Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana.(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of  Mycobacterium tuberculosis (MTB) isolates to primary treatment antibiotics is a major step in efforts to control the disease. Various microbiological methods are available for the susceptibility determination of MTB to treatment with drugs/antibiotics, such as streptomycin, rifampicin, ethambutol, isoniazid, and pyrazinamide [MTB strains showing varying levels of resistance to the drugs has frustrated efforts at TB control, a situation that is particularly associated with endemic countries with limited resources. This phenomenon is present in Ghana [Pulmonary tuberculosis (TB) is a preventable disease with a huge global impact due to its mode of transmission. It remains one of the world’s leading causes of death, killing more than 2 million people every year ,2,3. Acczinamide ,9. Thesein Ghana , where din Ghana .MTB involve the use of an isolate culture in a drug-incorporated mycobacterial medium. Frequently used media include Lowenstein-Jensen (L-J) egg based, Middlebrook 7H11 solid media, and Middlebrook 7H9 liquid medium. Methods for DST determination include the automated BACTEC MGIT 960 broth culture system and the Lowenstein-Jensen solid culture assay. Among these culture methods, the automated BACTEC assay is the most sensitive; this approach is suitable for growing MTB even in AFB negative samples within relatively shorter detection periods (7–14 days) [Methods for determining drug susceptibility tests (DST) for 14 days) , though 14 days) . The L-J14 days) ,6,7.The need for drug susceptibility methods that are cheap, rapid, convenient, and easily applicable in rural health posts of TB-endemic areas cannot be overemphasized. Such methods will provide useful information on suitable antibiotics and their effective therapeutic doses, as well as on the current prevalence of circulating MDR-TB in endemic areas. These efforts will contribute immensely to effective disease control. As emphasized by some researchers, a viable method is one that would incorporate functionally-suitable aspects of microscopy and culture, and also consider robustness, while being technically less demanding ,13,14.MTB growth in broth, in addition to typical cord (pellicle) formation. This method has been recommended by several studies as being relatively rapid, cost effective, and less technically demanding [The microscopic observation drug susceptibility (MODS) assay is one such qualitative test which fits into these criteria. It involves the visualization of emanding ,17,18,19emanding . The adaemanding .MTB disease, they are limited by their inability to provide results on the status of drug resistance [MTB isolates. Thus, the MODS and BACTEC MGIT 960 methods were evaluated for their usefulness in DST determination of MTB, using clinical MTB isolates. Specifically, the study sought to evaluate the feasibility of applying these methods in resource-constrained areas by evaluating their turnaround periods, rapidity, ease of use, and cost effectiveness.Though many tools exist for the diagnosis of sistance ,21,22,23MTB patients who consented to be enrolled in the study. After initial screening using Zeihl-Neelsen smear microscopy, 130 samples with smears showing no acid fast bacilli (MTB) were excluded. Therefore, a total of 403 samples qualified to be used for the study. Out of the 403 samples, 54 participants decided to opt out of the study. Thus, a total of 349 samples were finally available for analyses. Ethical approval was given by the Scientific and Ethical review boards of Ministry of Health and the Korle-Bu Teaching hospital.This was a cross-sectional study conducted at the chest clinic of the Korle-Bu teaching hospital, the premier public tertiary hospital and the largest referral hospital in Ghana. The study was carried out in a Class II biosafety cabinet in a biosafety level 3 (BSL3) laboratory. All safety precautions were appropriately observed. Subjects who consented to participate in the study were instructed on sputum collection, as recommended by the World Health Organization (WHO) . Each paN-acetyl-l-cysteine decontamination method [MTB growth.A minimum of one (1) and a maximum of three (3) sputum samples were obtained from each subject. These were processed to reduce fast growing bacteria contaminants by the n method . The decn method , as welln method  and the n method  for MTB g for 20 min), and resuspended in 1000 µL of sterile saline. The turbidity of the suspension was adjusted to a McFarland number 1 standard and then used to inoculate for cultures, as well as DST by BACTEC MGIT 960 and direct MODS methods. The isolated cultures from processed samples were then used for the indirect MODS DST method. The procedures used were adapted from Caviedes et al. [Sputum samples were decontaminated by the N-A-L-C-sodium hydroxide method as described in , pelletes et al.  and Musas et al. .For culture on LJ media, 250 μL of decontaminated bacterial suspension was inoculated. The LJ slants were incubated at 37 °C and examined twice a week for 42 days. Growth on the LJ slants was checked with the Zeihl-Neelsen smear microscopy  for the presence of AFB. Growth was recorded at 28 days and at 42 days with the following descriptions: +++ for confluent growth, ++ for >100 colonies, and 1–100 actual numbers of colonies.BACTEC MGIT 960, 500 μL of bacterial suspension was inoculated in MGIT tubes and incubated at 37 °C in the BACTEC MGIT 960 system, which monitors for fluorescence every day for up to 42 days by automated mechanisms.For culture with Middlebook 7H9 broth and incubated at 37 °C for 42 days. The wells were examined daily after day five under an inverted microscope for growth; bacterial growth was seen as granular serpentine cords. All negative cultures were further confirmed with Z-N microscopy after the 42 days’ incubation.With the MODS culture method, 130 μL of decontaminated bacterial suspension was inoculated in BACTEC MGIT 960 susceptibility method, as well as the direct and indirect MODS methods. The procedures for BACTEC MGIT 960, as well as the direct and indirect MODS methods, are briefly described below.Drug susceptibility tests were performed by the MTB inoculum was dispensed into each test tube. Control tubes contained no antibiotics, whiles 1:100 inoculum dilutions were set up as microbial controls; this was to ensure growth in the controls before tests. DST was performed for all positive MGIT on Isoniazid 0.2 μg/mL, Streptomycin 4 μg/mL, Rifampicin 40 μg/mL, Ethambutol 2 μg/mL, and Pyrazinamide 200 μg/mL, according to WHO guidelines [Drug susceptibility tests set up by the BACTEC system required five tubes for the test drugs and two others for pyrazinamide (PZA). Streptomycin, isoniazid, rifampicin, and ethambutol (SIRE) were prepared in dilutions of 1:100 (1%), whilst pyrazinamide (PZA) was diluted to 1:10 (10%). Control tubes contained broth without drugs. Each test contained 7 mL of broth with 800 µL of the supplement and 100 µL of specified antibiotic. Then, 500 µL of idelines .Tests and controls were arranged in the BACTEC MGIT 960 instrument for a 14-day incubation period. Tests with growth units of less than 200 were classified as indeterminate, and therefore, repeated. Controls usually attained 400 growth units before flagging for end point. Tests with fewer than 100 growth units were indicative of susceptibility. Smears were made from controls and Z-N stained to establish purity.The MODS susceptibility assay was conducted by the direct and indirect methods. The tests were done in a 12-well culture plates. Each test well contained 2 mL Middlebrook 7H9 broth, 200 µL of suspension, and 34 µL of the appropriate test antibiotic. A McFarland 0.5 (approximately 104 CFU/mL) suspension of isolates was used. The controls were set up in the first row of the plate. For each plate, two antibiotics in combinations of streptomycin and isoniazid (S&I) and rifampicin and ethambutol (R&E) were set up with the control. The drug concentrations were similar for the BACTEC MGIT 960 method according to WHO guidelines .Each plate was set up with a complement of a positive and a negative control in addition to the test wells. Tests for pyrazinamide were set up on a separate plate, as this required conditions that were different from those of the other test drugs, including a relatively low pH and so a higher dose of inoculum. Each PZA test well contained 2 mL of Middle brook 7H9 broth, 200 µL of supplement, and 34 µl of reconstituted PZA. All tests and controls were appropriately labeled.MTB colony suspension. A negative control well contained only MODS media. A positive control contained the MODS media and MTB inoculum at a dilution of 1:100 and 1:10 for PZA.Tests for the direct method were inoculated with 140 µL of decontaminated sputum sample prepared in pelleted dilutions as indicated. An indirect test was inoculated with appropriate diluents of MTB growth observed under an inverted microscope at 40× magnification and confirmed by AFB microscopy was indicative of positive culture. The growth observed in both the drug-free and the drug-containing wells was indicative of resistance, whilst tests with growth in drug-free and no growth in drug-incorporated media indicated susceptibility of the test isolates to the test drugs [Plates were incubated at 37 °C and examined twice weekly after an initial daily check for contamination for up to 42 days. Detectable serpentine clusters (pellicle formation) of st drugs . If growThe broth from the MGIT tubes was poured into the MODS wells to find out whether cord formation in acidic medium could be easily visualized. The broth medium for the determination of DST for streptomycin, isoniazid rifampicin, and ethambutol (SIRE) was prepared in the laboratory. With respect to pyrazinamide testing, the quality could have been compromised if the media had been prepared in the laboratory.