Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular junctions. Cytokines play important roles in facilitating the immune response and augmenting the pathogenic antibody production. The current study aims to sensitively characterize the serum levels of cytokines with very low concentration in generalized MG (gMG).Using ultrasensitive single-molecule arrays (SIMOA), we measured serum IL-2, IL-4, IL-5 and IL-12p70 in 228 participants including 152 immunotherapy-naïve anti-acetylcholine receptor (AChR) subtype gMG from Huashan MG registry and 76 age-matched healthy controls. Subgroup analysis was then performed by stratifying patients according to the onset ages, MGFA classification, disease duration at baseline.P < 0.0001; 0.029 pg/mL versus 0.018 pg/mL, P = 0.0259; 0.215 pg/mL versus 0.143 pg/mL, P = 0.0007; 0.132 pg/mL versus 0.118 pg/mL, P = 0.0401). Subgroup analysis revealed that IL-2 levels were slightly elevated in gMG with MGFA II compared to MGFA III/IV , as well as elevated levels of IL-2  and IL-5  in late-onset gMG compared with the early-onset gMG. gMG patients with a long duration had a significant increased serum IL-12p70 than those with a short duration .Serum IL-2, IL-4, IL-5 and IL-12p70 levels were significantly elevated in gMG compared to controls (0.179 pg/mL versus 0.011 pg/mL, Serum IL-2, IL-4, IL-5 and IL-12p70 levels were increased in AChR subtype gMG using ultrasensitive measurement. Serum cytokines with very low concentrations may provide as potential biomarkers in stratifying gMG patients in future prospective cohort studies. Myasthenia gravis (MG) is characterized by fluctuating muscle weakness caused by autoantibodies against the acetylcholine receptor (AChR) and the muscle-specific tyrosine kinase (MuSK) , 2. RecePrevious studies on MG-relevant inflammatory cytokines mainly focused on Interleukin (IL)-17 (Th17 related), IL-21(Tfh related) and IL-6  –12. In cUltra-sensitive single-molecule arrays (SIMOA) provides an alternative and more sensitive method for detecting trace cytokines. , 17. HerA total of 228 participants were finally enrolled. Of these, 152 gMG patients  were immunotherapy naïve with positive anti-AChR antibodies and 76 age-matched participants were healthy controls . The median anti-AChR antibodies titer for MG is 5.86  nmol/L and thymoma concurrence is 9.9%. We further divided 152 AChR subtype gMG patients into subgroups: (1) Clinical severity according to the Myasthenia Gravis Foundation of America (MGFA): mild group with MGFA II (n = 100) and moderate to severe group with MGFA III/IV (n = 52); (2) Onset ages: early-onset MG  and late-onset MG ; (3) Disease duration: short disease duration group (n = 76) and long disease duration group (n = 76). Detailed information regarding each subgroup are listed in Table The levels for serum IL-2, IL-4 and IL-5 was higher in immunotherapy-naïve gMG in comparison to HCs (measurable in 98.7% (150/152) versus 82.9% (63/76) for IL-2; 99.3% (151/152) versus 78.9% (60/76) for IL-4; 99.3% (151/152) versus 85.5% (65/76) for IL-5, respectively. For IL-12p70, the levels were equally matched in gMG and HCs (measurable in 83.6% (127/152) versus 84.2% (64/76)).P < 0.0001; 0.029 [0.016–0.045] pg/mL versus 0.018 [0.011–0.044] pg/mL, P = 0.0259; 0.215 [0.129–0.360] pg/mL versus 0.143 [0.077–0.243] pg/mL, P = 0.0007; 0.132 [0.092–0.202] pg/mL versus 0.118 [0.073–0.167] pg/mL, P = 0.0401) . In contrast, there were no differences in serum IL-4 levels , IL-5 levels , IL-12p70 levels  between these two subgroups . In parallel, serum IL-5 was also elevated in patients with late-onset gMG . In contrast, there were no difference in serum IL-4 nor IL-12p70 levels between these two gMG groups with different onset age  , while no significant differences in IL-2 , IL-4  and IL-5   and a negative correlation with disease course , baseline MG-ADL , QMG  and MG-QoL15 . In parallel, serum IL-5 had a positive correlation with age  and a negative correlation with QMG . Serum IL-4 was negatively correlated with disease course , while IL-12p70 was positively correlated with disease course . Multivariate analyses indicated that IL-4 levels had positive impact on QMG scores and MMT scores , while cytokines IL-2, IL-5 and IL-12p70 showed no statistically significant difference  score, MGFA-quantitative MG test (MGFA-QMG), MG quality of life 15-item (QoL-15) questionnaire, and MG manual muscle test (MMT). Serum IL-2 had a positive correlation with age . The other cytokines showed no statistically significant difference.Moreover, the correlations between baseline cytokine levels and the short-term prognosis at 12 months after enrollment were assessed. Baseline IL-12p70 was positively correlated with MG-ADL score at 12 months-follow up , which have been demonstrated decreased and impaired in MG patients , 29. OngRecent clinical studies had emerged regarding the difference in presentations and outcomes between the EOMG and LOMG cohorts. The majority of LOMG had ocular phenotype. The presence of neither anti-titin nor anti-MuSK antibodies points to an unfavorable outcome . Still tAlthough the concordant elevation in type 1 and type 2 cytokines were demonstrated in gMG, the correlations between baseline cytokines with the short-term clinical outcome revealed some difference. IL-12p70 promotes Th1 immunity thus imposes positive effect in longitudinal autoimmune response for MG. However, the correlations are relatively weak to make any conclusions.+ T lymphocytes and B cells are required to better delineate the role of cytokines in the immunological network. Third, given the current cost and the availability, SIMOA may not be adopted for routine diagnostics in a short time. Ultrasensitive measurement of serum cytokines with very low concentrations in a large longitudinal prospective MG cohort is anticipated in future investigations to monitor the outcome and therapeutic response.Several limitations in this study are needed to be addressed. First, the normal datasets for all age groups are not available for these cytokines measured by ultrasensitive analysis. Second, the concurrent measurement of peripheral CD4We confirmed the high levels of serum IL-2, IL-4, IL-5 and IL-12p70 in a cohort of AChR subtype gMG patients. In particular, further analysis revealed significant difference in serum levels of these cytokines in gMG subgroups divided by clinical severity, onset ages and the prior disease duration. These inflammatory serum cytokines may provide as disease biomarkers in stratifying gMG patients in future prospective cohort studies.This study was conducted in accordance with the ethical standards established in the 1964 Declaration of Helsinki and was approved by the Medical Ethics Committee of Huashan Hospital affiliated to Fudan University . Written informed consent was obtained from each participant.There are 1662 MG patients registered in National center for neurological disorders, huashan hospital, Shanghai from August 8, 2012, through May 31, 2021. MG mimicking diseases including Lambert-Eaton myasthenic syndrome, peripheral neuropathy, myopathies, and motor neuron diseases were excluded before the recruiting. The patients who have already enrolled in previous study for 18 cytokine measurement have been excluded in current study  and group comparisons were analyzed using Mann Whitney test as the data were previously tested for not following the normal distribution. Categorical variables were presented as No. (%) and analyzed by Chi-square test. Correlations between serum cytokines and clinical variables of MG cohort were analyzed using Spearman’s method. Multiple linear-regression analyses were then further conducted. We considered a two-tailed adjusted


Itraconazole is first-line treatment for mild-moderate blastomycosis and consolidation of moderate-severe disease. Itraconazole is metabolized to 3 metabolites, including an active metabolite hydroxy-itraconazole. The sum of itraconazole and hydroxy-itraconazole levels > 1.0 mcg/mL is guideline recommended for treatment of invasive fungal infections; conversely, some experts suggest targeting a parent compound level alone > 1.0 mcg/mL. This study aims to compare clinical outcomes and adverse drug reactions (ADRs) of combined itraconazole and hydroxy-itraconazole levels > 1.0 mcg/mL versus itraconazole parent compound alone > 1.0 mcg/mL in patients with blastomycosis.Blastomyces infection who received itraconazole with at least one documented serum itraconazole level. The primary outcome was rate of partial or complete treatment response in patients with a combined itraconazole and hydroxy-itraconazole level > 1.0 mcg/mL versus itraconazole parent compound > 1.0 mcg/mL for > 75% of measured levels. ADRs attributable to itraconazole were compared between the groups. Treatment response rates and ADRs were compared between groups using two proportion z-tests.This study is a single-center, retrospective chart review of patients ≥ 18 years with probable or proven Total of 80 patients were included: 36 = combined itraconazole and hydroxy-itraconazole > 1.0, 32 = itraconazole parent alone > 1.0, 12 = 75% of all levels < 1.0. No statistically significant difference was observed between groups for blastomycosis rate of partial or complete treatment response . Significantly higher mortality was observed in patients failing to achieve itraconazole or itraconazole/hydroxy-itraconazole > 1.0 . There was no significant difference in total ADRs between the three groups (p=0.56).This limited evidence supports an itraconazole therapeutic target combining itraconazole and hydroxy-itraconazole > 1.0 for blastomycosis treatment.Paschalis Vergidis, MD, AbbVie: DSMB|Cidara: Grant/Research Support|Scynexis: Grant/Research Support Christina G. Rivera, PharmD, Gilead: Grant/Research Support|Gilead: Honoraria|Insmed: Honoraria.


