CRISPR screens reveal convergent targeting strategies against evolutionarily distinct chemoresistance in cancer.

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

Inhibition of PEDV viral entry upon blocking N-glycan elaboration.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the global swine industry, demanding a thorough understanding of its cellular invasion mechanism for effective interventions. This study meticulously investigates the impact of O- and N-linked glycans on PEDV proteins and host cell interaction, shedding light on their influence on the virus's invasion process. Utilizing CRISPR-Cas9 technology to inhibit cell surface O- and N-linked glycan synthesis demonstrated no discernible impact on virus infection. However, progeny PEDV strains lacking these glycans exhibited a minor effect of O-linked glycans on virus infection. Conversely, a notable 40% reduction in infectivity was observed when the virus surface lacked N-linked glycans, emphasizing their pivotal role in facilitating virus recognition and binding to host cells. Additionally, inhibition studies utilizing kifunensine, a natural glycosidase I inhibitor, reaffirmed the significant role of N-linked glycans in virus infection. Inhibiting N-linked glycan synthesis with kifunensine substantially decreased virus entry into cells and potentially influenced spike protein expression. Assessment of the stability and recovery potential of N-linked glycan-deficient strains underscored the critical importance of N-glycans at various stages of the virus lifecycle. In vivo experiments infecting piglets with N-glycan-deficient strains exhibited milder clinical symptoms, reduced virus excretion, and less severe pathological lesions compared to conventional strains. These findings offer promising translational applications, proposing N-glycosylation inhibitors as potential therapeutic interventions against PEDV. The utilization of these inhibitors might mitigate virus invasion and disease transmission, providing avenues for effective antiviral strategies and vaccine development. Nonetheless, further research is warranted to elucidate the precise mechanisms of N-linked glycans in PEDV infection for comprehensive clinical applications.

Oxygen self-supplying small size magnetic nanoenzymes for synergistic photodynamic and catalytic therapy of breast cancer.

In recent years, tumor catalytic therapy based on nanozymes has attracted widespread attention. However, its application is limited by the tumor hypoxic microenvironment (TME). In this study, we developed oxygen-supplying magnetic bead nanozymes that integrate hemoglobin and encapsulate the photosensitizer curcumin, demonstrating reactive oxygen species (ROS)-induced synergistic breast cancer therapy. Fe3O4 magnetic bead-mediated catalytic dynamic therapy (CDT) generates hydroxyl radicals (˙OH) through the Fenton reaction in the tumor microenvironment. The Hb-encapsulated Fe3O4 magnetic beads can be co-loaded with the photosensitizer/chemotherapeutic agent curcumin (cur), resulting in Fe3O4-Hb@cur. Under hypoxic conditions, oxygen molecules are released from Fe3O4-Hb@cur to overcome the TME hypoxia, resulting in comprehensive effects favoring anti-tumor responses. Upon near-infrared (NIR) irradiation, Fe3O4-Hb@cur activates the surrounding molecular oxygen to generate a certain amount of singlet oxygen (1O2), which is utilized for photodynamic therapy (PDT) in cancer treatment. Meanwhile, we validated that the O2 carried by Hb significantly enhances the intracellular ROS level, intensifying the catalytic therapy mediated by Fe3O4 magnetic beads and inflicting lethal damage to cancer cells, effectively inhibiting tumor growth. Therefore, significant in vivo synergistic therapeutic effects can be achieved through catalytic-photodynamic combination therapy.

Intrinsic targeting of host RNA by Cas13 constrains its utility.

Cas13 can be used for the knockdown, editing, imaging or detection of RNA and for RNA-based gene therapy. Here by using RNA immunoprecipitation sequencing, transcriptome profiling, biochemical analysis, high-throughput screening and machine learning, we show that Cas13 can intrinsically target host RNA in mammalian cells through previously unappreciated mechanisms. Different from its known cis/trans RNA-cleavage activity, Cas13 can also cleave host RNA via mechanisms that are transcript-specific, independent of the sequence of CRISPR RNA and dynamically dependent on the conformational state of Cas13, as we show for several Cas13-family effectors encoded in one-vector and two-vector lentiviral systems. Moreover, host genes involved in viral processes and whose transcripts are intrinsically targeted by Cas13 contribute to constraining the lentiviral delivery and expression of Cas13. Our findings offer guidance for the appropriate use of lentiviral Cas13 systems and highlight the need for caution regarding intrinsic RNA targeting in Cas13-based applications.

