy in a significant minority of patients; for this reason, it has been suggested that diabetic patients with DAA-induced SVR should be carefully monitored regarding their glycometabolic profile in order to avoid hypoglycemic episodes . HoweverDuring the IFN era, some studies ,49,50 shp < 0.001), advanced nephropathy , stroke , and retinopathy  in SVR patients compared to noncured or untreated patients. However, some methodological weaknesses  and the short duration of follow-up (2.4–2.7 years) suggest caution regarding the reproducibility of the study. Moreover, in a previous study [Since IR and DM are now considered extrahepatic manifestations of HCV, a reduction in complications of DM can be expected in diabetic patients with viral clearance. A Taiwanese study  on diabeus study , the autA possible explanation could derive from the observation that viral clearance with DAAs determines a significant improvement of extrahepatic diseases such as cardiovasculopathies and nephropathies, regardless of the presence of DM. A recent American study  showed aIn conclusion, it is still unclear whether the reduction in complications of diabetic disease observed after viral eradication is mainly due to glycometabolic improvement or to the direct beneficial effect of viral clearance on extrahepatic sites. Further studies with appropriate follow-up are needed to assess which is the key mechanism and whether this beneficial effect is maintained over the long term. However, the data show that no improvement in DM is observed in patients with advanced fibrosis or with long-term DM history, suggesting that there is a point of no return for the modifications in glucose metabolism and, conversely, DM clinical outcome. For this reason, early identification of HCV-positive diabetic patients is strongly recommended in order to start DAA therapy as early as possible.Previous studies ,63,64 coData from follow-up studies on patients with SVR after therapy with DAAs ,79,80,81Arase and colleagues  showed tIn conclusion, the magnitude of risk reduction in HCC incidence found among diabetic patients with SVR is probably comparable to that observed in nondiabetics; however, the discrepancy between the data of the IFN period compared with the DAA period is puzzling and deserves further consideration. It is likely that the difference in follow-up, which is generally longer in the IFN-based studies, may have influenced the final results. However, the metabolic influence of DM on the residual HCC risk in DAA-cured patients should be reconsidered thanks to recent studies ,87 showiAccording to the vast majority of studies considered, the incidence of IR and DM appears to decrease in CHC patients achieving SVR after DAA treatment, but the extent of this reduction is questionable. In sustained responders with established DM, glycometabolic improvement is often found with concomitant reduction of antidiabetic therapy in a significant subset of patients. However, whether these beneficial effects are long lasting or just transient is still an unresolved issue; conversely, the long-term clinical implications in terms of diabetic and hepatic outcomes of these biochemical and pharmacological variations remain largely unknown. Therefore, a careful longitudinal monitoring of these patients is needed before claiming any consolidated efficacy. Obviously, in order to arrive at meaningful conclusions, it is necessary to await the results of long-term follow-up prospective studies with an adequate number of patients. These results should include not only the biochemical but also the clinical aspect; in other words, the main endpoint to be tested must focus on the question of whether any improvement in glycemic control is going to have a major impact on the outcome of liver and diabetic disease.


