Fast and sensitive CRISPR detection by minimized interference of target amplification.

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10-10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp µl-1, making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp µl-1. The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection.

Helicobacter pylori infection induces autophagy via ILK regulation of NOXs-ROS-Nrf2/HO-1-ROS loop.

Reactive oxygen species (ROS) can regulate the occurrence of autophagy, and effective control of the balance between ROS and autophagy may be an important strategy for Helicobacter pylori induced gastric-related diseases. In this study, infection with H. pylori led to a lower level of ILK phosphorylation and increased ROS generation. Knockdown of ILK enhanced total ROS generation, and upregulated NADPH oxidase (NOX) subunit p22-phox levels. Inhibition of NOXs affected total ROS generation. The inhibition of NOX and ROS generation reduced Nrf2 and HO-1 levels, and knockdown of ILK significantly enhanced Nrf2 levels in H. pylori-infected GES-1 cells. Activation of Nrf2 by DMF decreased ROS levels. Therefore, NOX-dependent ROS production regulated by ILK was essential for activation of Nrf2/HO-1 signaling pathways in H. pylori-infected GES-1 cells. Beclin1, ATG5 and LC3B-II levels were higher both in H. pylori-infected and ILK-knockdown GES-1 cells. In NAC-pretreated GES-1 cells infected with H. pylori, the LC3B-II level was decreased compared to that in cells after H. pylori infection alone. Stable low expression of ILK with further knockdown of Beclin1 or ATG5 significantly reduced LC3B-II levels in GES-1 cells, while with the addition of the autophagy inhibitor chloroquine (CQ), LC3B-II and p62 protein levels were both remarkably upregulated. H. pylori accelerated the accumulation of ROS and further led to the induction of ROS-mediated autophagy by inhibiting ILK levels. Together, these results indicate that H. pylori infection manipulates the NOX-ROS-Nrf2/HO-1-ROS loop to control intracellular oxygen stress and further induced ROS-mediated autophagy by inhibiting ILK levels.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

Helicobacter pylori regulates ILK to influence autophagy through Rac1 and RhoA signaling pathways in gastric epithelial cells.

The ability of Helicobacter pylori to manipulate host autophagy is an important pathogenic mechanism. We found an inverse correlation between the expression of ILK and the autophagy marker protein LC3B in H. pylori-positive human samples, H. pylori-infected mice models and H. pylori-infected GES-1 cell lines. When the ILK-knockdown GES-1 cells were infected by H. pylori, CagA were significantly degraded, autophagosomes accumulation and autolysosomes formation were significantly increased, and LC3B protein levels and ratio of LC3BII to LC3BI were also remarkably upregulated. And chloroquine treatment increased LC3B levels in ILK-knockdown GES-1 cells. The expression levels of both Rac1 and RhoA were downregulated in GES-1 cells after H. pylori infection and were decreased in ILK-knockdown GES-1 cells. The mRNA and protein levels of PAK1, MLC, and LIMK were significantly decreased and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the Rac1 inhibitor NSC 23766. The mRNA and protein levels of ROCK1, ROCK2, MLC, and LIMK1 were significantly reduced and cofilin mRNA and protein levels were significantly increased in GES-1 cells treated with the RhoA inhibitor CCG-1423. F-actin was significantly reduced in Rac1- or RhoA-inhibited GES-1 cells. F-actin depolymerization induced autophagosomes accumulation, autolysosomes formation, and the increase of LC3B levels in GES-1 cells. Therefore, these findings revealed that ILK could serve as a novel regulator to affect Rac1/PAK1 and RhoA/ROCKs signaling pathways, thereby influencing H. pylori-induced autophagy.

Regulation of ß1-integrin in autophagy and apoptosis of gastric epithelial cells infected with Helicobacter pylori.