® Office Professional 2007, Microsoft Corporation, Redmond, WA, USA) and GraphPad software version 8.0. . The concordance of susceptibility results was determined based on the sensitivity, specificity, and positive and negative predictive values for the detection of resistance with 95% confidence intervals (CIs), as well as with kappa values [p-value less than 0.01 was considered statistically significant. For the estimations of the sensitivity, specificity, and predictive value of detection for the methods, a positive reference result was defined as a positive culture according to at least one method for which cross contamination had been conclusively ruled out. A negative reference result was defined as any sample in which all three culture methods yielded negative results, or when two were negative and the third indeterminate, owing to repeated bacterial or fungal overgrowth as defined in an earlier study [Data obtained from this study was stored and analyzed using the Microsoft office Excel 2007 software .A total of 349 samples were analyzed. Two hundred and eighty three samples were tested for susceptibility to SIRE and 66 to pyrazinamide by the MODS method. Seventeen (17) samples were lost after the MODS method, thus, two hundred and sixty six samples were tested for susceptibility to streptomycin, isoniazid, rifampicin and ethambutol (SIRE) and 66 to pyrazinamide, all by the MGIT method. The average duration from culture to drug susceptibilities by MODS was 12.5  for the direct method and 9.3 days  for the indirect method, whilst for BACTEC MGIT 960, it was 10.1 days for culture , with a further 12 days to detect resistance . TherefoMTB isolates to tested antimicrobials by the various methods.Growth in the tubes or wells containing any of the antimicrobials was interpreted as a sign of resistance to that particular antimicrobial. Susceptible samples were those that showed no growth in the antimicrobial tubes or wells, even though there was growth in the corresponding control test tube or well. True resistance was defined as resistance by the two methods, and true susceptibility as those that were susceptible to the two methods. Indeterminate represents samples in which that the MGIT instrument flagged positive but no AFB was seen in their smears; thus, a possible contamination of culture was considered to have occurred, or growth was detected in only one control well for MODS-DST. The sensitivity, specificity, and predictive values of all the antimicrobials are displayed in MTB isolates resistance to the primary antimicrobials by the MODS method were streptomycin (27.9%), isoniazid (27.9%), rifampin (8.1%), ethambutol (8.1%), and pyrazinamide (6.1%). Meanwhile, those for the MGIT method were streptomycin (19.6%), isoniazid (24.5%), rifampicin (7.2%), ethambutol (4.2 %), and pyrazinamide (7.6%) (Tested e (7.6%) . Sensitie (7.6%) .Concordance between MODS and MGIT for the individual antimicrobials as calculated by kappa coefficients (κ) were streptomycin (0.87), isoniazid (0.79), rifampin (0.90), ethambutol (0.63), and pyrazinamide (1.0). Kappa coefficients values that were excellent (perfect agreement) were observed for streptomycin, rifampin, and pyrazinamide (0.87–1.0) across the two tested methods, giving a nearly completely concordant result. However, low substantial agreement was apparent for ethambutol. MTB isolates. Therefore, the MODS and BACTEC MGIT 960 methods were evaluated for their usefulness in DST determination of MTB, using clinical MTB isolates. This specifically involved a comparison of the drug susceptibility outcome for MODS to BACTEC MGIT 960, and of the ease of recognizing cord formation using each of the two methods.The current study purposed to identify which, among the most commonly used (in Ghana) methods, can conveniently be applied at health centers located in rural areas for effective DST determination of MTB isolates to streptomycin, rifampicin, isoniazid, ethambutol, and pyrazinamide, the primary antimicrobials for TB treatment by the BACTEC MGIT method, as well as by the MODS method. Tested MTB isolates showed varying resistance and susceptibility to the primary antimicrobials by the MODS (6.1–27.9 and 68.2–93.9 respectively) and MGIT (4.2–24.5 and 61.9–86.4 respectively) methods. Meanwhile, the concordance between MODS and MGIT with respect to streptomycin was 0.87, isoniazid (0.79), rifampin (0.90), ethambutol (0.63), and pyrazinamide (1.0).The study determined the susceptibility of There were, however, a few discrepancies in the results of the two methods with respect to ethambutol and, to a lesser extent, isoniazid (correlation between the two methods was 63% for ethambutol and 79% for isoniazid); however, these discrepancies were not enough to make the results obtained by the two methods statistically different. The correlation between the two methods for rifampin (90%) and isoniazid (79%) was in accordance with a previous work which compared MODS to the proportion method . Other rIn contrast, this study recorded levels that were higher. All the same, the extent of disagreement was not statistically significant . AnotherIn the current study, however, the observed differences in the results of MGIT and MODS could be attributed to any of the following three factors: inoculum size, method of reading the results, or failure rates with respect to the MGIT method. These reasons are interrelated. MGIT variation in bacterial concentration could be reduced, since the results were not manually read. On a given day of positivity, the bacterial concentrations are usually fairly the same. A manual reading of MODS makes it subjective, indicating that the concentration on the day of positivity may not be the same. Unlike the MGIT, that measures the unit of growth as well, there are no discrete colonies to count in the case of MODS, and therefore, any growth in a drug-containing well was deemed to reflect resistance to that drug. The high number of indeterminate cases (13.6%) with respect to the MGIT method could account for the differences observed in the results of susceptibility testing by the two methods. Regarding resistance, other studies have reported varying level of resistance in different parts of Ghana ,31,33. AThe marked increase in resistance to virtually all the primary antimicrobials within the study period represents a major health concern. For instance, the isoniazid resistance of 27.9% observed in this study could have negative implications for TB patients, since such resistance is associated with poor clinical outcomes , with reConsidering the individual antimicrobials separately, there were rises in resistance to isoniazid (5.0 to 6.9%) and rifampicin (1.0 to 1.2%), but not to ethambutol and pyrazinamide. In Belarus, out of 163 TB isolates that were tested against the antibiotics, 42 (68.5%) were identified as being resistant to isoniazid, rifampicin, and streptomycin, and eight (28%) were resistant to ethambutol . MonoresMTB isolates to primary antibiotics for TB treatment. This method was observed to be cost effective and easily applicable in health centers of TB-endemic, rural communities in Ghana. It could therefore be introduced alongside the BACTEC MGIT system, with little training being required due to its ease of use.This study determined that the MODS method is efficient in determining the susceptibility of


It was declared a pandemic by the World Health Organization on March 11, 2020, almost ninety days after the first cases appeared. With high transmissibility, vertiginous increase in the number of cases, and clinical severity, it is impossible to disregard its psychological effects.Covid-19 is a respiratory infection caused by coronavirus- initially detected in China, in December 2019, which progresses to pneumonia in 81% of the cases, with an average fatality rate of 2.3%Despite being a recent pathology, studies addressing the theme of emotional stress in today’s scenario already emerged. The bibliographic research conducted on March 22, 2020, using the search strategies “Psychological Stress AND covid 19”, “Estresse Psicológico AND covid 19”, and “mental disorder AND covid 19” in Pubmed and BIREME, found seven articles, all with equivalence in both databases. No other filters have been defined. Two articles in Chinese were not evaluated. After reading the other five articles in full, two were originals; the others represented brief communications.2. Frontline nurses were the least affected by emotional stress, condition linked to strategies to cope with emotional stress developed by these professionals. However, the accumulation of work overload may change this scenario, and the continuous exposure to emotional stress may trigger other disorders3. Regardless of the group, emotional stress was present in more than 60%2. In the general population , 28.8% exhibited symptoms of moderate to severe anxiety and 53.8% reported psychological impact of moderate to severe intensity, because of the epidemic4.Both original articles with primary data were conducted in China. One of these papers evaluated three groups: general population, frontline nurses and general nurses 5. In this study, they evaluated individuals in social isolation due to oncological disease, and observed symptoms of depression (92.3%), anxiety (76.9%), sleep disorders (61.5%) and hallucinations in 38.5% of the subjects, among other alterations5.Considering the epidemiological behavior of covid-19, the period of social isolation may be necessary indefinitely. Regardless of the covid-19 pandemic, social isolation itself is capable of triggering psychotic symptoms, as Kellerman et al. described in 1977Besides the covid-19 pandemic, health professionals and the general population are at risk of mental illness. We must develop psychological support strategies to promote mental health, emphasizing the use of online technologies to maintain the necessary physical distancing. Keep in mind those already suffering from psychiatric disorders, which can be worsened by the current scenario. Surveillance will be needed to control the “pandemic of fear” and treat mental illness even after the covid-19 pandemic is resolved.


