Genera that were significantly associated with ENL production  were unique to each group. Bacteria  previously associated with colorectal cancer and cardiovascular disease, both diet-related chronic diseases, were unique to either AA or EA and were significantly reduced in the FS intervention. This study suggests that ethnic variation in ENL metabolism may be linked to gut microbiota composition, and its impact on disease risk deserves future investigation.Lignans are phytochemicals studied extensively as dietary factors in chronic disease etiology. Our goal was to examine associations between the gut microbiota and lignan metabolism and whether these associations differ by ethnicity. We conducted a flaxseed (FS) dietary intervention in 252 healthy, postmenopausal women of African ancestry (AA) and European ancestry (EA). Participants consumed ~10 g/d ground flaxseed for 6 weeks and provided overnight urine collections and fecal samples before and after intervention. The gut microbiota was characterized using 16S rRNA gene sequencing and differences in microbial community composition compared by ethnicity and intervention status. We observed a significant difference in the composition of the microbiota measured as beta diversity ( Bacteroides and Klebsiella ), whole grains, vegetables and fruits ,2, and hebsiella ,11,12. Abacillus ,12. ED cbacillus . Efficiebacillus  and may bacillus . In factbacillus ,16,17,18Given the contribution of individual characteristics to variation in lignan metabolism, ethnicity may also be an important effect modifier, as ethnicity can incorporate genetic diversity, microbial diversity, and sociocultural and lifestyle factors, including diet. As mentioned above, as lignans have structural and functional similarities to endogenous estrogens as well as anticarcinogenic activities, determination of influences on lignan production are relevant to postmenopausal women who are at higher risk for hormone-related cancers. Therefore, we conducted a FS dietary intervention study in healthy postmenopausal women to determine how variation in GMC affects the production of the bacterially derived enterolignans, ENL and ED, and how these associations differ for non-Hispanic US women of African ancestry (AA) and European ancestry (EA).n = 93; 52 EA, 41 AA), or group (2) maintenance of usual diet , and after a 2-month washout crossed over to the other condition for an additional 6 weeks. Overnight urine collections and fecal samples were collected at the beginning and end of each 6-week intervention period, for a total of 4 collections per participant over the 5-month study period. Overnight urine collections were obtained for determination of SECO, ENL, and ED. Fecal samples were self-collected at home using the Commode Specimen Collection system  and a feces collection tube with scoop in the lid , glass beads  and 5 ml RNAlater  for preservation of microbial nucleic acids. All biospecimens were frozen at −80 °C in the DataBank and BioRepository (DBBR) laboratories at Roswell Park Comprehensive Cancer Center  until assay. This study was registered at clinicaltrials.gov as NCT01698294. The study protocol was conducted according to established ethical guidelines, approved by the Roswell Park Institutional Review Board, and women provided signed, informed consent before participation.The study design and methodology were described in detail elsewhere . BrieflyAccrual and retention goals were 100 each EA and AA completing all study activities. Participants self-identified as either non-Hispanic African American or White. Ancestry informative markers confirmed that participants were genetically homogenous with self-reported ancestry (hereafter referred to as ‘ethnicity’) . Of 933 Ground whole FS was provided as a single lot from a single source . Heartland Flax stored the whole FS and ground (milled through a 14 mesh US Standard sieve screen) and packaged it for the duration of the study. Participants were provided with one 500 g bag of ground FS and standardized scoops with instructions to consume one scoop per day (~10 g/d) mixed into water or juice. Any unused FS was returned to the study coordinator at the end of the 6-week period to monitor adherence. During the 5-month study period, women were instructed to avoid other dietary sources of FS such as breads and cereals with flaxseed added. In addition to returning non-consumed FS, adherence was assessed through determination of urinary lignan levels. Compliance was good with 91% (AA) and 95% (EA) of planned FS doses consumed.2) and body composition (lean and fat mass) were assessed at each visit by a bioimpedance analysis (BIA) system . Dietary data were collected via 12 telephone-administered 24-hour dietary recalls administered randomly throughout the 5-month active intervention period and analyzed with the University of Minnesota Nutrient Data Systems for Research (NDSR) dietary analysis program. Study participants completed interviewer-administered questionnaires including basic demographic information, medical and reproductive histories, medication and dietary supplement use, family history of chronic disease, physical activity, cigarette smoking history, and other epidemiologic data relevant to diet and cancer. BMI  as described previously . All ligTo assign bacterial taxonomy, sequences were processed using QIIME v.1.8  and clashttps://cran.ism.ac.jp/web/packages/vegan/vegan.pdf). Additional summary statistics, tests, and visualizations for alpha and beta diversity were performed by ethnicity and intervention status. Statistical analyses and comparisons to detect differential abundance of genera were carried out using pscl package . This methodology implements a likelihood ratio test using a hurdle generalized linear model (hurdle GLM) assuming the outcome variable, in this case genera, has a negative-binomial distribution. The choice of using a hurdle model was based on the sparseness of the genera counts (2), total dietary fiber (g/d), energy intake , and total FS consumption during the intervention period (g/intervention period). Post-intervention models were further adjusted for baseline ENL excretion. Genera with <1% zeros across samples were fit with a negative-binomial generalized linear model (GLM); otherwise, a hurdle model was used. Genera with >85% zeros across samples were considered low quality and removed. From 115 genera, 8 and 6 were removed when fitting AA and EA models, respectively. Forest plots were used to visualize important genera (FDR < 0.05) which were then color-coded for phylum and displayed by ethnicity. Statistical analyses were performed in R (3.6.1) and related programs specific to running the programs listed above. 16S sequence data were first summarized to operational taxonomic units (OTUs) at the species level (97% similarity). We identified 10 phyla, 115 genera and 689 OTUs from 16S rRNA gene sequences in the samples. Shannon diversity index was estimated for alpha diversity scores using phyloseq package v1.28.0) . For bet.0 . For a counts . Differehttps://github.com/jbisanz/ElenMatchR) [ber) and its putative transcriptional regulator responsible for ENL production from lignans. We analyzed the genomes for two Senegalimassilia species, nine Slackia species, and two Collinsella species. We set parameters for a clustering threshold of 30% amino acid identity and 50% coverage (We used ElenMatchR (nMatchR) , a randocoverage .Demographic and personal characteristics by ethnicity were described using basic descriptive statistics. Differences in enterolignan excretion by randomization group across visits were assessed with repeated measures mixed models adjusting for age, BMI, and amount of flax consumed during intervention and stratified by ethnicity and randomization group.p < 0.05).We previously observed no statistically significant differences in age, BMI, cigarette smoking status, education, or lignan excretion between women who completed the study vs. women with baseline data only . As showp < 0.001, AA and EA), ED , and SECO . p < 0.001, AA and EA), ED , and SECO . Interestingly, at each time point, excretion was higher among EA compared to AA: ENL , ED  and SECO .Urinary lignan excretion by ethnicity, randomization group, and visit are shown in p = 0.019), but not after FS consumption (2 = 0.01). Community structure did not differ by intervention status for AA or EA women (Alpha diversity (Shannon Diversity Index) did not differ between AA and EA women, nor from baseline to post-intervention (data not shown). As shown in sumption b. AlthouEA women c,d, nor p < 0.05). At baseline in AA women, Alistipes (adj. p = 0.0003), a Lachnospiraceae genera (adj. p = 0.000008), and Slackia (adj. p = 0.04) were enriched and Lachnospira (adj. p < 0.0001) and Tyzzerella (adj. p < 0.0001) were reduced. Fusobacterium (adj. p = 0.01) and Paraprevotella (adj. p = 0.004) were reduced and a Puniceicoccaceae genera (adj. p < 0.0001) was enriched in EA at baseline. In the AA women after FS, Klebsiella (adj. p < 0.0001) was enriched in participants who excreted ENL whereas Fusobacteria (adj. p = 0.009), Odoribacteria (adj. p = 0.035), and Pyramidobacter (adj. p = 0.007) were reduced. In contrast, Lactobacillus (adj. p = 0.02), two Ruminiclostridium genera (adj. p < 0.01) and Victivalis (adj. p = 0.001) were enriched and Lachnoclostridium (adj. p = 0.005) was inversely associated with ENL in EA after FS. At baseline, 12 genera associated with ENL excretion were found in both AA and EA. In contrast, after FS intervention nine bacteria were found in common in AA and EA participants who excreted ENL . The difSlackia  was enriched at baseline and Senegalimassilia (adj. p = 0.0005) was enriched after the FS intervention (Collinsella (adj. p = 0.01) was enriched in the baseline samples, although not after the FS intervention  convert SDG to SECO [Lactobacillus was significantly enriched after FS. Gram-positive bacteria including Lactobacillus have been associated with deconjugation of lignans and three strains of Lactobacillus were shown to produce ED and ENL from flax extracts [Lactobacillus strains have strong substrate specificity for SECO [Ruminococcus groups in both EA and AA, although different genera were enriched in each group. Ruminococcus are an integral part of consortia involved in the anaerobic fermentation of phytochemicals including conversion of ED to ENL [Inter-individual variation in ENL production by bacterial metabolism may be a result of different patterns of microbial composition, wherein either a single genus or a consortium of multiple genera can metabolize plant lignans to ENL. ,6,13,47.rvention . This fa to SECO . In the for SECO ,47. CoriD to ENL . Slackia was associated with ENL at baseline and Senegalimassilia after FS. The reference genomes for both genera contain genes associated with ENL production (ber) and its putative transcriptional regulator responsible for ENL production from a lignan [Collinsella and an uncultured genus in Coriobacteriaceae were enriched in EA women. In contrast, our bioinformatic analysis suggested that species related to Collinsella did not contain genes involved in ENL production in the EA although they are members of the family Coriobacteriaceae  and other metabolites. Both EA and AA showed enrichment in genera in Christensenellaceae, Prevotelleaceae, Ruminococcaeceae, and some Lachnospiraceae ,50, whic and CO2 . In our ostasis. ,60 Lachnostasis. , a genusur study . Pyramidobobacter, Odoribacter and Fuosbacteria, associated with genotoxic sulfide production and CRC, [Lachnoclostridium, a genus associated with secondary bile acid metabolism, was inversely associated with ENL production in EA. Although many of the bacteria in common between AA and EA after FS supplementation are associated with saccharolytic fermentation and phytochemical metabolism, some of the genera unique to each race are associated with diet-related chronic diseases. Our data suggest that observed differences in ENL production and microbiome composition between AA and EA could be relevant to population-specific differences in chronic disease prevalence. For example, cross-sectional studies have reported an inverse relationship between ENL excretion and circulating secondary bile acids [Lachnoclostridium was inversely associated with ENL production in EA. Lachnoclostridium includes species involved in the 7α-dehydroxylation of cholic acid to the secondary bile acid dehydroxycholic acid (DCA) [Our findings suggest that differential metabolism of lignans in AA and EA women has implications for disparities in the prevalence of diet-related chronic diseases. and CRC,  were redle acids ,64, bactle acids , and CRCle acids ,64. In oid (DCA) , and a rid (DCA) . FurtherWe present herein one of the few studies to examine differences in ENL exposure in a dietary intervention with FS and associations with the gut microbiome that vary by ethnicity. To our knowledge, our FS intervention study is the largest to date that includes similar numbers of AA and EA women, thus providing important insight into potential population-specific differences in the impact of dietary components on microbial-host metabolism. However, since the study was limited to postmenopausal women, we do not know if our results are applicable to AA and EA men. Finally, using taxon-based methodology, we were unable to directly identify species or strain differences and genome content linked to functional gene pathways involved in ENL production. Future analyses should include both sexes as well as functional analysis of species and strains in the intervention response.


