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Objective: To study the role of CK19 in the survival of patients diagnosed with DPHCC in Fujian, China, which has a high incidence of hepatocellular carcinoma (HCC). Methods: Patients with DPHCC ( n =84) who had undergone surgical interventions at the 900th Hospital of Joint Logistic Support Force between 2013 and 2019 were retrospectively analyzed using the log-rank test and Kaplan-Meier method. Univariate and multivariate Cox model analyses were also conducted to further understand the correlation between CK19 and patient survival. Results: (1)Tumor size, differentiation, peripheral hepatic fibrosis, liver capsule invasion, microvascular invasion (MVI) and serum CA-199 level showed a correlation with CK19, as per the outcomes of Chi-squared tests. (2)According to the univariate analysis, the expression of CK19, MVI, the number of tumor lesions, necrosis, differentiation, peripheral hepatic fibrosis, and serum levels of both alpha-fetoprotein (AFP) and CA-199 showed a strong correlation with overall survival. (3)Necrosis and serum AFP levels were strongly related to an increased risk of death, according to the multivariate analysis. Conclusions: The expression of CK19 may correlate with the survival of patients with DPHCC and could potentially serve as a prognostic predictor of survival.

Colibacillosis, a bacterial disease caused by avian pathogenic E. coli (APEC), is a prevalent condition in the poultry industry, resulting in substantial economic losses annually. Previously, we identified PTEN as a crucial candidate gene that may play a significant role in chicken's immune response to APEC infection. Bioinformatics analysis indicated that the PTEN protein was unstable, hydrophilic and nuclear localization, with multiple putative phosphorylation sites and a high degree of similarity to duck and goose PTEN. Moreover, PTEN exhibited high expression levels in various tissues such as the stomach, cecum, small intestine, spleen, thymus, harderian gland, muscle, cerebrum, cerebellum, lung, and liver in comparison to heart tissue. Overexpression of PTEN resulted in a significant promotion of the expression level of pro-apoptosis genes and inflammatory mediators, as well as the production of NO, with or without APEC infection, which led to cellular injury. Furthermore, overexpression of PTEN was found to regulate the expression levels of autophagy related genes, regardless of APEC infection. Additionally, PTEN was a target gene of gga-miR-20a-5p and regulated by gga-miR-20a-5p upon APEC infection. Taken together, these findings establish a foundation for investigating the biological function of chicken PTEN, providing a potential target for future treatments against APEC infection as well as the breeding of genetically resistant poultry., Competing Interests: DISCLOSURES The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The occurrence of ventricular tachycardia (VT) in patients with acute myocardial infarction (AMI) is associated with poor prognosis. Drug therapy and implantable cardioverter-defibrillators (ICDs) are effective methods to prevent sudden death. Radiofrequency (RF) catheter ablation can map the matrix and mechanism of VT, thereby effectively reducing the occurrence of ICD discharge. This paper reports on the case of a middle-aged man who underwent emergency percutaneous coronary intervention for AMI and developed VT and ventricular fibrillation on day 7 after reperfusion. An ICD was implanted. On day 19, he received catheter ablation because of refractory monomorphic ventricular tachycardia and frequent discharge of the ICD. After three months, the patient had not experienced any further ventricular tachycardia attacks. The conclusion is that RF catheter ablation can resolve the ES after myocardial infarction and significantly reduce the occurrence of ICD discharges., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate the clinical phenotypes and genotypes of children with congenital fibrinogen disorder (CFD)., Methods: A retrospective analysis was conducted on the clinical data of 16 children with CFD. Polymerase chain reaction was used to amplify all exons and flanking sequences of the FGA , FGB , and FGG genes, and sequencing was performed to analyze mutation characteristics., Results: Among the 16 children, there were 9 boys (56%) and 7 girls (44%), with a median age of 4 years at the time of attending the hospital. Among these children, 9 (56%) attended the hospital due to bleeding events, and 7 (44%) were diagnosed based on preoperative examination. The children with bleeding events had a significantly lower fibrinogen activity than those without bleeding events ( P <0.05). Genetic testing was conducted on 12 children and revealed a total of 12 mutations, among which there were 4 novel mutations, i.e., c.80T>C and c.1368delC in the FGA gene and c.1007T>A and C.1053C>A in the FGG gene. There were 2 cases of congenital afibrinogenemia caused by null mutations of the FGA gene, with relatively severe bleeding symptoms. There were 7 cases of congenital dysfibrinogenemia mainly caused by heterozygous missense mutations of the FGG and FGA genes, and their clinical phenotypes ranged from asymptomatic phenotype to varying degrees of bleeding., Conclusions: The clinical phenotypes of children with CFD are heterogeneous, and the severity of bleeding is associated with the level of fibrinogen activity, but there is a weak association between clinical phenotype and genotype.