Rapeseed meal (RSM) is a by-product of rapeseed oil production. Owing to its lower cost and abundant sulfur-containing amino acids, RSM can be used for replacing soybean meal in broiler diets. However, its use is limited by the presence of numerous anti-nutritional factors. As an ancient technique to convert the complex substrates into simple compounds by a number of microorganisms, microbial solid-state fermentation (SSF) has been shown as an effective way to eliminate or reduce anti-nutritional factors in RSM and improve growth performance when fed to animals. This improvement is not yet clear; in particular, the understanding of the feeding nutritional value of fermented rapeseed meal (FRSM) is not very well studied. Hence, the trial is conducted to investigate the effects of fermentation on standardized ileal digestibility (SID) of amino acids and apparent metabolizable energy (AME) in RSM fed to broiler chickens. According to our findings, fermentation had a significant effect on the chemical composition of RSM. In comparison to RSM, FRSM had greater nitrogen-corrected apparent metabolizable energy (AMEn) values and SID of amino acids. FRSM was nutritionally superior to RSM for use in broiler diets.p < 0.05) apparent ileal digestibility (AID) and SID of alanine, valine, isoleucine, leucine, tyrosine, lysine, arginine, and phenylalanine. No significant differences between RSM and FRSM were found for AID and SID of asparagine, histidine, threonine, serine, glutamine, praline, glycine, methionine, and cystine. FRSM had greater AMEn values and SID of amino acids compared to RSM, therefore, FRSM was nutritionally superior to RSM in broiler diets.Rapeseed meal (RSM) is a common protein ingredient in animal diets, while the proportion of RSM in diets is limited because of its anti-nutritional factors. Fermentation based on mixed microbial strains appears to be a suitable approach to improve the nutritive value of rapeseed meal in animal feed. In this study, we evaluated the effects of fermentation on the apparent metabolizable energy (AME) values and standardized ileal digestibility (SID) of amino acids in RSM fed broilers. The AME and nitrogen-corrected apparent metabolizable energy (AMEn) values of RSM and fermented rapeseed meal (FRSM) were determined by the substitution method, with RSM and FRSM proportionally replacing the energy-yielding components of the basal diet by 30%. Results show that fermentation improved AME and AMEn of RSM from 7.44 to 8.51 MJ/kg and from 7.17 to 8.26 MJ/kg, respectively. In the second experiment, two experimental diets were formulated, with RSM and FRSM being the sole sources of amino acids. A nitrogen-free diet (NFD) was also formulated to determine endogenous amino acids losses (EAAL). Feeding on FRSM resulted in higher ( Rapeseed meal (RSM) is a by-product of rapeseed oil production, containing 35–40% crude protein (CP) with abundant sulfur-containing amino acids, and a potential substitute for soybean meal in broiler diets ,2,3. HowLactobacilli fermentum increased 58% when rapeseed meal was fermented by mixed strains of Lactobacillus fermentum, Bacillus subtilis, Saccharomyces cerevisiae, and Enterococcus calcium. Fermented rapeseed meal (FRSM) has a positive effect on growth performance when fed to animals. In a study by Drazbo et al. (2018) [Pichia pastoris, had reduced levels of glucosinolates and phytate-phosphorus, and the use of FRSM in diets increased the final body weight of turkeys. Ashayerizadeh et al. (2018) [Fermentation is an ancient technique to convert the complex substrates into simple compounds by a number of microorganisms, and it is widely used in food processing and pharmaceutical industries ,11. In r. (2018) , RSM fer. (2018)  stated t. (2018)  also repThese studies suggest that greater quantities of fermented rapeseed meal (FRSM) can be used in broiler diets to reduce the cost of broiler production. To study the potential for greater use of FRSM in broiler diets, a better understanding of the nutritional value of FRSM is necessary. Based on previous research about FRSM, we hypothesized that FRSM had greater AMEn values and standardized ileal digestibility (SID) of amino acids in comparison to RSM. All animal management and experimental procedures for this study were approved by the Animal Ethics Committee of the Chinese Academy of Agricultural Sciences, and performed according to the guidelines for animal experiments set by the National Institute of Animal Health (Statement no. AEC-CAAS-20181208). The authors confirm that they have followed European Union (EU) standards for the protection of animals used for scientific purposes .The RSM and FRSM  samples Lactobacillus acidophilus, Bacillus subtilis, and Saccharomyces cerevisiae were used for fermentation. The concentration of strains used in this study was Lactobacillus acidophilus, 1.5 × 109 CFU (counting flora unite)/mL; Bacillus subtilis, 5.6 × 108 CFU/mL; Saccharomyces cerevisiae, 2 × 108 CFU/mL. The ratio of the mixed strains for FRSM was Lactobacillus acidophilus: Bacillus subtilis: Saccharomyces cerevisiae in the ratios: 1:3:2. The fermentation conditions were constant temperature 33 °C, feed water ratio 1:1, time 84 h, and inoculum volume 6%. Ultimately, FRSM was dried for 3 days at 55 °C, and then the dried samples were ground to pass through a 0.5 mm sieve, and kept at room temperature until mixed in the diets. The total dry matter losses were about 8% in our previous study. All of the processes were completed in the Nankou Experiment Base of the Chinese Academy of Agricultural Sciences. The mixing machine, a constant temperature fermented room, and the fermentation bag (25 kg/bag), with one way degassing valve, were used during the fermentation process. The total cost was in the range of $22–30 USD/ton. Strains of 2) was added to diets at 0.4%, as an exogenous digestibility marker. The proportional replacement of the energy-yielding components was essential for the calculation of AME for FRSM and RSM [A total of 72 Arbor Acres (AA) male broilers (45 ± 3 g/bird) were used to determine the AME and AMEn values of RSM and FRSM. All of the birds were raised together from 0 to 21 day and received a starter maize–soybean meal basal diet. The basal diet was formulated to meet or exceed the National Research Council (NRC) 1994)  energy a and RSM ,19. Tabl94  energ and RSM . Birds wAll of the broilers were fed the same basal diet for 21 day and were subsequently provided with the experimental diets. After a diet acclimation period from 22 to 25 day, total excreta was collected daily from 26 to 30 day and stored in a −20 °C freezer. Spilled feed and feathers were removed from the excreta samples. At the end of excreta collection, all of the excreta were dried, ground, passed through a 0.5 mm screen, and stored in a 4 °C freezer prior to analysis. The average daily feed intake for each treatment was recorded.A total of 72 AA male broilers were used to determine the apparent ileal digestibility (AID) and SID of the amino acids in RSM and FRSM. All of the birds were raised together from 0 to 25 day and received maize–soybean basal diet. The basal diet was formulated to meet or exceed the NRC (1994) energy and nutrient requirements of broilers. On day 25, the birds were allocated to 3 treatments in a completely randomized design. Each treatment had 6 replicate cages with 4 birds per cage, the mean body weight among replicates was 3509 ± 61 g. A nitrogen-free diet (NFD) was formulated to determine the endogenous amino acids losses (EAAL) . The proOn day 25, the birds were acclimatized to their new environments and diets during the first 48 h. After 48 h of ad libitum feed intake, the birds were deprived of food for 6 h and then resumed feed intake ad libitum. Three hours later, all birds were euthanized by intravenous injection of pentobarbitone. The contents of the ileum from the Meckel’s diverticulum to a point approximately 50 mm anterior to the ileocecal junction were collected in a plastic culture dish. Ileal digesta of all birds collected in a cage were pooled in the same plastic culture dish, immediately stored at −20 °C, and subsequently freeze-dried. Dried ileal digesta were then ground using a micro grinding machine to pass through a 0.5-mm sieve and stored in plastic tubes at −4 °C for subsequent chemical analyses.Samples of RSM, FRSM, diets, excreta, and ileal digesta were dried at 105 °C in a drying oven to determine the dry matter (DM) content  2000) . RSM and2), g/kg, DM;The AME and AMEn values of RSM and FRSM were determined by the substitution method using the following equations ,29:ADMD 2/g feed; Tofc is Tio2/g feces; Nfc is nitrogen/g feces. ME is the apparent metabolizable energy of a diet. MEn is the apparent metabolizable energy of diets corrected by N. Econfd and Econcdg are the energy of feed and excreta, MJ/kg, DM. RN is total nitrogen retained to correct the AME. The correction factor for broilers was 8.22. The RN was calculated as:The AME and AMEn of the RSM and FRSM samples were calculated asThe AID and SID of RSM and FRSM were determined by the NFD method using the following equations described by Ullah et al. (2017) .Apparen2;The endogenous ileal amino acid losses were used to calculate the SID of amino acids by the following equations; the marker in diet and ileal digesta wasTiOp < 0.05.The data for both experiments were analyzed using SPSS 19.0 statistical software  under a completely randomized design. Significant differences among treatments were determined using an independent sample t-test. Data are presented as means ± SEM. Statistical significance was set at The chemical compositions of RSM and FRSM are presented in p < 0.05) AME and AMEn values than RSM. The fermentation of RSM increased AME from 7.44 to 8.51 MJ/kg (p = 0.04) and AMEn from 7.17 to 8.26 MJ/kg (p = 0.03).The data for AME and AMEn of RSM and FRSM are shown in p < 0.05) SID of Ala, Val, Ile, Leu, Tyr, Lys, Arg, and Phe. No significant differences were observed between RSM and FRSM on the AID of Asp, His, Thr, Ser, Glu, Pro, Gly, Met, and Cys, and the SID of Asp, His, Thr, Ser, Glu, Pro, Gly, Met, and Cys.In this experiment, the nitrogen-free diet (NFD) was formulated to determine endogenous amino acids losses (EAAL). Lactobacillus acidophilus, Bacillus subtilis, and Saccharomyces cerevisiae were used for the fermentation of RSM. Previous studies have demonstrated that fermentation can improve the quality of RSM by reducing the levels of antinutritional factors [In modern commercial poultry farming systems, the production of broiler feed contributes up to 70% of the total production cost. Due to increases in the global feed prices, there is now a tendency in the poultry industry to develop new, high-quality protein sources, which can maximize animal growth performance and maintain body health ,30. FRSM factors . Vig and factors  showed t factors . In the  factors . Accordi factors , the inc factors  also notPrevious studies also reported that fermentation can increase the level of polypeptides in RSM, which is consistent with our finding ,37. The Lactobacillus acidophilus fermentation of rapeseed meal, a considerable amount of amylases, proteases, phytases, and β-glucanases may be produced and activated. These enzymes consumed the insoluble fibers in RSM and produced soluble matters with simple structure; carbohydrate composition was modified as a result of microbial metabolism and, to some extent, due to activation of inherent enzymes in the cereal protein feedstuff [β-glucanase, 0.5 g/kg) barley had significantly higher AMEn values than no enzyme treatment when fed to cockerels. Studies have shown that the addition of enzymes to chicken diets degrades endosperm cell walls, resulting in increased digestibility of nutrients, which otherwise may be encapsulated in the cell structures [The AME values of RSM and FRSM were consistent with data from the Feed Composition and Nutritive Values in China   is limited, and the values for the AID and the SID of amino acids in FRSM is also not known. In the present study, we used a nitrogen-free diet method to correct the endogenous amino acid flux in broilers. Our results indicated that broiler digestibility of amino acids was improved when they were fed FRSM diets in comparison with RSM. The increased AID and SID of amino acids in FRSM, compared with RSM, are likely due to the reduced anti-nutritional factors, low-molecular-size proteins, and L-lactic acid contents in FRSM. Ullah et al.  showed tFermentation had a significant effect on the chemical composition of RSM. It reduced the concentration of glucosinolates and increased the concentrations of CP, gross energy, polypeptides, and lactic acid. In comparison to RSM, FRSM had greater AMEn values and SID of amino acids. Therefore, FRSM was nutritionally superior to RSM for use in broiler diets. The basic data and theories for the research of fermented rapeseed meal in this study would provide a reference for the application of fermented feed in poultry nutrition.


