miR-214-PTEN pathway is a potential mechanism for stress-induced immunosuppression affecting chicken immune response to avian influenza virus vaccine.

Stress-induced immunosuppression (SIIS) is one of common problems in the intensive poultry industry, affecting the effect of vaccine immunization and leading to high incidences of diseases. In this study, the expression characteristics and regulatory mechanisms of miR-214 in the processes of SIIS and its influence on the immune response to avian influenza virus (AIV) vaccine in chicken were explored. The qRT-PCR results showed that serum circulating miR-214 was significantly differentially expressed (especially on 2, 5, and 28 days post immunization (dpi)) in the processes, so had the potential as a molecular marker. MiR-214 expressions from multiple tissues were closely associated with the changes in circulating miR-214 expression levels. MiR-214-PTEN regulatory network was a potential key regulatory mechanism for the heart, bursa of Fabricius, and glandular stomach to participate in the process of SIIS affecting AIV immune response. This study can provide references for further understanding of stress affecting immune response.

Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.

The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.

Preparation of Self-Coating Al2O3 Bonded SiAlON Porous Ceramics Using Aluminum Dross and Silicon Solid Waste under Ambient Air Atmosphere.

Al2O3-bonded SiAlON ceramic with self-coating was prepared using aluminum dross and silicon solid waste as starting materials under ambient air conditions. The changes in phase, microstructure, and physical properties of the ceramic with temperature were analyzed and the formation mechanism of the SiAlON phase was elucidated. The results showed that higher temperature was more suitable for the preparation of SiAlON ceramics. As the temperature increased from 1400 to 1600 °C, the main phases in the ceramic transformed from mullite, Al2O3, and SiAlON to Al2O3 and SiAlON. An Al2O3-rich layer spontaneously coated the surface of the porous ceramic as Al melted and oxidized at high temperature. The thickness of this layer decreased as the temperature increased. The presence of Al2O3-rich coating layer impeded air flow, allowing nitriding of Si and Al, and the formation of the SiAlON phase in ambient air conditions. This study not only presents a strategy to successfully recycle aluminum dross and silicon solid waste but also offers a straightforward approach to preparing SiAlON material in air atmosphere.

Multi-omics joint analysis reveals how Streptomyces albidoflavus OsiLf-2 assists Camellia oleifera to resist drought stress and improve fruit quality.

Camellia oleifera (C. oleifera) is a unique edible oil crop in China cultivated in the hilly southern mountains. Although C. oleifera is classified as a drought-tolerant tree species, drought remains the main factor limiting the growth of C. oleifera in summer and autumn. Using endophytes to improve crop drought tolerance is one effective strategy to meet our growing food crop demand. In this study, we showed that endophyte Streptomyces albidoflavus OsiLf-2 could mitigate the negative impact of drought stress on C. oleifera, thus improving seed, oil, and fruit quality. Microbiome analysis revealed that OsiLf-2 treatment significantly affected the microbial community structure in the rhizosphere soil of C. oleifera, decreasing both the diversity and abundance of the soil microbe. Likewise, transcriptome and metabolome analyses found that OsiLf-2 protected plant cells from drought stress by reducing root cell water loss and synthesizing osmoregulatory substances, polysaccharides, and sugar alcohols in roots. Moreover, we observed that OsiLf-2 could induce the host to resist drought stress by increasing its peroxidase activity and synthesizing antioxidants such as cysteine. A multi-omics joint analysis of microbiomes, transcriptomes, and metabolomes revealed OsiLf-2 assists C. oleifera in resisting drought stress. This study provides theoretical and technical support for future research on endophytes application to enhance the drought resistance, yield, and quality of C. oleifera.

"Repaired and Activated" DNAzyme Enables the Monitoring of DNA Alkylation Repair in Live Cells.

