Regulation of water balance as a function of the excretory system of the filariform larvae of Nippostrongylus muris and Ancylostoma caninum

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1. The excretory system in the filariform larvae of N. muris and A. caninum is concerned with the regulation of water balance.     

Studies on cestode metabolism. II. The utilization of glycogen by Schistocephalus solidus in vitro

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1. The amount of endogenous glycogen used by Schistocephalus plerocercoids, when cultured to maturity, has been measured in a range of media and physical conditions.     

Aspects of the carbohydrate metabolism of a mutant of Neurospora crassa requiring acetate for growth

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1. Two mutant genes controlling the activities of different enzyme systems in Neurospora are described. One controls the activity of the enzyme pyruvic carboxylase, the other an enzyme system involved in the oxidation of pyruvic acid.     

Comparison of Effects of Trichuris muris and Spontaneous Colitis on the Proximal Colon Microbiota in C3H/HeJ and C3Bir IL10–/– Mice

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host''s microbial community are not fully understood. We hypothesized that Trichuris muris-infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4^−/− IL10^−/− and C3H/HeJ TLR4^−/− IL10^+/+ mice) with and without T. muris infection, in uninfected C3BirIL10^−/− mice with and without spontaneous colitis, and in normal C3H/HeJ mice. T. muris-infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Approximately 80% of infected IL10^−/− mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10^−/− and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10^−/− and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10^−/− mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10^−/− mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10^−/− mice or noncolitic C3H/HeJ mice. Therefore, Trichuris-infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.roup method with arithmetic averaging; OTU, operational taxonomic unit; qPCR, quantitative real-time PCR; SIMPER, similarity percentage; T-RFLP, terminal restriction fragment length polymorphism