Coir fiber (CF), a renewable natural plant fiber, is more competitive in improving poor toughness and crack resistance of magnesium phosphate cement (MPC) than artificial fibers, due to its slight energy consumptions and low costs in production and waste treatment. In this paper, a typical three-point bending test was carried out to study the effects of CF length on MPC flexural properties. A total of forty-two cuboid specimens were employed to investigate the flexural strength, load-deflection behavior, and flexural toughness of MPC, with CF lengths varying from 0 to 30 mm at the curing age of 7 days and 28 days. Results showed that, at both two curing ages, MPC flexural strength first increased with CF length increasing, and then deceased when CF length exceeded the threshold. However, with the increase of CF length, MPC flexural toughness increased continuously, while MPC elastic modulus displayed a decreasing trend. Additionally, Modern micro testing techniques, such as scanning electron microscope (SEM) and X-ray diffraction (XRD), were also used to study the microstructure and phase compositions of specimens for further explaining the themicroscopic mechanism. Magnesium phosphate cement (MPC), as a new inorganic cementitious material, has great potential in rapidly repairing roads, bridges, and airstrips, due to its advantages of fast setting, high early strength, good durability, and perfect fire resistance ,2. HowevReis et al.  found th2/kg, a density of 2650 kg/m3, and a particle size of 45 μm, was produced by Zhengyang Casting Material Company , its chemical compositions listed in 2PO4 and borax from Jiang Hua Chemical Glass Co., Ltd. in Nanjing, China, had been ground for six hours and dried for at least 24 h, before their mixing with MgO. American Society of Testing Materials  C618 class F FA was employed to replace partial MgO to improve the workability of MPC mortar, with its chemical compositions listed in All specimens had a basic mix proportion employed in the authors’ previous studies ,18,19, aCF from Sri Lanka, provided by Jia Gao Cheng (Import and Export) Trading Co., Ltd. in Shangrao, China, was adopted in this study, its physical properties being presented in 2PO4), borax, and fly ash (FA) were first mixed before water was poured in. Then, magnesium oxide (MgO) was added into the homogeneous black slurry, comprising water and the mixture. When the slurry turned from black to tawny, CF was dusted into the slurry and stirred for about 1 min. Finally, the mixed MPC slurry was pumped into the molds within 3–4 min. When the MPC slurry was solidified completely (after approximately 30–60 min), specimens were demolded and then cured in the time corresponding to the test requirement. The specimens’ preparation process was as follows: potassium dihydrogen phosphate  shown in A specimen was preloaded firstly at a rate of 2.0 mm/min according to the loading cell displacement, until loading force increased to 0.01 kN, when the load cell, the specimen, and the base had been center aligned, as shown in After that, sample pieces were taken from failed specimens and tested using SEM and XRD. Thus, the microstructure and phase constitution of MPC were characterized for understanding the changing mechanism of MPC performance with CF length.For group T7, specimens presented a typical brittle failure when they were not mixed with CF (set CF0-T7). Before the specimens failed, no obvious failure signs could be noticed, except for a fissure, which occurred at the midspan of the specimens’ bottom shortly after loading. Then, the fissure expanded fleetly through the specimens and broke them in seconds with a rock-fissuring sound. Few pieces fell off from the specimens. However, some symptoms of ductile failure appeared when CF was added into specimens, with a tendency to be more distinct accompanying the increase of CF length. For example, in the sets of CFL5-T7--CFL15-T7 in T28 presented a similar effect of CF on failure modes as T7, i.e., the modes shifted from typical brittleness to typical ductility, as shown in sK of specimens shown in In branch O--A--B, as in When curves dropped to point B, as in When loading force increased to point C, however, the tension of CF exceeded its tensile strength or the bonding force, leading to the rupture or pulling out of CF. As a result, specimen capacity declined again, until the specimen was broken (point D). Additionally, branch C--D declined more gradually in the case of longer CF. For example, as in Although CF length had similar effects on the L-D behavior of specimens at two curing ages, it had a lower effectiveness in improving specimen ductility at longer curing age. As in clf is the flexural strength, P is the peak load, L is the support distance (100 mm), b is the specimen width (40 mm), and d is the specimen depth (40 mm). As well as the stress-strain behavior, AFS was improved after adding CF and increased with CF length increasing for all specimens at both curing ages. However, this increase of AFS was cut off when CF length grew to a certain value, and then declined in turn as CF length increased continuously. For example, As in 4·6H2O, MKP)”  around them, as shown in These phenomena could be ascribed to the following reasons. In fact, MPC matrix, as a space network assembled by hydration crystals comprising unreacted MgO particles and a compound named “potassium phosphate magnesium  and the dihydrogen phosphate ion (H2PO4−) first, as in 2PO4− is further dissolved into the hydrogen ion (H+) and the phosphate ion (PO43−). Meanwhile, MgO is converted into the hydroxyl ion (OH−) and the magnesium ion (Mg2+) by the chemical reaction with water. This Mg2+ finally combines K+, PO43−, and the bound water coming from the reaction of H+ and OH−, to produce the hydration product MKP. Additionally, the excess bound water continues to engage in the hydrolysis reaction of KH2PO4 and MgO together with poured water. However, this cycle hydration process will be disturbed by adding CF. CF will absorb lots of water in the hydration reaction, slowing down the hydration. Furthermore, the longer CF is, the more water is absorbed, due to the increased CF surface area. As a result, MgO and KH2PO4 cannot obtain sufficient water to keep the reaction going on to produce more MKP with CF length increasing, which can also be demonstrated by the growing MgO shown in Moreover, the XRD result in From the analysis for SEM and XRD described above, it can be known that longer CF will lower the compactness of MPC and reduce the amount of MKP, although it contributes to a stronger connection between the MPC matrix. Consequently, the improvement of MPC flexural strength and toughness caused by adding CF will decline when CF is longer than a threshold (20 mm in this study), as reported in (1)CF length presents similar effects on the flexural performances of MPC at different curing ages. However, specimens at the curing age of 28 days exhibit higher flexural strength, stiffness, and flexural toughness than specimens at the curing age of 7 days.(2)The flexural strength of MPC can be improved by increasing the CF length within a certain length range, but begins to decrease when CF is longer than 20 mm (in this study). However, further tests are needed to capture a more precise threshold of CF length.(3)Longer CF is more beneficial to the improvement of MPC ductility, but reduces MPC stiffness continuously. In addition, MPC performance displays a secondary rise after its softening first as CF length increases.(4)CF contributes to improving MPC toughness. However, this improvement slows down after CF is longer than a threshold. In this study, the threshold is 20 mm for T7 and 25 mm for T28. Moreover, overlong CF has slight extra help in strengthening MPC’s crack-resistance in its elastic stage than CF with the threshold length.(5)Longer CF reduces the amount of MKP and degrades the compactness of MPC matrix.(6)The threshold of CF length suggested in this study may change with the scaling of specimens, which will be investigated in further tests.A typical three-point bonding test was employed in this study to investigate the effect of CF length on the flexural performances of MPC, including flexural strength, loading-deflection behaviors, and flexural toughness. Additionally, the microstructure analysis by means of SEM and XRD were conducted for further explaining the microscopic mechanism. Conclusions are summarized as follows:


No significant differences were found in the prevalence of obesity or being overweight at seven years of age in children with early or timely AR . Ex-preterm infants have an increased risk of EAR. Since EAR may lead to long-term detrimental health effects with the onset of various chronic diseases , healthcare providers should be prepared to counteract its occurrence, especially in delicate sub-populations of infants.Adiposity rebound (AR) refers to the second rise of the body mass index (BMI) curve that usually occurs physiologically between five and seven years of age. AR timing has a great impact on patients’ health, since early adiposity rebound (EAR) is associated with the development of metabolic syndrome later in life. We aimed to investigate the prevalence of EAR in a cohort of inborn preterm infants admitted to the Neonatal Intensive Care Section of the Policlinico University Hospital of Bari, Italy. Secondarily, we assessed whether some determinants such as (1) gender; (2) delivery mode; (3) birth weight and classification into small, normal, or large for gestational age; (4) type of feeding; (5) breastfeeding duration; (6) timing of introduction of solid food; (7) parental education; and (8) parental pre-pregnancy body mass index (BMI) influenced EAR in this cohort. The tertiary aim was to evaluate the prevalence of obesity or being overweight at seven years of age in children according to early versus timely AR. This is a prospective, population-based longitudinal study conducted at the Neonatal Intensive Care Section of the Policlinico University Hospital of Bari, Italy. Inborn preterm infants admitted to the neonatal ward between 2009 and 2011 were eligible. Enrolled preterm infants were evaluated at birth and at 1, 3, 6, 9, 12, 15, 18, and 24 months and 3, 4, 5, 6, and 7 years of age. Weight and height data were analyzed, and BMI was calculated. AR was assessed in the growth trajectory in a body mass index (BMI) plot. Of the 250 preterm newborns included, 100 completed the seven-year follow-up and entered the final analysis, 138 were lost during the seven-year follow-up, and in 12 cases parents withdrew over the course of the study. The prevalence of EAR in our cohort of preterm newborns was 54% at seven years of age. Early adiposity rebound was associated with being large for gestational age (LGA) at birth. No other factors were associated with EAR. Early adiposity rebounders had a significantly higher BMI at seven years compared to children with timely AR (17.2 ± 2.7 vs. 15.6 ± 2.05,  Obesity in childhood is a major concern for public health worldwide and its prevalence is expected to increase in the near future . To counRolland-Cachera et al. identified a typical growth trajectory of the body mass index (BMI) plot. Physiologically, BMI rapidly increases during the first year of life, then it decreases and reaches a nadir between five and seven years of life; thereafter it increases again throughout childhood. The exact point of the BMI curve nadir with the immediate subsequent increase represents the so-called adiposity rebound (AR) . If the Early adiposity rebound has recently been a matter of research, however its pathophysiology as