Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

Realization of digital twin for dynamic control toward sample variation of ion exchange chromatography in antibody separation.

Digital twin (DT) is a virtual and digital representation of physical objects or processes. In this paper, this concept is applied to dynamic control of the collection window in the ion exchange chromatography (IEC) toward sample variations. A possible structure of a feedforward model-based control DT system was proposed. Initially, a precise IEC mechanistic model was established through experiments, model fitting, and validation. The average root mean square error (RMSE) of fitting and validation was 8.1% and 7.4%, respectively. Then a model-based gradient optimization was performed, resulting in a 70.0% yield with a remarkable 11.2% increase. Subsequently, the DT was established by systematically integrating the model, chromatography system, online high-performance liquid chromatography, and a server computer. The DT was validated under varying load conditions. The results demonstrated that the DT could offer an accurate control with acidic variants proportion and yield difference of less than 2% compared to the offline analysis. The embedding mechanistic model also showed a positive predictive performance with an average RMSE of 11.7% during the DT test under >10% sample variation. Practical scenario tests indicated that tightening the control target could further enhance the DT robustness, achieving over 98% success rate with an average yield of 72.7%. The results demonstrated that the constructed DT could accurately mimic real-world situations and perform an automated and flexible pooling in IEC. Additionally, a detailed methodology for applying DT was summarized.

Structure and stability analysis of antibacterial substance produced by selenium enriched Bacillus cereus BC1.

Microorganisms can produce many antibiotics against bacteria and fungi, which have been used as a potential choice of new antibiotics. In this paper, we studied the characteristics of antibacterial substances by Bacillus cereus BC1. The results showed that the acid-precipitated substance played the main role in antibacterial activity, and further characterization indicated that the antibacterial substance might be a lipopeptide substance. Then the antibacterial spectrum suggested that the antibacterial substance had an inhibitory effect on Gram-positive bacteria and fungi, while selenium-riched antibacterial substance of Bacillus cereus BC1 could significantly enhance the inhibition. Then the morphological effects of the antibacterial substance to indicator bacteria were determined. The effects of different treatment methods on the stability of antibacterial substances were studied and the results showed that the antibacterial substance was stable to heat, ultrasonic, and ultraviolet treatment, and their antibacterial activity would not be greatly affected. However, they were sensitive to pepsin. The optimum pH range of antibacterial activity was 3-5. This study may contribute to reusing the fermentation supernatant often discarded in the previous fermentation process. At the same time, the lipopeptide antibacterial substance extracted from the fermentation broth of selenium-enriched Bacillus cereus BC1 can be used in the development of antibiotics and biopesticides, and open up a new way for the control of plant diseases.

Salicylic Acid Is Required for Broad-Spectrum Disease Resistance in Rice.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.

Process development and optimization of continuous capture with three-column periodic counter-current chromatography.

Continuous capture with affinity chromatography is one of the most important units for continuous manufacturing of monoclonal antibody (mAb). Due to the complexity of three-column periodic counter-current chromatography (3C-PCC), three approaches (experimental, model-based, and simplified approaches) were studied for process development and optimization. The effects of residence time for interconnected load (RT C ), breakthrough percentage of the first column for interconnected load (s) and feed protein concentration (c 0 ) on productivity and capacity utilization were focused. The model-based approach was found superior to the experimental approach in process optimization and evaluation. Two phases of productivity were observed and the optimal RT C for the maximum productivity was located at the boundary of the two phases. The comprehensive effects of the operating parameters (RT C , s, and c 0 ) were evaluated by the model-based approach, and the operation space was predicted. The best performance of 34.5 g/L/h productivity and 97.6% capacity utilization were attained for MabSelect SuRe LX resin under 5 g/L concentration at RT C = 2.8 min and s = 87.5%. Moreover, a simplified approach was suggested to obtain the optimal RT C for the maximum productivity. The results demonstrated that model-assisted tools are useful to determine the optimum conditions for 3C-PCC continuous capture with high productivity and capacity utilization.

Exosomal MMP2 derived from mature osteoblasts promotes angiogenesis of endothelial cells via VEGF/Erk1/2 signaling pathway.