Cassia occidentalis L. (Coffee senna), Sesbania sesban (L.) Merr. (Common sesban) and Melilotus alba Medik. (White sweetclover) against seed germination and seedling growth of some broadleaved weed species. Firstly, N-hexane and aqueous extracts of these species were assessed for their phytotoxic effect against lettuce (Lactuca sativa L.). The extracts found more potent were further tested against germination and seedling growth of four broadleaved weed species, i.e., Parthenium hysterophorus L. (Santa-Maria), Trianthema portulacastrum L. (Pigweed), Melilotus indica L (Indian sweetclover). and Rumex dentatus L. (Toothed dock) in Petri dish and pot experiments. Aqueous extracts of all species were more toxic than their N-hexane forms for seed germination and seedling growth of lettuce; therefore, aqueous extracts were assessed for their phytotoxic potential against four broadleaved weed species. Aqueous extracts of all species proved phytotoxic against T. portulacastrum, P. hysterophorus, M. indica and R. dentatus and retarder their germination by 57, 90, 100 and 58%, respectively. Nevertheless, foliar spray of C. occidentalis extract was the most effective against T. portulacastrum as it reduced its dry biomass by 72%, while M. alba was effective against P. hysterophorus, R. dentatus and M. indica and reduced their dry biomass by 55, 68 and 81%, respectively. It is concluded that aqueous extracts of M. alba, S. sesban and C. occidentalis could be used to retard seed germination of T. portulacastrum, P. hysterophorus, M. indica and R. dentatus. Similarly, aqueous extracts of C. occidentalis can be used to suppress dry biomass of T. portulacastrum, and those of M. alba against P. hysterophorus, R. dentatus. However, use of these extracts needs their thorough testing under field conditions.Weed infestation is a persistent problem for centuries and continues to be major yield reducing issue in modern agriculture. Chemical weed control through herbicides results in numerous ecological, environmental, and health-related issues. Moreover, numerous herbicides have evolved resistance against available herbicides. Plant extracts are regarded as an alternative to herbicides and a good weed management option. The use of plant extracts is environmentally safe and could solve the problem of herbicide resistance. Therefore, laboratory and wire house experiments were conducted to evaluate the phytotoxic potential of three Fabaceae species, i.e.,  Weeds exist everywhere and obstruct the germination, growth, and yield of field crops. Furthermore, weeds compete with crop plants and suppress their growth by releasing different allelochemicals –3. WeedsHerbicides are the most effective and quickest weed management option; however, they reduce food quality and impose many adverse effects on environment, animal and human health ,14. FurtTrianthema portulacastrum L., Parthenium hysterophorus L., Melilotus indica L. and Rumex dentatus L. are broadleaved obnoxious weed species that are widely distributed throughout Pakistan  and extraction methods, i.e., A = Aqueous and N = N-hexane.A laboratory bioassay was carried out for preliminary screening of N-hexane and aqueous extracts of A filter paper (10 cm diameter) was placed in pre-washed and dried Petri dishes. Subsequently, 3 ml of all plant extracts was added to each dish. Twenty seeds of lettuce were placed in each dish. Consequently, Petri dishes were packed in the polythene bags to avoid moisture loss. The experiment was carried out at room temperature.Cassia occidentalis (L.), Sesbania sesban (L.) Merr., and Melilotus alba Medik. on seed germination and seedling growth of parthenium (Parthenium hysterophorus L.), horse purslane (Trianthema portulacastrum L.), sweet clover (Melilotus indica L.) and toothed dock (Rumex dentatus L.). Distilled water was applied as control.This experiment evaluated phytotoxic effects of aqueous extracts of 2), root and shoot length (cm) were taken.Twenty seeds were placed in sterilized Petri dishes having round discs of filter paper as described above. Extracts were applied and dishes were sealed with paraffin. For seedling growth plants were grown in plastic pots of 250 ml volume. Soil was taken from College of Agriculture, University of Sargodha. Soil was thoroughly mixed with sand at 1:1 ratio. Afterwards, 3–4 seeds of each weed species were sown in pot and pots were irrigated. The aqueous extracts were sprayed by hand sprayer on seedlings at 2–4 leaf stage. Second spray was done after five days of first spray. The data related to chlorophyll contents, dry weight (mg), seedling vigor index, leaf area (cmSeed germination percentage (GP) was recorded by counting the germinated seeds on each day for 20 days. Seeds were considered germinated when growing plumule extended to length of about 2 mm, respectively. The GP was calculated by Germination index (GI) w was calculated by 50) was calculated according to Time to obtain 50% germination  was calculated by using th day of sowing to the total number of emerged seeds till 15th day. It was determined by using Germination energy (GE) is the ratio of germinated seeds at 42.Three seedlings were randomly selected and their root and shoot lengths were measured and averaged. All seedlings from each Petri dish or pot were taken and wrapped in tissue paper and placed in oven at 70°C for 48 hours and then weighed on electric balance. Chlorophyll contents of three plants were determined with the help of chlorophyll meter  and average. Three plants from each treatment were chosen and washed. Afterwards, their leaf area was determined with the help of leaf area meter in cmFirst experiment was laid out according to randomized complete block design with factorial arrangement, while second experiment was laid out in completely randomized design and all treatments were replicated four times. Data were analyzed statistically using Fisher’s Analysis of Variance (ANOVA) technique (two-way for first experiment and one-way for second experiment) and treatment means were compared at 5% significance level by least significant difference post hoc test . NormaliM. alba and C. occidentalis extracts showed the highest phytotoxicity against seed germination and seedling growth of lettuce.Data of various germination and seedling growth parameters of lettuce in response to aqueous and N-hexane extracts of plant species proved that aqueous extracts of all Fabaceae plants were phytotoxic for lettuce germination and growth (data not shown). Among Fabaceae plants, M. alba plant extract exhibited the highest inhibitory effect on seed germination of T. portulacastrum (31.25%), while S. sesban and M. indica reduced seed germination of P. hysterophorus by 6.25 and 0.00%. However, C. occidentalis extract reduced seed germination of R. dentatus by 26.2%.Data regarding seed germination percentage of four broadleaved weed species in response to aqueous extracts of Fabaceae plants is given in Tables C. occidentalis significant delayed time to 50% germination of T. portulacastrum and M. indica. Germination energy of T. portulacastrum was significantly reduced by all plant extracts, while M. indica remained unaffected  in germination index of P. hysterophorus.Aqueous extracts of affected . All plaC. occidentalis and M. alba extracts resulted in the lower root length of T. portulacastrum  and M. indica . All extracts caused significant reduction in root length  of P. hysterophorus. As compared to DWTC, M. alba extract caused the highest reduction (61% and 40%) in shoot length of T. portulacastrum and R. dentatus, whereas S. sesban extract resulted in the lowest shoot length (1.13 cm) in P. hysterophorus  of seedling vigor index in P. hysterophorus. However, C. occidentalis extract resulted in the lowest seedling vigor index of R. dentatus (85.00) and M. indica (77.32). The M. alba extract resulted in the lowest dry weight (1.55 mg) of T. portulacastrum, while C. occidentalis extract resulted in the lowest dry weights (2.30 mg and 1.32 mg) of R. dentatus and M. indica, respectively.The least value of seedling vigor index (87.62) T. portulacastrum with foliar application of C. occidentalis extract, whereas the contents were P. hysterophorus (21.02), R. dentatus (8.70) and M. indica (22.0) with the application of M. alba extract. Statistically similar values of chlorophyll contents in P. hysterophorus and R. dentatus were observed with the application of S. sesban and C. occidentalis extracts, respectively (Data relating to pot experiment is given in Tables ectively .T. portulacastrum (3.07 cm) with C. occidentalis extract. The lowest root length of R. dentatus (2.64 cm and 3.09 cm) was recorded in response to M. alba and C. occidentalis extracts, respectively. The C. occidentalis extract caused the highest reduction (50%) in shoot length of T. portulacastrum, while M. alba in shoot lengths of P. hysterophorus (42%) and M. indica (47%) as compared to DWTC. However, both C. occidentalis and M. alba extracts produced lower values of shoot lengths (3.62 and 4.87 cm) in M. indica seedlings. Foliar application of C. occidentalis extract resulted in the lowest dry weight (49 mg) of T. portulacastrum seedlings. Data indicated that C. occidentalis extract resulted in the lowest leaf area (122.6 cm2) of T. portulacastrum, while S. sesban extract in the lowest leaf areas of P. hysterophorus (171.3 cm2), R. dentatus (187.3 cm2) and M. indica (61.3 cm2) (The lowest root lengths were recorded for 1.3 cm2) .Cassia occidentalis (coffee senna), Sesbania sesban (Egyptian river hemp) and Melilotus alba (Yellow sweet clover). Fabaceae synthesize greater diversity of secondary metabolites compared to other plant families [Soon valley is known as central salt range, located in the north-west side of the district Khushab, and has rich floristic diversity. The plants naturally found here have significant medicinal value ,52. Abbafamilies . These ifamilies .C. occidentalis is a well-known medicinal plant, rich in fedegoso that contains minute amount of anthraquinones that gives it substantial purgative and laxative properties [S. sesban is a commonly used leguminous fodder as well as valuable green manure crop. It contains one of the toxic non-protein amino acids that is called canavanine [M. alba is considered highly allelopathic. Various allelochemicals, including the highest quantity of coumarin content followed by coumarin, flavonoids, phenolic acids, triterpenes and saponins are found in M. alba [The operties . In addinavanine . The M.  