Objective: To summarize the clinical, imaging, and pathological characteristics of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS) to improve the diagnosis of this rare disease. Methods: A retrospective case series was conducted to collect the clinical data and results of genetic testing, muscle biopsy, and imaging studies including computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance spectroscopy (MRS) of 35 patients with MELAS admitted to the Nanjing Drum Tower Hospital from 2012 to 2021. Descriptive statistical analysis including mean, standard deviation, and frequency percentage were carried out. Results: The average age of onset of the patients was 30.2±2.3 years; the prevalence of family history was 20%. The two main initial symptoms were limb weakness and convulsions. The clinical manifestations of the neuromuscular system were proximal muscle weakness and exercise intolerance. The endocrine system is the most affected outside the neuromuscular system, with diabetes being the most common condition. Among the five patients who underwent brain CT, four showed hypodense lesions and two had calcified lesions. Brain MRI in 26 patients showed that the lesions more often affected the parietal lobe, basal ganglia, temporal lobe, occipital lobe, and frontal lobe than the infratentorial areas. Twelve of these individuals exhibited different levels of brain atrophy. Among the 10 patients who underwent 1 H-MRS, nine showed a decrease in N-acetylaspartate (NAA) levels, eight exhibited abnormal lactate elevation (Lac peaks), whereas six had both reduced NAA levels and the presence of Lac peaks. Thirty-one patients underwent genetic testing; among them, 25 were found to have the mt.3243A>G mutation, while the remaining six exhibited rare gene alterations. Muscle biopsies were performed in 21 patients, and 15 showed abnormal mitochondrial proliferation manifested by ragged red fibers and defective oxidative phosphorylation manifested by cytochrome C oxidase (COX) enzyme-deficient muscle fibers. Conclusion: The clinical manifestations of MELAS syndrome are variable and complex, and early atypical symptoms could be missed or misdiagnosed. A detailed clinical history, imaging MRS analysis, muscle biopsy, and genetic testing are necessary to confirm the accurate diagnosis of MELAS.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Aims: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown., Methods: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay., Results: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway., Conclusion: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke., (© 2024 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Plant reproduction is a complex, highly-coordinated process in which a single, male germ cell grows through the maternal reproductive tissues to reach and fertilise the egg cell. Focussing on Arabidopsis thaliana, we review signalling between male and female partners which is important throughout the pollen tube journey, especially during pollen tube reception at the ovule. Numerous receptor kinases and their coreceptors are implicated in signal perception in both the pollen tube and synergid cells at the ovule entrance, and several specific peptide and carbohydrate ligands for these receptors have recently been identified. Clarifying the interplay between these signals and the downstream responses they instigate presents a challenge for future research and may help to illuminate broader principles of plant cell-cell communication., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Japanese sawyer beetle Monochamus alternatus (Coleoptera: Cerambycidae) is a pest in pine forests and acts as a vector for the pine wood nematode Bursaphelenchus xylophilus, which causes the pine wilt disease. We assembled a high-quality genome of M. alternatus at the chromosomal level using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled genome is 767.12 Mb, with a scaffold N50 of 82.0 Mb. All contigs were assembled into ten pseudo-chromosomes. The genome contains 63.95% repeat sequences. We identify 16, 284 protein-coding genes in the genome, of which 11,244 were functionally annotated. The high-quality genome of M. alternatus provides an invaluable resource for the biological, ecological, and genetic study of this beetle and opens new avenues for understanding the transmission of pine wood nematode by insect vectors., (© 2024. The Author(s).)

Pulmonary nodules are a common complication in solid organ transplant recipients, and may have various underlying causes, with Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) being one of them. Given the rarity of this entity, we describe the diagnosis and therapeutic interventions for post-transplant EBV-SMT in two individuals. Both cases involved female patients who were diagnosed with multiple pulmonary nodules 60 months and 116 months, respectively, after receiving living-related kidney transplantation. Pathological examination revealed a spindle cell tumor, with immunophenotype and EBV in situ hybridization supporting the diagnosis of EBV-SMT. After diagnosis, these two patients underwent intervention by decreasing their intake of immunosuppressants. As of the latest follow-up, the patients' lesion size remained stable, and their overall condition was favorable. We also reviewed literature about the morphological and molecular pathological features of EBV-SMT and highlighted the diagnosis and differential diagnosis of pulmonary spindle cell lesions especially in the setting of immunosuppression.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P 2 fluorescent marker protein 2xmCHERRY-2xPH PLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P 2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Intrahepatic cholangiocarcinoma (iCCA) can originate from the large bile duct group (segment bile ducts and area bile ducts), small bile duct group (septal bile ducts and interlobular bile ducts), and terminal bile duct group (bile ductules and canals of Hering) of the intrahepatic biliary tree, which can be histopathological corresponding to large duct type iCCA, small duct type iCCA and iCCA with ductal plate malformation pattern, and cholangiolocarcinoma, respectively. The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies, tissue structures, growth patterns, invasive behaviors, immunophenotypes, molecular mutations, and surgical prognoses. For these reasons, this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA, mainly based on the 5 th edition of the World Health Organization Classification of Tumours of the Digestive System., Competing Interests: The authors have no conflict of interests related to this publication., (© 2023 Authors.)