Chaenomeles sinensis polysaccharide and its carboxymethylated derivative alleviate dextran sulfate sodium-induced ulcerative colitis via suppression of inflammation and oxidative stress.

Chaenomeles sinensis fruit polysaccharide (CSP) and carboxymethylated CSP (CSP-M) were prepared using ultrasound extraction and the sodium hydroxide-chloroacetic acid method. Structural analysis revealed that both CSP and CSP-M mainly consisted of glucose, arabinose, rhamnose, glucuronic acid, galactose, and xylose, and the introduction of carboxymethyl did not damage the polymer chain of CSP. In vivo studies verified that both CSP and CSP-M could remarkably alleviate the symptoms of ulcerative colitis (UC) mice and reduce intestinal epithelial cell depletion, along with the infiltration of inflammatory cells in colon tissue, by mediating the expression of myeloperoxidase (MPO), inflammatory factors [tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6], and oxidative stress factors [malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and nitric oxide (NO)]. Most importantly, the introduction of carboxymethyl significantly enhanced the anti-UC activity of CSP, confirming the efficacy of carboxymethylation as a method to enhance the biological activities of CSP, thereby suggesting the potential of CSP-M as a therapeutic option for UC.

Suppression of hollow droplet rebound on super-repellent surfaces.

Droplet rebound is ubiquitous on super-repellent surfaces. Conversion between kinetic and surface energies suggests that rebound suppression is unachievable due to negligible energy dissipation. Here, we present an effective approach to suppressing rebounds by incorporating bubbles into droplets, even in super-repellent states. This suppression arises from the counteractive capillary effects within bubble-encapsulated hollow droplets. The capillary flows induced by the deformed inner-bubble surface counterbalance those driven by the outer-droplet surface, resulting in a reduction of the effective take-off momentum. We propose a double-spring system with reduced effective elasticity for hollow droplets, wherein the competing springs offer distinct behavior from the classical single-spring model employed for single-phase droplets. Through experimental, analytical, and numerical validations, we establish a comprehensive and unified understanding of droplet rebound, by which the behavior of single-phase droplets represents the exceptional case of zero bubble volume and can be encompassed within this overarching framework.

Probiotic Escherichia coli Nissle 1917 protect chicks from damage caused by Salmonella enterica serovar Enteritidis colonization.

As a foodborne pathogen of global importance, Salmonella enterica serovar Enteritidis (S. Enteritidis) is a threat to public health that is mainly spread by poultry products. Intestinal Enterobacteriaceae can inhibit the colonization of S. Enteritidis and are regarded as a potential antibiotic substitute. We investigated, in chicks, the anti-S. Enteritidis effects of Escherichia coli (E. coli) Nissle 1917, the most well-known probiotic member of Enterobacteriaceae. Eighty 1-d-old healthy female AA broilers were randomly divided into 4 groups, with 20 in each group, namely the negative control (group P), the E. coli Nissle 1917-treated group (group N), the S. Enteritidis-infected group (group S) and the E. coli Nissle 1917-treated and S. Enteritidis-infected group (group NS). From d 5 to 7, chicks in groups N and NS were orally gavaged once a day with E. coli Nissle 1917 and in groups P and S were administered the same volume of sterile PBS. At d 8, the chicks in groups S and NS were orally gavaged with S. Enteritidis and in groups P and N were administered the same volume of sterile PBS. Sampling was conducted 24 h after challenge. Results showed that gavage of E. coli Nissle 1917 reduced the spleen index, Salmonella loads, and inflammation (P < 0.05). It improved intestinal morphology and intestinal barrier function (P < 0.05). S. Enteritidis infection significantly reduced mRNA expression of angiotensin-converting enzyme 2 (ACE2) and solute carrier family 6-member 19 (SLC6A19) in the cecum and the content of Gly, Ser, Gln, and Trp in the serum (P < 0.05). Pretreatment with E. coli Nissle 1917 yielded mRNA expression of ACE2 and SLC6A19 in the cecum and levels of Gly, Ser, Gln, and Trp in the serum similar to that of uninfected chicks (P < 0.05). Additionally, E. coli Nissle 1917 altered cecum microbiota composition and enriched the abundance of E. coli, Lactobacillales, and Lachnospiraceae. These findings reveal that the probiotic E. coli Nissle 1917 reduced S. Enteritidis infection and shows enormous potential as an alternative to antibiotics.

