Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe ...a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.
Black walnuts (Juglans nigra L.) are highly valued for producing phenolic-enriched nuts. The objectives of this study were to identify and characterize the phenolic contents of 11 different black ...walnut cultivars and compare the levels of these phenolics between black walnuts and English walnut (Juglans regia L.). Totally, 16 phenolics including phenolic acids, flavonoids, and catechins were identified in the black walnut kernels, with ellagic acid predominating over the other phenolics. Significant differences were noted for the levels of quinic acid, gallic acid, 1,3,6-trigalloylglucose, catechin, and penta-O-galloyl-β-d-glucose between the studied black walnuts and English walnut. Through principal component analysis, 51.54% of the variance in the phenolic data was explained. The hierarchical cluster analysis results showed three groups to which each walnut sample belongs. Most of the phenolics identified in this study have been reported to exert potential health-promoting activities. The findings of this study will provide critical information for consumers, nutritional therapy practitioners, researchers, and producers.
The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response ...to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca
-imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.
Our recent studies have demonstrated multiple health-promoting benefits from black walnut kernels. These biological functions of black walnuts are likely associated with their bioactive constituents. ...Characterization of phenolic compounds found in black walnut could point out underexplored bioactive activities of black walnut extracts and promote the development of novel applications of black walnut and its by-products. In the present study, we assessed bioactivity profiles of phenolic compounds identified in the kernels of black walnuts using a high-throughput screening (HTS) approach. Black walnut phenolic compounds were evaluated in terms of their total antioxidant capacity, antioxidant response element (ARE) induction, and anticancer activities. The anticancer activities were identified by evaluating the effects of the phenolic compounds on the growth of the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). Out of 16 phenolic compounds tested, several compounds (penta-
-galloyl-
-d-glucose, epicatechin gallate, quercetin, (-)-epicatechin, rutin, quercetin 3-
-d-glucoside, gallic acid, (+)-catechin, ferulic acid, syringic acid) exerted antioxidant activities that were significantly higher compared to Trolox, which was used as a control. Two phenolic compounds, penta-
-galloyl-
-d-glucose and quercetin 3-
-d-glucoside, exhibited antiproliferative activities against both the tumorigenic alveolar epithelial cells (A549) and non-tumorigenic lung fibroblast cells (MRC-5). The antioxidant activity of black walnut is likely driven not only by penta-
-galloyl-
-d-glucose but also by a combination of multiple phenolic compounds. Our findings suggested that black walnut extracts possibly possess anticancer activities and supported that penta-
-galloyl-
-d-glucose could be a potential bioactive agent for the cosmetic and pharmaceutical industries.
Bacterial transposons propagate through either non-replicative (cut-and-paste) or replicative (copy-and-paste) pathways, depending on how the mobile element is excised from its donor source. In the ...well-characterized E. coli transposon Tn7, a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each transposon end during the excision step. Whether a similar pathway is involved for RNA-guided transposons, in which CRISPR-Cas systems confer DNA target specificity, has not been determined. Here, we apply long-read, population-based whole-genome sequencing (WGS) to unambiguously resolve transposition products for two evolutionarily distinct transposon types that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results show that RNA-guided transposon systems lacking functional TnsA primarily undergo copy-and-paste transposition, generating cointegrate products that comprise duplicated transposon copies and genomic insertion of the vector backbone. Finally, we report natural and engineered transposon variants encoding a TnsAB fusion protein, revealing a novel strategy for achieving RNA-guided transposition with fewer molecular components.
Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and ...challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
As a critical issue in drug development and postmarketing safety surveillance, drug-induced liver injury (DILI) leads to failures in clinical trials as well as retractions of on-market approved ...drugs. Therefore, it is important to identify DILI compounds in the early-stages through in silico and in vivo studies. It is difficult using conventional safety testing methods, since the predictive power of most of the existing frameworks is insufficiently effective to address this pharmacological issue. In our study, we employ a natural language processing (NLP) inspired computational framework using convolutional neural networks and molecular fingerprint-embedded features. Our development set and independent test set have 1597 and 322 compounds, respectively. These samples were collected from previous studies and matched with established chemical databases for structural validity. Our study comes up with an average accuracy of 0.89, Matthews’s correlation coefficient (MCC) of 0.80, and an AUC of 0.96. Our results show a significant improvement in the AUC values compared to the recent best model with a boost of 6.67%, from 0.90 to 0.96. Also, based on our findings, molecular fingerprint-embedded featurizer is an effective molecular representation for future biological and biochemical studies besides the application of classic molecular fingerprints.
