Expression pattern and activity of six glutelin gene promoters in transgenic rice*

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with β-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity

Symptomatic and Asymptomatic Protist Infections in Hospital Inpatients in Southwestern China

Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, and Blastocystis sp. infections have been frequently reported as etiological agents for gastroenteritis, but also as common gut inhabitants in apparently healthy individuals. Between July 2016 and March 2017, stool samples (n = 507) were collected from randomly selected individuals (male/female ratio: 1.1, age range: 38-63 years) from two sentinel hospitals in Tengchong City Yunnan Province, China. Molecular (PCR and Sanger sequencing) methods were used to detect and genotype the investigated protist species. Carriage/infection rates were: Blastocystis sp. 9.5% (95% CI: 7.1-12.4%), G. duodenalis 2.2% (95% CI: 1.1-3.8%); and E. histolytica 2.0% (95% CI: 0.9-3.6%). Cryptosporidium spp. was not detected at all. Overall, 12.4% (95% CI: 9.7-15.6) of the participants harbored at least one enteric protist species. The most common coinfection was E. histolytica and Blastocystis sp. (1.0%; 95% CI: 0.3-2.2). Sequence analyses revealed that 90.9% (10/11) of the genotyped G. duodenalis isolates corresponded to the sub-assemblage AI. The remaining sequence (9.1%, 1/11) was identified as sub-assemblage BIV. Five different Blastocystis subtypes, including ST3 (43.7%, 21/48), ST1 (27.1%, 13/48), ST7 (18.8%, 9/48), ST4 (8.3%, 4/48), and ST2 (2.1%, 1/48) were identified. Statistical analyses confirmed that (i) the co-occurrence of protist infections was purely random, (ii) no associations were observed among the four protist species found, and (iii) neither their presence, individually or jointly, nor the patient's age was predictors for developing clinical symptoms associated with these infections. Overall, these protist mono- or coinfections are asymptomatic and do not follow any pattern.This research was supported by the fund of the 13th Five-Year National Science and Technology Major Project for Infectious Diseases (No. 2017ZX10305501-002, No. 2018ZX10725-509), the fund of Chinese traditional medicine for treating the novel Coronavirus pneumonia patients in convalescence (No. JJ202002), the Emergency Project of Shanghai for the prevention and treatment of COVID-19 in traditional Chinese medicine (Grant No. 2020NCP001), the fund of the China Postdoctoral Science Foundation (No.2020T130022ZX), the fund of National Natural Science Foundation of China (No. 81473022). In addition, E.S. was a recipient of a Ramon y Cajal agreement (RYC-2016-21120) funded by the Spanish Ministry of Economy and Competitiveness (MINECO).S

Protective Effects of Li-Fei-Xiao-Yan Prescription on Lipopolysaccharide-Induced Acute Lung Injury via Inhibition of Oxidative Stress and the TLR4/NF- κ

Li-Fei-Xiao-Yan prescription (LFXY) has been clinically used in China to treat inflammatory and infectious diseases including inflammatory lung diseases. The present study was aimed at evaluating the potential therapeutic effects and potential mechanisms of LFXY in a murine model of lipopolysaccharide- (LPS-) induced acute lung injury (ALI). In this study, the mice were orally pretreated with LFXY or dexamethasone (positive drug) before the intratracheal instillation of LPS. Our data indicated that pretreatment with LFXY enhanced the survival rate of ALI mice, reversed pulmonary edema and permeability, improved LPS-induced lung histopathology impairment, suppressed the excessive inflammatory responses via decreasing the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokine (MIP-2) and inhibiting inflammatory cells migration, and repressed oxidative stress through the inhibition of MPO and MDA contents and the upregulation of antioxidants (SOD and GSH) activities. Mechanistically, treatment with LFXY significantly prevented LPS-induced TLR4 expression and NF-κB (p65) phosphorylation. Overall, the present study suggests that LFXY protected mice from acute lung injury induced by LPS via inhibition of TLR4/NF-κB p65 activation and upregulation of antioxidative enzymes and it may be a potential preventive and therapeutic agent for ALI in the clinical setting