The aim of this study was to evaluate retinal and choriocapillaris perfusion, using optical coherence tomography angiography (OCT-A), and visual function in healthy subjects following dark chocolate ingestion. (2) Methods: This prospective randomized study was carried out on 18 healthy young subjects at the St. Andrea Hospital, Sapienza, University of Rome. Visual acuity assessment and a complete ophthalmologic examination were carried out at baseline. In session one, each subject was randomized to eat either a 100 g dark chocolate bar or a 100 g white chocolate bar. In session two, the opposite chocolate was given to each participant. OCT-A and best corrected visual acuity (BCVA) were performed before the chocolate was eaten and repeated 1, 2, and 3 h after that. Retinal vessel density and choriocapillaris flow area were assessed. (3) Results: 18 patients with a mean (SD) age of 26.3 (1.5) years were included. No significant differences between dark or white chocolate were found when evaluating foveal density (%), whole density (%), choriocapillaris flow area, and BCVA. (4) Conclusions: Dark chocolate did not result in significant changes in retinal perfusion and choriocapillaris flow area. However, given the results of other studies showing the positive effects of flavonoids on visual function, further studies are warranted using pure chocolate without other components such as caffeine that can potentially affect results. Furthermore, we cannot rule out the possible benefits of higher doses of flavonoids in dietary supplementation over a more extended period and in a larger patient population. Polyphenols are organic compounds naturally occurring in plants and constitute a group of more than 8000 molecules with similar chemical structures. Flavonoids are part of the polyphenol family and are generally present in chocolate, cereals, fruits, vegetables, olives, legumes, and in some of the most common beverages such as tea, wine, and beer . Various studies have evaluated the association between consumption of polyphenol-rich foods and the incidence of chronic diseases, such as hypertension, dyslipidemias, heart failure, hypercholesterolemia, and diabetes ,7,8,9,10These effects have been associated with the natural antioxidant properties of polyphenols and their impact in improving endothelial function, decreasing coagulation by inhibiting platelet activation and aggregation ,13,14,15Within the ophthalmology field, different studies have been conducted on polyphenols. Terai et al. , in a stOn the effect of polyphenols on visual function, there have been contrasting reports ,19,20. ROptical coherence tomography (OCT) has been a groundbreaking imaging method in ophthalmology, as it enables rapid in vivo evaluation of individual retinal layers. It is used routinely in the diagnosis and follow-up of retinal diseases ,22. SuccThe choroid is the principal source of nutrients and oxygen to the retinal pigment epithelium-photoreceptor layer, as the outer retinal layers do not have a vascular network ,31. The Therefore, it may be hypothesized that perfusion changes could be better evidenced in the choroid due to its predominantly vascular nature. Choriocapillaris flow area has not been previously evaluated following chocolate ingestion. Thus, the aim of our study was to determine both retinal and choriocapillaris perfusion alterations following dark chocolate ingestion using optical coherence tomography angiography (OCT-A).This was a prospective randomized, study on healthy subjects who were enrolled at the St. Andrea Hospital, Sapienza University of Rome. The study was approved by the Institutional Review Board (reference number 5606_2019) and was conducted in accordance with the tenets of the Declaration of Helsinki. All the subjects gave informed consent.Exclusion criteria were (1) subjects with history of any systemic disease, including diabetes, hypertension, and renal disease, which may affect ocular circulations, (2) subjects with a history of recent or chronic use of medications, including antihistamines or analgesics, (3) subjects with a history of ocular pathology or surgery, (4) subjects with a refractive spherical equivalent above ± 3 diopters, and (5) subjects with intraocular pressure (IOP) above 18 mmHg.n = 18.The sample size was calculated at 18 subjects based on Siedlecki et al. ,35 who dDuring the first visit randomization was carried out with forced equal distribution into two groups to determine which type of chocolate patients would start with. On the subsequent visit, the opposite chocolate was given. All healthy participants underwent a complete ophthalmologic examination at baseline, before the ingestion of both dark and white chocolate, including BCVA assessment with the Early Treatment Diabetic Retinopathy Study (ETDRS) chart, rebound tonometry , slit laEach participant was required to participate in two sessions of OCT-A testing, at least 72 h apart. Only one investigator was responsible for randomizing patients and distributing the allocated chocolate. Each patient ingested the chocolate in a separate room and was instructed not to inform the investigators performing the examinations about the type of chocolate they consumed. The subjects were not masked to the type of chocolate, as this was evident on ingestion due to its color and taste. However, the ophthalmologist performing OCT-A and visual acuity assessment was not informed of the type of chocolate consumed by the participants. Blood pressure was not measured before OCT-A examinations as Siedlecki et al.  did not In session one, each subject was randomized to eat either a 100 g dark chocolate bar  with a composition of 447 mg epicatechin, 59 mg catechin, 14 mg quercetin, and 108.8 mg caffeine  or a 100An Optovue RTVue XR Avanti instrument  based on split spectrum amplitude decorrelation angiography was used to detect macular blood flow. This OCT-Angiography device has a scanning velocity of 70.000 A-scans per second obtaining volumetric 304 × 304 A-scans scans utilizing a light source of 840 nm and an axial resolution of 5 microns. Motion correction technology removes artifacts due to loss of fixation and saccades.All subjects were requested to fast from midnight before the day of testing, as some foods and beverages, such as caffeine, can affect perfusion. OCT-A imaging was performed between 10:30 to 13:30 to avoid bias due to diurnal variations. Baseline OCT-A was performed on the day of testing before chocolate ingestion.Three-dimensional OCT scans were acquired over a 3 mm × 3 mm macular region. With the use of the DualTrac Motion Correction Technology, which combines real-time tracking, a high-speed infrared camera (30 frames/sec.), and patented post-processing, saccades and minor fixation losses were removed. Scans with low quality were excluded and repeated until good quality was achieved with a minimum cutoff of signal strength ≥ 8/10 ,39. Autop value less than 0.05 was considered statistically significant.Statistical analysis was performed with SPSS software package V.21 . Demographic and clinical characteristics were summarized by standard descriptive summaries . The normality of data was determined with the Kolmogorov-Smirnov normality test. If normality was denied, a nonparametric test was used. The primary outcome measure was the change in retinal and choriocapillaris perfusion measured with OCT-A attributable to dark chocolate or white chocolate consumption as measured by OCT-A. The secondary outcome was the change in BCVA as measured with ETDRS charts. The change in DCP foveal density (%), SCP foveal density (%), DCP whole density (%), SCP whole density (%), choriocapillaris flow area, and BCVA 1, 2, and 3 h following the consumption of dark or white chocolate was evaluated with the non-parametric Wilcoxon test for paired data. Results are presented as the mean (standard deviation), as the median interquartile range (IQR), or as the frequency (percentage). A A total of 18 patients were included. Thus, 36 eyes of 18 patients  with a mean (SD) age of 26.3 (SD 1.5) years (range 24–30 years) were included. Mean BCVA was 61.0 (SD 2.07), mean spherical equivalent was 0.5 (range −2.0–+3.0), mean IOP was 13.83 mmHg (SD 0.88). All 18 patients completed the study and were fully available for analysis. There were no adverse events. The demographic and clinical characteristics of patients are shown in p value > 0.05 for all mean differences, data not shown).p value > 0.05 for all mean differences, data not shown).No significant differences between dark chocolate or white chocolate consumption were found in the DCP foveal density (%), SCP foveal density (%), DCP whole density (%), SCP whole density (%), or choriocapillaris flow area.No significant differences in BCVA were observed following the ingestion of dark or white chocolate.In this study, we did not find any significant differences following dark chocolate or white chocolate consumption in the superficial vascular plexus, deep vascular plexus, and choriocapillaris flow area. No significant changes in BCVA were observed. Our results are similar to the results obtained by Siedlecki et al.  as regarIn ophthalmology, consumption of flavonoid-rich cocoa has been associated with visual function and contrast sensitivity enhancement ,19,20. TAnother explanation could also be that automated choriocapillaris perfusion only evaluates the choriocapillaris, and not the whole choroidal circulation. Thus, improved technology to reliably evaluate a larger section of the choroid could perhaps provide more significant results and allow a better