Interactions between pectin, starch and linoleic acid and their effects on starch structure, digestion and release properties.

This study aimed at gaining insight into the mechanism of interactions between pectin (PE), starch and unsaturated fatty acids (UFAs) in relation to structure, in vitro digestibility and release properties of starch. Due to the barrier and encapsulation effects of PE, the complexing behavior of potato starch (PtS) with linoleic acid (LOA) was enhanced, which increased the complexing index, the compactness of network structure, short-range ordered structure and relative crystallinity of PtS-LOA-PE films. These structural changes resulted in the increases of slowly digestible starch and resistant starch and in the decreases of first-order rate coefficient in PtS-LOA-PE films. Besides, the in vitro release results also showed that the release properties of PtS-LOA could be controlled by the PE addition with the decreases in LOA release rate and increase in LOA bioavailability under simulated gastrointestinal conditions. Notably, at different PtS-LOA:PE ratios, the PtS-LOA-PE film with the PtS-LOA:PE ratio of 5:1 showed the better complexing degree, structural order, anti-digestibility and colon-targeted release properties than other PtS-LOA-PE films. These results indicated that PE influenced the release properties of the PtS-LOA-PE films, which was closely related to their complexing degree, structural order, and digestibility. This study provided new insights into the design of resistant films for delivery of UFAs to colon.

Effects of OSA-starch-fatty acid interactions on the structural, digestibility and release characteristics of high amylose corn starch.

This study investigated the effects of octenyl succinic anhydride (OSA)-starch-fatty acid (FA) interactions on the structural, digestibility and release characteristics of high amylose corn starch (HAS). FTIR and XRD analysis showed that the hydrophobic interaction between HAS and FA promoted the covalent binding between OSA and HAS. With the increasing of the FA chain length, the complex index, degree of substitution, R1047/1022 and relative crystallinity of OSA-HAS-FA increased first and then decreased, whereas the first-order rate coefficient and percentage of digested in infinite time showed an opposite trend. Structural changes and the molecular interactions of OSA-HAS-FA with 12­carbon FA resulted in highest resistant starch content (45.43%) and encapsulation efficiency of curcumin (Cur) (47.98%). In vitro release test revealed that Cur could be gradually released from OSA-HAS-FA in simulated gastric, intestinal and colonic fluids. Results provided novel insights into HAS-FA complex grafted with OSA as carrier for colon-specific of functional materials.

Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs.

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs. Highlights: Measurement of >30,000 human 5' UTRs reveals a 250-fold range of translation outputSystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elementsN1-methylpseudouridine alters translation initiation in a sequence-specific mannerOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines.

Assessment of sedation by automated pupillometry in critically ill patients: a prospective observational study.

BACKGROUND: Quantitative measurement of pupil change has not been assessed against the Richmond Agitation and Sedation Scale (RASS) and spectral edge frequency (SEF) during sedation. The aim of this study was to evaluate pupillometry against these measures in sedated critically ill adult patients. METHODS: In ventilated and sedated patients, pupillary variables were measured by automated pupillometry at each RASS level from -5 to 0 after discontinuation of hypnotics, while processed electroencephalogram variables were displayed continuously and SEF was recorded at each RASS level. Correlations were made between percentage pupillary light reflex (%PLR) and RASS, and between %PLR and SEF. The ability of %PLR to differentiate light sedation (RASS ≥-2), moderate (RASS =-3), and deep sedation (RASS ≤-4) was assessed by areas under receiver operating characteristic (ROC) curves. RESULTS: A total of 163 paired measurements were recorded in 38 patients. With decreasing sedation depth, median %PLR increased progressively from 20% (interquartile range 17-25%) to 36% (interquartile range 33-40%) (P<0.001). Strong correlations were found between %PLR and RASS (Rho=0.635) and between %PLR and SEF (R=0.641). Area under the curve (AUC) of 0.87 with a %PLR threshold of 28% differentiated moderate/light sedation from deep sedation with sensitivity of 83% and specificity of 83%. An AUC of 0.82 with a threshold of 31% distinguished light sedation from moderate/deep sedation with a sensitivity of 81% and a specificity of 75%. CONCLUSIONS: Quantitative assessment of %PLR correlates with other indicators of sedation depth in critically ill patients.

Characterization, in vitro digestibility and release properties of starch-linoleic acid-sodium alginate composite film.