These insect-resistant inbred lines can be used as parents in maize breeding programs to develop new varieties.Maize seedlings contain high amounts of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and the effect of DIMBOA is directly associated with multiple insect-resistance against insect pests such as Asian corn borer and corn leaf aphids. Although numerous genetic loci for multiple insect-resistant traits have been identified, little is known about genetic controls regarding DIMBOA content. In this study, the best linear unbiased prediction (BLUP) values of DIMBOA content in two ecological environments across 310 maize inbred lines were calculated; and their phenotypic data and BLUP values were used for marker-trait association analysis. We identified nine SSRs that were significantly associated with DIMBOA content, which explained 4.30–20.04% of the phenotypic variation. Combined with 47 original genetic loci from previous studies, we detected 19 hot loci and approximately 11 hot loci  supported pleiotropy for their association with two or more insect-resistant traits. Within the 19 hot loci, we identified 49 candidate genes, including 12 controlling DIMBOA biosynthesis, 6 involved in sugar metabolism/homeostasis, 2 regulating peroxidases activity, 21 associated with growth and development [(auxin-upregulated RNAs (SAUR) family member and v-myb avian myeloblastosis viral oncogene homolog (MYB)], and 7 involved in several key enzyme activities . The synergy and antagonism interactions among these genes formed the complex defense mechanisms induced by multiple insect pests. Moreover, sufficient genetic variation was reported for DIMBOA performance and SSR markers in the 310 tested maize inbred lines, and 3 highly (DIMBOA content was 402.74–528.88 μg g Zea mays), is an important agro-economical crop that is utilized globally as food, animal feed, and biofuel products with production of more than 1.14 billion tons in 2018 [Ostrinia furnacalis; Lepidoptera, Pyralidae) is one of the most destructive insect pests in maize. Newly hatched ACB larvae primarily feed on leaves during the whorl stage; subsequently, the 3rd or 4th instars bore into the stalk. This may cause yield losses of 10 to 30% in the outbreaks recorded in China [Rhopalosiphum maidis; Homoptera, Aphididae), especially in tropical and warmer temperature regions [Maize  provides a fine-mapping method that enables researchers to identify functional variation in a broader germplasm background . Both QT−1 FW (ZY19-Jiu1101) with an overall mean of 94.21μg g−1 FW; similarly, the average DIMBOA content of these seedlings in the E2 environment varied from 8.84 (T58) to 493.40 μg g−1 FW (RX20-1006) with an overall mean of 93.26 μg g−1 FW , umc2314  and umc2031  exhibited the maximum number of alleles  . It is wIn addition, the population structure of these inbred lines was analyzed by STRUCTURE software. When various groups  ranging from 1 to 12 were compared, the ΔK reached the maximum value when K = 5, thus the 310 maize inbred lines were divided into five optimal groups A. The in−1 FW; accounted for 0.97%) had three genotypes, which were 19LX-1230, RX20-1006, and ZY19-Jiu1101; they were defined as high insect-resistant germplasms. Type II  had 15 inbred lines including F1227, M1005, F0501, LongF1008, 1201, M1009, PH1CRW, 1512, F3202, F1220, F3208, F2260, F1233, F2303, and F3210, and they were defined as moderate insect-resistant germplasms. Type III  had 27 genotypes, including ShanM3304, M1001, MeizaS2–3, Jizaoyu, M0803, F0306, M10202, ly6305, F2211, M1202, F0311, M1409Ying, F1501, M0124, M0822, M0105, F2502, M0505, PHHJC, 8723-2, M0306, F2222, F2213, M0824, M0125, 747, and F3226, and they were defined as insect-resistant germplasms. Type IV  included 52 moderate insect-susceptible germplasms. Type V  included 213 insect-susceptible germplasms . Type I (DIMBOA content in E1/E2: 467.11–528.88/402.74–493.40 μg grmplasms C. Furthermplasms B. The dahttps://www.maizegdb.org/data_center/map (accessed on 18 September 2022)) (p < 0.01) SSR loci associated with DIMBOA content in both environments (E1 and E2) by GLM and MLM, respectively; these SSR loci were in Bin 1.04, Bin 1.11, Bin 2.01, Bin 4.00, Bin 4.01, Bin 6.02, Bin 8.04, and Bin 10.04. The phenotypic variation explained by these SSR loci ranged from 4.30%  in the E1 environment to 20.04%  in the E2 environment via GLM, and ranged from 4.74%  in the E2 environment to 10.57%  in the E1 environment via MLM (p < 0.01) associated with BLUP values by GLM and MLM, respectively, which explained 5.41% –19.42%  by GLM, and explained 9.29% –13.70%  by MLM  of 186 SSRs on an IBM2 2008 Neighbors map frame (r 2022))  to buildr 2022)) . Then, t via MLM ; Table 1) by MLM ; Table 1Next, we attempted to obtain hot genetic loci for multiple insect-resistant traits of ACB and CLA to lay a foundation for fine mapping and candidate gene prediction, verification, and breeding application. First, we collected 47 original genetic loci for ACB-/CLA-resistant traits including DIMBOA content, tunnel length of corn borer (TL), aphid incidence rate (AIR), average aphid incidence grade (AIG), LFR, HO, tunnel length/number of holes of corn borer (TL/HO), and aphid resistance (AR) from previous studies , and comGRMZM2G085381 (bx1), GRMZM6G617209 (bx6), GRMZM2G441753 (bx7), GRMZM2G311036 (bx10), and GRMZM2G336824 (bx11) to examine their relative expression levels in maize seedlings of RX20-1006 (high insect-resistant line) and T58 (insect-susceptible line) in the E1 environment at the V6 stage using real-time PCR (RT-qPCR). The results showed that the relative expression levels of the five genes were significantly correlated with the DIMBOA content in the two maize genotypes , DIMBOA, and 6-methoxy-3h-1,3-benzoxazol-ne (MBOA) are multifunctional defense metabolites that can protect maize against insect pests feeding and pathogens ,32,33. Df 42–96% . Indeed,f 42–96% ,34,35, sf 42–96%  also repf 42–96%  further f 42–96% . Understanding the genetic basis of the multiple insect resistance of maize is critical to the control of combinatorial attacks of ACB and CLA in the field. In this study, we observed a key trait for multiple insect resistance, i.e., the DIMBOA content in two ecological environments and their BLUP values, to detect nine significant associated SSR markers using association mapping via GLM and MLM across 310 diverse maize inbred lines from Gansu Province, China; these nine SSR markers were located in Bin 1.04, Bin 1.11, Bin 2.01, Bin 4.00, Bin 4.01, Bin 6.02, Bin 8.04, and Bin 10.04, respectively ; Table 1r); r = 0.252) and TL/HO (r = 0.229) in 162 F3 maize population to ACB resistance [Interestingly, we identified 19 hot loci (Loci 1-Loci 19) involved in maize multiple insect resistance in the present study ; Table 3According to the physical interval of the above 19 hot loci controlling 8 insect-resistant traits and the GO annotations of corresponding genes in these hot intervals, a total of 49 candidate genes were identified ; they maGRMZM2G311036 (bx10), GRMZM2G336824 (bx11), and GRMZM2G023325 (bx12) were detected in Loci 1 (Bin 1.04), and they encoded DIMBOA-glucoside O-methyltransferase; GRMZM2G046163  was mapped in Loci 3 (Bin 1.11); GRMZM2G167549 , GRMZM2G172491 ), GRMZM2G063756 ), GRMZM6G617209 , GRMZM2G085054 -one 2-D-glucosyltransferase), GRMZM2G085381 , and GRMZM2G085661  were identified in Loci 8 (Bin 4.00–4.03); and GRMZM2G441753 -one (TRIBOA-Glc) O methyl transferase) was located in Loci 9 (Bin 4.04)  converted free indole to 2,4-dihydroxy-1,4-benoxazin-3-one (DIBOA) [bx1 was a modified form of the tryptophan synthase alpha subunit, and it was expressed constitutively in young seedlings, while igl was induced in more advanced stages of plant development and contributed to the blend of odors that attracted beneficial parasitoids [bx8 and GRMZM2G161335 (bx9) were involved in the glucosylation of DIBOA [bx6 was responsible for the hydroxylation step that converted DIBOA-Glc to TRIBOA-Glc, and this conversion likely took place in the cytosol [bx7, making DIMBOA-Glc [bx10, bx11, and bx12 were likely candidates for catalyzing the conversion of DIMBOA-Glc to HDMBOA-Glc [The 12 candidate genes were identified within three hot loci; namely, in 4.04) . The lgls larvae ,39,40. T (DIBOA) . The bx1asitoids ,39,40. T cytosol ,43. MethMBOA-Glc . In addiGRMZM2G150256 (mir2) and GRMZM2G150276 (mir1) were mapped within Loci 14 and encoded a maize insect resistance-cysteine protease (key defensive protein) against chewing insect pests in maize , Loci 5 (Bin 2.03–2.04), Loci 12 (Bin 5.03), Loci 17 (Bin 