Flavokawain B is an effective natural peroxisome proliferator-activated receptor γ-selective agonist with a strong glucose-lowering effect.

Rosiglitazone, a full PPARγ agonist and a classical insulin sensitizer, was once used as a powerful weapon in the treatment of T2DM. However, its applications have been restricted recently because of its multiple side effects. Here, a natural compound, flavokawain B (FKB), which was screened in our previous experiments, was investigated for its potential as a preferable insulin sensitizer because it has no or few side effects. Using the surface plasmon resonance (SPR) technique, we confirmed that FKB is a natural ligand for PPARγ with high binding affinity. In in vitro experiments, FKB significantly increased 2-NBDG uptake in HepG2 and 3T3-L1 cells, which partially stimulated PPARγ transcriptional activity. Compared with rosiglitazone, FKB had little effect on the adipose differentiation of 3T3-L1 cells, and all of these features suggest that FKB is a selective modulator of PPARγ (SPPARγM). Moreover, FKB increased the mRNA expression levels of most genes related to insulin sensitivity and glucose metabolism but had no obvious effect on those related to adipose differentiation. In vivo experiments confirmed that FKB effectively decreased abnormal fasting blood glucose and postprandial blood glucose levels and reduced glycated hemoglobin levels, similar to rosiglitazone, in HFD-fed/STZ-treated and db/db mice, two T2DM animal models, but did not cause side effects, such as weight gain or liver or kidney damage. Further investigation revealed that FKB could inhibit PPARγ-Ser273 phosphorylation, which is the key mechanism involved in improving insulin resistance. Together, FKB is a well-performing SPPARγM that exerts a powerful glucose-lowering effect without causing the same side effects as rosiglitazone, and it may have great potential for development.

GLUTAMYL-tRNA SYNTHETASE 1 deficiency confers thermosensitive male sterility in rice by affecting reactive oxygen species homeostasis.

Temperature is one of the key environmental factors influencing crop fertility and yield. Understanding how plants sense and respond to temperature changes is, therefore, crucial for improving agricultural production. In this study, we characterized a temperature-sensitive male sterile mutant in rice (Oryza sativa), glutamyl-tRNA synthetase 1-2 (ers1-2), that shows reduced fertility at high temperatures and restored fertility at low temperatures. Mutation of ERS1 resulted in severely delayed pollen development and meiotic progression at high temperatures, eventually leading to male sterility. Moreover, meiosis-specific events, including synapsis and crossover formation, were also delayed in ers1-2 compared with the wild type. However, these defects were all mitigated by growing ers1-2 at low temperatures. Transcriptome analysis and measurement of ascorbate, glutathione, and hydrogen peroxide (H2O2) contents revealed that the delayed meiotic progression and male sterility in ers1-2 were strongly associated with changes in reactive oxygen species (ROS) homeostasis. At high temperatures, ers1-2 exhibited decreased accumulation of ROS scavengers and overaccumulation of ROS. In contrast, at low temperatures, the antioxidant system of ROS was more active, and ROS contents were lower. These data suggest that ROS homeostasis in ers1-2 is disrupted at high temperatures but restored at low temperatures. We speculate that ERS1 dysfunction leads to changes in ROS homeostasis under different conditions, resulting in delayed or rescued meiotic progression and thermosensitive male fertility. ers1-2 may hold great potential as a thermosensitive material for crop heterosis breeding.

Antibiotic resistance of Escherichia coli isolated from food and clinical environment in China from 2001 to 2020.

Antibiotics are widely used in China's aquaculture, agricultural, and clinical settings and can lead to antibiotic resistance in various pathogens. Although the pooled prevalence estimate (PPE) and antibiotic resistance of Escherichia coli (E. coli) in food and clinical settings has been extensively studied, a comprehensive analysis of the published literature is lacking. We conducted a comprehensive search for research indicators for 2001-2020 in eight major Chinese and English literature databases. Antibiotic PPE and resistance trends of 5933 and 29,451 E. coli isolates were screened and analysed in 35 food studies (total 1821) and 62 clinical studies (total 5159). E. coli strains derived from food had the highest antibiotic resistance rate to tetracycline (TET, 71.3 %), followed by trimethoprim-sulfamethoxazole (SXT, 62.5 %) and cefazolin (CFZ, 36.2 %). E. coli strains isolated from clinical environments were highly resistant to piperacillin (PIP, 71.7 %), TET (68.3 %) and CFZ (60.9 %), consistent with foodborne E. coli drug resistance patterns. E. coli strains isolated from food and clinical samples collected in laboratories carry multiple antibiotic resistance genes (ARGs), such as blaTEM, gryA, gryB, sul1, and tetA, making E. coli a reservoir of ARGs. This study highlights the presence of drug-resistant E. coli pathogens and ARGs in food and clinical environments.