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation

CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function

A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation. We have found that the rate of homology-directed recombination mediated by guideRNA (gRNA) complementary to the deletion mutant is significantly higher than the one mediated by gRNA complementary to the wild-type (WT) donor. In addition, the rate of homology-directed recombination mediated by gRNA complementary to the WT donor is significantly higher than that of gRNAs complementary to the 5′ or 3′ side of the deletion mutant. Interestingly, the frequency of mutations introduced by gRNA complementary to the deletion mutant is significantly higher than with gRNA complementary to WT donor. However, combination of gRNAs complementary to both WT donor and deletion mutant decreased the rate of homology-directed recombination, but did not significantly decrease the mutation rate introduced by this system. Thus, the data presented here provide guidance for designing of gRNA and donor DNA to do genome editing, especially to correct the mutations with three mismatched nucleotides, such as three-nucleotide deletion or insertion

Sulforaphene Ameliorates Neuroinflammation and Hyperphosphorylated Tau Protein via Regulating the PI3K/Akt/GSK-3β Pathway in Experimental Models of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia characterized by progressive loss of cognitive functions due to neuronal death mainly in the hippocampal and cortical brain. Sulforaphene (SF) is one of the main isothiocyanates isolated from a Chinese herb Raphani Semen. In this study, we aimed to investigate the neuroprotective effects of SF using in vitro and in vivo models of AD. Streptozotocin (STZ) was intracranially injected into the rats; then, SF (25 and 50 mg/kg) was given orally once a day for 6 consecutive weeks. After drug treatment, the cognitive functions were assessed using the Morris Water Maze Test (MWMT). After the MWMT, the rats were euthanized and brain tissues were collected. In the in vitro test, BV-2 microglia were pretreated with SF (1 and 2 μM) for 1 h and then stimulated with lipopolysaccharide (LPS) for another 23 h. Both molecular and histological methods were used to unravel the action mechanisms and elucidate the signaling pathway. The MWMT results showed that SF treatment significantly improved the STZ-induced cognitive deficits in rats. SF treatment markedly suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) but increased the release of IL-10 in the STZ-treated rats. In addition, SF significantly inhibited the phosphorylation of tau protein at Thr205, Ser396, and Ser404 sites, while enhancing the ratios of p-Akt (Ser473)/Akt and p-GSK-3β (Ser9)/GSK-3β in the hippocampus of the STZ-treated rats. On the other hand, SF (1 and 2 μM) treatment also markedly attenuated the cytotoxicity induced by LPS in BV-2 cells. In addition, SF treatment obviously suppressed the releases of nitric oxide (NO), TNF-α, and IL-6 in the LPS-stimulated BV-2 cells. Moreover, SF treatment significantly mitigated the nuclear translocation of p-NF-κB p65 and the ratio of p-GSK-3β (Ser9)/GSK-3β in LPS-stimulated BV-2 cells. Taken together, SF possessed neuroprotective effects against the STZ-induced cognitive deficits in rats and LPS-induced neuroinflammation in BV-2 cells via modulation of the PI3K/Akt/GSK-3β pathway and inhibition of the NF-κB activation, suggesting that SF is a promising neuroprotective agent worthy of further development into AD treatment

Evidence on Integrating Pharmacokinetics to Find Truly Therapeutic Agent for Alzheimer’s Disease: Comparative Pharmacokinetics and Disposition Kinetics Profiles of Stereoisomers Isorhynchophylline and Rhynchophylline in Rats

Isorhynchophylline (IRN) and rhynchophylline (RN), a pair of stereoisomers, are tetracyclic oxindole alkaloids isolated from Uncaria rhynchophylla, a commonly used Chinese medicinal herb. These two compounds have drawn extensive attention due to their potent neuroprotective effects with promising therapeutic potential for the treatment of Alzheimer’s disease (AD). However, IRN and RN can interconvert into each other in vivo after oral administration. The present study aimed to elucidate the pharmacokinetic profiles and disposition kinetics of the administered and generated stereoisomers in the brain and cerebrospinal fluid (CSF) after oral administration of equal dose of IRN or RN to rats. Our study demonstrated that after oral administration, RN showed significantly higher systemic exposure (6.5 folds of IRN, p < 0.001) and disposition in the brain (2.5 folds of IRN, p < 0.01) and CSF (3 folds of IRN, p < 0.001) than IRN. The results indicated that interconversion between IRN and RN occurred. Notably, regardless of the orally administered IRN or RN, RN would always be one of the major or predominant forms present in the body. Our results provided sound evidence supporting further development of RN as a potential therapeutic agent for the treatment of AD. Moreover, the present study sets a solid example that integrating pharmacokinetics is crucial to identify the truly therapeutic agent