Melamine (ML) is a common environmental contaminant, commonly used in food fraud, representing a serious health hazard and jeopardizing human and animal health. Recently, nootkatone (NK), a naturally occurring sesquiterpenoid, has garnered considerable attention due to its potential therapeutic advantages. We investigated the potential mechanisms underlying the protective effects of NK against ML-induced liver injury in rats. Five groups were utilized: control, ML, NK10, ML-NK5, and ML-NK10. ML induced substantial hepatotoxicity, including considerable alterations in biochemical parameters and histology. The oxidative distress triggered by ML increased the generation of malondialdehyde (MDA) and nitric oxide (NO) and decreased levels of reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) activities. In addition, decreased expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and increased nuclear factor kappa beta (NF-κB) expression levels were observed in hepatocytes, which indicated the occurrence of inflammatory changes following ML exposure. These alterations were alleviated by NK supplementation in a dose-dependent manner. The data revealed that the favorable effects of NK were attributed, at least in part, to its antioxidant and anti-inflammatory properties. Moreover, our results were supported by molecular docking studies that revealed a good fit and interactions between NK and antioxidant enzymes. Thus, the current study demonstrated that NK is a potential new food additive for the prevention or treatment of ML-induced toxicity. Melamine  is a triazine heterocyclic chemical, frequently employed for the production of resins, plastics, enamel dyes, commercial filters, glues, dishware, and kitchenware , causing2•–; hydrogen peroxide, H2O2; and hydroxyl radical, OH•; as well as reactive nitrogen species such as nitric oxide (NO) [Many compelling publications have supported the presence of oxidative stress following ML exposure, primarily due to the depletion of the intracellular antioxidant defense systems and the uncontrolled formation of reactive oxygen species (ROS) such as superoxide anion, Oide (NO) ,13. Theside (NO) ,14,15. Aide (NO) . Currentide (NO) . Consequ15H22O) is a sesquiterpenoid extracted from several plants including grapefruit and rhizomes of Cyperus rotundas, as well as essential oil derived from citrus [Antioxidants of plant origin have garnered global attention recently and are frequently used due to their phytochemical content . Among tm citrus . Due to m citrus . NK has m citrus ,21. In am citrus ,24, rotem citrus , D-galacm citrus , lipopolm citrus , and watm citrus .Therefore, a literature review revealed that even though numerous studies have been performed regarding ML toxicity, research focused on ML-induced liver injury and its alleviation is minimal. Therefore, this study evaluated whether NK exhibited a modulatory effect on ML-triggered oxidative stress and inflammatory liver damage. Hepatic biochemical parameters, oxidative status, histological alterations, and expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa beta (NF-κB) were investigated.Melamine and nootkatone were obtained from Sigma Chemical Company . The analytical kits used to assay the biochemical parameters and oxidative cascade markers were purchased from Bio-diagnostics Co., Giza, Egypt.Male Wister albino rats weighing 150–170 g were used to complete this experimental protocol. Rats were procured from the Medical Experimental Research Center of Mansoura University (MERC) for experimental research, Faculty of Veterinary Medicine, Mansoura, Egypt. Before the trial, the rats were confined in comfortable, standard hygienic conditions for two weeks . During the trial, all rats were fed a standard baseline diet and had unrestricted access to water. ad libitum without any treatment. The ML group was the positive toxic group, in which rats received 700 mg/kg body weight ML [After being acclimated, animals were sorted into five equivalent groups (five rats/group). The control group received food and water eight ML . In the eight ML . In the v/v; 0.15 mL/100 g body weight). Blood specimens were gathered from the heart of each animal. The serum was collected after centrifugation of the coagulated blood at 2000× g for 10 min and preserved at −20 °C until further analysis. Immediately following blood collection, the liver was rapidly extracted, washed in ice-cold physiological saline, and sliced into several pieces. One piece was placed in neutral buffered formalin (10%) for subsequent histopathological evaluation. The other fresh tissue samples were preserved at −80 °C for oxidative cascade marker investigation.At the end of the trial, the rats were euthanized by intraperitoneal injection of a xylazine and ketamine mixture  using kits from Bio-diagnostics Co. The analysis of all markers was performed according to the manufacturer’s directions.Liver homogenates from each rat were prepared by centrifuging the tissue samples at 8000 rpm for 15 min at 4 °C in 400 mL of phosphate-buffered saline (PBS). The supernatants were collected in cold Eppendorf tubes for enzymatic analyses. The non-enzymatic oxidative markers  and the enzymatic oxidative marker (CAT) were purchased from Bio-diagnostics Co, and measured according to the manufacturers’ guidelines.Formalin-fixed liver samples were dehydrated in a graded alcohol series. Subsequently, xylene clearing was performed followed by routine paraffin embedding. The tissue was cut into 5 μm sections and stained with hematoxylin and eosin (H&E) for histological evaluation. Representative images were captured using an integrated digital scanning camera system .Following the manufacturer’s directions, immunohistochemical staining was used to evaluate the hepatic expression of NF-κB and Nrf2. Liver sections (4 mm) on microscope slides were incubated in 0.3% hydrogen peroxide for 20 min to prevent endogenous peroxidase activity. The sections were blocked for 30 min with 2% bovine serum albumin (BSA) for 10 min before incubation in a water bath at 100 °C. Then, the sections were incubated overnight at 4 °C in rat antibodies directed against a rat-targeted antigen. Subsequently, the sections were washed and then incubated with horseradish peroxidase (HRP) secondary antibody at 37 °C for 1 h. Then, the sections were exposed to a diaminobenzidine (DAB) working solution for 4 min and counterstained with Mayer’s hematoxylin. Finally, the sections were evaluated and photographed. Images were taken using a Nikon integrated digital imaging system , at an original magnification of ×400.https://www.uniprot.org/; accessed on 15 June 2023) and AlphaFold  protein structure databases. Proteins were prepared for docking using Molecular Operating Environment software . In addition, the three-dimensional structures of ML and NK were retrieved from the PubChem  database. Furthermore, the molecular docking, protein–ligand interactions, and visualization were carried out using MOE software (https://www.chemcomp.com/ accessed on 15 June 2023).ML was docked with rat superoxide dismutase , catalase (CAT), glutathione peroxidase-1 (GPx-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione reductase (GR), and glutathione synthase (GS). NK was docked against tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), transforming growth factor beta-activated kinase 1 (TAK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKB), interleukin-1 receptor type 1 (IL-1R1), interleukin-1 receptor type 1 (IL-1R2), caspase-3, and inducible nitric oxide synthase (iNOS). The three-dimensional structures of target proteins were retrieved from UniProt (p ≤ 0.05 and expressed as means ± standard error of the mean (SE). GraphPad Prism version 8 was used for data analysis and the generation of column charts. Multivariate principal component analysis (PCA), the variable importance in projection (VIP) score, and clustering heatmaps were created using MetaboAnalyst software .One-way analysis of variance (ANOVA) was used to analyze the results, and differences between the groups were revealed using Duncan’s post-hoc multiple tests. All values were judged to be statistically significant at As shown in The lipid peroxidation (MDA) index and oxidative status  following ML and/or NK administration are presented in The hepatic histoarchitecture of the control and NK groups revealed normal hepatic cords radially arranged around central veins and normal portal areas and sinusoids A,C. HepaExpression of Nrf2 and NF-κB in liver sections following treatment by ML and/or NK are displayed in In addition, immunohistochemical evaluation of the liver sections revealed changes in the inflammatory state of the hepatic tissue after ML exposure, with up-regulated NF-κB expression B. In conThe clustering heatmap depicted in PCA was implemented to analyze the correlations between different treatments and variables. Based on PCA, all variables were sorted into three principal dimensional components , which accounted for 98.6% of the variance. PC1 distinguished most of the evaluated variables and represented a greater proportion of the variation (95.1%), while PC2 (2.2%) and PC3 (1.3%) represented less of the percentage of the variance. The PCA revealed that the control, NK, and ML-NK10 groups were clustered on the left side and separated from the groups treated with ML B. FurtheML interacted with the binding sites of SOD1, SOD2, SOD3, CAT, GPx-1, GCLC, GR, and GS with binding energies of −3.81, −3.90, −4.23, −4.65, −4.16, −3.99, −4.34, and −4.92 kcal/mol, respectively A–H. ML bNK interacted with energy values of −4.67, −5.30, −5.71, −5.52, −5.22, −5.34, −5.63, and −5.15 kcal/mol with TNFRSF1A, TNFRSF1B, TAK1, IKKB, IL-1R1, IL-1R2, iNOS, and caspase-3 binding sites A–H, respML is a common pollutant and carcinogen that threatens human and animal health, and has sparked major global concerns . It is w− loss and produces significant quantities of O2− [2− and NO radicals react together, resulting in the generation of nitrogen radical species that cause oxidative stress and have deleterious consequences on living cells. Interestingly, SOD, an endogenous antioxidant enzyme, functions as the first line of enzymatic antioxidant defense necessary for the dismutation of O2− to O2 and H2O2 [•, the most detrimental radical, because it degrades membrane lipids resulting in increased LPO, as indicated by elevated MDA levels [Ample evidence strongly indicates that LPO, cellular antioxidant insufficiency, and disruption of mitochondria are key pathogenic pathways associated with ML exposure . Accordis of O2− . Furtherand H2O2 ,34. Notaand H2O2 ,35. AlteA levels . More imA levels . According to our findings and those of others ,13, hepaExpectedly, the enhanced LPO and disruption of hepatic cell membranes detected following ML exposure might have enhanced the release of liver enzymes  into the circulation, resulting in increased levels in the serum, as observed in the current study. These results corroborate our earlier reports that demonstrated a positive association between MDA levels and elevated levels of hepatic enzymes ,14,15. EBesides LPO and oxidative damage, ML-induced hepatic injuries have been associated with increased production of pro-inflammatory cytokines and significant inflammatory cell mechanisms involved in ML hepatotoxicity . This fiNK is a naturally occurring sesquiterpenoid, found in grapefruit peels and can be synthetically produced through biotransformation of valencene . NK has Overwhelming evidence confirms the idea that NK supplementation boosts antioxidant enzyme activity by modulating the expression of oxidative stress biomarkers, such as Nrf2 , along wBesides its antioxidant activity, NK has demonstrated robust anti-inflammatory activity, which might be partly ascribed to the suppression of the NF-κB pathway , and IL1We performed multivariate statistical analysis, depicted by PCA, to assess the multiple contributions from different treatments on liver tissue. Each treatment was primarily identified on the PC1 axis (95.1%). ML-exposed animals were distinguished from the other treated groups because they clustered on the left side, apart from other treatment groups. The ML-NK10 co-treated group, on the other hand, was located close to the control and NK groups. In contrast to the other treatment groups, the clustering heatmap exhibited variations in all variable levels with respect to ML exposure. Thus, these findings robustly emphasize the potential protective effect of NK on ML toxicity. Taken together, our results revealed that the liver is one of the principal target organs for ML toxicity. ML exposure caused substantial hepatocellular injury which is attributed to increased oxidative stress, LPO, and inflammatory reactions. In a dose-dependent manner, NK supplementation ameliorated the ML-inflicted oxidative and inflammatory changes in the liver tissue. NK corrected biomarkers of hepatic toxicity and relieved oxidative stress and inflammation induced by ML. This hepatoprotective impact of NK is ascribed to its antioxidant and anti-inflammatory properties, along with its potential to restore the normal structure of the injured liver tissues. Molecular docking dynamics confirmed the anti-inflammatory and antioxidant proteins targeted by NK and ML. We thereby propose that NK supplementation could be an effective preventive approach for diseases or toxicants that trigger hepatic degeneration.