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops., Competing Interests: Declaration of interests A patent application (no. 202311098597.3) for the pRALFs and their potential applications in achieving distant hybridization in plants has been submitted to the State Intellectual Property Office of China., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: N-methyl-D-aspartic acid (NMDA) receptors play subunit-specific role in central neuronal development. However, insights into the pharmacological modulation of NMDA receptors were mainly lack of subunit and synaptic selectivity. The purpose of the present study was to develop a novel strategy to rapidly recognize NMDA subunit 2A (NMDA-2A) ligands from natural products and provide subunit-selective drug candidates for Alzheimer's disease (AD)., Methods: The recombinant NMDA-2A containing a tag of epidermal growth factor receptor (EGFR) was expressed in Escherichia coli cells and immobilized on ibrutinib-modified microspheres based on the specific reaction between EGFR and its inhibitor ibrutinib. A novel affinity stationary phase was synthesized to screen NMDA-2A ligands from Gardenia jasminoides Ellis., Results: The immobilized receptor column exhibited excellent receptor selectivity and ligand-binding activity. Crocetin was screened by using this method. In a cellular model of AD, the protein level of NMDA-2A was significantly decreased compared with the control group, while treatment with crocetin significantly increased NMDA-2A level in a concentration-dependent manner, confirming that crocetin could bind to NMDA-2A in vitro., Conclusion: In the present study, we developed a reliable method for the rapid identification of NMDA-2A ligands from natural products, which may be used as a platform for new drug discovery to generate high-quality drug candidates., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Epigenetic reprogramming in the germline contributes to the erasure of epigenetic inheritance across generations in mammals but remains poorly characterized in plants. Here we profiled histone modifications throughout Arabidopsis male germline development. We find that the sperm cell has widespread apparent chromatin bivalency, which is established by the acquisition of H3K27me3 or H3K4me3 at pre-existing H3K4me3 or H3K27me3 regions, respectively. These bivalent domains are associated with a distinct transcriptional status. Somatic H3K27me3 is generally reduced in sperm, while dramatic loss of H3K27me3 is observed at only ~700 developmental genes. The incorporation of the histone variant H3.10 facilitates the establishment of sperm chromatin identity without a strong impact on resetting of somatic H3K27me3. Vegetative nuclei harbor thousands of specific H3K27me3 domains at repressed genes, while pollination-related genes are highly expressed and marked by gene body H3K4me3. Our work highlights putative chromatin bivalency and restricted resetting of H3K27me3 at developmental regulators as key features in plant pluripotent sperm., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objective: To analyze the level of PCDD/Fs exposure of occupational workers in the waste incineration industry and explore the risk of occupational exposure. Methods: In September 2021, literature on environmental PCDD/Fs exposure in waste incineration plants published from the establishment of the database to February 10, 2021 was retrieved from CNKI database. A total of 1365 literatures were retrieved, and 7 met the criteria for inclusion. The US Environmental Protection Agency (EPA) inhalation risk model was used to assess and analyze carcinogenic and non-carcinogenic risks of PCDD/Fs exposure among occupational workers in the waste incineration industry. Results: A total of 86 sampling sites were included in incineration plants in 7 regions. The study of Wuhan area showed that the concentration of working environment near the waste incinerator in the same factory was the highest, followed by the rest and office area in the factory. The concentration of PCDD/Fs in waste incinerators was the highest in Southwest China (4880.00-24880.00 pg TEQ/m(3)), and the lowest in Shenzhen (0.02-0.44 pg TEQ/m(3)). According to the cancer risk assessment, with the increase of exposure years, the risk of cancer increased. The highest risk of cancer was found in the waste incineration plants in Southwest China. When the exposure period was 1 year, the risk was moderate (22.40×10(-6)-114.20×10(-6)). When the exposure time was more than 5 years, the risk of cancer was high. In Jinan, workers working near the incinerator had a moderate risk of cancer after five years of exposure. In Zhejiang, workers were at medium risk of cancer after exposure for more than 20 years. Workers in Wuhan, Shanghai, Zhejiang Province, Shenzhen and the Pearl River Delta were still at low risk of cancer after 40 years of occupational exposure. HQ>1 of workers working near the waste incinerators in Jinan, Zhejiang Province and Southwest China, and the qualitative evaluation results showed that the non-carcinogenic risk was unacceptable. Conclusion: There are great differences in PCDD/Fs of occupational exposure in waste incineration industry, and the occupational exposure exceeding the occupational exposure limit has higher carcinogenic and non carcinogenic risks.