M. alba –59.Euphorbia dracunculoides, Dicliptera bupleuroides and Justicia adhatoda exhibited greater allelopathic activity than their organic solvents  against germination and seedling growth of chickpea, wheat and broadleaved weeds, respectively. Our observations are supported by Oudhia & Tripathi [Lantana camara aqueous extracts on germination and seedling growth of T. aestivum and many vegetable species. Oyun et al. [Gliricidia sepium and Acacia auriculiformis had pronounced phyto-inhibitory effect on germination and seedling vigor of maize. Wakjira et al. [P. hysterophorus significantly inhibited germination index of lettuce. Tanveer et al. [Achyranthes aspera significantly inhibited germination index of Sorghum bicolor.Initial screening indicated that aqueous extracts of all plants were more phytotoxic against lettuce. The possible reasoning of higher allelopathic activity of aqueous extracts in comparison to their N-hexane fractions seems to be the higher concentrations of water soluble phenolics that caused more inhibitory effect –62. TanvTripathi  and SahiTripathi  who repon et al.  demonstra et al.  stated tr et al.  also repT. portulacastrum and R. dentatus was noted with M. alba and C. occidentalis extracts. Similarly, S. seban extract reduced the germination percentage of P. hysterophorus and M. indica by 90% and 100%, respectively. The reduction in germination percentage of weeds might be the result of presence of phytotoxic compounds that limited the underlying germination processes. Our results are supported by Randhawa et al. [T. portulacastrum in response to sorghum water-soluble extract. Delayed germination of weeds by application of plant extracts was attributed to their phyto-inhibitory effect that hindered germination process. Our results are supported by Mubeen et al. [Oryza sativa.In Petri dish germination bioassay, differential phyto-inhibitory effect on germination and seedling growth of different weed species were observed in response to aqueous extracts of different plants. The highest reduction in germination percentage of a et al.  who repon et al.  who obseA. aspera and P. hysterophorus. The decreased seedling vigor index is the result of reduced germination percentages and seedling lengths. The reduction in dry weight might be attributed to reduced root and shoot growth due to the presence of toxic compounds in studied extracts. These results are supported by Komal [occidentalis on dry weight of maize. The reduction in leaf area of weeds in response to foliar application of plant extracts seems to be the consequence of lower shoot growth and chlorophyll content. These results are supported by Musyimi et al. [Sesbania sesban (L.) Merrill extract significantly influenced the leaf area of Solanum nigrum L.The decrease in shoot lengths of weeds seems to be the phyto-inhibitory effect of allelochemicals in these extracts. Our results are supported by Safdar et al.  who obseby Komal  who obsei et al.  who repoM. alba, S. sesban and C. occidentalis extracts showed higher phytotoxicity against seed germination of T. portulacastrum, P. hysterophorus, M. indica and R. dentatus. Similarly, application of C. occidentalis on the seedlings had the highest inhibitory effect on T. portulacastrum, and M. alba against P. hysterophorus. Hence, these extracts cab ne recommend for the management of these weed species. However, their thorough understanding under field conditions is necessary before making a general recommendation.The S1 Dataset(XLSX)Click here for additional data file.


Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16 S rRNA genes in samples aspirated from the descending duodenum of 47  individuals who were screened for gastric cancer. Samples were analysed using 16 S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection.The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection changed the relative mean abundance of three phyla  and ten genera . Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways.Thirteen and 34 participants tested positive and negative for H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.The online version contains supplementary material available at 10.1186/s12866-022-02437-w. Helicobacter pylori infection can cause chronic gastritis or peptic ulcers and is associated with the development of certain gastric cancers [H. pylori infection [H. pylori infections and various other extra-gastroduodenal diseases remain obscure [ cancers . Recent nfection –6. Howev obscure , 7, 8.The theory that the bioactivities of commensal gut microbiota greatly influence host biofunction has attracted considerable attention in elucidating the pathophysiology of various diseases. In addition, the duodenum plays a key role in the crosstalk between the gut and the central nervous system, particularly as the release of brain-gut hormones and neurotransmitters in the small intestine, including the duodenum, are regulated by eating stimuli and information arising from the intraluminal environment. These hormones regulate widespread biofunctions, such as metabolism, biosynthesis, feeding behaviour, and gastrointestinal functions , 10.H. pylori infection alters the duodenal microbiota based on evidence from reverse-transcribed 16 S rRNA and that the same results were derived from duodenal biopsies and aspirates. These findings suggest that alteration in the duodenal microbiota induced by H. pylori infection is related to the onset of various extra-gastroduodenal diseases. This is because some degradation products of digestion, attributed to duodenal microbial biofunction, act as chemical effectors on host biofunctions [Schulz et al.  found thunctions , 13.H. pylori infection on the structure of commensal duodenal microbiota and their biofunctions using conventional analyses of microbial taxonomic diversity and the novel linear discriminant analysis (LDA) effect size (LEfSe) algorithm method to discover metagenomic biomarkers that could explain differences among microbial communities [Therefore, we aimed to elucidate the impact of munities . We alsomunities –17, whicH. pylori-positive and negative, respectively. Considering the Kimura–Takemoto classification of endoscopic atrophy, 30 of the 34 participants (88.2%) in the H. pylori-negative group had C0 (non-atrophy)–C2 atrophy, and most of the remaining four participants had a history of H. pylori eradication therapy. In contrast, C0–C2 atrophy was observed in five of the 13 participants (38.5%) in the H. pylori-positive group .MiSeq sequencing produced 3,312,084 reads with a mean of 70,470 ± 14,833 sequences per sample. These analyses were based on a rarefied table determined from 8,804 gene reads per sample. We identified 1,404 bacterial operational taxonomy units (OTU) from 23 phyla and 253 genera. The numbers of bacterial OTU per participant were 263.86 ± 78.46 and 284.53 ± 67.20 in the H. pylori-positive and -negative groups  and TM7 (negative 3.6%: positive 1.5%) was significantly higher in the H. pylori-negative group than in the H. pylori-positive group. In contrast, the abundance of Proteobacteria (negative 11.3%: positive 23%) was significantly higher in the H. pylori-positive group, and Acidobacteria and Planctomycetes were evident only in the H. pylori-negative group. The relative mean abundance of 14 phyla did not significantly differ between the H. pylori-negative and -positive groups. The results for the other four phyla were invalid for statistical analysis  was significantly higher in the H. pylori-negative group. Furthermore, 188 and 143 genera were detected in the H. pylori-negative and -positive groups, respectively. These differences were attributed to the different microbial community structures of each group. Specifically, 60 and 15 genera were found only in the H. pylori-negative and -positive groups, respectively. Helicobacter was detected only in the H. pylori-positive group, with an abundance of 2.79 ± 6.82% , one class (Gammaproteobacteria), one order , one family (Enterobacteriaceae), three genera , and three species  of bacteria. Figure Gammaproteobacteria (phylum Proteobacteria): Pasteurellales, Pseudomonas, Moraxella, Actinobacillus, H. segnis, and Enterobacteriaceae. Moraxella and H. segnis are notable intraoral bacteria. Two taxa, S. porcinus and P. durum, belonged to the class Bacilli (belonging to Firmicutes). These results suggest Gammaproteobacteria can affect duodenal microbial features.The LDA score derived from LEfSe analyses indicated that 12 taxa significantly influenced the biological features of the duodenal microbiota in the H. pylori infection . This suggests that H. pylori generally upregulates metabolic functional activities in duodenal microbes. The remaining pathways comprised those associated with genetic information processing (n = 16), environmental information processing (n = 21), cellular processes (n = 19), organismal systems (n = 48), and human diseases (n = 60). Among these 164 pathways, 18 significantly differed between the individuals with and without H. pylori. However, it was impossible to infer whether these pathways were functioning for microbial biofunctions and affecting biofunctions of the host.Among 327 investigated KEGG pathways  in the H. pylori-positive group. However, since the phylum to which H. pylori belongs has recently been changed from Proteobacteria to the newly established Epsilonbacteraeota [H. pylori infection altered the microbial features by increasing the abundance of Neisseria and enhancing the impact of Gammaproteobacteria in the duodenum  to the number of reads for the entire sequence. These results do not indicate the actual number of bacteria because the method aligns the DNA concentrations between samples to equal concentrations. LEfSe is designed to increase the detection power compared to community structure analysis, which compares simple ratios [Although a study comparing biopsied gastric tissue with and without iversity , the preteraeota , the resum Table ; Fig. 2.e ratios . TherefoNeisseria in the duodenum is probably related to the gastric acid output owing to atrophic gastritis induced by H. pylori. Intraoral indigenous bacteria in the genus Neisseria are not generally highly pathogenic, except for Neisseria gonorrhoeae and Neisseria meningitidis, which cause gonorrhoea and meningitis, respectively [Neisseria proliferation in the duodenum may be pathogenic through changes in microbial community structure [Gammaproteobacteria comprises several medically important bacteria, such as The increase in ectively . Howevertructure , 22. TheH. pylori infections and extra-gastric diseases have identified increased short-chain fatty acid (SCFA) production induced by the proliferation of Bacteroidetes [H. pylori did not significantly change the abundance of Bacteroidetes in the duodenum  and ether lipid metabolism (ko00565), are important for degradation of fatty acids, butyrate and acetic acid synthesis, and the production of phosphocholine or seminolipid, which functions in the maintenance of mucosal integrity and immune homeostasis [The KEGG pathway analysis showed that 12 bacterial metabolic pathways were affected by the presence or absence of eostasis , 26.H. pylori-positive group, suggesting that an abnormal tryptophan supply from the intestine impaired serotonin production. Serotonin