8.06), and Loci 18 (Bin 9.01), respectively . SimilarLOX genes, i.e., GRMZM2G017616 (LOX9) within Loci 1 (Bin 1.01–1.02), GRMZM2G040095 (LOX6) within Loci 4 (Bin 2.00–2.01), and GRMZM2G102760 (LOX5) within Loci 12 (Bin 5.03)  plays critical roles in plant defense against multiple insect pests and pathogens ,45,46; ain 5.03) . LOX6 wain maize .MYB transcription factor can interact with mRNA/proteins to form a fine regulatory network to activate the expression of downstream defense genes and induce insect-resistance defense response. Interestingly, the previous findings [MYB genes were validated within Loci 2 (Bin 1.04), Loci 5 (Bin 2.03–2.04), Loci 10 (Bin 4.05), Loci 13 (Bin 5.05–5.07), Loci 14 (Bin 6.02), Loci 15 (Bin 8.01–8.03), Loci 16 (Bin 8.04–8.05), and Loci 19 (Bin 10.04) in this study, respectively , GRMZM2G018716 (incw7), and GRMZM2G018692 (incw6) within Loci 4 (Bin 2.00–2.01), GRMZM2G139300 (incw1) within Loci 13 (Bin 5.05–5.07), and GRMZM2G123633 (incw3) within Loci 19 (Bin 10.04) were identified in this study  within Loci 16 (Bin 8.04–8.05)  catalyze the irreversible hydrolysis of sucrose into glucose and fructose and play important roles in sucrose partitioning, plant development, and defense responses to biotic stresses . The hydis study . Essmann04–8.05) . Thereby2O2 induces POD activity, which then oxidizes and polymerizes p-coumaryl/coniferyl-/sinapyl-alcohol into lignin monomers on the cell wall [ZmPrx35 as the prevailing POD was involved in defense against pathogens and insects. Similarly, we also identified Zm00001d024752 (POD21) and Zm00001d052335 (POD23) within Loci 19 (Bin 10.04) and Loci 11 (Bin 4.08), respectively  responsible for ubiquitin-conjugating enzyme 4, RMZM2G130224 responsible for restriction endonuclease type II-like superfamily protein, and GRMZM2G504910 responsible for tetratricopeptide repeat protein 27 homolog, were identified within Loci 4 (Bin 2.00–2.01), Loci 6 (Bin 2.04), and Loci 7 (Bin 2.05) in the present study, respectively (GRMZM2G130225 (near rs658849) and GRMZM2G102471 (near rs624256) were also identified to be involved in TL of ACB using GWAS analysis [In addition, three other candidate genes, i.e., ectively . Interesanalysis . In summary, according to the above studies, a possible molecular network underlying maize multiple insect-resistance to ACB and CLA was constructed , which cWhen assessing the genetic diversity in maize genotypes, SSR remains the preferred choice due to their co-dominant and multi-allelic nature, abundance, and loci specificity . In the In this study, the collected 310 elite maize inbred lines from different ecological environments  in Gansu Province, China . A totalv/v) was added to each tube. The tubes were rotated and placed in the dark for 12 h and then centrifuged at 12,000 rpm  for 20 min at 4 °C. Supernatants (600 μL) were slowly passed the corresponding Millex® needle filter and transferred into auto-sample vials for analysis by HPLC. Standard DIMBOA (CAS No.: 15893-52-4) was purchased  and was used to optimize the mass spectrometric parameters and fragment spectra.The DIMBOA content was determined using high-performance liquid chromatography . Namely, freeze-dried leaves (0.2 g per sample) were homogenized and weighted into screw-capped 10 mL polypropylene centrifuge tubes, and 5 mL of HPLC grade methanol-methanoic acid solution ) were used to perform SSR analysis. Fragments were separated using polyacrylamide gel electrophoresis. The polymorphism information content (PIC) value and Shannon-Wiener’s index (I) value were determined as follows [http://web.stanford.edu/group/pritchardlab/structure_software/release_versions/v2.3.4/html/structure.html (accessed on 10 August 2022)) for the assessment of groups and genetic relationships among the 310 maize inbred lines. The project was run with the set parameters of the population admixture model, and the allele frequency correlated. The optimum group number was determined by ΔK value [https://www.maizegdb.org/data_center/map (accessed on 18 September 2022)) using BioMercator v. 4.2 software (http://www.bioinformatics.org/mqtl/wiki/ (accessed on 18 September 2022)). The linkage disequilibrium (LD) values for r2 [https://tassel.bitbucket.io/ (accessed on 10 August 2022)), following a permutation test of 10,000. The K matrix and marker-DIMBOA content association mapping was completed in Tassel 3.0 software using the genotypic data of 186 polymorphic SSRs and phenotypic data on DIMBOA content for a set of 310 inbred lines. The association analysis was conducted by a GLM with Q matrix  [2) of the marker at p < 0.01 level and with the lowest false discovery rate (FDR). Using MaizeGDB (http://www.maizegdb.org/ (accessed on 20 September 2022)) and nucleotide and primer blast tools, the physical locations of associated SSR markers for DIMBOA content were determined on chromosomes.Genomic DNA was extracted from the 6th leaf of 310 inbred lines using the cetyltrimethyl ammonium bromide (CTAB) method . Then, a follows :(1)PIC=1tructure  was analΔK value . A linkas for r2  and D′ [s for r2  between ulation)  and an Mulation) . The asshttp://www.maizegdb.org/ (accessed on 23 September 2022)), NCBI (http://www.ncbi.nlm.nih.gov (accessed on 23 September 2022)), and CNKI (http://www.cnki.net (accessed on 23 September 2022)), we collected information on corresponding QTLs and associated markers for multiple insect-resistant traits from our results and previous studies via QTL analysis, GWAS, and association mapping. The hot genetic loci were the overlapping regions combining multiple genetic loci responsible for multiple insect-resistant traits or single genetic loci that explained the large R2 (10%) in 10 Mb physical intervals. Further, the physical map of all hot genetic loci and candidate genes involving multiple insect-resistant traits in these hot genetic loci regions was developed by BioMercator v. 4.2 software (http://www.bioinformatics.org/mqtl/wiki/ (accessed on 28 September 2022)) [https://agbase.arizona.edu/ (accessed on 6 October2022)) [Using public databases, i.e., MaizeGDB (r 2022)) . The funer2022)) .http://www.premierbiosoft.com/ (accessed on 9 October 2022)) , and cDNA was made using a kit , according to the manufacturer’s instructions. RT-PCR was conducted using TransStart Tip Green qPCR SuperMix . The primers for five candidate genes ,64 were r 2022)) . Relativnce gene .http://www.R-project.org/ (accessed on 16 December 2021)) to calculate the BLUP of DIMBOA content values [http://www.ibm.com/products/spss-statistics (accessed on 16 December 2021)). The significance of the total and residual variances of DIMBOA content in 310 inbred lines under both ecological environments was estimated by a GLM for univariate data and by one-way analysis of variance (ANOVA). The broad-sense heritability , and r was the number of replications (r = 5). The CVg [The average DIMBOA content in 310 inbred lines from five biological replicates in each ecological environment were analyzed, respectively. A mixed linear model was fitted using the lmer function in lme4 package of R (t values . These d follows ,66,67:HB The CVg  of DIMBOIn summary, the DIMBOA confers significant resistance to ACB and CLA. In this study, SSR analysis revealed a wide genetic diversity in the 310 tested maize inbred lines from four type regions of China’s Gansu Province, which is the largest maize seed production and breeding area in China. Population structure indicated that 294 inbred lines were successfully assigned to one or another group at a membership probability of ≥ 0.500. DIMBOA performance evaluation screened out 3 high and 15 moderate insect-resistant inbred lines, which can be used as parents in breeding programs to develop new maize varieties with multiple insect resistance. Using linkage mapping, we detected nine significant SSRs associated with DIMBOA content in both environments. We then combined the 47 original genetic loci for 8 multiple insect-resistant traits from previous studies to detect 19 hot loci. Among them, 11 hot loci were located in Bin 1.04, Bin 2.00–2.01, Bin 2.03–2.04, Bin 4.00–4.03, Bin 5.03, Bin 5.05–5.07, Bin 8.01–8.03, Bin 8.04–8.05, Bin 8.06, Bin 9.01, and Bin 10.04 regions, and they supported pleiotropy for their association with two or more insect-resistant traits. Further, the 49 candidate genes involved in DIMBOA biosynthesis, sugar metabolism/homeostasis, and other multiple insect-resistant defense mechanisms in maize were identified in all 19 hot loci, and their highly interconnected network may form complex maize, multiple insect, pest-induced defense mechanisms.