Objective: This study aimed to analyze the conventional surface electrocardiogram (ECG) characteristics of premature ventricular contractions (PVCs) originating from the tricuspid annulus and to investigate the efficacy of locating their origins according to ECG results., Methods: Eight patients who underwent radiofrequency ablation in the First Hospital of Shanxi Medical University (China) were included in the study. Pace mapping (PM) was used to analyze the characteristics of the PVCs originating from the tricuspid annulus recorded via 12-lead body surface ECGs., Results: An R-wave was found in leads I, V 5 , and V 6 . The QRS wave was narrower when the PVCs originated from the septum and shifted in lead V 3 (R-wave amplitude/S-wave amplitude in the precordial lead-1). The QRS wave was broadest when the PVCs originated from the 7 to 9 o'clock position. The augmented vector left lead showed RS, QS, or RSR-type waves with a low amplitude when the PVCs originated from the upper part of the annulus. When the PVCs originated from the lower part of the annulus, the augmented vector right lead reflected multidirectional and QS-type waves., Conclusion: The ECG-lead characteristics related to the origin of PVCs in the tricuspid annulus indicate some level of significance and can be used to formulate a specific diagnosis., (© 2022 The Authors. Annals of Noninvasive Electrocardiology published by Wiley Periodicals LLC.)

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P 4 ) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N 6 -methyladenosine (m 6 A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P 4  signaling. Conditional deletion of methyltransferase-like 3 ( Mettl3 ), encoding the m 6 A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter ( Pgr-Cre ) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m 6 A modification. A luciferase assay revealed that the m 6 A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P 4 signaling during embryo implantation via m 6 A-mediated translation control of Pgr mRNA.

The activation of the monocyte-macrophage system and the damage to the renal and pancreatic tissue are common complications in patients with diabetes induced by hyper-glycemia. This study aimed to evaluate the effect and mechanism of butyrate (NaB), a metabolite of intestinal flora, on inhibiting the inflammatory response of human monocyte-macrophages (THP-1 cells) induced by high glucose and the damage of pancreatic and renal tissue in diabetic mice. The results showed that high concentration glucose significantly up-regulated the expressions of IL-1β, TNF-α, and NLRP3 in THP-1 cells and mouse spleen, and that NaB could inhibit the overexpression of those genes. The abundance of Beclin-1, LC3B and reactive oxygen species (ROS) in THP-1 cells is increased due to the high glucose concentration, and NaB can inhibit the genes responsible for upregulating the expression. In diabetic mice, vacuolar degeneration of renal tubules was observed. Then we observed that some of the epithelial cells of the renal tubules were exfoliated and some formed tubules. NaB could alleviate these pathological lesions, but NaB cannot alleviate pancreatic injury. Our results indicated that NaB could be used for the prevention and adjuvant treatment of diabetic kidney injury., (© 2023 Man Yan et al., published by Sciendo.)

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

In flowering plants (angiosperms), fertilization of the egg cell by one sperm cell produces an embryo, whereas fusion of a second sperm cell with the central cell generates the endosperm. In most angiosperms like Arabidopsis, a pollen grain contains two isomorphic sperm cells required for this double fertilization process. A long-standing unsolved question is whether the two fertilization events have any preference. A tool to address this question is the usage of the cyclin-dependent kinase a1 (cdka;1) mutant pollen, which produces a single sperm-like cell (SLC). Here, we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC. Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell, precursor of the two sperm cells. Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2% of the ovules, single fertilization of the egg cell occurred. Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery, which fertilized the central cell, resulting in 20.7% double-fertilized ovules. This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell. Taken together, our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