The skeletal system is a dynamic organ that continuously undergoes coupled trabeculae and blood vessels remodeling, indicating the possible existence of molecular crosstalk between endothelial and osteoblastic cells. Since the cross-talk between bone-forming osteoblasts (OBs) and vessel-forming endothelial cells (ECs) have progressively gained investigators' attention, few studies focused on the regulatory function of extracellular vesicles derived from OBs on ECs. In this study, the effect of the exosomes derived from mature osteoblasts (MOBs) on the ECs was investigated. Firstly, exosomes derived from mature osteoblasts (MOB-Exos) were isolated and identified by NanoSight light scatter technology, electron microscopy and Western bolting. Fluorescent labeling of MOB-Exos revealed its internalization by ECs. RNA interference technique was used to knock down matrix metalloproteinase-2 (MMP2) in MOB-Exos. Then ECs were co-cultured with MOB-Exos and MMP2 knockdown MOB-Exos. Wound healing migration assay, transwell migration assay, CCK-8 assay and tube formation assay of ECs were conducted to determine the angiogenic capability of ECs. Then the VEGF/Erk1/2 pathway markers were detected by Western blot. Our results showed that MOB-Exos could promote the proliferation, migration and tube formation of ECs. Meanwhile, the promoted angiogenetic capacities of ECs were impaired when MMP2 in MOB-Exos was knocked down. In addition, immunoblotting indicated that MOB-Exos could promote the activation of the VEGF/Erk1/2 pathway of ECs; whereas the activation of the VEGF/Erk1/2 pathway was attenuated when the ECs were co-cultured with the MMP2 knockdown MOB-Exos. In conclusion, the MMP-2 existing in exosomes derived from MOBs could promote the angiogenesis of ECs in vitro, which might be realized through VEGF/Erk1/2 signaling pathway.

Ketamine affects the integration of developmentally generated granule neurons in the adult stage.

BACKGROUND: Ketamine has been reported to cause neonatal neurotoxicity in a variety of developing animal models. Various studies have been conducted to study the mechanism of neurotoxicity for general anesthetic use during the neonatal period. Previous experiments have suggested that developmentally generated granule neurons in the hippocampus dentate gyrus (DG) supported hippocampus-dependent memory. Therefore, this study aimed to investigate whether ketamine affects the functional integration of developmentally generated granule neurons in the DG. For this purpose,the postnatal day 7 (PND-7) Sprague-Dawley (SD) rats were divided into the control group and the ketamine group (rats who received 4 injections of 40 mg/kg ketamine at 1 h intervals). To label dividing cells, BrdU was administered for three consecutive days after the ketamine exposure; NeuN+/BrdU+cells were observed by using immunofluorescence. To evaluate the developmentally generated granule neurons that support hippocampus-dependent memory, spatial reference memory was tested by using Morris Water Maze at 3 months old, after which the immunofluorescence was used to detect c-Fos expression in the NeuN+/BrdU+ cells. The expression of caspase-3 was measured by western blot to detect the apoptosis in the hippocampal DG. RESULTS: The present results showed that the neonatal ketamine exposure did not influence the survival rate of developmentally generated granule neurons at 2 and 3 months old, but ketamine interfered with the integration of these neurons into the hippocampal DG neural circuits and caused a deficit in hippocampal-dependent spatial reference memory tasks. CONCLUSIONS: In summary, these findings may promote more studies to investigate the neurotoxicity of ketamine in the developing brain.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

Alternative Splicing of Rice WRKY62 and WRKY76 Transcription Factor Genes in Pathogen Defense.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Pseudomonas syringae Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 Gene Required for Antibacterial Immunity in Arabidopsis.

Many bacterial pathogens of plants and animals deliver effector proteins into host cells to promote infection. Elucidation of how pathogen effector proteins function not only is critical for understanding bacterial pathogenesis but also provides a useful tool in discovering the functions of host genes. In this study, we characterized the Pseudomonas syringae pv tomato DC3000 effector protein Avirulence Protein E (AvrE), the founding member of a widely distributed, yet functionally enigmatic, bacterial effector family. We show that AvrE is localized in the plasma membrane (PM) and PM-associated vesicle-like structures in the plant cell. AvrE contains two physically interacting domains, and the amino-terminal portion contains a PM-localization signal. Genome-wide microarray analysis indicates that AvrE, as well as the functionally redundant effector Hypersensitive response and pathogenicity-dependent Outer Protein M1, down-regulates the expression of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13) gene in Arabidopsis (Arabidopsis thaliana). Mutational analysis shows that NHL13 is required for plant immunity, as the nhl13 mutant plant displayed enhanced disease susceptibility. Our results defined the action site of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene.

