Genomes on the edge: programmed genome instability in ciliates.

Ciliates are an ancient and diverse group of microbial eukaryotes that have emerged as powerful models for RNA-mediated epigenetic inheritance. They possess extensive sets of both tiny and long noncoding RNAs that, together with a suite of proteins that includes transposases, orchestrate a broad cascade of genome rearrangements during somatic nuclear development. This Review emphasizes three important themes: the remarkable role of RNA in shaping genome structure, recent discoveries that unify many deeply diverged ciliate genetic systems, and a surprising evolutionary "sign change" in the role of small RNAs between major species groups.

MicroRNA Clustering Assists Processing of Suboptimal MicroRNA Hairpins through the Action of the ERH Protein.

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.

Piwi-interacting RNAs protect DNA against loss during Oxytricha genome rearrangement.

Genome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, whereas the germline contributes DNA to the next sexual generation. Somatic nuclear development removes all transposons and other so-called "junk" DNA, which comprise ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally deleted regions leads to their retention in later generations. Our findings highlight small RNAs as powerful transgenerational carriers of epigenetic information for genome programming.

A sheep pangenome reveals the spectrum of structural variations and their effects on tail phenotypes.

Structural variations (SVs) are a major contributor to genetic diversity and phenotypic variations, but their prevalence and functions in domestic animals are largely unexplored. Here we generated high-quality genome assemblies for 15 individuals from genetically diverse sheep breeds using Pacific Biosciences (PacBio) high-fidelity sequencing, discovering 130.3 Mb nonreference sequences, from which 588 genes were annotated. A total of 149,158 biallelic insertions/deletions, 6531 divergent alleles, and 14,707 multiallelic variations with precise breakpoints were discovered. The SV spectrum is characterized by an excess of derived insertions compared to deletions (94,422 vs. 33,571), suggesting recent active LINE expansions in sheep. Nearly half of the SVs display low to moderate linkage disequilibrium with surrounding single-nucleotide polymorphisms (SNPs) and most SVs cannot be tagged by SNP probes from the widely used ovine 50K SNP chip. We identified 865 population-stratified SVs including 122 SVs possibly derived in the domestication process among 690 individuals from sheep breeds worldwide. A novel 168-bp insertion in the 5' untranslated region (5' UTR) of HOXB13 is found at high frequency in long-tailed sheep. Further genome-wide association study and gene expression analyses suggest that this mutation is causative for the long-tail trait. In summary, we have developed a panel of high-quality de novo assemblies and present a catalog of structural variations in sheep. Our data capture abundant candidate functional variations that were previously unexplored and provide a fundamental resource for understanding trait biology in sheep.

Unique microbiome in organic matter-polluted urban rivers.

Approximately half of the global annual production of wastewater is released untreated into aquatic environments, which results in worldwide organic matter pollution in urban rivers, especially in highly populated developing countries. Nonetheless, information on microbial community assembly and assembly-driving processes in organic matter-polluted urban rivers remains elusive. In this study, a field study based on water and sediment samples collected from 200 organic matter-polluted urban rivers of 82 cities in China and Indonesia is combined with laboratory water-sediment column experiments. Our findings demonstrate a unique microbiome in these urban rivers. Among the community assembly-regulating factors, both organic matter and geographic conditions play major roles in determining prokaryotic and eukaryotic community assemblies, especially regarding the critical role of organic matter in regulating taxonomic composition. Using a dissimilarity-overlap approach, we found universality in the dynamics of water and sediment community assembly in organic matter-polluted urban rivers, which is distinctively different from patterns in eutrophic and oligotrophic waters. The prokaryotic and eukaryotic communities are dominated by deterministic and stochastic processes, respectively. Interestingly, water prokaryotic communities showed a three-phase cyclic succession of the community assembly process before, during, and after organic matter pollution. Our study provides the first large-scale and comprehensive insight into the prokaryotic and eukaryotic community assembly in organic matter-polluted urban rivers and supports their future sustainable management.

The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes.

MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base pairing throughout the remainder of the stem, and a narrow stem-length preference of 35 ± 1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence yet processed more efficiently than natural pri-miRNAs.