comparison of our results with other studies.The rationale of Field et al.  as to whAs the choroid is a prevalently vascular structure and had not been previously analyzed with OCT-A following chocolate consumption, we had hypothesized that the choriocapillaris could be an additional, possibly more sensitive, parameter to evaluate changes in perfusion, but we did not find any alterations in the flow area. The caffeine present in the chocolate could have affected this result, especially since the choroid, being a predominantly vascular structure, could be more sensitive to caffeine. It has been suggested that introducing a moderate amount of cocoa in a healthy diet can be beneficial not only on the cardiovascular and cerebral level, but also for the ocular system . The opt


The clinical condition COVID-19, caused by SARS-CoV-2, was declared a pandemic by the WHO in March 2020. Currently, there are more than 5 million cases worldwide, and the pandemic has increased exponentially in many countries, with different incidences and death rates among regions/ethnicities and, intriguingly, between sexes. In addition to the many factors that can influence these discrepancies, we suggest a biological aspect, the genetic variation at the viral S protein receptor in human cells, ACE2 (angiotensin I-converting enzyme 2), which may contribute to the worse clinical outcome in males and in some regions worldwide. We performed exomics analysis in native and admixed South American populations, and we also conducted in silico genomics databank investigations in populations from other continents. Interestingly, at least ten polymorphisms in coding, noncoding and regulatory sites were found that can shed light on this issue and offer a plausible biological explanation for these epidemiological differences. In conclusion, there are ACE2 polymorphisms that could influence epidemiological discrepancies observed among ancestry and, moreover, between sexes. At the end of 2019, a new outbreak caused by SARS-CoV-2 (a coronavirus) started in Hubei Province, China. The clinical condition, COVID-19, probably arose from natural selection in bat reservoirs [The lethality rate is influenced by the speed of contagion, idiosyncrasies of the affected populations according to the containment policies adopted, socioeconomic conditions and the absorption limit of the health system . Due to Although mortality in men is significantly higher , represeStudies indicate that cell-virus interaction is mediated by the connection of the transmembrane glycoprotein spike (S), present in the form of trimers on the viral surface, to angiotensin I-converting enzyme 2  .The level and expression pattern of ACE2 in different tissues and cells can be critical to the susceptibility and symptoms resulting from SARS-CoV-2 infection . Zhou anCurrently, investigations of potential genetic variations that may favor or hinder interactions between the virus and the host have been conducted –16, showConsidering the above, we sought explanations for an intrinsic factor that differed between sexes and populations that may justify the differences observed in the incidence and lethality of SARS-CoV-2 infection among the different regions of the world, as well as between sexes. We analyzed global data in the 1000 Genomes Database and, in addition, we conducted studies of exomes in two population groups in the Brazilian Amazon (Indians and miscegenated), without description in public genomic banks, and we compared this information with a public databank from a population in southeastern Brazil. These comparisons are important because Brazil has a continental size and an admixed population in the North , Northeast (more Africans), and South and Southeast (more Europeans) .The analysis was performed on data from the 1000 Genomes Phase 3 database (1000G), which comprises 84,4 million variants in 2,504 individuals from 26 different populations . These pThrough the complete sequence of the X chromosome, a region between nucleotides 15620281 and 15512643 was selected in revision GRCh37.p13 (15602158 and 15494520 in GRCh38.p13) since the angiotensin I-converting enzyme 2 gene  is located on the complementary strand, covering 107639 bp. Additionally, a region of ten thousand base pairs upstream to the gene was included in the analysis so that the initial search site became 15630281 (GRCh37.p13) [For the allelic comparison between the populations cataloged in the 1000 Genomes database and the population not described in the respective project, we investigated a population composed of 64 Amerindians and 82 admixed individuals from the Amazon region of northern Brazil (data available at Compared with other Native Americans (from different countries and even different regions of Brazil), the Amazonian indigenous groups