Infected groups  revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 10 Low-pathogenic avian influenza (LPAI) H9N2 virus has been endemic in Bangladesh since 2006 and causes significant economic losses in the poultry industry . H9N2 viSince the first outbreak in 2006, H9N2 viruses of the G1 lineage have been frequently identified in clinical samples from commercial and backyard poultry and in surveillance samples from live bird markets (LBMs) in Bangladesh ,9,10. MoIn recent years, H9N2 virus has attained further importance due to its involvement as a donor in the reassortment of zoonotic AIV of subtypes H5, H7, and H10 . H9N2 viIn Bangladesh, commercial poultry farming has been growing rapidly since the early 1990s . PoultryTrachea and lung tissue samples  obtained at necropsy of affected chickens were collected from four commercial poultry farms in Mymensingh division in the North of Bangladesh during 2020 and 2021. All sampled flocks had a history of respiratory distress, and hemorrhage, and congestion in the trachea, lung, and pancreas . Tissue ® universal probe one-step RT-PCR kit  was used to prepare 12.5 µL volume of reaction mixture containing the following: 6 µL master mix, 1 µL forward primer (10 pmol/µL), 1 µL reverse primer (10 pmol/µL), 0.5 µL enzyme mix, 1.5 µL RNase-free water, and 2.5 µL of extracted RNA. The reaction was conducted in a 7500 fast real-time PCR machine  using the following thermal profile: reverse transcription at 55 °C for 2 min, initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation  and annealing .Viral RNA was extracted using the QIAamp Viral RNA Mini Kit  following the manufacturer’s instructions and quantified using a Nanodrop One . RNA was then tested for the Matrix (M) gene of AIV and for HA H9 and NA N2 subtype-specific segments by an established RT-qPCR utilizing a set of gene-specific primers ,19. LunaSelected RT-qPCR-positive H9N2 samples were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. The eggs were incubated at 37 °C for 96 h. Subsequently, allantoic fluid (AF) was harvested and stored at −80 °C until use. The harvested AF was subjected to a hemagglutination test (HA) using 1% chicken red blood cells (RBCs) using standard procedures . The prehttps://mafft.cbrc.jp/alignment/server/; accessed on 13 September 2022). Phylogenetic analysis was performed using the neighbor joining method and Jukes-Cantor substitution model incorporated in the MAFFT package, with the confidence intervals estimated by applying a 1000 bootstrap algorithm [http://tree.bio.ed.ac.uk/software/figtree/; accessed on 14 September 2022) and Inkscape 1.0 . The obtained full genome sequences of four Bangladeshi H9N2, A/chicken/Bangladesh/2458-LT2/2020 (LT_2), A/chicken/Bangladesh/2462-LT14/2020 (LT_14), A/chicken/Bangladesh/2465-LT56/2021 (LT_56), and A/chicken/Bangladesh/2474-LT86/2021 (LT_86) were deposited in the GISAID database  of the virus stocks was calculated based on serial 10-fold dilutions in PBS and culture in 10-day-old embryonated chicken eggs following standard procedures [Among the respective four H9N2 field isolates, LT_2 and LT_56 were selected for the experimental pathogenesis study as they were obtained in 2020 and 2021. The 50% egg infectious dose (EIDocedures .n = 70) and broiler (n = 70) breeds were obtained from a leading, trusted commercial company and raised with food and water ad libitum. At the age of 42  and 21 (broiler) days, AIV-seronegative chickens were divided into three groups for each bird type: the LT_2 infected Sonali group (LT_2-So1) (n = 30), LT_56 infected Sonali group (LT_56-So2) (n = 30), and control Sonali group (So3) (n = 10); similarly, broilers were distributed to LT_2 infected broiler (LT_2-Br1) (n = 30); LT_56 infected broiler (LT_56-Br2) (n = 30) and a control broiler group (Br3) (n = 10)  . The difEID50/mL ,26 of thThe birds in each group were inspected twice a day for any behavioral changes or clinical manifestations. A full necropsy was performed on each bird. At necropsy, tissue samples  were collected in 10% neutral buffered formalin for microscopic examination and in sterile tubes for virus detection. Subsequently, the formalin-fixed tissues were embedded in paraffin. The tissue sections were prepared and stained with hematoxylin and eosin (H&E) stain as per the standard method . The staTo assess the viral distribution and shedding in the trachea, lungs, and intestine of necropsied birds as well as in oropharyngeal and cloacal swabs of dpi 1, 3, 5, 7, 10, and 15, tissue suspensions or swabs were prepared in 2 mL of minimal essential medium supplemented with penicillin and streptomycin. A single stainless-steel bead (5 mm) was added to each organ sample and homogenized in a 2 mL collection tube for 2 min using a Tissue Lyser instrument . RNA was extracted from both tissue suspensions and swab samples. RT-qPCR was performed as described above. Cycle threshold (Ct) value was used to semi-quantify the viral load after RT-qPCR targeting the H9 gene. A Ct value of <40 was used as a threshold for positive results. Hemagglutination inhibition (HI) assays were performed following standard guidelines . Sera weStatistically significant differences between Ct values from the H9-specific RT-qPCR obtained at different organs of experimentally infected LT_2 and LT_56 in Sonali and broiler chickens were determined by ANOVA. A one-way ANOVA with Tukey’s multiple comparison test was performed to elicit whether the antibody titers varied significantly among the pre- and post-infected groups. Results were plotted using GraphPad Prism 5 .Four strains  of low-pathogenic H9N2 viruses were successfully propagated in embryonated chicken eggs, identified by RT-qPCR, and fully sequenced. All eight genome segments were characterized. Phylogenetic analysis based on the HA and NA gene sequences revealed characteristics of the G1 lineage of H9N2 viruses in Bangladesh . The stuNo mortality or clinical signs were observed in both infected and control Sonali or broiler chickens throughout the experimental periods. One control and five infected birds were euthanized at 1, 3, 5, 7, 10, and 15 dpi and examined at necropsy. At necropsy, the tracheas of infected Sonali chickens (LT_2-So1 and LT_56-So2) showed slight hemorrhages along the tracheal rings up to 15 dpi . In the Histopathological findings in the tracheas of infected Sonali chickens (LT_2-So1 and LT_56-So2) revealed slight proliferation of goblet cells from the beginning of the experiment, focal loss of lining epithelium and cilia, and slight infiltration of inflammatory cells at 3 dpi b,c. In SIn the intestine of Sonali chickens, no histological lesions were observed on 1 dpi; however, at 3 dpi, intestinal villi were severely lost and crypts were visible. The desquamated epithelial cells were accumulated in the lumen of the intestine b. At 5 dAltogether, both infected groups of broiler chickens (LT_2-Br1 and LT_56-Br2) showed similar histopathological changes in the trachea and lungs as the Sonali chickens. The detailed histopathological scoring of different parameters of the trachea, lungs, and intestine of Sonali and broiler chickens infected with H9N2 is shown in p < 0.05) in the cases of LT_2-So1 and LT_56-So2 compared with the LT_2-Br1 group of birds, according to a two-way ANOVA with Bonferroni post hoc testing. The infected chickens shed viruses via oropharyngeal and cloacal routes. Viruses were detected in the oropharyngeal swabs between 1 and 10 dpi and in cloacal swabs between 3 and 10 dpi  were studied by RT-qPCR targeting the H9 gene segment. In LT_2 and LT_56 infected Sonali chickens, viral RNA was detected in the trachea, lungs, and intestine from 1 dpi to 10 dpi. The highest viral load was detected at 3 and 5 dpi in the trachea, lung, and intestine. The viral RNA load decreased progressively from 7 dpi, and no RNA was detected after 15 dpi. Similar viral dissemination kinetics were observed in the tissues of infected broiler chickens. The overall viral load in the lungs and intestine of Sonali chickens was incomparable to broiler chickens . Howeverd 10 dpi . No signAll chickens were considered susceptible to AIV before infection. The kinetics of antibody formation by means of the hemagglutination inhibition (HI) test are presented in Since 2006, the low-pathogenic avian influenza (LPAI) H9N2 virus has been endemic in Bangladesh, causing huge economic losses in the poultry industry. The current study describes the genetic characteristics of recently circulating H9N2 avian influenza viruses and their pathogenicity in crossbred Sonali and Cobb 500 broiler chickens under experimental conditions. Genetic analysis of H9N2 LPAIV has identified several distinct lineages, with the G1 lineage being the most prevalent and diversified. The G1 lineage has spread to different countries in Asia, Africa, and the Middle East , whereasResults of previous in vitro studies were unequivocal as to whether the evolution of a tri-basic HACS already induced phenotypic changes in vivo or whether a true furin-sensitive HACS motif such as R-X-K/R-R was required for trypsin-independent, systemic replication . Here weAlthough no clinical signs were observed in any group of infected chickens, moderately severe pathological lesions were evident specifically in their respiratory systems (trachea and lungs), similar to previous studies ,34, and Replication of LPAIVs is limited to trypsin-expressing epithelial cells lining the respiratory and gastrointestinal tracts ,38. In tThe avian influenza H9N2 viruses prevailing in commercial poultry in Bangladesh belong to the G1 lineage and express a tri-basic HACS and multiple mutations at antigenic sites. The pathogenicity of two LPAI H9N2 subtype viruses isolated recently in Bangladesh was assessed in Sonali (crossbreed) and Cobb 500 broiler chickens. Under experimental conditions, both virus isolates did not produce any mortality or clinical signs in either of the infected bird types. The viruses demonstrated tropism mainly for the respiratory and gastrointestinal tracts, while more virus was shed through the oropharyngeal route up to 10 dpi. Virus shedding was found to be more prolonged in Sonali chickens. The presence of a tri-basic HACS, although only one mutation step away from a bona fide highly pathogenic HACS, did not increase the pathogenicity of the viruses.