Spatial Distribution, Bioconversion and Ecological Risk of PCBs and PBDEs in the Surface Sediment of Contaminated Urban Rivers: A Nationwide Study in China.

Surface sediments of polluted urban rivers can be a reservoir of hydrophobic persistent organic pollutants (POPs). In this study, we comprehensively assessed the contamination of two groups of POPs, that is, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), in 173 black-odorous urban rivers in China. Spatial distribution of PCBs and PBDEs showed similar patterns but very different contamination levels in surface sediments, that is, average concentrations of 10.73 and 401.16 ng/g dw for the ∑PCBs and ∑PBDEs, respectively. Tetra-/di-CBs and deca-BDE are major PCBs and PBDEs and accounted for 59.11 and 95.11 wt % of the ∑PCBs and ∑PBDEs, respectively. Compared with the persistence of PBDEs, the EF changes of chiral PCBs together with previous cultivation evidence indicated indigenous bioconversion of PCBs in black-odorous urban rivers, particularly the involvement of uncharacterized Dehalococcoidia in PCB dechlorination. Major PCB sources (and their relative contributions) included pigment/painting (25.36%), e-waste (22.92%), metallurgical industry (13.25%), and e-waste/biological degradation process (10.95%). A risk assessment indicated that exposure of resident organisms in urban river sediments to deca-/penta-BDEs could pose a high ecological risk. This study provides the first insight into the contamination, conversion and ecological risk of PCBs and PBDEs in nationwide polluted urban rivers in China.

A near complete genome for goat genetic and genomic research.

BACKGROUND: Goat, one of the first domesticated livestock, is a worldwide important species both culturally and economically. The current goat reference genome, known as ARS1, is reported as the first nonhuman genome assembly using 69× PacBio sequencing. However, ARS1 suffers from incomplete X chromosome and highly fragmented Y chromosome scaffolds. RESULTS: Here, we present a very high-quality de novo genome assembly, Saanen_v1, from a male Saanen dairy goat, with the first goat Y chromosome scaffold based on 117× PacBio long-read sequencing and 118× Hi-C data. Saanen_v1 displays a high level of completeness thanks to the presence of centromeric and telomeric repeats at the proximal and distal ends of two-thirds of the autosomes, and a much reduced number of gaps (169 vs. 773). The completeness and accuracy of the Saanen_v1 genome assembly are also evidenced by more assembled sequences on the chromosomes (2.63 Gb for Saanen_v1 vs. 2.58 Gb for ARS1), a slightly increased mapping ratio for transcriptomic data, and more genes anchored to chromosomes. The eight putative large assembly errors (1 to ~ 7 Mb each) found in ARS1 were amended, and for the first time, the substitution rate of this ruminant Y chromosome was estimated. Furthermore, sequence improvement in Saanen_v1, compared with ARS1, enables us to assign the likely correct positions for 4.4% of the single nucleotide polymorphism (SNP) probes in the widely used GoatSNP50 chip. CONCLUSIONS: The updated goat genome assembly including both sex chromosomes (X and Y) and the autosomes with high-resolution quality will serve as a valuable resource for goat genetic research and applications.

Highly Mineralized Biomimetic Polysaccharide Nanofiber Materials Using Enzymatic Mineralization.

Many biological high-performance composites, such as bone, antler, and crustacean cuticles, are composed of densely mineralized and ordered nanofiber materials. The mimicry of even simplistic bioinspired structures, i.e., of densely and homogeneously mineralized nanofibrillar materials with controllable mechanical performance, continues to be a grand challenge. Here, using alkaline phosphatase as an enzymatic catalyst, we demonstrate the dense, homogeneous, and spatially controlled mineralization of calcium phosphate nanostructures within networks of anionically charged cellulose nanofibrils (CNFs) and cationically charged chitin nanofibrils (ChNFs)-both emerging biobased nanoscale building blocks for sustainable high-performance materials design. Our study reveals that anionic CNFs lead to a more homogeneous nanoscale mineralization with very high mineral contents up to ca. 70 wt % with a transition from amorphous to crystalline deposits, while cationic ChNFs yield rod-like crystalline morphologies. The bone-inspired CNF bulk films exhibit a significantly increased stiffness, maintain good flexibility and translucency, and have a significant gain in wet state mechanical properties. The mechanical properties can be tuned both by the enzyme concentration and the mineralization time. Moreover, we also show a spatial control of the mineralization using kinetically controlled substrate uptake in a dialysis reactor, and by spatially selectively incorporating the enzyme into 2D printed filament patterns. The strategy highlights possibilities for spatial encoding of enzymes in tailored structures and patterns and programmed mineralization processes, promoting the potential application of mineralized CNF biomaterials with complex gradients for bone substitutes and tissue regeneration in general.