Trichuris spp. are gastrointestinal nematodes that dwell in close association with a complex bacterial community in the host''s colon. After ingestion, embryonated eggs hatch in the cecum or colon releasing first-stage larvae that penetrate the epithelium and undergo 4 molts before becoming sexually mature. Both larval and adult Trichuris form syncytial tunnels in the colonic epithelium^21,30 that anchor the organisms in the proximal colon, where females produce eggs that pass in feces and embryonate in the environment.T. suis excretory secretory products (ESP) condition the colonic environment for enhanced worm survival, including effects on intestinal bacteria. Previous work demonstrated that T. suis ESP had dose-dependent effects on the tight junctions of epithelial cells.¹ The ESP fraction below a molecular weight of 10,000 kDa was mainly composed of an antimicrobial moiety² with bactericidal activity against gram-negative (Campylobacter jejuni, C. coli, and Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. In addition, due to several enzymatic activities, T. suis ESP have been demonstrated to aid the worms in burrowing into the host''s colonic epithelium and in feeding.^1,10,12 In addition to a 20-kDa diagnostic antigen,^10,11 higher molecular-weight fractions of ESP harbored a 42-kDa zinc metalloprotease that likely functions to provide nutrition for the worms through collagenase and elastase activities.¹⁰ Furthermore, a serine protease inhibitor (TsCEI) was purified from adult-stage T. suis by using acid precipitation, affinity chromatography, and reverse-phase HPLC.³³ This 6.43-kDa TsCEI inhibited chymotrypsin, pancreatic elastase, neutrophil elastase, and cathepsin G and was suggested to function as a parasite defense mechanism by modulating host immune responses. Indeed, exposure of cultured epithelial cells to T. suis ESP elicited IL6 and IL10 cytokine responses.³¹Trichuris has also been reported to interact with bacteria in vivo. Early studies demonstrated development of diarrhea in weaning age pigs concurrently harboring T. suis and various bacteria.³⁵ A mixed inoculum of T. suis and cecal scrapings containing Brachyspira, Campylobacter spp., or Salmonella spp. were implicated in this diarrhea by means of passive transfer to SPF pigs.³⁵ Interactions between this helminth and enteric bacteria were also explored by antibiotic treatment of T. suis-infected pigs.^20,27 Results of both passive transfer and antibiotic treatment experiments in pigs showed that Trichuris and various bacterial strains were necessary to produce the type of diarrhea and colonic lesions seen in weaning aged pigs in production, but did not implicate a single bacterial agent. In 2003, synergism between T. suis and C. jejuni was proven to cause mucohemorrhagic colitis in that germ-free piglets inoculated with both agents developed disease, whereas those infected with a single agent did not.²⁵ Recent studies in T. suis-infected pigs show changes in the microbial community of the colon with some accompanying metabolic changes.^22,45 Similar interactions have been found in extensive studies of captive rhesus monkeys with chronic enterocolitis. In these monkeys, severe disease was associated with presence of Trichuris trichiura and several enteric pathogens including C. coli, C. jejuni, Shigella flexneri, Yersinia enterocolitica, adenovirus, and Strongyloides fulleborni.³⁸ Therefore, Trichuris interacts with and may demonstrate synergy in disease production with the host''s colonic microflora.Interactions between Trichuris and bacteria have also been studied in mice.^9,20,36 One study found 100% morbidity in C57BL/6 IL10^−/− and congenic IL10^−/− IL4^−/− mice after challenge with T. muris.³⁶ The authors hypothesized that this high morbidity was due to an overgrowth of opportunistic invasive bacteria that use the mechanical damage caused by T. muris larvae to breach the intestinal tract. Adding the broad-spectrum antibiotic neomycin sulfate to the drinking water of IL10^−/− IL4^−/− mice and then infecting them with T. muris resulted in a statistically significant increase in the percentage of mice that survived infection.³⁶ The authors concluded that growth of opportunistic bacteria may have contributed to the previously observed morbidity and mortality. Most recently, another group⁹ found that increased levels of colonic microflora favor increased numbers of T. muris and chronic infections. The group also demonstrated that T. muris eggs hatched more efficiently in vitro when incubated with explants of mouse cecum containing 5 isolates of bacteria (E. coli, Staphylococcus aureus, Salmonella typhimurium, or Pseudomonas aeruginosa) and the yeast Saccharomyces cerevisiae, with the greatest effects seen at 37 °C. Similarly, work from our laboratory²⁰ demonstrated that treatment of T. muris-infected C57BL/6 IL10^−/− mice with metronidazole but not prednisolone increased survival.²⁰ Most recently, chronic infections with T. muris in C57BL/6 mice have been shown to decrease the diversity of intestinal microbiota,¹³ increase the abundance of Lactobacillus spp., and alter the metabolome.¹⁴Taken together, these data suggest an important microbial component to the pathogenesis of Trichuris infections in a variety of species. Given that Trichuris suis has been administered to patients with inflammatory bowel disease (IBD), and in some studies appeared to diminish IBD symptoms^42,43 we sought to understand the community-wide interactions of this worm with enteric bacteria in a mouse model of colitis. We hypothesized that the microbiota of the proximal colon would differ significantly in mice infected with T. muris as compared with uninfected mice. We theorized that these effects would occur due to the worm''s immunomodulatory properties in the host and may contribute to the successful outcomes of Trichuris treatment in patients with IBD.     

Prenylated acetophenones from Melicope obscura and Melicope obtusifolia ssp. obtusifolia var. arborea and their distribution in Rutaceae

Four prenylated acetophenones 2,6-dihydroxy-4-geranyloxyacetophenone (1), 4-geranyloxy-2,6,β-trihydroxyacetophenone (2), 2,6-dihydroxy-4-geranyloxy-3-prenylacetophenone (3), and 4-geranyloxy-3-prenyl-2,6,β-trihydroxyacetophenone (4) have for the first time been isolated from Melicope obscura (1 and 2) and Melicope obtusifolia ssp. obtusifolia var. arborea (3 and 4). The distribution of prenylated acetophenones in Rutaceae is reviewed and the results, including the new records, indicate that prenylated acetophenones are valuable as chemotaxonomic markers for the subfamily Rutoideae, tribe Xanthoxyleae sensu Engler.     

Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

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Highlights

• •db/db β-cells restores appropriate insulin stores and normalize secretory function.
• •Numerous changes in the phosphorylation and sialylation states by euglycemic rest.
• •Restoration of numerous dysfunctional biological processes following euglycemic rest.
• •β-cell adaptive flexibility may lead to improvement in endogenous β-cell function.