Exosomal miR-423-5p Derived from Cerebrospinal Fluid Pulsation Stress-Stimulated Osteoblasts Improves Angiogenesis of Endothelial Cells via DUSP8/ERK1/2 Signaling Pathway.

Exosomes secreted from osteoblasts (OBs) can regulate the angiogenesis of endothelial cells (ECs); however, whether cerebrospinal fluid pulsation (CSFP) stress, a special mechanical stimulation, can influence the cell's communication in the context of angiogenesis remains unknown. In this study, the effect of exosomes derived from CSFP stress-stimulated OBs on facilitating the angiogenesis of ECs was investigated. First, OBs were cultured in a CSFP bioreactor, and exosomes derived from OBs were isolated and identified. Cell Counting Kit 8 assay, transwell migration assay, wound healing migration assay, and tube formation assay were conducted to assess the effects of CSFP stress-stimulated OBs-derived exosomes (CSFP-Exos) on the angiogenesis of ECs. Then high-throughput RNA sequencing was used to determine the miRNA profiles of Non-CSFP stress-stimulated OBs-derived exosomes (NCSFP-Exos) and CSFP-Exos, and the luciferase reporter gene assay was performed to confirm the binging of miR-423-5p to DUSP8. In addition, the Matrigel plug assay was performed to explore whether exosomal miR-423-5p has the same effects in vivo. Our results suggested that CSFP-Exos can promote the angiogenesis of ECs, and miR-423-5p was enriched in CSFP-Exos. Moreover, miR-423-5p could promote the effect of angiogenesis via directly targeting dual-specificity phosphatase 8 (DUSP8), which inhibited the ERK1/2 signaling pathway. In conclusion, exosomal miR-423-5p derived from CSFP stress-stimulated OBs could promote the angiogenesis of ECs by the DUSP8/ERK1/2 signaling pathway.

Exosomal miR-423-5p Derived from Mineralized Osteoblasts Promotes Angiogenesis of Endothelial Cells by Targeting CXCL10.

BACKGROUND: Bone tissue engineering offers a new approach for the treatment of bone defects, with angiogenesis being critical to the survival and development of tissue-engineered bone. Mineralized osteoblasts (MOBs) have been reported to promote vascular formation by endothelial cells (ECs) through the secretion of exosomes containing a variety of angiogenic factors. The aim of the present study was to investigate the effect of miR-423-5p contained within exosomes derived from MOBs (MOB-Exos) on EC angiogenesis. METHODS: The Cell Counting Kit-8 (CCK-8), scratch wound healing, Transwell migration, and tube formation assays were conducted to assess the in vitro effects of MOB-Exos on EC proliferation, migration, and tubule-forming capabilities. The miR-423-5p level in MOB-Exos was quantified using quantitative polymerase chain reaction (qPCR). Co-culture experiments were used to study the exosomal transport of miR-423-5p and its angiogenic effects. High-throughput sequencing was used to identify differentially expressed genes, and a dual luciferase reporter assay to determine whether CXCL10 was a direct target gene for miR-423-5p. Furthermore, the in vivo effect of MOB-Exos-derived miR-423-5p on angiogenesis was evaluated using a subcutaneous xenograft model. RESULTS: MOB-Exos significantly promoted the in vitro proliferation, migration, and tubule formation of ECs. A high level of miR-423-5p was found in MOB-Exos and promoted the angiogenesis of ECs. The CXCL10 gene was significantly downregulated in ECs upon miR-423-5p mimic transfection. Dual luciferase reporter assay confirmed the direct binding of miR-423-5p to the CXCL10 gene. miR-423-5p derived from MOB-Exos upregulated expression of the vascular markers CD31 and vascular endothelial growth factor (VEGF) in vivo, thus underscoring its angiogenic potential. CONCLUSION: This study found that miR-423-5p derived from MOB-Exos could potentially enhance EC angiogenesis via the regulation of CXCL10. Therefore, exosomes are promising therapeutic candidates for clinical bone defects.