Organohalide-Respiring Bacteria in Polluted Urban Rivers Employ Novel Bifunctional Reductive Dehalogenases to Dechlorinate Polychlorinated Biphenyls and Tetrachloroethene.

Polluted urban river sediments could be a sink of persistent and toxic polychlorinated biphenyls (PCBs) in urban areas and provide desired growth niches for organohalide-respiring bacteria (OHRB). In this study, microcosms were set up with surface sediments of nationwide polluted urban rivers in China, of which 164 cultures could dechlorinate tetrachloroethene (PCE) to dichloroethenes (DCEs) and to vinyl chloride and/or ethene. Further in vivo tests showed extensive PCB dechlorination with different pathways in 135 PCE pregrown cultures. Taking reductive dechlorination of PCB180 (2345-245-CB) as an example, 121 and 14 cultures preferentially removed flanked para- and meta-chlorines, respectively. Strikingly, all in vitro assays with the 135 PCE pregrown cultures showed identical PCB dechlorination pathways with their living cultures, implying the involvement of bifunctional reductive dehalogenases (RDases) to dechlorinate both PCBs and PCE. Further 16S rRNA and RDase gene-based analyses, together with enantioselective dechlorination of chiral PCBs, suggested that Dehalococcoides and Dehalogenimonas in the 135 cultures largely employed distinctively different novel bifunctional RDases to catalyze PCB/PCE dechlorination. Quantitative assessment of the community assembly process with the modified stochasticity ratio (MST) indicated three different stages in enrichment of OHRB. The second stage, as the only one controlled by stochastic processes (MST > 0.5), required extra attention in monitoring community successional patterns to minimize stochastic variance for enriching the PCB/PCE-dechlorinating OHRB.

Raising the Bar in Aromatic Donor-Acceptor Interactions with Cyclic Trinuclear Gold(I) Complexes as Strong π-Donors.

Aromatic donor-acceptor interactions are of high importance in supramolecular chemistry, materials science and biology. Compared to other noncovalent interactions, such as hydrogen bonding, the binding is often weak. Here we show that strong donor-acceptor interactions between planar aromatics with binding free energies down to -10.1 kcal mol-1 and association constants of up to 2.34 × 107 L mol-1 for 1:1 complexes can be realized using cyclic trinuclear complexes of gold(I) with pyridinate, imidazolate, or carbeniate ligands. Data were obtained through NMR and UV/vis absorption spectroscopic studies and supported by quantum chemical calculations for a variety of acceptors. By using a specifically designed bridged naphthalene diimide-based acceptor with only one binding site, we furthermore show that a 1:2 (donor:acceptor) binding model is best suited to quantify the donor and acceptor/complex equilibrium. Scanning electron microscopy on selected donor-acceptor pairs shows crystalline supramolecular assemblies. We anticipate this study to be relevant for the future design of supramolecular systems and chemical sensors and the determination of binding energies between planar donors and acceptors.

Elastic and pH-Responsive Hybrid Interfaces Created with Engineered Resilin and Nanocellulose.

We investigated how a genetically engineered resilin fusion protein modifies cellulose surfaces. We characterized the pH-responsive behavior of a resilin-like polypeptide (RLP) having terminal cellulose binding modules (CBM) and showed its binding to cellulose nanofibrils (CNF). Characterization of the resilin fusion protein at different pHs revealed substantial conformational changes of the protein, which were observed as swelling and contraction of the protein layer bound to the nanocellulose surface. In addition, we showed that employment of the modified resilin in cellulose hydrogel and nanopaper increased their modulus of stiffness through a cross-linking effect.

