An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants.

The type V-I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

Discovery and Optimization of WDR5 Inhibitors via Cascade Deoxyribonucleic Acid-Encoded Library Selection Approach.

The DNA-encoded library (DEL) is a powerful hit generation tool for chemical biology and drug discovery; however, the optimization of DEL hits remained a daunting challenge for the medicinal chemistry community. In this study, hit compounds targeting the WIN binding domain of WDR5 were discovered by the initial three-cycle linear DEL selection, and their potency was further enhanced by a cascade DEL selection from the focused DEL designed based on the original first run DEL hits. As expected, these new compounds from the second run of focused DEL were more potent WDR5 inhibitors in the protein binding assay confirmed by the off-DNA synthesis. Interestingly, selected inhibitors exhibited good antiproliferative activity in two human acute leukemia cell lines. Taken together, this new cascade DEL selection strategy may have tremendous potential for finding high-affinity leads against WDR5 and provide opportunities to explore and optimize inhibitors for other targets.

Chicken speckle-type POZ protein (SPOP) negatively regulates MyD88/NF-κB signaling pathway mediated proinflammatory cytokine production to promote the replication of Newcastle disease virus.

The speckle-type POZ protein (SPOP) is demonstrated to be a specific adaptor of the cullin-RING-based E3 ubiquitin ligase complex that participates in multiple cellular processes. Up to now, SPOP involved in inflammatory response has attracted more attention, but the association of SPOP with animal virus infection is scarcely reported. In this study, chicken MyD88 (chMyD88), an innate immunity-associated protein, was screened to be an interacting partner of chSPOP using co-immunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry methods. This interaction was further confirmed by fluorescence co-localization, Co-IP, and pull-down assays. It was interesting that exogenous recombinant protein HA-chSPOP or endogenous chSPOP alone was mainly located in the nucleus but was translocated to the cytoplasm upon co-expression with chMyD88 or lipopolysaccharide stimulation. In addition, chSPOP reduced chMyD88 expression by ubiquitination in a dose-dependent manner, and the regulation of NF-κB activity by chSPOP was dependent solely on chMyD88. Importantly, chSPOP played a negative regulatory role in the MyD88/NF-κB signaling pathway and the production of proinflammatory cytokines. Moreover, we found that velogenic Newcastle disease virus (NDV) infection changed the subcellular localization of chSPOP and the expression patterns of chSPOP and chMyD88, and overexpression of chSPOP decreased the production of proinflammatory cytokines to enhance velogenic and lentogenic NDV replication, while siRNA-mediated chSPOP knockdown obtained the opposite results, thereby indicating that chSPOP negatively regulated MyD88/NF-κB signaling pathway mediated proinflammatory cytokine production to promote NDV replication. These findings highlight the important role of the SPOP/MyD88/NF-κB signaling pathway in NDV replication and may provide insightful information about NDV pathogenesis.

Genome editing in rice using CRISPR/Cas12i3.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Multifunctionality of matrix protein in the replication and pathogenesis of Newcastle disease virus: A review.

As an important structural protein in virion morphogenesis, the matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to be a nuclear-cytoplasmic trafficking protein and plays essential roles in viral assembly and budding. In recent years, increasing lines of evidence have indicated that the M protein has obvious influence on the pathotypes of NDV, and the interaction of M protein with cellular proteins is also closely associated with the replication and pathogenicity of NDV. Although substantial progress has been made in the past 40 years towards understanding the structure and function of NDV M protein, the available information is scattered. Therefore, this review article summarizes and updates the research progress on the structural feature, virulence and pathotype correlation, and nucleocytoplasmic transport mechanism of NDV M protein, as well as the functions of M protein and cellular protein interactions in M's intracellular localization, viral RNA synthesis and transcription, viral protein synthesis, viral immune evasion, and viral budding and release, which will provide an in-depth understanding of the biological functions of M protein in the replication and pathogenesis of NDV, and also contribute to the development of effective antiviral strategies aiming at blocking the early or late steps of NDV lifecycles.