Objective: To analyze the clinical characteristics of children with Burkitt lymphoma (BL) and to summarize the mid-term efficacy of China Net Childhood Lymphoma-mature B-cell lymphoma 2017 (CNCL-B-NHL-2017) regimen. Methods: Clinical features of 436 BL patients who were ≤18 years old and treated with the CNCL-B-NHL-2017 regimen from May 2017 to April 2021 were analyzed retrospectively. Clinical characteristics of patients at disease onset were analyzed and the therapeutic effects of patients with different clinical stages and risk groups were compared. Survival analysis was performed by Kaplan-Meier method, and Cox regression was used to identify the prognostic factors. Results: Among 436 patients, there were 368 (84.4%) males and 68 (15.6%) females, the age of disease onset was 6.0 (4.0, 9.0) years old. According to the St. Jude staging system, there were 4 patients (0.9%) with stage Ⅰ, 30 patients (6.9%) with stage Ⅱ, 217 patients (49.8%) with stage Ⅲ, and 185 patients (42.4%) with stage Ⅳ. All patients were stratified into following risk groups: group A ( n =1, 0.2%), group B1 ( n =46, 10.6%), group B2 ( n =19, 4.4%), group C1 ( n =285, 65.4%), group C2 ( n =85, 19.5%). Sixty-three patients (14.4%) were treated with chemotherapy only and 373 patients (85.6%) were treated with chemotherapy combined with rituximab. Twenty-one patients (4.8%) suffered from progressive disease, 3 patients (0.7%) relapsed, and 13 patients (3.0%) died of treatment-related complications. The follow-up time of all patients was 24.0 (13.0, 35.0) months, the 2-year event free survival (EFS) rate of all patients was (90.9±1.4) %. The 2-year EFS rates of group A, B1, B2, C1 and C2 were 100.0%, 100.0%, (94.7±5.1) %, (90.7±1.7) % and (85.9±4.0) %, respectively. The 2-year EFS rates was higher in group A, B1, and B2 than those in group C1 (χ 2 =4.16, P =0.041) and group C2 (χ 2 =7.21, P =0.007). The 2-year EFS rates of the patients treated with chemotherapy alone and those treated with chemotherapy combined with rituximab were (79.3±5.1)% and (92.9±1.4)% (χ 2 =14.23, P <0.001) respectively. Multivariate analysis showed that stage Ⅳ (including leukemia stage), serum lactate dehydrogenase (LDH)>4-fold normal value, and with residual tumor in the mid-term evaluation were risk factors for poor prognosis ( HR =1.38,1.23,8.52,95% CI 1.05-1.82,1.05-1.43,3.96-18.30). Conclusions: The CNCL-B-NHL-2017 regimen show significant effect in the treatment of pediatric BL. The combination of rituximab improve the efficacy further.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ-1 and PRC2 acting in synergy, a process that is vital for seed germination., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: The aim of this study was to investigate whether 11q loss of heterozygosity (LOH) aberration would impact the response of the primary tumor to neoadjuvant chemotherapy or to the degree of surgical resection in neuroblastoma (NB) patients with MYCN amplification., Methods: The clinical data of 42 NB patients with MYCN amplification who were newly diagnosed and received treatments at our hospital from 2011 to 2020 were retrospectively analyzed. According to the results of the segmental chromosome aberration analysis, the patients enrolled were assigned to an 11qLOH positive group and an 11qLOH negative group., Results: There was no significant difference in the mean number of chemotherapy courses completed before surgery between the 11qLOH positive and 11qLOH negative groups ( p = 0.242). Each of the 42 patients had metaiodobenzylguanidine (MIBG) scans both before and after neoadjuvant chemotherapy. The percentage of patients who had a clinical MIBG change in the 11qLOH positive group was lower than the percentage in the 11qLOH negative group (27.27 vs. 66.67%, p = 0.030). The 11qLOH negative group seemed to have a higher rate of surgical resection (≥90%); however, the difference between the two groups was not statistically significant ( p = 0.088). Furthermore, the 11qLOH negative group did not show significantly superior event-free survival and overall survival rates compared with the 11qLOH positive group., Conclusions: This study showed that patients with NB and MYCN amplification in combination with 11qLOH might be less likely to respond to neoadjuvant chemotherapy when compared with patients with NB and MYCN amplification without 11qLOH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Qu, Duan, Zuo, Sai, Rui, Gong, Ding and Gao.)

Background: This study determined the predictive value of CRMP4 promoter methylation in prostate tissues collected by core needle biopsies for a postoperative upgrade of Gleason Score (GS) to ≥8 in patients with low-risk PCa., Method: A retrospective analysis of the clinical data was conducted from 631 patients diagnosed with low-risk PCa by core needle biopsy at multiple centers and then underwent Radical Prostatectomy (RP) from 2014-2019. Specimens were collected by core needle biopsy to detect CRMP4 promoter methylation. The pathologic factors correlated with the postoperative GS upgrade to ≥8 were analyzed by logistic regression. The cut-off value for CRMP4 promoter methylation in the prostate tissues collected by core needle biopsy was estimated from the ROC curve in patients with a postoperative GS upgrade to ≥8., Result: Multivariate logistic regression showed that prostate volume, number of positive cores, and CRMP4 promoter methylation were predictive factors for a GS upgrade to ≥8 (OR: 0.94, 95% CI: 0.91-0.98, P =0.003; OR: 3.16, 95% CI: 1.81-5.53, P <0.001; and OR: 1.43, 95% CI: 1.32-1.55, P <0.001, respectively). The positive predictive rate was 85.2%, the negative predictive rate was 99.3%, and the overall predictive rate was 97.9%. When the CRMP4 promoter methylation rate was >18.00%, the low-risk PCa patients were more likely to escalate to high-risk patients. The predictive sensitivity and specificity were 86.9% and 98.8%, respectively. The area under the ROC curve (AUC) was 0.929 (95% CI: 0.883-0.976; P <0.001). The biochemical recurrence (BCR)-free survival, progression-free survival (PFS), and cancer-specific survival (CSS) were worse in patients with CRMP4 methylation >18.0% and postoperative GS upgrade to ≥8 than in patients without an upgrade ( P ≤ 0.002)., Conclusion: A CRMP4 promoter methylation rate >18.00% in prostate cancer tissues indicated that patients were more likely to escalate from low-to-high risk after undergoing an RP. We recommend determining CRMP4 promoter methylation before RP for low-risk PCa patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Qin, Lu, Yang, Wang, Chen, Liu, Chen, Zhou, Pan, Li, Ren, Liu, Liu, Qian, Yi, Lai, Qu, Gao, Xu, Chen and Zhuo.)