Noncovalent Dispersion and Functionalization of Cellulose Nanocrystals with Proteins and Polysaccharides.

Native cellulose nanocrystals (CNCs) are valuable high quality materials with potential for many applications including the manufacture of high performance materials. In this work, a relatively effortless procedure was introduced for the production of CNCs, which gives a nearly 100% yield of crystalline cellulose. However, the processing of the native CNCs is hindered by the difficulty in dispersing them in water due to the absence of surface charges. To overcome these difficulties, we have developed a one-step procedure for dispersion and functionalization of CNCs with tailored cellulose binding proteins. The process is also applicable for polysaccharides. The tailored cellulose binding proteins are very efficient for the dispersion of CNCs due to the selective interaction with cellulose, and only small fraction of proteins (5-10 wt %, corresponds to about 3 µmol g(-1)) could stabilize the CNC suspension. Xyloglucan (XG) enhanced the CNC dispersion above a fraction of 10 wt %. For CNC suspension dispersed with carboxylmethyl cellulose (CMC) we observed the most long-lasting stability, up to 1 month. The cellulose binding proteins could not only enhance the dispersion of the CNCs, but also functionalize the surface. This we demonstrated by attaching gold nanoparticles (GNPs) to the proteins, thus, forming a monolayer of GNPs on the CNC surface. Cryo transmission electron microscopy (Cryo-TEM) imaging confirmed the attachment of the GNPs to CNC solution conditions.

Photoluminescent Nanocellulosic Film for Selective Hg2+ Ion Detection.

We developed a highly sensitive solid-state sensor for mercury detection by stabilizing red-sub-nanometric fluorescent gold nanoclusters (AuNC, 0.9 ± 0.1 nm diameter) with bovine serum albumin in a matrix composed of cellulose nanofibrils (CNF) (BSA-AuNC/CNF). The main morphological and optical features of the system were investigated via atomic force/transmission electron microscopy and UV-Vis/fluorescence spectroscopy. The hybrid film (off-white and highly transparent) showed strong photoluminescene under UV irradiation. The latter is assigned to the AuNC, which also increase the ductility of the emitting film, which was demonstrated for high sensitivity Hg2+ detection. When used as a sensor system, following AuNC printing on CNF hybrid films, a limit of detection <10 nM was confirmed. What is more, nanocellulose films have a high pore structure and selective separation properties, showcasing a wide range of potential applications in many fields such as water treatment and oil-water separation.

Quantitative determination of prothioconazole in wheat grain, soybean, and pond water based on a polyclonal antibody.

Prothioconazole and its metabolite are considered a potential threat to human health and environmental safety. Thus, the development of a sensitive and rapid detection method for prothioconazole is crucial to ensure the safety of agricultural products. In this study, a new hapten of prothioconazole was designed and synthesized, and a selective polyclonal antibody with high affinity against prothioconazole was produced, which was obtained from immunized New Zealand white rabbits. Based on the polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) were developed for detecting prothioconazole pesticides. Under optimized experimental conditions, the limit of quantification (LOQ) values for ic-CLEIA and ic-ELISA were 1.8 and 10.7 ng mL-1, respectively. The results demonstrated that the sensitivity (LOQ) achieved by ic-CLEIA was more than five times higher compared to that obtained with ic-ELISA. In addition, the recoveries obtained by adding standard prothioconazole to wheat grain, soybean, and pond water samples were in the range of 81.9 to 104.7% for ic-ELISA and 89.0 to 118.0% for ic-CLEIA.

(-)-Epicatechin ameliorates type 2 diabetes mellitus by reshaping the gut microbiota and Gut-Liver axis in GK rats.