are similar to each other. Commonly, there are quite differences between distinct ethnic groups of native Americans. However, in comparison to populations of other ancestry backgrounds (Europeans or Africans), the Native Americans of the Brazilian Amazon may be grouped as a homogeneous population –24.The 82 admixed individuals (Brazilian Admixed Population—BAP) live in Belém city, located in northern Brazil, where, due to the colonization process, are characterized by three ancestral genetic components: European, Native American and African. This sample group is also enrolled in a broad project. Furthermore, we also compared our findings to a database of variants analyzed in a Southeast Brazilian population, the Online Archive of Brazilian Mutations  .The Brazilian population is one of the populations with the broadest genetic diversity in the world due to the high degree of miscegenation in its formation –28. DiffThe DNA was extracted as described by Sambrook and collaborators . The genhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and the samples were filtered to eliminate low-quality readings (fastx_tools v.0.13 - http://hannonlab.cshl.edu/fastx_toolkit/). The sequences were aligned with the reference genome (GRCh37) using the BWA v.0.7 tool (http://bio-bwa.sourceforge.net/). The file was indexed and sorted (SAMtools v.1.2 - http://sourceforge.net/projects/samtools/). Subsequently, the alignment was processed  (Picard Tools v.1.129 - http://broadinstitute.github.io/picard/), and mapping quality recalibration and local realignment (GATK v.3.2 - https://www.broadinstitute.org/gatk/) were performed. The results were processed to determine the variants (GATK v.3.2) from the reference genome. SnpEff v.4.3t, Ensembl Variant Effect Predictor (Ensembl release 99) and ClinVar (v.2018-10) were used for variant annotations.The quality of the FASTQ reads was analyzed . Another three polymorphisms  appear mainly in the Brazilian population. Nine of these exonic polymorphisms are present in the most common isomorphs (v1 and v2) and show important differences in populational frequency .As noted, some of these polymorphisms have very significant differences between MAFs from different populations. rs35803318 is absent in Asians  and has an average MAF of 0.05 in EUR and ABM, increased to 0.074 in BAP, and has the highest allele frequency in the indigenous population among all populations (MAF = 0.121).On the other hand, rs4646179 is absent in indigenous, miscegenated people from the Amazon, Asians and Europeans and is found in the population of southeastern Brazil and Americans, with a MAF = 0.023 and an even greater frequency in Africans (MAF = 0.074).Interestingly, rs1027571965 and rs889263894 presented allele frequencies exclusively in indigenous people and are not being found in any other world population of 1000G, neither in BAP nor in the Brazilian ABM database, with MAF = 0.095 and 0.034, respectively.rs147464721 has MAF = 0.014 in the miscegenated population of the Amazon (BAP); however, they did not present any allelic frequency among the indigenous population, as well as in Asians and Europeans, presenting a slightly lower MAF difference when compared to AFR and AMR.Differences greater than 1% of MAF in the region 10,000 base pairs 5´ upstream to the gene were observed in 57 polymorphisms .Among the highlights, rs5934250, with a change from G to T at approximately 5700 bp upstream to the gene, presented a difference of up to 0.47 in the AFR (0.10), AMR (0.29), EAS (0.01), EUR (0.47) and SAS (0.22) populations for the T allele. This means that the T allele is almost zero in the East Asian population, while it has a MAF in almost half of Europeans.It is also worth mentioning that the rs2097723 SNP presents a very heterogeneous distribution, oscillating between 7% in Africans, 32% in Americans, 42% in East Asians, 28% in Europeans and 22% in South Asians.In intronic polymorphisms, many of them present a very relevant MAF interpopulational difference (up to 0.46). Two of them deserve to be highlighted, rs2285666 and rs4646140, because they are near exons.It is important to mention that rs2285666 has the highest frequency of the rarest allele (MAF = 0.71) in the indigenous population, with very important MAF differences of 0.17 (EAS), 0.23 (SAS), 0.36 (BAP), and 0.37 (AMR) and an average difference of 0.48 for the others . In contrast, rs4646140 has a MAF ranging from zero in Indians to 0.13 in Africans through EUR, AMR, BAP, EAS, ABM and SAS . FurtherThe main findings of our study are concatenated in Considering the regions where the virus commonly binds to ACE2 ,16, our The ACE2 gene is mainly composed of two isoforms with 18 or 19 exons (v1 and v2) that encode the same protein (805 amino acids) and three other smaller variants: x1, x2 