This study aimed to improve the complexing degree, digestibility and controlled release properties of the potato starch (PS)-linoleic acid (LA) complexes by encapsulating PS-LA complexes to sodium alginate (AG) beads. The results revealed that AG had a positive effect on the complexing index, R1047/1022 values, relative crystallinity, enthalpy and morphological structure of PS-LA-AG films, especially for PS-LA-AG film with the PS-LA: AG of 5:1. The in vitro digestion and hydrolysis kinetic analysis indicated that AG addition reduced the digestibility of PS-LA-AG films to a higher slowly digestible starch content and resistant starch content and a lower equilibrium hydrolysis percentage and kinetic constant. Furthermore, in vivo release study of PS-LA-AG films indicated a restrained release in simulated gastrointestinal conditions. Consequently, the results indicated that AG addition significantly improved the inclusion efficiency for the complex formation between PS and LA, which was beneficial for the design of resistant films to entrap and control release of unsaturated fatty.

Structural basis for translation inhibition by MERS-CoV Nsp1 reveals a conserved mechanism for betacoronaviruses.

All betacoronaviruses (ß-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the ß-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across ß-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict ß-CoVs.

HIV-1 Gag Binds the Multi-Aminoacyl-tRNA Synthetase Complex via the EPRS Subunit.

Host factor tRNAs facilitate the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 uses human tRNALys3 as the primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by matrix (MA) domain-tRNA interactions. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytosol and consists of eight aminoacyl-tRNA synthetases (ARSs) and three other cellular proteins. Proteomic studies to identify HIV-host interactions have identified the MSC as part of the HIV-1 Gag and MA interactomes. Here, we confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC, mapped the primary interaction site to the linker domain of bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA-EPRS interaction was RNA dependent. MA mutations that significantly reduced the EPRS interaction reduced viral infectivity and mapped to MA residues that also interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments did not affect susceptibility to HIV-1 infection, and knockdown of EPRS reduced both a control reporter gene and HIV-1 protein translation. EPRS knockdown resulted in decreased progeny virion production, but the decrease could not be attributed to selective effects on virus gene expression, and the specific infectivity of the virions remained unchanged. While the precise function of the Gag-EPRS interaction remains uncertain, we discuss possible effects of the interaction on either virus or host activities.

Aminoacylation-defective bi-allelic mutations in human EPRS1 associated with psychomotor developmental delay, epilepsy, and deafness.

Aminoacyl-tRNA synthetases are enzymes that ensure accurate protein synthesis. Variants of the dual-functional cytoplasmic human glutamyl-prolyl-tRNA synthetase, EPRS1, have been associated with leukodystrophy, diabetes and bone disease. Here, we report compound heterozygous variants in EPRS1 in a 4-year-old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.

Assessment of Combination of Automated Pupillometry and Heart Rate Variability to Detect Driving Fatigue.

Objectives: Approximately 20~30% of all traffic accidents are caused by fatigue driving. However, limited practicability remains a barrier for the real application of available techniques to detect driving fatigue. Use of pupillary light reflex (PLR) may be potentially effective for driving fatigue detection. Methods: A 90 min monotonous simulated driving task was utilized to induce driving fatigue. During the task, PLR measurements were performed at baseline and at an interval of 30 min. Subjective rating scales, heart rate variability (HRV) were monitored simultaneously. Results: Thirty-two healthy volunteers in China participated in our study. Based on the results of subjective evaluation and behavioral performances, driving fatigue was verified to be successfully induced by a simulated driving task. Significant variations of PLR and HRV parameters were observed, which also showed significant relevance with the change in Karolinska Sleepiness Scale at several timepoints (|r| = 0.55 ~ 0.72, P < 0.001). Furthermore, PLR variations had excellent ability to detect driving fatigue with high sensitivity and specificity, of which maximum constriction velocity variations achieved a sensitivity of 85.00% and specificity of 72.34% for driving fatigue detection, vs. 82.50 and 78.72% with a combination of HRV variations, a nonsignificant difference (AUC = 0.835, 0.872, P > 0.05). Conclusions: Pupillary light reflex variation may be a potential indicator in the detection of driving fatigue, achieving a comparative performance compared with the combination with heart rate variability. Further work may be involved in developing a commercialized driving fatigue detection system based on pupillary parameters.

A-DA'D-A fused-ring small molecule-based nanoparticles for combined photothermal and photodynamic therapy of cancer.

New nanoparticles (Y6 NPs) based on the A-DA'D-A fused-ring conjugated small molecule Y6 have been prepared for the combined photothermal and photodynamic therapy of cancer. Y6 NPs show excellent light absorption from 300 to 900 nm, a good photothermal conversion efficiency of 57% and reactive oxygen species generation capability. The high photothermal conversion ability and superior photodynamic activity of Y6 NPs endow them with great potential for cancer therapy.