CYP3A4*22 (rs35599367), CYP3A5*3 (rs776746) single nucleotide polymorphism (SNP) with response to simvastatin in hypercholesterolemia patients conducted at King Abdulaziz University hospital (KAUH) in Jeddah, Saudi Arabia.Inter-individual variability in response to statin was mainly due to genetic differences. This study aimed to investigate the association of A total of 274 participants were registered in the current study. Hypercholesterolemic patients taking simvastatin 20 mg (n = 148) and control subjects (n = 126) were tested for rs35599367 and rs776746 genotypes using Custom Taqman ® Assay Probes. Response to simvastatin in these patients was assessed by determination of low density lipoprotein (LDL-C), total cholesterol (TC) and by measuring statin plasma levels using Liquid Chromatography-Mass Spectrometry (LC-MS).CYP3A4*22 mutant genotype, while 12 (4.4%) individuals had a heterozygous genotype and 262 (95.6%) had a wild homozygous genotype. The CYP3A5*3 allele was detected in the homozygous mutant form in 16 (5.8%) individuals, while 74 (27.0%) individuals carried the heterozygous genotype and 184 (67.2%) carried the wildtype homozygous genotype. Of the patient group, 15 (11%) were classified as intermediate metabolizers (IMs) and 133 (89%) as extensive metabolizers (EMs). Plasma simvastatin concentrations for the combined CYP3A4/5 genotypes were significantly (P<0.05) higher in the IMs group than in the EMs group. TC and plasma LDL-C levels were also significantly (P<0.05) higher in IMs than in EMs.None of the participants carried a homozygous CYP3A4*22 (rs35599367) and CYP3A5*3 (rs776746) SNP combination genotypes with response to statins in hypercholesterolemia. Patients who had either a mutant homozygous allele for CYP3A5*3 or mutant homozygous and heterozygous alleles for CYP3A4*22 showed increased response to lower TC and LDL-C levels.The present study showed associations between  Hypercholesterolemia and its clinical manifestations such as cardiovascular diseases (CVDs) are the leading cause of death worldwide, both in developed industrialized and developing countries , 2. AccoThe main group of drugs used as standard and first-line therapy in patients with hypercholesterolemia are statins, also known as 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors , 18. AccP450 3A5 (CYP3A5) and cytochrome P450 3A4 (CYP3A4), [CYP3A4 and CYP3A5) is responsible for statin metabolism depends on the dose of statin administered and the patient [CYP3A4 is thought to affect the blood level of the drug, the kinetic disposition of statin metabolites and subsequently the lipid response [CYP3A4 polymorphisms have reported interindividual variations in statin response associated with genetic differences, and occurs when a variant allele replaces one or both wild-type alleles [CYP450 enzyme that has reduced or no activity. Persons with two copies of variant alleles are poor metabolizers, those with one wild-type and one variant allele have reduced activity, whereas those who inherit multiple copies of wild-type alleles have excessive enzyme activity.Among the available statins in the market, simvastatin, atorvastatin, and lovastatin are metabolized by cytochrome CYP3A4), . The ext patient . Since iresponse , 31. Sev alleles , 33. VarCYP3A4*22 (rs35599367), CYP3A5*3 (rs776746) SNPs and combined CYP3A4/5 genotypes on the response to statins in hypercholesterolemic patients treated at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia.In Saudi Arabia, studies showed high rates of hypercholesterolemia in both Saudis and Non-Saudis residents. As a result, statins were highly prescribed to lower LDL-C levels. From the latest annual statistics book of the Ministry of Health in Saudi Arabia (MOH) for 2018 G, the number of emergency admissions due to heart disease in Saudi Arabia was 149,090 cases, with Saudi patients accounting for 122,477 cases. The number of deaths due to circulatory diseases was reported to be 8,924 cases in 2012 G . Al-NozhCYP3A4*22 and CYP3A5*3, and grouped into phenotypes because simvastatin is metabolized by both CYP3A4 and CYP3A5 due to high amino acid sequence similarity. With comparable expression of both CYPs in some individuals, the interaction between the two enzyme polymorphisms should be considered [CYP3A4*22 and CYP3A5*3 alleles, patients in the current study were classified into three categories: poor metabolizers (PMs), intermediate metabolizers (IMs), and extensive metabolizers (EMs) [Two hundred and seventy-four subjects were enrolled in this study and divided into two groups, either control subjects (126) (non-statin users) or hypercholesterolemic patients (148) taking simvastatin (Zocor® 20mg) and followed up at KAUH. All participants were of Arab ethnicity (Saudi and non-Saudi) having similar type of diet, life style and habit. Patients were excluded from lipid profile testing for genotyping: if they were taking other lipid-lowering medications such as fibrates, bile acid sequestrants, nicotinic acids, or ezetimibe; or if they were not taking the same simvastatin dose for at least six weeks prior to blood collection; or if they were taking medications that interact with simvastatin. Participants were genotyped for nsidered . Dependirs (EMs) , 39.CYP3A4*22, (GG) allele genotype was considered as expresser allele, while, (AA) and (GA) allele genotypes were considered as a decrease of function (DOF) alleles. For CYP3A5*3, (CC) and (CT) allele genotypes were considered as expresser alleles, while, (TT) allele genotype was considered as the loss of function (LOF) allele . PMs are individuals who carry the reduced expresser allele for CYP3A4*22 and non-expresser allele for CYP3A5*3. They are the individuals who carry a DOF for CYP3A4*22 (either AA or GA) and LOF for CYP3A5*3 which is the (TT) allele. IMs are individuals who carry the expresser allele of either CYPs but not both. They are individuals who carry expresser allele of CYP3A4*22 (GG) and non-expresser allele of CYP3A5*3 (TT) allele, or those who carry DOF allele for CYP3A4*22 (either AA or GA) and the expresser alleles for CYP3A5*3 (either CC or CT). EMs are individuals who carry the expresser alleles of both CYPs. They are individuals who carried expresser allele of CYP3A4*22 (GG) and CYP3A5*3 (either CC or CT).The classification of alleles expresser status for each CYP was done according to the following: for The protocol of the present study was approved by KAUH, Biomedical Ethics Research Committee. Written informed consents were obtained from all participants.Five milliliters (ml) of venous blood samples were obtained from each patient (2–4 hours after dosing with simvastatin) into tubes containing ethylene diamine tetraacetic acid (EDTA). The blood was immediately centrifuged for 15 minutes at 4,000 g and the collected plasma was preserved at -80°C until used for genotype and phenotype study.DNA was extracted from blood samples leukocytes by DNA mini kit QIAamp .DNA was quantified by spectrophotometer Nanodrop-2000 .CYP3A4*22 rs35599367 C>T) and (CYP3A5*3 rs776746 G>A) genotypes were detected by Custom Taqman Assay Probes using Real time PCR system 7500 fast . TaqMan Drug Metabolism Genotyping Assay (40X) was obtained from . PCR Pre- and post-PCR fluorescence measurements and genotype calls were made using 7500 fast Real time PCR system.Single nucleotide polymorphism (DNA was used from each patient in each 25 μl reaction (10–20 ng) according to standard PCR thermocycling conditions. This consisted of an initial cycle of 10 minutes at 95° C, followed by 40 cycles of denaturation at 92° C for 15 seconds, annealing and extension at 60° C for 1 minute.The high-performance liquid chromatography (HPLC) system comprised of Agilent 1200 system, autosampler, quaternary pump, solvent delivery mode, and column compartment . Detector utilized an Agilent 6420 triple quad mass spectrometer (TQ-MS) which was coordinated by MassHunter software. The screw-capped (PTFE/silicon) total recovery 1-ml autosampler vial, 12 millimeter (mm) was used. The injection volume was 20 μL . ChromatThe levels of TC and LDL-C were determined by standard kit methods using fully Automated COBAS ® 8000 modular analyzer series at KAUH, Jeddah, Saudi Arabia.Data analysis was performed using the software Statistical Package for the Social Sciences (SPSS) version 20 . Results were expressed as mean +/- standard error or number (%). Differences in the mean of the variables studied, including clinical and demographic characteristics, were tested using an unpaired Student "t" test, analysis of variance (ANOVA), or chi-square (χ2) test, depending on the type and number of variables studied. A P value less than 0.05 was considered significant.The demographic data of the patients are summarized in CYP3A5*3) alleles and for rs35599367 (CYP3A4*22) are summarized in Tables CYP3A4*22, the G allele was the most frequently observed in both Saudi (97.8%) and non-Saudi (97.6%) participants. Of all participants, 262 carried CYP3A4*22 wild homozygous (GG) with a genotype frequency of 95.60% and 12 participants carried heterozygous (GA) with a genotype frequency of 4.40%, with MAF 0.022. For the CYP3A5*3 SNP, C allele was the most frequent among both Saudi (77.0%) and non-Saudi (89.0%) participants. 184 participants carried wild homozygous (CC) with a genotype frequency of 67.20%; 16 participants carried mutant homozygous (TT) with a genotype frequency of 5.80% and 74 participants carried heterozygous (CT) with a genotype frequency of 27.00%, with MAF 0.193. The loci for rs35599367 (CYP3A4*22) was consistent with HWE (P = 0.701), indicating that the mutant allele was normally distributed in the population studied. While the loci for rs776746 (CYP3A5*3) was not consistent with HWE (P = 0.040), which meant that the mutant allele was not normally distributed in the studied population. In Saudi and non-Saudi subjects, the CYP3A5*3 allele was mutant homozygous (TT) in 13 and 3 participants with genotype frequencies of 6.80% and 3.53%, respectively, and wild homozygous (CC) in 114 and 70 participants with genotype frequencies of 60.40% and 82.35% and heterozygous (CT) and in 62 and 12 participants with genotype frequencies of 32.80% and 14.12%, respectively, with MAF of 0.233 and 0.106 respectively. In Saudi and non-Saudi subjects, the CYP3A4*22 allele was wild homozygous (GG) in 181 and 81 participants with genotype frequencies of 95.80% and 95.30%, respectively, and heterozygous (GA) in 8 and 4 participants with genotype frequencies of 4.20% and 4.70% with MAF of 0.021 and 0.024, respectively.TaqMan assay genotyping data for all individuals including mutant and wild-type genotypes for rs776746  genotype allele for CYP3A5*3 , heterozygous (GA) for CYP3A5*3 . HoweverCYP3A5*3 .CYP3A4/5 genotypes on LDL-C and TC and simvastatin plasma levels in patients with hypercholesterolemia is shown in The effect of combined CYP3A4*22 (rs35599367) for the wild type homozygous (GG) in all studied groups, in Saudi participants and in non-Saudi participants were similar to those reported in the 1000 Genomes database. Our results showed allele frequencies for G = 0.978 and A = 0.022. Allele frequencies for other populations showed that African , American , European  and South Asians . Meanwhile, the homozygous mutant allele (AA) was not detected in either Saudi or non-Saudi participants which could be explained by its low allele frequency and its occurrence mainly in the Caucasian population [In this study, the high frequencies of pulation , 42.CYP3A5*3 (rs776746) reported in the 1000 Genomes database varied among different populations. The frequencies of wild homozygous (CC) ranged from 0.01 to 0.89, and mutant homozygous (TT) ranged from 0.02 to 0.78 among different populations [CYP3A5*3 (rs776746) were 0.255 in Korean, 0.085 in European-American, 0.198 in African-American, 0.344 in Han Chinese and 0.260 in Japanese [Global genotype frequencies of ulations . Our resCYP3A4/5 genotype frequencies, it was considered that 90% of participants were EMs and only 10% were IMs. This is similar to previous studies that reported that most of the African population and African-Americans were classified as 90% EMs and 10% IMs [In our study, based on the combined  10% IMs , 42.Depending on this study a higher dose should be prescribed for EMs patients, which supports the earlier study by Kitzmiller et al. which found that most PMs patients took lower doses of simvastatin, while, most EMs patients took higher doses of the statin .CYP3A5*1/*1 allele than in carriers of the mutant homozygous CYP3A5*3/3*, and similarly for the combined CYP3A4/5 genotypes, plasma simvastatin concentration were found to be highest in PMs than IMs and least in EMs [We observed a significant difference in plasma statin concentrations in hypercholesterolemia patients between EMs and IMs in our study. In EMs the statin concentrations were 41.19 7.20 ng/ml which were significantly (P<0.05) decreased from 65.16 12.00 ng/ml in IMs. This resulted in a significant (P<0.05) increase in both LDL-C and TC. Other studies have also shown lower simvastatin concentrations in African-American carriers of the homozygous wild-type t in EMs .CYP3A4*22 mutant homozygous allele (AA) had a better lipid lowering response to simvastatin than homozygous wild-type allele (GG) [CYP3A5*3 (TT or CT) than expresser allele (CC) carrier. Similar observations were shown by Kivisto et al. in Caucasian patients receiving simvastatin and reported that LDL-C and TC levels were higher in patients with CYP3A5*3 wild-type homozygous CC or heterozygous CT than in patients with mutant homozygous TT [CYP3A*33 and rs776746 in CYP3A5*3 and phenotypes, and reported no significant differences between these SNPs and lipid-lowering response to simvastatin [CYP3A4*22 allele and lipid-lowering response to simvastatin and atorvastatin and explained their findings due to the effects of some confounding or uncontrolled factors [In addition, Elens et al. performed a study on patients on simvastatin, and found that patients who carried the ele (GG) . They alygous TT . Howevervastatin . This wa factors .Our work has some limitations. The number of non-Saudi participants in the study was limited. Further work with more Saudi and non-Saudi participants (with similar Arab ethnicity) need to be conducted to support our findings. Further information on the functional implications of the different alleles in these SNPs is also lacking. Functional studies are important to clarify the exact role of these SNP’s in relation to drug response.CYP3A4/5 in Saudi and non-Saudi populations of Arab ethnicity, as these two CYPs are involved in the metabolism of various drugs including statins. A lower simvastatin dose should be administered to patients who have either a mutant homozygous allele for CYP3A5*3 or mutant homozygous and heterozygous alleles for CYP3A4*22. On the other hand, a higher dose of simvastatin should be administered to patients with either a homozygous wild-type allele for CYP3A5*3 or a homozygous wild-type allele for CYP3A4*22.In conclusion, the current study shed light on the knowledge of two important SNPs in S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Plot(PDF)Click here for additional data file.