and x3, which have rarely been studied ,33. ThusFifteen SNPs showed MAF differences greater than 1% among the studied populations, mainly belonging to exons 17, 18, 19, 20 and 21; the last two are terminal exons belonging only to rare isoforms [Among these, rs41303171 is a missense SNP, causing the replacement of an asparagine  with aspartic acid (electronegative amino acid) at codon 720, which can culminate in a conformational disorder of this protein that, directly or indirectly, can change viral interactions. This polymorphic variant C is practically exclusive to Europeans (MAF = 0.018), a fact corroborated by Cao and collaborators , mainly The change from leucine to phenylalanine in codon 731 (rs147311723) results in the exchange of two nonpolar amino acids that are structurally different (with the presence of an aromatic ring in this last), which may culminate in functional modifications in the ACE2 protein, with the prediction that this will occur equally to 0.941, according to the PolyPhen algorithm . This poIn a context focused on the Brazilian Amazon, unprecedented data showed the presence of two SNPs absent in all populations of the 1000G. One of them is rs1027571965, which is characterized by an exchange of G>C in exon 16, leading to a substitution of alanine for glycine at codon 673 (MAF = 0.095), and the other, rs889263894, an exchange of T>A in exon 13 (MAF = 0.034), causing an alteration of lysine to isoleucine in codon 541; thus, this last SNP should cause structural differences by the exchange of a polar and electropositive amino acid with a hydrophobic amino acid, possibly resulting in functional changes in ACE2, with a probability of 0.958 that this event will occur . Both SNIt is also noteworthy that in codons 351 (rs147464721), 690 (rs4646179) and 749 (rs35803318), there are just synonymous alterations, without any study of modifications of this enzyme in a genotype-dependent manner.Our data point to a series of polymorphisms in the region upstream of the ACE2 gene, which oscillate markedly among populations and, therefore, in individuals, although some of them may not have relevant frequency at a global level. Among them, there are rs2097723 and rs5934250, with population allele differences of up to 0.35 and 0.47, respectively.The lower frequency allele (C) of rs2097723 has a normalized effect size (NES) of up to 0.36  for incrAnother interesting observation involves the exon 19 polymorphisms, which are contained in the 3´ UTR region of the two most important isoforms, the canonical miRNA binding site for translation control dependent on this epigenetic mechanism ,35. In tThe rs142017934 site includes the connection site for miR-610 and miR-3646, both with 8mer of match in ACE2 , in addition to the seed regions of miR-3609 and miR-548ah-5p, both with a match of 7mer-m8 and scores of 84 and 74, respectively . This poAmong the intronic polymorphisms, rs2285666 draws much attention because it presents the highest frequency of the rarest allele (MAF = 0.71) in the indigenous population, with differences ranging from 0.48 to 0.17 for the other populations, with the Asians being the most similar to the Indians, mainly the Chinese. The high MAF observed in the Chinese population in the present study corroborates the data of Cao and collaborators  using thAnother intronic polymorphism, rs4646140, has no MAF in the indigenous population, reaching 0.13 in Africans, mainly in Nigerians (0.17). Some studies show the influence of these two intronic SNPs with hypertension ,40, sincIn conclusion, considering this genetic aspect involving ACE2 in the complex relationship between SARS-CoV-2 and humans, we emphasize the following:There are genetic markers that influence in ACE2 alterations and potentially to the unequal rates of aggravation and death observed between men and women. If considering some of the polymorphisms as harmful when in homozygosity  or hemizygosity , men would have this conditions from 1·4 to 77 times more  than women . Thus, aThe rates of contagion and death fluctuate greatly; in this sense, ACE2 polymorphisms could contribute to these differences. The rs182366225 and rs2097723 polymorphisms that potentially may increase the expression of the ACE2 are more frequent in the East Asian population. These allele frequencies are even higher in Chinese and Vietnamese populations. Such markers are on the order of 30% to 180% more frequently in East Asians than in other populations.Indigenous populations from Amazon have exclusive genetic polymorphisms (rs1027571965 and rs889263894) or with higher frequencies (rs2285666 and rs35803318) than other populations. These polymorphisms are related to increased expression of the ACE2 gene in brain tissues, among others. As supported previously , this isAfricans have higher