Helicobacter pylori infection is an essential factor in the development of human gastric diseases, but its pathogenic mechanism is still unclear. In this work we have showed that, the LC3II levels were increased and ß1-integrin levels were decreased in H. pylori-positive human gastric tissue samples and H. pylori co-cultured GES-1 cells. There was significant upregulation of LC3II levels and downregulation of P62 levels in GES-1 cells after ß1-integrin knockdown co-cultured with H. pylori. This indicated that ß1-integrin downregulation promoted autophagy in GES-1 cells after H. pylori infection. The cell apoptosis rate and poly ADP-ribose polymerase (PARP) and caspase-3 activities were increased in GES-1 cells pretreated with 3-methyladenine (3-MA ) after H. pylori infection. Furthermore, there was a significant decrease in apoptosis of ß1-integrin knockdown GES-1 cells co-cultured with H. pylori; apoptosis was also downregulated in ß1-integrin knockdown- and 3-MA-treated GES-1 cells co-cultured with H. pylori. Correspondingly, PARP and caspase-3 activities were decreased in ß1-integrin knockdown cells co-cultured with H. pylori and ß1-integrin knockdown-3-MA-treated-1 cells with H. pylori infection. Thus, ß1-integrin is a novel autophagy and apoptosis regulator during H. pylori infection. However, inhibition of autophagy did not reverse the decrease in apoptosis caused by downregulation of ß1-integrin.

Mucin1 as a potential molecule for cancer immunotherapy and targeted therapy.

Mucin1 is a highly glycosylated type 1 transmembrane mucin that ranks second among 75 tumor-related antigens published by the National Cancer Institute, and has been identified as a possible therapeutic target over the past 30 years. MUC1 plays an important role in malignant transformation and disease evolution, including cell proliferation, survival, self-renewal, and metastatic invasion. MUC1 has been shown to interact with diverse effectors such as ß-catenin, receptor tyrosine kinases, and cellular-abelsongene, which are of importance in the pathogenesis of various malignant tumors. Targeting MUC1 has been shown to be an effective way to induce tumor cell death in vivo and in vitro models. In recent years, a number of therapeutic strategies targeting MUC1 have been developed and their value for tumor therapy have been demonstrated experimentally. This review summarizes recent findings on the structure of MUC1, its expression in different tumors and its involved mechanism pathways, with emphasis on new progress in cancer therapy which related MUC1 in the past decade and evaluates their therapeutic effect.

Elevated dorsal medial prefrontal cortex to lateral habenula pathway activity mediates chronic stress-induced depressive and anxiety-like behaviors.

The medial prefrontal cortex (mPFC) sends projections to numerous brain regions and is believed to play a significant role in depression and anxiety. One of the key downstream targets of the mPFC, the lateral habenula (LHb), is essential for chronic stress (CS)-induced depressive and anxiety-like behaviors. Nevertheless, whether the mPFC-LHb pathway mediates the co-occurrence of depression and anxiety and the underlying mechanism remain incompletely understood. Here, using chemogenetics, we first determined that activation of LHb-projecting mPFC neurons is essential for the development of depressive and anxiety-like behaviors induced by CS. Subsequently, we identify the extent and distribution of LHb-projecting neurons originating from the mPFC subregion. Through circuit-specific in vivo fiber photometry, we found that Ca2+ activity in dorsal mPFC (dmPFC) axon terminals within the LHb was increased during exposure to stressful and anxiety-related stimuli, highlighting the potential role of LHb-projecting dmPFC neurons in conveying stressful and anxiety-related information to the LHb. Finally, we observed that activation of both LHb-projecting dmPFC neurons and their postsynaptic counterparts in the LHb was necessary for CS-induced depressive and anxiety-like behaviors. Overall, this study provides multiple lines of evidence demonstrating that activation of the dmPFC-LHb pathway is a crucial neural circuitry for CS-induced depressive and anxiety-like behaviors.

Burst firing in Output-Defined Parallel Habenula Circuit Underlies the Antidepressant Effects of Bright Light Treatment.

