Highly Aromatic Norditerpenoid Heterodimers and Monomers from Trigonostemon fragilis

Four new norditerpenoid heterodimers with different dimerization patterns-namely,trigofragiloids A-C(denoted as compounds 1-3)and(+)-and(-)-trigofragiloid D(compound 4)-and three new phenanthrenone norditerpenoids-namely,trigofragiloids E-G(compounds 5-7)-were isolated from Trigonostemon fragilis.Compounds 1 and 2 feature a novel heterodimeric carbon skeleton formed by the conjugation of a tetra-norditerpenoid and an ennea-norditerpenoid;they have been identified as class 2 atropisomers by means of quantum chemical calculations.Compound 3 is an unprecedented phenylpropanoid-norditerpenoid adduct with a new dimerization pattern.Compounds(+)-and(-)-4 are the first example of S-shaped 1,4-dioxane-fused norditerpenoid dimers.Inspired by the structure elucidation of compound 4,two co-occurring analogues,actephilol A and epiactephilol A,were structurally revised as a pair of geometrical isomers and were identified as two pairs of enantiomers,(+)-and(-)-8 and(+)-and(-)-9,respectively.Their structures were characterized using a combined method.Notably,compound 7 exhibits remarkable adenosine triphosphate-citrate lyase(ACLY)inhibition with a halfmaximal inhibition concentration(IC50)value of(0.46±0.11)lmol·L^(-1),as active as the positive control BMS-303141,and a molecular docking study offers deep insight into the interaction between compound 7 and ACLY.

K.fragilis β-半乳糖苷酶催化合成低聚半乳糖的组成分析

采用SephadexG-15与DTF-02树脂对K.fragilisβ-半乳糖苷酶催化生产低聚半乳糖的反应液中的糖类物质进行了分离纯化。分别应用高效液相色谱测定了分离的低聚半乳糖的纯度，气相色谱分析了该糖的化学组成。酶反应液经过SephadexG-15色谱柱分成低聚半乳糖、乳糖和单糖(葡萄糖、半乳糖)3个组分。由SephadexG-15色谱柱收集的低聚半乳糖组分上DTF树脂柱出现2个洗脱峰，分别为低聚半乳糖和乳糖。经过上述2步纯化的低聚半乳糖组分在高效液相色谱(HPLC)上为一个单峰，其保留时间为10.481min。该低聚半乳糖是由1分子葡萄糖和2分子半乳糖组成的三糖。

Kluyveromyces fragilis LFS-8611 β-D-半乳糖苷酶的摇瓶培养条件

脆壁克鲁维酵母(Kluyveromycesfragilis)LFS 8611合成的β D 半乳糖苷酶具有较高的催化半乳糖基转移反应活力.脆壁克鲁维酵母(K.fragilis)LFS 8611细胞生长和β D 半乳糖苷酶的合成同步.该菌株生长和产酶的最适碳源为半乳糖,乳糖次之;最适氮源为蛋白胨F403;最适培养条件为:发酵培养基的初始pH值为7.0,摇床的转速为200r/min.培养基中碳源和氮源质量浓度对菌体生物量和β D 半乳糖苷酶活力有重要影响,以12mg/mL乳糖为碳源,16mg/mL蛋白胨(F403)为氮源,在最适培养条件下培养32h后,菌体生物量和β D 半乳糖苷酶活力分别为7.56g/L和18.83U/mL.

影响kluyveromyces fragilisβ-D-半乳糖苷酶催化合成低聚半乳糖的因素

研究了酶反应的温度、pH值、底物浓度和反应时间等对kluyveromycesfragilisβ-D-半乳糖苷酶催化合成低聚半乳糖的影响.结果显示,以乳糖为底物,kluyveromycesfragilisβ-D-半乳糖苷酶催化合成低聚半乳糖的最适温度为60℃,最适pH值为6.0.酶法合成低聚半乳糖的产率随着底物浓度增加而增加.kluyveromycesfragilisβ-D-半乳糖苷酶催化低聚半乳糖合成的初速度较快,随着反应时间延长,反应速率降低,反应20h酶反应达到平衡.以660mg/mL乳糖为底物,在最适酶反应条件下,酶法合成低聚半乳糖的产率为207.12mg/mL.

Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 mu g), and a model + high-dose toxin (ADHT; n = 20, 20 mu g) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. RESULTS Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 +/- 0.68 vs 23.23 +/- 0.91, P < 0.05) and the ADHT group (21.82 +/- 0.68 vs 23.57 +/- 1.06, P < 0.05). More tumors were found in the AD group than in the ADLT and ADHT groups (19.75 +/- 3.30 vs 6.50 +/- 1.73, P < 0.05; 19.75 +/- 3.30 vs 6.00 +/- 2.16, P < 0.05). Pathology showed that 12 mice had adenocarcinoma and 6 cases had adenoma in the AD group. Five mice had adenocarcinoma and 15 had adenoma in the ADLT group. Four mice had adenocarcinoma and 16 had adenoma in the ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group (P < 0.05, P < 0.05). The positive rate of Ki-67 in the ADLT group and the ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P < 0.05, P < 0.05). Caspase-3 expression in the ADLT group and the ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). CONCLUSION Oral administration with lower-dose biologically active recombinant BFT-2 inhibited colorectal tumorigenesis in mice.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-,mitogen-activated protein kinase-,and Nrf2-dependent pathway

BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Molecular detection and genetic diversity of Dientamoeba fragilis and Enterocytozoon bieneusi in fecal samples submitted for routine parasitological examination

Objective:To detect Dientamoeba(D.)fragilis and Enterocytozoon(E.)bieneusi and to assess their genetic characteristics in stool samples submitted for routine examination in a clinical laboratory in Southeastern Brazil.Methods:In this survey,348 stool samples from female and male individuals with age ranging from 0 to≥55 years were analyzed by PCR amplifying and sequencing based on the small subunit ribosomal RNA(SSU r RNA)gene of D.fragilis and the internal transcribed spacer of E.bieneusi.Results:D.fragilis and E.bieneusi isolates were observed in 2.29%(8/348)and 4.59%(16/348)of the samples,respectively.These parasites were detected in stool samples from individuals of both genders,including young children under nine until adults over 55 years old.No statistically significant differences were found.All D.fragilis isolates were classified as genotype 1 and E.bieneusi isolates included genotypes D(n=15)and A(n=1).Conclusions:The findings provide relevant findings on occurrence and genetic diversity of D.fragilis and E.bieneusi,pointing to the need for the diagnosis of these parasites in routine examinations in clinical laboratories.In addition to sensitive diagnostic methods,it is mandatory that these parasites be considered relevant for physicians and laboratory staff.

Physico-chemical Characterization of the Lipopolysaccharide (Endotoxin) Isolated from Bacteriodes fragilis NCTC 9343

The lipopolysaccharide (LPS, endotoxin) isolated from Bacteriodes fragilis NCTC 9343 ex-hibted an endogenous heterogencity consisting of R (rough) and SR (semi-rough ) LPS fractionsupon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LPS was chemically consti-tuted by D-glucosamine, D-glucose. L-rhamnose, D-galactose. fatty acids and phosphate in amolar ratio of 2: 1: 1: 4: 5. 4.: 2.7 The fatty acids were predominated by 3-hydroxypentadecanoic(3-OH-15: 0), 3-hydroxyhcxadecanoic (3-OH-16: 0), 3-hydroxyheptadecanoic (3-OH-17: 0), 3-hydroxy-15-methyl hexadecanoic (3-OH-15-Me-16: 0) and 13-methyl-letradecanoic acid (13-Mc-14:0). Approximately 1 mol of 2-Keto-3-deoxy-D-mannooctonatc (KDO) was detected after treat-ment of LPS with 48% of hydrofluoric acid (HF), indicating it was present as a phosphorylatedmolecule.

