Unveiling the Role of RNA Recognition Motif Proteins in Orchestrating Nucleotide-Binding Site and Leucine-Rich Repeat Protein Gene Pairs and Chloroplast Immunity Pathways: Insights into Plant Defense Mechanisms.

In plants, nucleotide-binding site and leucine-rich repeat proteins (NLRs) play pivotal roles in effector-triggered immunity (ETI). However, the precise mechanisms underlying NLR-mediated disease resistance remain elusive. Previous studies have demonstrated that the NLR gene pair Pik-H4 confers resistance to rice blast disease by interacting with the transcription factor OsBIHD1, consequently leading to the upregulation of hormone pathways. In the present study, we identified an RNA recognition motif (RRM) protein, OsRRM2, which interacted with Pik1-H4 and Pik2-H4 in vesicles and chloroplasts. OsRRM2 exhibited a modest influence on Pik-H4-mediated rice blast resistance by upregulating resistance genes and genes associated with chloroplast immunity. Moreover, the RNA-binding sequence of OsRRM2 was elucidated using systematic evolution of ligands by exponential enrichment. Transcriptome analysis further indicated that OsRRM2 promoted RNA editing of the chloroplastic gene ndhB. Collectively, our findings uncovered a chloroplastic RRM protein that facilitated the translocation of the NLR gene pair and modulated chloroplast immunity, thereby bridging the gap between ETI and chloroplast immunity.

First Report of Bacterial Leaf Streak of Rice Caused by Pantoea ananatis in Guangdong Province, China.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 µL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

Osa-miR11117 Targets OsPAO4 to Regulate Rice Immunity against the Blast Fungus Magnaporthe oryzae.

The intricate regulatory process governing rice immunity against the blast fungus Magnaporthe oryzae remains a central focus in plant-pathogen interactions. In this study, we investigated the important role of Osa-miR11117, an intergenic microRNA, in regulating rice defense mechanisms. Stem-loop qRT-PCR analysis showed that Osa-miR11117 is responsive to M. oryzae infection, and overexpression of Osa-miR11117 compromises blast resistance. Green fluorescent protein (GFP)-based reporter assay indicated OsPAO4 is one direct target of Osa-miR11117. Furthermore, qRT-PCR analysis showed that OsPAO4 reacts to M. oryzae infection and polyamine (PA) treatment. In addition, OsPAO4 regulates rice resistance to M. oryzae through the regulation of PA accumulation and the expression of the ethylene (ETH) signaling genes. Taken together, these results suggest that Osa-miR11117 is targeting OsPAO4 to regulate blast resistance by adjusting PA metabolism and ETH signaling pathways.

[Effects of sorafenib and liposome doxorubicin on human poorly differentiated thyroid carcinoma xenografts in nude mice].

OBJECTIVE: To evaluate the therapeutic effects of sorafenib and liposome doxorubicin on poorly differentiated thyroid carcinoma (PDTC) xenografts in nude mice. METHODS: Sorafenib and liposome doxorubicin were applied to PDTC xenografts in nude mice. The mice were randomized into seven groups: blank control (A), vehicle control (B), single liposome doxorubicin (C), single sorafenib group (D), liposome doxorubicin combined with low dose sorafenib group (E), combined group with medium dosage of sorafenib (F), combined group with high-dose of sorafenib(G). The volume, weight and growth inhibition rate of tumours were measured to evaluate the therapeutic effects of drugs. RESULTS: Sorafenib and liposome doxorubicin showed significant antitumor activity in the PDTC xenografts. The mean tumor volumes of seven groups were (1274.13 ± 393.76) mm(3), (1060.00 ± 469.05) mm(3), (726.76 ± 488.22) mm(3), (451.54 ± 97.75) mm(3), (518.37 ± 164.44) mm(3), (310.51 ± 210.53) mm(3), and (228.44 ± 129.21) mm(3), respectively. The mean tumor weights of the seven groups were (1.13 ± 0.42)g, (0.91 ± 0.39)g, (0.78 ± 0.45)g, (0.55 ± 0.17) g, (0.52 ± 0.19) g, (0.34 ± 0.21) g, and (0.19 ± 0.09) g separately. The tumor inhibition rates of group C to G were 30.8%, 40.8%, 42.3%, 62.9%, 72.6% separately. CONCLUSIONS: Sorafenib and liposome doxorubicin, no matter for single agent or in combination, showed significant antitumor activity in the PDTC PDTC xenografts in vivo. The tumour-inhibited effect of single sorafenib is better than that of single liposome doxorubicin. Liposome doxorubicin combined with medium dosage of sorafenib had a better therapeutic effect and less side effects.