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, development, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postulated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain components of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana , the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Accurate communication at the stigma surface is required to promote plants' own pollen and reject foreign pollen. Liu et al. have now discovered an autocrine signaling pathway at the surface of arabidopsis stigmatic papillae, accumulating ROS. Downregulation of ROS production via an antagonistic peptide from the pollen coat promotes pollen hydration and germination., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The present study aimed to explore associations between brain activity in the auditory cortex and clinical and psychiatric characteristics in patients with migraine without aura (MwoA) during interictal periods. Resting-state data were acquired from patients with episodic MwoA (n = 34) and healthy controls (n = 30). Independent component analysis was used to extract and calculate the resting-state auditory network. Subsequently, we analyzed the correlations between spontaneous activity in the auditory cortex and clinical and psychiatric features in interictal MwoA. Compared with healthy controls, patients with MwoA showed increased activity in the left superior temporal gyrus (STG), postcentral gyrus (PoCG) and insula. Brain activity in the left STG was positively correlated with anxiety scores, and activity in the left PoCG was negatively correlated with anxiety and depression scores. No significant differences were found in intracranial volume between the two groups. This study indicated that functional impairment and altered integration linked to the auditory cortex existed in patients with MwoA in the interictal period, suggesting that auditory-associated cortex disruption as a biomarker may be implemented for the early diagnosis and prediction of neuropsychiatric impairment in interictal MwoA patients., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Male germline development in flowering plants involves two distinct and successive phases, microsporogenesis and microgametogenesis, which involve one meiosis followed by two rounds of mitosis. Many aspects of distinctions after mitosis between the vegetative cell and the male germ cells are seen, from morphology to structure, and the differential functions of the two cell types in the male gametophyte are differentially needed and required for double fertilization. The two sperm cells, carriers of the hereditary substances, depend on the vegetative cell/pollen tube to be delivered to the female gametophyte for double fertilization. Thus, the intercellular communication and coordinated activity within the male gametophyte probably represent the most subtle regulation in flowering plants to guarantee the success of reproduction. This review will focus on what we have known about the differentiation process and the functional diversification of the vegetative cell and the male germ cell, the most crucial cell types for plant fertility and crop production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The small nucleolar RNA host gene 12 (SNHG12) has been reported to play an important role in the tumorigenesis and progression of PCa, but the functional underlying mechanism has not been studied clearly. We detected the expression level of SNHG12 in PCa tissues and matched adjacent normal tissues that were collected from 85 patients. Then, colony formation assays, MTT experiments, and flow cytometry were used to examine the effect of SNHG12 on proliferation, cell cycle distribution, and apoptosis of DU145 cells. Further, Transwell invasion assay was utilized to assess whether SNHG12 participates in PCa cell invasion and affects the secretion of VEGF secretion in DU145 cells. Finally, we investigated the effect of SNHG12 on tumor growth in vivo . We found that SNHG12 promoted cell proliferation and suppressed apoptosis in PCa cells, which suggests that SNHG12 is probably a novel PCa biomarker and therapy target of PCa., Competing Interests: The authors have declared that no competing interest exists., (Copyright © 2020 Zheng Chen et al.)