is a paracrine messenger expressed primarily in enterochromaffin cells and enteric neurons. This information would help to clarify the causal relationship between H. pylori infection, the duodenal microbiota, and the pathophysiology of functional dyspepsia [Notably, the ko-abundance of the tryptophan metabolic pathway (ko00380) was significantly greater in the yspepsia –29. In ayspepsia .H. pylori-positive group , was upregulated in the H. pylori-positive group [H. pylori-positive group. This could extensively affect host functions because terpenoids are steroid precursors and closely related to cytochrome P450 that functions as an oxidase in terpenoid biosynthesis [The pathways of linoleic (ko00591) and α-linolenic ko00592) acid metabolism were also upregulated in the  acid metve group , 33. Theynthesis , 35.H. pylori eradication. The affected metabolites were mapped to various biochemical pathways, including tryptophan metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. Although whether these alterations affect host biofunctions remains obscure, our findings confirmed that the metabolomic findings reported by Yap et al. originated from microbial metabolic pathways affected by H. pylori infection. Notably, KEGG analysis is only a prediction due to the presence of DNA. Transcriptome analysis will be needed to determine if these metabolic pathways are indeed upregulated.Yap et al.  found 45H. pylori-negative group might have already experienced significant changes in the structure and biofunctions of the commensal duodenal microbiota due to previous H. pylori infections. In fact, nine patients with a history of infection and eradication were included in the H. pylori-negative group  inhabit the mucosa , 39. OurFinally, differential abundance (DA) analysis methods for microbiome data are controversial in terms of consistency and reliability. For example, some have pointed out that the false discovery rate could sometimes not be controlled in LEfSe analysis, which was used in this study . The proH. pylori infection changed the aspects of the microbiota in the descending part of the duodenum. This dysbiosis, characterised primarily by the upregulation of microbial metabolic pathways, altered commensal microbial biofunctions, which may affect host biofunctions. The gut microbiota can be regarded as an independent organ within the gut lumen, and an investigation of biofunctions originating from this “commensal bacterial organ” would help elucidating the aetiology of various diseases.In conclusion, H. pylori eradication , who were screened for gastric cancer. We obtained information from all participants about treatment with gastric acid inhibitors, antibiotics, and medical history of The design of this study proceeded without public involvement. Patient involvement was restricted to sample collection at the time of enrolment. Patients were neither consulted to interpret the results nor invited to contribute to the writing or editing of this article.Duodenal fluids were collected from the descending part of the duodenum using a PW-2 L-1 fluororesin tube  under standard video endoscopy with the Olympus GIF-XQ260 or GIF-XP260N video gastroscope . The tube was sterilised and changed for each patient. Duodenal fluid was aspirated immediately after injecting 5 mL of saline into the descending part of the duodenum, and the aspirate was immediately cryopreserved at −80 °C. One certified endoscopist (HZ) conducted all endoscopic procedures and sampling to avoid bias. The endoscopic findings were recorded simultaneously, and the extent of atrophic gastritis was evaluated in accordance with Kimura–Takemoto classification for endoscopic atrophy .Genomic DNA (gDNA) was extracted from duodenal fluid using PowerFecal DNA Isolation Kit  as described in the protocol provided by the manufacturer.H. pylori. The participants were then assigned to groups based on the presence or absence of H. pylori.We applied conventional nested polymerase chain reactions (PCR) of extracted gDNA  and thenhttps://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). The PCR amplicons were purified using Wizard SV Gel and PCR Clean-Up System , then sequenced and quantified using the MiSeq system [Sequencing libraries were prepared for the Illumina MiSeq platform . The V3 and V4 regions of the 16 S rRNA gene were targeted using the primer pair 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′) , and a 1q system .H. pylori-positive and -negative groups were compared based on unrarefied OTU tables using Welch’s t-tests in Microsoft Excel for Windows 10.Sequencing data were processed using CLC Genomic Workbench 10.0.1 and CLC Microbial Genomics Module 2.5 . Overlapping paired-end reads were merged and trimmed, and chimeric reads were filtered using default parameters. The remaining reads were clustered into OTU with 97% identity using the Greengenes database (version 13.5) as the reference . We evalhttps://huttenhower.sph.harvard.edu/galaxy/) to support high-dimensional class comparisons with a focus on metagenomic analysis.The LEfSe algorithm can identify genomic taxa with a relative abundance that differs between groups. We computed LEfSe using the Galaxy web application and workflow framework (https://piphillin.secondgenome.com/) with the KEGG Orthology (KO) database as the reference genomic database [The biofunctions of the duodenal microbiota were inferred via metagenomic functional annotation. The OTU abundance table was uploaded to the Piphillin server (database . ThereafH. pylori-positive and -negative groups using Mann–Whitney U tests in Microsoft Excel for Windows 10. All values with P < 0.05 were considered statistically significant.The KEGG pathway analysis results identified the ko-abundances that corresponded to the abundance of specific KEGG pathways and quantitatively represented microbial biofunction characteristics. Then, pathways classified under metabolism were investigated in detail because the gut microbiota could be regarded as an independent organ that produces various metabolites with metabolic functions in the gut lumen . Each koAdditional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Purpose: This study aimed to investigate the potential of computed tomography (CT) imaging features and texture analysis to distinguish bronchiolar adenoma (BA) from adenocarcinoma in situ (AIS)/minimally invasive adenocarcinoma (MIA).Materials and Methods: Fifteen patients with BA, 38 patients with AIS, and 36 patients with MIA were included in this study. Clinical data and CT imaging features of the three lesions were evaluated. Texture features were extracted from the thin-section unenhanced CT images using Artificial Intelligence Kit software. Then, multivariate logistic regression analysis based on selected texture features was employed to distinguish BA from AIS/MIA. Receiver operating characteristics curves were performed to determine the diagnostic performance of the features.Results: By comparison with AIS/MIA, significantly different CT imaging features of BA included nodule type, tumor size, and pseudo-cavitation sign. Among them, pseudo-cavitation sign had a moderate diagnostic value for distinguishing BA and AIS/MIA . Further, a total of 396 quantitative texture features were extracted. After comparation, the top six texture features showing the most significant difference between BA and AIS or MIA were chosen. The ROC results showed that these key texture features had a high diagnostic value for differentiating BA from AIS or MIA, among which the value of a comprehensive model with six selected texture features was the highest  for BA and AIS or MIA. These results indicated that texture analyses can effectively improve the efficacy of thin-section unenhanced CT for discriminating BA from AIS/MIA.Conclusion: CT texture analysis can effectively improve the efficacy of thin-section unenhanced CT for discriminating BA from AIS/MIA, which has a potential clinical value and helps pathologist and clinicians to make diagnostic and therapeutic strategies. Accordima (MIA) . As subtma (MIA) –7. HowevClinically, biopsy is used to preoperatively confirm the diagnosis when lung cancer is suspected. This process requires an invasive procedure, which has risks including bleeding, pneumothorax, and infection , 9. And Texture analysis is a novel imaging post-processing technique used for the quantification of image grayscale distribution features, pixel interrelationships, and spectral properties of images –13. CompThis study was approved by the ethics committee of Daping Hospital, and the informed consent requirement was waived. In this retrospective study, from October 2017 to June 2020, 86 patients underwent surgical resection of tumors presenting as solitary pulmonary nodule, including solid nodule, GGN, or subsolid GGN, on CT images and pathologically diagnosed as BA, AIS, or MIA. AIS and MIA were diagnosed according to the World Health Organization 2015 criteria as confirmed by surgery , 22. TisAll CT images were obtained on a 64-detector CT scanner  with a breath-held helical acquisition of the entire thorax. CT parameters were as follows: tube voltage = 120 kVp; tube current = 150 mAs; detector collimation = 0.5 mm × 64; pitch = 0.625; rotation time = 0.5 s; reconstruction slice thickness = 1 mm; matrix = 512 × 512; field of view = 407 mm. All CT images were analyzed by two radiologists  independently. Both radiologists were informed of the location of each lesion but were blinded to the pathological diagnosis. Lung nodules were divided into three types, containing pure GGNs, subsolid GGNs, and solid nodules based on thin-section unenhanced CT images. A pure GGN was defined as a nodule occupied by ground-glass opacity without solid regions. A subsolid GGN was defined as a nodule that obscured underlying vascular signs and where <50% of the nodule was observed at the mediastinal window. When more than 50% of a nodule was seen at the mediastinal window, a solid nodule was defined . Pseudo-www.itksnap.org) at the lung window. Two experienced radiologists  blinded to the clinical outcomes were involved in region of interest (ROI) segmentation. The whole tumor volume was determined by manually drawing an ROI along the border of the tumor on each consecutive slice covering the whole lesion. Therefore, a three-dimensional VOI was finally obtained. The texture features were automatically calculated by the AK software . A total of 396 texture features were extracted, including six types: histogram, gray-level co-occurrence matrix (GLCM), gray-level size zone matrix (GLSZM), gray-level run-length matrix (GLRLM), form factor features, and Haralick features. The extracted texture features were standardized to remove the unit limits of each feature. The histogram described the distribution of voxel intensities within the image region defined by the mask through commonly used and basic metrics. GLRLM depicts