Enhanced detection of ligand-PPARγ binding based on surface plasmon resonance through complexation with SRC1- or NCOR2-related polypeptide.

A previous study reported the use of a biosensing technique based on surface plasmon resonance (SPR) for the ligand binding detection of peroxisome proliferator activator receptor gamma (PPARγ). This detection was designed based on the structural properties of PPARγ. Because of cross-linked protein inactivation and the low molecular weight of conventional ligands, direct ligand binding detection based on SPR has low stability and repeatability. In this study, we report an indirect response methodology based on SPR technology in which anti-His CM5 chip binds fresh PPARγ every cycle, resulting in more stable detection. We developed a remarkable improvement in ligand-protein binding detectability in vitro by introducing two coregulator-related polypeptides into this system. In parallel, a systematic indirect response methodology can reflect the interaction relationship between ligands and proteins to some extent by detecting the changes in SA-SRC1 and GST-NCOR2 binding to PPARγ. Rosiglitazone, a PPARγ agonist with strong affinity, is a potent insulin-sensitizing agent. Some ligands may be competitively exerted at the same sites of PPARγ (binding rosiglitazone). We demonstrated using indirect response methodology that selective PPARγ modulator (SPPARM) candidates of PPARγ can be found by competing for the binding of the rosiglitazone site on PPARγ, although they may have no effect on polypeptides and PPARγ binding.

Celastrol Stabilizes Glycolipid Metabolism in Hepatic Steatosis by Binding and Regulating the Peroxisome Proliferator-Activated Receptor γ Signaling Pathway.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been increasing. Obesity, insulin resistance, and lipid metabolic dysfunction are always accompanied by NAFLD. Celastrol modulates the Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) signaling pathways, thereby promoting lipolysis in 3T3-L1 adipocytes. In the present study, oleic-acid-induced NAFLD and differentiated 3T3-L1 preadipocytes were used as models of NAFLD and obesity to investigate the protective effect of celastrol. We investigated the impact of celastrol on hepatic steatosis caused by oleic acid (OA), as well as the associated underlying molecular pathways. To address the aforementioned questions, we used a cellular approach to analyze the signaling effects of celastrol on various aspects. These factors include the improvement in fatty liver in HepG2 cells, the differentiation of 3T3-L1 preadipocytes, glucose uptake, and the modulation of key transcriptional pathways associated with PPARγ. The administration of celastrol effectively mitigated lipid accumulation caused by OA in HepG2 cells, thereby ameliorating fatty liver conditions. Furthermore, celastrol suppressed the impacts on adipocyte differentiation in 3T3-L1 adipocytes. Additionally, celastrol exhibited the ability to bind to PPARγ and modulate its transcriptional activity. Notably, the ameliorative effects of celastrol on hepatic steatosis were reversed by rosiglitazone. According to our preliminary findings from in vitro celastrol signaling studies, PPARγ is likely to be the direct target of celastrol in regulating hepatic steatosis in HepG2 cells and adipocyte differentiation in 3T3-L1 cells.

Detection of Various Traditional Chinese Medicinal Metabolites as Angiotensin-Converting Enzyme Inhibitors: Molecular Docking, Activity Testing, and Surface Plasmon Resonance Approaches.

Angiotensin-converting enzyme 1 (ACE1) is a peptide involved in fluid and blood pressure management. It regulates blood pressure by converting angiotensin I to angiotensin II, which has vasoconstrictive effects. Previous studies have shown that certain compounds of natural origin can inhibit the activity of angiotensin-converting enzymes and exert blood pressure-regulating effects. Surface Plasmon Resonance (SPR) biosensor technology is the industry standard method for observing biomolecule interactions. In our study, we used molecular simulation methods to investigate the docking energies of various herbal metabolites with ACE1 proteins, tested the real-time binding affinities between various herbal metabolites and sACE1 by SPR, and analyzed the relationship between real-time binding affinity and docking energy. In addition, to further explore the connection between inhibitor activity and real-time binding affinity, several herbal metabolites' in vitro inhibitory activities were tested using an ACE1 activity test kit. The molecular docking simulation technique's results and the real-time affinity tested by the SPR technique were found to be negatively correlated, and the virtual docking technique still has some drawbacks as a tool for forecasting proteins' affinities to the metabolites of Chinese herbal metabolites. There may be a positive correlation between the enzyme inhibitory activity and the real-time affinity detected by the SPR technique, and the results from the SPR technique may provide convincing evidence to prove the interaction between herbal metabolites and ACE1 target proteins.