Advanced atherosclerotic lesions were observed in the left coronary artery of C57BL/6N apo E−/− TAC mice but not in C57BL/6N TAC mice. Pressure overload resulted in markedly increased cardiac hypertrophy and more pronounced heart failure in C57BL/6N apo E−/− TAC mice in comparison to C57BL/6N TAC mice. Pathological hypertrophy, as evidenced by increased myocardial fibrosis and capillary rarefaction, was more prominent in C57BL/6N TAC apo E−/− than in C57BL/6N TAC mice and led to more marked cardiac dysfunction. In conclusion, TAC in apo E deficient mice induces coronary atherosclerosis and aggravates the development of pathological cardiac hypertrophy and heart failure.Murine coronary arteries are very resistant to the development of atherosclerosis, which may be related to their intramyocardial course. Blood pressure promotes atherosclerotic plaque formation by acting as a physical force that potentiates the migration of pro-atherogenic lipoproteins across the endothelium. C57BL/6N apolipoprotein (apo) E deficient mice have increased remnant lipoproteins that are a risk factor for coronary atherosclerosis. In this study, our aim was to quantify coronary atherosclerosis and artery remodeling following transverse aortic constriction (TAC) in C57BL/6N apo E All pro-atherogenic lipoproteins contain apolipoprotein (apo) B. Retention of pro-atherogenic lipoproteins within the vessel wall is the obligatory initiating event of atherogenesis ,2. The rDistinct cardiovascular risk factors may have a differential effect on the endothelial cells and vascular smooth muscle cells at distinctive parts of the vascular tree . ObservaA particular observation is that the coronary arteries of mice are very resistant to the development of atherosclerosis . AtherosPatients with hypertension have increased presence, extent, and severity of coronary atherosclerosis quantified by coronary computed tomographic angiography . Blood p−/− TAC mice and 2) to evaluate the impact of increased remnant lipoproteins on the development of pressure overload-induced cardiac hypertrophy and heart failure.Since transverse aortic constriction (TAC) between the origin of the right innominate artery and the left common carotid artery induces a rise in blood pressure proximal to the constriction, this procedure raises coronary pressure. Notwithstanding this, coronary atherosclerosis is completely absent in several murine models of hypercholesterolemia or dyslipidemia following TAC ,22,23. I−/− mice, originally purchased from Taconic , were locally bred in the animal unit at Gasthuisberg and were fed standard chow diet . Apo E deficient mice from Taconic had been backcrossed to the C57BL/6N background for 11 generations and were backcrossed for one additional generation. All experiments were performed on female mice. In order to induce pressure overload, transverse aortic constriction (TAC) was performed at the age of 14 weeks, as described in detail before [−/− mice were assigned for hemodynamic quantification and morphometric and histological analyses 8 weeks following the sham or TAC operation. The second experimental layer consisted of mice that did not undergo perfusion fixation and that were used for unbiased quantification of tissue and organ weights 8 weeks after the TAC or sham procedure.All investigations were performed in accordance with the European legislation on protection of animals used for scientific purposes (Directive 2010/63/EU), and all experimental procedures on animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven . Mice were housed in filter top type II cages containing 5 mice or less in the semi-specific pathogen-free facility of KU Leuven at Gasthuisberg. A semi-natural 12 h cycle of light and dark was maintained, and temperature was kept in a range from 21 °C to 22 °C. C57BL/6N mice and C57BL/6N apo El before ,27. The g for 10 min and immediately stored at −80 °C.Blood was collected by puncture of the vena cava inferior at the end of the experiment just before euthanasia. Anticoagulation was performed with 0.1 volume of 136 mmol/L trisodium citrate. Subsequently, plasma was isolated by centrifugation at 1100× −/− mice as described previously [TM Red Kit .Murine lipoproteins were fractioned by fast performance liquid chromatography gel (FPLC) filtration  of 100 µL plasma obtained from C57BL/6N mice or from C57BL/6N apo Eeviously . CholestLipid peroxidation was analyzed by measurement of Thiobarbituric Acid Reactive Substances (TBARS). Quantification of TBARS was performed according to the instructions of the manufacturer .Invasive hemodynamic measurements were executed 8 weeks after sham operation or TAC using a 1.0 French Millar pressure catheter  as described. In order to perform these measurements, anesthesia was induced by intraperitoneal administration of 1.2 g/kg urethane . Hemodynamic data were registered with Powerlab Bridge Amplifier and Chart Software .Histological analysis was performed as described before . Mice thSirius Red staining was performed to quantify interstitial collagen content . Sirius Immunostaining for 3-nitrotyrosine with rabbit anti-nitrotyrosine antibodies  was applied to evaluate nitro-oxidative stress .Morphometric analyses of the left coronary artery were performed on Sirius Red-stained sections at 130 μm spaced intervals.t-test using GraphPad Instat . When indicated, a logarithmic transformation, a square root transformation, or a non-parametric Mann–Whitney test was conducted. The assumption of Gaussian distribution was checked using the method Kolmogorov and Smirnov. Parameters between four groups were compared by one-way analysis of variance followed by Bonferroni multiple comparisons post-test for comparing the sham groups, the TAC groups, and the sham versus respective TAC groups using GraphPad Instat. When the assumption of sampling from populations with identical standard deviations was not met, a logarithmic transformation or a square root transformation was applied. When the assumption of sampling from populations with Gaussian distributions was not met, a Kruskal–Wallis test was executed, followed by Dunn’s multiple comparisons post-test. Kaplan–Meier survival curves were analyzed by log-rank test using Prism4 (GraphPad Software). A two-sided p-value of less than 0.05 was considered statistically significant.At the completion of the study, data of all surviving mice were included in the analysis. All investigators who performed different endpoint analyses were blinded to group allocation. Unblinding of animal numbers corresponding to specific allocation groups was carried out at completion of measurements. All data are expressed as means ± standard error of the means (SEM). Parameters between two groups  were compared using Student’s −/− TAC mice compared to C57BL/6N TAC mice  during a follow-up period of 8 weeks .Transverse aortic constriction (TAC) was performed at the age of 14 weeks to induce pressure overload. Comparison of Kaplan–Meier survival curves by log-rank test demonstrated a pronounced and highly statistically significant increased mortality rate in C57BL/6N apo E 8 weeks . Eight wre alive . Sham op−/− mice. Since the genetic background may have a profound effect on cholesterol levels in apo E deficient mice [−/− mice backcrossed for 12 generations to the C57BL/6N background and wild-type C57BL/6N mice (p < 0.0001) higher in C57BL/6N apo E−/− mice than in C57BL/6N mice (−/− mice were 61.8% (p < 0.0001) lower than in C57BL/6N mice  increase in the area within the external elastic lamina. In addition, pressure overload in C57BL/6N TAC mice resulted in media hypertrophy, as evidenced by a 1.73-fold (p < 0.001) increase in the medial area compared to C57BL/6N sham mice. The luminal area was 2.50-fold (p < 0.001) higher in C57BL/6N TAC mice than in C57BL/6N sham mice. These changes reflect the increased distending pressure in the coronary arteries of C57BL/6N TAC mice.Data on coronary artery remodeling and coronary atherosclerosis are provided in −/− sham mice. In contrast, advanced lesions were observed in the left coronary artery of C57BL/6N apo E−/− TAC mice  in C57BL/6N apo E−/− TAC mice compared to C57BL/6N TAC mice. However, because of outward remodeling, the luminal area was 1.73-fold (p < 0.01) higher in C57BL/6N apo E−/− TAC mice than in C57BL/6N apo E−/− sham mice , area within the internal elastic lamina (IEL), medial area, intimal area, luminal area, and percentage stenosis in the left coronary artery in the four experimental conditions.p < 0.001) in C57BL/6N TAC mice and by 2.62-fold (p < 0.001) in C57BL/6N apo E−/− TAC mice compared to respective sham groups. Cardiac weight was 1.30-fold (p < 0.001) higher in C57BL/6N apo E−/− TAC mice than in C57BL/6N TAC mice (p < 0.001) higher in C57BL/6N TAC mice (p < 0.001) and 2.59-fold (p < 0.001) higher in C57BL/6N apo E−/− TAC mice than in respective sham groups (p < 0.05) in C57BL/6N apo E−/− TAC mice compared to C57BL/6N TAC mice (p < 0.001) in C57BL/6N TAC mice and by 1.92-fold (p < 0.001) in C57BL/6N apo E−/− TAC mice compared to respective sham groups (p < 0.001) higher in C57BL/6N TAC mice and 2.29-fold (p < 0.001) higher in C57BL/6N apo E−/− TAC mice than in respective sham groups (p < 0001) in C57BL/6N apo E−/− TAC mice compared to C57BL/6N TAC mice, indicating much more pronounced heart failure in the former  in C57BL/6N TAC mice and by 25.6% (p < 0.001) in C57BL/6N apo E−/− TAC mice compared to respective sham groups (p < 0.05) lower in C57BL/6N apo E−/− TAC mice than in C57BL/6N TAC mice  in C57BL/6N apo E−/− TAC mice compared to C57BL/6N TAC mice (p < 0.001) higher in C57BL/6N apo E−/− TAC mice than in C57BL/6N TAC mice  decrease in the absolute value of the maximal rate of isovolumetric relaxation and a 1.12-fold (p < 0.05) increase in the time constant of isovolumetric relaxation (p < 0.05) increase in end-diastolic pressure compared to wild-type TAC mice  increase in the time constant of isovolumetric relaxation (Diastolic dysfunction was observed in C57BL/6N apo Elaxation . PressurTAC mice . Diastollaxation .p < 0.01) in C57BL/6N apo E−/− sham mice compared to C57BL/6N sham mice (p < 0.01) higher in C57BL/6N TAC mice and 1.57-fold (p < 0.05) higher in C57BL/6N apo E−/− TAC mice compared to respective sham groups. The level of lipid peroxidation products was increased by 1.61-fold (p < 0.001) in C57BL/6N apo E−/− TAC mice compared to C57BL/6N TAC mice (p < 0.001) and in TAC mice  (Lipid peroxidation was quantified by the measurement of Thiobarbituric Acid Reactive Substances (TBARS) in plasma. TBARS were increased by 1.85-fold (ham mice a. PressuTAC mice a. The saTAC mice b. The ef< 0.001) b. Repres−/− TAC mice is much more marked than in wild-type mice as evidenced by the increased end-diastolic pressure and the remarkable augmentation of the lung weight; (4) increased oxidative stress is not only observed in TAC mice but also in C57BL/6N apo E−/− sham mice, indicating that apo E deficiency is a model of increased oxidative stress.The main findings of this study are that (1) increased coronary artery pressure induced by TAC on the one hand and increased remnant lipoproteins, on the other hand, interact to produce coronary atherosclerosis in apo E deficient mice; (2) pressure overload induced by TAC leads to much more pronounced cardiac hypertrophy and much more prominent features of pathological hypertrophy in apo E deficient mice compared to wild-type mice; (3) heart failure in C57BL/6N apo E−/− mice as well as in low-density lipoprotein receptor (LDLr) deficient mice are the aortic root, the lesser curvature of the aortic arch, and the brachiocephalic artery [Predilection sites for atherosclerosis in apo Ec artery ,34,35,36c artery . This mac artery . Similarc artery . Thus, ic artery . In specc artery . A singlc artery ,38,39. Oc artery ,38. WhenCoronary atherosclerosis in humans develops in the epicardial coronary arteries and not in the intramyocardial arteries . Therefo−/− TAC mice. Moreover, increased pressure is the cause of media hypertrophy [−/− TAC mice. In contrast to the presence of extensive lesions in female C57BL/6N apo E−/− TAC mice, no coronary atherosclerosis at all has previously been observed in female C57BL/6J LDLr−/− TAC mice on standard chow [−/− mice on a 0.2% cholesterol 10% coconut oil are higher than in C57BL/6N apo E−/− mice fed standard chow. Therefore, differences in the development of coronary atherosclerosis must be related to differences in the nature of pro-atherogenic lipoproteins between apo E deficient mice and LDLr deficient mice, properties of apo E independent of its effects on lipid levels, or differences in the genetic background (C57BL/6N versus C57BL/6J). Apo E deficient mice are characterized by increased concentration of chylomicron remnants and VLDL remnants, which are contained in β-VLDL particles and intermediate density lipoprotein (IDL) particles [TAC in mice will increase coronary artery pressure and the distending pressure in the coronary artery. Changes in coronary pressure affect coronary dimensions . The incertrophy . The strard chow  or on a ard chow . This caarticles ,47,48. Larticles ,50. Furtarticles . Therefoarticles ,25,52.