Research highlights the significance of increased bursting in lateral habenula (LHb) neurons in depression and as a focal point for bright light treatment (BLT). However, the precise spike patterns of LHb neurons projecting to different brain regions during depression, their roles in depression development, and BLT's therapeutic action remain elusive. Here, LHb neurons are found projecting to the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and median raphe nucleus (MnR) exhibit increased bursting following aversive stimuli exposure, correlating with distinct depressive symptoms. Enhanced bursting in DRN-projecting LHb neurons is pivotal for anhedonia and anxiety, while concurrent bursting in LHb neurons projecting to the DRN, VTA, and MnR is essential for despair. Remarkably, reducing bursting in distinct LHb neuron subpopulations underlies the therapeutic effects of BLT on specific depressive behaviors. These findings provide valuable insights into the mechanisms of depression and the antidepressant action of BLT.

Gastric mechanosensitive channel Piezo1 regulates ghrelin production and food intake.

Ghrelin, produced mainly by gastric X/A-like cells, triggers a hunger signal to the central nervous system to stimulate appetite. It remains unclear whether X/A-like cells sense gastric distention and thus regulate ghrelin production. Here we show that PIEZO1 expression in X/A-like cells decreases in patients with obesity when compared to controls, whereas it increases after sleeve gastrectomy. Male and female mice with specific loss of Piezo1 in X/A-like cells exhibit hyperghrelinaemia and hyperphagia and are more susceptible to overweight. These phenotypes are associated with impairment of the gastric CaMKKII/CaMKIV-mTOR signalling pathway. Activation of PIEZO1 by Yoda1 or gastric bead implantation inhibits ghrelin production, decreases energy intake and induces weight loss in mice. Inhibition of ghrelin production by Piezo1 through the CaMKKII/CaMKIV-mTOR pathway can be recapitulated in a ghrelin-producing cell line mHypoE-42. Our study reveals a mechanical regulation of ghrelin production and appetite by PIEZO1 of X/A-like cells, which suggests a promising target for anti-obesity therapy.

Prophylactic nicotinamide mononucleotide (NMN) mitigates CSDS-induced depressive-like behaviors in mice via preserving of ATP level in the mPFC.

Depression is a prevalent psychiatric disorder with accumulating evidence implicating dysregulation of extracellular adenosine triphosphate (ATP) levels in the medial prefrontal cortex (mPFC). It remains unclear whether facilitating endogenous ATP production and subsequently increasing extracellular ATP level in the mPFC can exert a prophylactic effect against chronic social defeat stress (CSDS)-induced depressive-like behaviors and enhance stress resilience. Here, we found that nicotinamide mononucleotide (NMN) treatment effectively elevated nicotinamide adenine dinucleotide (NAD+) biosynthesis and extracellular ATP levels in the mPFC. Moreover, both the 2-week intraperitoneal (i.p.) injection and 3-week oral gavage of NMN prior to exposure to CSDS effectively prevented the development of depressive-like behavior in mice. These protective effects were accompanied with the preservation of both NAD+ biosynthesis and extracellular ATP level in the mPFC. Furthermore, catalyzing ATP hydrolysis by mPFC injection of the ATPase apyrase negated the prophylactic effects of NMN on CSDS-induced depressive-like behaviors. Prophylactic NMN treatment also prevented the reduction in GABAergic inhibition and the increase in excitability in mPFC neurons projecting to the lateral habenula (LHb). Collectively, these findings demonstrate that the prophylactic effects of NMN on depressive-like behaviors are mediated by preventing extracellular ATP loss in the mPFC, which highlights the potential of NMN supplementation as a novel approach for protecting and preventing stress-induced depression in susceptible individuals.

A ferroptosis amplifier based on triple-enhanced lipid peroxides accumulation strategy for effective pancreatic cancer therapy.

