Dietary fiber-and antioxidant-enriched cookies prepared by using jackfruit rind powder and ascorbic acid.

The demand for dietary fiber-rich cookies has increased due to customer awareness about the importance of dietary fiber in human health. In addition, the urge of creating food sustainability has led to the need to reuse food by-products. In this study, dietary fiber-rich cookies were developed by incorporating jackfruit rind (JFR) powder, a by-product of jackfruit processing, as a replacement for wheat flour. The study aimed to evaluate the effects of different replacement levels (0, 10, 20, 30 and 40 %) on the proximate composition, physical properties and overall sensory acceptability of the cookies. While JFR powder addition led to a significant increase in dietary fiber and antioxidant (phenolics, flavonoids and carotenoids) contents of the cookies, the physical properties and overall acceptability of the cookies were adversely affected. The total dietary fiber and total phenolic content of the cookies at 40 % JFR powder addition were 5 and 5.5 times as much as those of the cookies with 0 % JFR powder addition. To address the adverse effects of JFR addition, various concentrations of ascorbic acid (AA), a dough improver agent, were added to the blended dough, and their effects on dough and cookie properties were investigated. With the addition of ascorbic acid at concentrations of 200 mg ascorbic acid per 100 g of the blend flour, the cookie density and cookie hardness reduced by 16 % and 31 %, respectively while the overall acceptability increased by 37 % compared to those of the cookies without ascorbic acid addition.

Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cß on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Functional and Promiscuity Studies of Three-Residue Cyclophane Forming Enzymes Show Nonnative C-C Cross-Linked Products and Leader-Dependent Cyclization.

Enzymes catalyzing peptide macrocyclization are important biochemical tools in drug discovery. The three-residue cyclophane-forming enzymes (3-CyFEs) are an emerging family of post-translational modifying enzymes that catalyze the formation of three-residue peptide cyclophanes. In this report, we introduce three additional 3-CyFEs, including ChlB, WnsB, and FnnB, that catalyze cyclophane formation on Tyr, Trp, and Phe, respectively. To understand the promiscuity of these enzymes and those previously reported (MscB, HaaB, and YxdB), we tested single amino acid substitutions at the three-residue motif of modification (Ω1X2X3, Ω1 = aromatic). Collectively, we observe that substrate promiscuity is observed at the Ω1 and X2 positions, but a greater specificity is observed for the X3 residue. Two nonnative cyclophane products were characterized showing a Phe-C3 to Arg-Cß and His-C2 to Pro-Cß cross-links, respectively. We also tested the leader dependence of selected 3-CyFEs and show that a predicted helix region is important for cyclophane formation. These results demonstrate the biocatalytic potential of these maturases and allow rational design of substrates to obtain a diverse array of genetically encoded 3-residue cyclophanes.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs.

Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C(sp2)-C(sp3) cross-link between the (hetero)aromatic ring to Cß. In addition to 3-CyFEs, we functionally validated an Fe(II)/α-ketoglutarate-dependent hydroxylase, resulting in ß-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Post-translational formation of strained cyclophanes in bacteria.

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence-function space.

Folding Kinetics of G-Quadruplexes: Duplex Stem Loops Drive and Accelerate G-Quadruplex Folding.

The folding kinetics is an important parameter affecting the relevance of DNA and RNA G-quadruplex (G4) structures in biological processes. Previous studies of the G4 folding kinetics mainly depended on fast measurement techniques. In previously available examples of G4s with loops up to three residues, the folding kinetics spanning several orders of magnitude has been reported, ranging from milliseconds to over 100 s. It is difficult to systematically and fundamentally understand the effect of multiple parameters, especially the loop properties, on the G4 folding kinetics, as the G4 fold is often altered when the sequence is varied. In this study, judicious choices of multiple parameters allowed us to bring various systems into the measurable window of a simple UV absorption technique. Using a well-controlled parallel-stranded G4 fold, we were able to systematically investigate the effect of seven different parameters of the environment and loop properties (temperature, K+ concentration, ionic strength, co-solute, loop length, loop sequence, and loop structure) on the G4 folding kinetics. We found that structured loops can drive up the G4 folding: for a long loop, the fast folding of a stem loop can guide the G4 folding and accelerate its folding kinetics by several orders of magnitude compared to an unstructured loop counterpart.