Glioblastoma multiforme (GBM) has high mortality and recurrence rates. Malignancy resilience is ascribed to Glioblastoma Stem Cells (GSCs), which are resistant to Temozolomide (TMZ), the gold standard for GBM post-surgical treatment. However, Nitric Oxide (NO) has demonstrated anti-cancer efficacy in GBM cells, but its potential impact on GSCs remains unexplored. Accordingly, we investigated the effects of NO, both alone and in combination with TMZ, on patient-derived GSCs. Experimentally selected concentrations of diethylenetriamine/NO adduct and TMZ were used through a time course up to 21 days of treatment, to evaluate GSC proliferation and death, functional recovery, and apoptosis. Immunofluorescence and Western blot analyses revealed treatment-induced effects in cell cycle and DNA damage occurrence and repair. Our results showed that NO impairs self-renewal, disrupts cell-cycle progression, and expands the quiescent cells’ population. Consistently, NO triggered a significant but tolerated level of DNA damage, but not apoptosis. Interestingly, NO/TMZ cotreatment further inhibited cell cycle progression, augmented G0 cells, induced cell death, but also enhanced DNA damage repair activity. These findings suggest that, although NO administration does not eliminate GSCs, it stunts their proliferation, and makes cells susceptible to TMZ. The resulting cytostatic effect may potentially allow long-term control over the GSCs’ subpopulation. Glioblastoma multiforme (GBM) is the most prevalent primary malignant brain tumor in adults, with an exceptionally poor prognosis. With the disease essentially incurable, most patients, despite receiving a comprehensive multimodal treatment approach—encompassing surgery, radiation, and chemotherapy—inevitably succumb to tumor recurrence . The relclinicaltrials.gov website).Nitric oxide (NO), a signaling molecule generated by the family of nitric oxide synthases , plays a plethora of physiological and pathophysiological roles in the human body and in tumor biology ,14. DepeN-acetylpenicillamine (SNAP) has been shown to inhibit the growth of GBM cells both in vitro and in vivo [N-methylamino)benzoyloxy]phenyl}1-diazen-1-ium-1,2-diolate) in combination with TMZ leads to the resensitization of TMZ-resistant GBM cells [2- 1-[(4-ethoxycarbonyl) piperazin-1-yl]diazen-1-ium-1,2-diolate) reduces GBM cells’ radioresistance when administered prior to radiation exposure. Various mechanisms underpin these effects, including apoptosis activation in response to DNA damage [In the context of cancer, the influence of NO extends to cell cycle, apoptosis, mitogenic pathways, angiogenesis, invasion, and DNA integrity ,28. Than in vivo . FurtherBM cells ,46. JS-KA damage . In addiA damage ,49. HoweTherefore, in this study, we investigated the effects of experimentally selected concentrations of NO donor diethylenetriamine/nitric oxide adduct  and TMZ, administered both as single treatments and in combination, on patient-derived GSCs and, when appropriate, on U87MG glioblastoma cells. Our results demonstrate that sub-lethal NO concentration can reversibly inhibit GSC proliferation and self-renewal. This finding aligns with NO-promoted DNA damage, increasing the proportion of non-proliferating cells. Intriguingly, NO also heightens the sensitivity of GSCs to TMZ treatment, primarily by amplifying cytostatic effects and inducing cell death, potentially allowing long-term control over the GSCs’ population. To the best of our knowledge, this is the first documentation of the effect of exogenous NO on GSCs, bolstering the case for NO as a potential adjunct to current GBM treatments.Under appropriate culture conditions  was also present , whereas the number of NO-treated cells did not increase over time A. Consis present E.− cells increased to +60% after 14 days of NO administration. To further investigate the NO-dependent GSCs’ cell cycle regulation, cells were also analyzed with FACS. Consistent with the distribution of Ki67 foci, accumulation of GSCs in the S phase after 3 and 7 days of NO treatment was revealed . According to the experimental design, after 3, 7, 14, and 21 days, spheres were dissociated and single cells were stained with 0.4% Trypan blue to evaluate the number of both alive and dead cells through the use of a Burker counting chamber. To reveal the presence of necrotic cells in intact spheres, untreated and NO-treated neurospheres were stained with Trypan blue (1:4 in stem cell medium) for 4 h, then placed in PBS and photographed using a phase contrast microscope. IC50 value was calculated using GraphPad Prism v5.01 . U87MG cells were seeded at about 8000 cells/cmThe morphology of both floating neurospheres and adherent U87MG cells was observed under an Axiovert 40 inverted microscope . Cell pictures were taken with a ZOE cell imager .GSCs were seeded at 150,000 cells/mL of culture medium. On the cells treated with NO or vehicle for 3, 7, and 14 days (as described above) immunostaining for SOX2, Ki67, and γH2AX was performed. Moreover, on GSCs treated for 7 days with TMZ alone and in combination with NO (as described above), immunostaining for Ki67 and γH2AX was also performed. Briefly, at each time point, neurospheres were dissociated, and GSCs were fixed in 4% paraformaldehyde and cytocentrifuged onto a clean slide using a Shandon cytocentrifuge at 800 rpm for 5 min, permeabilized in 0.3% Triton in PBS for 5 min, and then incubated in PBS containing 10% BSA for 30 min. Samples were immunoassayed by using the mouse anti-SOX2 , rabbit anti-γH2AX, and mouse anti-Ki67  antibodies, all diluted 1:100 in PBS, O/N at RT. Primary antibodies were detected by 1 h incubation with FITC-conjugated anti-mouse  or Cy3-conjugated anti-rabbit . The slides were mounted in Vectashield with DAPI (Vector) to stain the DNA and reduce fluorescence fading. All images were captured using a CoolSnap HQ CCD camera  connected to a Zeiss Axioplan fluorescence microscope equipped with an HBO 100 W mercury lamp. At least 15 fields and 100–150 cells for each sample were analyzed by three independent individuals.In all the experiments, 150,000 GSCs/mL of culture medium were seeded. Protein expression was analyzed in GSCs, following treatment with NO and TMZ, singly and in combination, as described above, as well as in U87MG cells after NO treatment. Intact neurospheres were pelleted at 400 rpm for 20 min and solubilized in Laemmli buffer on ice (5000 cells/µL of buffer). Adherent U87MG cells were scraped and solubilized in Laemmli buffer on ice (5000 cells/µL of buffer). Before lysis, the number of cells in each sample was obtained by counting a parallel series of replicates. Cell lysates were boiled for 10 min and the supernatants were collected after centrifugation at 13,000 rpm for 30 min at 4 °C. Equal volumes of protein extracts were subjected to 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes which were probed with primary antibodies. Anti-SOX2 and -p53 antibodies were from Santa Cruz Biotechnology, anti-PCNA was from Abcam , anti-γH2AX and -p21 were from Cell Signaling Technology, anti-caspase 3 was from Millipore , anti-PARP was from DB Pharmingen , and anti-β-actin was from Sigma-Aldrich. After incubation with the appropriate secondary antibody conjugated to peroxidase (Sigma-Aldrich), SuperSignal West Pico plus or SuperSignal West Femto chemiluminescent systems (Thermo Fisher Scientific) were used for band detection. Images were captured at a ChemiDoc XRS+ Gel Imaging System (Bio-Rad). Band densitometry was performed by ImageJ 1.47v software.GSCs were seeded at 150,000 cells/mL of culture medium. The next day, cells were treated with NO or vehicle, and grown as described above. At the adequate time points, control and NO-treated spheres were dissociated, then the cells were counted and resuspended in PBS and fixed for 2 h at 4 °C in cold 70% ethanol. Thereafter, GSCs were incubated for 30 min at room temperature with 50 µg/mL propidium iodide (PI) in PBS containing 0.5 mg/mL RNAse (Sigma-Aldrich) and immediately analyzed using a FACSCanto . Analysis of the cell cycle distributions was performed using the Dean–Jett–Fox model in FlowJo V10 Cytometric Analysis Software (BD Biosciences).Apoptosis occurrence was investigated by the Annexin V-FITC apoptosis detection kit (Abcam), according to the manufacturer’s instructions. Briefly, 150,000 GSCs were seeded and treated with NO alone and in combination with TMZ, as described above. After 21 days of NO treatment and NO/TMZ cotreatment, neurospheres were dissociated and 100,000 cells were resuspended in 500 µL of Binding Buffer. Five µL of Annexin V-FITC antibody and 5 µL of PI were added. Cells were incubated 5 min in the dark, then placed onto a glass slide, covered with a glass coverslip and observed under an Axio-observer3 inverted microscope .t-test, while the unpaired t-test was used when two different treatment groups were compared. p values of <0.05 were considered significant.Data were analyzed using GraphPad Prism v5.01 software and are presented as mean values ± SEM from at least three independent experiments. Statistical significance between treated and control cells was determined using the paired Student’s Given the multifaceted challenge of GBM, the need for optimizing existing therapies and identifying additional treatments to enhance current therapeutic strategies is clear. Therefore, the primary focus of our study revolved around deciphering the pathways that confer GSCs’ resistance to treatments. To this aim, we evaluated the effects of prolonged NO exposure, which elicited significant cytostatic effects leading to the curtailment of GSCs’ expansion. Notably, GSCs conditioned with NO displayed sensitivity to TMZ, exhibiting further suppression of cell proliferation, expansion of the quiescent cell subpopulation, and increased cell death. Our findings suggest that, while administering NO alone may not eliminate the GSCs’ subpopulation, it could be a valuable therapeutic tool when used with TMZ. Indeed, this combination might effectively overcome TMZ resistance in the stem-like cell subpopulation, thus dramatically limiting GSC proliferation. Furthermore, given that differentiated glioblastoma cells also displayed sensitivity to both NO and TMZ, the combined treatment could be effective against the whole tumor. However, further in-depth molecular studies are necessary to explore the mechanisms underpinning the combined NO/TMZ treatment to better understand its antiproliferative effects. Moreover, our hypothesis could be further investigated by developing tools for site-specific NO release or hydrogel-mediated NO delivery ,88. In a