Background and Aims: Microvascular invasion (MVI) is a key pathological factor that severely affects the postoperative prognosis of patients with hepatocellular carcinoma (HCC). However, no MVI classification schemes based on standardized gross sampling protocols of HCC are available at present., Methods: 119 HCC specimens were sampled at multiple sites (3-, 7-, and 13 points) for the optimum MVI detection rate. 16,144 resected HCCs were graded as M0, M1 or M2 by adopting three-tiered MVI grading (MVI-TTG) scheme based on the seven-point sampling protocol (SPSP). Survival analyses were performed on 2573 patients to explore the advantages of MVI-TTG., Results: The MVI detection rate determined by SPSP was significantly higher than that determined by the 3-point sampling method (34.5% vs. 47.1%, p = 0.048), but was similar to that determined by the 13-point sampling method (47.1% vs. 51.3%, p = 0.517). Among 16,144 resected HCCs, the proportions of M0, M1 and M2 specimens according to SPSP were 53.4%, 26.2% and 20.4%, respectively. Postoperative survival analysis in 2573 HCC patients showed that the 3-year recurrence rates in M0, M1 and M2 MVI groups were 62.5%, 71.6% and 86.1%, respectively (p < 0.001), and the corresponding 3-year overall survival (OS) rates were 94.1%, 87.5% and 67.0%, respectively (p < 0.001). M1 grade was associated with early recurrence, while M2 grade was associated with both early and late recurrence. MVI-TTG had a larger area under the curve and net benefit rate than the two-tiered MVI grading scheme for predicting time to recurrence and OS., Conclusions: SPSP is a practical method to balance the efficacy of sampling numbers and MVI detection rates. MVI-TTG based on SPSP is a better prognostic predictor than the two-tiered MVI scheme. The combined use of SPSP and MVI-TTG is recommended for the routine pathological diagnosis of HCC.

Aberrant functional connectivity of brain networks has been demonstrated in migraine sufferers. Functional magnetic resonance imaging (fMRI) may illustrate altered connectivity in patients suffering from migraine without aura (MwoA). Here, we applied a seed-based approach based on limbic regions to investigate disrupted functional connectivity between spontaneous migraine attacks. Resting-state fMRI data were obtained from 28 migraine patients without aura and 23 well-matched healthy controls (HC). The functional connectivity of the limbic system was characterized using a seed-based whole-brain correlation method. The resulting functional connectivity measurements were assessed for correlations with other clinical features. Neuropsychological data revealed significantly increased connectivity between the limbic system (bilateral amygdala and right hippocampus) and left middle occipital gyrus (MOG), and a positive correlation was revealed between disease duration and connective intensity of the left amygdala and the ipsilateral MOG. There was decreased functional connectivity between the right amygdala and contralateral orbitofrontal cortex (OFC). In addition, resting-state fMRI showed that, compared to HC, patients without aura had significant functional connectivity consolidation between the bilateral hippocampus and cerebellum, and a negative correlation was detected between scores on the headache impact test (HIT) and connectivity intensity of the right hippocampus and bilateral cerebellum. There was decreased functional connectivity between the left hippocampus and three brain areas, encompassing the bilateral inferior parietal gyri (IPG) and contralateral supplementary motor area (SMA). There were no structural differences between the two groups. Our data suggest that migraine patients have disrupted limbic functional connectivity to pain-related regions of the modulatory and encoding cortices, which are associated with specific clinical characteristics. Disturbances of resting-state functional connectivity may play a key role in neuropathological features, perception and affection of migraine. The current study provides further insights into the complex scenario of migraine mechanisms. .

Precursor mRNA (pre-mRNA) splicing is essential for gene expression in most eukaryotic organisms. Previous studies from mammals, Drosophila, and yeast show that the majority of splicing events occurs co-transcriptionally. In plants, however, the features of co-transcriptional splicing (CTS) and its regulation still remain largely unknown. Here, we used chromatin-bound RNA sequencing to study CTS in Arabidopsis thaliana. We found that CTS is widespread in Arabidopsis seedlings, with a large proportion of alternative splicing events determined co-transcriptionally. CTS efficiency correlated with gene expression level, the chromatin landscape and, most surprisingly, the number of introns and exons of individual genes, but is independent of gene length. In combination with enhanced crosslinking and immunoprecipitation sequencing analysis, we further showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding, frequently to exonic sequences. Notably, this general effect of RZ-1B/1C on splicing promotion is mainly observed at the chromatin level, not at the mRNA level. RZ-1C promotes CTS of multiple-exon genes in association with its binding to regions both proximal and distal to the regulated introns. We propose that RZ-1C promotes efficient CTS of genes with multiple exons through cooperative interactions with many exons, introns, and splicing factors. Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

In flowering plants, each pollen tube delivers two sperm cells into the ovule to complete double fertilization. During the process, pollen tubes need to be navigated into the ovule, where accurate and complex pre-ovule guidance and ovule guidance are required. In recent years, different methods have been established to study those genes involved in the regulation of pollen tube guidance. Semi-in vivo ovule targeting mimics in vivo pollen tube micropylar guidance, and the semi-in vivo ovule targeting assay has been used to investigate function of genes involved in micropylar guidance. Moreover, the ovule targeting assay is the best way to do live cell imaging, which facilitates observation of pollen tube reception, synergid cell degeneration, and semi-in vivo gamete fusion. Meanwhile, semi-in vivo pollen tube attraction assay is another useful method to directly determine whether a certain molecule has pollen tube attraction activity.