In this study, we assessed the anti-inflammatory properties of spent coffee grounds. Methanolic extracts of spent coffee grounds obtained from 3 Arabica cultivars possess compounds that exerted ...inhibitory effects on the secretion of inflammatory mediators (TNF-α, IL-6, and IL-10) induced by a human pro-monocytic cell line differentiated with PMA and stimulated with lipopolysaccharide (LPS). Our results indicated that the cytokine suppressive activities of the spent coffee ground (SCG) extracts were different among coffee cultivars tested. Hawaiian Kona extracts exhibited inhibitory effects on the expression of 3 examined cytokines, Ethiopian Yirgacheffe extracts reduced the secretion of TNF-α and IL-6, and Costa Rican Tarrazu extracts decreased the secretion of IL-6 only. Untargeted metabolomics analyses of SCG extracts led to the putative identification of 26 metabolites with known anti-inflammatory activities. Multiple metabolites (i.e., chrysin, daidzein, eugenol, naringenin, naringin, oxyresveratrol, pectolinarin, resveratrol, tectochrysin, theaflavin, vanillic acid, and vitexin rhamnoside) identified in the SCGs represent possible novel anti-inflammatory compounds. Of the 26 identified metabolites, the 12 compounds that had high relative intensities in all of the extracts were successfully quantified using liquid chromatography-tandem mass spectrometry analyses. Results from the targeted analyses indicated that caffeine and 5-caffeoylquinic acid (CQA) were the most abundant compounds in the SCG extracts. The contents of caffeine ranged from 0.38 mg/g (Ethiopian Yirgacheffe) - 0.44 mg/g (Costa Rican Tarrazu), whereas 5-CQA concentrations were in the range of 0.24 mg/g (Costa Rican Tarrazu) - 0.34 mg/g (Ethiopian Yirgacheffe). The presence of multiple anti-inflammatory compounds in SCGs provides a promising natural source for cosmetic and pharmaceutical industries.
Children are particularly vulnerable to many classes of the volatile organic compounds (VOCs) detected in indoor environments. The negative health impacts associated with chronic and acute exposures ...of the VOCs might lead to health issues such as genetic damage, cancer, and disorder of nervous systems. In this study, 40 VOCs including aldehydes and ketones, aliphatic hydrocarbons, esters, aromatic hydrocarbons, cyclic terpenes, alcohols, and glycol ethers were identified and qualified in different locations at the University of Missouri (MU) Child Development Laboratory (CDL) in Columbia, Missouri. Our results suggested that the concentrations of the VOCs varied significantly among classrooms, hallways, and playground. The VOCs emitted from personal care and cleaning products had the highest indoor levels (2‐ethylhexanol‐1, 3‐carene, homomenthyl salicylate with mean concentration of 5.15 µg/m3, 1.57 µg/m3, and 1.47 µg/m3, respectively). A cancer risk assessment was conducted, and none of the 95th percentile dose estimates exceeded the age‐specific no significant risk levels (NSRL) in all classrooms. Dimensionless toxicity index scores were calculated for all VOCs using a novel web‐based framework called Toxicological Prioritization Index (ToxPi), which integrates multiple sources of toxicity data. According to the method, homomenthyl salicylate, benzothiazole, 2‐ethylhexyl salicylate, hexadecane, and tridecane exhibited diverse toxicity profiles and ranked as the five most toxic indoor VOCs. The findings of this study provide critical information for policy makers and early education professionals to mitigate the potentially negative health impacts of indoor VOCs in the childcare facilities.
The rise of hydraulic fracturing and unconventional oil and gas (UOG) exploration in the United States has increased public concerns for water contamination induced from hydraulic fracturing fluids ...and associated wastewater spills. Herein, we collected surface and groundwater samples across Garfield County, Colorado, a drilling-dense region, and measured endocrine bioactivities, geochemical tracers of UOG wastewater, UOG-related organic contaminants in surface water, and evaluated UOG drilling production (weighted well scores, nearby well count, reported spills) surrounding sites. Elevated antagonist activities for the estrogen, androgen, progesterone, and glucocorticoid receptors were detected in surface water and associated with nearby shale gas well counts and density. The elevated endocrine activities were observed in surface water associated with medium and high UOG production (weighted UOG well score-based groups). These bioactivities were generally not associated with reported spills nearby, and often did not exhibit geochemical profiles associated with UOG wastewater from this region. Our results suggest the potential for releases of low-saline hydraulic fracturing fluids or chemicals used in other aspects of UOG production, similar to the chemistry of the local water, and dissimilar from defined spills of post-injection wastewater. Notably, water collected from certain medium and high UOG production sites exhibited bioactivities well above the levels known to impact the health of aquatic organisms, suggesting that further research to assess potential endocrine activities of UOG operations is warranted.
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•Tested endocrine bioactivities in water near unconventional oil/gas (UOG) operations•Antagonist activities increased near and correlated with density of UOG operations.•Geochemistry supports UOG produced water contamination at some sites.•Organic chemistry analysis suggests UOG contamination at other sites.•UOG-impacted samples disrupted nuclear receptors at environmentally relevant concentrations.
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