Correction: BMC Nephrol 23, 348 (2022)https://doi.org/10.1186/s12882-022-02986-2Following publication of the original article , the autThe original article has been corrected.Additional file 1 Table 1. Summary of literature on renal biopsy after intravitreal injection of vascular endothelial growth factor inhibitors.


Sphaerotilus natans and Pseudomonas aeruginosa strains. Its genome consists of 61,858 bp (64.3% GC) and 89 genes, including 32 with predicted functions. SN1 genome is very similar to Pseudomonas phage M6, which contains hypermodified thymidines. Genome analyses revealed similar base-modifying genes as those found in M6.Phage SN1 infects  S. natans ATCC 13338 as the host  using the host . An earlthe host . Host rand OT684 .S. natans ATCC 13338 in nutrient broth  and agitated at 30°C (P. aeruginosa PAO1 (HER1153) in TSB/TSA medium at 30°C using both plaque assays and lysis of liquid cultures. Species identification of the above two host strains was confirmed by 16S sequencing.Here, phage SN1 was amplified with its host  at 30°C . Cell deS. natans as host) using the phenol-chloroform extraction method (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Phage genomic DNA was purified from lysate (n method . Libraryn method . Illuminn method . Trimmedn method . Two assn method  in combiast.cgi) , 9 usingast.cgi) . BioinfoPseudomonas phages with 95 to 98% nucleotide identity (73 – 96% query cover). Interestingly, phage SN1 has 96.76% nucleotide identity (91% query cover) with Pseudomonas phage M6 genome, which contains hypermodified thymines . Half oON165687. Raw sequence reads are available under SRA number SRR18758685. Phage SN1 is available at www.phage.ulaval.ca.Genome sequence is available under GenBank number


Globularia L. have been used as healing agents for various ailments, with utilization of Globularia alypum L. being most frequently reported. The aim of this study was to evaluate the antidiabetic, antioxidant, anti-inflammatory, antibacterial and anticancer potential of G. alypum and three related species, G. punctata Lapeyr., G. cordifolia L. and G. meridionalis (Podp.) O.Schwarz, in relation to their phytochemical compositions. Globularin and verbascoside were identified using LC-PDA-ESI-MSn as the major metabolites of G. alypum with known biological activities. G. alypum demonstrated the greatest α-glucosidase inhibitory activity and DPPH radical scavenging activity (IC50 = 17.25 μg/mL), while its anti-inflammatory activity was not significantly different from those of related species. All investigated species showed considerable antibacterial activity against methicillin-resistant Staphylococcus aureus in the broth microdilution method (MIC = 1.42–3.79 mg/mL). G. punctata also showed antibacterial activities against Escherichia coli (MIC = 1.42 mg/mL), Bacillus subtilis (MIC = 1.89 mg/mL), B. cereus (MIC = 2.84 mg/mL) and Enterococcus faecalis (MBC = 5.68 mg/mL). G. punctata, G. cordifolia and G. meridionalis showed greater anticancer potential than G. alypum. Obtained results indicate investigated Globularia species could serve as sources of diverse bioactive molecules, with G. punctata having the greatest antibacterial potential.Species from the genus  According to the World Health Organization (WHO) statistics for 2021, the leading causes of global premature mortality from non-communicable diseases include cancer, cardiovascular diseases, diabetes, and chronic respiratory diseases. In 2019, 33.2 million people died solely from these diseases, which is 28% more deaths caused by the same four diseases than in 2000. Taken individually, there has been a 25% increase in the total global mortality from cardiovascular diseases (17.9 million deaths), a 37% increase in cancer mortality (9.3 million deaths), and a 10% increase in chronic respiratory diseases mortality (4.1 million deaths), while diabetes mortality has grown by 72% − and 153 [M−H−anhydroglucose]−. In MS2, compound 50 was characterized by product ions at m/z 161 (−330 Da) and 175 (−316 Da). In MS3 of the first product ion, further loss of 28 Da (−CO) was observed. The first loss in MS2 was attributed to simultaneous loss of hydroxytyrosol glucoside and a methyl group and the second to hydroxytyrosol glucoside. Loss of a methyl group was previously observed for the reference standard of ferulic acid −. The compound was tentatively identified as galypumoside C (6′-O-menthiafoloylverbascoside), whose presence in leaves of G. alypum was already reported −. The formed radical aglycone product ion was further subjected to loss of one methyl group (−15 Da) in MS3 and major loss of 84 Da in MS4, attributed to the cleavage of three CO −, 209 [M–H–anhydroglucose−benzoylglucose]−, 227 [M–H–anhydroglucose−benzoylanhydroglucose]− and 371 [M–H–anhydroglucose−benzoic acid]−, indicated a similar structure to previously identified C-4 carboxylated iridoids. Second- and third-order mass spectra of compound 25 were comparable to MS3 and MS4 of compound 21, while major loss of 30 Da (−CH2O) from the MS3 product ion present at m/z 165 was observed in MS4 together with minor fragment ions present at m/z 121 (−C2H4O), 137 (−CO) and 147 (−H2O). These compounds were tentatively identified as 6′-O-benzoyldeacetylasperulosidic acid glucoside and 6′-O-benzoyldeacetylasperulosidic acid, keeping in mind other observed asperuloside-type iridoids and benzoylation of glucose at C-6′ position, which was recorded for dumuloside in G. dumulosa –, 163 [p-coumaric acid–H]–, 293 [M–H–glucose–CO2]– and 355 [M–H–anhydroglucose]–. Further MS/MS fragmentation pattern obtained in MS3 and MS4 were in accordance with those of asperuloside and besperuloside, which have acetic/benzoic acid attached at C-10 position. This compound, with UV λmax present at 191, 232 and 315 nm, comparable to those previously observed for asperuloside (191 and 239 nm) and p-coumaric acid  [O-(p-coumaroyl)-deacetylasperuloside.Compound G. alypum methanolic leaf extract indicated not only the previously described high globularin  content [G. alypum were glycosides of 6-hydroxyluteolin, the same as for other investigated species, while vicenin-2 was observed only in G. alypum, as well as the lignan diglucoside liriodendrin. Leaf extract of G. punctata contained high relative amounts of iridoids asperuloside, besperuloside, globularin, deacetylasperuloside, asperulosidic acid, and scandoside, while verbascoside, its isomers  and glycosylated derivatives  were observed as the major phenylethanoids. Solely G. punctata was characterized by acylated derivatives of 6-hydroxyluteolin, as previously reported [G. cordifolia and G. meridionalis were characterized by iridoids asperuloside and monomelittoside and their often benzoylated derivatives, with globularifolin (10-O-benzoylmonomelittoside) as the major compound, as previously reported [O-glucoside and demethoxycentaureidin 6,4′-dimethyl ether were recognized as characteristic flavonoids. Other major constituents observed in investigated Globularia species included mannitol, sucrose, quinic acid  and fatty acid oxidation products that have already been reported [Obtained chromatogram of  content , but als content , do not reported ,40,42. Greported ,26,43. Mreported .Globularia species obtained by Soxhlet extraction was performed using LC-PDA-ESI-MSn -sophoroside, trichosanthoside B, trichosanthoside A, arenarioside, and besperuloside, with the iridoid asperuloside as the major compound. Other more prominent peaks included deacetylasperuloside and 10-O-(p-coumaroyl)-deacetylasperuloside, present also in G. cordifolia and G. meridionalis, as well as gardoside, caffeoylglucoside isomer and globusintenoside isomer present in all four species. For G. cordifolia and G. meridionalis, the major compounds were mannitol, sucrose, asperuloside and several phenylethanoids, including verbascoside, methoxyverbascoside isomer, isoverbascoside, globusintenoside isomer and benzoylrossicaside A isomer, while their distinctive peaks could be attributed to monomelittoside, 6′-O-benzoylmonomelittoside and globularifolin. The major identified flavonoid characteristic for these two species was demethoxycentaureidin 6,4′-dimethyl ether. Besides 6-hydroxyluteolin 7-O-glucoside, apigenin was detected in Soxhlet extracts of all investigated species.Identification of major constituents of methanolic aerial parts extracts of investigated -ESI-MSn , Table 4-ESI-MSn  or thoseG. alypum  using two different mobile phases . Blue zone of besperuloside, present only in G. punctata, was not clearly visible due to its overlapping with the brown zone of globularin. Shared yellow zones may be attributed to phenylethanoids present in all investigated species with verbascoside as the major shared component, while additional yellow zones observed for G. alypum and G. punctata correspond to the presence of their major unique phenylethanoids  that were detected using LC-MS  and glutathione peroxidase (GPx)) and two non-enzymatic biomarkers of oxidative stress (free thiol groups (-SH) and reduced glutathione (GSH)) in human hepatocellular carcinoma Hep G2 cells exposed to hyperglycemic conditions. Cell