Human respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection and hospitalization, especially in children. Highly mutagenic nature and antigenic diversity enable the RSV to successfully survive in human population. We conducted a molecular epidemiological study during 2017–2021 to investigate the prevalence and genetic characteristics of RSV.A total of 6499 nasopharyngeal (NP) swabs were collected from hospitalized children at Department of Pediatrics, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, China. All NP swab specimens were preliminary screened for common respiratory viruses and then tested for RSV using specific PCR assays. Partial G genes of RSV were amplified for phylogenetic analysis and genetic characterization.The overall detection rate for common respiratory viruses was 16.12% (1048/6499). Among those, 405 specimens  were found positive for RSV. The monthly distribution of RSV and other respiratory viruses was variable, and the highest incidence was recorded in Autumn and Winter. Based on the sequencing of hypervariable region of G gene, 93 RSV sequences were sub-grouped into RSV-A  and RSV-B . There was no coinfection of RSV-A and RSV-B in the tested samples. Phylogenetic analysis revealed that RSV-A and RSV-B strains belonged to ON1 and BA9 genotypes respectively, indicating predominance of these genotypes in Guangzhou. Several substitutions were observed which may likely change the antigenicity and pathogenicity of RSV. Multiple glycosylation sites were noticed, demonstrating high selection pressure on these genotypes.This study illustrated useful information about epidemiology, genetic characteristics, and circulating genotypes of RSV in Guangzhou China. Regular monitoring of the circulating strains of RSV in different parts of China could assist in the development of more effective vaccines and preventive measures. Respiratory syncytial virus (RSV) is considered a serious respiratory tract infection vulnerable of humans especially infants and children , 2. AccoPneumoviridae family of respiratory viruses . While other 36 sites showed less than 1% frequency of amino acid change. Amino acid position 258 , and 266  showed the highest amino acid changes  lost N glycosylation due to T320A substitutions. Analysis of the second variable region predicted different pattern of O-glycosylation sites with 35–46 potential O-glycosylation sites among genotype ON1 sequences when compared to the Ontario reference strain. The amino acid positions most likely to have O-glycosylation in ON1 strains of present study are T211, T219, T220, T220, P222, L226, T227, T228, T231, P234, T235, T238, T239, T241, R244, T245, T246, T249, S250, T252, K253, T259, S260, T264, S267, T268, T269, S270, S275, P276, S277, T281, T282, S283, S299, S301, T305, T306, S307, S311, S313, S315, S316, S317, T319, T320 (refer to OR456347).We also aligned and compared the second variable region of protein G of RSV-B strains (BA9 genotype) of our study with their respective reference sequences BA AY333364). There were 396 amino acid substitutions in all 37 sequences of second variable region of the G protein of RSV-B genotype (BA9), compared with BA reference sequences (AY333364). The amino acid variations analyses revealed 10 sites  with more than 5% frequency in amino acid changes and 102 sites with less than 5% frequency of amino acid changes in second variable region of the G protein (aa200 to aa312)  showed potential N-glycosylation sites at residue 310. Similarly, three BA9 strains  lost potential N-glycosylation sites due to N296Y substitution. Analysis of the second variable region predicted different patterns of O-glycosylation with 27–50 potential O-glycosylation sites among genotype BA9 sequences when compared to the BA9 reference strain (KT765097). The amino acid positions most likely to have O-glycosylation in BA9 strains of present study are T211, P216, K218, T218, T222, T227, T228, I229, P231, T232, T236, T239, T240, R242, T244, S245, T246, S249, T250, T254, T255, T256, S257, T260,T264, S265, T266, S267, S269, T274,T275, T276, S277, T280, I281, S285, S288, T289, T290, T294, S297, T298, T300, T302, S304,S307,T308, S309, S311 and T312 for genotype BA9 (refer to OR456403).Interestingly, we observed several differences in amino acid frequency at position 254, 275, 290. 302 and 312 when compared reference strain of BA9 from China . Deduced amino acid analysis revealed two Respiratory viruses usually cause high morbidity and mortality with acute respiratory infections, especially in children. Viral infections involving respiratory tract get out of hand quickly via sneezing and coughing in contrast to other type of infections. For better therapeutic and preventive measures, it is imperative to understand the epidemiology and genotypic characteristics of respiratory viruses. RSV is a key respiratory virus worldwide and imposes a serious burden on health care settings by causing severe morbidity and hospitalization in children. Knowledge of circulating RSV genotypes can help us to better understand RSV infection and to implement suitable preventive measures , 25, 29.p < 0.05) when compared to RSV negative patients. In our study, we found 1048 specimens positive for respiratory viruses  indicating the role of these respiratory viruses in causing respiratory diseases. These findings align with previously published reports  with more than 5% frequency in amino acid changes and 102 sites with less than 5% frequency of amino acid changes in second variable region of the G protein (aa200 to aa312). These findings are consistent with previously published reports in China [N and O-linked glycans on the G protein could play a critical role for the virus to evade host immune responses. It has been established that N and O linked glycosylation in the G protein can alter the attachment and antigenicity properties of RSV [N-glycosylation sites revealed two potential N glycosylation sites at amino acid positions of 237 and 318 in second variable region of the G protein of ON1 strains. Analysis of the second variable region of the G protein predicted different pattern of O-glycosylation sites with 35–46 potential O-glycosylation sites among genotype ON1 Sequences when compared to the Ontario reference strain. Similarly, deduced amino acid analysis revealed two N-glycosylation sites at 296 and 310 amino acid positions of genotype BA9 sequences. Analysis of the second variable region of the G protein predicted different pattern of O-glycosylation with 16–27 potential O-glycosylation sites among genotype BA9 sequences when compared to the BA9 reference strain (KT765097). These mutations indicated a high selection pressure as described previously [N linked glycosylation and O linked glycosylation may change the antigenicity and virus fitness of RSV and could potentially lead to more serious respiratory infections in people.s of RSV . The anaeviously . These vOur study did not reveal any association among specific RSV types, genotypes, lineages, and diseases severity. A great variation in clinical and laboratory parameters were observed among patients. There is conflicting data regarding virulence of RSV and disease severity , 53.Our study had some limitations. First, our study only included patients who visited hospital for treatment, therefore cannot draw a conclusion for the overall RSV infection in a community and may not represent regional epidemic pattern. In addition, several factors such as usage of antibiotic or antivirals prior to visiting hospital for treatment or failure in collection of consecutive samples in holidays may affect outcome of the study. Usually, number of patients visiting hospital for treatment become low during spring festival holidays that could possibly lead to failure in consecutive sample collection. Chinese Lunar year occurs every year in between January and February and most of the people go home to visit their families during these holidays. Second, we could not perform sequencing analysis for specimens which were collected in 2021 due to limited time and funding. Third, we performed genotyping of RSV on second variable region of G gene and could not utilize whole gene sequencing for genotyping. Furthermore, we did not include data about bacterial coinfections, which might also influence disease severity. Coinfection with respiratory viruses and bacteria have been commonly observed in the past , 40. ZhaIn summary, detection rate of RSV was statistically associated with age and season. Therefore, appropriate knowledge and understanding about RSV infections is highly desired to implement better preventive and therapeutic strategies. Molecular characterization of RSV in Guangzhou China confirmed the co circulation of ON1 and BA9 genotypes of RSV. Deduced amino acid sequence analysis revealed several substitutions which may likely change antigenicity and pathogenicity of RSV. Multiple glycosylation sites were observed in the hypervariable region of the G protein, demonstrating high selection pressure on these genotypes. Due to emergence of several lineages of ON1 and BA9 genotypes, it is quite possible that new lineages will emerge in near future due to rapid evolution in RSV. Continuous and long-term surveillance programs coupled with clinical data must be initiated in Guangzhou to better understand the pattern of seasonal distribution of circulating genotypes of RSV and find any association between emerging genotypes and disease severity.