Endophthalmitis caused by Bacteroides fragilis after pars plana vitrectomy and treatment approach

Rationale:Endophthalmitis is an uncommon but serious ocular infection often resulting in probable visual loss.Bacteroides fragilis is a rare cause of endophthalmitis.Patient concerns:A 46-year-old male patient complained of eye pain and low vision after pars plana vitrectomy.Diagnosis:Bacteroides fragilis endophthalmitis after pars plana vitrectomy was diagnosed.Interventions:Pars plana vitrectomy and silicone oil implantation were performed.Outcomes:Early treatment and choice of tamponade in endophthalmitis after pars plana vitrectomy may possibly prevent evisceration and progression of endophthalmitis.Lessons:Bacteroides fragilis can be seen in cases of endophthalmitis after pars plana vitrectomy.

Bacteroides fragilis Supernatant Extracts Enriched in Phenylacetic Acid Induce a Cytotoxic Effect in Mammalian Cells

Bacteroides species are nearly half of the fecal flora community and some are host symbionts crucial to host nutrition and systemic immunity. Among Bacteroides species B. fragilis strains are considered to be the opportunistic ones, being the most isolated anaerobic bacteria in clinical samples. Cell-free supernatants of 65 B. fragilis strains were assayed and they were capable of inducing vacuolating phenotype on Vero cells lineage. The supernatant of the Bacteroides fragilis ATCC 23745 strain was elicited to have the strongest vacuolating effect on Vero cells monolayers and peritoneal macrophages. Some drastic cell alterations were observed, such as a general disorganization of cytoplasm and chromatin condensation, evidencing cell death. By transmission electron microscopy it was confirmed that the vacuoles observed were, in fact, swollen mitochondria. An immunocytochemical assay, TUNEL, was used to confirm this hypothesis and showed that Vero cells and peritoneal macrophages were dying by apoptotic process after exposition of B. fragilis cell-free supernatant. Physical analysis of the apoptotic factor has revealed properties similar to short-chain fatty acids. After gas chromatography and mass spectrometry analysis, phenylacetic acid (PA) was characterized as the major compound present in the most purified active fraction. We believe that the PA is responsible for the pro-apoptotic effect elicited by the supernatant of B. fragilis cultures.

Kluyveromyces fragilis β-D-半乳糖苷酶催化合成低聚半乳糖

低聚半乳糖具有促进肠道有益菌增殖、抗龋齿、低能量等特定的生理功能。研究了酶反应条件对脆壁克鲁维酵母(kluyveromyces fragilis)β-D-半乳糖苷酶催化合成低聚半乳糖的影响。以乳糖为底物,k.fragilis β-D-半乳糖苷酶催化合成低聚半乳糖的最适温度为40℃,最适pH值为7.0,反应20h低聚半乳糖的产率达到最大。底物初始浓度影响k.fragilis β-D-半乳糖苷酶催化合成低聚半乳糖的产率,乳糖起始浓度由110mg/mL增加到660mg/mL,低聚半乳糖的含量从43mg/mL增加到207mg/mL。

离子交换树脂DOEX~ 50WX8固定化K.fragilis β-D-半乳糖苷酶

K.fragilisβ-D-半乳糖苷酶通过离子吸附固定在离子交换树脂DOEX50WX8上,用于催化牛乳中的乳糖水解。研究了pH、酶的初始浓度、时间等因素对酶固定化效率的影响,探讨了固定化酶的操作稳定性及其水解乳糖的效率。结果表明:在pH7.0、初始酶浓度10.0 mg/mL、吸附时间2.0 h的条件下,吸附在DOEX50WX8 UPW上的K.fragilisβ-D-半乳糖苷酶的酶量与酶活回收率均达到最大值,分别为45.7 mg/g和83.2%。该固定化酶连续使用5次,其活力保留82.6%,固定化酶对乳糖的水解率为87.2%。

Two furano-sesquiterpenes from the South China Sea Sponge Dysidea fragilis

A new sesquiterpene, ent-furodysin (1), has been isolated from the South China Sea Sponge Dysidea fragilis, along with a known sesqui-terpene, ent-furodysinin (2). The structure and relative stereochemistry of 1 and 2 were established by spectral analysis.