the amount of homogeneity in specific directions. GLCM and Haralick features provide information about the gray-level value distribution of pixel pairs in all directions. GLSZM is efficient for characterizing texture homogeneity, non-periodicity, or a speckle-like texture  was considered significant after Bonferroni correction. Feature dimension reduction was performed as follows: first, a t-test or Mann–Whitney U-test was performed; second, univariate logistic analysis was conducted, and statistically significant features (P < 0.05) were chosen; third, the minimal-redundancy maximal-relevance method (mRMR) was used to remove the redundant and less-relevant features. Multivariate logistic regression analyses were performed to establish a comprehensive model with the most valuable parameters to distinguish BA from AIS or MIA. The diagnostic accuracy of different CT imaging features, textural features, and comprehensive models were evaluated by a receiver operating characteristic (ROC) analysis to obtain area under curves (AUCs), sensitivity, and specificity. The MedCalc Statistical Software  was used to compare differences in AUCs. Two-tailed P-values were calculated with a 0.05 significance level.Statistical analyses were performed using the SPSS software . Kolmogorov–Smirnov and Levene tests were used for the assessment of normal distribution and equal variance. With regard to the reproducibility of volumetric and texture analysis, inter-observer reliability was assessed by an intraclass correlation coefficient (ICC) test. In general, an ICC <0 indicates no agreement, 0–0.20 slight agreement, 0.21–0.40 fair agreement, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement, and 0.81–1 almost perfect agreement. A one-way analysis of variance (ANOVA) test was applied to assess the ability of CT imaging features to differentiate between BA and AIS/MIA. The level of significance for intergroup differences was set at The clinical characteristics of 15 BA, 35 AIS, and 38 MIA patients are summarized in P < 0.001), but no significant difference was found between BA and AIS . Notably, a pseudo-cavitation sign was observed more frequently in BA , compared to AIS  or MIA  (P = 0.002). No statistically significant differences with respect to tumor shape (P = 0.216), margin (P = 0.358), tumor–lung interface (P = 0.265), and distance to pleura (P = 0.623) were observed among BA, AIS, and MIA. Furthermore, ROC analysis was performed to ascertain relevant CT imaging features in differentiating BA from AIS/MIA. The results showed that the pseudo-cavitation sign had a moderate diagnostic value . For BA and MIA, the nodule type, tumor size, and pseudo-cavitation sign had moderate diagnostic values , LongRunLowGreyLevelEmphasis_angle45_offset7 (P = 0.00028), GLCMEnergy_AllDirection_offset1_SD (P = 0.00042), ShortRunEmphasis_angle0_offset1 (P < e−4), VoxelValueSum (P = 0.00310), and Quantile0.975 (P = 0.00348), were calculated between BA and AIS , ClusterShade_angle0_offset1 (P < e−4), LongRunLowGreyLevelEmphasis_angle0_offset7 (P < e−4), ClusterShade_angle45_offset1 (P < e−4), VoxelValueSum (P < e−4), and ClusterShade_angle90_offset7 (P < e−4) were the top six texture features showing the most significant difference  or MIA . However, a pseudo-cavitation sign had a moderate diagnostic value for distinguishing BA and AIS (AUC: 0.741) or MIA (AUC: 0.708), while texture features had a high diagnostic value for differentiating BA from AIS (highest AUC: 0.912) or MIA (highest AUC: 0.876). These results indicate that the diagnostic values of texture features are higher than those of CT imaging features for differentiating BA from AIS/MIA.To improve the diagnostic value for BA and AIS/MIA, we performed the CT texture analysis of these lesions. A total of 396 texture features were extracted from unenhanced CT images. The ICC values of the inter-observer of our research were 0.82–0.98, which suggests great accordance between two readers and the reliability of VOI sketching. Three representative sets of CT texture features of patients with BA, AIS, and MIA are shown in  and AIS . The diafference . The ROCfference , among wfference , 4. FurtZ = 3.153, P = 0.0016; BA vs. MIA: Z = 3.508, P = 0.0005).Multivariate logistic regression analyses were performed to establish a comprehensive model with six selected texture features to distinguish BA from AIS or MIA. The sensitivity, specificity, and AUC for differentiating BA from AIS were 93.3, 92.1, and 0.977 (95% CI 0.893–0.999), respectively . While tThe present study is the first to distinguish BA from AIS/MIA using texture analysis on thin-section unenhanced CT images. In our study, we evaluated the role of CT imaging and texture analysis in differentiating BA from AIS/MIA. Pseudo-cavitation, one of the CT imaging features, could help differentiate BA from AIS and MIA. Key texture features showing the most significant difference between BA and AIS or MIA have a better distinguishing effect on disease than CT imaging features. Our data indicate that CT texture analysis demonstrates great potential in differentiating BA from AIS/MIA pre-operation.BA, a rare benign tumor appearing as a peripherally solitary small lung nodule, was first reported in 2018 by Chang et al. , 26. MisTexture analysis is an emerging imaging-based post-processing method that allows for quantification of tissue heterogeneity . There hThere are some limitations in our study. First, this is a single-institution retrospective analysis, and the sample size is rather small because of the low morbidity of BA. Second, potential selection biases cannot be excluded since this is a retrospective study. Third, manual segmentation of GGN ROIs has a higher risk of observer bias compared to delineation with semi-automatic regression. However, the ICC values of the inter-observer of this study were 0.82–0.98, suggesting great accordance between two readers and the reliability of VOI sketching. Therefore, a multicenter program to include more BA patients may be needed, and a validation to confirm the potential value of CT texture analyses in discriminating BA from AIS/MIA may also be needed in the future.In conclusion, our study indicated that CT texture analysis can effectively improve the efficacy of thin-section unenhanced CT for discriminating BA from AIS/MIA, which has a potential clinical value and helps pathologist and clinicians to make diagnostic and therapeutic strategies.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Research Ethics Committee of Daping Hospital, Army Medical University, Chongqing, China. The ethics committee waived the requirement of written informed consent for participation.XC and JF conceived and designed the study. JS and YLu collected the CT and clinical data. JF and XL analyzed the CT data. HT and JS extracted the CT texture features. HL analyzed the CT texture data. KL performed the statistics. YLi and YY prepared the figures and tables. XC and KL wrote the manuscript. JF and RJ edited the manuscript. All authors revised the manuscript and read and approved the submitted version.HL was employed by company GE Healthcare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Claviceps purpurea produces many pharmacologically important ergot alkaloids (EAS), which are widely used to treat migraine and hypertension and to aid childbirth. Although an EAS biosynthetic cluster of C. purpurea has been discovered more than 20 years ago, the complete biosynthetic pathway of EAS has not been fully characterized until now. The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C. purpurea. The conventional gene manipulation method for C. purpurea relies on homologous recombination (HR), although the efficiency of HR in C. purpurea is very low (∼1–5%). Consequently, the disruption of target genes is laborious and time-consuming. Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently, their gene-disruption efficiency is still very low. Here, we developed an efficient genome-editing system in C. purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes. As proof of principle, three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system, with editing efficiencies ranging from 50% to 100%. Inactivation of the three genes, which are closely related to uridine biosynthesis (ura5), hypha morphology (rac), and EAS production (easA), resulted in a uridine auxotrophic mutant, a mutant with a drastically different phenotype in axenic culture, and a mutant that did not produce EAS, respectively. Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C. purpurea, which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C. purpurea. It produces pharmacologically important ergot alkaloids (EAS), such as ergosine, ergocristine, ergotamine, and ergonovine, which have been used to treat migraine, hypertension, or to aid childbirth . Dis. DisdmaWpurpurea . Thus, we strain . These rin vitro assembled Cas9/gRNA RNP effectively stimulates HR in C. purpurea. Compared with the in vivo strategy in C. purpurea, this system has obvious advantages in terms of the process and editing efficiency (C. purpurea 3.1003 used here, it is difficult to establish a CRISPR/Cas9 system via an in vivo strategy, since neither codon-optimized Cas9 genes nor RNA polymerase III promoters can be produced without genomic information. The RNP-based method established here overcomes these difficulties via in vitro transcription of gRNA and purification of Cas9 protein. Because of the transient existence of the RNP complex in cells, in vitro assembled and delivered Cas9/gRNA-based genome editing reduces the risk of unnecessary gene targeting and avoids the integrated mutations that may result from gene-delivery strategies [In summary, our CRISPR/Cas9 system based on ficiency . Moreoverategies .Table 1CC. purpurea based on gRNA in vitro transcription. Target genes were efficiently disrupted by CRISPR/Cas9-mediated HR. Based on genomic information, the candidate genes involved in EAS pathway could be deleted efficiently using our developed method. With the advantage of counter selection using 5-FOA, ura5 could be utilized as the bidirectional selection marker for recyclable genome editing. These strategies rapidly achieved simultaneous or stepwise deletion of multiple candidate genes. This work will be useful for metabolic engineering and gene regulation of C. purpurea, as well as elucidating the EAS pathway.In conclusion, we developed a simple, efficient CRISPR/Cas9 system in Lu Yu: Conceptualization, Investigation, Writing – original draft. Meili Xiao: Conceptualization, Investigation, Writing – original draft. Zhihua Zhu: Investigation. Yinmei Wang: Investigation. Zhihua Zhou: Project administration, Conceptualization, Supervision. Pingping Wang: Conceptualization, Writing – review & editing, Supervision. Gen Zou: Conceptualization, Writing – review & editing, Supervision.The authors have no interests to declare.


