Gold nanoparticles (AuNPs) can rapidly deliver artificial microRNA (AmiRNA)-ATG6 to silence ATG6 expression in Arabidopsis.

KEY MESSAGE: We establish a fast and efficient transient silencing system that facilitates functional studies of some genes, whose knockout leads to plant lethality. In plants, the generation of loss-of-function mutants is crucial for studying gene function. Artificial microRNA (AmiRNA) technology is a more targeted and effective tool for gene silencing. Gold nanoparticles (AuNPs) can bind nucleic acids and deliver them into animal cells. Here, AuNPs are used in combination with AmiRNA technology in plants. We found that AmiRNA-autophagy-related proteins (ATG6) can be delivered to cells by AuNPs to achieve the effect of ATG6 silencing. It is worth noting that on the 10th day there is still a silencing effect. Similar to the atg5 lines, silencing of ATG6 significantly reduced plant resistance to Pseudomonas syringae pv.maculicola (Psm) ES4326/AvrRpt2. Interestingly, ATG6 silencing and ATG5 mutation in NPR1-GFP (nonexpressor of pathogenesis-related genes) lines significantly reduced plant resistance to Psm ES4326/AvrRpt2, suggesting that autophagy is also involved in NPR1-regulated plant immune responses. In summary, we establish a fast and efficient transient silencing system that facilitates functional studies of some genes, whose knockout leads to plant lethality.

Aptamer-based self-assembled nanomicelle enables efficient and targeted drug delivery.

Nucleic acid aptamer-based nanomicelles have great potential for nanomedicine and nanotechnology applications. However, amphiphilic aptamer micelles are known to be inherently unstable upon interaction with cell membranes in the physiological environment, thus potentially compromising their specific targeting against cancer cells. This flaw is addressed in the present work which reports a superstable micellar nanodelivery system as an amphiphilic copolymer self-assembled micelle composed of nucleic acid aptamer and polyvalent hydrophobic poly(maleic anhydride-alt-1-octadecene) (C18PMH). Using Ce6 as a drug model, these C18-aptamer micelles exhibit efficient tumor-targeting and -binding ability, facilitating the entry of Ce6 into targeted cells for photodynamic therapy. In addition, they can be loaded with other hydrophobic drugs and still demonstrate favorable therapeutic effects. As such, these C18-aptamer micelles can serve as a universal platform for loading multiple drugs, providing a safer and more effective solution for treating cancer.

Research Progress of ATGs Involved in Plant Immunity and NPR1 Metabolism.

Autophagy is an important pathway of degrading excess and abnormal proteins and organelles through their engulfment into autophagosomes that subsequently fuse with the vacuole. Autophagy-related genes (ATGs) are essential for the formation of autophagosomes. To date, about 35 ATGs have been identified in Arabidopsis, which are involved in the occurrence and regulation of autophagy. Among these, 17 proteins are related to resistance against plant pathogens. The transcription coactivator non-expressor of pathogenesis-related genes 1 (NPR1) is involved in innate immunity and acquired resistance in plants, which regulates most salicylic acid (SA)-responsive genes. This paper mainly summarizes the role of ATGs and NPR1 in plant immunity and the advancement of research on ATGs in NPR1 metabolism, providing a new idea for exploring the relationship between ATGs and NPR1.

[Development of a Forensic Multiplex Amplification STR Kit for 15 Autosomal STR Loci and 10 Y-STR Loci].

OBJECTIVE: To establish a multiplex STR genotyping method for autosomal STR and Y-STR loci in forensic biological practice. METHODS: Widely used autosomal STR loci and Y-STR loci were selected. A set of PCR primers was designed, and a 5-dye fluorescent labeled STR multiplex PCR reagent kit was developed. RESULTS: A kit was developed which can simultaneously detect 15 autosomal STR loci, 10 Y-STR loci, and an Amelogenin. CONCLUSION: The 15 autosomal STR plus 10 Y-STR kit in combination with capillary electrophoresis method was used to STR genotyping with accurate and reliable results. The new one-step testing kit can potentially be widely used in forensic cases and DNA databank in the future.

A Dual and Rapid RPA-CRISPR/Cas12a Method for Simultaneous Detection of Cattle and Soybean-Derived Adulteration in Goat Milk Powder.

The adulteration of goat milk powder occurs frequently; cattle-derived and soybean-derived ingredients are common adulterants in goat milk powder. However, simultaneously and rapidly detecting cattle-derived and soybean-derived components is still a challenge. An efficient, high-throughput screening method for adulteration detection is needed. In this study, a rapid method was developed to detect the adulteration of common cattle-derived and soybean-derived components simultaneously in goat milk powder by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). A dual DNA extraction method was employed. Primers and crRNA for dual detection were designed and screened, and a series of condition optimizations were carried out in this experiment. The optimized assay rapidly detected cattle-derived and soybean-derived components in 40 min. The detection limits of both cattle-derived and soybean-derived components were 1% (w/w) for the mixed adulteration models. The established method was applied to a blind survey of 55 commercially available goat milk powder products. The results revealed that 36.36% of the samples contained cattle-derived or soybean-derived ingredients, which revealed the noticeable adulteration situation in the goat milk powder market. This study realized a fast flow of dual extraction, dual amplification, and dual detection of cattle-derived and soybean-derived components in goat milk powder for the first time. The method developed can be used for high-throughput and high-efficiency on-site primary screening of goat milk powder adulterants, and provides a technical reference for combating food adulteration.