Direct measurement of DNA repair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a "repaired and activated" DNAzyme (RADzyme) by incorporating a single methyl lesion (O6 MeG, 3MeC, or 1MeA) at designated positions through systematic screening. We found that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme-mediated DNA repair. Benefit from these findings, a fluorogenic RADzyme sensor was developed for the monitoring of MGMT-mediated repair of O6 MeG lesion. Importantly, the sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore, another RADzyme sensor was engineered for the monitoring of ALKBH2-mediated repair of 3MeC lesion. This strategy provides a simple and versatile tool for the study of the basic biology of DNA repair, clinical diagnosis and therapeutic assessment.

Programming DNA cascade circuits on live cell membranes for accurate cancer cell recognition and gene silencing.

A dual-aptamer based AND logic cascade circuit is activated on cell membranes in response to the receptor-aptamer binding, affording enhanced specificity for cell subtype recognition and gene silencing.

RNA imaging in living mice enabled by an in vivo hybridization chain reaction circuit with a tripartite DNA probe.

RNA imaging in living animals helps decipher biology and creates new theranostics for disease treatment. Due to their low delivery efficiency and high background, however, fluorescence probes for in situ RNA imaging in living mice have not been reported. We develop a new cell-targeting fluorescent probe that enables RNA imaging in living mice via an in vivo hybridization chain reaction (HCR). The minimalistic Y-shaped design of the tripartite DNA probe improves its performance in live animal studies and serves as a modular scaffold for three DNA motifs for cell-targeting and the HCR circuit. The tripartite DNA probe allows facile synthesis with a high yield and demonstrates ultrasensitive RNA detection in vitro. The probe also exhibits selective and efficient internalization into folate (FA) receptor-overexpressed cells via a caveolar-mediated endocytosis mechanism and produces fluorescence signals dynamically correlated with intracellular target expressions. Furthermore, the probe exhibits specific delivery into tumor cells and allows high-contrast imaging of miR-21 in living mice. The tripartite DNA design may open the door for intracellular RNA imaging in living animals using DNA-minimal structures and its design strategy can help future development of DNA-based multi-functional molecular probes.

Self-Tracking Multifunctional Nanotheranostics for Sensitive miRNA Imaging Guided Photodynamic Therapy.

A theranostic system that enables monitoring the delivery process and amplified detection of low-abundance biomarkers is significant for bioimaging and biomedicine. We here develop a graphitic carbon nitride (g-C3N4) nanosheet based self-tracking multifunctional nanotherapeutic system for sensitive miRNA guided photodynamic therapy in living cells. The g-C3N4 nanosheets are employed not only as a nanocarrier for an intracellular hybridization chain reaction for sensitive miRNA imaging but also as an excellent photosensitizer for tumor therapy. Moreover, the fluorescent g-C3N4 nanosheets allow assessing the transfection efficiency to mitigate the false negative signals. In vitro experiments showed that the proposed strategy had high signal amplification efficiency. The live cell studies revealed that the g-C3N4@H1/H2 could distinguish different miRNA expression levels from tumor to normal cells. The cell viability assay demonstrated the good photodynamic therapy efficiency of g-C3N4@H1/H2. Therefore, the developed self-tracking multifunctional nanotheranostics hold great potential for biomarker detection and imaging guided photodynamic therapy in living cells.

Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep.

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of -1912 bp to -1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (-1786 to -1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (-1786 to -1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.

Aggregation-Induced Emission-Based Fluorescence Probe for Fast and Sensitive Imaging of Formaldehyde in Living Cells.

Formaldehyde (FA), as a reactive carbonyl species and signaling molecule, plays an important role in living systems. Here, an FA-responsive probe with fast response and great selectivity is designed based on aggregation-induced emission. The probe is prepared by functionalizing tetraphenylethene (TPE) with two amine groups. FA is detected based on the solubility differences between the amine-functionalized TPE and the corresponding Schiff bases after reaction with FA. The probe exhibits a limit of detection of 40 nM and a response time of ∼90 s. Furthermore, its ability to detect both endogenous and exogenous FA is demonstrated in living cells with high specificity. Moreover, the probe is also introduced to image endogenous FA in real time with fast response. These results suggest that our probe holds great potential for tracking FA in living systems under various physiological conditions as well as related biomedical applications.