中国南海柳珊瑚Junceella fragilis中一个新的briarane型二萜

综合利用硅胶、凝胶柱色谱及半制备HPLC等分离技术,对中国南海西瑁岛的脆灯芯柳珊瑚Junceella fragilis Ridley的丙酮提取物进行化学成分的研究,共分离得到6个化合物。根据其理化性质和各种波谱数据分析,分别鉴定为:fragilide Y(1)、fragilide D(2)、胆甾醇(3)、过氧化麦角甾醇(4)、2’-脱氧胸腺嘧啶核苷(5)和cis-thyminenol(6)。其中化合物1为新的briarane型二萜;生物活性结果表明,化合物1~6均未表现明显的抗炎及肿瘤细胞毒性作用。

Cloning and expression of Kluyveromyces fragilis LAC4 gene

The genomic library of Kluyveromyces fragilis was constructed in E.coli TG1,and 5β-galactosidase gene(LAC4)clones have been obtained from the library by complementation of theKluyveromyces lactis lac4-8 mutation.The studies on the structure and the function of the LAC4 gene revealedthat(i)the gene can also complement E.coli lacZ mutation;(ii)the physical map of the K.fragilis LAC4gene was very similar to that of K.lactis;(iii)the β-galactosidase levels expressed by the clone strains weremuch higher than that expressed by the original strain;(iv)the variation of the β-galactosidase level of differ-ent clone strains induced by lactose or galactose was related to the retained degree of the 5’ flanking region ofLAC4 gene,suggesting that there migth he a lactose specific transcription activating element in the region.

渗透化K.fragilis细胞水解牛乳中乳糖的研究

应用渗透化K.fragilis细胞中的β-D-半乳糖苷酶水解牛乳中的乳糖,研究了影响乳糖水解率的因素、乳糖的水解动力学,以及酶的稳定性。结果表明:以牛乳中的乳糖为底物,4℃条件下水解75 min,乳糖的水解率为62.66%。该酶的Km值为3.18 mmol/L,Vmax为3.32μmol/min。乳糖的水解产物(葡萄糖)对K.fragilisβ-D-半乳糖苷酶具有反馈抑制作用,反应体系中存在5%葡萄糖时,对该酶活性的抑制率为36.88%。水解牛乳中乳糖连续使用4个批次后,酶活力仍保持初始酶活力的65%以上。渗透化K.fragilis细胞在低温(4℃)下具有良好的贮存稳定性,贮存14 d,β-D-半乳糖苷酶活力保存为95.64%。

Cryo-EM structure of L-fucokinase/GDP-fucose pyrophosphorylase(FKP)in Bacteroides fragilis

Dear Editor,L-Fucose(6-deoxy-L-galactose,fucose)is the basic compone nt of a variety of glyca n structures.The fucosylated oligosaccharides participate in a variety of cellular activities,like the cell-cell recognition,selectin-mediated leukocyteendothelial adhesion and the formation of Lewis blood group antigens(Ma et al.,2006).GDP-fucose is an important fucose donor in the process of fucosylated oligosaccharides formation.Two pathways of GDP-fucose synthesis are present in the cytosol of mammalian cells,including the de novo pathway and the salvage pathway(Becker et al.,2003).In the salvage pathway,cells use fucose from the extracellular or lysosomal sources to synthesize GDP-fucose.