well as related risk factors are still under debate . GrowingFurthermore, the first few months after birth may be regarded as a crucial period in terms of later effects on weight gain and childhood obesity . This maPreterm infants are defined by the WHO as “born alive before 37 weeks of pregnancy” and the prevalence of preterm birth ranges from 5% to 18%. According to the programming theories, preterm infants during early stages of life show different postnatal patterns of growth (catch-up growth) than term infants, with a greater likelihood of developing obesity, cardiovascular disease, and diabetes in adulthood.Due to the significant improvements of neonatal intensive care and the high survival of preterm infants, their lifelong management has become an important field of research with impacts for the primary care provider. Primary care pediatricians have to screen all ex-preterm patients for obesity and should advise behavioral interventions and provide intensive counseling to children at high risk. So far, there are no clear recommendations for ex-preterm infants on the timing of initiation of complementary feeding, and only few data are available regarding the optimal timing of solid food introduction in ex-preterm infants and the effect on being overweight and having obesity later in life .The main aim of this population-based longitudinal study is to evaluate the rate of EAR in preterm newborns. The secondary aim is to investigate the role of parental and neonatal factors and nutritional characteristics in EAR development. The tertiary aim is to calculate rates of obesity and being overweight at seven years of age in infants with and without EAR.This study is a prospective, population-based longitudinal study, conducted in the Neonatal Intensive Care Section of the Department of Biomedical Science and Human Oncology of Aldo Moro University in Bari, Italy. All inborn preterm infants between 2009 and 2011 were eligible for the study. Inclusion criteria were (a) Italian language-speaking parents and (b) gestational age (GA) at birth between 23 and 36 weeks. Exclusion criteria included genetic syndromes, congenital and/or malformation disorders; any kind of surgery; major neurological, immune, metabolic, cardiac, or renal diseases; monozygotic twins; and absence of parental consent.All consecutive newborns were recruited over the first week of life, during hospitalization. Clinical visits were scheduled at 3, 6, 9, 12, 15, 18, and 24 months of corrected age and at 3, 4, 5, 6, and 7 years of age. Up to 3 years of age, infants’ growth was evaluated according to the adjusted postnatal age  ; whereasThe primary outcome was the prevalence of EAR in a cohort of Italian preterm newborns.BMI was calculated based on the anthropometrical data collected over the 7-year follow-up. The timing of AR was defined as the age at the lowest BMI . An AR oThe secondary outcomes aimed to evaluate any associations between EAR and neonatal/infant factors, such as (1) gender ; (2) type of delivery ; (3) birth weight and classification into small, normal, or large for gestational age; (4) type of feeding (breast milk/formula/mixed); (5) duration of breastfeeding; (6) timing of introduction of solid food; (7) parental education; and (8) parental BMI. Such variables were selected for their potential role in increasing the risk for adiposity rebound or obesity in adult age, or both.All newborns were classified according to birth weight corrected for gestational age for Italian newborns in normal for gestational age , small for gestational age , and large for gestational age  .The timing of the introduction of solid foods was regarded as the corrected age (in months) at the earliest administration of any solid food: before 4 months (corresponding to 16 weeks), between 4 and 6 months (corresponding to 24 weeks), and after 6 months.Breastfeeding and its duration were categorized as (a) no breastfeeding, (b) less than 6 months, (c) from 6 to 12 months, and (d) more than 12 months.Parental and social determinants were also investigated. Parental education level was defined as high  or low.2), normal weight (BMI 18.5–24.9 kg/m2), overweight (BMI 25.0–29.9 kg/m2), or obese (BMI ≥ 30.0 kg/m2).Parental BMI calculation was based on self-reported height and pre-pregnancy weight, and was categorized as underweight , overweight , or obese (≥97th percentile) according to the WHO BMI-for-age sex-specific growth charts .A simple description of data was made in terms of mean, standard deviation, and percentage depending on the type of data.2 test. Subsequently, a multivariate logistic regression model was created. Gestational age at birth, duration of breastfeeding, time at the first food introduction, and BMI were compared between children with or without EAR by Student’s unpaired t-test. For all tests, a p-value of <0.05 was considered statistically significant. SPSS v23  was employed to analyze the data.The association between EAR and (1) sex, (2) type of delivery, (3) birth weight classification, (4) type of feeding at 6 months of age (breast milk/formula/mixed), (5) duration of breastfeeding, (6) timing of introduction of solid food, (7) parental education, and (8) parental BMI was assessed by contingency tables and χOut of 411 eligible preterm infants, 161 were excluded after not meeting inclusion criteria; 250 children entered the study but 138 were lost during the seven-year follow-up and in 12 cases parents withdrew over the course of the study. Most of these newborns were lost at one year of life, hence the data were insufficient to carry out a statistical analysis. One hundred (40%) completed the seven-year follow-up and entered the final analysis .Demographical and nutritional characteristics of the sample according to timing of adiposity rebound are described in AR age (years) is shown in p = 0.85), duration of breastfeeding (p = 0.49), or timing of first solid food introduction (p = 0.98).No significant differences between children with or without EAR were observed for gestational age at birth (p = 0.75), type of delivery (p = 0.40), birth weight (p = 0.09), type of feeding (p = 0.48), duration of breastfeeding (p = 0.83), timing of introduction of solid food (p = 0.97), maternal education (p = 0.27) and BMI (p = 0.98), or paternal education (p = 0.28) and BMI (p = 0.18). The results of the multivariate analysis for risk factors associated with EAR are shown in EAR was not significantly associated with gender . Furthermore, the prevalence of obesity in subjects with EAR was higher, although not statistically significant .At the seven-year follow-up visit, 6% of children were overweight and 23% obese. Ex-preterm infants with EAR have a significantly higher BMI at seven years compared to those with normal AR  is associated with excessive weight and obesity in children, with contrasting results ,21,22. BSGA neonates are more prone to early adiposity rebound, obesity, and metabolic syndrome, especially those featuring the early catch-up growth phenotype . In our Conversely, LGA newborns had increased odds of EAR compared to their NGA counterparts, confirming that LGA status is a risk factor for being overweight or obese in early childhood, as demonstrated by previous studies ,29.In our sample of preterm infants, parental BMI and educational status did not show a significant relationship with EAR, in contrast with other studies ,30.To date, breastfeeding is widely advocated as a protective factor against childhood obesity. Breastfed infants feature an ideal growth model in which growth occurs regularly without rapid weight gain. Recent literature has widely shown that infants who are exclusively breastfed are less prone to having excess weight during late infancy, in part because breastfeeding in comparison to formula milk determines lower insulin response, which prevents excessive fat deposition and decreases adiposity, thus helping prevent chronic diseases later in adulthood. Breast milk also features a high amount of bifidobacteria, which have been proven scarce in the gut of overweight or obese children. Lastly, children who were breastfed in the early stages of life show healthier food habits afterwards, preferring fruit and vegetables as compared to their formula-fed counterparts .The switch from exclusively milk-based nutrition to solid foods represents a delicate moment for the infant. This timepoint was defined as “weaning” until some years ago, but today the term “complementary feeding” is preferred as it underlines the role of nutritional integration to breast milk. The age of four to six months is regarded as the most appropriate for the introduction of complementary feeding, since after six months of life breast milk is not sufficient to meet the growing child’s needs, with special regard to proteins, iron, zinc, and vitamins .Over the last two decades several studies have assessed the impact of timing of complementary feeding ,40,41,42Our study failed to demonstrate any different prevalence of EAR according to type of breastfeeding, formula or mixed. Our results show only a weak association between breastfeeding and normal adiposity rebound, possibly as a consequence of the limited number of exclusively breastfed infants in our cohort (15%).Furthermore, there are few data regarding the effect of the timing of complementary feeding on the risk of being overweight or obese in ex-preterms. A recent study showed that weaning of these infants is characterized by great heterogeneity ,45. In oHowever, a recent systematic review assessing the relationship between the timing of complementary feeding in preterm infants and the incidence of being overweight failed to draw clear conclusions due to the shortage of large randomized controlled trials .We are aware of some limitations of our study. First, our inclusion criteria could represent a bias since preterm infants with significant comorbidities have been excluded. Secondly, our follow-up stopped at seven years, possibly missing or underestimating any association of EAR with obesity or being overweight that occurred after seven years of age . FurtherThis paper is the first to show EAR prevalence in a cohort of ex-preterm newborns and to evaluate the role of different neonatal factors on EAR with a follow-up up to seven years.We demonstrated a high prevalence of EAR in ex-preterm newborns that is not influenced by several neonatal factors, confirming that prematurity per se represents a high-risk condition. Our data suggest that pediatricians should carefully follow ex-preterm children to screen for excessive weight or obesity very early because of their confirmed predisposition .Further studies are needed to confirm our data in ex-preterm babies, focusing on the role of other factors such as feeding type and quality of solid foods during weaning, with the aim of developing new preventive strategies. A case-control study on ex-preterm infants matched with their term counterparts would confirm the role of prematurity per se on EAR.The timing of adiposity rebound has an impact on adverse health outcomes. More than 50% of preterm newborns in our cohort showed early adiposity rebound, revealing that prematurity per se represents a high-risk condition. Determinants such as gender, type of delivery, birth weight, feeding type and breastfeeding duration, timing of introduction of solid food, parental education, and parental BMI are not associated with EAR. Since EAR may lead to long-term detrimental health effects with the onset of various chronic diseases , healthcare providers should be prepared to counteract its occurrence, especially in delicate sub-populations of infants.