ZmST genes were identified from the maize genome and classified into eight major groups based on phylogenetic relationship. Gene structure analysis revealed that members within the same group shared similar exon numbers. Synteny analysis indicated that ZmSTs underwent 15 segmental duplication events under purifying selection. Three-dimensional structure of ZmSTs demonstrated the formation of a compact helix bundle composed of 8–13 trans-membrane domains. Various development-related cis-acting elements, enriched in promoter regions, were correlated with the transcriptional response of ZmSTs during kernel development. Transcriptional expression profiles exhibited expression diversity of various ZmST genes in roots, stems, leaves, tassels, cobs, embryos, endosperms and seeds tissues. During kernel development, the expression of 24 ZmST genes was significantly upregulated in the early stage of grain filling. This upregulation coincided with the sharply increased grain-filling rate observed in the early stage. Overall, our findings shed light on the characteristics of ZmST genes in maize and provide a foundation for further functional studies.Sugar transporters (STs) play a crucial role in the development of maize kernels. However, very limited information about STs in maize is known. In this study, sixty-eight  In higher plants, sugars, including monosaccharide and sucrose, play a crucial role in enhancing yield . In many+/inositol symporters  and sugar will eventually be exported transporters (SWEET) family, have been identified in various species . MFS is mporters , plastidmporters , and twomporters . These emporters . The SWEmporters . Due to STs in the transportation of sugars to sink tissues, which is crucial for crop yield and quality. In Arabidopsis, there are 62 identified AtST genes. Mutants of AtSUC2, which have decreased sucrose transport in the phloem, accumulate excessive starch in the leaves, leading to severe growth inhibition and reduced fertility  is a significant global food crop with important economic and social value, as well as applications in the bioenergy industries /23 °C (dark) with a 16 h light/8 h dark photoperiod  with default parameters (http://pfam.xfam.org/) and utilized for HMMER 3.0 searches against the potential ST proteins in maizeGDB (https://www.ncbi.nlm.nih.gov/cdd/) and SMART (https://smart.embl.de/).To investigate putative rameters . AdditiomaizeGDB . All potST genes from various plants, including Arabidopsis  phylogenetic method with 1,000 bootstrap replications  (The gene structure of g/meme/) . The maxZmST genes , with ChamQ Universal SYBR qPCR Master Mix , as previously described  according to the manufacturer’s instruction. About 1-2 µg of RNA was using to reverse transcribe with HiScript® III All-in-one RT SuperMix reverse kit reagents . In order to eraser gDNA and synthesize cDNA, 4 µl of 5 × All-in-one RT SuperMix, 1 µl of Enzyme Mix, 1–2 µg of RNA and appropriate volume of RNase-free ddHescribed . Finallyhmittgen .ZmSTs were obtained with TBtools software for promoter analysis. The cis-acting regulatory elements were identified by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and presented with TBtools.The promoter regions of p I), the grand averages of hydropathicity (GRAVY), and localization prediction, were characterized , isoelectric point  through the cytoplasm or inner membrane, except SUT clade. This SUT clade comprised a conserved GPH_sucrose superfamily domain, which might export sucrose from photosynthetic sources to the phloem or import sucrose into sucrose sinks . Furthercis-elements are the binding sites for transcription factors, carrying information to regulate the gene expression in biological pathways. Thus, we extracted the promoter regions of 68 ZmST genes and examined their cis-acting elements using PlantCARE database 5.0. A total of sixty-six cis-elements were identified and categorized into four main groups: photoresponse, hormonal response, stress response and development. Among these cis-elements, thirty were related to light-responsive pathway, indicating their role in responding to light, which aligned with the function of sugar transporters in the distribution of photosynthetic products. Additionally, fourteen were associated with hormone response, eleven with abiotic and biotic stress response, and eleven with plant growth and development  showed constitutive expression (log2 (FPKM+1) ≥ 1). Among these genes, ZmpGlcT1, ZmpGlcT2, ZmpGlcT4, ZmSFP5, ZmTST1, ZmTST2, ZmTST4, ZmSUT2 and ZmSUT4 showed the highest expression levels across all tested tissues and organs (log2(FPKM+1) ≥ 3)  showed no expression across all tested tissues and organs (ZmST genes (ZmPMT1 and ZmPMT7), four genes , and four genes  exhibited specific expression only in old leaves, roots, and tassels, respectively, with hardly detected expression in other organs. Additionally, we found that ZmSTP and ZmPMT groups displayed high expression in vegetative organs, whereas ZmPMT group members were scarcely detected in developing seeds. However, ZmTST group members showed higher transcription level in the developing seeds (We downloaded the transcriptome data for +1) ≥ 3) . On the d organs . Furtherng seeds .ZmST genes during stages of embryo, endosperm, and seed development suggested a significant role of ZmSTs in maize kernel development. To explore the probable functions of ZmST genes, we randomly selected 24 ZmST genes representing eight groups and executed qRT-PCR analysis across embryo and endosperm developmental stages as well as seed maturity stages. ZmpGlcT2, ZmSFP5, ZmSTP3, ZmTST1, ZmTST2, ZmVGT1, ZmVGT2 and ZmSUT2 showed up-regulation during embryo development and down-regulation during endosperm development. Conversely, ZmSTP7 were down-regulated during embryo development but up-regulated during endosperm development. Some genes like ZmINT4, ZmPMT4, ZmPMT8, ZmSFP10, ZmSTP15 and ZmSUT1 were down-regulated, while ZmSFP7 was up-regulated, during both embryo and endosperm development (ZmST genes, such as ZmINT4, ZmpGlcT4, ZmPMT4, ZmPMT5, ZmPMT8, ZmPMT9, ZmPMT13, ZmSFP5, ZmSFP7, ZmSFP9, ZmSFP10, ZmSTP7, ZmSTP15, ZmVGT1 and ZmSUT2, increased gradually in the early stage of grain filling, reaching a peak at 8-12 days after pollination, and then declined gradually (ZmST genes in the embryonic, endospermic, and seed development stages.As shown in elopment . In partradually . As suppradually , we inveradually . These rZmST genes in the maize genome and analyzed their physicochemical properties (ST genes in Arabidopsis (ST genes in grape (ST genes in strawberry (ST genes in rice (ST genes in apple (ST genes in pear (ST genes in maize was similar to those found in other plant species, suggesting that the functions of ST genes were relatively conserved in functions and crucial for plant growth and development throughout different species. Additionally, the SWEET genes, as a newly characterized group of sugar transporters, were identified 20 genes in maize, 23, 22, 24, 21, 29 and 19 genes in sorghum, pearl millet, foxtail millet, rice, barley and Brachypodium, respectively (SWEET genes was comparable to those observed in other plants. These findings indicated that sugar transporters gene family as well as the SWEET gene family in maize, didn’t participate in gene expansion, maintaining the number of genes unchanged.The structures, phylogenetic relationship, and functional evolutions of sugar transporters have been extensively studied in various plant species . Howeveroperties . As we kbidopsis , sixty-fin grape , sixty-srawberry , sixty-n in rice , seventyin apple , seventy in pear . The numectively . The numArabidopsis were primarily distributed in the cell membrane and tonoplast (ZmSWEETs in maize are mainly localized in the plasma membrane, vacuolar membrane, and chloroplast thylakoid membrane (Arabidopsis and rice, ZmSTs were clustered into eight groups (ST genes were relatively conserved, especially in graminaceous crops.The subcellular localization of sugar transporters is crucial to explore their potential functions in various biological processes. It is known that sugar transporters are typically localized in membrane system of cells, facilitating the transport of sugar from source leaves to sink tissues, such as developing seeds, providing essential carbon and energy sources for plant growth and development. The sugar content is higher in sink tissues, which requires the proton pumps on the membrane to counteract sugar concentration gradient. The AtSTs in onoplast . The ZmSmembrane . Consistmembrane . Based ot groups , consistZmST genes were located on 10 chromosomes in the maize genome (ZmST gene family, we only observed 15 gene pairs with segmental duplication (ST gene family in maize. Several duplicated gene pairs, such as ZmpGlcT1 and ZmpGlcT2, ZmPMT7 and ZmPMT10, ZmSFP3 and ZmSFP4, ZmSTP5 and ZmSTP20, ZmSUT1 and ZmSUT7, exhibited similar exon-intron structures and conserved motifs, indicating a certain degree of functional redundancy (ZmST genes.In this study, we identified that 68 e genome . In the lication , Table 3dundancy –3D. DuriMdERDL6 and OsTST1 gene play important roles in plant development and kernel maturation in tomato and rice (SWEETs showed increased expression levels during maize seed germination, indicating their involvement in nutrient supply to the embryo axis (ZmSWEET family