Cryptococcus, a notorious pathogen since the advent of the HIV/AIDS-pandemic. A cryptococcal infection usually manifests itself as a pneumonia and/or meningitis. The genus includes ten species, most of them belonging to the C. gattii/C. neoformans species complexes, and some non-pathogenic species  [Naganishia albida, Naganishia diffluens and Papilliotrema laurentii) [Cryptococcosis is the collective heading of infections caused by members of the basidiomycetous yeast genus  luteus) . Previouurentii) , 2.Cryptococcus neoformans sensu stricto (previously C. neoformans variety grubii) is globally the major cause of systemic cryptococcosis among immunocompromised individuals. It is strongly associated with bird excreta, especially pigeon droppings [Cryptococcus deneoformans (formerly C. neoformans variety neoformans) is less common but remain a major cause of cryptococcosis in Europe [C. deneoformans × C. neoformans, which occurs predominantly in Mediterranean Europe [C. neoformans and C. deneoformans is that the latter is associated with skin-infections and more often seen in elderly patients [roppings . Cryptocn Europe . This apn Europe . Relevanpatients , 4.Cryptococcus gattii sensu lato (formerly C. neoformans variety gattii) became notorious due to the unprecedented outbreak on Vancouver Island . Until then, C. gattii was recognized as a pathogen exclusive to tropical and subtropical regions [C. gattii is –like C. neoformans– a species complex, it comprises six lineages of which five are recognized as species while the sixth needs to be named [C. gattii VGII-outbreak lineage’ is now called C. deuterogattii. The environmental niches are trees and plants, and it is globally distributed like its sibling C. gattii sensu stricto. As both species occur in the environment of temperate climate zones it is obvious that they cannot longer be regarded as strict (sub)tropical pathogens. The remaining three pathogenic species have a predilection for the immunocompromised host. C. bacillisporus and C. decagattii infections are mainly reported from the America’s, with some inexplicable cases outside these continents [Cryptococcus tetragattii is, like C. neoformans, a frequent cause of cryptococcal meningitis among HIV-positive patients from sub-Saharan African and the Indian subcontinent  species-level identification is of importance and could contribute to adequate treatment.PCR-fingerprinting, AFLP genotyping, microsatellite typing, multi-locus sequencing typing and whole genome sequencing are widely used to investigate the molecular epidemiology , 6–9 Fi. The bipC. gattii sensu lato infections need to be more aggressively treated than C. neoformans sensu lato, as the former produce cryptococcoma’s that are difficult to eradicate [Recommended treatment is a 2-weeks induction therapy with amphotericin B plus 5-fluorocytosine, followed by fluconazole as suppressive therapy . C. gattradicate . Antifunradicate .


Porphyromonas gingivalis, can directly increase the progression of atherosclerosis and the risk of cardiovascular disease. Observational studies found that patients with periodontal disease were at higher risk of developing atherosclerotic cardiovascular disease. Moreover, periodontal treatment leads to a reduction in cardiovascular risk therefore taking care of oral hygiene should be an important cardiovascular disease preventive measure.Periodontal disease and atherosclerotic cardiovascular disease are very common around the world. Coronary artery disease is the leading cause of death. The main factor involved in the pathogenesis of atherosclerosis is inflammation. Therefore, a number of studies have indicated that periodontal disease (causes chronic inflammation) is a risk factor for the progression of atherosclerosis. The presence of periodontal pathogens has been found in human atherosclerotic plaques. A number of pathomechanisms have been demonstrated, thanks to which periodontal pathogens, especially Tannerella forsythia Treponema denticola and Porphyromonas gingivalis responsible for severe clinical manifestation of PD. ASCVD and PD share a number of risk factors, and it is difficult to establish a causal relationship between these diseases. The influence of PD on ASCVD should be treated as a factor increasing the risk of atherosclerotic plaque destabilization and cardiovascular events. The results of observational studies indicate that PD significantly increases the risk of ASCVD. In interventional studies, PD treatment was found to have a beneficial effect in the prevention and control of ASCVD. This comprehensive review summarizes the current knowledge of the relationship between PD and ASCVD.Atherosclerotic cardiovascular disease (ASCVD) and periodontal disease (PD) are global health problems. High frequency of ASCVD is associated with the spread of many risk factors, including poor diet, sedentary lifestyle, diabetes, hyperlipidemia, obesity, smoking, hypertension, chronic kidney disease, hypertension, hyperhomocysteinemia, hyperuricemia, excessive stress, virus infection, genetic predisposition, etc. The pathogenesis of ASCVD is complex, while inflammation plays an important role. PD is a chronic, multifactorial inflammatory disease caused by dysbiosis of the oral microbiota, causing the progressive destruction of the bone and periodontal tissues surrounding the teeth. The main etiological factor of PD is the bacteria, which are capable of activating the immune response of the host inducing an inflammatory response. PD is associated with a mixed microbiota, with the evident predominance of anaerobic bacteria and microaerophilic. The “red complex” is an aggregate of three oral bacteria:  Atherosclerotic cardiovascular disease (ASCVD) and periodontal disease (PD) are global health problems. ASCVD is defined as coronary artery disease (CAD), cerebrovascular disease (stroke), or peripheral arterial disease (PAD) of atherosclerotic origin. ASCVD represents the number one cause of morbidity and mortality worldwide ,2. The ip = 0.005). It was found that the incidence of PD increased with age   may be important for microbial persistence within tissues [Periodontal pathogens are characterized by the ability to directly infect vascular endothelial cells (EC) [P. gingivalis into EC is positively associated with bacterial load and some virulent proteins such as gingipains, fimbriae and haemagglutinin A [P. gingivalis may be synergized by F. nucleatum and T. forsythia [P. gingivalis infection induced mitochondrial fragmentation, increased the mitochondrial reactive oxygen species levels, and decreased the mitochondrial membrane potential and adenosine triphosphate concentration in vascular EC. Researchers indicate that mitochondrial dysfunction may represent the mechanism by which P. gingivalis accelerates the progression of atherosclerosis [P. gingivalis), with human HSP60 on EC may subsequently result in endothelial dysfunction and the development of atherosclerosis [P. gingivalis infection also affects the permeability of the vascular endothelium. A study by Farrugia et al. assessed the effect of outer membrane vesicles (OMVs) produced by P. gingivalis on vascular endothelium. It was shown that human microvascular EC displayed an OMV-associated, gingipain-dependent decrease in cell surface levels of the intercellular adhesion molecule platelet endothelial cell adhesion molecule-1, leading to increased vascular endothelial permeability [P. gingivalis significantly reduces plasminogen activator inhibitor-1 levels in human EC. They found that plasminogen activator inhibitor-1 was proteolyzed by lysine-specific gingipain-K, leading to deregulation of endothelial homeostasis, thus contributing to permeabilization and dysfunction of the vascular endothelial barrier [P. gingivalis on EC was also demonstrated by Xie et al. These researchers found that P. gingivalis can impair endothelial integrity by inhibiting cell proliferation and inducing endothelial mesenchymal transformation and apoptosis of EC, which reduce the cell levels and cause the endothelium to lose its ability to repair itself. Moreover, the use of toll-like receptor (TLR) antagonist or nuclear factor-κB (NF-κB) signaling inhibitor significantly reduced the adverse effect of P. gingivalis on the EC, which indicates a significant role of TLR-NF-κB signaling pathway plays in the pathogenesis of EC damage by this periodontal pathogen [P. gingivalis not only leads to EC damage but also reduces the regenerative capacity of the vascular endothelium. A study by Kebschull et al. found that P. gingivalis mediated TLR-2 to increase peripheral endothelial progenitor cells (EPCs) counts and decrease EPCs pools in the bone marrow, thereby possibly reducing overall endothelial regeneration capacity [+/KDR+) compared to healthy subjects (p < 0.001) [P. gingivalis, the vicious circle effect develops. P. gingivalis stimulates the TLRs-NF-κB signaling pathway, which then leads, inter alia, to downregulation of brain and muscle Arnt-like 1 protein releases CLOCK, which phosphorylates p65 and further enhances NF-κB signaling, elevating oxidative stress and inflammatory response in human aortic EC [P. gingivalis up-regulates IL-1β and tumor necrosis factor α expression, which consequently causes cell death of brain EC through the ROS/NF-κB pathway [P. gingivalis is also characterized by the ability to directly increase the expression of adhesion molecules such as ICAM-1 in EC [P. gingivalis induced a pro-inflammatory and pro-oxidative EC response including up-regulation of tumor necrosis factor α, interleukin 6 and interleukin 8 as well as an altered expression of inducible and endothelial nitric oxide synthase at both mRNA and protein level. An increase of vascular cell adhesion protein 1 and ICAM-1 mRNA expression was also observed after P. gingivalis infection [A study by Li et al. showed that lectin-type oxidized LDL receptor 1 (LOX-1) plays a key role in human EC . Moreove tissues . Periodolls (EC) . The entutinin A . Moreoveorsythia ,63. The clerosis . A very clerosis . P. gingeability . Probableability . Importa barrier . The dirpathogen . P. gingcapacity . This wa< 0.001) . Vascula< 0.001) . It is wortic EC . In turn pathway . P. ging-1 in EC . Buguenonfection .P. gingivalis is the proliferation and migration of smooth muscle cells (SMCs). The study by Cao et al. found that gingipains can promote phenotypic transformation and proliferation of rat SMCs [P. gingivalis-mediated proliferation of human aortic SMCs [P. gingivalis can promote neointimal formation in mice [P. gingivalis infection leads to vascular calcification [Another stage of atherogenesis that may be potentiated by rat SMCs . Moreovetic SMCs . In turn in mice . The resfication ,81.P. gingivalis directly promotes the formation of foam cells. It has been suggested that lipopolysaccharides (LPS) P. gingivalis leads to acetyl-coenzyme A acetyltransferase 1 up-regulation [P. gingivalis led, via heme oxygenase-1, to upregulation of scavenger receptor class B member 3 and down-regulation of ATP binding cassette subfamily A member 1 protein, which also favored the accumulation of cholesterol in macrophages [P. gingivalis enhanced expression of macrophage migration inhibitory factors in the EC. Increasing the expression of macrophage migration inhibitory factor stimulates macrophages to secrete pro-inflammatory cytokines, prolongs the survival time of macrophages and thus maintains inflammation [It should also be mentioned that gulation . Moreoverophages . Moreoveammation . macrophammation .P. gingivalis can also destabilize atherosclerotic plaque. In the study by Mubarokah et al., it was found that P. gingivalis induced macrophage to secrete matrix metallopeptidase 9 that led to fragmentation of vascular type IV collagen. This mechanism plays an important