+ at the price of oxidation of NADH oxidation and proton re-entry into the mitochondrial matrix. Functional loss of this enzyme due to this spontaneous mutation in C57BL/6J mice results in mitochondrial redox abnormalities that result in a poor capacity to sustain NADP and glutathione in their reduced states. This mutation may be relevant both for the development of atherosclerosis [−/− TAC mice and the absence of coronary atherosclerosis in C57BL/6J LDLr−/− TAC mice. However, since the mutation in the nicotinamide nucleotide transhydrogenase gene is only one of many genetic factors that may affect cardiovascular phenotypic differences between C57BL/6J and C57BL/6N mice [Apart from its effects on lipoprotein metabolism and lipoprotein levels, lipoprotein-independent functions have also been attributed to apo E . These fclerosis ,58 and f/6N mice ,57, we c−/− TAC mice compared to C57BL/6N apo E−/− sham mice. Therefore, TAC in C57BL/6N apo E−/− mice remains a model of non-ischemic cardiomyopathy. The direct impact of hypercholesterolemia on cardiac hypertrophy and cardiac function has previously been demonstrated both in the absence of pressure overload [TAC leads to much more marked cardiac hypertrophy and much more prominent features of pathological hypertrophy in apo E deficient mice compared to wild-type C57BL/6N mice. Notwithstanding the presence of coronary atherosclerosis, the luminal area was increased in C57BL/6N apo Eoverload ,60,61 anoverload ,22. Hypeoverload ,62 and ioverload . Oxidatioverload ,65,66. Hoverload ,68.−/− TAC mice resulted in pronounced heart failure, as evidenced by the very marked increase in the wet lung weight, which is much more prominent compared to other models of TAC-induced heart failure [−/− sham mice [Pathological hypertrophy and cardiac dysfunction in C57BL/6N apo E failure ,22. This failure ,69,70,71 failure . This ma failure  and oxid failure  contribuham mice .TAC in C57BL/6N apo E deficient mice resulted in higher mortality than in C57BL/6N mice. This may partially reflect a higher incidence of death caused by acute heart failure induced by the hemodynamic effects of pressure overload but may also reflect an increased incidence of lethal arrhythmias. The pro-arrhythmic effects of hypercholesterolemia may be mediated through direct interactions between cholesterol and the membrane proteins, through alterations of membrane fluidity, and/or the impact of hypercholesterolemia on lipid rafts ,75.−/− TAC mice also constitute a model of severe oxidative stress and heart failure and may be used to evaluate novel therapeutic heart failure strategies in mice.The current study highlights the importance of interaction between different risk factors in the development of coronary atherosclerosis and potentially also in the development of heart failure. In clinical medicine, the effects of two causes are frequently not additive, but the impact of one causal variable on an outcome may be dependent on the state of a second causal variable. In particular, interactions between genotype and environmental factors should be considered, and the impact of genetic risk factors may therefore be conditional on specific environmental exposures. Murine atherosclerosis models are generally hampered by the absence of coronary atherosclerosis. The model investigated in this study offers perspectives for evaluating the effect of pharmacological interventions in a robust model of coronary atherosclerosis in mice. The C57BL/6N apo E−/− sham mice.In conclusion, the interaction between increased remnant lipoproteins and increased coronary pressure induced by TAC produces advanced coronary atherosclerosis in apo E deficient mice. Pressure overload-induced cardiomyopathy in apo E deficient mice is characterized by a very severe phenotype with prominent features of pathological remodeling and very pronounced heart failure. Apo E deficient mice are a model of increased oxidative stress, which may explain diastolic dysfunction in C57BL/6N apo E


Moreover, Sal-H, Sal-Gd(III), and Sal-Mn(II) were able to induce perturbations in the cell cycle of treated colorectal and breast human cancer cell lines . The relaxivity (r1) values of both complexes as well as of the loaded BGs, were higher or comparable to the relaxivity values of the clinically applied contrast agents gadopentetate dimeglumine and gadoteridol. Conclusion: This research is the first assessment that demonstrates the potential of Gd(III) and Mn(II) complexes of Sal as theranostic agents for MRI. Due to the remarkable selectivity and mode of action of Sal as part of the compounds, they could revolutionize cancer therapy and allow for early diagnosis and monitoring of therapeutic follow-up.Combining therapeutic with diagnostic agents (theranostics) can revolutionize the course of malignant diseases. Chemotherapy, hyperthermia, or radiation are used together with diagnostic methods such as magnetic resonance imaging (MRI). In contrast to conventional contrast agents (CAs), which only enable non-specific visualization of tissues and organs, the theranostic probe offers targeted diagnostic imaging and therapy simultaneously. Methods: Novel salinomycin -based theranostic probes comprising two different paramagnetic metal ions, gadolinium(III) (Gd(III)) or manganese(II) (Mn(II)), as signal emitting motifs for MRI were synthesized and characterized by elemental analysis, infrared spectral analysis (IR), electroparamagnetic resonance (EPR), thermogravimetry (TG) differential scanning calorimetry (DSC) and electrospray ionization mass spectrometry (ESI-MS). To overcome the water insolubility of the two Sal-complexes, they were loaded into empty bacterial ghosts (BGs) cells as transport devices. The potential of the free and BGs-loaded metal complexes as theranostics was evaluated by in vitro relaxivity measurements in a high-field MR scanner and in cell culture studies. Results: Both the free Sal-complexes (Gd(III) salinomycinate  and Mn(II) salinomycinate ) and loaded into BGs demonstrated enhanced cytotoxic efficacy against three human tumor cell lines  relative to the free salinomycinic acid  and its sodium complex  applied as controls with IC Cancer theranostics, the combination of a diagnostic with a therapeutic, pave the way for personalized medicine, allowing for simultaneous and precise diagnosis and treatment of malignant diseases. Current theranostic concepts aim at (i) facilitating the observation and monitoring of anticancer drugs, (ii) providing targeted and personalized cancer therapy, and (iii) realizing simultaneous diagnosis and treatment in (early-stage) cancer patients . MagnetiThe design of new CAs plays an essential role in contrast-enhanced MRI today. Currently used CAs are predominantly low-molecular-weight gadolinium(III) (Gd(III))-based complexes, which can provide outstanding positive MR images with high resolution . Unfortu2+, Cu2+, Co2+, and Ni2+, which exerted superior anticancer activity in vitro compared to the free ligand +, calcd: 906.40 (100%), 908.40 (88.0%), 904.40 (82.4%), 905.40 (63%); 1657.56 [C42H69O11)2Gd]+, calcd: 1656.89 (100%); 1657.90 (90.9%); 1658.90 (88%); 1654.89 (82.4%); 1659.90 (80%); 1655.89 (74.9%); 1655.89 (63%).The metal salt  was added to the ligand solution. The reaction mixture was stirred at room temperature for 30 min. The solution slowly evaporated, and the resulting light-brownish precipitate was washed with water, filtered off, and dried over P4O10 for three days. Yield: 329 mg, 83%. Anal. Calcd. for C84H142O24Mn (MW = 1590.94 g/mol): H, 8.93%; C, 63.36%; Mn, 3.45%. Found: H, 9.35%; C, 63.05%; Mn, 3.24%. ESI-MS, m/z: 804.76 [(C42H69O11)Mn]+, calcd.: 804.42 (100%); 805.43 (45.4%); 1578.55 [(C42H69O11)2MnNa]+, calcd: 1576.90 (100%); 1577.90 (90.9%); 1578.90 (40.8%).Salinomycin sodium  was dissolved in mixed solvents (20 mL MeOH + 2 mL MeCN). The solution of Mn(II) chloride  and Mn(II) complexes of salinomycin are insoluble in water and soluble in organic solvents, such as MeOH, MeCN, EtOH, CHClEscherichia coli Nissle 1917 (EcN1917) and Escherichia coli NM522 BGs (NM522) were successfully loaded by simple resuspension of BGs  with each Sal-complex solutions , Sal-Mn(II), SalNa, and SalH) in methanol. Ten mg of each Sal-based compound were used for loading EcN1917 and 5 mg for NM522. The BGs suspensions were stirred for two hours at RT. Subsequently, the loaded BGs were collected by centrifugation at 11 300 g for 15 min, and the pellets were washed three times with Milli-Q® water. One mL aliquots were stored at −20 °C for further analyses.Non-pathogenic ® H2O (1:1) and immediately centrifuged at 11,300× g for 15 min at 4 °C. FPLC analysis was performed using an AKTÄ Purifier 10 System®  equipped with a Superdex 10/300 GL column , and UV and conductivity detectors. MeOH-acetate (65:35) was used as an eluent. The quantification was carried out using the peak area method applying free Sal-Mn(II) complex as an external standard.In order to determine the amount of Sal-complex within the BGs, 4 mg loaded BGs were resuspended in 0.5 mL of 96% ethanol , followed by 5 min of ultrasonication. Subsequently, the ethanolic extract was diluted equally with Milli-QThe infrared spectra of both compounds were recorded on a Specord-75IR  in a nujol mull.The EPR analysis was performed on a Bruker EMX PremiumX instrument . All measurements were carried out in the X-band at a frequency of microwave electromagnetic radiation 9.45 GHz. Quantitative analysis was performed using Bruker software (Version 1.1b 119) equipped with a spin count option. For temperature variation, a variable temperature unit, ER4141VTM, was used.2O (10% H2O), positive mode, in the range of 0–2000 m/z.ESI-MS spectra were recorded on Waters Micromass ZQ2000 Single Quadrupole Mass spectrometer  in MeOH/H® STA-449 F3 Jupiter instrument from 40 to 800 °C under airflow of 20 mL·min−1 as carrier gas with a heating rate of 10 °C min−1. Simultaneous thermal analyses allow the measurement of mass changes and thermal effects in the range of 150 °C to 2400 °C. The percentage of mass loss was estimated in the temperature range of 40–800 °C.Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) was executed using a Netzsch®) and gadoteridol (ProHance®) as positive controls. Serial dilutions of BGs loaded with the salinomycin complexes were carried out in ultra-pure Milli-Q® water ranging from 0.02 to 0.1 mM for Mn(II) salinomycinate or Gd(III) salinomycinate, respectively. For the free salinomycinate complexes, six dilutions ranging from 0.01 mM to 3.3 mM (for the Gd(III) salinomycinate) and from 0.02 mM to 1.4 mM (for the Mn(II) salinomycinate) in MeOH with a volume of 0.5 mL were prepared. All probes were measured in Eppendorf safe-lock polypropylene tubes with a diameter of 0.4 cm. The tubes were placed in the center of a plastic box and measured at ambient temperature.As a reference substance, pure MeOH was used as a negative control, and gadopentetate dimeglumine .Relaxivity measurements were conducted on a high-field MRI scanner . T1 spin-lattice relaxation times, spin echo inversion recovery (IR) sequences with inversion times from 0 to 3500 ms  were used. An adiabatic pulse was applied for B1-insensitive inversion. The other parameters were TR/TE = 5000/8.1 ms; flip angle 180°; 8 turbo factor 11; FOV 180 × 180 mm; resolution matrix 192 × 192; bandwidth 260 Hz/pixel; and slice thickness 3 mm. For all measurements, the relaxation rates (R1) and the relaxivities (r1) were calculated for each substance. The relaxivities  were calculated as the slope of the linear regression of R1 and R2 as a function of the contrast agent concentration.To determine the v/v) non-essential amino acid solution, and 10% (v/v) heat-inactivated fetal calf serum (from BioWest) in 75 cm² flasks at 37 °C under a humidified atmosphere containing 5% CO2 in the air. MCF-7 cells were cultured in High Glucose Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% (v/v) fetal calf serum , L-glutamine (4 mM) and 0.01 mg/mL of human insulin. All cell culture media, supplements, and assay reagents were purchased from Sigma-Aldrich, and all plastic ware from Starlab unless stated otherwise.Four adherently growing human cancer cell lines were used for this study: CH1/PA-1 (ovarian teratocarcinoma) cells were a gift from Lloyd R. Kelland , whereas A549  and SW480 (colon carcinoma) cells were kindly provided by the Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria, and MCF-7 (mammary carcinoma) cells by the Department of Pharmaceutical Sciences, University of Vienna. The first three cell lines were grown in Eagle’s minimal essential medium (MEM) supplemented with L-glutamine (4 mM), sodium pyruvate (1 mM), 1% , serially diluted in supplemented MEM not to exceed a final DMSO content of 0.5% (v/v) and added in 100 μL per well. After 96 h, the drug-containing medium was replaced with 100 μL of an RPMI 1640/MTT mixture  per well. After incubation for four hours, the MTT-containing medium was replaced with 150 μL DMSO per well to dissolve the formazan product formed by viable cells. Optical density at 550 nm (and at a reference wavelength of 690 nm) was measured with a microplate reader . The 50% inhibitory concentrations (IC50) relative to untreated controls were interpolated from the concentration-effect curves. At least three independent experiments were performed, each in triplicate per concentration level.The antiproliferative activity of the compounds was determined by the colorimetric MTT assay -2,5-diphenyl-2H-tetrazolium bromide). The 1 × 104 cells per well in 1 mL of the corresponding medium. After a recovery time of 24 h, cells were treated with different concentrations of Sal-H, Sal-Mn(II), and Sal-Gd(III). For this purpose, the test substances were dissolved in DMSO and diluted in a medium such that the maximum concentration of DMSO in the cells did not exceed 0.5%. For assay validation, the well-known cell cycle inhibitors etoposide and gemcitabine were applied as positive controls. Plates were incubated with test compounds for 24 h at 37 °C, 5% CO2. Following the exposure, the medium was completely removed, and adherent cells were gently washed twice with ice-cold PBS. Propidium iodide  and HFS-buffer (0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate in Milli-Q® water) were mixed for staining to yield a final PI concentration of 40 μg/mL. The ice-cold staining solution was added to the cells (500 μL/well). The staining was carried out overnight at 4 °C in the dark. To prepare the samples for measurement, the staining solution was vigorously pipetted against the surface to achieve sufficient resuspension of the cells. For each sample, 200 μL of the stained cell suspensions were added to a 96-well round-bottom FACS plate . The fluorescence of all samples stained with a DNA-intercalating agent was measured no longer than 24 h after staining using a flow cytometer . GuavaSoft™ software was used in the InCyte-modus, and 10,000 events/probes were counted.Colon carcinoma cells (SW480) and breast adenocarcinoma cells (MCF-7) were seeded in 12-well plates  in density of 8 × 10The flow cytometry data sets were evaluated in FlowJo software (v 10.8.1). The single-cell populations were gated based on forward and side scatters characteristics. The Watson Pragmatic model was applied as a standard method to analyze the resulting histograms: the recorded red fluorescence intensities of cells in the S phase were located between normally distributed G1/G0 and G2/M peaks. The number of cells in each phase was calculated from the model by means of integration. Means and standard deviations were calculated from at least three independently performed experiments. The figures were adjusted in GIMP freeware (v 2.10.24).42H69O11)3(H2O)3] and [Mn(C42H69O11)2(H2O)2] respectively. The ESI-MS spectra of both complexes corresponded to the elemental analysis and contained several signals due to the dissociation of water molecules, ligand anions, and/or complexation with Na+  and Mn(II) to form homometallic mononuclear complexes of composition 2+ was known to be 9.4 mT and, thus, approximated the corresponding constant found for the Mn(II) salinomycinate complex , [Ni(C42H69O11)2(H2O)2], [Cu(C42H69O11)2(H2O)2], [Zn(C42H69O11)2(H2O)2], and [Cd(C42H69O11)2(H2O)2] discussed previously [The IR spectra of salinomycin with Mn(II) and Gd(III) reported in this study were similar to the IR spectra of [Co6]2+ is 9.4 mT 4−), in which the relaxation enhancement arises from the Mn(II) ion released from the complex in vivo  reported for Mangafodipir®. The contrast efficiency of the loaded BGs with Sal-Gd(III) (r1 = 6.674 1/mM·s) improved approximately 1.8 times compared to the clinically applied Magnevist® (r1 = 3.76 [1/mM·s]).All Sal-complexes loaded into BGs showed a superior signal enhancement in MRI, with The summarized proton relaxivities of our Gd(III) and Mn(II) complexes of salinomycin with regard to various published low-molecular-weight paramagnetic complexes ere shown in T1 values and the calculated relaxivities (r1), it is known that, at higher field strength, the relaxivity values tend to decrease slightly .,31.30,3150 value for [Mn2(H2O)2] was from eight to 984 times lower compared to IC50 values for the other Mn(II) complexes. This enormous difference in the cytotoxicity of Mn(II) complexes against the A549 cell line could not be attributed to the differences in the experimental protocols. We found only one study where the cytotoxicity data for Gd(III) complexes with other ligands against A549 were reported [50 value was 733 times lower compared to the value for the published complex) [The antiproliferative activity of Mn(II) salinomycinate against the more chemo-resistant A549 cell line was much more pronounced compared to that of Mn(II) with other ligands ,49,50,51reported . The cytcomplex) .The results demonstrate that salinomycinic acid and its metal-based complexes Sal-Gd(III) and Sal-Mn(II) may induce cell cycle perturbations in both breast cancer MCF-7 and colon cancer SW480 cells, shifting the cell number distribution from G1/G0- towards the S- and G2/M-phases. In line with our data, Niwa et al. reported that a 24 h treatment of MCF-7 cells with 20 μM salinomycin significantly increased cell numbers in the G2/M-phase at the expense of cells in the G0/G1-phase .The combination of MRI contrast agents and a highly effective anticancer drug into a single construct, such as a Sal-based theranostic complex formulated within bacterial ghosts as a transport and targeting device, may offer a powerful “all in one” tool for diagnosis, therapy, and monitoring of various cancers.EcN1917 and NM522 BGs as transport vehicles was successfully carried out. The higher payload of paramagnetic metal ions and an anticancer drug within the BGs may provide better biocompatibility, as well as low/no toxicity or undesired side effects, making these new formulations suitable for clinical applications. To the best of our knowledge, this is the first study that demonstrates the potential use of salinomycin complexes with Gd(III) and Mn(II) as theranostic agents for MRI. This is the first report that a natural, polyether ionophorous antibiotic can act as a chelator of paramagnetic metals and an anticancer drug, exerting a superior cytotoxic effect and favorable MRI properties.In this study, we synthesized and characterized, for the first time, the Gd(III) complex of salinomycin. We provide new data about the structure and cytotoxicity of Mn(II) salinomycinate. Further loading of both Sal-complexes into


CYP2D6*10 has the most clinical relevance in tramadol treatment. Other SNPs such as rs7439366 (UGT2B7), rs1641025 (ABAT) and rs1718125 (P2RX7) though significant have limited pharmacogenetic implications due to insufficient evidence. OPRM1 rs1799971, COMT rs4680 and ABCB1  need to be further explored in future for relevance in Asian populations.In advanced cancer, pain is a poor prognostic factor, significantly impacting patients’ quality of life. It has been shown that up to 30% of cancer patients in Southeast Asian countries may receive inadequate analgesia from opioid therapy. This significant under-management of cancer pain is largely due to the inter-individual variability in opioid dosage and relative efficacy of available opioids, leading to unpredictable clinical responses to opioid treatment. Single nucleotide polymorphisms (SNPs) cause the variability in opioid treatment outcomes, yet their association in Asian populations remains unclear. Therefore, this review aimed to evaluate the association of SNPs with variability in opioid treatment responses in Asian populations. A literature search was conducted in Medline and Embase databases and included primary studies investigating the association of SNPs in opioid treatment outcomes, namely pharmacokinetics, opioid dose requirements, and pain control among Asian cancer patients. The results show that  Pain is the most prevalent symptom experienced by patients with advanced cancer. Affecting up to 96% of patients , cancer Since the introduction of the three-step analgesic ladder by the World Health Organization (WHO) , strong Clinical evidence shows that patients need variable opioid doses, and that the relative efficacy of available opioids varies between patients ,12. SeveCYP2D6, CYP3A4, CYP3A5), membrane drug transport proteins (ABCB1), opioid receptors (OPRM1), and pain receptors (COMT) associated with the efficacy, dose, and toxicity of opioids . Th. ThCYP2DFentanylCYP2D6*10 polymorphism on 207 Chinese gastric cancer