[Anti-tumor efficacy of interventional chemotherapy with liposomal doxorubicin for hepatic metastasis of pancreatic cancer in nude mouse model].

OBJECTIVE: To investigate the anti-tumor effect of interventional chemotherapy with liposome doxorubicin for hepatic metastasis of pancreatic tumor in nude mice. METHODS: After the establishment of hepatic metastatic model of pancreatic tumor, the nude mice received various formulations via a spleen injection to imitate the interventional chemotherapy. In each of two following experiments, 42 nude mice were randomly divided into 6 groups. They received liposomal doxorubicin (including high, intermittent and low-dose), free doxorubicin, gemcitabine plus cisplatin and control respectively. In the first experiment, the doses were 6, 3, 1.5, 3, 3 mg/kg and 100 µl 10% glucose for each group respectively. And in the second experiment, 9, 6, 3, 6, 6 mg/kg, and 100 µl 10% glucose respectively. The efficacies of interventional injection of liposomal doxorubicin with different doses were examined in terms of tumor growth retardation for the hepatic metastatic foci of pancreatic tumor. RESULTS: In the first experiment, the difference of median hepatic tumor volume was significant among the three groups of mice receiving liposomal doxorubicin with incremental doses in a dose-dependent manner [high dose: (3 ± 1) mm(3), middle dose: (55 ± 18) mm(3), low dose: (90 ± 23) mm(3), P < 0.05]. The liposomal doxorubicin led to a substantial delay of tumor growth as compared to the free drug or gemcitabine plus cisplatin at the same dose (both P < 0.05). In addition, all animals were well-tolerated with no obvious acute toxicity. In the second experiment, significant difference was obtained for the mice injected with different doses of liposomal doxorubicin [(11 ± 4) mm(3), (13 ± 4) mm(3), (50 ± 18) mm(3), P < 0.05]. It was correlated with tumor growth delay. The mice administered with either 9 mg/kg or 6 mg/kg were more efficacious to retard tumor growth than those given 3 mg/kg (P < 0.05). Despite its enhanced effectiveness as compared to mice in gemcitabine plus cisplatin group (P < 0.05), the liposomal doxorubicin at a dose of 6 mg/kg resulted in a marginally delayed tumor growth compared to those of free doxorubicin at the same dose (P > 0.05). No evident acute toxic response was observed for each group of mice receiving liposomal doxorubicin. In contrast, approximately half of the animals receiving either free doxorubicin or gemcitabine plus cisplatin died of toxic responses. CONCLUSION: Liposomal doxorubicin may be a potential interventional chemotherapeutic agent for hepatic metastasis of pancreatic tumor because of improved anti-tumor efficacy and reduced toxicity in comparison to free doxorubicin and gemcitabine plus cisplatin.

Antimetastatic activities of pegylated liposomal doxorubicin in a murine metastatic lung cancer model.

To determine whether pegylated liposomal doxorubicin (PLD) could control the metastasis beyond primary tumor, the efficacy of PLD was evaluated in terms of metastatic growth inhibition and increasing life span in a murine lung metastasis model. As early as 20 days after C57BL/6 mice were inoculated with 2 x 10(6) murine Lewis lung carcinoma cells into the right legs, metastases could be observed in the lungs. Comparing the metastatic status of the PLD treatment conducted one day with one week after the tumor implantation, pathological study of the metastasis in the lungs indicated that without the removal of primary tumor, PLD could prevent metastasis by suppressing the growth of primary tumor. To evaluate the direct antimetastasis ability of PLD with clinical relevance, a surgery was performed to resect the tumor-bearing limb. The treatment was started 3 days after the amputation with free doxorubicin or PLD. In this therapeutic model, PLD targeted directly to the metastasis in the lungs, which resulted in substantially longer survival time than free doxorubicin. Despite the superiority of PLD over free doxorubicin in treating pulmonary metastasis, our observation suggested that without the removal of primary tumor, the effect of PLD was only modest, and surgery plus multiple injections of PLD will be the best choice for patients with metastatic disease.

[Effect of irradiation-induced pcDNA3.1-Egr.1p-p16 on cell apoptosis and cell cycle of JF305 cells].