Discovery, SAR Study of GST Inhibitors from a Novel Quinazolin-4(1H)-one Focused DNA-Encoded Library.

The DNA-encoded library (DEL) is a powerful hit-generation tool in drug discovery. This study describes a new DEL with a privileged scaffold quinazolin-4(3H)-one developed by a robust DNA-compatible multicomponent reaction and a series of novel glutathione S-transferase (GST) inhibitors that were identified through affinity-mediated DEL selection. A novel inhibitor 16 was subsequently verified with an inhibitory potency value of 1.55 ± 0.02 µM against SjGST and 2.02 ± 0.20 µM against hGSTM2. Further optimization was carried out via various structure-activity relationship studies. And especially, the co-crystal structure of the compound 16 with the SjGST was unveiled, which clearly demonstrated its binding mode was quite different from the known GSH-like compounds. This new type of probe is likely to play a different role compared with the GSH, which may provide new opportunities to discover more potent GST inhibitors.

Mutation of Basic Residues R283, R286, and K288 in the Matrix Protein of Newcastle Disease Virus Attenuates Viral Replication and Pathogenicity.

The matrix (M) protein of Newcastle disease virus (NDV) contains large numbers of unevenly distributed basic residues, but the precise function of most basic residues in the M protein remains enigmatic. We previously demonstrated that the C-terminus (aa 264-313) of M protein interacted with the extra-terminal (ET) domain of chicken bromodomain-containing protein 2 (chBRD2), which promoted NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. However, the key amino acid sites determining M's interaction with chBRD2/ET and their roles in the replication and pathogenicity of NDV are not known. In this study, three basic residues-R283, R286, and K288-in the NDV M protein were verified to be responsible for its interaction with chBRD2/ET. In addition, mutation of these basic residues (R283A/R286A/K288A) in the M protein changed its electrostatic pattern and abrogated the decreased expression of endogenic chBRD2. Moreover, a recombinant virus harboring these mutations resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chickens due to the decreased viral RNA synthesis and transcription. Our findings therefore provide a better understanding of the crucial biological functions of M's basic residues and also aid in understanding the poorly understood pathogenesis of NDV.

The rare complication caused by thrombus shedding during arteriovenous fistula thrombolysis: a case report.

BACKGROUND: Arteriovenous fistula is the lifeline of maintenance for patients requiring hemodialysis, with thrombosis being a common complication of this procedure. Traditionally, thrombi have been removed via thrombectomy. In recent years, it has been reported that newly occurring thrombi can be treated by urokinase thrombolysis. However, the thrombus shedding in the process should be valued, which may cause the distal limb ischemia syndrome. The complication of thrombolysis is rare but serious. Patients will experience pain and numbness, and possibly even extremity necrosis may occur without diagnosed or treated timely. There are not any reports about the occurrence or treatment of distal limb ischemia syndrome caused by thrombus shedding during thrombolysis. CASE DESCRIPTION: Considering the thrombosis volume and texture of this case, we attempted to use urokinase thrombolysis to resolve the thrombus in fistula. During thrombolysis, thrombus shedding occurred in the distal limb. The patient's fingers of the limbs on the side of the internal fistula were pale, numb, and painful in the case. Fortunately, we solved the problem ultimately by continuously pumping urokinase. The heparin, urokinase, infusion pumps, ultrasound, and infrared therapy devices were obtained from the Affiliated Hospital of Chuanbei Medical College. Their usage and dosage are described in the relevant literature and China's 2020 blood purification standard operating procedures. CONCLUSIONS: In the process of thrombolysis of arteriovenous fistula, attention should be paid to thrombus shedding. Distal limb ischemia syndrome is a rare but serious complication of thrombus shedding. Continued pumping of urokinase may be effective for this complication.

Cas12a-based on-site, rapid detection of genetically modified crops.

A CRISPR/LbCas12a-based nucleic acid detection method that uses crude leaf extracts as samples and is rapid (≤40 min for a full run) and highly sensitive (0.01%) can be used to monitor genetically modified organisms in the field.