As an iron dependent regulatory cell death process driven by excessive lipid peroxides (LPO), ferroptosis is recognized as a powerful weapon for pancreatic cancer (PC) therapy. However, the tumor microenvironment (TME) with hypoxia and elevated glutathione (GSH) expression not only inhibits LPO production, but also induces glutathione peroxidase 4 (GPX4) mediated LPO clearance, which greatly compromise the therapeutic outcomes of ferroptosis. To address these issues, herein, a novel triple-enhanced ferroptosis amplifier (denoted as Zal@HM-PTBC) is rationally designed. After intravenous injection, the overexpressed H2O2/GSH in TME induces the collapse of Zal@HM-PTBC and triggers the production of oxygen and reactive oxygen species (ROS), which synergistically amplify the degree of lipid peroxidation (broaden sources). Concurrently, GSH consumption because of the degradation of the hollow manganese dioxide (HM) significantly weakens the activity of GPX4, resulting in a decrease in LPO clearance (reduce expenditure). Moreover, the loading and site-directed release of zalcitabine further promotes autophagy-dependent LPO accumulation (enhance effectiveness). Both in vitro and in vivo results validated that the ferroptosis amplifier demonstrated superior specificity and favorable therapeutic responses. Overall, this triple-enhanced LPO accumulation strategy demonstrates the ability to facilitate the efficacy of ferroptosis, injecting vigorous vitality into the treatment of PC.

Cas12a-based one-pot SNP detection with high accuracy.

CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant with enhanced sensitivity against SNP, named seCas12a (sensitive Cas12a). SeCas12a-based one-pot SNP detection system is a versatile platform that could use both canonical and non-canonical PAM, and was almost not limited by mutation types to distinguish SNPs located between position 1 to 17. The use of truncated crRNA further improved SNP specificity of seCas12a. Mechanistically, we found only when the cis-cleavage was at low level between 0.01min-1 and 0.0006 min-1, a good signal-to-noise ratio can be achieved in one-pot test. SeCas12a-based one-pot SNP detection system was applied to detect pharmacogenomic SNPs in human clinical samples. Of thirteen donors tested in two different SNPs, the seCas12a mediated one-pot system could faithfully detect the SNPs in 30 min with 100% accuracy.

Metformin Ameliorates D-Galactose-Induced Senescent Human Bone Marrow-Derived Mesenchymal Stem Cells by Enhancing Autophagy.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are promising candidates for stem cell therapy in clinical trials. Applications of hBMSCs in clinical therapy are limited by cellular senescence due to long-term ex vivo expansion. Metformin, an oral hypoglycemic drug for type 2 diabetes, has been shown to have antiaging effects. However, the mechanisms of metformin in antiaging treatment remain controversial. Here, we used D-galactose (D-gal) to establish an appropriate model of senescent hBMSCs to explore the antiaging effects of metformin. Following metformin treatment with a low concentration range, senescence phenotypes induced by D-gal significantly changed, including generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and cell cycle arrest. In contrast, no apparent change was found in unsenescent hBMSCs. Furthermore, the results show that activation of 5'AMP-activated protein kinase (AMPK) by metformin enhances cell autophagy in senescent hBMSCs. These findings suggest that metformin exerts antiaging function within the low concentration range by enhancing autophagy and exhibits potential benefits for clinical stem cell therapy by ameliorating the ex vivo replicative senescence of hBMSCs.

A Dual Stimuli-Responsive Nanoplatform Loaded PtIV -Triptolide Prodrug for Achieving Synergistic Therapy toward Breast Cancer.