Exploring the factors effecting on carbon emissions in each province in China: A comprehensive study based on symbolic regression, LMDI and Tapio models.

Carbon emission (CE) has led to increasingly severe climate problems. The key to reducing CE is to identify the dominant influencing factors and explore their influence degree. The CE data of 30 provinces from 1997 to 2020 in China were calculated by IPCC method. Based on this, the importance order of six factors included GDP, Industrial Structure (IS), Total Population (TP), Population Structure (PS), Energy Intensity (EI) and Energy Structure (ES) affecting the CE of China's provinces were obtained by using symbolic regression, then the LMDI and the Tapio models were established to deeply explore the influence degree of different factors on CE. The results showed that the 30 provinces were divided into five categories according to the primary factor, GDP was the most important factor, followed by ES and EI, then IS, and the least TP and PS. The growth of per capita GDP promoted the increase of CE, while reduced EI inhibited the increase of CE. The increase of ES promoted CE in some provinces but inhibited in others. The increase of TP weakly promoted the increase of CE. These results can provide some references for governments to formulate relevant CE reduction policies under dual carbon goal.

Devising novel near-infrared aggregation-induced-emission luminogen labeling for point-of-care diagnosis of Mycobacterium tuberculosis.

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required.

Liver Protection of a Low-Polarity Fraction from Ficus pandurata Hance, Prepared by Supercritical CO2 Fluid Extraction, on CCl4-Induced Acute Liver Injury in Mice via Inhibiting Apoptosis and Ferroptosis Mediated by Strengthened Antioxidation.

Ficus pandurata Hance (FPH) is a Chinese herbal medicine widely used for health care. This study was designed to investigate the alleviation efficacy of the low-polarity ingredients of FPH (FPHLP), prepared by supercritical CO2 fluid extraction technology, against CCl4-induced acute liver injury (ALI) in mice and uncover its underlying mechanism. The results showed that FPHLP had a good antioxidative effect determined by the DPPH free radical scavenging activity test and T-AOC assay. The in vivo study showed that FPHLP dose-dependently protected against liver damage via detection of ALT, AST, and LDH levels and changes in liver histopathology. The antioxidative stress properties of FPHLP suppressed ALI by increasing levels of GSH, Nrf2, HO-1, and Trx-1 and reducing levels of ROS and MDA and the expression of Keap1. FPHLP significantly reduced the level of Fe2+ and expression of TfR1, xCT/SLC7A11, and Bcl2, while increasing the expression of GPX4, FTH1, cleaved PARP, Bax, and cleaved caspase 3. The results demonstrated that FPHLP protected mouse liver from injury induced by CCl4 via suppression of apoptosis and ferroptosis. This study suggests that FPHLP can be used for liver damage protection in humans, which strongly supports its traditional use as a herbal medicine.

Salvianolic acid A alleviates atherosclerosis by inhibiting inflammation through Trc8-mediated 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation.