The matrix protein of Newcastle disease virus inhibits inflammatory response through IRAK4/TRAF6/TAK1/NF-κB signaling pathway.

The matrix (M) protein of several cytoplasmic RNA viruses has been reported to be an NF-κB pathway antagonist. However, the function and mechanism of NDV M protein antagonizing NF-κB activation remain largely unknown. In this study, we found that the expression levels of IRAK4, TRAF6, TAK1, and RELA/p65 were obviously reduced late in NDV infection. In addition, the cytoplasmic M protein rather than other viral proteins decreased the expression of these proteins in a dose-dependent manner. Further indepth analysis showed that the N-terminal 180 amino acids of M protein were not only responsible for the reduced expression of these proteins, but also responsible for the inhibition of NF-κB activation and nuclear translocation of RELA/p65, as well as the production of inflammatory cytokines. Moreover, small interference RNA-mediated knockdown of IRAK4 or overexpression of IRAK4 markedly enhanced or reduced NDV replication by decreasing or increasing inflammatory cytokines production through the IRAK4/TRAF6/TAK1/NF-κB signaling pathway. Strangely, there were no interactions detected between NDV M protein and IRAK4, TRAF6, TAK1 or RELA/p65. Our findings described here contribute to a better understanding of the innate immune antagonism function of M protein and the molecular mechanism underlying the replication and pathogenesis of NDV.

Water-soluble non-conjugated polymer dots with strong green fluorescence for sensitive detection of organophosphate pesticides.

Water-soluble non-conjugated polymer dots (PDs) have been synthesized using hyperbranched polyethyleneimine (PEI) and dihydroxybenzaldehyde (DHB) for the first time via the Schiff base reaction at room temperature. The yielded non-conjugated PDs of PEI-DHB could display the well-defined spheric structure and good water solubility. In contrast to the common PDs otherwise showing blue emission, the PEI-DHB PDs could give out strong green fluorescence in aqueous media. Especially, the fluorescence of the PEI-DHB PDs could be specifically quenched by MnO2 nanosheets through the inner filter effects and further restored by the thiocholine that could reduce MnO2 nanosheets into Mn2+. Herein, thiocholine could be produced in hydrolysis reaction of acetylthiocholine catalyzed by the acetylcholinesterase (AChE), of which the catalytic activity could be irreversibly inhibitted by the introduction of organophosphates. A highly selective fluorimetric method was thereby been developed for the detection of organophosphorus pesticides using dimethyl-dichloro-vinyl phosphate as a model. The linear concentrations ranges from 0.050 to 2.5 µM. Importantly, the non-conjugated PDs probes with strong green fluorescence and high water solubility may promise the extensive applications in the environmental, food, and clinical analysis fields.

The association of ribosomal protein L18 with Newcastle disease virus matrix protein enhances viral translation and replication.

Numerous studies have shown that viruses can utilize or manipulate ribosomal proteins to achieve viral protein biosynthesis and replication. In our recent studies using proteomics analysis of virus-infected cells, we found that ribosomal protein L18 (RPL18) was the highest up-regulated differentially expressed protein, along with the increasingly expressed viral proteins later in Newcastle disease virus (NDV) infection. However, the association of RPL18 with viral protein biosynthesis and NDV replication remains unclear. In this study, we found that the expression and transcription levels of RPL18 was reduced early in NDV infection but increased later in NDV infection. In addition, the presence of cytoplasmic NDV matrix (M) protein was responsible for the increased expression of RPL18 in both virus-infected cells and plasmid-transfected cells. Moreover, cytoplasmic M protein increased RPL18 expression in a dose-dependent manner, even though they did not interact with each other. Furthermore, siRNA-mediated knockdown of RPL18 or overexpression of RPL18 dramatically reduced or enhanced NDV replication by decreasing or increasing viral protein translation rather than viral RNA synthesis and transcription. Taken together, these results suggested that the increased expression of RPL18 might be associated with the physical clumping together of the M protein, which in turn promoted viral protein biosynthesis and NDV replication. RESEARCH HIGHLIGHTSThe increased expression of RPL18 is associated with the presence of cytoplasmic M protein.Cytoplasmic M protein increases RPL18 expression in a dose-dependent manner.Knockdown of RPL18 reduces NDV replication by decreasing viral protein translation.Overexpression of RPL18 enhances NDV replication by increasing viral protein translation.