patients, particularly those treated with fentanyl after undergoing radical gastrectomy ..CYP2D6*1CYP3A4, CYP3A5, CYP3A7 and CYP3A43, is one of the most versatile biotransformation systems that facilitate the elimination of drugs. CYP3A4 and CYP3A5 together account for approximately 30% of hepatic cytochrome P450, and approximately half of medications that are oxidatively metabolized by P450 are CYP3A substrates. Both CYP3A4 and CYP3A5 are expressed in the liver and intestine, with CYP3A5 being the predominant form expressed in extrahepatic tissues [The human CYP3A subfamily, consisting of  tissues .CYP3A5 on fentanyl pharmacokinetics among 60 Japanese cancer patients undergoing conversion to a transdermal fentanyl from a previous treatment of either oral morphine or oxycodone [CYP3A5*3*3 genotype than in those with the *1*1 and *1*3 genotypes [Takashina et al. investigated the impact of xycodone . In thisenotypes .UGT2B7 encodes for UDP-Glucuronosyltransferase-2B7 phase II metabolism isoenzyme. Morphine is primarily metabolized by the hepatic enzyme UGT2B7 to two active metabolites morphine-3-glucuronide (M3G) and morphine-6 glucuronide (M6G) [de (M6G) .MorphineUGT2B7 and the efficacy of morphine treatment in cancer pain was investigated in 120 Chinese cancer patients [UGT2B7, the plasma concentration of morphine for patients with the CC genotype was significantly lower than that in patients carrying the CT or TT genotypes (p < 0.05). The VAS score of patients with either CT or TT genotypes was significantly higher than those with CC genotype (p < 0.05) [UGT2B7 was also analyzed in this study. Carriers of the G allele (patients with GG and GT genotypes) had no significant difference in pain scores [The relationship between SNPs in patients . Morphin < 0.05) . G221T, n scores .OxycodoneUGT2B7 C802T (rs7439366) on pain relief in 47 Han Chinese patients with malignant tumors receiving prolonged-release oxycodone [p = 0.047). The authors also found that there were more patients with TT genotype in the refractory group (23.5%) compared to the remission group (2.5%)(60%) (p < 0.05) [Li et al. investigated the impact of xycodone . Patient < 0.05) .OPRM1 encodes for the mu (μ) opioid receptor. OPRM1 is the primary target of both endogenous and exogenous opiates including morphine and tramadol and has been shown to mediate both baseline nociception and response to OPRM1 agonists [agonists .MorphineOPRM1 SNP as a genetic factor in the optimization of cancer pain treatment was evaluated by Hajj et al. [p = 0.043) but increased with the duration of morphine treatment (p = 0.029). Patients carrying the AG genotype for OPRM1 c.118A > G (rs1799971) required significantly higher morphine doses than patients with the AA genotype (p < 0.001) [The added value of determining j et al. . A total< 0.001) .Tramadol/Paracetamol combinationOPRM1 A118G (rs1799971) polymorphism [p <0.001) [In 2011, Liu et al. explored the efficacy of tramadol/paracetamol combination in 96 Chinese patients with adenocarcinoma of the colon, rectum, or stomach and the morphism . Compare <0.001) .SufentanilOPRM1 SNPs on the analgesic effect and consumption of sufentanil after thoracoscopic-assisted radical resection of lung cancer were evaluated in 225 Han Chinese cancer patients [OPRM1 at three different time points (both p> 0.05) [The effects of patients . The autpatients . Howeverp> 0.05) .COMT encodes for the catechol-O-methyltransferase (COMT) enzyme which is responsible for catalyzing the transfer of a methyl group from S-adenosylmethionine to catecholamines, including neurotransmitters dopamine, epinephrine, and norepinephrine. In addition, COMT is known to be involved in pain modulation, through dopamine-mediated change in enkephalins neuronal content [ content , followe content ,43.MorphineCOMT on plasma concentration and dose requirements of morphine were evaluated in a prospective study conducted in 48 Japanese patients with cancer [p = 0.008 and 0.03, respectively). However, this significance was lost on day 8 of the treatment [The effects of the G472A (rs4680) SNP in h cancer . The autreatment .p = 0.013) [The significant difference was later replicated by the same group in a later study . This ti= 0.013) . However= 0.013) . Interes= 0.013) .ABAT codes for the γ-Aminobutyric acid (GABA)-transaminase enzyme, which is responsible for the metabolism of inhibitory neurotransmitter GABA. In addition, GABA neurons and receptors are found in supraspinal sites known to coordinate the perception and response to painful stimuli and this system has been shown to regulate sensory information processing in the spinal cord [nal cord ,45.Multiple opioidsABAT was significantly associated with opioid responsiveness in cancer pain [p < 0.001). Additionally, the CT genotype was found to have the lowest mean pain severity after the increment in the opioid dose (p < 0.001) [The rs1641025 SNP in cer pain . The aut< 0.001) .The protein encoded by this gene, P-glycoprotein (P-gp), is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs .FentanylABCB1 1236TT (rs1128503) on fentanyl pharmacokinetics among 60 Japanese cancer patients undergoing conversion to transdermal fentanyl from previous treatment of either oral morphine or oxycodone [p = 0.036) [Takashina et al. investigated the impact of xycodone . They sh= 0.036) .SufentanilABCB1 SNPs, rs2032582 and rs1128503 with the analgesic effect and dose of sufentanil taken for pain relief [ABCB1 did not show any significance (p > 0.05). There were no significant differences in the VAS scores at the three time points nor association with adverse effects [A study conducted among 225 Han Chinese patients with lung cancer reported the association of n relief . The dosn relief . However effects .Multiple opioidsABCB1 C3435T (rs1045642) polymorphism on opioid requirements among 112 Chinese patients [p = 0.057 and 0.028, respectively) [Gong et al. evaluated the influence of P2RX7, encodes the purinergic receptor P2X7 which is a ligand-gated cation channel that opens in response to ATP binding and leads to cell depolarization. P2X7 is expressed in peripheral and central nervous systems and the immune system, mediating and modulating pain. P2X7 function has been previously linked to chronic inflammatory and neuropathic pain [The purinergic receptor P2X, ligand-gated ion channel 7, hic pain .FentanylP2RX7 with postoperative fentanyl analgesia was investigated in an observational study among Han Chinese patients with lung cancer [p < 0.05). Patients carrying the GA and AA genotypes required more fentanyl doses for pain control within 48 h postoperatively (p < 0.05). The postoperative VAS score was also significantly higher in carriers of the GA genotype when compared to the GG genotype group in the period of analepsis after general anesthesia and 6 h post-surgery . Meanwhile, the postoperative VAS score was significantly higher in the A homozygotes than in the period of analepsis after general anesthesia (p < 0.001), at 6 (p = 0.006) and 24 h (p = 0.016) post-surgery [The correlation of rs1718125 in g cancer . This SN-surgery .CYP2D6, CYP3A5, UGT2B7), neurotransmitter-metabolizing enzymes , transporters (ABCB1) and drug receptor (OPRM1) and ion channels (P2RX7) in contributing to inter-individual variability in opioid treatment responses in Asian patients with cancer pain.The findings of this review have highlighted the role of genetic variants in drug-metabolizing enzymes  to serine leading to a decreased enzymatic activity [CYP2D6*10 polymorphism produces an intermediate metabolizer phenotype and is predominant and well-documented among Asian populations [Genetic polymorphisms in capacity . The CYPdol dose  and lowedol dose  due to adol dose . The CYPactivity . The redactivity ,51, leadulations ,53,54.CYP2D6*1/*5 and *2/*5 genotypes have been re-classified from NM to IM phenotype based on their activity scores [CYP2D6*5/*14 and *14/*14 have been re-classified from PM to IM phenotype. Based on this update, the carriers of the CYP2D6*1/*5 and *2/*5 genotypes would have to be re-classified from the NM to the IM + PM group [CYP2D6*5 variant has been reported to be relatively low at 6.3% in Japanese populations [CYP2D6 activity [CYP2B6 and CYP3A enzymes [CYP2D6*10 variant  has been associated with higher fentanyl dose and pain scores compared to the wild type CYP2D6*1  [CYP2D6*9 and *29 alleles were associated with impaired clearance of fentanyl in burn patients [In the study by Tanaka et al. , patienty scores . SimilarPM group ,53. As tulations , it is hactivity . The hig enzymes . Althoug allele) . Grimsrupatients . The metpatients ,57. It ipatients ,57.UGT2B7 are known to affect morphine glucuronidation to form M3G and M6G metabolites [UGT2B7*1) to histidine (UGT2B7*2). UGT2B7*2 homozygotes are lower in the Chinese population (9.2%) when compared to the Caucasian population (25.3%) (p < 0.001) [UGT2B7 was studied in patients receiving morphine and prolonged-release oxycodone [Polymorphisms in abolites ,58.rs743< 0.001) . In thisxycodone ,27. Carrxycodone . ConversUGT2B7 rs7439366 allele had a higher affinity for M3G and M6G compared to their wild type UGT2B7 counterparts [UGT1A9 polymorphisms, the homozygous mutant UGT2B7 allele carriers tend to produce M6G, which is known to be more potent than M3G [UGT2B7 rs7439366*T allele has been listed as insufficient (levels 3 to 4) to be used as a biomarker for treatment response in the PharmGKB database [These results are consistent with previous studies suggesting that the rs7439366*C allele may enhance analgesic efficacy ,59. M3G terparts . Paired than M3G ,60, and database .OPRM1 is the G472A (rs1799971) SNP, which brings about the substitution of asparagine at position 40  to aspartic acid  [Exogenous opioids including morphine bind to the μ-opioid receptor to bring about opioid-induced analgesia ,63. The  allele) . This SN allele) . The res allele) . Carrier allele) . These f allele) ,67.OPRM1 [Sufentanil, being a fentanyl analog, also binds to the μ-opioid receptor. Among sufentanil-treated patients, both sufentanil dose and analgesic effect were associated with the rs1799971 and rs1323040 in OPRM1 . The dosOPRM1  suggestiOPRM1 ,67.COMT SNP G472A (rs4680) has been widely associated with various neuropsychological phenotypes from the dopaminergic pathway, including pain modulation, hyperalgesia, anxiety disorders, opioid-related disorders, and substance addiction [COMT is responsible for degrading the majority of the dopamine in the prefrontal cortical regions of the brain . The COMddiction . This SNddiction . In thisddiction  comparedddiction . Two studdiction .COMT rs4680′s genotypes [However, these results are contradictory to other studies that have reported the A allele being associated with reduced analgesic efficacy ,72. The enotypes ,32.COMT rs4680 homozygous wild type G allele and mutant A allele in the first 24 h of treatment [Pooled data from a meta-analysis did not find any statistical difference in opioid consumption in patients carrying reatment . Howeverreatment .ABAT rs1641025 SNP represents a substitution of wild type T allele to the mutant C allele in the gene encoding for GABA transaminase. In this review, the opioid dose requirement was found to be lowest in patients with homozygous for the ABAT rs1621025*C allele, while pain scores were lowest in patients who were heterozygous for this SNP [ABAT rs1641025 may not influence the opioid dose requirement directly, the pain perception modulated by this SNP may be associated with a higher pain threshold and thus a lower opioid dose