viability was evaluated using lactate dehydrogenase (LDH) and 3--2,5-diphenyltetrazolium bromide (MTT) assay. Antioxidant potential of aerial parts extracts was evaluated based on spectrophotometric measurement of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and by performing thin layer chromatography (TLC) bioautography using the same free radical as spray reagent.p < 0.05), with G. alypum extracts being most effective (30.0–45.7% inhibition)  and reducing diabetic complications. This can partially be achieved by inhibiting the activity of α-glucosidase, the key enzyme responsible for hydrolytic cleavage of complex carbohydrates. Inhibition of α-glucosidase may retard the absorption of glucose and decrease postprandial blood glucose levels and, therefore, it is one of the key targets for treating type 2 diabetes mellitus (T2DM) . All fouibition) . This su50 = 0.52 mg/mL) and α-amylase (IC50 = 0.57 mg/mL) was reported for diethyl ether fraction of G. alypum aerial parts extract [G. alypum in the present and in related studies [Inhibition of α-glucosidase  (r = −0.791), was previously reported for G. trichosantha and G. orientalis [Clerodendrum bungei Stud., the phenylethanoids verbascoside (IC50 = 0.5 mM), leucosceptoside A (IC50 = 0.7 mM) and isoverbascoside (IC50 = 0.1 mM), which were found in all investigated Globularia species, exhibited stronger anti-α-glucosidase activities than the positive control (acarbose) [50 = 0.15 mM) [Globularia species. Hereby, many acylated phenylethanoids were only found in G. alypum, including galypumoside B, calceolarioside A, and calceolarioside B, of which the latter was recently identified also as a pan inhibitor of SARS-CoV-2 proteins [G. alypum, such as verminoside (caffeic acid) and specioside (p-coumaric acid). However, greater inhibition was observed for leaf extracts of G. alypum and G. meridionalis  . Inhibitientalis . In a stcarbose) . The obscoside IC = 0.5 mMproteins . Phenolidionalis , which cdionalis .-O-glucoside). In Origanum majorana L. leaves, 6-hydroxyapigenin (scutellarein) (IC50 = 12 μM) and 6-hydroxyluteolin (IC50 = 10 μM) were recognized as the most potent inhibitors of α-glucosidase, while weaker inhibitions were observed for 6-hydroxyluteolin 7-O-glucoside (IC50 = 300 μM) and flavones lacking the 6-hydroxyl substituent, apigenin and luteolin (IC50 > 500 μM) [G. alypum extract compared to those of extracts of related species may also be explained by presence of additional α-glucosidase inhibitors, such as vicenin-2 (IC50 = 270.53 μM) [The observed inhibitory effect of all four species may in part be connected to the presence of 6-hydroxyflavones  . The rec0.53 μM) .G. alypum [G. alypum as an antidiabetic could include prevention of diabetic complications through direct or indirect antioxidant activity, including radical scavenging activity [G. alypum methanolic leaf extract reduced glycemia and glycosylated hemoglobin levels, and improved the redox status, especially in the liver [The liver plays a vital role in blood glucose level regulation both in physiological and pathological states. In DM, liver is among the primary organs susceptible to hyperglycemia-induced oxidative stress, which may result in irreversible oxidative modifications of its macromolecules that may lead to abnormal glycogen deposition, non-alcoholic fatty liver disease, fibrosis, cirrhosis, hepatocellular carcinoma, and other liver abnormalities . Besides. alypum ,11,56, w. alypum , additioactivity ,57, enhaactivity ,59. For Globularia leaf extracts on biomarkers of oxidative stress have been evaluated in Hep G2 cells cultured under hyperglycemic conditions (p < 0.05). Increased GSH content was observed for G. alypum (+18.6%), G. punctata (+47.4%), and G. cordifolia extracts (+68.1%) at 1.0 mg/mL concentration and G. cordifolia extract at 0.5 mg/mL (+11.7%) (p < 0.05). All tested samples significantly increased free thiol groups content (+14.0–73.7%) and cell viability in the LDH assay (+22.2–76.7%) (p < 0.05), while in the MTT assay, a 21.6–25.4% increase was observed only for G. punctata (both concentrations) and G. cordifolia (c = 1.0 mg/mL) (p < 0.05). Overall, G. cordifolia reduced the pro-oxidant effects of hyperglycemic conditions observed in Hep G2 cells in all performed assays. Its favorable effect on oxidative status  has already been recorded in human keratinocytes [In the present study, effects of two different concentrations (0.5 and 1.0 mg/mL) of nditions . Treatmeinocytes .G. cordifolia was characterized by the highest condensed tannin content , and very good positive correlation between GPx activity and condensed tannin content    . Condens < 0.05) . The majpunctata , Table 3. alypum , was aspflavones , many ofr = 0.88, p < 0.01) as well as total phenolic content   than its phenylethanoids (IC50 =11.8–15.5 µM) and iridoids (IC50 = 28.2–76.0 µM), as well as than quercetin (IC50 = 7.8 µM) and butylated hydroxytoluene (IC50 = 30.0 µM) [Very good positive correlation was found between cell viability assessed by the LDH assay and flavonoid content ( < 0.05) . Accordiactivity ,35,64,65punctata . The latpunctata , contain30.0 µM) . In the G. alypum [Globularia [50 values ranged from 17.25 μg/mL  to 24.19 μg/mL (G. punctata). The obtained results are in accordance with our previous study, in which relatively higher antioxidant activities of both G. alypum leaf and flower extracts and relatively lower antioxidant activity of G. punctata flower extract were observed [50 value for G. alypum is comparable to previous reports for the diethyl ether fraction of its aerial parts (IC50 = 20.54 μg/mL) [50 = 15.58–27.54 μg/mL [50 = 25.65 μg/mL) and stems (IC50 = 22.11 μg/mL) [50 = 23.50 μg/mL) [50 value established for G. meridionalis is in accordance with that previously reported for methanolic extract of its aerial parts obtained by maceration (IC50 = 21.00 μg/mL), from whose methanolic fraction verbascoside (acteoside), isoverbascoside (isoacteoside) and apigenin 7-O-glucoside were isolated [50 values for DPPH radical scavenging activity and the estimated total phenolic content of aerial parts extracts  (50 = 22.9 µM) and calceolarioside B (desrhamnosyl isoacteoside) (IC50 = 26.2 µM) [G. alypum.Direct antioxidant activity of studied species was confirmed by the DPPH assay, which is the most frequently used assay for evaluation of antioxidant activity of . alypum ,67,68,69obularia ,30, to eobserved . Obtaine4 μg/mL) , methano54 μg/mL , IC50 = isolated . Excelle < 0.05) , which i26.2 µM) , found oGlobularia species’ constituents [Globularia [G. alypum [50 = 58.1 µM) than ascorbic acid [Globularia species [G. punctata showed the lowest radical scavenging activity, the second method revealed at least three zones characteristic for this species showing prominent antiradical activity . Their Rf values matched those of three orange, fluorescent zones observed at 365 nm after NP/PEG treatment, presumably of flavone origin [G. punctata (Globularia elongata Hegetschw. (syn. G. punctata) [-O-sophoroside, 6-hydroxyluteolin 7-O-(6′′′-O-caffeoyl)-sophoroside, and 6-hydroxyluteolin 7-O-(6′′′-O-(p-coumaroyl))-sophoroside.To identify the constituents responsible for observed activity, TLC bioautography with DPPH used as spray reagent was performed as in earlier studies of antioxidant activity of different tituents ,65,67. Bobularia , as well. alypum ,67, a mo. alypum . Two dombic acid . The for species ,39,40,64e origin . Based opunctata  and lite2, PGD2, PGF2α, and PGI2) and thromboxane A2 [2 (cyclooxygenase site), which is afterwards reduced to PGH2 (peroxidase site) [G. alypum, G. punctata, G. cordifolia and G. meridionalis methanolic extract of aerial parts obtained by Soxhlet extraction was evaluated spectrophotometrically using two methods, one based on inhibition of peroxidase activity of COX-1 using N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD assay) and the other based on inhibition of its cyclooxygenase activity assessed by the prostaglandin E2 assay (PGE2 assay). All tested species inhibited COX-1 activity  to 51.3%  in the TMPD assay (IC50 = 2.90 μM for indomethacin), and from 25.7%  to 40.6%  in the PGE2 assay (IC50 = 1.03 μM for indomethacin). The results obtained for G. alypum are comparable to those reported for methanolic leaf (5.33%) and flower extract (61.05%) of the same species tested at 33 μg/mL concentration using the TMPD assay [Cyclooxygenase isoenzymes COX-1 and COX-2 are first in the cascade of enzymes responsible for metabolism of arachidonic acid to prostaglandins (PGEoxane A2 . Due to se site) . Althougse site) . Anti-inactivity . ExtractPD assay .50 > 1 mM), one of the major metabolites found in all investigated species, was lower than that against COX-2 (IC50 = 0.69 mM) [Anisomeles indica (L.) Kuntze, of which verbascoside, isoverbascoside, calceolarioside A and apigenin were also recorded in investigated Globularia extracts, the latter compound gave the lowest docking score with COX-1 (−6.558) and COX-2 receptors (−8.441) in molecular docking analysis, which indicated its strong binding to the enzymes’ active sites [G. alypum, for which the