Sulodexide Prevents Peritoneal Fibrosis by Downregulating the Expression of TGF-ß1 and Its Signaling Pathway Molecules.

Peritoneal dialysis is one of the main renal replacement treatments. However, long-term peritoneal dialysis keeps the peritoneum in contact with the sugar-containing nonphysiological peritoneal fluid, which leads to recurrent peritonitis, peritoneal fibrosis, and failure of ultrafiltration. Transforming growth factor-ß1 (TGF-ß1), related cytokines, and inflammatory factors are closely related to peritoneal fibrosis. Sulodexide (SLX) is a new type of glycosaminoglycan preparation, which is involved in the formation of an anionic charge barrier and can maintain the selective permeability of vascular endothelial cells. In this study, the innovative analysis of SLX specifically prevents the process of peritoneal dialysis peritoneal fibrosis by downregulating the expression of TGF-ß1 and its signaling pathway molecules. We randomly divided 30 rats into three groups. The blank control group received no treatment. The peritoneal dialysis model group was injected with 4.25% peritoneal dialysate (PDF) 20 ml daily, and the SLX group was injected with 4.25% PDF 20 ml + sulodexide (SLX) 20 mg/kg daily. After 8 weeks of dialysis, the rats were sacrificed, and the peritoneal function test was performed to determine the amount of glucose transport and ultrafiltration. The thickness of peritoneal per unit area was observed under high magnification. The level of inflammation in peritoneal tissue and the expression of TGF-ß1/Smad were detected. The results showed that SLX can significantly improve peritoneal tissue thickening and inflammation, can downregulate the expression of TGF-ß1, Smad2, Smad3, and Smad7 in peritoneal tissue, and improve the progression of peritoneal fibrosis.

Effects of N-Acetylcysteine on the reproductive performance, oxidative stress and RNA sequencing of Nubian goats.

N-acetylcysteine (NAC) has been found to enhance the protective ability of cells to counter balance oxidative stress and inflammation. To investigate the effects of dietary NAC supplementation on the reproductive performance of goats, the reproductive performance and endometrial transcriptome of goats fed with diets with NAC (NAC group) and without NAC supplementation (control group) were compared. Results showed that the goats fed with 0.03% and 0.05% NAC had similar litter size, birth weight, nitric oxide (NO), sex hormones and amino acids levels compared with the goats of the control group. However, feeding with 0.07% NAC supplementation from day 0 to day 30 of gestation remarkably increased the litter size of goats. The goats of the 0.07% NAC group presented increased levels of NO relative to the control group, but their sex hormones and amino acids showed no differences. Comparative transcriptome analysis identified 207 differentially expressed genes (DEGs) in the endometrium between the control and the 0.07% NAC groups. These DEGs included 146 upregulated genes and 61 downregulated genes in the 0.07% NAC group. They were primarily involved in the cellular response to toxic substances, oxidoreductase activity, immune receptor activity, signalling receptor binding, cytokine-cytokine receptor interactions, PI3K-Akt signalling pathway and PPAR signalling pathway. In conclusion, results showed that dietary 0.07% NAC supplementation exerted a beneficial effect on the survival of goat embryos at the early pregnancy stage. Such positive outcome might be due to the increased NO production and affected expression of genes involved in the anti-inflammation pathways of the endometrium.

Chicken bromodomain-containing protein 2 interacts with the Newcastle disease virus matrix protein and promotes viral replication.

Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264-313) of the M protein and the extra-terminal (ET) domain (aa 619-683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.

miR-31-5p Regulates 14-3-3 ɛ to Inhibit Prostate Cancer 22RV1 Cell Survival and Proliferation via PI3K/AKT/Bcl-2 Signaling Pathway.