[Effectiveness analysis of posterolateral approach lumbar interbody fusion assisted by one-hole split endoscope for L4, 5 degenerative lumbar spondylolisthesis].

Objective: To compare the effectiveness of posterolateral approach lumbar interbody fusion assisted by one-hole split endoscope (OSE) and traditional posterior lumbar interbody fusion (PLIF) in the treatment of L4, 5 degenerative lumbar spondylolisthesis (DLS). Methods: The clinical data of 58 patients with DLS who met the selection criteria admitted between February 2020 and March 2022 were retrospectively analyzed, of which 26 were treated with OSE-assisted posterolateral approach lumbar interbody fusion (OSE group) and 32 were treated with PLIF (PLIF group). There was no significant difference between the two groups in terms of gender, age, body mass index, Meyerding grade, lower limb symptom side, decompression side, stenosis type, and preoperative low back pain visual analogue scale (VAS) score, leg pain VAS score, Oswestry disability index (ODI), and the height of the anterior and posterior margins of the intervertebral space (P>0.05). The operation time, intraoperative blood loss, postoperative hospital stay, and complications were compared between the two groups. The low back pain and leg pain VAS scores and ODI before operation, at 1 month, 6 months after operation, and last follow-up, the height of anterior and posterior margins of the intervertebral space before operation, at 6 months after operation, and last follow-up, the modified MacNab criteria at last follow-up after operation were used to evaluate the effectiveness; and the Bridwell method at last follow-up was used to evaluate the interbody fusion. Results: Both groups successfully completed the operation. Compared with the PLIF group, the OSE group showed a decrease in intraoperative blood loss and postoperative hospital stay, but an increase in operation time, with significant differences (P<0.05). In the OSE group, no complication such as nerve root injury and thecal sac tear occurred; in the PLIF group, there were 1 case of thecal sac tear and 1 case of epidural hematoma, which were cured after conservative management. Both groups of patients were followed up 13-20 months with an average of 15.5 months. There was no complication such as loosening, sinking, or displacement of the fusion cage. The low back pain and leg pain VAS scores, ODI, and the height of anterior and posterior margins of the intervertebral space at each time point after operation in both groups were significantly improved when compared with those before operation (P<0.05). Except for the VAS score of lower back pain in the OSE group being significantly better than that in the PLIF group at 1 month after operation (P<0.05), there was no significant difference in all indicators between the two groups at all other time points (P>0.05). At last follow-up, both groups achieved bone fusion, and there was no significant difference in Bridwell interbody fusion and modified MacNab standard evaluation between the two groups (P>0.05). Conclusion: OSE-assisted posterolateral approach lumbar interbody fusion for L4, 5 DLS, although the operation time is relatively long, but the postoperative hospitalization stay is short, the complications are few, the operation is safe and effective, and the early effectiveness is satisfactory.

Enhance antibiotic resistance and human health risks in aerosols during the COVID-19 pandemic.

Aerosols are an important route for the transmission of antibiotic resistance genes (ARGs). Since the 2019 (COVID-19) pandemic, the large-scale use of disinfectants has effectively prevented the spread of environmental microorganisms, but studies regarding the antibiotic resistance of airborne bacteria remain limited. This study focused on four functional urban areas (commercial areas, educational areas, residential areas and wastewater treatment plant) to study the variations in ARG abundances, bacterial community structures and risks to human health during the COVID-19 pandemic in aerosol. The results indicated the abundance of ARGs during the COVID-19 period were up to approximately 13-fold greater than before the COVID-19 period. Large-scale disinfection resulted in a decrease in total bacterial abundance. However, chlorine-resistant bacteria tended to be survived. Among the four functional areas, the diversity and abundance of aerosol bacteria were highest in commercial aera. Antibiotic susceptibility assays suggested elevated resistance of isolated bacteria to several tested antibiotics due to disinfection exposure. The potential exposure risks of ARGs to human health were 2 times higher than before the COVID-19 pandemic, and respiratory intake was the main exposure route. The results highlighted the elevated antibiotic resistance of bacteria in aerosols that were exposed to disinfectants after the COVID-19 pandemic. This study provides theoretical guidance for the rational use of disinfectants and control of antimicrobial resistance.

