Bifidobacterium longum LBUX23 Isolated from Feces of a Newborn; Potential Probiotic Properties and Genomic Characterization.

Bifidobacterium longum is considered a microorganism with probiotic potential, which has been extensively studied, but these probiotic effects are strain dependent. This work aims to characterize the probiotic potential, based on the biochemical and genomic functionality, of B. longum LBUX23, isolated from neonates' feces. B. longum LBUX23 contains one circular genome of 2,287,838 bp with a G+C content of 60.05%, no plasmids, no CRISPR-Cas operon, possesses 56 tRNAs, 9 rRNAs, 1 tmRNA and 1776 coding sequences (CDSs). It has chromosomally encoded resistance genes to ampicillin and dicloxacillin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and to some emergent pathogen's clinical strains. B. longum LBUX23 was able to utilize lactose, sucrose, fructooligosaccharides (FOS), and lactulose. The maximum peak of bacterial growth was observed in sucrose and FOS at 6 h; in lactose and lactulose, it was shown at 8 h. B. longum LBUX23 can survive in gastrointestinal conditions (pH 4 to 7). A decrease in survival (96.5 and 93.8%) was observed at pH 3 and 3.5 during 120 min. argC, argH, and dapA genes could be involved in this tolerance. B. longum LBUX23 can also survive under primary and secondary glyco- or tauro-conjugated bile salts, and a mixture of bile salts due to the high extracellular bile salt hydrolase (BSH) activity (67.3 %), in taurocholic acid followed by taurodeoxycholic acid (48.5%), glycocholic acid (47.1%), oxgall (44.3%), and glycodeoxycholic acid (29.7%) probably due to the presence of the cbh and gnlE genes which form an operon (start: 119573 and end: 123812). Low BSH activity was determined intracellularly (<7%), particularly in glycocholic acid; no intracellular activity was shown. B. longum LBUX23 showed antioxidant effects in DPPH radical, mainly in intact cells (27.4%). In the case of hydroxyl radical scavenging capacity, cell debris showed the highest reduction (72.5%). In the cell-free extract, superoxide anion radical scavenging capacity was higher (90.5%). The genome of B. longum LBUX23 contains PNPOx, AhpC, Bcp, trxA, and trxB genes, which could be involved in this activity. Regarding adherence, it showed adherence up to 5% to Caco-2 cells. B. longum LBUX23 showed in vitro potential probiotic properties, mainly in BSH activity and antioxidant capacity, which indicates that it could be a good candidate for antioxidant or anti-cholesterol tests using in vivo models.

Isolation and Identification of Multidrug-Resistant Klebsiella pneumoniae Clones from the Hospital Environment.

Global dispersion, hospital outbreaks, and lineage relationships between emerging antibiotic-resistant strains such as Klebsiella pneumoniae are of public health interest. This study aimed to isolate and identify K. pneumoniae clones from third-level healthcare hospitals in Mexico to establish their multidrug-resistant phenotype, phylogeny, and prevalence. Biological and abiotic surface samples were used to isolate K. pneumoniae strains and to test their antibiotic susceptibility to classify them. The housekeeping genes: gapA, InfB, mdh, pgi, phoE, ropB, and tonB were used for multilocus sequence typing (MLST). Phylogenetic networks were constructed with 48 strains. Isolated strains (93) were mainly from urine and blood, 96% were resistant to ampicillin as expected, 60% were extended-spectrum ß-lactamases (ESBL), 98% were susceptible to ertapenem and meropenem and 99% were susceptible to imipenem, 46% were multi-drug resistant (MDR), 17% were extensively-drug resistant (XDR), 1% were pan-drug resistant (PDR), and 36% were not classified. The tonB, mdh, and phoE genes were the most variable, and the InfB gene showed positive selection. The most prevalent sequence types (STs) were ST551 (six clones), ST405 (six clones), ST1088 (four clones), ST25 (four clones), ST392 (three clones), and ST36 (two clones). ST706 was PDR, and ST1088 clones were MDR; neither of these STs has been reported in Mexico. The strains analyzed were from different hospitals and locations; thus, it is important to maintain antibiotic surveillance and avoid clone dissemination to prevent outbreaks, adaptation to antibiotics, and the transmission of antibiotic resistance.

Genomic and Biochemical Characterization of Bifidobacterium pseudocatenulatum JCLA3 Isolated from Human Intestine.

Bifidobacteria have been investigated due to their mutualistic microbe-host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5-5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.

Synthesis and Cytotoxic Activity of Combretastatin A-4 and 2,3-Diphenyl-2H-indazole Hybrids.