南海脆灯蕊柳珊瑚化学成分的研究

从南海脆灯蕊柳珊瑚Junceella fragilis的二氯甲烷/乙醇提取物中分离得到13个化合物,通过光谱分析及文献对照,它们的结构分别被鉴定为:stigmasta-5,28-diene-3,24-diol(1),stigmasta-5,22-diene-3-ol(2),24α-methylcholest-7,22-dien-3,β5α,6-tβriol(3),cholesterol(4),1,2-O-[2’-hydroxyoctadecyl]-glycerol(5),ba-tyl alcohol(6),N-palmitoyloctadecasphinga-4(E)-ene(7),(2S,3S,4R,8E)-2-N-(2-hydroxytetracosanoyl)octa-decasphinga-8-ene(8),1-O--βD-glycopyranosyl-(2S,3S,4R,8Z)-2-N-(2-hydroxytetracosanoyl)octadecasphinga-8-ene(9),uracil(10),thymine(11),purine(12),adenosine(13);其中化合物1,3,5,7,8,9,13均为首次从该种海洋动物中分离得到。

Extended role for insertion sequence elements in the antibiotic resistance of Bacteroides

The Bacteroides species are important micro-organisms, both in the normal physiology of the intestines and as frequent opportunistic anaerobic pathogens, with a deeply-rooted phylogenetic origin endowing them with some interesting biological features. Their prevalence in anaerobic clinical specimens is around 60%-80%, and they display the most numerous and highest rates of antibiotic resistance among all pathogenic anaerobes. In these antibiotic resistance mechanisms there is a noteworthy role for the insertion sequence(IS) elements, which are usually regarded as representatives of ‘selfish' genes; the IS elements of Bacteroides are usually capable of up-regulating the antibiotic resistance genes. These include the cep A(penicillin and cephalosporin), cfx A(cephamycin), cfi A(carbapenem), nim(metronidazole) and erm F(clindamycin) resistance genes. This is achieved by outwardoriented promoter sequences on the ISs. Although some representatives are well characterized, e.g., the resistance gene-IS element pairs in certain resistant strains, open questions remain in this field concerning a better understanding of the molecular biology of theantibiotic resistance mechanisms of Bacteroides, which will have clinical implications.

Two-day enema antibiotic therapy for parasite eradication and resolution of symptoms

BACKGROUND Blastocystis hominis(B.hominis)and Dientamoeba fragilis(D.fragilis)are two protozoan parasites of human bowel that are found throughout the world.There is still debate about the pathogenicity of these protozoans,despite them being commonly associated with gastrointestinal symptoms and can cause health issue in both children and adults.These parasites are usually transmitted through faecal-oral contact particularly under poor hygiene conditions or food/water contamination.Once a person is infected,the parasites live in the large intestine and are passed in the faeces.AIM To investigate the effect of triple antibiotic therapy using enema infusion in the treatment of B.hominis and D.fragilis infections.METHODS This retrospective longitudinal study was conducted in a single medical centre,which included fifty-four patients(≥18 years)who were positive for D.fragilis,B.hominis or both between 2017 and 2018.The treatment consisted of triple antibiotics that were infused over two consecutive days through rectal enema.Faecal samples were collected from participants pre-and post-treatment and were tested for parasites using microscopy and polymerase chain reaction.Patients’symptoms were recorded prior and after the treatment as well as patient demographic data.RESULTS Patients(n=54),were either positive for B.hominis(37%),D.fragilis(35%)or both(28%).All patients completed the two-day treatment and no serious adverse effect was reported.The most common side effect experienced by the patients during the treatment was urine discolouration which was cleared by six weeks of followup.Common symptoms reported prior to treatment were diarrhoea,abdominal pain,constipation and fatigue.Other symptoms included abdominal discomfort,dizziness and blood in the stool.Eighty-nine percent of patients completed a final stool test post-treatment.At six weeks post-treatment,79%of patients cleared the parasites from their faeces.Symptoms such as abdominal discomfort,dizziness and blood in the stool decreased significantly at both seven days and six weeks post-treatment(P<0.040).The enema retention time,bowel preparation,previous antibiotic treatment or previous gastrointestinal problems had no significant effect on parasite eradication.CONCLUSION Overall,eradication of parasites and improvement of clinical outcomes were observed in treated patients,showing the efficacy of this combination to eradicate the parasites and provide positive clinical outcome.