[Imaging study and clinical application of unilateral biportal endoscopy technique for upper lumbar disc herniation via contralateral approach].

Objective: To investigate the relationships between the bony structures, nerve, and indentations of ligamentum flavum of the upper lumbar spine by using CT three-dimensional reconstruction technique, in order to guide the unilateral biportal endoscopy (UBE) technique via contralateral approach in the treatment of upper lumbar disc herniation (ULDH). Methods: Twenty-one ULDH patients who were admitted between June 2019 and July 2021 and met the selection criteria were selected as the research subjects. There were 12 males and 9 females with an average age of 62.1 years (range, 55-72 years). The disease duration was 1-12 years (mean, 5.7 years). There was 1 case of L 1, 2, 4 cases of L 2, 3, and 16 cases of L 3, 4. The CT myelography data of T 12-S 3 segment was saved in DICOM format and imported into Mimics21.0 software for three-dimensional reconstruction. The relationship between the intersection (point Q) of spinous process and the inferior margin of lamina, the indentation of superior margin of ligamentum flavum, the inferior margin of nerve root origin, intervertebral space, and foramen were observed. The Mimics21.0 software was used to create a 3-mm-diameter cylinder to simulate the UBE channel and measure its abduction angle (∠b1), as well as measure the following lumbar vertebra-related indicators: in L 1,2-L 3,4 segments, the vertical distance from the point Q to the inferior margin of the contralateral lumbar pedicle of the same lumbar vertebra (a1), the superior margin of the contralateral pedicle of the lower lumbar vertebra (a2), the lower endplate of the same lumbar vertebra (a3), the upper endplate of the lower lumbar vertebra (a4); the vertical distance from the lower endplate of lumbar vertebra to the inferior margin of the lumbar pedicle (c1), the vertical distance from the upper endplate of the lower lumbar vertebra to the superior margin of the lumbar pedicle (c2); the vertical distance from the inferior margin of the nerve root origin to the superior margin (d1) and the inferior margin (d2) of the lumbar pedicle, respectively; the vertical distance from the intersection (point P) of the indentation of superior margin of ligamentum flavum and the medial margin of the lumbar pedicle to the superior margin (e1) and the inferior margin (e2) of the lumbar pedicle, respectively; the horizontal distance from the lateral margin of the dural mater (f1) and the narrowest part of the lumbar isthmus (f2) to the facet joint space, respectively. Thirteen of the patients included in the study chose the UBE surgery via contralateral approach. There were 8 males and 5 females with an average age of 63.3 years (range, 55-71 years). The disease duration was 2-12 years, with an average of 6.2 years. There were 3 cases of L 2, 3 and 10 cases of L 3, 4. The perioperative complications and surgical decompression were recorded. And the effectiveness were evaluated by visual analogue scale (VAS) score, Oswestry disability index (ODI), and short form-36 health survey (SF-36) score. Results: The imaging results showed that there was no significant difference in a1, a3, a4, e1, e2, f1, and f2 between segments ( P>0.05), and there were significant differences ( P<0.05) in a2 and c2 between L 1, 2 and L 3, 4 segments, in ∠b1 and d2 between L 1, 2, L 2, 3 segments and L 3, 4 segments, and in c1 and d1 between L 1, 2 and L 2, 3, L 3, 4 segments. The 87.30% (110/126) of point Q of L 1, 2-L 3, 4 segments corresponded to the inferior articular process, and 78.57% (99/126) of the lower endplate corresponded to the level of the isthmus. All 13 patients completed the UBE surgery via contralateral approach, and none were converted to open surgery. All patients were followed up 12-17 months (mean, 14.6) months. The VAS score of low back pain and leg pain, ODI, and SF-36 score at 6 and 12 months after operation significantly improved when compared with those before operation ( P<0.05), and further improved at 12 months after operation when compared with 6 months after operation ( P<0.05). The imaging review results showed that the herniated disc was removed and the dura mater was decompressed adequately. Conclusion: The point Q, the superior margin of ligamentum flavum, and lumbar pedicle can be used as the markers for the treatment of ULBD with UBE surgery via contralateral approach, making the procedure safer, more precise, and more effective.