Natural product guvermectin inhibits guanosine 5'-monophosphate synthetase and confers broad-spectrum antibacterial activity.

Bacterial diseases caused substantial yield losses worldwide, with the rise of antibiotic resistance, there is a critical need for alternative antibacterial compounds. Natural products (NPs) from microorganisms have emerged as promising candidates due to their potential as cost-effective and environmentally friendly bactericides. However, the precise mechanisms underlying the antibacterial activity of many NPs, including Guvermectin (GV), remain poorly understood. Here, we sought to explore how GV interacts with Guanosine 5'-monophosphate synthetase (GMPs), an enzyme crucial in bacterial guanine synthesis. We employed a combination of biochemical and genetic approaches, enzyme activity assays, site-directed mutagenesis, bio-layer interferometry, and molecular docking assays to assess GV's antibacterial activity and its mechanism targeting GMPs. The results showed that GV effectively inhibits GMPs, disrupting bacterial guanine synthesis. This was confirmed through drug-resistant assays and direct enzyme inhibition studies. Bio-layer interferometry assays demonstrated specific binding of GV to GMPs, with dependency on Xanthosine 5'-monophosphate. Site-directed mutagenesis identified key residues crucial for the GV-GMP interaction. This study elucidates the antibacterial mechanism of GV, highlighting its potential as a biocontrol agent in agriculture. These findings contribute to the development of novel antibacterial agents and underscore the importance of exploring natural products for agricultural disease management.

The Chromosome-Scale Genomes of Exserohilum rostratum and Bipolaris zeicola Pathogenic Fungi Causing Rice Spikelet Rot Disease.

Rice spikelet rot disease occurs mainly in the late stages of rice growth. Pathogenicity and biological characteristics of the pathogenic fungus and the infestation site have been the primary focus of research on the disease. To learn more about the disease, we performed whole-genome sequencing of Exserohilum rostratum and Bipolaris zeicola for predicting potentially pathogenic genes. The fungus B. zeicola was only recently identified in rice.We obtained 16 and 15 scaffolds down to the chromosome level for E. rostratum LWI and B. zeicola LWII, respectively. The length of LWI strain was approximately 34.05 Mb, and the G + C content of the whole genome was 50.56%. The length of the LWII strain was approximately 32.21 Mb, and the G + C content of the whole genome was 50.66%. After the prediction and annotation of E. rostratum LWI and B. zeicola LWII, we predicted that the LWI strain and LWII strain contain 8 and 13 potential pathogenic genes, respectively, which may be related to rice infection. These results improve our understanding of the genomes of E. rostratum and B. zeicola and update the genomic databases of these two species. It benefits subsequent studies on the mechanisms of E. rostratum and B. zeicola interactions with rice and helps to develop efficient control measures against rice spikelet rot disease.

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

[Experimental study of constructing vertebral canal on rabbit model with bone marrow mesenchymal stem cells].

OBJECTIVE: To construct the artificial lamina of vertebral arch with bone marrow mesenchymal stem cells transplanted in collagen sponge on a rabbit model and observe the growth of new bone. METHODS: To draw out the bone marrow blood from the femur of 2 weeks old rabbit and get the bone marrow mesenchymal stem cells by centrifugal and adhesive effect. To induce the MSCs to osteoblasts and transplant the induced cells in collagen sponge to construct the tissue engineering bone. To divide 48 rabbits into 3 groups randomly, namely group A, group B and group C. All of the rabbits are taken laminectomy in L6, and to group B and C, collagen sponge and tissue engineering bone are implanted in the operation area respectively. The artificial lamina of vertebral arch is determined qualitatively and quantitatively by methods including imageology and histomorphometry. RESULTS: The artificial lamina of vertebral arch is successfully constructed 4 weeks after operation in group C, CT examination at 4 weeks shows that new lamina of vertebral arch is formed, and the vertebral canal is intact. CONCLUSIONS: The artificial lamina of vertebral arch can be constructed successfully with the usage of tissue engineering bone transplanted bone marrow mesenchymal stem cells.

OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus.

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin ß-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Exosomal let-7f-5p derived from mineralized osteoblasts promotes the angiogenesis of endothelial cells via the DUSP1/Erk1/2 signaling pathway.