Right-to left shunt (RLS) is regarded as a risk factor resulting in migraine, but the relevance between the RLS and migraine remains controversial. This paper aims at investigating the prevalence and RLS grade of patent foramen ovale (PFO) in cases of migraine (including migraine with and without aura) and evaluate the relationship between PFO and migraine.Synchronous test of contrast transthoracic echocardiography and contrast transcranial Doppler ultrasonography was performed in 251 cases of migraine, which contains 62 cases of migraine with aura (MA) and 189 cases without aura (MO) and 275 healthy adults. Among these cases, 25 cases with migraine and 14 healthy adults were evaluated through transesophageal echocardiography.P = .042, <.001, and.001, respectively). (2). Permanent RLS was detected as 7.93% of the cases in MO, 20.96% in MA, and 6.18% in controls. Total RLS was detected as 35.98% of the cases in MO, 48.38% in MA, and 23.64% in controls. Large RLS was detected as 13.76% of the cases in MO, 27.41% in MA, and 7.27% in controls. Compared with controls, the positive rate of total RLS and large RLS in MO increased , the that of permanent RLS, total RLS, and large RLS in MA also increased (P < .001 for each of the comparisons). The positive rate of permanent RLS and large RLS in MA was remarkably higher than that in MO . (3) The presence of large-size PFO (≥2.0 mm) of migraine showed higher than that of the controls (P = .048).(1). The prevalence of permanent RLS, total RLS, and large RLS in migraine was 11.16%, 39.04%, and 17.13%, respectively, which was significantly higher than that of the controls , when it is permanent RLS, large RLS, and large-size PFO (≥2.0 mm). The presence of PFO has been pointed out to be strongly related to various disease processes, which includes cryptogenic stroke, transient ischemic attack, migraine headaches, peripheral arterial embolism, platypnea-orthodeoxia syndrome, and decompression sickness.,4 Characterized by moderate or severe headache attacks and reversible neurological and systemic symptoms, migraine is a chronic neurological disorder, which is one of the most prevalent and disabling medical illnesses all over the world. Migraines influence about 13% of the population aged 20 to 64, with one third of migraineurs suffering migraine with aura (MA).,7 Furthermore, it appears to be more severe widespread in women. In a meta-analysis, PFO is associated with a 2.5-fold increase in the prevalence of migraine and a 3.4-fold increase in that of MA. Hence, Identifying PFO in migraine cases has great significance. Previous studies have found that contrast transthoracic echocardiography (c-TTE) and contrast transcranial Doppler ultrasonography (c-TCD) are simple, repeatable, and commonly used screening methods, and both positive results can enhance the diagnostic value of PFO. The purpose of this study is to evaluate the prevalence and shunt grade of PFO through synchronous test of c-TTE and c-TCD, and to investigate the correlation between PFO and migraine.The foramen ovale is an important fetal structure, which closes after birth in most individuals but remains open as a patent foramen ovale (PFO) in approximately 25% of the healthy people.22.1 251 cases diagnosed with migraine (aged 14 –74) were recruited from January 2018 to August 2019 at The First College of Clinical Medical Science, China Three Gorges University. Based on a questionnaire, the detailed clinical medical history of these cases was recorded through face-to-face interviews. The first part of the questionnaire, experienced neurologist collected fundamental information such as name, gender, age, height, body weight, and education level, smoking, hypertension, diabetes, dyslipidemia, high D-dimer, silent brain infarcts, and deep white matter lesions. The second part of the questionnaire captured the clinical characteristics of the diagnostic criteria of MA and migraine without aura (MO), and then cases with migraine were classified into 2 subgroups: MA (62 cases) and MO (189 cases). Also, 275 healthy controls (aged 14–81 years) were accrued. However, cases with carotid artery plaque, congenital heart disease, rheumatic heart disease, aortic dissection, malignant arrhythmia, hypercoagulability, and other possible cerebrovascular incidents were excluded. Without being aware of the patient's disease condition, 2 experienced ultrasound technicians implemented the synchronous detection of c-TTE and c-TCD. This study was authorized by the ethics committee . All cases provided informed consent to participate in the study.According to the International Classification of Headache Disorders III- beta,2.2 When TTE detected micro-bubbles (MES) in the left atrium within 3 to 5 cardiac cycles, and TCD detected more than 1 MES appeared within 10 seconds after VM was detected, the c-TCD results were regarded as positive. It is assumed that cardiac RLS passes through the PFO when both of c-TTE and c-TCD are positive.The test was performed with transthoracic echocardiography  and transcranial Doppler . In this test, participants lay down comfortably in the left lateral position, who were connected to ECG leads. Single channel TCD and double depth monitoring were selected, and the middle cerebral artery was observed through the right temporal bone window. The test was conducted during normal breathing and subsequently with Valsalva maneuver (VM). The contrast agent was produced with 8 mL saline solution, 1 mL air, and 1 ml participant's blood, which was vigorously mixed within the two 10 mL syringes via a 3-way stopcock at least 30 times and then rapidly injected into the right antecubital vein. The first injection was performed during the normal respiration. During VM, the contrast agent was injected 5 seconds before the start of VM. This was produced by the cases blew into a small soft plastic tube connected to the manometer device, and then the cases started the VM at the command of the inspector and held it for 5 seconds. The effective VM was assessed by monitoring the peak flow velocity of the middle cerebral artery. Doppler spectrum was decreased by at least 25%, and the manometer device reached and maintained a pressure of 40 mm Hg.2.3 The length of PFO tunnel was measured by the maximum overlap between the septum primum and septum secundum and a length ≥10 mm was defined as long-tunnel PFO  was utilized. The cases were fasting  for 4 hours, and oral dyclonine hydrochloride mucilage was taken 10 minutes before the examination. Then the operator was inserted by the operator about 30 to 35 cm away from the incisor. The integrity of the foramen ovale flap was observed at rest, by looking at whether there were fissures between the septum primum against the septum secundum, and whether there were color shunt observed by color Doppler. The height of PFO was measured by the maximum separation between the septum primum and septum secundum at the end of the systole, and a height ≥2 mm was defined as a large-size PFO.2.4 the degree of shunt in c-TTE was quantified based on detected micro-bubbles in the left atrium: grade 0 = no occurrence of micro-bubbles; grade I = 1 to 10 micro-bubbles; grade II = 11 to 25 micro-bubbles; grade III = over 25 micro-bubbles or left atrium nearly filled with micro-bubbles or left atrial opacity  for continuous variables and as frequency (n) and percentage (%) for categorical variables. The differences between these 2 groups were analyzed by the 33.1A total of 251 cases with migraine and 275 healthy volunteers were recruited. The characteristics of participants were studied in Table 3.2P = .042; P < .001; P = .001, respectively) , and that of permanent RLS, total RLS, and large RLS in MA also increased . The positive rate of permanent RLS and large RLS in MA increased indicated significantly higher than that in MO  , the presence of large-size PFO (≥2.0 mm) of migraine increased. No differences existed in the length of PFO and long-tunnel PFO (≥10.0 mm) among these groups  , both of which has advantages and limitations in diagnosing a patient with stroke, but in principle, they should be equivalent: they all detect RLS through MES, either visually (TTE and TEE) or by Doppler shift (TCD). Consequently, provided that the technique and visualization are adequate, and the physiological mechanism behind the presence of the shunt (at rest or with the VM) is similar, there should be no significant differences in diagnostic accuracy. C-TEE is considered to be the “gold standard” for diagnosing cardiac RLS. However, it is hard to detect small PFO by TEE, there is difficulty in performing the VM during TEE, especially for elderly patients with serious neurological deficits. Besides, other less invasive detection techniques, such as TTE and TCD, have also been improved, which have become the preferred methods for PFO detection. A total of 769 suspected PFO cases were collected in the previous study, and TEE was adopted as the diagnostic standard, and the diagnostic accuracy of synchronous test of c-TTE and c-TCD was 95.2%. Unfortunately, the study subjects involve indecipherable ischemic stroke, transient ischemic attack, migraine, and vertigo. Moreover, independent analyses of migraine patients were not available.RLS is an abnormal pathway between the venous and arterial circulations, which includes both intracardiac and extracardiac RLS. Intracardiac RLS are usually connected with PFO, which has been described as a “back door to the brain.”,24 In addition, current evidence demonstrated that obesity could increase the risk and severity of migraine, especially for women in childbearing age. This may be associated with several hormones such as leptin and adiponectin, and some migraine patients may be able to relieve migraine symptoms through weight loss interventions. The relationship between migraine and subclinical brain ischemic lesions including silent brain infarctions and white matter hyperintensities, is complicated and disputed. Sas et al proposed that migraine and stroke may have a common pathogenesis, both of which may be accompanied by neuronal dysfunction and neuronal vulnerability, thus resulting in neurodegeneration and apoptosis. In conclusion, comparable characteristics of the studied participants play an important role in this study.The pathogenesis of migraine is complicated, which is accompanied by various risk factors. Studies have revealed that