members exhibited specific expression patterns in embryo and surrounding regions and upregulated expression under abiotic stress (Arabidopsis, the gene AtPLT5 was primarily expressed in sink tissues (ZmPMT group were expressed in seeds and vegetative organs like old leaves, roots and stems, indicating their involvement in sugar transport to specific sink tissues in plants. Neither AtSTPs in Arabidopsis nor ZmSTPs in maize were found in the female gametophyte or developing seeds (STP family may not be involved in the seed maturation process. This suggestion was supported indirectly by previous research, demonstrating the involvement of ZmSTP2 and ZmSTP20 in maize disease resistance (AtERDL6 orthologs showed higher expression levels in fleshy fruits that accumulated a large amount of sugar, such as tomatoes (ZmSFPs in maize showed lower expression levels than fruits, indicating that the role of SFPs may not be prominent in crops. AtSUC2 was expressed in source-leaf companion cells of phloem (OsSUT1 in rice played a role in sucrose transport during grain filling (ZmSUTs showed high expression levels in leaves and developing seeds, implying conserved functions of SUTs across Arabidopsis, rice and maize. It was reported that the zmsut2 mutant plant displayed slower growth, smaller ears, and reduced yield compared to WT (AtTMT1 gene was strongly expressed in young developing tissues (ZmTSTs in maize exhibited higher expression levels in the young leaves, suggesting their involvement in sugar transport during rapid tissue expansion of cells in young tissues. The expression levels of OsTMT1 and OsTMT2 in rice were high in various organs excluding the endosperm (ZmTSTs in maize were highly expressed in leaves, roots, stems, tassels, embryos and seeds, with lower expression in endosperm. Moreover, ZmTSTs showed higher expression levels in the early stage of grain filling compared to the late stage of grain filling, indicating their importance in early seed development. AtVGT1 was detected in all developmental stages and organs except roots in Arabidopsis (ZmVGT genes were expressed in roots. Overall, most ZmPMT, ZmSFP and ZmSTP genes were predominantly expressed in vegetative organs and barely expressed in developing kernels, whereas, most ZmpGlcT, ZmVGT and ZmTST genes were highly expressed in seeds (ZmST genes exhibited an increase in expression during the early stage of grain filling and a subsequent decline during seed development in maize, suggesting their potential role in grain filling and seed maturation. Overall, our findings deepen our understanding of the pivotal roles of these transporters during maize kernels development and maturation, with the potential to contribute to improving maize yield.Sugar transporters, divided into MFS-type sugar transporters and SWEET family, are critical for the proper functioning and coordination of various physiological processes during plants growth and development. Previous studies have highlighted the significant roles of MFS-type sugar transporters in different species. and rice . ZmSWEETryo axis . Moreovec stress . So far, tissues , while ing seeds , suggestsistance . AtERDL6tomatoes , orangestomatoes  and appltomatoes . Howeverf phloem , while O filling . Similared to WT . This re tissues , and ZmTndosperm , and simbidopsis , while Zin seeds , 8 and 9ZmST genes were identified and systematically analyzed in maize. Gene family analyses were conducted to investigate their physicochemical properties, chromosomal localizations, gene structures and biological functions in maize. The expression pattern analyses suggested that these ZmST genes may play a vital role in maize kernel development and could be potential candidates for improving maize yield. These significant findings will serve as valuable references for further research on ZmSTs and provide new genetic resources for high-yield maize breeding.In summary, sixty-eight 10.7717/peerj.16423/supp-1Figure S1Click here for additional data file.10.7717/peerj.16423/supp-2Table S1Click here for additional data file.10.7717/peerj.16423/supp-3Table S2Click here for additional data file.10.7717/peerj.16423/supp-4Table S3Click here for additional data file.10.7717/peerj.16423/supp-5Supplemental Information 1Click here for additional data file.10.7717/peerj.16423/supp-6Dataset S1Click here for additional data file.


KRAS-mutant driven cancers. In this review, we focus on recent strategies to inhibit RAS-signaling via disrupting PPIs associated with SOS1, RAF, PDEδ, Grb2, and RAS.Within the MAPK/RAS pathway, there exists a plethora of protein–protein interactions (PPIs). For many years, scientists have focused efforts on drugging KRAS and its effectors in hopes to provide much needed therapies for patients with  These surfaces are frequently devoid of well formed binding pockets, channels, and grooves in which small molecules typically bind  Th Th2, 12)SOS1 PPI . The cloV600E inhibitors that target the orthosteric/ATP-binding site has resulted in three approved B-RAFV600E inhibitors: vemurafenib [15–17) [V600E inhibitors has given rise to acquired resistance mechanisms within treated patients [V600E inhibitors surprisingly activate the MAPK pathway in KRAS mutant driven cancers [V600E inhibitors can increase dimerization potential with uninhibited RAF monomers in the cell, thus driving pathway activation when KRAS is in its active form. Recently, it has been demonstrated that type II “pan-RAF” inhibitors, such as LY3009120 [18–20), do not cause activation of the MAPK pathway [V600E inhibition, these pan-RAF inhibitors also inhibit wild-type B-RAF and C-RAF.RAS in its active, GTP-bound, form promotes the dimerization and phosphorylation of the RAF family of kinases , which in turn leads to the phosphorylation of MEK1/2 and further downstream signaling within the RAS/MAPK pathway ,64. The urafenib ,65,66,67urafenib ,69,70,71 [15–17) ,73,74. Opatients ,76,77. A cancers . It has  cancers ,80,81,82Y3009120 ,84,85,86Y3009120 ,88, and Y3009120   was originally thought to behave as an RAS mimetic, binding to the RAS binding domain (RBD) of C-RAF and B-RAF  and disrupting the RAS:RAF PPI [An alternative approach to inhibit the action of the RAF kinases is to disrupt the protein–protein interactions between RAF and RAS. The small molecule rigosertib C, 21 was:RAF PPI . However:RAF PPI ,92,93, a:RAF PPI ,95 or or:RAF PPI  compoundAfter translation, RAS proteins are frequently modified with lipids. Broadly, there are two types of post-translational lipid modifications that occur on the RAS proteins: irreversible farnesylation and subsequent reversible palmitoylation. KRAS is not reversibly palmitoylated in its hypervariable region. Upon farnesylation, KRAS undergoes rapid mediated transport to the Golgi (and subsequently the plasma membrane). This critical spatial organization of KRAS to the plasma membrane contributes to the protein’s activity in signal transduction . PDEδ biD) and disrupt the protein–protein interaction between KRAS:PDEδ [22, 23, PDEδ KD = 38 nM, 24, 25, 26, 27 .In 2013, Waldmann and coworkers harnessed a high-throughput Alpha Screen using farnesylated KRAS4B and His-tagged PDEδ to discover benzimidazole-based compounds that bound to the farnesyl-binding pocket of PDEδ. These compounds could effectively bind to PDEδ with nanomolar binding potencies  was elucidated through X-ray crystallography efforts . When tested in vitro, these compounds were found to inhibit cellular viability in the 10–100 μM range [Additional putative molecular glues were initially identified via in silico techniques by Vargas and coworkers. Their group harnessed virtual library screening and molecular dynamics simulations to identify aryl amide compounds B, 34–36.μM range ,111. HowG12C-targeted drugs adagrasib [34) and sotorasib [35) nor the several additional compounds working their way through the clinic behave like traditional PPI inhibitors that bind on the surface of one protein and compete with the binding of a partner protein , can bind KRASG12D in both the GTP-bound and GDP-bound states. X-ray crystal structures show that even when in the GTP-bound state, the Switch-II loop is perturbed and is not competent to bind RAF   protein A. Ratherbind RAF B. There   protein37, 15N-labeled KRAS and subsequent X-ray crystallography . This resulted in the design of 42 . Molecular modeling helped to explain the cellular data, by revealing that the binding of 42 to KRAS overlaps with the binding sites of effectors RAF, PI3K, and RALGDS.An additional set of compounds that bind in the Switch-I/Switch-II surface pocket were discovered by Rabbitts et al., who performed a fragment library SPR screen against HRASfragment . The antgn of 42 F, which G12V was used to discover two hits 43 and 44  concomitant tight binding of the protein partners, and the necessity for a well-defined groove or pocket in close proximity to the PPI of the protein partners. Herein, several approaches have been discussed to disrupt PPIs within the RAS/MAPK pathway, harnessing innovative platforms or strategies to find the first toehold of chemical matter. However, there are still plenty of opportunities for growth and improvement to these methodologies. Within this ever-growing field, we look forward to the new technologies and approaches that researchers will utilize to help combat KRAS-mutant driven cancers in the future.