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response.

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNAGlu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.

Bioguided isolation, identification and bioactivity evaluation of anti-MRSA constituents from Morus alba Linn.

ETHNOPHARMACOLOGY RELEVANCE: The root bark of Morus alba Linn. (M. alba), a traditional folk medicine, has been documented in the Chinese Pharmacopoeia, which has been widely used for asthma, fever, pneumonia, edema, vomit, colitis, bronchitis and keratitis diseases. Some of the diseases may be related to respiratory, digestive, urinary tract infections. Although Diels-Alder adducts (DAAs), flavonoids, 2-arylbenzofurans and stilbene compounds have been isolated from the root bark of M. alba, few compounds are reported for their antimicrobial efficacy in vivo and the mechanism. AIM OF THE STUDY: The aim of the study was to isolate and identify compounds of the root bark of M. alba in view of their anti-MRSA bioactivity, evaluate the anti-MRSA bioactivity of compounds and 60% ethanol elution (MA-6) in vitro and in vivo, and explore preliminary antibacterial mechanism in order to provide natural resources against MRSA infection. MATERIALS AND METHODS: Systematic phytochemical investigations were carried out according to the thin layer chromatography (TLC) of the active fraction MA-6 to find more anti-MRSA ingredients. The compounds of the root bark of M. alba were separated by column chromatography and identified by LC-MS/MS and NMR spectroscopy. The anti-MRSA efficacy of the active ingredients were evaluated by broth microdilution method and a murine infection model. The mode of action of compounds was explored by time-kill curve and post-contact effect. The preliminary mechanism of compounds against MRSA was explored by drug efflux pumps and bacterial biofilms. RESULTS: Chemical isolation resulted in twenty-nine known compounds, most with one or more geranyl and prenyl units exhibited superior anti-MRSA bioactivity, with MIC values of 2-16 µg/mL. In addition, the mode of action indicated that compounds presented persistent antimicrobial effect, which also produced concentration-dependent and time-dependent killing activity or property. Preliminary mechanism showed that the compound kuwanon O (29) damaged the bacterial cell membranes, leading to the accumulation of antibiotics inside bacterial cells, moreover, MA-6 and kuwanon O (29) inhibited the efflux of drugs by combining with methicillin or ethidium bromide (EtBr), resulting in the MICs of EtBr and methicillin were obviously decreased three-fold. The anti-MRSA efficacy in vivo indicated that the active fraction MA-6 could reduce bacteria in spleen, liver, kidney and mortality of acutely infectious mice, which was better than the positive drug berberine chloride. CONCLUSION: Experimental investigation showed that the MA-6 and compound 29 have promising bioactivity against MRSA in vitro and in vivo, which might be used as a potential source of new antibacterial medicine or a potential efflux pump inhibitor against MRSA infection.

Bioactivity Ingredients of Chaenomeles speciosa against Microbes: Characterization by LC-MS and Activity Evaluation.

Chaenomeles speciosa (Sweet) Nakai is a dual-purpose Chinese herbal medicine and functional food favored by minorities in Southwest China, and its fruits are used for the treatment of dyspepsia, dysentery, enteritis, and rheumatism inflammation. Some diseases may be related to microbial infection; however, it is not known how the fruits possess antimicrobial activity. We evaluated the antimicrobial bioctivity of different evaluation extracts of C. speciosa fruits by in vitro and in vivo with colony-forming unit assays, and the strongest bioactive-guided fraction was selected for column chromatography (CC), UHPLC-QTOF-MS/MS, and NMR spectroscopy to confirm the chemical constituents. The most possible antimicrobial mechanism of C. speciosa fruits was explored by metabolomics approach, fluorescence microscopy imaging, and scanning electron microscopy (SEM). Thirty compounds, which were major characteristic ions of the bioactive fraction, were determined precisely. The bioactive fraction could inhibit 18 pathogenic microorganisms, significantly reduced, especially drug-resistant bacteria, compared to ampicillin sodium salt, fluconazole, and berberine chloride form; and the minimum inhibitory concentration (MIC) or minimum fungicidal concentration (MFC) values were in the range of 0.1-1 mg/mL. The compounds 2'-methoxyaucuparin (1) and oleanolic acid (20) not only have antibacterial activity but also may have synergistic effects. Further, the bioactive fraction might inhibit the biofilm formation, enhance immunity, and restore bacterial infection damage in vitro and in vivo to kill microorganisms. The data indicated that C. speciosa fruits' major bioactive fraction enriched with triterpenes, flavonoids, and phenolics could be developed as a functional supplement for individuals to prevent and treat microbial infection.