A Rapid RPA-CRISPR/Cas12a Detection Method for Adulteration of Goat Milk Powder.

Because of the serious adulteration of goat milk, the rapid on-site detection of goat milk powder adulteration is needed. In this study, the CRISPR/Cas12a detection system combined with recombinase polymerase amplification (RPA) was employed to qualitatively detect the adulteration of goat milk powder with cattle-derived components. Specific primers and crRNA were designed and screened. After the optimization of RPA and the Cas system, the RPA-CRISPR/Cas12a detection method was established. The detection can complete the rapid identification of cattle-derived components in 45 min, without the assistant of large equipment. The absolute detectability of the RPA-CRISPR/Cas12a assay could reach 10-2 ng/µL for cattle genomic DNA, and 1% (w/w) for cattle milk powder, which is suitable to meet the testing requirements for on-site detection. In total, 55 commercial goat milk powder products were collected for blind testing. The results showed that 27.3% of the samples were adulterated with cattle ingredients, revealing a serious adulteration situation in goat milk powder market. The RPA-CRISPR/Cas12a assay established in this research exhibited its potential for practical use of on-site detection to detect cow milk powder in goat milk powder and can provide reliable technical reference for combating food fraud of adulteration of goat milk products.

Salicylic acid accelerates carbon starvation-induced leaf senescence in Arabidopsis thaliana by inhibiting autophagy through Nonexpressor of pathogenesis-related genes 1.

In plants, leaf senescence is regulated by several factors, including age and carbon starvation. The molecular mechanism of age-regulated developmental leaf senescence differs from that of carbon starvation-induced senescence. Salicylic acid (SA) and Nonexpressor of pathogenesis-related genes 1 (NPR1) play important roles in promoting developmental leaf senescence. However, the relationship between SA signaling and carbon starvation-induced leaf senescence is not currently well understood. Here, we used Arabidopsis thaliana as material and found that carbon starvation-induced leaf senescence was accelerated in the SA dihydroxylase mutants s3hs5h compared to the Columbia ecotype (Col). Exogenous SA treatment significantly promoted carbon starvation-induced leaf senescence, especially in NPR1-GFP. Increasing the endogenous SA and overexpression of NPR1 inhibited carbon starvation-induced autophagy. However, mutation of NPR1 delayed carbon starvation-induced leaf senescence, increased autophagosome production and accelerated autophagic degradation of the Neighbor of BRCA1 gene 1 (NBR1). In conclusion, SA promotes carbon starvation-induced leaf senescence by inhibiting autophagy via NPR1.

Label-Free Sequence-Specific Visualization of LAMP Amplified Salmonella via DNA Machine Produces G-Quadruplex DNAzyme.

Salmonella is one of four key global causes of diarrhea, and in humans, it is generally contracted through the consumption of contaminated food. It is necessary to develop an accurate, simple, and rapid method to monitor Salmonella in the early phase. Herein, we developed a sequence-specific visualization method based on loop-mediated isothermal amplification (LAMP) for the detection of Salmonella in milk. With restriction endonuclease and nicking endonuclease, amplicons were produced into single-stranded triggers, which further promoted the generation of a G-quadruplex by a DNA machine. The G-quadruplex DNAzyme possesses peroxidase-like activity and catalyzes the color development of 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) as the readouts. The feasibility for real samples analysis was also confirmed with Salmonella spiked milk, and the sensitivity was 800 CFU/mL when observed with the naked eye. Using this method, the detection of Salmonella in milk can be completed within 1.5 h. Without the involvement of any sophisticated instrument, this specific colorimetric method can be a useful tool in resource-limited areas.

Suppression of AGTR1 Induces Cellular Senescence in Hepatocellular Carcinoma Through Inactivating ERK Signaling.