Determination of Tumor Marker Carcinoembryonic Antigen with Biosensor Based on Optical Quantum Weak Measurements.

A phase-sensitive weak measurement biosensor was proposed for the detection of carcinoembryonic antigen (CEA), one common category of tumor markers. The total internal reflection (TIR) at the interface of the prism without precious metal coating was exploited to introduce the phase delay between horizontal and vertical polarizations, which can be determined through the central wavelength shift of output spectra for the sensing of the refractive index of the sample. In the weak measurement analysis, the specific binding reaction of tumor markers with a refractive index change on the surface of the prism can be monitored in real time through the central wavelength shift. With the specific absorption measurement, the feasibility of this weak measurement-based biosensor was experimentally demonstrated. We provide a low cost and convenient approach for tumor marker detection.

Surface Enhanced Laser Desorption Ionization of Phospholipids on Gold Nanoparticles for Mass Spectrometric Immunoassay.

High-throughput and sensitive detection of proteins are essential for clinical diagnostics and biomarker discovery. We develop a novel high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay method, which utilizes antibody-modified phospholipid bilayer coated gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized magnetic beads as the capture reagent. This method enables magnetic enrichment of the PBL-AuNPs label specific to target protein, allowing sensitive surface enhanced laser desorption ionization (SELDI)-TOF MS detection of the protein via its specific label. AuNPs act as not only the support but also the matrix for the phospholipids in SELDI TOF MS detection. Moreover, with phospholipids with varying molecular weights as the encoded MS reporters, this method allows multiplexed detection of multiple proteins. With the use of a predefined phospholipids internal standard, this method also affords excellent reproducibility in protein quantification. We have demonstrated this method using the assays of two tumor biomarkers, and the results reveal that it provides a sensitive platform for multiplexed protein detection with detection limits in the picomolar ranges. This method may provide a useful platform for high-throughput and sensitive detection of protein biomarkers for clinical diagnostics.

Identification of Rubisco rbcL and rbcS in Camellia oleifera and their potential as molecular markers for selection of high tea oil cultivars.

Tea oil derived from seeds of Camellia oleifera Abel. is high-quality edible oil in China. This study isolated full-length cDNAs of Rubisco subunits rbcL and rbcS from C. oleifera. The rbcL has 1,522 bp with a 1,425 bp coding region, encoding 475 amino acids; and the rbcS has 615 bp containing a 528 bp coding region, encoding 176 amino acids. The expression level of the two genes, designated as Co-rbcL and Co-rbcS, was determined in three C. oleifera cultivars: Hengchong 89, Xianglin 1, and Xianglin 14 whose annual oil yields were 546.9, 591.4, and 657.7 kg ha(-1), respectively. The Co-rbcL expression in 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was greater than 'Hengchong 89'. The expression levels of Co-rbcS in 'Xianglin 1' and 'Xianglin 14' were similar but were significantly greater than in 'Hengchong 89'. The net photosynthetic rate of 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was higher than 'Hengchong 89'. Pearson's correlation analysis showed that seed yields and oil yields were highly correlated with the expression level of Co-rbcL at P < 0.001 level; and the expression of Co-rbcS was correlated with oil yield at P < 0.01 level. Net photosynthetic rate was also correlated with oil yields and seed yields at P < 0.001 and P < 0.01 levels, respectively. Our results suggest that Co-rbcS and Co-rbcL in particular could potentially be molecular markers for early selection of high oil yield cultivars. In combination with the measurement of net photosynthetic rates, the early identification of potential high oil production cultivars would significantly shorten plant breeding time and increase breeding efficiency.