Recent advances in the development of metal-organic framework-based gas-releasing nanoplatforms for synergistic cancer therapy.

Gas therapy as a burgeoning and promising research field has attracted considerable attention in biomedicine due to its high therapeutic efficacy, biocompatibility, and biosafety. However, the lack of tumor site accumulation and controlled release of therapeutic gas molecules limited the therapeutic efficacy. Therefore, the development of gas-releasing nanoplatforms to realize tumor targeting and controllable release is highly desired. The structural diversity and tailorability and ultrahigh surface area make metal-organic frameworks (MOFs) find potential applications in the delivery and release of gas or gas releasing molecules (GRMs). In this Frontier article, we provide an overview of the recent developments achieved in gas-involving cancer therapy using MOFs or MOF-based materials. The main emphasis is focused on the design of multifunctional MOF-based nanoplatforms for the delivery and release of therapeutic gas molecules, and emphasizing their synergistic mechanism against tumor. Moreover, the challenges, future trends, and prospects of gas-related cancer therapy are also discussed.

Neothalfine, a potent natural anti-tumor agent against metastatic colorectal cancer and its primary mechanism.

Neothalfine is a natural bisbenzylisoquinoline alkaloid with the abundant resource in medicinal plants and has not been reported its anti-tumor efficacy. In the present study, the anti-tumor efficacy was investigated and it showed broad-spectrum activity against several cancer cell lines, especially metastatic colorectal cancer (HCT116, SW620, T84) with the IC50 values of 7.2, 5.9, 8.2 nM, respectively, roughly equal to well-known anti-tumor agent docetaxel (4.0, 4.7, 2.7 nM) and nearly 1000 folds than CPT-11 (4.4, 5.1, 6.9 µM). Furthermore, neothalfine inhibited colorectal cell proliferation by resulting in cell cycle arrest at the G2/M phase and induced apoptosis through the dysfunction of mitochondria to trigger intrinsic apoptotic pathway by untargeted metabolomic method, mitochondrial membrane potential, and caspase-3/7 activity assay. Moreover, neothalfine damaged colorectal cancer clonal spheres expansion significantly at the concentration of 3.5 nM with nearly 1000 folds efficacy than CPT-11 (3.0 µM). The results supported that neothalfine might be an anti-tumor lead for further investigation.

Phylogeny and taxonomy of Podosphaera filipendulae (Erysiphaceae) revisited.

The phylogeny and taxonomy of Podosphaera filipendulae (including P. filipendulensis, syn. nov.) have been examined. Asian, European and North American collections were examined and the nucleotides sequences of their partial rDNA region were determined. In particular, the relationship between P. filipendulae and P. spiraeae was analysed. The results confirmed P. filipendulae and P. spiraeae as two separate, morphologically similar species. The phylogenetic analysis revealed a similar phylogeny to that of the host genera. Although ITS sequences retrieved from Asian, European and North American specimens of P. filipendulae on various Filipendula spp. are identical to sequences from P. macularis on hop, there is consistently one base substitution at the 5'-end of 28S rRNA gene between the species. This result provides evidence that the hop powdery mildew and P. filipendulae are biologically and morphologically clearly distinguished, and should be maintained as two separate species.

Australia: A Continent Without Native Powdery Mildews? The First Comprehensive Catalog Indicates Recent Introductions and Multiple Host Range Expansion Events, and Leads to the Re-discovery of Salmonomyces as a New Lineage of the Erysiphales.

In contrast to Eurasia and North America, powdery mildews (Ascomycota, Erysiphales) are understudied in Australia. There are over 900 species known globally, with fewer than currently 60 recorded from Australia. Some of the Australian records are doubtful as the identifications were presumptive, being based on host plant-pathogen lists from overseas. The goal of this study was to provide the first comprehensive catalog of all powdery mildew species present in Australia. The project resulted in (i) an up-to-date list of all the taxa that have been identified in Australia based on published DNA barcode sequences prior to this study; (ii) the precise identification of 117 specimens freshly collected from across the country; and (iii) the precise identification of 30 herbarium specimens collected between 1975 and 2013. This study confirmed 42 species representing 10 genera, including two genera and 13 species recorded for the first time in Australia. In Eurasia and North America, the number of powdery mildew species is much higher. Phylogenetic analyses of powdery mildews collected from Acalypha spp. resulted in the transfer of Erysiphe acalyphae to Salmonomyces, a resurrected genus. Salmonomyces acalyphae comb. nov. represents a newly discovered lineage of the Erysiphales. Another taxonomic change is the transfer of Oidium ixodiae to Golovinomyces. Powdery mildew infections have been confirmed on 13 native Australian plant species in the genera Acacia, Acalypha, Cephalotus, Convolvulus, Eucalyptus, Hardenbergia, Ixodia, Jagera, Senecio, and Trema. Most of the causal agents were polyphagous species that infect many other host plants both overseas and in Australia. All powdery mildews infecting native plants in Australia were phylogenetically closely related to species known overseas. The data indicate that Australia is a continent without native powdery mildews, and most, if not all, species have been introduced since the European colonization of the continent.