BACKGROUND: Atherosclerosis is the most prevalent cardiovascular disease and remains the major contributor to death and mortality globally. Salvianolic acid A (SalA) is a water-soluble phenolic acid that benefits atherosclerosis. However, the mechanisms of SalA protecting against atherosclerosis remain unclear. PURPOSE: We aimed to determine whether SalA prevents atherosclerosis by modulating 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) degradation via the ubiquitin-proteasomal pathway. METHODS: The animal and cellular models of atherosclerosis were established by subjecting apolipoprotein E (ApoE) knockout mice to a high-fat diet (HFD) and exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (ox-LDL), respectively. RESULTS: Our results showed that similar to atorvastatin, SalA suppressed atherosclerotic plaque formation, improved serum lipid accumulation, and reduced cholesterol levels in HFD-fed ApoE-/- mice. Moreover, SalA protected HUVECs from ox-LDL-caused cell viability reduction and lipid accumulation. The mechanism study revealed that SalA reduced the production of proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, and augmented the generation of the anti-inflammatory cytokine IL-10 in ApoE-/- mice and HUVECs, accompanied by increased HMGCR ubiquitination and degradation via translocation in renal carcinoma on chromosome 8 (Trc8), insulin-induced gene (Insig)1 and Insig2. Furthermore, the knockdown of Trc8 abolished the SalA-induced HMGCR degradation and anti-atherosclerosis activity. CONCLUSION: SalA rescues atherosclerosis by inhibiting inflammation through the Trc8-regulated degradation of HMGCR. These findings underscore Trc8 as a potential target of atherosclerosis.

Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches.

A major challenge in the application of the CRISPR-Cas13d system is to accurately predict its guide-dependent on-target and off-target effect. Here, we perform CRISPR-Cas13d proliferation screens and design a deep learning model, named DeepCas13, to predict the on-target activity from guide sequences and secondary structures. DeepCas13 outperforms existing methods to predict the efficiency of guides targeting both protein-coding and non-coding RNAs. Guides targeting non-essential genes display off-target viability effects, which are closely related to their on-target efficiencies. Choosing proper negative control guides during normalization mitigates the associated false positives in proliferation screens. We apply DeepCas13 to the guides targeting lncRNAs, and identify lncRNAs that affect cell viability and proliferation in multiple cell lines. The higher prediction accuracy of DeepCas13 over existing methods is extensively confirmed via a secondary CRISPR-Cas13d screen and quantitative RT-PCR experiments. DeepCas13 is freely accessible via http://deepcas13.weililab.org .

Exploratory research on influential factors of China's sulfur dioxide emission based on symbolic regression.

The amount of China's sulfur dioxide emission remains significantly large in recent years. To further reduce sulfur dioxide emission, the key is to find out the leading factors affecting sulfur dioxide emission and then take measures to control it accordingly. In order to investigate the influential factors of sulfur dioxide emission of various provinces, the data of sulfur dioxide emission of 30 provinces in China from 2001 to 2020 were collected. We established the symbolic regression model to explore the relationship between the GDP (x1), total population (x2), total energy consumption (x3), thermal power installed capacity (x4), and sulfur dioxide emission (dependent variable) for each province. The results show that the amount of China's total sulfur dioxide emission and sulfur dioxide emission in most provinces meet the environmental Kuznets curve (EKC). The influential degree of the factors affecting China's sulfur dioxide emission are GDP, total energy consumption, thermal power installed capacity, and total population. The provinces with the primary factor of GDP have the lowest average total energy consumption and average thermal power installed capacity, and their average sulfur dioxide emissions are also relatively low. The provinces with the primary factor of GDP do not show obvious geographical characteristics, but the provinces with the primary factor of total energy consumption are all distributed in southern China. Based on the research results, some control measures are also put forward.

The Preparation of Gen-NH2-MCM-41@SA Nanoparticles and Their Anti-Rotavirus Effects.

Genistein (Gen), a kind of natural isoflavone drug monomer with poor water solubility and low oral absorption, was incorporated into oral nanoparticles with a new mesoporous carrier material, NH2-MCM-41, which was synthesized by copolycondensation. When the ratio of Gen to NH2-MCM-41 was 1:0.5, the maximum adsorption capacity of Gen was 13.15%, the maximum drug loading was 12.65%, and the particle size of the whole core-shell structure was in the range of 370 nm-390 nm. The particles were characterized by a Malvern particle size scanning machine, XRD, Fourier transform infrared spectroscopy, scanning electron microscopy, and nitrogen adsorption and desorption. Finally, Gen-NH2-MCM-41 was encapsulated by sodium alginate (SA), and the chimerism of this material, denoted as GEN-NH2-MCM-41@SA, was investigated. In vitro release experiments showed that, after 5 h in artificial colon fluid (pH = 8.0), the cumulative release reached 99.56%. In addition, its anti-rotavirus (RV) effect showed that the maximum inhibition rate was 62.24% at a concentration of 30 µM in RV-infected Caco-2 cells, and it significantly reduced the diarrhea rate and diarrhea index in an RV-infected-neonatal mice model at a dose of 0.3 mg/g, which was better than the results of Gen. Ultimately, Gen-NH2-MCM-41@SA was successfully prepared, which solves the problems of low solubility and poor absorption and provides an experimental basis for the application of Gen in the clinical treatment of RV infection.