Blood vessel formation is the prerequisite for the survival and growth of tissue-engineered bone. Mineralized osteoblasts (MOBs) have been shown to regulate angiogenesis through the secretion of exosomes containing various pro-angiogenic factors. However, whether the mineralized osteoblast-derived exosomes (MOB-Exos) containing let-7f-5p can regulate the angiogenesis of endothelial cells (ECs) is still unknown. In this study, the angiogenic capabilities of ECs respectively treated with MOB-Exos, let-7f-5p mimicked MOB-Exos (miR mimic group), and let-7f-5p inhibited MOB-Exos (miR inhibitor group) were compared through in vitro and in vivo studies. Moreover, the potential mechanism of MOB-Exo let-7f-5p regulating angiogenesis was explored by verifying the role of the Erk1/2 signaling pathway and target gene DUSP1. The results showed that MOB-Exos could significantly promote the angiogenesis of ECs, which could be enhanced by mimicked exosomal let-7f-5p and attenuated by inhibited exosomal let-7f-5p. Let-7f-5p could suppress the luciferase activity of wide-type DUSP1, and the mutation of DUSP1 could abrogate the repressive ability of let-7f-5p. Furthermore, the expression of DUSP1 exhibited a reversed trend to that of pErk1/2. The expression of pErk1/2 was significantly higher in the miR mimic group and lower in the miR inhibitor group than that in the MOB-Exos group, while inhibition of pErk1/2 could partly impair the angiogenic capabilities of ECs. In conclusion, we concluded that exosomal let-7f-5p derived from MOBs could promote the angiogenesis of ECs via activating the DUSP1/Erk1/2 signaling pathway, which might be a promising target for promoting the angiogenesis of tissue-engineered bone.

Converting a mAb downstream process from batch to continuous using process modeling and process analytical technology.

The biopharmaceutical market is driving the revolution from traditional batch processes to continuous manufacturing for higher productivity and lower costs. In this work, a batch mAb downstream process has been converted into an integrated continuous process with the combination of multiple techniques. For process intensification, two batch mode unit operations (protein A capture chromatography, ultrafiltration/diafiltration) were converted into continuous ones; for continuity, surge tanks were used between adjacent steps, and level signals were used to trigger process start or stop, forming a holistic continuous process. For process automation, manual operations (e.g., pH and conductivity adjustment) were changed into automatic operation and load mass was controlled with process analytical technology (PAT). A model-based simulation was applied to estimate the loading conditions for the continuous capture process, resulting in 21% resin capacity utilization and 28% productivity improvement as compared to the batch process. Automatic load mass control of cation exchange chromatography (CEX) was achieved through a customized in-line protein quantity monitoring system, with a difference of less than 1.3% as compared to off-line analysis. Total process time was shortened from 4 days (batch process) to less than 24 hours using the continuous downstream process with the overall productivity of 23.8 g mAb per day for the bench-scale system. Comparable yield and quality data were obtained in three test runs, indicating a successful conversion from a batch process to a continuous process. The insight of this work could be a reference to other similar situations.

Model-assisted process design for better evaluation and scaling up of continuous downstream bioprocessing.

Continuous process is a promising alternative for tradition batch process in biomanufacturing, which has higher productivity and lower material consumption. However, despite the maturation of necessary technologies for continuous process, there are few discussion about optimization of full continuous process. One possible reason is that the continuous process is such a complex and interacted process that the traditional experiment-based optimization approach is not sufficient anymore. To address that problem, the process simulation tool SuperPro Designer and continuous capture chromatography model were integrated into a model-assisted design platform for better development of continuous process. The influences of different continuous capture operation modes and sub-lot number under various upstream conditions were analyzed for pilot-scale production. The best combination of operation mode and sub-lot number were determined for the highest equipment utilization without any conflict. After the process optimization, the equipment utilization of continuous process was significantly improved to 27.3%. Then, a pilot-scale case study was carried out to validate the proposed approach. The results showed that the scaling up and process design of continuous process were successful. No time conflict and process failure happened and the final product met the release specification. Finally, the cost of goods was evaluated with SuperPro Designer, and the results showed a 17.4% cost reduction for optimized continuous downstream process compared to batch process, which is promoting for the future industrial applications.