Bioguided isolation, identification and activity evaluation of antifungal compounds from Acorus tatarinowii Schott.

ETHNOPHARMACOLOGY RELEVANCE: As a traditional folk medicine, Acorus tatarinowii Schott was used to treat digestive diseases, such as diarrhea, which may be related to Candida albicans infection; however according to literature surveys, there have been few studies of A. tatarinowii focusing on its antimicrobial activity, and almost all describe investigations using crude extracts or fractions. AIM OF THE STUDY: The aims of the current study were to isolate and identify antifungal fractions of A. tatarinowii based on their antifungal activity, explore the preliminary mechanism of 60% ethanol elution (AT60) by metabonomics, and evaluate the antifungal activity of AT60 in vivo and in vitro, to provide natural resources against fungal infections. MATERIALS AND METHODS: As a pilot evaluation of activity, A. tatarinowii fractions and compounds with antifungal bioactivity were isolated by bioactive-guided column chromatography, and identified by LC-QTOF-MS/MS and NMR spectroscopy. The antifungal effects of the active ingredients against resistant C. albicans were evaluated by in vivo and in vitro colony forming unit assays. The mechanism underlying the activity of AT60 against C. albicans was explored using an LC-QTOF-based metabonomics approach and fluorescence microscopy imaging. RESULTS: AT60 showed better activity against C. albicans than the same dose of the first line antifungal drugs, fluconazole and itraconazole (positive control drugs). Subsequent phytochemical investigation of AT60 identified twenty-five known compounds, six of which were isolated: asaraldehyde (7), 1-(2,4,5-trimethoxyphenyl)-1,2-propanediol (12), α-asarone (14), ß-asarone (15), γ-asarone (18), acotatarone C (19). Further, the compounds α-asarone (14) and acotatarone C (19) may be responsible for the antifungal activity, and exhibit synergistic effects. Metabonomics analysis indicated that AT60 can inhibit biofilm formation by regulating the C. albicans protein kinase C pathway. CONCLUSIONS: Our results show that A. tatarinowii has potent bioactivity against C. albicans in vitro and in vivo, and can be considered an antifungal botanic agent.

Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus.

BACKGROUND: Previous phylogenetic analyses of species within the genus Golovinomyces (Ascomycota, Erysiphales), based on ITS and 28S rDNA sequence data, revealed a co-evolutionary relationship between powdery mildew species and hosts of certain tribes of the plant family Asteraceae. Golovinomyces growing on host plants belonging to the Heliantheae formed a single lineage, comprised of a morphologically differentiated complex of species, which included G. ambrosiae, G. circumfusus, and G. spadiceus. However, the lineage also encompassed sequences retrieved from Golovinomyces specimens on other Asteraceae tribes as well as other plant families, suggesting the involvement of a plurivorous species. A multilocus phylogenetic examination of this complex, using ITS, 28S, IGS (intergenic spacer), TUB2 (beta-tubulin), and CHS1 (chitin synthase I) sequence data was carried out to clarify the discrepancies between ITS and 28S rDNA sequence data and morphological differences. Furthermore, the circumscription of species and their host ranges were emended. RESULTS: The phylogenetic and morphological analyses conducted in this study revealed three distinct species named, viz., (1) G. ambrosiae emend. (including G. spadiceus), a plurivorous species that occurs on a multitude of hosts including, Ambrosia spp., multiple species of the Heliantheae and plant species of other tribes of Asteraceae including the Asian species of Eupatorium; (2) G. latisporus comb. nov. (≡ Oidium latisporum), the closely related, but morphologically distinct species confined to hosts of the Heliantheae genera Helianthus, Zinnia, and most likely Rudbeckia; and (3) G. circumfusus confined to Eupatorium cannabinum in Europe. CONCLUSIONS: The present results provide strong evidence that the combination of multi-locus phylogeny and morphological analysis is an effective way to identify species in the genus Golovinomyces.