[Development Scenarios and Environmental Benefits Analysis of Future Power Generation Industry Under Two Modes in China].

In order to study the impact of the "carbon peak and neutrality" mode on future power generation and the environment in China, a Verhulst gray model was established to predict the development of the power generation industry from 2021 to 2060 under the non-"carbon peak and neutrality" mode. In addition, based on the "China 2030 Energy and Power Development Planning Research and 2060 Outlook Report," the development of the power generation industry from 2021 to 2060 under the "carbon peak and neutrality" mode was obtained, and the development scenarios of the future power generation industry in China under two models were compared and studied. The emission factors and emission reduction factors of CO2, SO2, NOx, PM, PM10, and PM2.5 were constructed through the conservation of elements and the generating performance standard, and then four environmental benefits A1-A4 were defined. The results showed that the installed capacity of thermal power will reach the carbon peak in 2026 under the "carbon peak and neutrality" mode. To achieve the carbon neutralization, the installed capacity of thermal power will be reduced by an average of 28 million kilowatts per year after 2026, and the installed capacity of renewable energy generated is required to increase by 154 million kilowatts per year after 2020. Compared with that in the non-"carbon peak and neutrality" mode, the installed capacity of thermal power generation will be greatly reduced, and the installed capacity of renewable energy power generation will be greatly increased under the "carbon peak and neutrality" mode, resulting in huge A1 and A2 environmental benefits. In the next four decades, the cumulative emission reductions in CO2, SO2, NOx, PM, PM10, and PM2.5 thermal power generation A1 are predicted to be 6.64×1010 tons, 1.54×107 tons, 1.55×107 tons, 3.18×106 tons, 1.71×106tons, and 2.23×105 tons, respectively. The cumulative emission reductions of renewable energy power generation A2 will be 5.77×1010 tons, 1.64×107 tons, 1.42×107 tons, 2.86×106 tons, 1.54×106 tons, and 2×105 t tons, respectively. Under the "carbon peak and neutrality" mode, compared with those from coal-fired power generation, the environmental benefits A3 and A4 produced by the relative cleanliness of renewable energy and nuclear power indicated that the cumulative emission reductions (A3+A4) in clean energy power generation of CO2, SO2, NOx, PM, PM10, and PM2.5 in the next four decades will be 3.014×1011 tons, 7.292×107 tons, 7.119×107 tons, 1.454×107 tons, 7.827×106tons, and 1.018×106 tons, respectively.

Effect of Transcranial Direct Current Stimulation on the Mismatch Negativity Features of Deviated Stimuli in Children With Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a devastating mental disorder in children. Currently, there is no effective treatment for ASD. Transcranial direct current stimulation (tDCS), which is a non-invasive brain stimulation neuromodulation technology, is a promising method for the treatment of ASD. However, the manner in which tDCS changes the electrophysiological process in the brain is still unclear. In this study, we used tDCS to stimulate the dorsolateral prefrontal cortex area of children with ASD (one group received anode tDCS, and the other received sham tDCS) and investigated the changes in evoked EEG signals and behavioral abilities before and after anode and sham stimulations. In addition to tDCS, all patients received conventional rehabilitation treatment. Results show that although conventional treatment can effectively improve the behavioral ability of children with ASD, the use of anode tDCS with conventional rehabilitation can boost this improvement, thus leading to increased treatment efficacy. By analyzing the electroencephalography pre- and post-treatment, we noticed a decrease in the mismatch negativity (MMN) latency and an increase in the MMN amplitude in both groups, these features are considered similar to MMN features from healthy children. However, no statistical difference between the two groups was observed after 4 weeks of treatment. In addition, the MMN features correlate well with the aberrant behavior checklist (ABC) scale, particularly the amplitude of MMN, thus suggesting the feasibility of using MMN features to assess the behavioral ability of children with ASD.