role in pathogenesis of atherosclerotic plaque rupture [ rupture .P. gingivalis infection led to the activation of platelets and their aggregation [P. gingivalis infection increased the expression of P-selectin and increased the binding of fibrinogen to platelets [An important factor involved in atherothrombosis is the activation and aggregation of platelets. Experimental and clinical studies showed that regation ,88,89. Platelets .Other periodontal pathogens are also characterized by properties favoring the progression of atherosclerosis ,91,92.VAMP3 and VAMP8) [In addition, there are common genetic factors leading to PD and ASCVD. A significant role is indicated as genetic variant at vesicle-associated membrane protein gene (d VAMP8) ,94.P. gingivalis, are directly involved in all major stages of atherosclerotic lesion formation, progression and instability [Thus, there is evidence that periodontal pathogens, mainly tability .p = 0.004) [The influence of the presence of PD on the risk of ASCVD has been assessed in a number of studies. A meta-analysis of 32 studies by Larvin et al. summarized the effect of PD on the risk of various ASCVDs. It was shown that the occurrence of PD was associated with a 23% increase in the risk of developing ASCVD . Subgroup analysis showed that PD increased the risk of ASCVD more in men than in women . The risk of ASCVD was found to be related to the severity of PD . Among all types of ASCVD, the risk of stroke was highest , the risk of CAD was also increased  . In turn= 0.004) .p. gingivalis and anti-A. actinomycetemcomitans) and CAD, with pooled OR of 1.23  and 1.25 , respectively [p < 0.001). Subgroup analysis showed that PD significantly increased the occurrence of lower extremity arterial disease and carotid artery disease  [p < 0.001). This risk depended on the severity of PD [p = 0.006 and OR = 1.5; 95% CI: 1.2–1.9, p = 0.001, respectively) [In meta-analysis by Joshi et al. was assessed the relationship between the occurrence of serum antibody response against periodontal bacteria and risk of CAD. It showed a significant association between elevated serum IgG antibody responses (anti-ectively . Moreoveectively . In a me< 0.001) . Zeng etty of PD . In the ctively) . An intectively) . It is actively) . In a stctively) . From a ctively) .P. melaninogenica, P. intermedia, P. nigrescens, and P. gingivalis) and the risk of cardiovascular disease mortality [p < 0.001) [In a study by Kotronia et al. involving 5222 elderly subjects who were followed for an average of 9–15 years, it was shown that the presence of PD increased the risk of cardiovascular mortality by 49%  . Similarortality . The importality . Moreoveortality . The rel< 0.001) .Thus, observational studies and their meta-analyzes have shown that PD increases the risk of ASCVD. At this point, however, it is worth noting that the results of observational studies only allow for the formulation of a research hypothesis, but cannot prove a cause-and-effect relationship. The cause and effect relationship between PD and ASCVD is currently under discussion. In Mendelian randomization studies by Zhou et al. did not provide evidence for dental caries and PD as the causes of ASCVD . Moreovep < 0.001) and leukocyte values (p < 0.001) [A primary goal of periodontal therapy (PT) is to reduce the burden of pathogenic bacteria and thereby reduce the potential for progressive inflammation and recurrence of PD . A syste< 0.001) . An earl< 0.001) . A numbe1C (p < 0.01) and CRP (p < 0.01) in patients with type 2 diabetes mellitus (T2DM) [1C was found [p = 0.001) in patients with T2DM [p = 0.001) and triglycerides (p < 0.00001) [p < 0.00001) and diastolic blood pressure  among prehypertensive/hypertensive patients. Moreover, significant reductions in CRP and improvement of endothelial function were found after PT in these patients [Thus, a number of clinical studies indicate that PT leads to a significant reduction ASCVD risk factors and biomarkers. It is worth citing the results of several more recent meta-analyzes that summarize the available data on the influence of PT on the control of ASCVD risk factors. A meta-analysis of nine randomized clinical trials (RCTs) by Baeza et al. showed that PT led to a significant reduction in HbAs (T2DM) . Similaras found . Moreoveith T2DM . The ben0.00001) . In a vepatients .P. gingivalis and citrulline, which may contribute to better control of this disease [It should also be mentioned that PT improves the control of other diseases that are a risk factor for ASCVD. For example, a meta-analysis by da Silva et al. showed that PD treatment, by increasing glomerular filtration rate, may improve renal function in patients with chronic kidney disease . Moreove disease . In turn disease .p = 0.007 vs. to good responders) [It is important to control the effectiveness of PT because, as demonstrated by Holmlund et al. patients who did not respond well to PT had an increased risk for future ASCVD  .The most important drugs in the treatment of ASCVD are statins with many pleiotropic effects . From thTo sum up, PT in many clinical trials and their meta-analyzes had a very positive effect on the control of the most important risk factors of ASCVD. Moreover, PT may be beneficial in controlling other diseases that increase cardiovascular risk. From the clinical point of view, it is also important that statins reduce periodontal inflammation.The authors of the paper, after analyzing the cited publications, came to the conclusion that at present it is not possible to unequivocally define PD as an independent factor generating ASCVD. This is due to the poor spectrum of biochemical inflammatory markers selectively referring to the oral cavity, and clinical periodontal examination remains the most reliable and unambiguous.P. gingivalis, can directly increase the progression of atherosclerosis at all its stages. The pathophysiological rationale is reflected in observational studies, which found that PD significantly increases the risk of various ASCVDs. Genetic analyzes do not confirm a cause-and-effect relationship between PD and ASCVD. The results of randomized clinical trials and their meta-analyzes stand in opposition to them, which clearly indicate that PD treatment significantly improves the control of the most important risk factors for ASCVD, contributing to the improvement of cardiovascular health. Taking all this into account, in the latest 2021 guidelines of the European Society of Cardiology (ESC) Guidelines on cardiovascular disease prevention in clinical practice, the inclusion of periodontal infection in the chapter “gap of evidence” indicates the need for further research [Experimental studies show that periodontal pathogens, especially research .H. pylori, pneumonia pathogens, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), cytomegalovirus (CMV), herpes simplex virus (HSV), hepatitis-C virus (HCV) and HIV [It is worth noting that not only periodontal pathogens contribute to the development of atherosclerosis, but also infections with such pathogens as:  and HIV .p < 0.001 and OR = 0.93; 95% CI: 0.91–0.96, p < 0.001, respectively) [There is no doubt that taking care of oral health should be the basis of ASCVD prevention. For this purpose, avoiding risk factors for PD should be recommended . An impoctively) . These dctively) . Patientctively) . Moreovectively) . Therefoctively) ,160. A vctively) .Taking into account that lifestyle modifications are the cornerstones in the prevention and treatment of ASCVD, in the assessment of cardiovascular risk, PD should be sought, considered and prevented, and, if necessary, periodontal treatment recommended.


Vulpia myuros in Denmark. However, farmers often report variable efficacy of these herbicides on V. myuros compared to other grass weeds. Dose–response experiments were conducted to evaluate the performance of prosulfocarb and mesosulfuron + iodosulfuron on V. myuros. Prosulfocarb was sprayed at different plant growth stages to study the influence of plant growth stage on the performance of prosulfocarb on V. myuros in comparison with the more susceptible grass weed species Apera spica-venti. Doses causing 50% reduction in response variable (ED50) were estimated from the dose–response analysis. The ED50 values revealed a higher tolerance of V. myuros to prosulfocarb and mesosulfuron + iodosulfuron than A. spica-venti. The relative difference in the effectiveness of prosulfocarb between V. myuros and A. spica-venti was constant among plant growth stages studied. The highest levels of V. myuros control were achieved when prosulfocarb was sprayed pre-emergence (BBCH 00), while the control substantially declined at later growth stages. The results from the current study document the tolerance of V. myuros to prosulfocarb and mesosulfuron + iodosulfuron and highlight the importance of optimization of prosulfocarb spray timing for achieving maximum control of V. myuros.Due to natural tolerance to most widely used herbicides for grass weed control, prosulfocarb as pre-emergence or early post-emergence herbicide and mesosulfuron + iodosulfuron as post-emergence herbicide are the mainstays of any chemical control program for  Vulpia myuros (L.) C.C. Gmel.) is considered a problematic grass weed in parts of Northern Europe including Denmark, where its recent increase in abundance is due to a high frequency of winter cereals in crop rotations and wide adoption of no tillage practices [V. myuros has been reported at densities of several hundred plants/m2 in winter cereals in Denmark [V. myuros also has been reported in other EU countries, for example, in United Kingdom, France, Germany, Romania, and Switzerland, however it is still considered a minor weed in those countries [Rattail fescue  and acetolactate synthase (ALS) inhibitors are the most widely used herbicides to control grass weeds in winter cereals. However, V. myuros is naturally tolerant to ACCase inhibitors, and many ALS inhibitor herbicides are not highly effective against this weed species [V. myuros to low spay retention on the narrow leaf blades of this species [ species ,8,9. Pre species ,11.V. myuros are limited compared with other grass weeds. In Denmark, farmers rely on residual herbicides such as prosulfocarb applied pre-emergence or early post-emergence and post-emergence application of mesosulfuron + iodosulfuron in autumn and/or spring for the control of V. myuros in winter cereals. However, some reports suggest that the typically used rates of prosulfocarb and mesosulfuron + iodosulfuron provide more inconsistent control of V. myuros than of other grasses [V. myuros. For example, previous studies have shown that some level of V. myuros control could be achieved if prosulfocarb was applied pre-emergence, but effectiveness tended to decrease rapidly when sprayed at later growth stages [V. myuros is limited. To optimize herbicide application timings, information on prosulfocarb effectiveness on V. myuros at different growth stages is required. Reports suggest that V. myuros can tolerate the recommended rates of mesosulfuron + iodosulfuron better than other grass weed species such as A. spica-venti [V. myuros against mesosulfuron + iodosulfuron. The objective of this study was to assess the performance of prosulfocarb and mesosulfuron + iodosulfuron and evaluate the impact of plant growth stages on the performance of prosulfocarb on V. myuros. Apera spica-venti was used as a susceptible control species in the study.Due to natural tolerance to most widely used herbicides, the options for chemical control of  grasses ,8,12. Thh stages . HoweverV. myuros were collected from a non-agricultural area at Flakkebjerg Research Centre, Denmark, where there has been no known history of herbicide application. Seeds of susceptible populations of