requirement. Further studies are needed to explore the potential pharmacogenetic role this SNP may play in pain control and opioid analgesic responsiveness and possibly its usefulness as a biomarker.The this SNP . These fthis SNP . To datethis SNP . Hence, ABCB1 that will remove drugs [ABCB1 is in exon 26, C3435T (rs1045642), while the others are present in exon 21, G2677T (rs2032582) and exon 12, C1236T (rs1128503). Of the three, only rs2032582 SNP results in the change in amino acid . Previous studies have demonstrated that patients homozygous for the mutant rs1045642*T allele display two to three folds lower expression of P-gp relative to the wild type CC genotype [The bioavailability of administered opioids is affected by P-gp, an ATP-binding cassette (ABC) family of efflux transporters encoded by ve drugs . One of genotype ,75. Althgenotype .ABCB1 and activity of P-gp [In the current review, rs1128503, rs2032582 and rs1045642 were studied in patients receiving fentanyl , and mul of P-gp ,75,76. I of P-gp , which m of P-gp ,75,76. H of P-gp . Pain sc of P-gp .P2X7, encoded by P2RX7, has been linked to chronic inflammatory and neuropathic pain. The expression of P2X7 receptors is upregulated in both dorsal root ganglia and injured nerves, and monocytes and lymphocytes in patients with neuropathic pain. In animal models of inflammatory and neuropathic pain, P2RX7 disruption reduces hypersensitivity [P2RX7 rs1718125 SNP was significantly associated with postoperative pain and fentanyl dose in patients with lung cancer [P2RX7 polymorphisms and opioid dose requirements, pharmacokinetics, or pain control. The potential pharmacogenetic relevance of this SNP needs to be further investigated as pre-clinical studies have shown that P2X7 may contribute to pain modulation both by its effect on peripheral tissue and through alterations in central nervous system processing [The function of purinergic receptor sitivity . In thisg cancer . Among pg cancer . The fing cancer . To dateocessing .A vast majority of the studies included in this review had patients of East Asian ancestry . As a result, the pharmacogenetic studies from the mentioned Asian populations may not be representative of other types of Asian populations such as those from Central, Southern, and Southeast Asia. In addition, outcome measures such as opioid dose requirements, duration of treatment, and assessment of pain control implemented in the included studies were different, limiting the ability to compare findings between studies. Compared to pharmacogenomic studies in Caucasian and European populations, pharmacogenetic studies in Asian patients with cancer pain treated with opioids tend to have smaller sample sizes lacking adequate statistical power. Additionally, genetic association studies have focused on a limited number of genes and a few SNPs within each gene, which does not take into account other facets of pain manifestation such as a combination of SNPs (haplotypes), epigenetic factors, and drug-drug interactions, as well as other possible confounding factors. This review only included articles published in English, which may have resulted in publication bias, where some relevant non-English studies could have been missed.CYP2D6*10 SNP is relevant to tramadol pharmacokinetics and opioid dose requirement, while SNPs within UGT2B7 (rs7439366), ABAT (rs1641025), and P2RX7 (rs1718125) have demonstrated some level of potential clinical relevance to cancer pain pharmacogenomics in Asian patients. SNPs within OPRM1, COMT, and ABCB1 were found to have low clinical significance due to several studies failing to replicate associations reported by others. Among these are the OPRM1 rs1799971 implicated in morphine pharmacokinetics and pain control and COMT rs4680 implicated in morphine pharmacokinetics, opioid dose requirement, and pain control. These SNPs do not have consistent findings between Caucasian and Asian populations. Multiple ABCB1 SNPs have been reported to have significance to opioid treatment. These include ABCB1 rs1045642 associated with a dose of multiple opioids, rs1128503 associated with a dose of fentanyl and sufentanil, and rs2032582 associated with a dose of sufentanil. Further investigations are required to unravel the role of these SNPs in opioid treatment outcomes.This review provides insights into the association of genetic variants and treatment responses in Asian patients treated with opioids for cancer pain. The CYP2D6, UGT2B7), neurotransmitter-metabolizing enzymes , transporters (ABCB1) and drug targets, including receptors (OPRM1) and ion channels (P2RX7). The clinical significance of pharmacogenetic biomarkers OPRM1 rs1799971, COMT rs4680 and ABCB1  should be further explored in future studies to improve precision pain management amongst Asian patients with cancer pain.Inter-individual variability in opioid responses has been associated with polymorphisms in genes encoding drug-metabolizing enzymes (


These suggested that purinergic receptors would be promising targets to control the inflammation of DED in the future.Inflammation is one of the potential factors to cause the damage of ocular surface in dry eye disease (DED). Increasing evidence indicated that purinergic A Dry eye disease (DED) is one of the most common ocular surface diseases in the world. DED is caused by a variety of etiologies. It is characterized by loss of tear film homeostasis with tear film instability and high osmolarity, ocular surface inflammation and injury, and neurosensory abnormalities . The inc1, A2A, A2B, and A3), two types of P2 receptors (P2X and P2Y), seven types of P2X receptors (P2X1–7), and eight types of P2Y receptors . Adenosine triphosphate (ATP) binds to these receptors and induces cell-to-cell communication and propagation of inflammation [+, K+, Ca2+) to control rapid responses. ATP and adenosine diphosphate (ADP) activate P2Y receptors; this is a second messenger system that acts through G proteins to control the inflammatory release of various neurotransmitters and hormones [Purinergic receptor was formally proposed as a medical term in 1978. There are four types of P1 receptors  and mal cells –15. The al cells . Adenosial cells , 18, andal cells . Undoubtal cells . These dnd so on –24. The 1R, A2AR, and A3R are mainly involved in the mechanism of inflammation regulation. The biological function of adenosine is mediated through the activation of different G-coupled receptors, A1R, A2AR, and A3R, which inhibit or promote the generation of cyclic adenosine monophosphate (cAMP) in multiple ways. A1R and A3R downregulate CAMP levels and inhibit adenylate cyclase (AC), while A2AR activate AC and promote CAMP production [1R promoted the expression of pro-inflammatory factors, while A2AR and A3R inhibited the expression of pro-inflammatory factors.Adenosine is an endogenous nucleoside that is widely distributed in the body and regulates a variety of functions. Among the P1 receptors, Aoduction . Activat1R knockout (A1KO) and corresponding wild-type (A1WT) mice and demonstrated that A1R is a systemic pro-inflammatory factor [1R have focused on retinal diseases [1R antagonist has little prospect as a DED candidate because it increases Intraocular pressure in mice and rabbits [Ponnoth Dovenia S et al. observed the effects of nebulized adenosine inhalation on vascular reactivity and inflammatory responses in Ay factor . Furthery factor . Studiesdiseases , and becdiseases , it was  rabbits .2AR exerts its anti-inflammatory effects primarily by inhibiting the secretion of pro-inflammatory cytokines after the activation of inflammatory responses. Ko Il-Gyu proposes that A2AR may be involved in the activation of the MAPK/nuclear factor-kappa B (NF-kB) pathway as an important neuromodulator. NF-kB is stimulated by various factors such as cytokines and cellular stress, and activation (phosphorylation) of mitogen-activated protein kinase (MAPK)/NF-kB may promote the expression of multiple pro-inflammatory cytokines, resulting in inflammation, which is one of the mechanisms [2AR agonist treatment significantly reduced the expression of inflammatory mediators  and microglial marker IBA-1 [2AR activation in diabetic retinal inflammation has been very well documented, and studies suggest that A2AR agonists produce anti-inflammatory effects by blocking ERK activation and subsequent cytokine release [2AR antagonist, decreased choroidal neovascularization [2AR antagonist in human, and it facilitates to develop ophthalmic drugs about A2 for the future.Achanisms , 31. In er IBA-1 .The prot release , 33. It rization . US FDA 3R is involved in a number of different intracellular signaling pathways, and the A3R was reported by Avni Isaac et al. to be highly upregulated in inflammatory cells [3R as a regulator of inflammation has become clear, making it an attractive target for the treatment of inflammatory diseases. A3R is linked to the inhibition of adenylate cyclase and the regulation of MAPK pathways, leading to the regulation of transcription [3R agonist for the treatment of inflammatory diseases, downregulates the NF-kB signaling pathway, thereby inhibiting pro-inflammatory cytokines  and macrophage inflammatory proteins , resulting in apoptosis of inflammatory cells [Ary cells . Recentlcription . CF101  and permeability. Rapid opening of potassium-selective channels results in a rapid decrease in intracellular potassium levels and the release of proinflammatory cytokines [2+ ions compensates for the outward flow of K+ ions, resulting in P2X7R-induced intracellular K+ ion loss or increased cytoplasmic Ca2+ ions thereby activating caspase-1. This activated caspase-1 further leads to rapid activation and release of the pro-inflammatory cytokine IL-1β. The subsequent increase in the concentration of IL-1β induces inflammation, which in turn induces other inflammatory mediators such as procysteine aspartate-1, nitric oxide synthase, cyclooxygenase-2, TNF-α, phospholipase D, phospholipase A2, NF-kB, and MAPK [P2X7R is an important target in the pathogenesis and treatment of DED. The ATP-gated P2X7R is a twice-transmembrane ion channel that is permeable to Nand IL-18 , 43. Wheytokines . The inwand MAPK . Furtherand MAPK . DED is 1, P2Y2, and P2Y4 receptors are found in the corneal epithelium and endothelium [2+ mobilization after being activated by ATP or uridine 5′-triphosphate (UTP), which results in downstream signaling to promote inflammation. According to the G protein coupling properties of P2YRs, P2Y1R, P2Y2R, and P2Y4R can be coupled to GQ to activate the phospholipase C (PLC)/IP3/Ca2+ signaling pathway. P2Y1R is involved in inflammatory sensitization [1R signaling [2R agonist induced IL-8 release and immune-mediated inflammation of the mucosa characterized by the release of interleukin IL-8[4R signaling is involved in the production of pro-inflammatory cytokines via PI3K/Akt and ERK1/2-dependent pathways, and inhibition of P2Y4R expression significantly suppresses the production of pro-inflammatory cytokines [1, P2Y2, and P2Y4 receptors may be involved in the pathogenesis of inflammation in DED. On the other hand, P2Y2R promotes mucus secretion from conjunctival gland vesicle cells by activating intracellular signals and specific transcription factors, and upregulates the expression of mucus genes [2R promotes an increase in Ca2+ concentration in cell membranes, which results in the secretion of mucin [2R agonist, activating P2Y2R on the ocular surface to improve the quantity and quality of tear fluid in patients with DED through a novel mechanism that promotes aqueous humor transport, conjunctival epithelial mucus secretion, lipid production, and cell survival [2R in conjunctival epithelial cells, increasing fluid flow and allowing Cl− secretion from the cell membrane to the mucosal side [P2Yothelium , 14. Thetization . High exignaling . P2Y2R aukin IL-8. P2Y4R sytokines . Furtherytokines . Thus, tus genes . Mucus pus genes , 54. Stuof mucin , 56  may have higher efficacy and can reduce the disadvantages usually associated with the use of combination therapies , 61. Bevtagonist . Hou Xiydiseases . Therefo