age, smoking, hypertension, hyperlipidemia, and diabetes could increase the risk of migraine, possibly because of increased risk factors of blood vessels, blood hypercoagulability, and vascular dysfunction. pointed out that 18 (41%) of these 44 cases with migraine presented RLS compared with 8 (16%) of 50 controls (P < .005), however, the limitation of this study was that the volume of the sample is small. In the study of Yang, 217 consecutive migraine in total were contained, which found that the prevalence of RLS showed significantly higher in Chinese migraineurs than that in healthy controls. Meanwhile, it revealed that the detection rates of migraine and control group were 28.0% and 44.2%, respectively, and the prevalence of RLS was 66.1% and 36.1% in the MA and MO, respectively, which indicated significantly higher than that of the healthy group and higher than the detection rate in this study. In this study, the prevalence of permanent and total RLS in migraine showed higher than that of controls. Additionally, compared with controls, the positive rate of RLS in both MA and MO increased (P < .05). This coincides with the study of Yang et al, which proposed that PFO-related may be associated with migraine (MA and MO). However, Yang only channeled c-TCD into evaluating the positive rate of RLS, which was difficult to determine the anatomic origin of intracranial MES, namely, it can not exclude extracardiac RLS, and to some extent it affects the accuracy of the results. However, c-TTE can offset the shortcomings of c-TCD, which has advantages in judging intracardiac RLS, so the results are expected to be more accurate.At present, the relationship between RLS and migraine is still being debated, the prevalence of RLS between both migraineurs and healthy individuals varies. In 1998, Del Sette et al Nozari et al observed in mice experiments that small particulate or air emboli injected into the carotid artery could induce a cortical spreading depression without causing ischemia. This study revealed that abnormal microembolism and ischemia may be the trigger factor for migraine.–31 Furthermore, the correlation between PFO and migraine may be related to genetics. A recent report pointed out that the occurrence of atrial shunt was in line with autosomal dominant inheritance to some families with aura migraine.The mechanism of PFO that causes migraine is unknown. The possible causes are coughing, straining to defecate, the VM, and lifting heavy objects which can lead to right atrial pressure raised exceeding the left atrial pressure, thus making it easier for RLS to pass through a PFO. MES and metabolite products from venous circulation enter into the intracranial artery, generating brain stimulation. reported a follow-up study of 67 migraineurs who had migraine symptoms after transcatheter PFO closure, which adopted migraine relief (>50% reduction in frequency) as the endpoint. It turned out that migraineurs with aura were 4.5 times more likely to relieve migraine than those without aura. Even though some patients have RLS shunt, no statistically significant difference was represented in migraine symptom relief between the complete and the incomplete group . In a world, migraine may be relieved despite of residual RLS after transcatheter PFO closure, which may indicate that the RLS burden is reduced below a neuronal threshold that triggers migraine. Some scholars have stated that migraineurs with RLS were connected with impairment of dynamic cerebral autoregulation. Guo et al separated 66 migraine cases into the RLS group (n = 30) and the non-RLS group (n = 36). It could be observed that phase difference of patients in the RLS group showed significantly lower than those in the non-RLS group (P < .001), Besides, the PD in the large RLS group was significantly lower than that of the small RLS group (P < .01) and non-RLS group (P < .001). Dynamic cerebral autoregulation was impaired in migraineurs with large RLS, which may represent a potential mechanism linked to RLS and migraine. Transcatheter PFO closure has recently become an effective therapy to improve migraine symptoms and reduce ischemic events. It is essential to analyze PFO characteristics and identify high-risk PFO. In contrast to controls, the prevalence of large-size PFO (≥2.0 mm) increased (P = .048), but the length of PFO and long-tunnel PFO (≥10.0 mm) showed no difference among these groups . It was proved that PFO may cause migraine related to the large-size PFO, which may be because of the increasing increased risk of paradoxical embolism in large PFO.In this study, compared with controls, the proportion of large shunt in the MA group and the MO group rise , which indicated that the correlation between PFO and migraine may be related to large shunt. Larger RLS may increase the possibility of migraine, which suggests that a “neuronal threshold” exceeding this threshold will trigger migraine. Jesurum et al Whether the relationship between PFO and migraine is causal or symbiotic remains to be researched.These discoveries of a possible connection between migraine and PFO is still controversial. Generally, embolic events showed an unpredictable hemispheric distribution, while migraine pain is typically lateralized, often periodic and predictable like menstrual migraine. Furthermore, the headache symptoms of patients with PFO combined with migraine can be eased with age.Our study may have some limitations: first of all, some of the researched subjects were outpatients, which may have bias in the selection. Secondly, in this research, the healthy adults tried to be chosen to match the migraine group with respect to characteristics and no significant differences in characteristics between the 2 groups, but other indicators like blood biochemistry, drug use, and treatment status are not available to be obtained. Lastly, the diagnostic method of PFO is TEE. Due to the invasive examination and difficulty with VM, only some patients have finished TEE.5PFO is associated with migraine , when the PFO is permanent RLS, large RLS, and large-size PFO (≥2.0 mm).Data curation: Qiuxia Zhao, Zhizhi Dong.Formal analysis: Qiuxia Zhao, Rong Liu.Funding acquisition: Qiuxia Zhao.Investigation: Qiuxia Zhao.Methodology: Yue Chen, Qiuxia Zhao, Jun Zhou, Zhizhi Dong.Project administration: Rong Liu, Jun Zhou.Resources: Rong Liu.Writing – original draft: Qiuxia Zhao, Yue Chen.Writing – review & editing: Qiuxia Zhao, Jun Zhou, Rong Liu.


Arg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host’s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis that a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNAMet that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12–13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae.Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNA Viruses rely on most or all of their host’s translation machinery to synthesize their proteins. A conflict for viruses occurs when they have a codon usage bias (CUB) different from their host. However, some viruses have genes that help adapt to the host’s CUB in favor of their own CUB by encoding tRNAs. These include some large dsDNA viruses infecting eukaryotic organisms  with a lytic lifestyle [Chlorella variabilis NC64A (referred to as NC64A viruses), viruses that only infect Chlorella variabilis Syngen 2–3 (referred to as Osy viruses), viruses that infect Chlorella heliozoae SAG 3.83 (referred to as SAG viruses), and viruses that infect Micractinium conductrix Pbi (referred to as Pbi viruses).Among the group of tRNA-encoding viruses are large viruses that infect algae ,4,5,6, iifestyle ,11,12,13ifestyle . They inC. variabilis NC64A [M. conductrix Pbi [C. heliozoae, which has not been sequenced, has a similar GC content.The genomes of more than 40 chloroviruses, representing 3 of the 4 clades, have been sequenced, assembled and annotated , and refis NC64A  and M. ctrix Pbi , both haTwo NC64A chloroviruses, PBCV-1 and CVK2, were reported to have the interesting property of clustered tRNA genes ,9. ClustC. variabilis NC64A, and likewise, if there was a CUB among the chloroviruses. iv) To evaluate whether the chlorovirus clades acquired their tRNA gene clusters before or after their divergences from their most recent common ancestor (MRCA). (v) To investigate the role that tRNA isoleucine lysidine synthase, an enzyme encoded by almost all chloroviruses, might play in overcoming CUB. (vi) To examine the incidence of tRNA genes and gene clustering within other members of the Phycodnaviridae and in some other large DNA viruses.This current in silico investigation had several purposes: (i) To evaluate chlorovirus tRNA genes with respect to clustering. (ii) To compare and contrast the tRNA genes within and among the three clades of chloroviruses. (iii) To determine if there was a CUB in the algal host C. variabilis NC64A, which is the host of the NC64A viruses, was previously sequenced and annotated [M. conductrix, the host of the Pbi virus, has also recently been sequenced and annotated [In total, the genomes of 41 sequenced, assembled (in some cases to draft genomes) and annotated chlorovirus genomes that represent three of the four known chlorovirus clades were used in this study: 14 NC64A viruses, 13 SAG viruses, and 14 Pbi viruses . The Graphics link was used to identify the tRNA genes and their locations, as well as the surrounding non-tRNA genes. The putative tRNA sequences were verified using tRNAscan-SE [A tRNA gene cluster was previously reported for the chlorovirus PBCV-1 ,22, as wAscan-SE ,24. CommFor the chloroviruses examined herein, the non-tRNA genes immediately 5′ and 3′ to the tRNA clusters were sought in order to give insight as to whether the 41 chloroviruses acquired their tRNA gene clusters prior to or after their divergence into the three clades. The source of the data was the same NCBI website, and the data were processed as described above.C. variabilis NC64A using Geneious 11.1.5 .Two NC64A viruses, PBCV-1 and AN69C, were selected to compare their codon usage to that of their host, Tyr, tRNAGly, and tRNAArg. The following chloroviruses were not included in this analysis because they lacked one of the three tRNA genes that were concatenated: PBCV-1, NE-JV-1, AR158, NE-JV-4, GM0701.1, and MN0810.1. The tRNA sequences were retrieved from NCBI. The concatenated sequences were aligned by MUSCLE and curated by the Gblocks method; phylogenies were constructed