Vaccine of RANKL mutant conjugated with KLH effectively stabilizing bone metabolism and preventing trabecular microstructural degeneration in osteoporotic rats.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is one of the key factors regulating the maturation of osteoclasts and an important target for osteoporosis treatment. A monoclonal antibody against RANKL showed effective therapeutic activity against osteoporosis by inhibiting bone resorption by osteoclasts. However, being an exogenous protein, its efficacy decreases after long-term use, and its discontinuation increases the risk of vertebral fractures. Here, we aimed to design an active immunotherapeutic agent to induce a T-cell dependent primary response. The agent, a mutant RANKL vaccine (mRv), was produced by cross-linking mutant RANKL, lacking the ability to stimulate osteoclast maturation, with the carrier protein keyhole limpet hemocyanin, a neo-antigen with a large molecular mass. Subcutaneous injection of mRv stimulated rats with ovariectomy-induced osteoporosis to produce high titers of anti-RANKL antibodies. The mutant RANKL vaccine decreased serum CTX-1 and BALP levels and inhibited the microstructural degeneration of trabecular bone in osteoporotic rats. mRv overcame immune system tolerance, stimulated rats to produce therapeutic antibodies, stabilized bone metabolism, and inhibited trabecular microstructural degeneration. These findings confirm the potential of the mutant RANKL vaccine to be developed into an effective preventive and therapeutic agent for osteoporosis.

21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells.

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and play a role in regulating male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. These targets include 435 protein-coding genes and 71 transposons that show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells.

Oryza sativa RNA-Dependent RNA Polymerase 6 Contributes to Double-Strand Break Formation in Meiosis.

RNA-dependent RNA polymerase 6 (RDR6) is a core component of the small RNA biogenesis pathway, but its function in meiosis is unclear. Here, we report a new allele of OsRDR6 (Osrdr6-meiosis [Osrdr6-mei]), which causes meiosis-specific phenotypes in rice (Oryza sativa). In Osrdr6-mei, meiotic double-strand break (DSB) formation is partially blocked. We created a biallelic mutant with more severe phenotypes, Osrdr6-bi, by crossing Osrdr6-mei with a knockout mutant, Osrdr6-edit In Osrdr6-bi meiocytes, 24 univalents were observed, and no histone H2AX phosphorylation foci were detected. Compared with the wild type, the number of 21-nucleotide small RNAs in Osrdr6-mei was dramatically lower, while the number of 24-nucleotide small RNAs was significantly higher. Thousands of differentially methylated regions (DMRs) were discovered in Osrdr6-mei, implying that OsRDR6 plays an important role in DNA methylation. There were 457 genes downregulated in Osrdr6-mei, including three genes, CENTRAL REGION COMPONENT1, P31 comet , and O. sativa SOLO DANCERS, related to DSB formation. Interestingly, the downregulated genes were associated with a high level of 24-nucleotide small RNAs but less strongly associated with DMRs. Therefore, we speculate that the alteration in expression of small RNAs in Osrdr6 mutants leads to the defects in DSB formation during meiosis, which might not be directly dependent on RNA-directed DNA methylation.

Advances in drug development for targeted therapies for glioblastoma.

Glioblastoma is the most aggressive primary brain tumor in adults. The prognosis of patients with primary glioblastoma treated with the current standard of care, tumor resection followed by radiation therapy and auxiliary temozolomide, remains poor. Integrative genomic analyses have identified essential core signaling pathways and frequent genetic aberrations, which provide potential drug targets for glioblastoma treatment. Drugs against these therapeutic targets have been developed rapidly in recent years. Although some have shown promising effects on models in preclinical studies, many have shown only modest efficacy in clinical trials. New therapeutic strategies and potent drugs are urgently needed to improve the prognosis of patients with glioblastoma. The goal of this review is to summarize the current advances in drug development for targeted glioblastoma therapies and to reveal the major challenges encountered in clinical trials or treatment. This study will provide new perspectives for future studies of targeted therapeutic drug development and provide insights into the clinical treatment of glioblastoma.

[Construction of IL-6/STAT3 reporter gene system for screening and validation of traditional Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway].