To strengthen the antitumor efficacy and avoid toxicity to normal cells of cisplatin and triptolide, herein, an acid and glutathione (GSH) dual-controlled nanoplatform for enhanced cancer treatment through the synergy of both "1+1" apoptosis and "1+1" ferroptosis is designed. Remarkably, ZIF8 in response to tumor microenvironment enhances drug targeting and protects drugs from premature degradation. Meanwhile, the PtIV  center can be easily reduced to cisplatin because of the large amount of GSH, thus liberating the triptolide as the coordinated ligand. The released cisplatin and hemin in turn boost the tumor cell "1+1" apoptosis through chemotherapy and photodynamic therapy, respectively. Furthermore, GSH reduction through PtIV  weakens the activation of glutathione peroxidase 4 (GPX4) effectively. The released triptolide can inhibit the expressions of GSH by regulating nuclear factor E2 related factor 2 (Nrf2), further promoting membrane lipid peroxidation, thus "1+1" ferroptosis can be achieved. Both in vitro and in vivo results demonstrate that the nanosystem can not only perform superior specificity and therapeutic outcomes but also reduce the toxicity to normal cells/tissues of cisplatin and triptolide effectively. Overall, the prodrug-based smart system provides an efficient therapeutic strategy for cancer treatment by virtue of the effect of enhanced "1+1" apoptosis and "1+1" ferroptosis therapies.

Characterization of a thermostable Cas12a ortholog.

CRISPR-Cas12a has been used for genome editing and molecular diagnosis. The well-studied Cas12a orthologs have a T-rich PAM and are usually categorized as non-thermally stable enzymes. Here, we identified a new Cas12a ortholog from Clostridium thermobutyricum, which survives at 60 °C. This Cas12a ortholog is named as CtCas12a and exhibits low sequence similarity to the known Cas12a family members. CtCas12a is active in a wide temperature range from 17 to 77 °C. Moreover, this ortholog has a relaxed PAM of YYV (Y=C or T, V = A or C or G). We optimized the conditions for trans-cleavage and enabled its detection of nucleic acids. CtCas12a executed genome editing in human cells and generated up to 26% indel formation in the EGFP locus. With the ability to be active at high temperatures as well as having a relaxed PAM sequence, CtCas12a holds potential to be further engineered for pathogen detection and editing a wide range of genomic sequences.

Lipopolysaccharide-induced endotoxaemia during adolescence promotes stress vulnerability in adult mice via deregulation of nuclear factor erythroid 2-related factor 2 in the medial prefrontal cortex.

RATIONALE: Sepsis is a severe inflammatory response to infection that leads to long-lasting cognitive impairment and depression after resolution. The lipopolysaccharide (LPS)-induced endotoxaemia model is a well-established model of gram-negative bacterial infection and recapitulates the clinical characteristics of sepsis. However, whether LPS-induced endotoxaemia during adolescence can modulate depressive and anxiety-like behaviours in adulthood remains unclear. OBJECTIVES: To determine whether LPS-induced endotoxaemia in adolescence can modulate the stress vulnerability to depressive and anxiety-like behaviours in adulthood and explore the underlying molecular mechanisms. METHODS: Quantitative real-time PCR was used to measure inflammatory cytokine expression in the brain. A stress vulnerability model was established by exposure to subthreshold social defeat stress (SSDS), and depressive- and anxiety-like behaviours were evaluated by the social interaction test (SIT), sucrose preference test (SPT), tail suspension test (TST), force swimming test (FST), elevated plus-maze (EPM) test, and open field test (OFT). Western blotting was used to measure Nrf2 and BDNF expression levels in the brain. RESULTS: Our results showed that inflammation occurred in the brain 24 h after the induction of LPS-induced endotoxaemia at P21 but resolved in adulthood. Furthermore, LPS-induced endotoxaemia during adolescence promoted the inflammatory response and the stress vulnerability after SSDS during adulthood. Notably, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and BDNF in the mPFC were decreased after SSDS exposure in mice treated with LPS during adolescence. Activation of the Nrf2-BDNF signalling pathway by sulforaphane (SFN), an Nrf2 activator, ameliorated the effect of LPS-induced endotoxaemia during adolescence on stress vulnerability after SSDS during adulthood. CONCLUSIONS: Our study identified adolescence as a critical period during which LPS-induced endotoxaemia can promote stress vulnerability during adulthood and showed that this effect is mediated by impairment of Nrf2-BDNF signalling in the mPFC.