using three tree building algorithms that included 100 bootstraps each: maximum likelihood, parsimony and BioNJ, a distance method (http://phylogeny.fr). The trees were saved in Newick format and MEGAX was used to present the final trees [Three concatenated tRNA genes common to all three chlorovirus clades were analyzed by phylogenetic analysis: tRNAal trees . PhylogeAll 41 chloroviruses encode tRNA genes, and the tRNA genes were located in clusters, usually with intergenic spacers of 1 to ~30 nucleotides . HoweverAsn genes, in which the encoded tRNAs recognize the same codon AAC.The 14 NC64A viruses had from 7 (virus AR158) to 14  tRNA genes in their clusters ; the 13 Arg gene was found in all 41 chloroviruses, while 39/41 chloroviruses had the tRNAGly gene.The order of the chlorovirus tRNA genes within a cluster is also reported in Due to the short intergenic spacers between clustered chlorovirus tRNA genes, we suspected that the tRNA gene clusters were transcribed as one transcript. Indeed, Nishida et al.  reportedDifferences and similarities in tRNA gene content were observed among individual chloroviruses within each of the three clades of chloroviruses. In some chloroviruses, not all of the tRNA genes within a gene cluster were immediately contiguous to one another, leading to interrupted tRNA-gene clusters in which one or several non-tRNA genes were interspersed within the tRNA-gene cluster. This was especially true for the Pbi viruses in which at least four member viruses had interrupted tRNA-gene clusters, as indicated by large nt intergenic spacer numbers in Additionally, all of the chlorovirus-encoded tRNAs lacked the 3′ terminal three nucleotides (CCA) necessary to be aminoacylated. Since fully functional tRNAs were reported for the NC64A chlorovirus CVK2 , the chlThr gene located ~30 kb beyond the 3′ end of the tRNA gene cluster, placing the tRNAThr genes near the center of their respective genomes.The tRNA clusters in the NC64A and Pbi viruses were located near the center of their genomes except for the Pbi virus NE-JV-1, which was located in the last third of its genome; NE-JV-1 is unusual in many other aspects including the fact that it only has 3 tRNA genes . The tRNThr gene in the SAG clade’s MRCA. Indeed, if a translocation event did take place, then the original location of the cluster in the ancestral SAG virus would have been more towards the center of its genome. In support of this notion, all of the Pbi viruses have a tRNAThr at the 3’ end of their respective clusters.One possible explanation is that the tRNA cluster in the SAG viruses was translocated in the 5′ direction but without the tRNAThr genes in all 13 SAG viruses located approximately 30 kb downstream of their respective tRNA clusters  were the most common, while the two most common in PBCV-1 and AN69C ended in A and U (GGA and GGU) , as well as other amino acids with more than two synonymous codons. Furthermore, the isoleucine codon AUA (AU-rich) occurred in 2.44% of all PBCV-1 codons, while it occurred in only 0.20% of codons in C. variabilis NC64A, a 12-fold difference , while it was a moderately common codon used by PBCV-1   were the two most common in C. variabilis NC64A , while the same two codons in the two viruses were 0.75% and 0.70%, respectively.Because nuclear genomic codon usage data are available for n usages . Codon us 67% GC , while ts 67% GC . For exaand GGU) . A paralfference ; hence,  codons) . As a fiAsn genes whose tRNAs only recognize the codon AAC; none of the 41 chloroviruses encode a tRNAAsn that recognizes the alternate codon AAU, which was 10 times more common in the two viruses than in C. variabilis .  In therus CVK2 . The samhttp://lowelab.ucsc.edu/tRNAscan-SE/) predicts that all of the encoded chlorovirus tRNAs are functional, with the noted exceptions of several tRNA genes whose mutations have rendered them as pseudogenes, appropriate laboratory-based verification of functionality was beyond the scope of this study. However, Nishida et al. (1999) did demonstrate that some of the tRNAs encoded by CVK2, an NC64A virus, are functional. While we think it is unlikely, we cannot rule out the possibility that some or all of the viral-encoded tRNAs discussed herein are nonfunctional.Although the tRNA analysis software  . EF-3 plAsn genes, in which the encoded tRNAs recognize the codon AAC. There were, however, considerable differences in composition and order between clades, unlike the similar composition and synteny within each of the three clades  were concatenated . While the intron lengths and sequences were nearly identical within a clade, there were slight differences in intron length between the three clades: NC64A 13–14 nt; Pbi 13 nt; SAG 10–11 nt. Because introns within tRNATyr genes are relatively rare, it is unlikely that all of these tRNATyr genes with introns were acquired independently. Rather, from an Occam’s razor line of inference, the evidence supports a single origin for the chlorovirus tRNATyr gene and, therefore gene clusters, prior to the divergence of the three clades from the MRCA.A fourth line of inquiry focused on the predicted intron in the tRNAIt is also noted that all 410 tRNA genes of all 41 chloroviruses are on the same identical DNA strand and none on the alternate DNA strand. Separate acquisition events over time would likely have resulted in at least some tRNA genes being acquired on the alternate DNA strand.Asn genes that all recognized the same ACC codon, while almost all of the other Pbi viruses had just two copies. Likewise, the NC64A virus MA-1E had three tRNALys genes that all recognized the same AAG codon, while others in the same clade had just one, and virus AR158 had none. There are other similar examples among the three chlorovirus clades.The ability of tRNA genes to proliferate is thought to be similar to the mechanism by which mobile elements can lead to intragenomic gene duplications . DuplicaC. variabilis NC64A; however, the viruses probably had at least one other host through evolutionary time; (ii) bacteria, because some of the chlorovirus genes appear to be of bacterial origin; and, (iii) from other host-competing viral species. Plastids and mitochondria might also have contributed to the viruses via HGT, at least for the tRNA gene clusters, because those organelles also have tRNA gene clusters [C. variabilis, C. heliozoae, and M. conductrix. Morgado and Vicente [Pope et al.  implicatclusters ,20,21. TPhycodnaviridae family also encode tRNAs that are in gene clusters (Micromonas pusilla virus 12T (MpV12T) encodes six tRNA genes, five of which are clustered. The sixth tRNA gene, tRNAThr, is an orphan ~30 kb beyond the tRNALeu gene, the 3′ member of the tRNA cluster. The position of these two tRNA genes and the distance between them is the same as in the chlorovirus SAG clade that also has a 30 kb gap between the genes that encode tRNALeu and tRNAThr. MpV12T also encodes a tRNATyr, as do the chloroviruses, but unlike the chloroviruses, MpV12T does not have an intron in its tRNATyr gene. On the other hand, Ostreococcus lucimarinus virus 7 (OlV7) [Tyr that does have a 15 nt intron located in the same position as the chloroviruses. In fact, the tRNA genes in the Micromonas and Ostreococcus virus clusters are similar to some of the tRNA gene clusters in the NC64A viruses, suggesting the two groups of viruses might have a common evolutionary ancestor. The only phycodnavirus that lacks tRNA encoding genes is Ectocarpus siliculosus virus 1 (EsV1) (Most of the other viruses in the clusters . Micromo7 (OlV7) , which h1 (EsV1) ; howeverPhycodnaviridae family are proposed to have an ancient, common evolutionary ancestry with some other large dsDNA viruses in the Poxviridae, Iridoviridae, Ascoviridae, Asfarviridae, Mimiviridae, Marseillivirdae, and Pithoviridae families that infect eukaryotes. However, many more large DNA viruses are rapidly being discovered, including Pandoraviruses, Faustoviruses, Mollivirus, Kaumoebavirus, Cetratvirus, Pacmanvirus, and Orpheoviruses, and the evolutionary relationships among these viruses are just starting to be analyzed [Viruses in the analyzed ,41. Collanalyzed ,43,44.Mimiviridae viruses that were examined encoded tRNA genes and 8/9 had tRNA gene clusters. Most of the remaining NCLDVs, with few exceptions, encoded 0–3 tRNA genes, and none of them had 2 or more tRNA genes in clusters. A few baculoviruses and herpes viruses were also examined for tRNA-encoding genes. Nine of the baculoviruses examined encoded 1 tRNA gene. The 6 herpes viruses that were examined contained from 1 to 19 tRNA genes, but only one of the viruses had clustered tRNA genes (Phycodnaviridae viruses, along with many viruses in the Mimiviridae family, appear to be the exceptions among the large DNA viruses by having clustered tRNA genes.Therefore, we examined a few representatives from all of these groups to see if they encoded tRNAs and, if so, did the tRNA genes occur in clusters . All of NA genes . Thus, tTyr due to Wobble at the 5′ G of the tRNA, which can base pair with the U or C at the 3′ position of the codon. Wobble can also facilitate some of the other encoded amino acids including the codons for glutamic acid, cysteine, histidine, phenylalanine, etc. All the chloroviruses examined to date have clustered tRNA genes, as do many of their Phycodnaviridae relatives. With the exception of Mimiviridae viruses, most other large DNA viruses not only have few, if any, tRNA genes, but when a virus encodes two or more tRNA genes, they are not clustered.The chloroviruses overcome CUB of their algal hosts by encoding clusters of tRNA-encoding genes, with most of the tRNAs favoring the viruses. However, the evolutionary events associated with the tRNA clusters differed among the viruses, even within the same clade, because very few of the tRNAs were conserved among all of the chloroviruses. In addition, 39/41 chloroviruses encode a putative enzyme that modifies a tRNA to recognize a codon that is 12–13 times more prominent in the chloroviruses than their host, further acting to curb CUB. As well, it has not escaped our attention that Wobble, which has been extensively investigated and updated, may assist viral protein synthesis. For example, the two codons for tyrosine, UAU and UAC, can be recognized by the same tRNAFinally, we are aware that tRNAs are turning out to play other roles in cells besides protein synthesis , so it i