It is important to find monomers of traditional Chinese medicine that can inhibit the activation of IL-6/STAT3 signaling pathway to suppress the growth and deterioration of tumors. A novel expression vector containing STAT3 enhancer sequence and NanoLuc (NLuc) reporter gene sequence was constructed by gene recombination technology, then a cell line expressing NLuc luciferase and regulated by STAT3 was further established. The cell line was used to quantitatively detect the regulatory effects of various traditional Chinese medicine monomers on the IL-6/STAT3 signal pathway. The effects of the traditional Chinese medicine monomers inhibiting the IL-6/STAT3 signal pathway were verified by Western blotting immunoassay and Real-time PCR analysis. By enzyme digestion and DNA sequencing analysis, it showed that the reporter gene expression vector pQCXIP-STAT3-Nluc was constructed successfully. The addition of Interleukin-6 (IL-6), a stimulator of STAT3 transcription factor, to the constructed cell line specifically enhanced the luciferase enzymatic reaction in a dose dependent manner. These results demonstrated that the cell line expressing NLuc luciferase and regulated by STAT3 has been constructed successfully. We used the the cell line screened out dendrobiine and tetrandrine which could significantly inhibit IL-6/STAT3 signal pathway and down-regulated the expression of Bcl-2 and Bcl-x in a dose-dependent manner. In conclusion, we have constructed an efficient reporter gene system to detect the transcriptional activity of STAT3, by which the Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway were successfully screened out, which has some potential theoretical and practical values.

OsMTOPVIB is required for meiotic bipolar spindle assembly.

The organization of microtubules into a bipolar spindle is essential for chromosome segregation. Both centrosome and chromatin-dependent spindle assembly mechanisms are well studied in mouse, Drosophila melanogaster, and Xenopus oocytes; however, the mechanism of bipolar spindle assembly in plant meiosis remains elusive. According to our observations of microtubule assembly in Oryza sativa, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum, we propose that a key step of plant bipolar spindle assembly is the correction of the multipolar spindle into a bipolar spindle at metaphase I. The multipolar spindles failed to transition into bipolar ones in OsmtopVIB with the defect in double-strand break (DSB) formation. However, bipolar spindles were normally assembled in several other mutants lacking DSB formation, such as Osspo11-1, pair2, and crc1, indicating that bipolar spindle assembly is independent of DSB formation. We further revealed that the mono-orientation of sister kinetochores was prevalent in OsmtopVIB, whereas biorientation of sister kinetochores was frequently observed in Osspo11-1, pair2, and crc1 In addition, mutations of the cohesion subunit OsREC8 resulted in biorientation of sister kinetochores as well as bipolar spindles even in the background of OsmtopVIB Therefore, we propose that biorientation of the kinetochore is required for bipolar spindle assembly in the absence of homologous recombination.

OsHOP2 regulates the maturation of crossovers by promoting homologous pairing and synapsis in rice meiosis.

Meiotic recombination is closely linked with homologous pairing and synapsis. Previous studies have shown that HOMOLOGOUS PAIRING PROTEIN2 (HOP2), plays an essential role in homologous pairing and synapsis. However, the mechanism by which HOP2 regulates crossover (CO) formation has not been elucidated. Here, we show that OsHOP2 mediates the maturation of COs by promoting homologous pairing and synapsis in rice (Oryza sativa) meiosis. We used a combination of genetic analysis, immunolocalization and super-resolution imaging to analyze the function of OsHOP2 in rice meiosis. We showed that full-length pairing, synapsis and CO formation are disturbed in Oshop2 meiocytes. Moreover, structured illumination microscopy showed that OsHOP2 localized to chromatin and displayed considerable co-localization with axial elements (AEs) and central elements (CEs). Importantly, the interaction between OsHOP2 and a transverse filament protein of synaptonemal complex (ZEP1), provided further evidence that OsHOP2 was involved in assembly or stabilization of the structure of the synaptonemal complex (SC). Although the initiation of recombination and CO designation occur normally in Oshop2 mutants, mature COs were severely reduced, and human enhancer of invasion 10 (HEI10)10 foci were only present on the synapsed region. Putting the data together, we speculate that OsHOP2 may serve as a global regulator to coordinate homologous pairing, synapsis and meiotic recombination in rice meiosis.

HEIP1 regulates crossover formation during meiosis in rice.