Microaerobacter geothermalis Nad S1T is a rare Bacillaceae thermophile that grows optimally at 55°C and circumneutral pH. Although strain Nad S1T was discovered >10 years ago, its genome is yet to be described. The release of the Nad S1T genome sequence serves as reference genetic information for subsequent use. Microaerobacter geothermalis Nad S1T (= DSM 22679T = JCM 16213T) is the only described type strain of the genus Microaerobacter  NaCl . A paired-end library was prepared using the NEBNext Ultra II DNA library preparation kit for Illumina , according to the manufacturer’s instructions. Sequencing was performed using the NovaSeq 6000 system  with 150-bp paired-end reads. Sequence adaptors and low-quality reads were filtered using Trimmomatic v0.39 . Based on the earlier study, strain Nad S1T is able to use nitrate and nitrite as electron acceptors under anaerobic conditions, and the cells can reduce nitrate (T transports nitrate and nitrite via its NarK family nitrate/nitrite major facilitator superfamily (MFS) transporter.The sequencer generated a total of 1.2 Gb in 3.9 million paired-end reads. Upon removal of the low-quality reads, the assembled genome has a size of 3,132,374 bp, contributed by 85 contigs, with up to 350× coverage, an Microaerobacter geothermalis Nad S1T shared 90 to 92% sequence identity with those of other species affiliated with the family Bacillaceae  with accession number SRX13800198. The 16S rRNA gene sequence of Microaerobacter geothermalis Nad S1T has been deposited in NCBI GenBank with accession number FN552009.1.The whole-genome shotgun sequence of


Reovirideae) strains cause acute viral gastroenteritis in piglets , resulting in significant economic losses. In this study, we analyzed the VP7 and VP4 genes of PoRVA isolated between 2014 and 2018 from domestic pigs in South Korea to investigate the prevalence of predominant circulating genotypes (G and P types). The prevalence of the PoRVA antigen in the diarrheic fecal samples was 14.1% (53/377). Further genetic characterization of the VP7 and VP4 genes of 53 PoRVA isolates identified six different G-genotypes and five different P genotypes. The G4 and G9 genotypes were the most common (each 39.6%) in PoRVA-positive pigs, followed by P[7] and P[6] . Because the G5 and G9 genotype vaccines are currently mainly used in South Korea, this result provides valuable epidemiological information about the genetic characteristics of PoRVA circulating on domestic pig farms. Development of a novel PoRVA vaccine that targets the current strains circulating in South Korea may be required for more effective virus control on pig farms.Porcine group A rotavirus (PoRVA; family,  Reoviridae (genus Rotavirus), are important pathogens that cause viral gastroenteritis in piglets worldwide  was added. The inoculated cell plates were incubated at 37 °C under 5% CO2 and examined daily for significant cytopathic effects (CPE). When CPE appeared in more than 80% of cells (approximately 3 days after inoculation), the plates were frozen at −70 °C and thawed twice. The culture supernatants were used as viral seed stocks for the next blind passage. The isolated PoRVA strains were confirmed by PCR and in a direct immunofluorescence (IF) assay . If the CPE and PCR results were negative after five blind passages, virus isolation was considered negative. The culture scale was increased gradually until PoRVA strains could be cultured in T-75 flasks.PoRVA was isolated in TF-104 cells  as described previously, with some modifications ,17. BrieBetween 2014 and 2018, 377 diarrheic fecal samples collected from pig-producing regions across South Korea were tested for the PoRVA antigen; of these, 53 (14.1%) were positive . AccordiTo determine RV G and P genotypes, 53 PoRVA-positive fecal samples from pigs with suspected rotaviral enteritis were tested by partial nucleotide sequencing and phylogenetic analysis of the VP7 and VP4 genes .As a result, we identified 15 different G and P genotype combinations. A previous study of the distribution of G and P combinations of PoRVA strains during the year 2006–2007  showed tThe G4 (2014–2018) and G9 (2014–2017) genotypes were detected continuously across the country (the exceptions were G4 and G9 in CB province) every year . In the A comparison of the nucleotide sequences of a partial VP7 gene sequence (a 935 fragment) from the 53 Korean PoRVA strains examined in this study with representative PoRVA strains , OSU (G5), and A2 (G9)) was performed (data not shown). Twenty-one of the 53 Korean PoRVA strains showed 88.9–95.7% identity with the G9 genotype PoRVA vaccine strain A2 from the USA . In addition, 21 Korean PoRVA G4 genotype strains showed low identity (85.0–87.1%) at the nucleotide level with the G4 genotype PoRVA vaccine strain Gottfried from the USA .A comparison of the full-length nucleotide and deduced amino acid sequences of the VP7 gene of the RVA/pig/wt/KOR/GN-108/2017/G26 strain (accession no. OP3211073) detected in this study with reference strains from GenBank revealed that the GN-108 strain shares the closest similarity to the RVA/pig-wt/JPN/TJ4-1 strain (AB605258) from Japan both at the nucleotide (96.1%) and amino acid (97.7%) level. In addition, the GN-108 strain was homologous (86.5–93.8% at the nucleotide level and 90.1–97.4% at the amino acid level) with six strains (KT277523–KT277528) of the G26 genotype isolated from the Tripura and Assam regions in India.Phylogenetic analysis of the partial nucleotide sequences of the VP7 gene from the 53 Korean PoRVA strains, and the selected reference strains from GenBank, was performed using the maximum-likelihood method in MEGA X . The phyA comparison of the partial nucleotide sequence of the VP4 gene (an 816 fragment) from the 53 Korean PoRVA strains identified in this study and representative PoRVA strains , and A2 (P[7])) was performed. Eighteen of the 53 Korean PoRVA strains were 90.4–96.9% identical to the P[7] genotype PoRVA vaccine strain A2 from the USA . Sixteen Korean PoRVA P[6] genotype strains showed low nucleotide identity (79.1–81.9%) with the P[6] genotype PoRVA vaccine strain Gottfried from the USA . Phylogenetic analysis for the partial nucleotide sequences of the VP4 gene from the 53 Korean PoRVA strains and selected reference strains from GenBank was also performed using the maximum-likelihood method in MEGA X .The phylogenetic tree for VP4 revealed that 53 Korean PoRVAs belonged to five types , and that the P[7] type, which contains the 18 Korean PoRVAs, was closely related to the vaccine strains Porcilis, 10, A1, and A2 strains. Eight Korean PoRVAs, the China strain TM-a, and the USA strain A34 belonged to the P[23] type, whereas the P[13] type contained Korean PoRVAs that were first detected in South Korea and belonged to the same type as strains from various countries .Of the 53 PoRVA-positive fecal samples, seven were selected for viral isolation in the TF-104 cell line using the blind passage strategy. The seven samples (G9P[7]:JB-57 (2017), CN-67 (2017), G4P[6]:GB-68 (2017), CN-119 (2017), CN-145 (2017), G4P[13]:CN-202 (2017), and GN-368 (2018)) were selected based on the predominant G and P genotypes among relatively recent samples (2017 to 2018). The selected PoRVA-positive samples were diluted 10-fold and inoculated into TF-104 cells. The TF-104 cells were monitored for CPE after five continuous blind passages. Specific CPEs, characterized by the rounding and detachment of cell clusters, were observed in 10-passage cell cultures after 24–48 h. Finally, two Korean novel PoRVAs (CN-67:G9P[7] and CN-202:G4P[13]) adapted successfully to the TF-104 cells and propagated rapidly. These two novel viruses were confirmed by detecting PoRVA antigens by IFA using RVA VP6-specific MAbs .Previously, it has been reported that the prevalence of PoRVA in Korean pigs identified on 143 farms across six provinces between 2006 and 2007 was 20.6% (98/475) . The G5 However, 10 years later, the present study shows that various G types  and P types  remain prevalent in South Korea. We found that the most common combination of G and P genotypes had changed: G5P[7] had very low detection rates (1.9%), and G8P[7] and G8P[1] were not detected at all, whereas the detection rates of G9P[7] and G9P[23] were 17.0% and 11.3%, respectively. Since the newly emerged G4 genotype is the most prevalent, the detection rate of complexes containing P types [6], [7], [13], [23], and [26] is close to 40%, making them the most likely cause of diarrheal disease on Korean pig farms. This situation may make it more difficult to eradicate rotaviruses from pig farms, and increases the risk of severe and acute dehydrating diarrhea in piglets. Considering that the genetic diversity of PoRVA in South Korea has gradually increased, economic losses caused by the viruses in the Korean swine industry should not be underestimated.A novel PoRVA G26 genotype caused four different diarrheal outbreaks in Japan in 2009–2010, which affected almost all suckling pigs born to 20–30% of lactating sows . RecentlIn particular, the novel G4 genotype occurred in six regions (the exceptions were CB and JN). The G4 genotype first occurred in GW in 2014–2015, in GG in 2016, in JB, GB, and CN in 2017, and in GN in 2018. A new G4 genotype was generated through a combination of P[6] and P[23] on pig farms in 2014; it then recombined with P[7] and P[13] in 2017. As such, the G4 genotype combined with various P genotypes from 2014 to 2018 and comprises a great proportion (23.1–100% per year) of PoRVA-positive samples. Thus, the importance of the G4 genotype is based on the prediction that it will combine with new P genotypes in the future and cause economic damage to pig farms; therefore, continuous monitoring is required. Currently, the domestic bivalent vaccine (containing the G5P[7]-A1 and G9P[7]-10 strains), along with imported vaccines such as the ProSystem RCE vaccine, is used to protect against PoRVA in South Korea . AlthougTo isolate PoRVA from fecal samples of diarrheic piglets, previous studies attempted inoculation into MA-104 ,14,25 anIn this study, we tried to isolate G4P[6] and G9P[7], which were the most prevalent among the 53 PoRVAs. CN-67 (G9P[7]), which was isolated from diarrheic fecal samples from 30-day-old pigs, successfully adapted to TF-104 cells, but G4P[6] failed to adapt and could not be isolated. Instead, the CN-202 (G4P[13]) strain successfully adapted to TF-104 cells. In future studies, two novel Korean PoRVA (CN-67(G9P[7]) and CN-202 (G4P[13]) strains isolated from diarrheal samples will be subjected to virulence testing in piglets and to continuous passage in TF-104 cells in order to confirm their potential as attenuated vaccine candidates.The data presented herein provide valuable epidemiological information and genetic characteristics of the various field-type strains of PoRVA circulating on pig farms in South Korea. A novel G4 genotype vaccine targeting the current Korean circulating strains may be required for more effective control of PoRVA.