Preparation of lightweight daisy-like magnetic molecularly imprinted polymers via etching synergized template immobilization for enhanced rapid detection of trace 17ß-estradiol.

17ß-estradiol (E2), as one of the pharmaceutical and personal care product, frequently contaminates environmental water as estrogen pollutant and possesses great risk to human survival as well as the sustainable development of the ecosystem. Herein, to achieve an effective adsorbent system for the selective removal of E2 from the environmental water, Fe3O4 nanoparticles are subjected to chemical etching to reduce the overall mass and then employed as carriers to prepare a novel type of lightweight daisy-like magnetic molecularly imprinted polymers (LD-MMIPs) adopting template immobilization strategy. The LD-MMIPs based etched magnetic nanoparticles not only exhibit light mass but also have plentiful imprinted sites in the etched channels, which significantly increases the adsorption capacity for E2. The daisy-like LD-MMIPs own strong magnetic responsiveness, well crystallinity, fast binding kinetics, high adsorption amount, and excellent selectivity. Moreover, combining with HPLC, the LD-MMIPs as adsorbents have been successfully used to specifically recognize and detect trace E2 in environmental water. Thus, the proposed LD-MMIPs with high adsorption capacity hold great potential in monitoring water pollution. Additionally, this work also provides an alternative strategy for improving the adsorption capacity of magnetic molecularly imprinted polymers through a convenient chemical etching technology.

Establishment of a rotavirus-infected zebrafish model and its application in drug screening.

Rotavirus (RV) is one of the main pathogens that induce infantile diarrhea and by now no effective drugs are available for RV-induced infantile diarrhea. Thus the development of novel models is of vital importance for the pathological research of RV-induced infantile diarrhea, as well as the progress of the associated treatment strategy. Here we introduced for the first time that RV-Wa strain and RV-SA-11 strain could infect 5 dpf(day post fertilization) and 28 dpf larvae, to induce infantile diarrhea model that was highly consistent with the clinical infection of infants. RV infection significantly changed the signs, survival rate and inflammation of larvae. Some important indicators, including the levels of RV antigen VP4 and VP6, the in vivo RV tracking, and the RV particles were also analyzed, which collectively demonstrated that the model was successfully established. More importantly, we also determined the potentials of the proposed RV-infected zebrafish model for anti-viral drug assessment. In conclusion, we established a RV-infected zebrafish model with formulated relevant indicators both larvae and adult fish, which might be served as a high throughput platform for antiviral drug screening.

Host miR-4301 promotes rotavirus replication via PPP1R3D in Caco-2 cells.

To investigate the role of miR-4301 in rotavirus (RV)-infected Caco-2 cells. In this experiment, RNAs of RV-infected Caco-2 cells were extracted, and the high-throughput second-generation sequencing was performed to detect the expression profiles of host microRNAs (miRNAs). Synthetic miRNA mimics and inhibitors were examined (quantitative polymerase chain reaction [qPCR], crystalline violet, immunofluorescence and electron microscopy) to evaluate the effect on RV replication. Target genes of miR-4301 were predicted by software analysis. The expression of target genes was evaluated by qPCR and Western blot after transfected with miRNA inhibitor/mimic, and crystalline violet and qPCR were used to detect the downregulation effects of target genes on RV replication. By transfecting miRNA inhibitors/mimics and detecting downstream target genes, the mechanism of miRNA affecting RV replication was analyzed. There were 78 known miRNAs with significant differential expression, including 39 upregulated miRNAs and 39 downregulated miRNAs. The results showed that miR-4301 exerted a key role in enhancing RV replication. PPP1R3D protein which can inhibit RV replication was predicted as the target gene of miR-4301 by software analysis. While upregulating miR-4301 by RV, the expression of PPP1R3D and glycogen synthase (GS) is suppressed. For the first time, the effect of miR-4301 on RV infection, and its influence on GS was investigated. Specifically, RV inhibits host cell glycogen synthesis to utilize the host intracellular glucose for promoting its own replication.

A chemical-enhanced system for CRISPR-Based nucleic acid detection.

The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.