During meiosis, the number of double-strand breaks (DSBs) far exceeds the final number of crossovers (COs). Therefore, to identify proteins involved in determining which of these DSBs repaired into COs is critical in understanding the mechanism of CO control. Across species, HEI10-related proteins play important roles in CO formation. Here, through screening for HEI10-interacting proteins via a yeast two-hybrid system, we identify a CO protein HEI10 Interaction Protein 1 (HEIP1) in rice. HEIP1 colocalizes with HEI10 in a dynamic fashion along the meiotic chromosomes and specially localizes onto crossover sites from late pachytene to diplotene. Between these two proteins, HEI10 is required for the loading of HEIP1, but not vice versa. Moreover, mutations of the HEIP1 gene cause the severe reduction of chiasma frequency, whereas early homologous recombination processes are not disturbed and synapsis proceeds normally. HEIP1 interacts directly with ZIP4 and MSH5. In addition, the loading of HEIP1 depends on ZIP4, but not on MER3, MSH4, or MSH5. Together, our results suggest that HEIP1 may be a member of the ZMM group and acts as a key element regulating CO formation.

Ornithine δ-aminotransferase is critical for floret development and seed setting through mediating nitrogen reutilization in rice.

Nitrogen is one of the most important nutrient element that is essential for plant growth and development. Many genes have been reported to contribute to nitrogen absorption and transportation. However, genes involved in nitrogen reutilization are seldom reported. Ornithine δ-aminotransferase (δOAT) is the enzyme connecting arginine cycling and proline cycling. Here, we found that OsOAT, the homologue of δOAT in rice, is essential for nitrogen reutilization through mediating arginase activity. In the Osoat mutant, metabolic abnormality induced by nitrogen deficiency in floret causes malformed glumes, incapable glume opening and anther indehiscence. These defects in the mutant affect the pollination process and lead to a low seed setting rate as well as abnormal seed shape. Intriguingly, urea can rescue the phenotypes of the Osoat mutant. Therefore, OsOAT is crucial for nitrogen reutilization and plays a critical role in floret development and seed setting in rice.

Optimized application of the secreted Nano-luciferase reporter system using an affinity purification strategy.

Secreted Nano-luciferase (secNluc) is a newly engineered secreted luciferase that possesses advantages of high structural stability, long half-life, and glow-type kinetics together with high light emission intensity, and thus would become one of the most valuable tools for bioluminescence assays. However, like other secreted luciferases, secNluc has to mix with the components in the conditioned medium surrounding test cells, or in the biological samples such as blood or urine after being secreted. These components may interfere with secNluc-catalyzed bioluminescence reactions and thus limit the application of the secNluc reporter system. In this study, we first examined the effects of three factors, pH, serum and residual reagents, on secNluc-catalyzed bioluminescence reactions, finding that these factors could interfere with bioluminescence reactions and result in background signal. To resolve these problems, we applied a simple affinity purification strategy in which secNluc was fused with a FLAG-tag, and anti-FLAG magnetic beads were used to catch and transfer the fusion protein to PBST, an optimal buffer for secNluc-catalyzed bioluminescence reactions that was identified in this study. The results indicated that this strategy could not only negate the interferences from serum or residual reagents and enhance the stability of light emission but also greatly increase signal intensity through enzyme enrichment. This strategy may contribute to biomedical studies that utilize secNluc and other secreted luciferases, especially those requiring superior sensitivity, low background noise and high reproducibility.

OsSPL regulates meiotic fate acquisition in rice.

In angiosperms, the key step in sexual reproduction is successful acquisition of meiotic fate. However, the molecular mechanism determining meiotic fate remains largely unknown. Here, we report that OsSPOROCYTELESS (OsSPL) is critical for meiotic entry in rice (Oryza sativa). We performed a large-scale genetic screen of rice sterile mutants aimed to identify genes regulating meiotic entry and identified OsSPL using map-based cloning. We showed that meiosis-specific callose deposition, chromatin organization, and centromere-specific histone H3 loading were altered in the cells corresponding to pollen mother cells in Osspl anthers. Global transcriptome analysis showed that the enriched differentially expressed genes in Osspl were mainly related to redox status, meiotic process, and parietal cell development. OsSPL might form homodimers and interact with TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor OsTCP5 via the SPL dimerization and TCP interaction domain. OsSPL also interacts with TPL (TOPLESS) corepressors, OsTPL2 and OsTPL3, via the EAR motif. Our results suggest that the OsSPL-mediated signaling pathway plays a crucial role in rice meiotic entry, which appears to be a conserved regulatory mechanism for meiotic fate acquisition in angiosperms.