tendency of greater COX-1 inhibition was observed. Anti-inflammatory potential of investigated species could also be connected to their possible diminishing effects on other pro-inflammatory mediators , and cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [No correlation between the obtained results and assessed total phenolic or flavonoid content was found . Inhibit0.69 mM) . Among tve sites . Based ove sites , apigeni (TNF-α) ,79,80,81G. alypum and G. punctata, was recently reported to protect against glucose-induced podocyte injury by ameliorating apoptosis and inflammation through reduction of TNF-α, IL-6 and IL-1β at 1–10 μM concentrations, which would suggest its preventive potential against diabetic nephropathy [G. punctata, G. cordifolia and G. meridionalis, significantly decreased the production of NO, PGE2, TNF-α, and IL-6, and inhibited the expression of inducible NO synthase (iNOS), COX-2, TNF-α, and IL-6 mRNA in LPS-induced RAW 264.7 cells [-O-benzoyl-monomelittoside), the major metabolite of G. cordifolia and G. meridionalis, was reported to significantly suppress expression of nuclear factor-κB (NF-κB) in LPS-stimulated cells at 200 μM concentration [G. alypum. The same compound, together with other above-mentioned compounds , as well as other major compounds identified in this study , were also reported for G. trichosantha [Catalpol, the iridoid present together with its many esters and/or derivatives (including globularin) in hropathy . The samhropathy . Similarhropathy , as wellhropathy . Asperul.7 cells . On the ntration , a lignahosantha ,64,83, whosantha , which mStaphylococcus aureus, a Gram-positive bacterium, is the main causative pathogen in diabetic foot infections, with methicillin-resistant S. aureus (MRSA) being the major multidrug resistant bacterium. Other pathogens may include Gram-positive bacteria, such as Streptococcus spp. and Enterococcus faecalis, and Gram-negative bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Proteus spp., Enterobacter spp., and Klebsiella spp. [G. alypum, G. punctata, G. cordifolia and G. meridionalis was evaluated by testing the effect of methanolic extracts of aerial parts obtained by Soxhlet extraction against four Gram-positive  and three Gram-negative bacterial strains  using two complementary methods; well diffusion and serial broth microdilution method. In the first method, all four species showed notable inhibitory activity against S. aureus, with the greatest zone of inhibition recorded for G. alypum (25.0 mm), as well as low inhibitory activity against B. cereus  and 2.84 mg/mL . Low inhibitory activity of G. alypum against P. aeruginosa (MIC = 22.73 mg/mL) and moderate to good bactericidal/inhibitory activity of G. punctata against E. faecalis (MBC = 5.68 mg/mL) and E. coli (MIC = 1.42 mg/mL) were also confirmed. Comparable antibacterial activity of G. alypum methanolic extract against S. aureus (MIC = 2–4 mg/mL) was reported by previous studies [P. aeruginosa (MIC = 8 mg/mL) [The results of the serial broth microdilution method followed by sub-cultivation on agar plates mainly coincided with those of the first method . Assesse MBC = 5. mg/mL an studies , while g8 mg/mL) .G. punctata showed good antibacterial activity against B. cereus (MIC = 2.84 mg/mL) and B. subtilis (MIC = 1.89 mg/mL), while much lower inhibition against B. cereus was observed for related species (MIC = 11.36 mg/mL). The latter method possibly accounted for the antibacterial activity of less hydrophilic compounds of G. punctata, which might not have been observed earlier due to their lower solubility and consequential lower diffusion into the hydrophilic (agar) medium [r = −0.998, p < 0.01) was found between MIC values against B. cereus and flavonoid content , were equal to 8, 8 and 64–400 μg/mL, respectively, while against S. aureus no inhibitory activity was observed [Opposite to the results of the diffusion method, in the second method, ) medium . Excelle content , indicatlucoside   and five clinical isolates of MRSA, were also tested (p > 0.05). Keeping in mind that these are the most common causative pathogens found in diabetic foot infections [G. alypum in the treatment of diabetes-associated foot ulcers. The anti-staphylococcal activity could partly be attributed to verbascoside, for which the assessed inhibitory activity (MIC = 227.2 μg/mL) corresponded to values previously reported against five MRSA strains (MIC = 64–256 μg/mL) [S. aureus of all investigated species may also indicate contribution of other common constituents, such as isoverbascoside, rossicaside A, forsythoside A and 6-hydroxyluteolin 7-O-glucoside. Verbascoside (MIC = 60 μg/mL) was observed to have greater anti-staphylococcal activity than isoverbascoside (MIC = 130 μg/mL), while both compounds possessed lower inhibitory activities against E. faecalis (MIC = 100–150 μg/mL), E. coli (MIC = 100–250 μg/mL) and P. aeruginosa (MIC = 250 μg/mL) [G. alypum, also possess antibacterial potential against Gram-positive and Gram-negative bacteria [G. alypum against P. aeruginosa. The antibacterial potential of G. punctata against E. coli and E. faecalis, as well as S. aureus, may be connected to its characteristic phenylethanoids  and/or flavonoids. Excellent negative correlations were found between flavonoid content and MICs against MSSA MFBF 505  and MRSA MFBF 154 .Nine additional o tested . Observefections , the obs6 μg/mL) . Compara0 μg/mL) . Other pbacteria , which mGlobularia species was evaluated using the MTT assay based on their effects against MDA-MB-231 breast cancer cell line and A1235 glioblastoma cell line. Cytotoxic effect against MDA-MB-231 cell line was observed for extracts of G. punctata (34.48%), G. cordifolia (63.41%) and G. meridionalis (82.96%) (at the highest concentration used), while cytotoxic effect against A1235 cell line was observed for all investigated species (p < 0.05)  and the lowest for G. alypum (IC50 = 231.43 μg/mL)  . Greates3 μg/mL) .G. meridionalis, G. cordifolia and G. punctata may be connected to asperuloside and its derivatives (Oldenlandia diffusa (Willd.) Roxb. that contained asperuloside and deacetylasperulosidic acid also reduced MDA-MB-231 and MDA-MB-453 cell viability and suppressed their colony formation capacities [50 = 0.7 μg/mL), HL60 cells (IC50 = 11.0 μg/mL) and KB cells (IC50 = 104.2 μg/mL) was recorded for asperuloside [50 = 6.1 μg/mL) [50 = 10 μM) was reported [G. cordifolia and G. meridionalis. Very good negative correlations were established between observed cell viability and concentrations of flavonoids , iridoids , and total phenolics , while excellent correlation was found between MDA-MB-231 cell viability and concentration of condensed tannins  (50 = 0.159–0.258 μM) [The observed cytotoxic effects against MDA-MB-231 cells that were shown by ivatives  [102,103s IC50 = .2 μg/mL reported , could b< 0.001) . Cytotox.258 μM)  and a ra.258 μM) , the majCallicarpa nudiflora Hook. and Arn. against HeLa cells, A549 lung adenocarcinoma cells and MCF-7 cells resulted in isolation of flavonoids, phenylethanoids and iridoids, which included those found in investigated species, 6-hydroxyluteolin 7-O-glucoside, verbascoside and catalpol [50 = 1530 μg/mL) was also reported for G. alypum [Moreover, investigation of cytotoxic activity of catalpol . Weak cy. alypum .r = −0.95, p < 0.001), flavonoids  and condensed tannins , while very good negative correlation was observed between A1235 cell viability and iridoid concentrations   . Common thanoids ,111. Thi87 cells .2EDTA), 5,5′-dithiobis(2-nitrobenzoic acid) , Dulbecco’s modified Eagle’s medium (DMEM), Type I α-glucosidase (isolated from Saccharomyces cerevisiae), glutathione (GSH), hematin, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), p-nitrophenyl-α-d-glucopyranoside (PNG), quercetin, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), trichloroacetic acid, 2,3,5-triphenyltetrazolium chloride (TTC), trypsin-EDTA and vanillin from Sigma-Aldrich, St. Louis, MO, USA; and sodium diethyldithiocarbamate trihydrate (DDC) from VWR International, Radnor, PA, USA.Adrenalin bitartrate and indomethacin were purchased from Acros Organics, Geel, Belgium; glacial acetic acid from Alkaloid, Skopje, North Macedonia; asperuloside and catalpol from Carl Roth, Karlsruhe, Germany; hydrochloric acid from Carlo Erba, Emmendingen, Germany; arachidonic acid from Cayman Chemical Company, Ann Arbor, MI, USA; (+)-catehin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), diphenylboric acid-β-ethylamino ester  and LC-MS grade formic acid from Fluka, Buchs, Schwitzerland; fetal bovine serum (FBS), minimum essential medium (MEM), penicillin-streptomycin, and trypan blue from Gibco, Gaithersburg, MD, USA; absolute ethanol from Gram-Mol, Zagreb, Croatia; verbascoside from HWI Analytik, Rülzheim, Germany; ethyl acetate, Folin-Ciocalteu’s reagent, D-(+)-glucose, polyethylene glycol 4000 (PEG 4000), potassium chloride, potassium phosphate monobasic, sodium carbonate decahydrate, sodium hydroxide, sodium phosphate dibasic heptahydrate, sulfuric acid and Tween from Kemika, Zagreb, Croatia; gentamicin sulfate and norfloxacin