Cancer is the second leading cause of death, after cardiovascular diseases. Different strategies have been developed to treat cancer; however, chemotherapy with cytotoxic agents is still the most widely used treatment approach. Nevertheless, drug resistance to available chemotherapeutic agents is still a serious problem, and the development of new active compounds remains a constant need. Taking advantage of the molecular hybridization approach, in the present work we designed, synthesized, and tested the cytotoxic activity of two hybrid compounds and seven derivatives based on the structure of combretastatin A-4 and 2,3-diphenyl-2H-indazole. Practical modifications of reported synthetic protocols for 2-pheny-2H-indazole and 2,3-dipheny-2H-indazole derivatives under microwave irradiation were implemented. The cytotoxicity assays showed that our designed hybrid compounds possess strong activity, especially compound 5, which resulted even better than the reference drug cisplatin against HeLa and SK-LU-1 cells (IC50 of 0.16 and 6.63 µM, respectively), and it had similar potency to the reference drug imatinib against K562 cells. Additionally, in silico and in vitro studies strongly suggest tubulin as the molecular target for hybrid compound 5.

Design, Synthesis and Anticandidal Evaluation of Indazole and Pyrazole Derivatives.

Candidiasis, caused by yeasts of the genus Candida, is the second cause of superficial and mucosal infections and the fourth cause of bloodstream infections. Although some antifungal drugs to treat candidiasis are available, resistant strains to current therapies are emerging. Therefore, the search for new candicidal compounds is certainly a priority. In this regard, a series of indazole and pyrazole derivatives were designed in this work, employing bioisosteric replacement, homologation, and molecular simplification as new anticandidal agents. Compounds were synthesized and evaluated against C. albicans, C. glabrata, and C. tropicalis strains. The series of 3-phenyl-1H-indazole moiety (10a-i) demonstrated to have the best broad anticandidal activity. Particularly, compound 10g, with N,N-diethylcarboxamide substituent, was the most active against C. albicans and both miconazole susceptible and resistant C. glabrata species. Therefore, the 3-phenyl-1H-indazole scaffold represents an opportunity for the development of new anticandidal agents with a new chemotype.

Identification and Quantification of a Phytotoxic Metabolite from Alternaria dauci.

Alternaria dauci is the causal agent of Alternaria leaf blight (ALB) in carrot (Daucus carota) crops around the world. However, to date, A. dauci has received limited attention in its production of phytotoxic metabolites. In this investigation, the bioassay-guided isolation of the extract from liquid cultures of A. dauci resulted in the isolation of two metabolites identified as α-acetylorcinol (1) and p-hydroxybenzoic acid (2), based on their spectroscopic data and results from chemical correlation reactions. Testing of both metabolites in different assays showed an important phytotoxic activity for p-hydroxybenzoic acid (2) when tested in the leaf-spot assay on parsley (Petroselinum crispum), in the leaf infiltration assay on tobacco (Nicotiana alata) and marigold (Tagetes erecta), and in the immersion assay on parsley and parsnip (Pastinaca sativa) leaves. Quantification of the two metabolites in the crude extract of A. dauci kept at different times showed that p-hydroxybenzoic acid (2) is one of the first metabolites to be synthesized by the pathogen, suggesting that this salicylic acid derivative could play an important role in the pathogenicity of the fungus.

Secondary Metabolites from the Leaves of the Medicinal Plant Goldenseal (Hydrastis canadensis).

The study presented herein constitutes an extensive investigation of constituents in Hydrastis canadensis L. (Ranunculaceae) leaves. It describes the isolation and identification of two previously unknown compounds, 3,4-dimethoxy-2-(methoxycarbonyl)benzoic acid (1) and 3,5,3'-trihydroxy-7,4'-dimethoxy-6,8-C-dimethyl-flavone (2), along with the known compounds (±)-chilenine (3), (2R)-5,4'-dihydroxy-6-C-methyl-7-methoxy-flavanone (4), 5,4'-dihydroxy-6,8-di-C-methyl-7-methoxy-flavanone (5), noroxyhydrastinine (6), oxyhydrastinine (7) and 4',5'-dimethoxy-4-methyl-3'-oxo-(1,2,5,6-tetrahydro-4H-1,3-dioxolo-[4',5':4,5]-benzo[1,2-e]-1,2-oxazocin)-2-spiro-1'-phtalan (8). Compounds 3-8 have been reported from other sources, but this is the first report of their presence in H. canadensis extracts. A mass spectrometry based assay was employed to demonstrate bacterial efflux pump inhibitory activity against Staphylococcus aureus for 2, with an IC50 value of 180 ± 6 µM. This activity in addition to that of other bioactive compounds such as flavonoids and alkaloids, may explain the purported efficacy of H. canadensis for treatment of bacterial infections. Finally, this report includes high mass accuracy fragmentation spectra for all compounds investigated herein which were uploaded into the Global Natural Products Social molecular networking library and can be used to facilitate their future identification in H. canadensis or other botanicals.