     

Specific inactivation of an antifungal bacterial siderophore by a fungal plant pathogen

Bacteria and fungi secrete many natural products that inhibit each other’s growth and development. The dynamic changes in secreted metabolites that occur during interactions between bacteria and fungi are complicated. Pyochelin is a siderophore produced by many Pseudomonas and Burkholderia species that induces systemic resistance in plants and has been identified as an antifungal agent. Through imaging mass spectrometry and metabolomics analysis, we found that Phellinus noxius, a plant pathogen, can modify pyochelin and ent-pyochelin to an esterification product, resulting in reduced iron-chelation and loss of antifungal activity. We also observed that dehydroergosterol peroxide, the fungal metabolite, is only accumulated in the presence of pyochelin produced through bacteria–fungi interactions. For the first time, we show the fungal transformation of pyochelin in the microbial interaction. Our findings highlight the importance of understanding the dynamic changes of metabolites in microbial interactions and their influences on microbial communities.Subject terms: Microbial ecology, Metabolomics

Microorganisms use various strategies to establish themselves within an ecological niche while facing keen competition in the environment. Natural products such as antibiotics, quorum sensing molecules, and siderophores are crucial in microbial interactions [1–3]. Certain microorganisms are equipped with uptake systems that enable them to acquire siderophores, even by those that may not produce them [4]. For example, pyochelin is a siderophore produced by many Pseudomonas and Burkholderia strains. Such bacterial strains are commonly found in soils, as endophytes, and from the rhizosphere where they may inhibit plant pathogens [5, 6].Burkholderia cenocepacia 869T2 was isolated as an endophyte and showed beneficial abilities to control banana Fusarium wilt [7]. It harbors many biosynthetic gene clusters of secondary metabolites, such as pyochelin, pyrrolnitrin, and pyrroloquinoline quinone [8]. Recently, we found that this strain could temporarily inhibit the growth of P. noxius, a fungal pathogen of brown root rot disease, which is prevalent in tropical and subtropical regions and has a wide host range covering over 200 plant species [9]. However, in the competition between fungi and bacteria, P. noxius can resist this inhibition and overwhelm bacterial colonies after 1–2 weeks under dual-culture conditions (Fig. S1). These results imply that fungi might have resistance responses and undergo metabolic changes in bacteria–fungi interactions [10]. Here we unveiled metabolic changes in the competitive interaction between B. cenocepacia 869T2 and P. noxius 2252 using the matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS) [11, 12].We specifically monitored the metabolites in the inhibition region of B. cenocepacia 869T2 and P. noxius 2252 dual-culture using MALDI-TOF IMS. Several induced or enzymatically modified metabolites were detected, including m/z 275, 362, 383, and 427 (Fig. 1A). In particular, pyochelin (m/z 325), surrounding the B. cenocepacia 869T2 colony, showed asymmetric distribution in dual-culture samples. Near the P. noxius 2252 mycelia, a new metabolite with m/z 383 was detected with a complementary distribution to pyochelin (Fig. 1A). In LC-MS/MS-based molecular networking analysis [13], we found that this new metabolite structure is an esterification product of pyochelin and glycolic acid, which we named pyochelin-GA (Fig. 1B). We then constructed a pchF-null mutant strain, ΔpchF, which cannot produce pyochelin, and then dual cultured it with P. noxius. Pyochelin and pyochelin-GA were not observed in the MALDI-TOF IMS and LC-MS analysis of dual-culture samples (Fig. 1A and Fig. S2). We further inoculated P. noxius 2252 with pyochelin-GA-free extract harvested from B. cenocepacia 869T2 single culture, and the complementary distribution of pyochelin and pyochelin-GA was observed by MALDI-TOF IMS again (Fig. S3). These results demonstrated that pyochelin-GA was transformed from pyochelin by P. noxius 2252, rather than produced by B. cenocepacia 869T2 under dual-culture conditions.Open in a separate windowFig. 1Metabolic changes in the bacteria–fungi interaction.A Spatial distribution of selected mass signals (m/z) in MALDI-TOF IMS analysis of Phellinus noxius 2252 (Pn2252) dual-cultured with Burkholderia cenocepacia 869T2 (869T2) and a pchF-null mutant strain (Δ pchF). B Molecular networking analysis of pyochelin and analogs from the dual-culture sample. The red node is pyochelin, and the green node is pyochelin-GA. The structures of pyochelin, pyochelin-GA, and dehydroergosterol peroxide (DHEP), together with their mass signals in MALDI-TOF IMS, are shown. C Iron-chelating abilities of pyochelin and pyochelin-GA were evaluated by Chrome Azurol S liquid assay using different concentrations (2.5, 1.25, 0.63, 0.31, and 0.16 mM, n = 3). Proportions of siderophore