Polyethylene microplastics cooperate with Helicobacter pylori to promote gastric injury and inflammation in mice.

Microplastics provide stable habitats for the colonization and survival of pathogenic microorganisms, and cooperate with microorganisms to pose a potential threat to human health. In this study, polyethylene microplastics (PE-MPs) in artificial gastric juice time-dependently decomposed and broke into small-diameter PE-MP fragments that were more stable than those in an aqueous solution. Helicobacter pylori adhered to the surfaces of the PE-MPs to form a biofilm. The gastric tissues of mice treated with PE-MPs first and mixture of PE-MPs and H. pylori were positive for H. pylori infection in the 10th and 14th weeks after treatment, whereas those infected with H. pylori first and H. pylori alone were positive only in the 14th week after treatment. PE-MPs were visible in the gastric, intestinal, and liver tissues of mice treated with PE-MPs. The average diameter of the PE-MP fragments in the liver was greater than those of fragments that entered the gastric or intestinal tissues, and the average diameter of PE-MPs in the PE-MPs only-treated mice was significantly smaller than those of PE-MPs entering the intestinal tissues of the other groups. The infiltration of inflammatory cells was most serious in the mice treated with the mixture of PE-MPs and H. pylori, or with PE-MPs first and then H. pylori. Of all the groups, the gastric organ index and MPO, IL6, and TNF-α levels were highest in the mice treated with the mixture of PE-MPs and H. pylori. These results indicate that the interaction between PE-MPs and H. pylori contributed to the rapid bacterial colonization of gastric mucosal epithelial cells, improved the efficiency of PE-MP entry into tissues, and promoted gastric injury and inflammation in mice. These findings suggest that microplastics may provide a stable habitat for H. pylori, and act synergistically with H. pylori to pose a potential threat to human health.

Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

Tissue distribution of polystyrene nanoplastics in mice and their entry, transport, and cytotoxicity to GES-1 cells.

With the widespread use of plastics and nanotechnology products, nanoplastics (NPs) have become a potential threat to human health. It is of great practical significance to study and evaluate the distribution of NPs in mice as mammal models and their entry, transport, and cytotoxicity in human cell lines. In this study, we detected the tissue distribution of fluorescent polystyrene nanoplastics (PS-NPs) in mice and assessed their endocytosis, transport pathways, and cytotoxic effects in GES-1 cells. We found that PS-NPs were clearly visible in gastric, intestine, and liver tissues of mice and in GES-1 cells treated with PS-NPs. Entry of PS-NPs into GES-1 cells decreased with the inhibition of caveolae-mediated endocytosis (nystatin), clathrin-mediated endocytosis (chlorpromazine HCl), micropinocytosis (ethyl-isopropyl amiloride), RhoA (CCG-1423), and F-actin polymerization (lantrunculin A). Rac1 inhibitors (NSC 23766) had no significant effect on PS-NPs entering GES-1 cells. F-actin levels significantly decreased in CCG-1423-pretreated GES-1 cells exposed to PS-NPs. GES-1 cell ultrastructural features indicated that internalized PS-NPs can be encapsulated in vesicles, autophagosomes, lysosomes, and lysosomal residues. RhoA, F-actin, RAB7, and LAMP1 levels in PS-NPs-treated GES-1 cells were remarkably up-regulated and the Rab5 level was significantly down-regulated compared to levels in untreated cells. PS-NPs treatment decreased cell proliferation rates and increased cell apoptosis. The formation of autophagosomes and autolysosomes and levels of LC3II increased with the length of PS-NPs treatment. The results indicated that cells regulated endocytosis in response to PS-NPs through the RhoA/F-actin signaling pathway and internalized PS-NPs in the cytoplasm, autophagosomes, or lysosomes produced cytotoxicity. These results illustrate the potential threat of NPs pollution to human health.

Structural Basis for Inhibiting Porcine Epidemic Diarrhea Virus Replication with the 3C-Like Protease Inhibitor GC376.

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.