Within this context of ‘united airway diseases’, nasal polyposis encompasses several nosological entities such as chronic rhinosinusitis with nasal polyps (CRSwNP) and aspirin-exacerbated respiratory disease (AERD). Because of the common pathogenic origins of asthma and nasal polyposis, it is not surprising that the more severe forms of both these disorders can be successfully treated by the same biologic drugs, targeting many molecular components  of the type 2 inflammatory trait.Asthma and nasal polyposis often coexist and are frequently intertwined by tight pathogenic links, mainly consisting of the cellular and molecular pathways underpinning type 2 airway inflammation. The latter is characterized by a structural and functional impairment of the epithelial barrier, associated with the eosinophilic infiltration of both the lower and upper airways, which can be driven by either allergic or non-allergic mechanisms. Type 2 inflammatory changes are predominantly due to the biological actions exerted by interleukins 4 (IL-4), 13 (IL-13), and 5 (IL-5), produced by T helper 2 (Th2) lymphocytes and group 2 innate lymphoid cells (ILC2). In addition to the above cytokines, other proinflammatory mediators involved in the pathobiology of asthma and nasal polyposis include prostaglandin D Asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) are widespread, often coexisting as inflammatory disorders affecting the lower and upper airways, respectively ,2. AsthmStaphylococcus aureus-derived enterotoxins . Th. Th10]. rotoxins . Similare asthma ,18.In addition to CRSwNP, asthma is also associated with nasal polyps in patients suffering from aspirin-exacerbated respiratory disease (AERD) , and it In asthma and nasal polyposis, type 2 pathomechanisms are responsible not only for upper and lower airway inflammation, but also for relevant structural changes. Within this context, bronchial remodeling is characterized by epithelial disruption, goblet cell hyperplasia, subepithelial fibrosis, smooth muscle thickening, and increased vascularization, whereas CRSwNP is marked by stromal edema, turbinate hypertrophy, collagen/fibrin deposition, and polyp formation ,24,25,26Notably, the recent advances in the common pathophysiologic processes underpinning asthma and nasal polyposis highlight the therapeutic importance of targeting shared pathogenic cells and molecules, including eosinophils, IgE, type 2 cytokines, and their receptors  27,28].,28.27,28On the basis of the above concepts, the purpose of this narrative review is to discuss the pathobiologic mechanisms implicated in type 2 inflammation responsible for asthma and nasal polyposis, as well as to outline the therapeutic impact of the current biologic treatments which can be used to manage the most severe forms of both these diseases.2 (PGD2), and cysteinyl leukotrienes.Type 2 inflammation is the predominant pathologic trait underpinning asthma and nasal polyposis, driven by either allergic or non-allergic mechanisms ,30,31,32+ T lymphocytes towards the Th2 immunophenotype . Wi. Wi89]. The humanized monoclonal antibody omalizumab binds to the two Cε3 domains of the constant region of human IgE, thus generating IgE/anti-IgE immune complexes which prevent IgE interactions with their high-affinity FcεRI and low-affinity FcεRII/CD23 receptors expressed by immune/inflammatory and airway structural cells ,91,92. TOmalizumab is also capable of effectively and safely treating CRSwNP . Indeed,Mepolizumab is a humanized monoclonal IgG1/k antibody that specifically interacts with the α-chain of IL-5, thus inhibiting its binding to the α subunit of the IL-5 receptor (IL-5Rα) ,105. SevThe phase 3, 52-week SYNAPSE trial showed that, in patients with CRSwNP, when compared with a placebo, mepolizumab induced significant improvements in both nasal obstruction assessed by a visual analogic scale (VAS) and total endoscopic nasal polyp score, as well as prolonged the time to the first needed nasal surgery . These dReslizumab is another humanized IgG4/k monoclonal antibody which behaves as an IL-5 inhibitor ,124. BotThe humanized and afucosylated IgG1/k monoclonal antibody benralizumab utilizes its Fab portions to occupy and blockade IL-5Rα, thus inhibiting the interaction between this receptor subunit and the natural ligand IL-5 . Further1 (forced expiratory volume in one second) in subjects with eosinophilic asthma and blood eosinophil levels of at least 300 cells/μL [Benralizumab has been extensively evaluated within the context of a wide program of RCTs named WINDWARD, including the phase 3 studies SIROCCO and CALIMA, which clearly showed that this biologic drug significantly lowered the number of exacerbations of severe eosinophilic asthma, and also had a positive impact on symptom control and lung function ,134. Thecells/μL . Both ZOcells/μL ,137. Morcells/μL . The rescells/μL ,140. Succells/μL . Taken tcells/μL ,144,145.In addition to confirming many of the above findings, the phase 3b RCT ANDHI also showed that benralizumab improved the symptoms of nasal polyposis, as indicated by the significant reduction in SNOT-22 score . In part1 and also improved the control of asthma symptoms, as well as decreasing the levels of relevant biomarkers of type 2 asthma such as FeNO and serum IgE concentrations [1 by more than 200 mL [Dupilumab binds to the α subunit of the IL-4 receptor (IL-4Rα) and prevents its dimerization with the other molecular components of both IL-4 (γC chain) and IL-13 (IL-13Rα1 subunit) receptors, thereby acting as a dual receptor antagonist of these two cytokines, whose biological effects are thus effectively inhibited by such a fully human IgG4 monoclonal antibody ,151. Thetrations . The LIBtrations . Indeed,n 200 mL . Among tn 200 mL . The clin 200 mL . In aduln 200 mL ,158,159.With regard to the biologic therapy of nasal polyposis, some adult CRSwNP patients refractory to intranasal corticosteroids, with or without comorbid asthma, were enrolled in one of the first double-blind, randomized, and placebo-controlled studies, which showed that dupilumab bettered the SNOT-22 score, endoscopic nasal polyp score, Lund-Mackay CT score, and the sense of smell, evaluated by UPSIT (University of Pennsylvania Smell Identification Test) score . DupilumPatients with asthma and nasal polyposis are frequently characterized by a predominant type 2 trait of airway inflammation . Type 2


Of these 10 cytokines with significant concentration alteration, protein–protein interaction analysis revealed that 8 established an intricate interaction network. Various immune mediators may contribute to the pathogenesis of simple DR. Attention should be given to the concentrations of immune mediators in ocular fluids even in simple DR. Large-scale studies are warranted to assess whether altered aqueous humor concentrations of these 10 immune mediators are associated with an increased risk of progression to advanced stages of DR.Various immune mediators identified to date are associated with the development of advanced forms of diabetic retinopathy (DR), such as proliferative DR and diabetic macular edema, although the exact pathophysiological mechanisms of early stages of DR such as simple DR remain unclear. We determined the immune mediator profile in the aqueous humor of eyes with simple DR. Fifteen eyes of fifteen patients with simple DR were studied. Twenty-two eyes of twenty-two patients with cataracts and no DR served as controls. Undiluted aqueous humor samples were collected, and a cytometric bead array was used to determine the aqueous humor concentrations of 32 immune mediators comprising 13 interleukins (IL), interferon-γ, interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation, normal T cell expressed and secreted (RANTES), monokine induced by interferon-γ, basic fibroblast growth factor (bFGF), Fas ligand, granzyme A, granzyme B, interferon-inducible T-cell alpha chemoattractant (ITAC), fractalkine, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), angiogenin, tumor necrosis factor-α, and CD40 ligand. Among the 32 immune mediators, 10 immune mediators, including bFGF, CD40 ligand, fractalkine, G-CSF, IL-6, IL-8, MIP-α, MIP-1β, and VEGF, showed significantly higher aqueous humor concentrations and the Fas ligand had significantly lower concentration ( Diabetic retinopathy (DR) is the major cause of blindness in the older population. Currently, 284.6 million people worldwide are estimated to have diabetes mellitus; approximately one third of those with diabetes are at risk of developing DR to some extent, and approximately one third of those with DR may advance to the vision-threatening stage . Two majHowever, there is no report to date which describes immune mediator concentrations in the ocular fluids of eyes with simple DR. Recently, two-color flow cytometry has been used for the simultaneous detection of many immune mediators using a very small volume of a sample such as aqueous humor . The preThe purpose of this study was to identify and quantify a wide spectrum of immune mediators including cytokines, chemokines, growth factors, and apoptosis-related molecules in aqueous humor samples collected from eyes with simple DR. Understanding how immune mediators are associated in the early stages of simple DR may shed light on the pathophysiology of DR progression and help to develop new biomarkers for early diagnosis.1C (HbA1C) of 7.5 ± 1.4 and mean duration of diabetes of 12.1 ± 7.5 years  with simple DR were identified from the medical records between September 2011 and May 2022, and 22 patients (22 eyes) with cataract and no diabetic mellitus (DM) were included as disease controls. The 15 patients  diagnosed with simple DR according to Davis classification were 65.8 ± 11.6 years of age; all had type 2 diabetes mellites (T2DM) with mean hemoglobin A.5 years . The 22 Undiluted aqueous humor samples (approximately 100 μL) were collected via an anterior chamber tap with a 25 needle from patients with simple DR during outpatient consultation, and from cataract patients with no DM before cataract surgery. All samples were stored at −80 °C until use. The CBA Flex immunoassay kit  was used to determine the aqueous humor concentrations of 32 immune mediators comprising interleukins (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-17A, and IL-21, interferon (IFN)-γ, interferon-γ-inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation, normal T-cell expressed and secreted (RANTES), monokine induced by interferon-γ (Mig), basic fibroblast growth factor (bFGF), Fas ligand, granzyme A, granzyme B, interferon-inducible T-cell alpha chemoattractant (ITAC), fractalkine, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), angiogenin, tumor necrosis factor (TNF)-α, and CD40 ligand. This method allows the simultaneous detection of many analytes with a very small volume of sample (100 µL), as described previously  in this study. This aligns with previous reports, indicating a significant upregulation of aqueous humor IL-8 concentration in diabetic macular edema and PDR relative to controls [Notably, the aqueous humor concentration of IL-8 in simple DR was significantly different compared to controls [13.47 0–153) pg/mL versus 3 (0–15.99) pg/mL, 3 pg/mL vcontrols . TherefoIn addition, we investigated the relationship among immune mediators with altered aqueous humor concentrations in simple DR. Among the 10 cytokines with significantly altered aqueous humor concentrations, a protein–protein interaction analysis using the STRING database revealed that eight were closely interconnected, forming a complex interaction network . These rThe etiology of DR remains largely unknown. Exposure to hyperglycemia over an extended period is considered to promote changes that cause vascular endothelial impairment. The early histologic features of DR are capillary basement membrane thickening, leukostasis, a loss of endothelial cells, and a loss of pericytes ,18. SustSustained hyperglycemia is considered to be the major initiating factor of DR. However, detailed mechanisms causing retinal abnormality remain unclear. The disease involves the progression of retinovascular damage with stepwise clinical progression from mild stages to advanced proliferative changes. The rate of progression varies among patients and depends mainly on systemic factors such as blood glucose level, blood pressure control, and blood lipid profile, which constitute independent risk factors of DR development . Our resDR tends to cause ocular inflammation. Although the precise mechanisms underlying such chronic inflammation are not yet known, macrophages and many immune mediators including angiogenic growth factors, cytokines, and chemokines are involved in the onset and progression of DR. In advanced stages of DR, such as PDR and diabetic macular edema, immune cells and mediators play important roles in disease progression ,23,24,25Among the nine immune mediators that were upregulated in simple DR in this study, fractalkine, also known as chemokine CX3C Receptor 1, is particularly interesting for its dual roles as a chemotaxin and an activator of retinal microglia and infiltrating macrophages, as well as being a physiologically active compound that potently increases vascular permeability and promotes angiogenesis ,38,39. IOf the ten cytokines that showed significant alterations in simple DR in our study, nine immune mediators form a tightly interwoven and complex interaction network. This finding suggests that many immune mediators act synergistically, interacting to trigger the development of DR. Further research is warranted to uncover the functional roles of these 10 cytokines in the initiation and progression of early-stage DR.This study has several limitations. First, because retrospectively collected samples were used, the time from HbA1c measurement or disease onset to aqueous humor collection varied among samples. This may suggest a variable degree of disease progression across the samples. Second, the single-center case–control study design may give rise to potential sampling or clinical biases, including collection bias, in specific periods related to geographic location, ethnicity, age, and sex distribution. Third, selection bias when using cataract patients instead of healthy controls is included as a limitation. Notably, the simple DR group in this study consisted of predominantly males, which is a potential bias. In addition, our sample size was small to detect the generalized alternation with simple DR. To address these concerns, future prospective multi-center studies on a global scale would be needed to validate the present findings.The aqueous humor concentrations of 10 immune mediators comprising bFGF, CD40 ligand, Fas ligand, fractalkine, G-CSF, IL-6, IL-8, MIP-1α, MIP-1β, and VEGF were altered in patients with simple DR. Although this is the first study of the aqueous humor immune mediator profile in simple DR, our results are mostly in agreement with previous studies showing that various immune mediators associated with inflammation and angiogenesis are related to the development of DR. Combinations of these immune mediators may also be useful biomarkers for the early detection of DR and the prediction of prognosis. Future large-scale studies are needed to evaluate whether altered aqueous humor concentrations of these cytokines or their interactions are associated with an increased risk of progression to advanced stages of DR.