units are shown in Fig. S14. D Fungal transformation of pyochelin and ent-pyochelin by treating P. noxius 2252 with ethyl acetate crude extracts of B. cenocepacia 869T2, Pseudomonas aeruginosa PAO1, and P. protegens Pf-5 for 8 h. LC-MS was used to monitor the signals of pyochelin (red), ent-pyochelin (blue), and transformation product 383 (black).The chemical structure of pyochelin-GA was further confirmed via total synthesis, NMR, and LC-MS/MS analysis (Supplementary Material and Methods, and Figs. S4–7). The purified pyochelin and pyochelin-GA were also evaluated for their iron-chelating ability. Chrome Azurol S assay indicated that pyochelin had the dose-dependent iron-chelating ability, but pyochelin-GA had lower iron-binding efficiency (Fig. 1C, Fig. S8). Pyochelin chelates iron in the extracellular medium and transports it into cells via the specific outer membrane transporter FptA. The X-ray structure of FptA-pyochelin-Fe indicated that the terminal carboxylic acid of pyochelin plays an essential role in the iron uptake ability [14, 15]. Our docking analysis suggested that the glycolic ester moiety of pyochelin-GA would affect the binding pocket shape of FptA and result in different binding properties compared to FptA-pyochelin (Fig. S9).Pyochelin and ent-pyochelin are produced independently by different biosynthetic gene clusters in Pseudomonas species [16]. To determine whether P. noxius 2252 can transform both enantiomers via this esterification process, we treated P. noxius 2252 with the extracts of pyochelin producers (P. aeruginosa PAO1 and B. cenocepacia 869T2) and an ent-pyochelin producer (P. protegens Pf-5). After 8 h of treatment, both pyochelin and ent-pyochelin were converted to pyochelin-GA (or ent-pyochelin-GA) (Fig. 1D), demonstrating this is a non-stereospecific transformation.To better understand the iron-chelating ability of pyochelin, we used pyochelin and pyochelin-GA to treat P. noxius 2252 under iron-deficiency conditions, by adding the iron chelator deferoxamine, and iron-rich conditions by adding FeCl₃ (Fig. 2). Pyochelin-GA did not affect the growth of P. noxius 2252 under all conditions. However, P. noxius 2252 was more sensitive to pyochelin in iron-deficient conditions and more resistant to pyochelin in iron-rich conditions, demonstrating that iron availability directly affected the tolerance of P. noxius 2252 to pyochelin. A similar phenomenon was reported previously for Aspergillus fumigatus [17].Open in a separate windowFig. 2Pyochelin inhibition of mycelial growth of Phellinus noxius 2252 is inversely associated with iron concentration.Pyochelin-GA did not have an inhibition effect on P. noxius 2252. Potato dextrose agar (PDA) with deferoxamine (DFO; 200 and 400 µM) was used to mimic iron-deficiency conditions. Iron-rich conditions was prepared by adding FeCl₃ (200 and 400 µM) in PDA. P. noxius 2252 was treated with 0.03, 0.06, 0.12, and 0.24 µmol of pyochelin or pyochelin-GA at 30 °C for 24 h. The antifungal assay was performed in two biological replicates.Using MALDI-TOF IMS analysis of the dual-culture of B. cenocepacia 869T2 and P. noxius 2252, we observed that several metabolites (e.g., m/z 275, 362, and 427) were only observed in the boundary of fungal mycelia (Fig. 1A). Although those metabolites were not detected in the dual-culture of ΔpchF and P. noxius 2252 (Fig. 1A), they were present when we treated P. noxius 2252 with pyochelin (Fig. S10). We identified the metabolite associated with m/z 427 as dehydroergosterol peroxide (DHEP) (Fig. S11), which was initially oxidized from ergosterol and dehydroergosterol [18]. Pyochelin can enhance intercellular reactive oxygen species (ROS) and ultimately disrupts membrane integrity, leading to cell death [17, 19, 20]. To clarify whether ROS induced the accumulation of DHEP, we treated P. noxius 2252 with pyochelin, pyochelin-GA, and 2,2′-bipyridyl (an iron chelator). Pyochelin and 2,2′-bipyridyl showed antifungal effects on P. noxius 2252 and induced ROS production (Fig. S12). However, the accumulation of DHEP in P. noxius 2252 was only associated with pyochelin treatment (Fig. S13). The induction of ROS in P. noxius 2252 by pyochelin and pyochelin-GA was not significantly different (Fig. S14). Therefore, we predict that pyochelin-induced accumulation of DHEP in P. noxius 2252 is independent of ROS production and iron-deficiency.Overall, we demonstrate that pyochelin transformation by fungi, in the interaction between pyochelin-producing bacteria and the plant pathogen P. noxius transforms pyochelin and ent-pyochelin into pyochelin-GA (and ent-pyochelin-GA). This product no longer functions as an iron chelator and no longer shows antifungal activity. The production of a fungal metabolite, dehydroergosterol peroxide, was induced explicitly by pyochelin through an unknown mechanism. These results highlight the importance of monitoring dynamic changes of metabolites in situ to better understand the functions and influences of metabolites on microbial community interactions.     