Objective: Cellular senescence is an effective barrier against tumorigenesis. Hence, it is of significance to characterize key features of cellular senescence and the induction of senescence in hepatocellular carcinoma (HCC) cells via pharmacological interventions. Our study determined the biological roles as well as mechanisms of angiotensin II type I receptor (AGTR1) on cellular senescence in HCC. Methods: Lentivirus vector-mediated overexpression or knockdown of AGTR1 was conducted in HCC cells, respectively. A volume of 8 µM sorafenib was used to induce cellular senescence, and ERK was activated by 30 ng/ml ERK agonist EGF. Proliferation was evaluated via clone formation assay. HCC cell senescence was examined by flow cytometry for cell cycle, senescence-associated ß-galactosidase (SA-ß-gal) staining, and senescence-associated heterochromatin foci (SAHF) analysis. AGTR1, p53, p21, extracellular signal-regulated kinase (ERK), and p-ERK expression were assessed through Western blot or immunofluorescence. Results: AGTR1-knockout HCC cells displayed the attenuated proliferative capacity, G2-M phase arrest, increased expression of p53 and p21, and elevated percentages of SA-ß-gal- and SAHF-positive cells. In sorafenib-exposed HCC cells, overexpressed AGTR1 enhanced the proliferative capacity and alleviated G2-M phase arrest as well as decreased p53 and p21 expression and the proportions of SA-ß-gal- and SAHF-positive cells. Moreover, AGTR1 knockdown attenuated the activity of p-ERK in HCC cells, and ERK agonist ameliorated AGTR1 knockdown-induced cellular senescence. Conclusion: This study demonstrates that suppression of AGTR1 induces cellular senescence in HCC through inactivating ERK signaling. The significant synergistic effect of AGTR1 suppression and sorafenib might represent a potential combination therapy for HCC.

Rapid, Visual, and Sequence-Specific Detection of Salmonella in Egg Liquid with vis-NEAA, a CRISPR/Cas12 Empowered New Strategy.

Salmonella is one of the main pathogenic factors that cause foodborne diseases. Rapid and accurate detection of Salmonella in food is of great importance to ensure food safety. Nicking enzyme-assisted amplification (NEAA) is one of the promising isothermal amplification methods finishing the in vitro amplification in ∼10 min; however, it suffers from nonspecific amplification a lot (∼70% products are noises). In this paper, we introduced CRISPR/Cas12a to specifically recognize the NEAA amplicons and transduce the signals into turned-on fluorescent visual readouts (vis-NEAA). Impressively, with this method, the high efficiency of NEAA has been taken great advantage and the nonspecific products were successfully bypassed at the same time. In comparison to NEAA-gel electrophoresis, vis-NEAA showed complete fidelity toward the presence of specific products, while for real-time PCR, it possesses equivalent sensitivity and specificity but saves ∼80% of the time. A level of 80 CFU/mL Salmonella in spiked eggs can be detected on-site in ∼20 min.

Genetic polymorphism analysis of 40 Y-chromosomal STR loci in seven populations from South China.

China is a multi-ethnic country. Due to its diverse terrain, many ethnic groups are geographically isolated within China. This phenomenon is especially prevalent in southern China. As Y-STR loci are paternally inherited, they can be used to effectively understand the genetic relationship between different populations and thus aid in forensic science. In this study, forty Y-STR loci were analysed in 2018 unrelated male individuals from the following seven ethnic populations in South China: Yao (n=147), Zhuang (n=225), Gelao (n=156), Miao (n=186), Maonan (n=133), Gin (n=160) and Guangxi Han (n=1011). Using both AGCU Y24 STR and GFS Y24 STR genotyping kits, a total of 335 alleles and 141 haplotypes of three multi-copy loci were observed in these seven populations. The highest GD value of the 40 Y-STR loci in the overall population was 0.9643 for DYS385a/b, while the lowest was 0.4101 for DYS438. Out of the 2018 samples analysed, 1935 distinct haplotypes and 1858 unique haplotypes were observed. The HD value of the total samples was up to 0.9994 and ranged from a low of 0.9908 in the Yao to a high of 0.9999 in the Han population. We found using population structure analysis that the genetic distance is smaller among the seven southern populations (Guangxi Han, Gelao, Yao, Miao, Zhuang, Jing and Maonan) than the northern populations (Tibetan, Korean, Mongolian, Uygur and Hui). We show that the 40 Y-STRs have a high level of polymorphism in the South China ethnic groups and there is a high degree of differentiation among ethnic groups located in geographically distributed and densely populated areas. These data may provide additional resources for forensic applications and population genetic studies.

Genetic variation analysis of 15 autosomal STR loci of AmpFlSTR Sinofiler PCR Amplification Kit in Henan (central China) Han population.

AmpFlSTR Sinofiler PCR Amplification Kit is specially developed for Chinese forensic laboratories, but there are little population-genetic indices about this kit as a whole. This kit contains 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D6S1043, D12S391, D5S818 and FGA. In order to evaluate this kit and to get basic population-genetic indices for its use in forensic practice in Chinese Han population, the DNA of 231 unrelated Han individuals from Henan (central China) were typed using the Kit. The most discriminating locus was D6S1043 while the least was D3S1358. The combined match probability was 9.81x10(-19) and the combined power of exclusion was 0.99999974. Statistical analysis of the generated data indicated no departure from expectation of Hardy-Weinberg Equilibrium (HWE) in all loci but D6S1043 and no linkage disequilibrium in all pairs of loci. The observed and expected heterozygosity, power of discrimination, polymorphic information content, the other population-genetic indices were calculated.