Polyphosphate-accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources.

Enhanced biological phosphorus removal (EBPR) is considered challenging in the tropics, based on a great number of laboratory-based studies showing that the polyphosphate-accumulating organism (PAO) Candidatus Accumulibacter does not compete well with glycogen accumulating organisms (GAOs) at temperatures above 25 °C. Yet limited information is available on the PAO community and the metabolic capabilities in full-scale EBPR systems operating at high temperature. We studied the composition of the key functional PAO communities in three full-scale wastewater treatment plants (WWTPs) with high in-situ EBPR activity in Singapore, their EBPR-associated carbon usage characteristics, and the relationship between carbon usage and community composition. Each plant had a signature community composed of diverse putative PAOs with multiple operational taxonomic units (OTUs) affiliated to Ca. Accumulibacter, Tetrasphaera spp., Dechloromonas and Ca. Obscuribacter. Despite the differences in community composition, ex-situ anaerobic phosphorus (P)-release tests with 24 organic compounds from five categories (including four sugars, three alcohols, three volatile fatty acids (VFAs), eight amino acids and six other carboxylic acids) showed that a wide range of organic compounds could potentially contribute to EBPR. VFAs induced the highest P release (12.0-18.2 mg P/g MLSS for acetate with a P release-to-carbon uptake (P:C) ratio of 0.35-0.66 mol P/mol C, 9.4-18.5 mg P/g MLSS for propionate with a P:C ratio of 0.38-0.60, and 9.5-17.3 mg P/g MLSS for n-butyrate), followed by some carboxylic acids (10.1-18.1 mg P/g MLSS for pyruvate, 4.5-11.7 mg P/g MLSS for lactate and 3.7-12.4 mg P/g MLSS for fumarate) and amino acids (3.66-7.33 mg P/g MLSS for glutamate with a P:C ratio of 0.16-0.43 mol P/mol C, and 4.01-7.37 mg P/g MLSS for aspartate with a P:C ratio of 0.17-0.48 mol P/mol C). P-release profiles (induced by different carbon sources) correlated closely with PAO community composition. High micro-diversity was observed within the Ca. Accumulibacter lineage, which represented the most abundant PAOs. The total population of Ca. Accumulibacter taxa was highly correlated with P-release induced by VFAs, highlighting the latter's importance in tropical EBPR systems. There was a strong link between the relative abundance of individual Ca. Accumulibacter OTUs and the extent of P release induced by distinct carbon sources (e.g., OTU 81 and amino acids, and OTU 246 and ethanol), suggesting niche differentiation among Ca. Accumulibacter taxa. A diverse PAO community and the ability to use numerous organic compounds are considered key factors for stable EBPR in full-scale plants at elevated temperatures.

A Dual-Specific Targeting Approach Based on the Simultaneous Recognition of Duplex and Quadruplex Motifs.

Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.

Joining of multiple duplex stems at a single quadruplex loop.

We report here the formation of stable DNA quadruplex-duplex hybrid complexes harboring multiple duplex stems within the same loop of a quadruplex structure. The folding topology of a two-stem quadruplex-duplex hybrid construct was validated using nuclear magnetic resonance (NMR) spectroscopy. This multi-stem incorporation principle is applicable at different loop positions of the same quadruplex construct and could be extended to three or more duplex stems, giving rise to a diverse range of possible structures. These multi-stem complexes offer new design principles for the assembly of DNA architectures. The potential existence of such complex motifs in genomic sequences could have biological implications and would represent novel targets for drug development.