A. spica-venti originated from several locations in Denmark and were mixed together in the same proportion to create a meta-population [Seeds of pulation .V. myuros and A. spica-venti were sown in 1 L pots filled with field soil (sandy loam). The pots were placed in an unheated glasshouse or on outdoor tables depending on the time of the year when the experiment was conducted. Groups of pots were sown on different dates to ensure different plant growth stages could be sprayed at the same time. After seedling emergence, the number of plants per pot were thinned to a pre-set number. Pots treated pre-emergence were not thinned. Pre-emergence treatments were carried out one day after sowing. Plants were treated with a range of prosulfocarb doses  . Herbicide application was carried out in a spray cabinet equipped with a boom fitted with two flat-fan nozzle (HARDI ISO F-110-02). The nozzles were operated at a pressure of 300 kilopascal and velocity of a 5.2 km h−1 delivering spray volume of a 152 L ha−1. A few hours after application of prosulfocarb, 30 mL of Milli Q water was applied to the soil surface of each pot to ensure a uniform distribution of prosulfocarb in the upper soil layer. The plants were sprayed at BBCH  00 to 13 (Twenty seeds of 00 to 13 . The pro00 to 13 . The fou00 to 13 . There ww/v) was boiled in a microwave and allowed to cool to 35–40 °C. A stock solution of mesosulfuron-methyl + iodosulfuron-methyl  in the commercial form was prepared. Using this stock solution, different concentrations of mesosulfuron-methyl + iodosulfuron-methyl were prepared. Mesosulfuron-methyl + iodosulfuron-methyl assay concentrations were 0, 0.009, 0.018, 0.036, 0.072, and 0.144 mg/L. The herbicide concentrations were added to agar medium using micropipette. Twenty milliliters of agar medium plus herbicide solution were poured into quadratic plastic dishes (245 mm × 245 mm). Fifty seeds of V. myuros and A. spica-venti were placed in each dish. The dishes were covered with lids and placed in the greenhouse. The experiment was arranged in a completely randomized design with three replicates. Coleoptile and radicle lengths were measured 10–15 days after treatment.The plant agar medium (Duchefa) (10 g/L) . The plastic trays were placed in an unheated glasshouse on the table with automatic watering system. The plant agar media and herbicide concentrations were prepared as described above in the seed germination assay. Seedlings of V. myuros and A. spica-venti at the 2–3 leaf stage (BBCH 12–13) were harvested from the plastic trays and washed under tap water to remove any particles of potting mixture from roots. The seedling tips (growing points of leaves and roots) were excised. Roots of seedlings were transplanted into plastic dishes (245 mm × 245 mm) containing a plant agar media using forceps. The plastic dishes were covered and placed in the heated glasshouse. The experiment was arranged in a completely randomized design with ten plant seedlings in each dish and three replications. Herbicide efficacy was recorded as seedling survival by visually assessing the new roots and shoots 15 days after transplanting.Plants of Y is the response variable ; c and d denote the lower and upper asymptote, respectively; e is the dose causing 50% reduction in response variable (ED50); and b is the slope of the curve. The model was checked by residual plots and with lack of fit test (p > 0.05). If the value of c was zero, the four-parametric model was reduced to a three-parametric one as:A nonlinear log-logistic model was used to analyze the prosulfocarb and mesosulfuron + iodosulfuron dose–response studies :(1)Y=d−chttp://R-project.org) with drc package [e (ED50) was compared between the species using post hoc t-tests. Tolerance index (TI) was estimated as V. myuros to A. spica-venti ED50 ratios to compare the responses from a population of V. myuros with those of A. spica-venti.The analyses were performed using R version 3.6.1 , BBCH 11, BBCH 12, and BBCH 13 stages, respectively, in Experiment 1. V. myuros exhibited 2.8-, 3.5-, 3.2-, and 1.4-fold tolerance at the BBCH 00, BBCH 10, BBCH 11, and BBCH 12 stages, respectively, in Experiment 2 , i.e., EDca-venti . TI’s va studied . V. myurriment 2 . The TI riment 3 . In ExpeV. myuros was lowest at BBCH 00 stage and, with a few exceptions, tended to increase at more advanced growth stages. Generally, a similar trend was observed for A. spica-venti. For instance, the estimated ED50 value was lower at the BBCH 00 stage than at BBCH 11, BBCH 10, and BBCH 13 in Experiments 1–3, respectively. In Experiment 4, the plants were sprayed only at two growth stages (BBCH 11 and BBCH 13), where the rates of prosulfocarb for 50% shoot biomass reduction of V. myuros was lower at BBCH 11 than at BBCH 13, although the difference was non-significant. For A. spica-venti, the estimated ED50 values were significantly lower at BBCH 11 than at BBCH 13 in Experiment 4. In general, moving towards more advanced growth stages, higher ED50 values were estimated than at early growth stages, and these results are consistent in the two species and four trials.Application rates of prosulfocarb required to suppress 50% shoot biomass of A. spica-venti than for V. myuros  higher than A. spica-venti for coleoptile and radicle growth, respectively.Coleoptile and radicle lengths were inhibited by herbicide treatments for both grass weed species, but the inhibition was significantly higher for . myuros A,B. Estiectively . The ED5V. myuros and A. spica-venti, where individuals from both V. myuros and A. spica-venti developed new roots and shoots. New roots and shoots of both species were suppressed more in dishes with herbicide compared to untreated controls, but the suppression of new roots and shoots was significantly higher in A. spica-venti than in V. myuros  more tolerant to mesosulfuron + iodosulfuron than A. spica-venti . Similar to our results, Hull et al., [The results show that weed growth stage at the time of spraying had a significant effect on plant sensitivity to prosulfocarb. Pre-emergence application of prosulfocarb at the rate of 800 g/ha resulted, on average, in an 80–90% biomass reduction of 50 doses required to control V. myuros and A. spica-venti were lower than the typically used field doses. Pot-grown weed plants are generally more susceptible to herbicides than plants grown in the field and, interestingly, results from the four experiments are consistent, indicating lower susceptibility of V. myuros to prosulfocarb, regardless of plant growth stage. Farmers often experience failures when applying prosulfocarb for the control of V. myuros under field conditions, and field observations have shown that significantly higher doses of prosulfocarb are needed for effective control. In contrast, prosulfocarb provided effective control of A. spica-venti with ED50 values of less than 100 g ha−1 at pre-emergence (BBCH 00) and BBCH 10 and less than 200 g ha−1 at BBCH 12. However, beyond BBCH 12, control of A. spica-venti with prosulfocarb tended to decline. Our findings are in line with those of Adamczewski et al. [A. spica-venti with recommended rates of prosulfocarb when applied pre-emergence under field conditions.In the current study, the estimated EDi et al.  who alsoV. myuros and A. spica-venti were suppressed by increasing doses of mesosulfuron-methyl + iodosulfuron-methyl. Abdurruhman et al. [Avena sterilis L.) populations treated with mesosulfuron-methyl + iodosulfuron-methyl in agar media. Previously, many studies have shown that the agar-based assay can be used to detect resistance in grass weeds such as Sorghum halepense, Alopecurus myosuroides, and Lolium rigidum [50 values for V. myuros were 3–4.4 times higher than for A. spica-venti. The results confirm that V. myuros is tolerant to mesosulfuron-methyl + iodosulfuron-methyl, as it is to other herbicides. Other researchers found that mesosulfuron-methyl + iodosulfuron-methyl is not highly effective in controlling V. myuros, and often higher than the maximum recommended rates were needed for satisfactory control of this weed species [V. myuros with post-emergence herbicides can be attributed to low spray retention due to its narrow and erect leaves [V. myuros is naturally tolerant to mesosulfuron-methyl + iodosulfuron-methyl and that differences in spray deposition can only partly be the cause for the observed differences in susceptibility. This assumption is further supported by the finding of Akhter, [V. myuros. Yu et al. [V. bromoides, proved its tolerance to ACCase and several ALS inhibitors and concluded that an insensitive ACCase and enhanced metabolism were the likely mechanisms of tolerance to the two modes of action. Here, we document for first the time that V. myuros, similar to V. bromoides, is naturally tolerant to mesosulfuron-methyl + iodosulfuron-methyl compared to a population of A. spica-venti. In the current study, we did not attempt to study the V. myuros tolerance mechanism against mesosulfuron-methyl + iodosulfuron-methyl, but the available information shows that the response of V. myuros and V. bromoides to various herbicides are similar. Hence, it can be speculated that the mechanism of tolerance in V. myuros to ALS inhibiting herbicides including mesosulfuron-methyl + iodosulfuron-methyl might be due the same mechanism as found for V. bromoides [The agar-based assay showed that the lengths of coleoptiles and radicles and seedling survival of n et al.  reported rigidum ,21,22. T rigidum ,23. The  species . It is ot leaves ,11. The  Akhter, , who shou et al. , studyinV. myuros control but prosulfocarb has a narrow window of application as the activity declined significantly at more advanced growth stages. Moreover, the soil half-life of some residual herbicides is short, for example, the half-life of prosulfocarb is around 10 days [V. myuros plants, particularly in years with mild winters and high dormancy. Changing from a single application to sequential applications of herbicides can overcome this shortcoming and provide more effective control of other grass weeds. Nonetheless, the results from the current study indirectly indicate that V. myuros control with sequential treatments of herbicides, i.e., combining a pre-emergence application of prosulfocarb with a post-emergence treatment of mesosulfuron-methyl + iodosulfuron-methyl, would be less than acceptable, especially on dense stands. In the absence of effective chemical control methods, V. myuros, due to its profound growth behavior, can quickly take over the field and cause significant grain yield losses [V. myuros will require other inputs in addition to herbicides. Integrated weed management strategies with several preventive and cultural control measures such as tillage, crop rotation, delayed crop sowing, and crop competition applied in combination with available herbicides can facilitate sustainable weed management of V. myuros.According to the current study, pre-emergence application of prosulfocarb can provide some level of  10 days , and, thd losses . It appeV. myuros is tolerant to prosulfocarb and mesosulfuron-methyl + iodosulfuron-methyl compared with A. spica-venti, and the relative differences in the effectiveness of prosulfocarb between V. myuros and A. spica-venti tend to remain constant among the plant growth stages studied. The results further show that the highest level of V. myuros control with prosulfocarb is achieved if the herbicide is applied pre-emergence or on germinating seedlings, whereas control tends to decline substantially beyond these growth stages. The information on the level of V. myuros tolerance to herbicides documented here will help for planning a proper chemical weed control program as part of integrated weed management strategies against this species.The current study revealed that