OBJECTIVE: To observe the effect of pcDNA3.1-Egr.1p-p16 plasmid on apoptosis and cell cycle of pancreatic carcinoma JF305 cell line. METHODS: JF305 cells were cultured and transfected with pcDNA3.1-Egr.1p-p16 plasmid via Lipofectamine(TM) 2000, followed by irradiation by 6MV-X at 4 Gy (dose rate 2.50 Gy/min). The cell cycle and cell apoptosis changes were analyzed by flow cytometry. RESULTS: In cells infected with pcDNA3.1-Egr.1p-p16 plasmid and those with pcDNA3.1-Egr.1p-p16 plasmid infection before 4 Gy irradiation, the percentages of viable apoptotic cells were 6.4% and 10.4%, and those of advanced stage apoptotic or dead cells were 16.8% and 33.8%, significantly higher than those in the control group (P<0.05). JF305 cell apoptosis in 4 Gy irradiation group was obviously increased in comparison with non-irradiated plasmid-infected cells (P<0.05). Irradiation resulted in a predominant G(2) arrest of the plasmid-infected JF305 cells. Both pcDNA3.1-p16 plasmid and pcDNA3.1-Egr.1p-p16 plasmid infections could induce G1 arrest of JF-305 cells, and irradiation did not produce significant changes in G(1)-arrested cells in the two plasmid infection groups, but cells arresting at G(2) phase increased. CONCLUSION: pcDNA3.1-Egr.1p-p16 can induce JF-305 cells apoptosis, which is enhanced by irradiation. pcDNA3.1-Egr.1p-p16 tranfection may result in G(1) arrest of the cells, and when combined with irradiation, the cells arrested at G(2) phase can increase.

Pharmacokinetic and cytotoxic studies of pegylated liposomal daunorubicin.

Pegylated liposomes have been studied for nearly two decades. However, fewer pharmacological studies about its application in daunorubicin (DNR) than those in doxorubicin have been reported. In order to conduct a complete pharmacokinetic study, radiolabeled DNR was encapsulated in pegylated liposomes. Its in vitro drug release kinetics was determined to be in a slow manner, which was reflected in its cytotoxic effect on four cell lines. The lethal dose, plasma pharmacokinetics as well as tissue distribution of the formulation were evaluated in comparison with free DNR. The results revealed that liposomal daunorubicin significantly reduced the toxicity of the drug, with a half lethal dose of 29.35 mg/kg, compared with 5.45 mg/kg for free drug. Pharmacokinetic study of liposomal DNR demonstrated a slower clearance rate, an elevated area under the concentration-time curve, as well as increased half-lives compared to free drug. In addition, an altered tissue distribution of liposomal DNR was observed, with lower cardiac accumulation. Taken together, pegylated liposome-loaded DNR may be a promising anticancer drug and worth further therapeutic study.

[Anti-tumor effect of pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in tumor-bearing nude mice].

OBJECTIVE: To investigate the optimal doses of X-ray irradiation and plasmid injection in the anti-tumor effect of the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in vivo. METHODS: We observed the anti-tumor effect of the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy with different doses of X-ray irradiation (2, 10, 20 Gy) and plasmid injection (10, 20, 30 microg) in nude mice with JF-305 pancreatic carcinoma, and detected the expression of p16 in tumor by RT-PCR. RESULTS: The tumor growth rate of the nude mice irradiated locally with 20 Gy X-rays after the plasmid injection was significantly lower (P < 0.05 ) than that of the nude mice irradiated locally with 2 Gy or 10 Gy X-ray 3 days after the irradiation. The tumor growth rate of the nude mice injected locally with 20 microg or 30 microg plasmid was significantly lower (P <0.05 ) than that of the nude mice injected locally with 10 microg plasmid. Both pcDNA3. 1-Egr. 1p-p16 group and pcDNA3. 1-Egr. 1p-p16 +20 Gy group had p16 mRNA expression, but the mRNA level of pcDNA3. 1-Egr. 1p-p16 +20 Gy group was higher than that of pcD- NA3. 1-Egr. 1p-p16 group. CONCLUSION: In the pcDNA3. 1-Egr. 1p-p16 gene combined with radiotherapy in vivo the optimal dose of X-ray irradiation was 20 Gy and the optimal dose of plasmid injection was 20 microg. The anti-tumor effect of pcDNA3. 1-Egr. 1p-p16 combined with radiotherapy is better than that of radiotherapy or gene therapy alone, which may be related with the enhanced p16 expression in tumor after the irradiation.