rates of three relevant polymorphisms . Polymorphism in rs142017934 is exclusive to this population and can influence the translation regulation of the ACE2 gene, thus enhancing the expression of this gene in individuals who carry this variant. However, Europeans and some Africans have a higher frequency of an allele (rs5934250) that seems to reduce the expression of ACE2 in some tissues.Therefore, the study highlights the importance of considering this genetic factor as facilitating or restricting infection and, especially, in potential clinical manifestations and outcomes. Investigation of these polymorphisms in patients affected by COVID-19, with different clinical conditions and outcomes, could potentially advance our understanding of this pandemic disease.S1 TableRS: Reference SNP; Ref: Reference allele; Alt: Alternative allele; EUR: European; EAS: East Asian; SAS: South Asian; AMR: Ad Mixed American; AFR: African; NAM: Native American; BAP: Brazilian Admixed Population; ABM: Online Archive of Brazilian Mutations; GLO: Global (1000 Genomes). Bold exons are contained in common isoforms.(DOCX)Click here for additional data file.S2 Table*Insertion of an Alu mobile element relative to the reference.(DOCX)Click here for additional data file.S3 Table2); M/W: risk ratio between sexes to be a carrier of the minor allele only.Population*: Population with greater MAF; RS: Reference SNP; Site: genic region; MAF*: minor allele frequency of population*; Subpopulation alert (MAF): Subpopulation with greater MAF; Others (MAF): MAF of the other populations (or populations without MAF = null); Modification (score): Type of molecular consequence and PolyPhen Score ; Hypothe(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


The first example of photocatalytic trifluoromethoxylation of arenes and heteroarenes under continuous-flow conditions is described. Application of continuous-flow microreactor technology allowed to reduce the residence time up to 16 times in comparison to the batch procedure, while achieving similar or higher yields. In addition, the use of inorganic bases was demonstrated to increase the reaction yield under batch conditions. Upon irradiation with blue light , the desired trifluoromethoxybenzene (2) was obtained in 40% yield after a residence time of 1 h . A yield of 73% could be achieved at 0.08 M, without the tedious degassing procedure .tR) for the reaction .Under the conditions shown in reaction . Yields 3–11 in good yields. An exception is represented by iodobenzene, which could be methoxylated in only 23% yield (product 6). Similar results were obtained with carbonyl-containing compounds. Different aromatic ketones, aldehydes, esters, and carboxylic acids were transformed to the corresponding trifluoromethoxylated compounds (12–23) with generally good to high yields, except for 13, 16, 19 and 22 which were obtained in moderate or poor yields. Other synthetically useful substituents, including cyano, nitro, phenylsulphonyl, (pinacolato)boron, and alkyl groups could be tolerated in the reaction (24–30), leading to a yield range between 26 and 65%. The trifluoromethoxylation of pyridine, pyrimidine, and thiophene derivatives also took place smoothly, giving products 31–37 in good yields (50–65%).With the optimized conditions in hands, we proceeded to investigate the trifluoromethoxylation of different arenes and heteroarenes in acetonitrile. Solvent mixtures and lower concentrations had to be used in some cases due to solubility issues. As can be seen in 3CN/CH2Cl2 mixture previously used [Overall, the yields obtained for different products were similar to those reported by Ngai  and Tognsly used , furthersly used  for the 2HPO4 and KH2PO4 led to increased yields, reaching up to 86% or 89% in batch . These experiments demonstrated that the addition of bases indeed represent an interesting modification of the previous protocol, despite being substrate-dependent.Under flow conditions, products In summary, we have developed the first continuous-flow trifluoromethoxylation of arenes and heteroarenes. The use of flow photochemistry allows the reaction to occur 16 times faster than in batch, while at the same time achieving similar yields. Thanks to the intrinsic properties of microflow systems, especially for the scale-up of photochemical processes, we anticipate this procedure could be useful in medicinal chemistry applications. In addition, although no beneficial effect could be achieved in flow by the addition of bases, we proved this has a significant effect on the reaction in batch, leading to higher yields. This discovery will be useful for the development of future trifluoromethoxylation methodologies.File 119F NMR spectra of compounds 2–37.Procedure for continuous-flow and batch trifluoromethoxylation reactions and