INTRODUCTION: Prostate cancer (PCa) is one of the most common malignancies, and almost all patients with advanced PCa will develop castration-resistant prostate cancer (CRPC) after receiving endocrine therapy. Effective treatment for patients with CRPC has not been established. Novel approaches are needed to identify therapeutic targets for CRPC. PURPOSE: Recent research studies have found that members of the 14-3-3 family play an important role in the development and progression of PCa. Previous results have shown that 14-3-3 ɛ is significantly upregulated in several cancers. This study aimed to identify novel miRNAs that regulate 14-3-3 ɛ expression and therapeutic targets for CRPC. METHODS: In this study, we used computation and experimental approaches for the prediction and verification of the miRNAs targeting 14-3-3 ɛ, and investigated the potential roles of 14-3-3 ɛ in the survival and proliferation of 22RV1 cells. RESULTS: We confirm that mir-31-5p is downregulated in 22RV1 cells and acts as a tumor suppressor by regulating 14-3-3 ɛ. Ectopic expression of miR-31-5p or 14-3-3 ɛ interference significantly inhibits cell proliferation, invasion, and migration in 22RV1 cells, as well as promotes cell apoptosis via the PI3K/AKT/Bcl-2 signaling pathway. Moreover, 14-3-3 ɛ is required for the miR-31-5p-mediated upregulation of the PI3K/AKT/Bcl-2 signaling pathway. CONCLUSION: Our findings provide information on the underlying mechanisms of miR-31-5p/14-3-3 ɛ in 22RV1 cell proliferation and apoptosis through the PI3K/AKT/Bcl-2 signaling pathway. These results suggest that miR-31-5p and 14-3-3 ɛ may potentially be utilized as novel prognostic markers and therapeutic targets for PCa treatment.

On-DNA Cross-Dehydrogenative Coupling Reaction toward the Synthesis of Focused DNA-Encoded Tetrahydroisoquinoline Libraries.

To expand the chemical space of DNA-encoded library (DEL) technology, we have developed the DNA-compatible cross-dehydrogenative coupling reaction between tetrahydroisoquinolines (THIQs) and a series of nucleophiles such as nitromethane, terminal alkynes, and so on. Both photo- and metal-promoted conditions have been successfully established for this C-1 derivation of THIQs, as exemplified by the addition of nitromethane. All of these new DNA-compatible transformations have paved the way for the divergent oriented synthesis of THIQ-focused DELs.

Roles of the Brassica napus DELLA Protein BnaA6.RGA, in Modulating Drought Tolerance by Interacting With the ABA Signaling Component BnaA10.ABF2.

Drought is a major threat to plant growth and crop productivity. Reduced level of the gibberellin would result in increased drought tolerance, but the underlying mechanism is still unclear. In Brassica napus, there are four BnaRGA genes that code for DELLA proteins, negative regulators of GA signaling. Among them, expression of BnaA6.RGA was greatly induced by drought and abscisic acid (ABA). Previously, we created the gain-of-function mutant of BnaA6.RGA, bnaa6.rga-D, and the loss-of-function quadruple mutant, bnarga by CRISPR/Cas9, respectively. Here we show that bnaa6.rga-D displayed enhanced drought tolerance, and its stomatal closure was hypersensitive to ABA treatment. By contrast, bnarga displayed reduced drought tolerance and was less sensitive to ABA treatment, but there is no difference in drought tolerance between single BnaRGA mutant and WT, suggesting a functional redundancy between the BnaRGA genes in this process. Furthermore, we found that BnaRGAs were able to interact physically with BnaA10.ABF2, an essential transcription factor in ABA signaling. The BnaA10.ABF2-BnaA6.RGA protein complex greatly increased the expression level of the drought responsive gene BnaC9.RAB18. Taken together, this work highlighted the fundamental roles of DELLA proteins in drought tolerance in B. napus, and provide desirable germplasm for further breeding of drought tolerance in rapeseed.