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds, Including Some That Originate from Endophytic Bacteria.

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.

Metabolites from the endophytic [corrected] fungus Sporormiella minimoides isolated from Hintonia latiflora.

An extract of the solid cultures of Sporormiella minimoides (Sporormiaceae) isolated as an endophytic fungus from Hintonia latiflora (Rubiaceae), yielded three polyketides, 3,6-dimethoxy-8-methyl-1H,6H-benzo[de]isochromene-1,9-dione, 3-hydroxy-1,6,10-trimethoxy-8-methyl-1H,3H-benzo[de]isochromen-9-one, and 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone, along with three known compounds, corymbiferone, ziganein, and brocaenol B. Their structures were characterized by spectrometric and spectroscopic methods. So as to be consistent the literature reports, 3,6-dimethoxy-8-methyl-1H,6H-benzo[de]isochromene-1,9-dione and 3-hydroxy-1,6,10-trimethoxy-8-methyl-1H,3H-benzo[de]isochromen-9-one were given the trivial names of corymbiferone C and corymbiferan lactone E, respectively. All isolates were tested as potential human calmodulin (hCaM) inhibitors using the fluorescent biosensor hCaM V91C-mBBr, but only 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone quenched significantly the extrinsic fluorescence of this biosensor, with a dissociation constant (Kd) value of 1.55 µM. Refined docking analysis predicted that 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone could also be bound to hCaM at site I displaying hydrophobic interactions with Phe19 and 68, Met51, 71, and 72, and Ile52 and 63 residues.

(+)-Ascosalitoxin and vermelhotin, a calmodulin inhibitor, from an endophytic fungus isolated from Hintonia latiflora.

Chemical investigation of the endophytic MEXU 26343, isolated from the medicinal plant Hintonia latiflora, yielded the known polyketide vermelhotin (1) and a new salicylic aldehyde derivative, namely, 9S,11R-(+)-ascosalitoxin (2). The structure and absolute configuration of the new compound were established through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental optical rotation values. In addition, chemical transformations of 2 yielded suitable derivatives for NOESY and (1)H-(1)H NMR coupling constant analyses, which reinforce the stereochemical assignment. The potential affinity of 1 and 2 with (Ca(2+))(4)-hCaM in solution was measured using the fluorescent biosensor hCaM M124C-mBBr. The results showed that 1 bound to the protein with a dissociation constant (K(d)) of 0.25 ± 0.04 µM, close to that of chlorpromazine (K(d) = 0.64 ± 0.03 µM), a classical CaM inhibitor. The stoichiometry ratio of 1 to (Ca(2+))(4)-hCaM was 1:4, similar to other well-known CaM ligands.

Synthesis, biological evaluation, and docking studies of gigantol analogs as calmodulin inhibitors.

Several analogs of gigantol (1) were synthesized to evaluate their effect on the complexes Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-CaM sensitive phosphodiesterase 1 (PDE1). The compounds belong to four structural groups including, 1,2-diphenylethanes (2-11), diphenylmethanes (13-15), 1,3-diphenylpropenones (16-18), and 1,3-diphenylpropanes (20-22). In vitro enzymatic studies showed that all compounds except 11 inhibited the complex Ca(2+)-CaM-PDE1 with IC(50) values ranging from 9 to 146 µM. On the other hand, all analogs but 11, 12 and 15 quenched the extrinsic fluorescence of the CaM biosensor hCaM-M124C-mBBr to different extent, then revealing different affinities to CaM; their affinity constants (K(m)) values were in the range of 3-80 µM. Molecular modeling studies indicated that all these compounds bound to CaM at the same site that the classical inhibitors trifluoperazine (TFP) and chlorpromazine (CPZ). Some of these analogs could be worthy candidates for developing new anti-tumor, local anesthetics, antidepressants, antipsychotic, or smooth muscle relaxant drugs, with anti-CaM properties due to their good affinity to CaM and the straightforwardness of their synthesis. In addition they could be valuable tools for the study of Ca(2+)-CaM functions.