As one of the most abundant plant polyphenols in the human diet, (-)-epicatechin (EC) can improve insulin sensitivity and regulate glucose homeostasis. However, the primary mechanisms involved in EC anti-T2DM benefits remain unclear. The present study explored the effects of EC on the gut microbiota and liver transcriptome in type 2 diabetes mellitus (T2DM) Goto-Kakizaki rats for the first time. The findings showed that EC protected glucose homeostasis, alleviated systemic oxidative stress, relieved liver damage, and increased serum insulin. Further investigation showed that EC reshaped gut microbiota structure, including inhibiting the proliferation of lipopolysaccharide (LPS)-producing bacteria and reducing serum LPS. In addition, transcriptome analysis revealed that the insulin signaling pathway may be the core pathway of the EC anti-T2DM effect. Therefore, EC may modulate the gut microbiota and liver insulin signaling pathways by the gut-liver axis to alleviate T2DM. As a diet supplement, EC has promising potential in T2DM prevention and treatment.

RNA-mediated epigenetic regulation of DNA copy number.

Increasing evidence suggests that parentally supplied RNA plays crucial roles during eukaryotic development. This epigenetic contribution may regulate gene expression from the earliest stages. Although present in a variety of eukaryotes, maternally inherited characters are especially prominent in ciliated protozoa, in which parental noncoding RNA molecules instruct whole-genome reorganization. This includes removal of nearly all noncoding DNA and sorting the remaining fragments, producing extremely gene-rich somatic genomes. Chromosome fragmentation and extensive replication produce variable DNA copy numbers in the somatic genome. Understanding the forces that drive and regulate copy number change is fundamental. We show that RNA molecules present in parental cells during sexual reproduction can regulate chromosome copy number in the developing nucleus of the ciliate Oxytricha. Experimentally induced changes in RNA abundance can both increase and decrease the levels of corresponding DNA molecules in progeny, demonstrating epigenetic inheritance of chromosome copy number. These results suggest that maternal RNA, in addition to controlling gene expression or DNA processing, can also program DNA amplification levels.

Tuning the water interactions of cellulose nanofibril hydrogels using willow bark extract.

Cellulose nanofibrils (CNFs) are increasingly used as precursors for foams, films and composites, where water interactions are of great importance. In this study, we used willow bark extract (WBE), an underrated natural source of bioactive phenolic compounds, as a plant-based modifier for CNF hydrogels, without compromising their mechanical properties. We found that the introduction of WBE into both native, mechanically fibrillated CNFs and TEMPO-oxidized CNFs increased considerably the storage modulus of the hydrogels and reduced their swelling ratio in water up to 5-7 times. A detailed chemical analysis revealed that WBE is composed of several phenolic compounds in addition to potassium salts. Whereas the salt ions reduced the repulsion between fibrils and created denser CNF networks, the phenolic compounds - which adsorbed readily on the cellulose surfaces - played an important role in assisting the flowability of the hydrogels at high shear strains by reducing the flocculation tendency, often observed in pure and salt-containing CNFs, and contributed to the structural integrity of the CNF network in aqueous environment. Surprisingly, the willow bark extract exhibited hemolysis activity, which highlights the importance of more thorough investigations of biocompatibility of natural materials. WBE shows great potential for managing the water interactions of CNF-based products.

Optimization of Dry-Jet Wet Spinning of Regenerated Cellulose Fibers Using [mTBDH][OAc] as a Solvent.

Superbase-based ionic liquids (ILs) have demonstrated excellent dissolution capability for cellulose, and employing the dry-jet wet spinning process, high-tenacity regenerated textile fibers have been made. Among a range of superbase-based ILs, [mTBDH][OAc] exhibited not only good spinnability but also exceptional recyclability, making it highly suitable for a closed-loop production of regenerated cellulose fibers. To further optimize the spinning process, we investigated the influence of the cellulosic raw materials and the IL with residual water on spinnability and fiber properties. In addition, single-filament spinning and multifilament spinning using spinnerets with different hole densities were investigated to reveal the upscaling challenges of the dry-jet wet spinning process. The air gap conditions, for example, temperature and moisture concentration were simulated using COMSOL multiphysics. The results indicate that the presence of a small amount of water (3 wt%) in the IL has a positive effect on spinnability, while the mechanical properties of the fibers remain unchanged.

Exploration of the Nuclear Proteomes in the Ciliate Oxytricha trifallax.

Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in Oxytricha trifallax, in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus.