cGAS-STING signaling pathway in gastrointestinal inflammatory disease and cancers.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway has emerged as a key DNA-sensing machinery in innate immunity. Activation of cGAS-STING signaling pathway mediates the production of interferons and proinflammatory cytokines. Although cGAS-STING signaling pathway shows critical function in the maintenance of gut homeostasis, overactive cGAS-STING signaling pathway leads to gastrointestinal (GI) inflammation. Harnessing the effect and mechanism of the cGAS-STING signaling pathway could be beneficial for the development of novel strategies for the treatment of GI diseases. This review presents recent advances regarding the role of cGAS-STING signaling pathway in GI inflammatory disease and cancers and describes perspective therapeutic strategies targeting the signaling pathway.

A one-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have been considered a next-generation molecular diagnosis tool. Single-readout mode has been extensively employed in massive CRISPR/Cas12a-based biosensors. In this work, we propose a one-tube dual-readout biosensor (CRISAT) for the first time for the detection of ultrasensitive nucleic acids and non-nucleic acids developed by harnessing CRISPR-ALP tandem assay. In the presence of a target, Cas12a is activated to randomly cut the single-stranded hyDNA sequence of MB@hyDNA-cALP, thus releasing abundant alkaline phosphatase (ALP) in the supernatant solution. By using 4-aminophenol phosphate as the substrate of ALP, p-aminophenol is produced, which then reacts with N-[3-(trimethoxysilyl)propyl]ethylenediamine or diethylenetriamine to generate silicon-containing polymer carbon dots (Si PCDs) or polymer carbon dots (PCDs) in situ, which can be observed by the naked eye or detected using a fluorescent device in the same solution. Using this strategy, a fluorescence and colorimetry dual-readout nanoplatform for CRISPR-based biosensors can be rationally developed. We ascertain the applicability of CRISAT by detecting the SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can also be employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work shows great potential for the detection of nucleic acids and non-nucleic acids.

CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.

CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.

In Vitro Infant Faecal Fermentation of Low Viscosity Barley ß-Glucan and Its Acid Hydrolyzed Derivatives: Evaluation of Their Potential as Novel Prebiotics.

Barley contains high level of ß-1,3-1,4-glucans (BBGs) which can be fermented by microbes and are a potential prebiotic. In the present study, native BBG with low viscosity and a MW of 319 kDa was depolymerized by acid hydrolysis to produce a series of four structurally characterized fragments with MWs ranging from 6⁻104 kDa. In vitro fermentation of these BBG samples by infant faecal microbiome was evaluated using a validated deep-well plate protocol as parallel miniature bioreactors. Microbial taxa were identified using 16S amplicon sequencing after 40 h of anaerobic fermentation. Bioinformatics analysis including diversity indexes, predicted metagenomic KEGG functions and predicted phenotypes were performed on the sequenced data. Short chain fatty acids and dissolved ammonia were quantified and the SCFAs/NH3 ratio was used to evaluate the eubiosis/dysbiosis potential. Correlation analysis showed that most of the parameters investigated showed a parabolic function instead of a monotonous function with the BBG samples having different MWs. Among the five BBGs, it was concluded that BBG with an intermediate MW of 28 kDa is the most promising candidate to be developed as a novel prebiotic.

Biphasic reduction of histone H3 phosphorylation in response to N-nitroso compounds induced DNA damage.

BACKGROUND: N-nitroso compounds (NOC) can cause cancers in a wide variety of animal species, and many of them are also potential human carcinogens. However, their underlying genotoxic mechanisms occurred within the context of chromatin, such as aberrant histone modifications, remained elusive. METHODS: We investigated the dynamic landscapes of histone modifications after N-nitroso compound N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitroso-urea (MNU) exposure. Among the altered histone modifications, we also investigated the control mechanisms of histone H3 phosphorylation changes and its possible implications on transcriptional repression. RESULTS: Significantly, we find a specific biphasic reduction of histone H3 phosphorylation at serine 10 (H3S10ph) and serine 28 (H3S28ph), and a rapid decrease of histone H4 acetylation upon MNNG and MNU exposure. Further investigations reveal that the first hypophosphorylation of H3 occurs in a poly(ADP-ribosyl)ation enzyme PARP-1 (Poly(ADP-Ribose) Polymerase 1) dependent manner, whereas the second decline of H3 phosphorylation is at least partially under the control of histone kinase VRK1 (vaccinia-related kinase 1) and dependent on the tumor suppressor protein p53. In addition, DNA damage induced down-regulation of H3S10/S28 phosphorylation also functions in transcriptional repression of genes, such as cell-cycle regulators. CONCLUSIONS: Alkylating damage induced by NOC elicits a biphasic reduction of histone H3 phosphorylation with distinct control mechanisms, which is contributing to DNA damage responses such as the repair-facilitated transcriptional repression. GENERAL SIGNIFICANCE: Identification of the dynamic changes and underlying mechanisms of histone modifications upon NOC exposure would be of great help in understanding the epigenetic regulations of NOC induced DNA damage responses.

Unique sequence characteristics account for good DGGE separation of almost full-length 18S rDNAs.

A major limiting factor for DGGE-based microbial community studies is that the fragments should not be much longer than 500 bp for successful analysis. However, relatively high-resolution was achieved based on DGGE of the long 18S rDNA fragment (>1500 bp), which might be surprising due to the known decrease in DGGE resolution of DNA molecules with large melted regions. A unique sequence characteristic was found in a specific region (ca. 275 bp, named the NS1-end region) of 18S rDNAs, and fungal communities separated from Hong Qu glutinous rice wine brewing system was used to reveal the relationship between high resolution capacity and the unique sequence characteristics. The results showed that DGGE separation of the long 18S rDNA fragments depended on their NS1-end regions. The region is composed of a sequence-variable and short-length GC-poor region (ca. 160 bp) and a GC-rich region (ca. 110 bp), which contribute to the high resolution capacity achieved for DGGE of the long 18S rDNA fragments. Thus DGGE of the long 18S rDNA fragment is recommended as a target fragment for studies of fungal communities whose 18S rDNAs possess similar sequence characteristics. Good resolution and almost full-length 18S rDNA sequences can thus be obtained to provide more accurate and reliable analysis of fungal communities. Since more sequences are obtained directly from the PCR product through the long rDNA fragment approach, this is a convenient and effective approach for sequence-based analysis without using other complementary methods such as an rDNA clone library method.

A one-pot CRISPR-RCA strategy for ultrasensitive and specific detection of circRNA.

Accurate and precise detection of circular RNA (circRNA) is imperative for its clinical use. However, the inherent challenges in circRNA detection, arising from its low abundance and potential interference from linear isomers, necessitate innovative solutions. In this study, we introduce, for the first time, the application of the CRISPR/Cas12a system to establish a one-pot, rapid (30 minutes to 2 hours), specific and ultrasensitive circRNA detection strategy, termed RETA-CRISPR (reverse transcription-rolling circle amplification (RT-RCA) with the CRISPR/Cas12a). This method comprises two steps: (1) the RT-RCA process of circRNA amplification, generating repeat units containing the back-splicing junction (BSJ) sequences; and (2) leveraging the protospacer adjacent motif (PAM)-independent Cas12a/crRNA complex to precisely recognize target sequences with BSJ, thereby initiating the collateral cleavage activity of Cas12a to generate a robust fluorescence signal. Remarkably, this approach exhibits the capability to detect circRNAs at a concentration as low as 300 aM. The sensor has been successfully employed for accurate detection of a potential hepatocellular carcinoma biomarker hsa_circ_0001445 (circRNA1445) in various cell lines. In conclusion, RETA-CRISPR seamlessly integrates the advantages of exponential amplification reaction and the robust collateral cleavage activity of Cas12a, positioning it as a compelling tool for practical CRISPR-based diagnostics.

CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.

Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a.

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

Geospatial characterization of rural settlements and potential targets for revitalization by geoinformation technology.

To better implement the Strategy of Rural Revitalization, it is essential to characterize the rural settlements and understand their roles in the socio-environmental interactive system. This paper is hence aimed at achieving such a study using different spatial analysis such as kernel density and spatial autocorrelation (SA) and modeling approaches, e.g., simple and multiple linear regression analyses taking Jiangxi, a province in China as an example. Remote sensing, topographic and socioeconomic data were employed for this purpose. Through these analyses, it is found that the rural settlements in the study area appear to have a spatial distribution pattern of "dense north and sparse south" as an "F" type, and are quantitatively characterized as low elevations, flat terrain, high river and road densities, rich cultivated land resources and susceptible to the impact of urban radiation with a R2 of 0.520-0.748. Based on this understanding, a new inequality evaluation indicator of rural development, i.e., socio-environmental evaluation index (SEI), was developed. Areas with SEI lower than 0.40 should be given a priority to implement the revitalization strategy in the province. This index can also be extended to study of the imbalance of rural development in other regions and countries.

miR-377-3p-Mediated EGR1 Downregulation Promotes B[a]P-Induced Lung Tumorigenesis by Wnt/Beta-Catenin Transduction.

Polycyclic aromatic hydrocarbons (PAHs), particularly benzo[a]pyrene (B[a]P), found in cigarette smoke and air pollution, is an important carcinogen. Nevertheless, early molecular events and related regulatory effects of B[a]P-mediated cell transformation and tumor initiation remain unclear. This study found that EGR1 was significantly downregulated during human bronchial epithelial cell transformation and mice lung carcinogenesis upon exposure to B[a]P and its active form BPDE, respectively. In contrast, overexpression of EGR1 inhibited the BPDE-induced cell malignant transformation. Moreover, miR-377-3p was strongly enhanced by BPDE/B[a]P exposure and crucial for the inhibition of EGR1 expression by targeting the 3'UTR of EGR1. MiR-377-3p antagomir reversed the effect of EGR1 downregulation in cell malignant transformation and tumor initiation models. Furthermore, the B[a]P-induced molecular changes were evaluated by IHC in clinical lung cancer tissues and examined with a clinic database. Mechanistically, EGR1 inhibition was also involved in the regulation of Wnt/ß-catenin transduction, promoting lung tumorigenesis following B[a]P/BPDE exposure. Taken together, the results demonstrated that bBenzo[a]pyrene exposure might induce lung tumorigenesis through miR-377-3p-mediated reduction of EGR1 expression, suggesting an important role of EGR1 in PAHs-induced lung carcinogenesis.

STAT3 activates MSK1-mediated histone H3 phosphorylation to promote NFAT signaling in gastric carcinogenesis.

Epigenetic abnormalities contribute significantly to the development and progression of gastric cancer. However, the underlying regulatory networks from oncogenic signaling pathway to epigenetic dysregulation remain largely unclear. Here we showed that STAT3 signaling, one of the critical links between inflammation and cancer, acted as a control pathway in gastric carcinogenesis. STAT3 aberrantly transactivates the epigenetic kinase mitogen- and stress-activated protein kinase 1 (MSK1), thereby phosphorylating histone H3 serine10 (H3S10) and STAT3 itself during carcinogen-induced gastric tumorigenesis. We further identified the calcium pathway transcription factor NFATc2 as a novel downstream target of the STAT3-MSK1 positive-regulating loop. STAT3 forms a functional complex with MSK1 at the promoter of NFATc2 to promote its transcription in a H3S10 phosphorylation-dependent way, thus affecting NFATc2-related inflammatory pathways in gastric carcinogenesis. Inhibiting the STAT3/MSK1/NFATc2 signaling axis significantly suppressed gastric cancer cell proliferation and xenograft tumor growth, which provides a potential novel approach for gastric carcinogenesis intervention by regulating aberrant epigenetic and transcriptional mechanisms.

Synbiotic-driven improvement of metabolic disturbances is associated with changes in the gut microbiome in diet-induced obese mice.

OBJECTIVE: The gut microbiota is an important influencing factor of metabolic health. Although dietary interventions with probiotics, prebiotics, and synbiotics can be effective means to regulate obesity and associated comorbidities, the underlying shifts in gut microbial communities, especially at the functional level, have not been characterized in great details. In this study, we sought to investigate the effects of synbiotics on the regulation of gut microbiota and the alleviation of high-fat diet (HFD)-induced metabolic disorders in mice. METHODS: Specific pathogen-free (SPF) male C57BL/6J mice were fed diets with either 10% (normal diet, ND) or 60% (high-fat diet, HFD) of total calories from fat (lard). Dietary interventions in the HFD-fed mice included (i) probiotic (Bifidobacterium animalis subsp. lactis and Lactobacillus paracasei subsp. paracasei DSM 46331), (ii) prebiotic (oat ß-glucan), and (iii) synbiotic (a mixture of i and ii) treatments for 12 weeks. Besides detailed characterization of host metabolic parameters, a multi-omics approach was used to systematically profile the microbial signatures at both the phylogenetic and functional levels using 16S rRNA gene sequencing, metaproteomics and targeted metabolomics analysis. RESULTS: The synbiotic intervention significantly reduced body weight gain and alleviated features of metabolic complications. At the phylogenetic level, the synbiotic treatment significantly reversed HFD-induced changes in microbial populations, both in terms of richness and the relative abundance of specific taxa. Potentially important species such as Faecalibaculum rodentium and Alistipes putredinis that might mediate the beneficial effects of the synbiotic were identified. At the functional level, short-chain fatty acid and bile acid profiles revealed that all dietary interventions significantly restored cecal levels of acetate, propionate, and butyrate, while the synbiotic treatment reduced the bile acid pools most efficiently. Metaproteomics revealed that the effects of the synbiotic intervention might be mediated through metabolic pathways involved in carbohydrate, amino acid, and energy metabolisms. CONCLUSIONS: Our results suggested that dietary intervention using the novel synbiotic can alleviate HFD-induced weight gain and restore gut microbial ecosystem homeostasis phylogenetically and functionally.

Comparison of the Composition and Antioxidant Activities of Phenolics from the Fruiting Bodies of Cultivated Asian Culinary-Medicinal Mushrooms.

The composition profile and the antioxidant properties of phenolics in water extracts obtained from the fresh fruiting bodies of 4 common cultivated Asian edible mushrooms-Agrocybe aegerita, Pleurotus ostreatus, P. eryngii, and Pholiota nameko were compared. The water extract from A. aegerita (AaE) had the highest total phenolic content (TPC) at 54.18 ± 0.27 gallic acid equivalents (µmol/L)/mg extract (P < 0.05), as measured by the Folin-Ciocalteu method, and consisted of the largest number (including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, and sinapic acid) and total amounts of phenolic acids identified by Fourier transform-ion cyclotron resonance mass spectrometry. The water extract of Ph. nameko was found to have the second-highest TPC (43.55 ± 0.10 gallic acid equivalents [µmol/L]/mg extract), followed by the water extract of P. eryngii and the water extract of P. ostreatus (39.55 ± 0.25 and 39.02 ± 0.30 gallic acid equivalents/mg extract, respectively). The scavenging activities of the water extracts from these mushrooms were evaluated against 2,2-diphenyl-l-(2,4,6-trinitrophenyl) hydrazyl diphenylpicrylhydrazyl (DPPH), superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide. Based on halfmaximal effective concentrations, AaE was more effective in scavenging hydrogen peroxide (<0.05), followed by DPPH (0.51 mg/mL), superoxide anion radicals (0.85 mg/mL) and hydroxyl radicals (5.94 mg/mL), then the other mushroom water extracts. The differences in the half-maximal effective concentrations of individual mushroom water extracts were probably the result of the different numbers and amounts of individual phenolic acids in the extracts. The antioxidant activities of the mushroom water extracts were correlated with their TPC. The strongest antioxidant properties of AaE were consistent with its highest TPC and with the largest number and amount of phenolics identified in the extract. These results indicated that cultivated edible mushrooms could be a potential source of natural antioxidants with free radical scavenging properties for application as a functional food ingredient.