from Krka, Novo mesto, Slovenia; chloroform, copper(II) sulfate pentahydrate, gallic acid, LC-MS grade acetonitrile, methanol, Müller–Hinton agar, Müller–Hinton broth, sodium chloride and Tryptic soy agar (TSA) from Merck, Darmstadt, Germany; Tris-HCl buffer from Santa Cruz Biotechnology, Dallas, TX, USA; formic acid from Scharlau, Scharlab, Barcelona, Spain; acarbose, aluminum chloride hexahydrate, aucubin, 1-chloro-2,4-dinitrobenzene (CDNB), cyclooxygenase-1 (COX-1) from sheep, dimethyl sulfoxide (DMSO), 3--2,5-diphenyltetrazolium bromide (MTT), disodium ethylenediamine-tetraacetic acid , Grobnik field , Baške Oštarije, Velebit  and Alan, Velebit . Plant material was identified by Prof. Kroata Hazler Pilepić. Voucher specimens are deposited in the Herbarium of the Department of Pharmaceutical Botany of the Faculty of Pharmacy and Biochemistry, University of Zagreb. Collection data are provided in Aerial parts of investigated v/v) methanol and extracted three times with chloroform. After chloroform removal, the remaining solvents were again evaporated at 30 °C and freeze-dried. All samples were stored at −20 °C until further use.Ultrasound-assisted extraction of powdered leaves (0.5–1.25 g) was performed using methanol (1:10) for 2 × 30 min. Obtained liquid extracts were filtered and evaporated to dryness using a rotavapor at 50 °C. Soxhlet extraction of powdered aerial parts (145–280 g) was performed using methanol (1:5) for 8 h. Obtained extracts were evaporated to dryness using a rotavapor at 30 °C, re-suspended in 50% , was used for the analysis of methanolic leaf and aerial parts extracts composition. Zorbax SB-C18 column   was used as a stationary phase. Separation of constituents and data collection were carried out under the previously described conditions [m/z 50–2000. Electrospray ionization (ESI) was performed in negative ionization mode. MSn spectra (MS2–MS4) were obtained by collision induced dissociation (CID) of the ion of the greatest intensity in the mass spectrum of lower order with helium as the collision gas and normalized collision energy set at 35%. Data acquisition and processing were performed using Thermo Xcalibur 2.2 . Compound identification was based on the comparison of chromatographic and spectral data to those of previously identified constituents found in methanolic extracts of aerial parts of the same Globularia species obtained by heating under reflux conditions [High-performance liquid chromatography-photodiode array detection-electrospray ionization-tandem mass spectrometry, HPLC-PDA-ESI-MSnditions . UV/Vis nditions .254 aluminum plates , 10 × 20 cm, thickness 0.20 mm, using chloroform-methanol-water = 61:32:7 (v/v/v), or ethyl acetate-methanol-water = 20:2:1 (v/v/v) as the mobile phase [w/v) ethanolic vanillin-5% (v/v) ethanolic sulfuric acid reagent (5–10 min at 100–105 °C), to those of asperuloside, aucubin and catalpol reference standards (c = 0.5 mg/mL).Constituents of methanolic aerial parts extracts obtained by Soxhlet extraction (c = 50 mg/mL) were separated on thin-layer chromatography (TLC) silica gel 60 Fle phase . Sample v/v) DMSO were mixed with 50 µL Type I α-glucosidase from Saccharomyces cerevisiae  and preincubated at 37 °C. After 10 min, 50 µL PNG  was added. Absorbance was measured for 5 min at 405 nm against a blank in which PNG was replaced with phosphate buffer. Results are expressed as enzyme activity % in comparison to control (2% (v/v) DMSO), which was considered to give 100% enzyme activity. Acarbose was used as positive control.Inhibition of α-glucosidase activity was assessed using PNG as previously described . Methanov/v) FBS, 20 IU/mL penicillin and 20 µg/mL streptomycin at 37 °C and 5% CO2. After removal of culture medium, cells were washed with phosphate buffered saline (PBS), trypsinized using 0.25% trypsin-EDTA solution for 5 min at 37 °C, counted under a light microscope (Leitz Diavert) after 0.04% trypan blue staining using the Bürker–Türk counting chamber and seeded into six-well plates (1.67 × 106) with FBS-free medium or 96-well plates (2 × 105) (MTT assay). After 24 h incubation at 37 °C, cells were washed with PBS and incubated with methanolic leaf extract solutions (c = 0.5 and 1.0 mg/mL) in MEM with 20 mM glucose (hyperglycemic conditions) for 24 h at 37 °C. Normal control cells were kept in MEM with 5.56 mM glucose. Prior to cell treatment, samples were filtered using sterile Nalgene filter units of pore size 0.2 μm . After incubation and washing with PBS, the cells were lysed with 1% (v/v) Tween in PBS with the help of ultrasound (4 W) (Cole-Parmer 4710) for 15 s and centrifuged for 20 min at 14,000 rpm at 4 °C. The supernatant was stored at −80 °C until further use.Hep G2 cells obtained from European Collection of Authenticated Cell Cultures  were cultured in MEM supplemented with 10% (−1M−1). Content of free thiol groups (-SH) was evaluated spectrophotometrically using Ellman’s reagent [−1M−1). For evaluation of GSH content, as previously described [v/v) trichloroacetic acid was added (100 µL), and the mixture was centrifuged . To obtained supernatant (100 µL), PBS  and DTNB (50 µL) dissolved in the same buffer were added. The production of yellow colored 5-thio-2-nitrobenzoic acid (TNB) was measured at 405 nm against reagent blank. Results were calculated using the molar extinction coefficient of the product . All results are expressed per mg protein. Protein concentrations were established fluorometrically using the Qubit Protein Assay Kit (Invitrogen).Glutathione peroxidase (GPx) activity was measured spectrophotometrically using the Glutathione Peroxidase Activity Colorimetric Assay Kit . Glutathione S-transferase (GST) activity was evaluated spectrophotometrically using CDNB as the substrate . The cel reagent . Reactioescribed , to depr−1M−1). Cell viability was also evaluated based on the assessment of their metabolic/mitochondrial function using the MTT assay as previously described [Viability of Hep G2 cells was evaluated spectrophotometrically using the cell lysate supernatant by measuring lactate dehydrogenase (LDH) activity as an indicator of cell membrane damage  with comescribed . After 250).Antiradical activity was assessed using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the previously described procedure . Methano254 glass plates (Merck), 20 × 20 cm, thickness 0.25 mm, using ethyl acetate-formic acid-glacial acetic acid-water  as the mobile phase [w/v) methanolic solution of DPPH [w/v) methanolic diphenylboric acid-β-ethylamino ester, and 5% (w/v) ethanolic polyethylene glycol 4000 (NP/PEG reagent) [Constituents of methanolic aerial parts extracts obtained by Soxhlet extraction (c = 20 mg/mL) were separated on TLC silica gel 60 Fle phase . Sample  of DPPH . Constitreagent) , to thatv/v) Tween and 300 μM DDC (c = 200 U/mL) and then in 0.1 M Tris-HCl buffer, pH 8.0 (1:100) just before the performed assay, while hematin (c = 1 mM) was dissolved in 0.01 M NaOH and afterwards diluted in 0.1 M Tris-HCl buffer, pH 8.0 (1:10). Sample  was preincubated with 0.1 M Tris-HCl buffer, pH 8.0 (20 μL), 72 mM adrenalin bitartrate (50 μL), 2 U/mL COX-1 (100 μL) and 100 μM hematin (10 μL) for 5 min at room temperature. The reaction was started by the addition of 100 μM arachidonic acid (10 μL). After 20 min incubation at 37 °C, the reaction was terminated by the addition of 10% (v/v) formic acid (10 μL). Concentration of produced PGE2 was evaluated using the Prostaglandin E2 EIA Kit-Monoclonal . After 18 h sample incubation with PGE2 tracer (PGE2-acethylcholinesterase conjugate) and PGE2 monoclonal antibody at 4 °C, wells were emptied and rinsed five times with wash buffer. After 90 min incubation with Ellman’s reagent, absorbance was read at 405 nm using the iEMS Reader MF type 1401  and corrected for the absorbance at 620 nm. Blank absorbance (Ellman’s reagent) and non-specific binding absorbance (absence of antibody) were subtracted from the readings. From absorbances obtained for samples and control (ethanol), PGE2 tracer binding % were calculated, which were inversely proportional to the PGE2 concentrations in the wells. Results are expressed as COX-1 inhibition %, calculated from established PGE2 concentrations. Indomethacin was used as positive control.Cyclooxygenase activity of COX-1 was evaluated spectrophotometrically according to previously described protocols ,119. COXPeroxidase activity of COX-1 was evaluated spectrophotometrically using the TMPD assay , with soBacillus cereus American Type Culture Collection  11778, B. subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 6538 and S. aureus ATCC 29213 among Gram-positive and Escherichia coli ATCC 10536, Klebsiella pneumoniae MFBF 10402 and Pseudomonas aeruginosa ATCC 27853 among Gram-negative bacterial species. Additionally, eight clinical isolates of S. aureus, including methicillin-resistant S. aureus  and methicillin susceptible S. aureus  were used. Bacterial strains were sourced from the Collection of Microorganisms, Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb. Inoculums were prepared from overnight cultures that were cultured on tryptic soy agar (TSA) at 37 °C, suspended in sterile physiological saline, with the optic density adjusted to 0.5 McFarland (1.5 × 108 colony-forming units (CFUs)/mL) using a densitometer . Right before cultivation in nutrient medium, 1 mL of each prepared suspension was dissolved with 9 mL physiological saline. Prior to cell treatment, sample stock solutions in distilled water (c = 50 mg/mL) were filtered through Chromafil cellulose acetate filters of pore size 0.22 μm .Antibacterial activity was evaluated using the following strains: d = 6 mm) and filled with 50 μL of methanolic aerial parts extract solution (c = 50 mg/mL) in distilled water. Plates were incubated at 37 °C for 18 h in the dark. Diameters of the zones of inhibition of bacterial growth (mm) were read from the diameters of transparent zones around the wells. Gentamicin and norfloxacin (c = 0.2 mg/mL) were used as positive controls.Well diffusion method was carried out according to the European Pharmacopoeia . Inoculuw/v) TTC (20 μL), which in the presence of metabolically active bacteria  gave red coloration/precipitate [Broth microdilution method was conducted in sterile plastic microtiter plates with 96 wells , in accordance with the Clinical and Laboratory Standards Institute (CLSI) recommendations . Two-folcipitate . This wa2, as previously described [3). Cells were incubated with methanolic leaf extract solutions in DMEM  for 24 h at 37 °C. Prior to cell treatment, samples were filtered through sterile filters of pore size 0.22 μm.Human MDA MB-231 breast cancer cells obtained from Dr. Sonja Levanat and human A1235 glioblastoma cells obtained from S.A. Aaronson  were culescribed . After rCell viability was assessed using the MTT assay as previously described . Prior tα-glucosidase activity, COX-1 inhibitory activity (PGE2 assay) and MDA-MB-231 and A1235 cell viability were performed in quadruplicate, and viability of Hep G2 cells in hyperglycemic conditions (MTT assay) in octuplicate, and the results are expressed as mean values and standard deviations. Concentrations that were observed to reduce the DPPH radical absorbance by 50% (IC50) were estimated using linear regression and those that reduced COX-1 activity and cell viability by 50% (IC50) using logarithmic regression.Phytochemical content, assessment of oxidative stress biomarkers and viability of Hep G2 cells in hyperglycemic conditions (LDH assay), antibacterial activity and COX-1 inhibitory activity (TMPD assay) were evaluated in triplicate and the results are expressed as mean values and standard deviations or, exceptionally, mean values . Assessment of r) was used to establish the relationship between observed biological activities and phytochemical content. In all tests, significance level α was set at 0.05. Analyses were performed using GraphPad Prism 6.01 .Statistically significant differences were evaluated by using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test (comparison between different species) or Dunnett’s post hoc test (comparison between samples and control/hyperglycemia). Pearson’s correlation coefficient (G. alypum and three related species, G. punctata, G. cordifolia, and G. meridionalis, considering different methods of extract preparation and different plant parts used. The bioactive compounds contained in investigated extracts of G. alypum and their observed biological activities are in accordance with the results of previous biological activity studies, as well as the reported traditional uses of this well-investigated medicinal plant, including its antidiabetic use, while those of related species suggest they too may have therapeutic potential. Observed antioxidant, anti-inflammatory, and antimicrobial activities of aerial parts extracts of investigated Globularia species support their use in cosmetics, while cytotoxicity assay results indicate further studies should preferably be carried out on G. cordifolia, G. meridionalis and G. punctata. The latter species also showed greater antimicrobial potential in comparison to G. alypum, which may be associated with its characteristic phenylethanoids and flavonoids. The paper displays how a combination of phytochemical and biological activity data obtained for extracts of several different species of the same genus may improve the understanding of their potential health benefits and facilitate the identification of compounds that are of possible interest for future biological activity studies. Future studies could focus more on the biological activities of major metabolites found in these species and elucidation of their underlying molecular mechanisms.The present study provides a greater insight into the phytochemical composition of


Proteus mirabilis to imipenem allows this pathogen evade surveillance for carbapenemases. Metallo-beta-lactamases of the VIM family are widespread among Enterobacterales in Europe. We identified an outbreak of P. mirabilis carrying 2 VIM enzymes among elderly patients from a medical center in Hungary.Nonsusceptibility of P. mirabilis isolates were received from Hungary during 2020 as part of the SENTRY Antimicrobial Surveillance Program. Isolates were susceptibility tested by the CLSI reference broth microdilution method. Carbapenem-nonsusceptible isolates were submitted to whole genome sequencing, screened for resistance mechanisms, and evaluated for core genome multi-locus sequence typing (cgMLST).A total of 16 P. mirabilis isolates from Hungary, 5 carbapenem-nonsusceptible, multidrug-resistant isolates (3 from urinary tract infections and 1 each from bloodstream infection and pneumonia) were identified from patients 66-92 years old. Four of these 5 isolates were listed as community acquired. All isolates were resistant to ceftriaxone , cefepime (16- > 32), imipenem ( > 8), gentamicin ( > 16), levofloxacin (16- > 32), nitrofurantoin ( > 64), trimethoprim/sulfamethoxazole ( > 4), and plazomicin ( > 128), but susceptible to meropenem (0.25-0.5) and ertapenem (0.03-0.25). Isolates carried blaVIM-4 and blaVIM-75 on a class 1 integron within IS26 and separated by aac(6’)-IIc. IS26 was located on a compound plasmid carrying other resistance-encoding genes, including armA. The integron structure is identical to the blaVIM-4 and blaVIM-1-carrying integron described from a Vibrio cholerae isolated from a seagull in France in 2015 (KR262557). blaVIM-75 is a single amino acid variant (Q60R) of blaVIM-1. All isolates carried mutations in the QRDR . Based on cgMLST analysis, these 5 P. mirabilis isolates were highly similar, with only 7-19 SNPs detected.Among the 16 P. mirabilis isolates carrying blaVIM-4 and blaVIM-75 in a Hungarian medical center is a cause of concern. Surveillance should be performed to understand the spread of and treatment options for infections caused by carbapenem-nonsusceptible P. mirabilis.The outbreak of multidrug-resistant Lalitagauri M. Deshpande, PhD, Melinta: Grant/Research Support|Pfizer: Grant/Research Support Katalin Burián, MD, JMI Laboratories: Grant/Research Support Ilona Dóczi Csányi, MD, JMI Laboratories: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.


Correction: BMC Med 20, 398 (2022)https://doi.org/10.1186/s12916-022-02595-8The labels of the two groups – response vs. non-response – were mistakenly switched.The HR in panel A was incorrectly stated as 0.38 instead of 0.308.The original article  containeThe figure has been updated in the original article to reflect these amendments.


Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that can cause life-threatening infections among immunocompromised populations. This report presents the complete 74,962-bp genome of S. maltophilia podophage Paxi, an N4-like phage sharing 85.3% nucleotide similarity to S. maltophilia podophage Pokken. Stenotrophomonas maltophilia is an environmentally ubiquitous and commensal bacterium, and it has also emerged as a nosocomial pathogen capable of causing life-threatening infections, especially among immunocompromised individuals  at 30°C, and phage propagation was performed using the soft agar overlay method (www.bioinformatics.babraham.ac.uk/projects/fastqc) and FASTX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/) to yield 84,373 trimmed reads, from which a contig was assembled with 80-fold coverage using SPAdes v3.5.0 (ATGGAGCCGGAGAGATCCTT-3′ (forward) and 5′-ACTTCATCAAGCGTGTCGGT-3′ (reverse). The CPT Galaxy-Apollo phage annotation platform  was utilized for genome annotation , using otation 68. Structtation 6912. Gene tation 69–18. The Stenotrophomonas phage Pokken (GenBank accession number NC_049463.1) (Enterobacteria phage N4 (NC_008720.1), sharing 43 similar proteins  such as virion RNA polymerase (NCBI protein accession number YP_950528.1) and an SAR endolysin N-acetylmuramidase (YP_950539.1). Paxi was predicted by PhageTerm to contain 538-bp direct terminal repeats.Phage Paxi was determined to have a podovirus-like morphology  by viewi49463.1) , and BLAMZ326856. The associated BioProject, SRA, and BioSample accession numbers are PRJNA222858, SRR14095256, and SAMN18509291, respectively.The genome sequence for Paxi was deposited in GenBank under accession number