Versatile CRISPR/Cas9-mediated mosaic analysis by gRNA-induced crossing-over for unmodified genomes

Mosaic animals have provided the platform for many fundamental discoveries in developmental biology, cell biology, and other fields. Techniques to produce mosaic animals by mitotic recombination have been extensively developed in Drosophila melanogaster but are less common for other laboratory organisms. Here, we report mosaic analysis by gRNA-induced crossing-over (MAGIC), a new technique for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9. MAGIC efficiently produces mosaic clones in both somatic tissues and the germline of Drosophila. Further, by developing a MAGIC toolkit for 1 chromosome arm, we demonstrate the method’s application in characterizing gene function in neural development and in generating fluorescently marked clones in wild-derived Drosophila strains. Eliminating the need to introduce recombinase-recognition sites into the genome, this simple and versatile system simplifies mosaic analysis in Drosophila and can in principle be applied in any organism that is compatible with CRISPR/Cas9.

Analysis of mosaic animals has been crucial in developmental and cell biology; this study describes a versatile, simple, and likely widely-applicable technique, MAGIC (mosaic analysis by gRNA-induced crossing-over), for generating mosaic animals based on DNA double-strand breaks produced by CRISPR/Cas9.     

Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping

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Highlights

• •Discovery of peptide biomarker candidates of respiratory tract pathogens S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus as target pathogens.
• •Peptide biomarker candidates were experimentally verified in clinical samples.
• •Targeted MS using promising peptide biomarker candidates shown as proof-of-concept.

     

THE PHYSIOLOGICAL EFFECTS OF l-TYROSINE AND l-PHENYL-ALANINE ON THE RATE AND QUALITY OF PULSATION IN THE SEVENTY-TWO HOUR EXTIRPATED EMBRYONIC CHICK HEART

1. 72 hour isolated chick hearts show an increase in pulsation rate when placed in M/1000, M/10,000, and M/50,000 l-tyrosine solutions. The optimal effect is seen in M/10,000 and M/50,000 l-tyrosine. 2. All hearts show disturbance of rhythm either in the form of irregular rhythm or heart block. 3. 62 hour isolated chick hearts are not susceptible to l-tyrosine while 96 hour hearts are markedly sensitive. 4. 72 hour isolated chick hearts placed in 1 part in 10,000 and 1 part in 50,000 l-epinephrine show approximately the same effects as were seen with l-tyrosine. 5. 72 hour isolated chick hearts placed in M/1000 and M/10,000 l-phenylalanine show an initial depression followed by an l-tyrosine effect.