ERG11 were postulated to be the predominant cause of antifungal drug resistance in the clinic. However, recent advances in whole genome sequencing have revealed that there are multiple mechanisms leading to the microevolution of resistance. In many fungal species, resistance can emerge through ERG11-independent mechanisms and through the accumulation of mutations in many genes to generate a polygenic resistance phenotype. In addition, genome sequencing has revealed that full or partial aneuploidy commonly occurs in clinical or microevolved in vitro isolates to confer antifungal resistance. This review will provide an overview of the mutations known to be selected during the adaptive microevolution of antifungal drug resistance and focus on how recent advances in genome sequencing technology have enhanced our understanding of this process.The mortality rates of invasive fungal infections remain high because of the limited number of antifungal drugs available and antifungal drug resistance, which can rapidly evolve during treatment. Mutations in key resistance genes such as  Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus and Pneumocystis jirovecii [Cryptococcus species complex (C. deneoformans and C. gattii), Candida genera  and Aspergillus genera  also cause human invasive infections [Fusarium, Scedosporium, Mucorales and Lomentospora genera can cause life-threatening invasive infections, which, although rarer, have high mortality, because of high resistance rates or inherent resistance [Coccidioides immitis, Coccidioides posadasii, Blastomyces dermatitidis and Histoplasma capsulatum (USA); Paracoccidioides brasiliensis and Paracoccidioides lutzii (Brazil); and Talaromyces marneffei (Southeast Asia) [C. neoformans, C. albicans, A. fumigatus and C. auris as a critical group of species requiring priority research development and public health action to improve responses and prevent the development of antifungal drug resistance [C. glabrata, Histoplasma spp., Mucorales, Fusarium spp., C. tropicalis and C. parapsilosis are classified as high priority by the WHO, and Scedosporium spp., Lomentospora prolificans, Coccidioides spp., C. krusei, C. gattii, T. marneffei, P. jirovecii and Paracoccidioides spp. are classified as medium priority [Fungal infections pose an escalating health problem; however, their contribution to the global burden of disease remains under-recognized. It has been estimated that 1.7 billion people are infected with fungi each year, resulting in 1.5 million deaths annually from invasive infections . Althougirovecii . Other sfections ,4,5,6. Ist Asia) ,9,10. ThInvasive fungal infections are difficult to treat and result in a high mortality rate, often surpassing 50% and increasing to up to 90% for some species if treatment is delayed ,7,11. ThFungal cells possessing mutations causing antifungal resistance are selected for in the clinic and become predominant in the population in a short timeframe in a process termed adaptive microevolution A. AdaptiThe limited numbers of antifungal drugs and the tapered pipeline for the development of novel antifungals are widely recognised challenges for clinical mycology . Many anAzoles are the largest and most widely used class of antifungal agents because of their broad-spectrum activity and oral administration, which is useful in resource-limited settings ,19. AzolERG11/cyp51A gene encoding the target enzyme of the ergosterol biosynthesis pathway, lanosterol 14 alpha-demethylase [ERG11 have been shown to lead to resistance in C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis and C. albicans clinical isolates, which typically over-express ERG11 [C. auris clinical isolates possess ERG11 sequence variants that account for their intrinsic multi-drug resistance to azoles [ERG11 mutations have also been associated with fluconazole resistance in C. neoformans clinical isolates [C. glabrata, the resistance of microevolved strains can occur via mutations in ERG3 and ERG4 [C. albicans, mutations in ERG3, ERG2 or ERG6 confer resistance by preventing the generation of toxic sterol intermediates; however, ERG3A and ERG3B mutants in A. fumigatus do not [Resistance to azole antifungals is commonly attributed to the selection of mutants that over-express or alter the ethylase ,27,28,29ss ERG11 ,27,28,30o azoles . ERG11 misolates ,33,34,35s do not ,37.cyp51A (ERG11 homologue) promoter, in combination with a specific substitution (TR34/L98H) [A. fumigatus environmental isolates were resistant to at least one azole [In aspergillosis patients where an azole-resistant isolate is obtained through environmental exposure, the most common mechanism resulting in resistance is the duplication of a 34 bp tandem repeat in the 34/L98H) ,38,39. T34/L98H) ,41. Agri34/L98H) ,41,42. Acyp51A promoter tandem repeat have since been discovered in both the clinic and the environment in various locations around the world [A. fumigatus isolates, obtained from the environment from regions all over the globe, predominately carried the cyp51A TR34/L98H (60.7%) or TR46/Y121F/T289A (15.0%) alleles [cyp51A mutations that occur during human infection are commonly missense mutations that prevent the azole from binding to the 14 α-demethylase enzyme azole target [More pan-azole resistance alleles arising from various alterations to the he world ,40,43. A alleles . cyp51A R34/L98H 0.7% or Te target ,40,44.C. neoformans (Sre1) and A. fumigatus (SrbA) activates genes required for ergosterol biosynthesis, and deletion results in increased susceptibility to azole antifungals [cyp51A promoter [atrR, also binds the tandem repeats in the cyp51A promoter and is required for normal tolerance to azoles [SRE1/srbA in C. albicans, CPH2, is not necessary for ergosterol biosynthesis [C. albicans, a different transcription factor, Upc2, regulates the expression of ergosterol biosynthesis genes [UPC2 in C. albicans increases azole susceptibility and overexpression or activating mutations cause azole resistance in vitro [C. albicans clinical isolates, gain-of-function UPC2 mutants contribute to the increased expression of ERG11 and fluconazole resistance [UPC2 in C. glabrata decreases the expression of ergosterol biosynthesis genes [Genes regulating the transcription of ergosterol biosynthesis genes and ergosterol production also play a role in azole susceptibility . The steifungals ,47,48. Spromoter ,48. An ao azoles . The homynthesis . Rather,in vitro . In C. asistance ,54,55,56is genes .cyp51A expression in A. fumigatus is also regulated by a transcriptional complex containing HapB, HapC and HapE and an additional factor, HapX [hapE was initially found in an azole-resistant clinical isolate via whole genome sequencing [HapB, HapC, HapE and HapX and the expression of HapEP88L result in increased resistance to azoles [hmg1 is a sterol-sensing protein bound to the endoplasmic reticulum that initiates ergosterol biosynthesis. hmg1 mutations generated in the sterol-sensing domain or identified in clinical isolates result in altered sterol levels and azole resistance [or, HapX . A mutatquencing . The delo azoles . HMG CoAsistance ,59. Mutasistance . Mutatiosistance .ERG1) confer resistance to terbinafine in clinical isolates of Trichophyton interdigitale and Trichophyton rubrum [A. fumigatus and C. albicans also confers terbinafine resistance [Similar to azoles, allylamine antifungals target ergosterol biosynthesis  Figure . Terbinan rubrum ,63. The sistance ,64.MDR1 genes encoding efflux pumps of the major facilitator superfamily (MFS) or CDR genes encoding efflux pumps of the ATP-binding cassette (ABC) superfamily occurs in azole-resistant clinical isolates of C. albicans, C. parapsilosis, C. krusei, C. auris and C. glabrata [CDR1, CDR2 and MDR1 genes [C. albicans and C. parapsilosis are Tac1, Mrr1 and Upc1 [C. albicans [MDR1 expression, respectively [C. albicans and C. parapsilosis gain-of-function mutations in TAC1 result in the constitutive expression of CDR1 and CDR2 in azole-resistant clinical isolates and in vitro [MRR1, UPC1 and CAP1 gain-of-function mutations result in the overexpression of MDR1 in azole-resistant clinical isolates (MRR1 and UPC1) or in vitro (CAP1) [TAC1 and UPC1 and the overexpression of CDR1 are also responsible for azole resistance in C. auris [The overexpression of glabrata ,65,66  ,70,71,75C. auris ,77.C. glabrata clinical isolates and those from in vitro evolution experiments possess activating mutations in the PDR1 gene, which encodes a transcription factor that induces the expression of CDR1 [HST1 and components of the mediator complex result in fluconazole resistance [Almost all azole-resistant  of CDR1 ,79,80,81sistance ,83.C. neoformans and C. gattii, the expression of the ABC and MFS transporter genes is induced upon treatment with fluconazole [S. cerevisiae-expressing C. gattii AFR1, AFR2 and MDR1 lead to higher resistance to fluconazole [AFR1 is overexpressed in a fluconazole-resistant C. neoformans clinical isolate, and mice infected with an AFR1 mutant respond better to treatment with fluconazole [AFR1 overexpression in a susceptible strain in vitro or gene deletion results in increased fluconazole resistance or susceptibility, respectively [AFR1 is regulated by the CRZ1 and yap1 transcription factors [In conazole ,85. S. cconazole . AFR1 isconazole ,87. AFR1atrI, cdr1B and mdr1 in A. fumigatus, occurs in azole-resistant clinical isolates and the overexpression of atrF, Afumdr1, Afumdr3 and Afumdr4 in vitro results in azole resistance [atrR and yap1, which regulate the expression of cdr1B and atrF, respectively, also confer resistance [The overexpression of efflux pumps encoded by sistance . Mutatiosistance ,89.FKS genes , which results in altered nucleotide metabolism, also confers resistance in C. neoformans by suppressing the toxicity of 5-FC and its derivative, 5-FU [5-FC is used only in combination with amphotericin B, as resistance to 5-FC emerges frequently. Mutations in oformans ,102. Mutve, 5-FU   pathway, although an MLH1 variant  has been found in C. auris [MSH2 has been discovered in clinical populations of C. deuterogattii, C. neoformans, C. glabrata and A. fumigatus [MSH2 mutators in clinical populations and their clinical relevance remains controversial, as a correlation with antifungal resistance is often not found. Challenges in measuring mutation rates have led to many studies reporting sequence variance without confirming an increased mutation rate, and some MSH2 alleles in C. glabrata previously called mutators have subsequently been shown not to result in a mutator phenotype [MSH2 are strongly associated with the emergence of resistance to azoles, polyenes, pyrimidine analogues and echinocandins in vitro [C. glabrata, resistance to azoles, amphotericin B and echinocandins in msh2Δ mutants can arise from mutations in PDR1 (azoles), ERG6 (amphotericin B), FKS1 and FKS2 (echinocandins) [msh2Δ mutants in C. deuterogattii revealed mutations in FUR1, FCY2 and UXS1 [msh2Δ mutants in C. neoformans revealed polygenic resistance, where mutations accumulate in genes that alter stress signalling, cellular efflux, membrane trafficking and epigenetic modification [The microevolution of antifungal resistance is significantly increased by an elevated mutation rate, which bestows the fungal population with higher genetic diversity upon which selection can act . Strainsumigatus ,106,107.C. auris . Non-synumigatus ,113,114.henotype . Neverthin vitro ,106,107.candins) . Whole gand UXS1 . Whole gfication .ERG11 and PDR1 appear to be the predominant mode of azole resistance in C. albicans and C. glabrata, respectively, this is likely not the case in other pathogenic fungi, where resistance is also driven by ERG11-independent mechanisms [C. neoformans clinical isolates lack any mutations in ERG11 [C. gattii Pacific Northwest outbreak also concluded that neither the overexpression of ERG11 nor mutations within the gene were responsible for the resistance to fluconazole in these isolates [A. fumigatus azole-resistant clinical isolates do not possess mutations in the regulatory or coding regions of cyp51A [Although mutations in single key genes such as chanisms ,117. Betin ERG11 ,33,34,35isolates . In addif cyp51A ,119,120.C. albicans clinical isolates from oral candidiasis patients revealed mutations in genes required for filamentous growth, cell adhesion, biofilm formation, cell cycle and stress, drug responses and carbohydrate binding, as well as changes in ploidy [C. glabrata clinical isolate over time from azole susceptibility to posaconazole resistance and clotrimazole resistance to fluconazole/voriconazole resistance showed that only the population with resistance to all azoles had a gain-of-function PDR1 mutation, whereas intermediate strains possessed alternative resistance mechanisms [C. auris, the mutational spectrum, coupled with an analysis of the transcriptome of fluconazole-resistant in vitro microevolved strains, suggests mutations commonly accumulate in genes encoding transcription factors , but there are a large number of different mechanisms that promote drug resistance, including changes in ploidy and multiple pathways leading to resistance, including efflux transporter upregulation and transcriptional changes in ribosome biogenesis, RNA metabolism and sugar transport [An analysis of the mutational profiles of n ploidy . Transcrchanisms . In C. aransport ,108,123.msh2 (mutator) isolates in C. neoformans has shown aneuploidy and mutations accumulating in the genes of some of the same biological processes shown by transcriptomic studies to be differentially expressed in response to azole exposure, such as stress signalling, transmembrane transport, epigenetic modification, translation, transcription and carbohydrate metabolism [KES1 and ALP3) and epigenetic modification , and the deletion of these genes resulted in fluconazole resistance [Whole genome sequencing of in vitro microevolved azole-resistant tabolism ,124. Azosistance .A. fumigatus showed resistant strains did not possess mutations in cyp51A, hmg1 or hapE, but transcriptomic analysis of these strains showed resistance was likely due to the overexpression of transcription factor asg1, which has been shown to regulate the expression of several ABC and MFS transporter genes and genes of the ergosterol biosynthesis pathway [In vitro microevolution experiments on voriconazole resistance in  pathway .C. neoformans in a process called heteroresistance [C. neoformans, chromosome 1 containing ERG11 and AFR1 is duplicated, followed by the subsequent duplication of chromosomes 4, 10 and 14 [Recent advances in whole genome sequencing and mutational profiling have also revealed that large-scale alterations to the genome, such as changes in ploidy (the number of chromosome sets), are a common mechanism utilized by fungi to adapt to environmental stress and generate azole resistance ,127,128.sistance ,130. Uposistance ,130. In 0 and 14 .C. neoformans clinical isolates exposed to azoles commonly have disomy of chromosome 1, which results in azole resistance [A. fumigatus chronic pulmonary aspergillosis clinical isolates, which do not possess a mutation in cyp51A, display aneuploidy of chromosomal regions containing genes associated with azole resistance, cyp51A, cyp51B or cyp51ec, as well as those encoding MFS and ABC transporters and transcription factors [In addition, whole genome sequencing has revealed many antifungal-resistant clinical isolates possess permanent aneuploidies. sistance ,132,133.sistance . Aneuplosistance ,138,139. factors .C. albicans clinical isolates, azole resistance can arise from large genome rearrangements, including the translocations of chromosomal arms; the duplication of the chromosomal region of the left arm of chr5 containing ERG11 and TAC1 to produce an isochromosome (i(5L)); trisomies of chr3, chr4, chr5 or chr7; and loss-of-heterozygosity in the chromosomal regions containing TAC1 (chr5) and MRR1 (chr3); and the formation of new chromosomes via the duplication of segments containing a centromere and the addition of telomeric ends [C. albicans genome; for example, the recombination of a long-inverted repeat sequence at the centromere of chr5 is required for the formation of i(5L) [CDR1, CDR2, CRZ1 (transcription factor) and MRR1 on chr3 and TAC1 or ERG11 on chr5 [C. albicans that evolved in fluconazole concentrations close to the MIC50 of their ancestor acquired aneuploidies and copy number variations, whereas lineages evolved above the ancestral MIC50 acquired mutational changes [C. albicans, resistance to posaconazole generated through in vitro experimental evolution also results in aneuploidy and cross-tolerance to fluconazole [C. albicans [In ric ends ,147,148.of i(5L) ,149. Res on chr5 . One stu on chr5 . Recentl on chr5  showed t changes . In C. aconazole . Loss ofalbicans ,152.A. flavus strains, selected for voriconazole resistance, contain duplications of chromosome 8 or a segmental duplication of chromosome 3, which contains atrA but no cyp51A mutations [C. auris that sequenced and compared the genomes of parental fluconazole-susceptible strains and experimentally evolved fluconazole-resistant strains showed the rapid generation of the aneuploidies of chromosome 5 (which contains the regulator TAC1B) or 3, segmental aneuploidy of chromosome 1 (contains ERG11), loss of subtelomeric regions, karyotype alterations and the generation of supernumerary chromosomes  [C. glabrata [C. neoformans MSH2 strains possess permanent aneuploidies of chromosomes 1 and 4 [Azole-resistant strains selected directly in vitro or during microevolution experiments also gain permanent aneuploidies . A. flavutations . Numerouosome 5) ,123,155.glabrata . Flucona 1 and 4 .ERG11 were once thought to be the predominant cause of antifungal drug resistance. However, the recent use of whole genome sequencing has shown that the microevolution of resistance is far more complicated, and there is still a long way to go to understand this process. Although mutations in single genes such as ERG11 and PDR1 are the predominant cause of resistance in Candida species, a large proportion of clinical isolates of other fungal species lack ERG11-dependent resistance mechanisms and instead possess accumulated mutations in many genes in order to generate a polygenic resistance phenotype. Currently, it is impossible to determine the precise contribution, if any, of every mutational change observed in the genomes of resistant strains. Sexual outcrossing is not possible for most pathogenic fungi, meaning that the association between mutations and resistance phenotypes is difficult to analyse. Their phenotypic contribution to resistance could be confirmed by regenerating the mutation in an antifungal-susceptible strain using gene editing technology; however, this process would be unrealistic to perform for such a large number of mutations. In addition, these complex mutational profiles are coupled with highly plastic genomes—where aneuploidy is rapidly generated either transiently or permanently—and transcriptional changes, which must be separated from adaptive responses. Understanding the many factors contributing to the emergence of resistance is crucial for the development of effective future treatment strategies.There is a critical need for the development of novel antifungals given the restricted number available, limitations on use and the emergence of resistance. Resistance is rapidly becoming an important issue that will worsen without the introduction of new antifungals for use in the clinic. Mutations in key resistance genes such as