Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using Endophytic Bacteria Isolated from Solanum elaeagnifolium Stems

Seven culturable bacterial isolates, obtained from the internal stem tissues of Solanum elaeagnifolium and successfully colonizing the internal stem tissues of tomato cv. Rio Grande, were screened for their in vivo antifungal activity against Fusarium oxysporum f.sp. lycopersici (FOL) and their growth‐promoting potential on tomato plants. SV101 and SV104 isolates, assessed on pathogen‐challenged tomato plants led to a significant decrease (77–83%) in Fusarium wilt severity and vascular browning extent (76%), as compared to the inoculated and untreated control. Isolates enhanced growth parameters on pathogen‐challenged and unchallenged tomato plants. SV104 and SV101 isolates were most effective in suppressing disease and enhancing plant growth. These two isolates were identified as Bacillus sp. str. SV101 ( KU043040 ) and B. tequilensis str. SV104 ( KU976970 ). They displayed antifungal activity against FOL; pathogen growth was inhibited by 64% and an inhibition zone (11.50 and 19.75 mm) against FOL could be formed using whole cell suspensions. SV101 and SV104 extracellular metabolites also inhibited FOL growth by 20 and 55%, respectively, as compared to control. B. tequilensis str. SV104 was shown to produce protease, chitinase, pectinase, IAA and siderophores. Bacillus sp. str. SV101 displayed pectinase activity and was found to be an IAA‐producing and phosphate‐solubilizing agent. To our knowledge, this is the first study reporting on S. elaeagnifolium use as a potential source of potent biocontrol and plant growth‐promoting agents.     

Parallel synthesis of chiral pentaamines and pyrrolidine containing bis-heterocyclic libraries. Multiple scaffolds with multiple building blocks: A double diversity for the identification of new antitubercular compounds

Combinatorial chemistry offers a unique opportunity for the synthesis and screening of large numbers of compounds and significantly enhances the prospect of finding new drugs. Collaborative efforts with the Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF), have led to the identification of submicromolar novel antitubercular hits. Chiral pentaamines and bis-heterocyclic compounds with 90–100% inhibition against Mycobacterium tuberculosis strain H₃₇R_v were identified. Some of the identified compounds are more active than the existing drug ethambutol.     

On resin amino acid side chain attachment strategy for the head to tail synthesis of new glutamine containing gramicidin-S analogs and their antimicrobial activity

The alarming increase in infections caused by multiple drug resistant bacteria including methicillin-resistant Staphylococcus aureus has prompted a desperate search for new antimicrobials. Augmenting the discoveries of completely new scaffolds with antimicrobial activity are efforts aimed at modifying existing molecules to optimize activity or reduce toxicity. We report herein the parallel solid-phase synthesis of analogues of the cationic antimicrobial peptide gramicidin S (GS) using amino acid side chain attachment strategy. The ornithine (Orn) residues were replaced by glutamine (Gln) and the aromatic d-phenylalanine (Phe) were replaced by different aromatic d-amino acids. Additional Gln containing GS analogues with all the possible combinations of the hydrophobic amino acids valine and leucine were also synthesized. In this work we also report the antibacterial activity of these analogs against several clinically-important drug-resistant Gram-positive and Gram-negative pathogens.     

Integrating computational and mixture-based screening of combinatorial libraries

Mixture-based synthetic combinatorial library (MB-SCL) screening is a well-established experimental approach for rapidly retrieving structure–activity relationships (SAR) and identifying hits. Virtual screening is also a powerful approach that is increasingly being used in drug discovery programs and has a growing number of successful applications. However, limited efforts have been made to integrate both techniques. To this end, we combined experimental data from a MB-SCL of bicyclic guanidines screened against the κ-opioid receptor and molecular similarity methods. The activity data and similarity analyses were integrated in a biometric analysis–similarity map. Such a map allows the molecules to be categorized as actives, activity cliffs, low similarity to the reference compounds, or missed hits. A compound with IC₅₀ = 309 nM was found in the “missed hits” region, showing that active compounds can be retrieved from a MS-SCL via computational approaches. The strategy presented in this work is general and is envisioned as a general-purpose approach that can be applied to other MB-SCLs.     

Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato

Nine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37?mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.     

A versatile access to new macrocyclic oligoheterocycles (MOH)

An efficient and straightforward methodology for the parallel solid-phase synthesis of a variety of new macrocyclic oligoheterocycles is described. Exhaustive reduction of resin-bound cyclic polyamides using borane generates polyamines. Treatment of separated pairs of amines with a variety of bifunctional reagents provides, following cleavage from the solid support, the desired macrocyclic oligoheterocyclic (MOH) compounds in good yields and purities.     

Small molecule functional analogs of peptides that inhibit λ site-specific recombination and bind Holliday junctions

Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors.     

Solid phase synthesis of mixture-based acyclic and heterocyclic small molecule combinatorial libraries from resin-bound polyamides.

The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described. Starting with a "toolbox" of various chemical transformations, including alkylations, reductions, acylations, and the use of a variety of bifunctional reagents, the "libraries from libraries" concept has been expanded to encompass the development of more than fifty positional scanning combinatorial libraries each composed of tens of thousands of low molecular weight acyclic and heterocyclic compounds.     

Identification of potent and highly selective chiral tri-amine and tetra-amine mu opioid receptors ligands: an example of lead optimization using mixture-based libraries

The generation of chiral polyamine libraries has been successfully accomplished in our laboratory following exhaustive reduction of resin-bound peptides. Herein, we report the synthesis and screening results of a positional scanning mixture-based library of chiral hepta-amines in a radioreceptor assay for the opioid receptor. The positional scanning hepta-amine library was generated by the exhaustive reduction of a library of 34,012,070 hexapeptides. Following screening of the entire library, combinations of the most active functionalities found at each position were used to synthesize and screen 40 individual hepta-amines and served as starting 'hits' for further SAR studies. The individual compounds showed IC(50) values ranging from 14 to 345 nM. As might be anticipated by the known studies of mu opiate antagonists, the identified active hepta-amines possessed aromatic rings derived from phenylalanine and tyrosine amino acid side chains. Following SAR studies, a truncation analog, reduced and permethylated YYF-NH(2), was found to be highly active (0.5 nM) as a selective mu antagonist in the guinea pig ileum bioassay.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.