Mutant phenotype observation is the most useful and important method to study which biological process a gene-of-interest is involved in. In flowering plants, excessive pollen grains land and germinate on the stigma, then pollen tubes grow through the transmitting tract to reach the ovules, eventually enter the micropyle to complete double fertilization. First, for mutants whose homozygotes could not be obtained due to pollen tube defects, it is difficult to observe the defect phenotype since the pollen grains of different genotypes are mixed together. Here, we provide a detailed protocol to pick out mutant pollen grains from the heterozygous mutant plants in Arabidopsis thaliana. By using this method, we could obtain sufficient mutant pollen grains for phenotypic analysis. Second, it is difficult to compare the pollen/pollen tube behavior of two different genotypes/species in vivo in a same pistil. Here, we develop a new dual staining method which combines GUS staining with aniline blue staining. By using this method, we can analyze the competence of the two different pollen tubes in the same pistil.

Background: Resting-state functional magnetic resonance imaging (fMRI) has confirmed disrupted visual network connectivity in migraine without aura (MwoA). The thalamus plays a pivotal role in a number of pain conditions, including migraine. However, the significance of altered thalamo-visual functional connectivity (FC) in migraine remains unknown. The goal of this study was to explore thalamo-visual FC integrity in patients with MwoA and investigate its clinical significance., Methods: Resting-state fMRI data were acquired from 33 patients with MwoA and 22 well-matched healthy controls. After identifying the visual network by independent component analysis, we compared neural activation in the visual network and thalamo-visual FC and assessed whether these changes were linked to clinical characteristics. We used voxel-based morphometry to determine whether functional differences were dependent on structural differences., Results: The visual network exhibited significant differences in regions (bilateral cunei, right lingual gyrus and left calcarine sulcus) by inter-group comparison. The patients with MwoA showed significantly increased FC between the left thalami and bilateral cunei and between the right thalamus and the contralateral calcarine sulcus and right cuneus. Furthermore, the neural activation of the left calcarine sulcus was positively correlated with visual analogue scale scores (r = 0.319, p = 0.043), and enhanced FC between the left thalamus and right cuneus in migraine patients was negatively correlated with Generalized Anxiety Disorder scores (r = - 0.617, p = 0.005)., Conclusion: Our data suggest that migraine distress is exacerbated by aberrant feedback projections to the visual network, playing a crucial role in migraine physiological mechanisms. The current study provides further insights into the complex scenario of migraine mechanisms.

Background: Isocitrate lyase (ICL) is a key enzyme in the glyoxylate cycle. In a previous study in rice, the expression of the ICL-encoding gene (OsICL) was highly induced by salt stress and its expression was enhanced in transgenic rice lines overexpressing OsCam1-1, a calmodulin (CaM)-encoding gene. CaM has been implicated in salt tolerance mechanisms in plants; however, the cellular mechanisms mediated by CaM are not clearly understood. In this study, the role of OsICL in plant salt tolerance mechanisms and the possible involvement of CaM were investigated using transgenic plants expressing OsICL or OsCam1-1., Results: OsICL was highly expressed in senesced leaf and significantly induced by salt stress in three OsCam1-1 overexpressing transgenic rice lines as well as in wild type (WT). In WT young leaf, although OsICL expression was not affected by salt stress, all three transgenic lines exhibited highly induced expression levels. In Arabidopsis, salt stress had negative effects on germination and seedling growth of the AtICL knockout mutant (Aticl mutant). To examine the roles of OsICL we generated the following transgenic Arabidopsis lines: the Aticl mutant expressing OsICL driven by the native AtICL promoter, the Aticl mutant overexpressing OsICL driven by the 35SCaMV promoter, and WT overexpressing OsICL driven by the 35SCaMV promoter. Under salt stress, the germination rate and seedling fresh and dry weights of the OsICL-expressing lines were higher than those of the Aticl mutant, and the two lines with the icl mutant background were similar to the WT. The F v /F m and temperature of rosette leaves in the OsICL-expressing lines were less affected by salt stress than they were in the Aticl mutant. Finally, glucose and fructose contents of the Aticl mutant under salt stress were highest, whereas those of OsICL-expressing lines were similar to or lower than those of the WT., Conclusions: OsICL, a salt-responsive gene, was characterized in the transgenic Arabidopsis lines, revealing that OsICL expression could revert the salt sensitivity phenotypes of the Aticl knockout mutant. This work provides novel evidence that supports the role of ICL in plant salt tolerance through the glyoxylate cycle and the possible involvement of OsCam1-1 in regulating its transcription.

RALFs are secreted peptides that are perceived by various CrRLK1L-LRE/LLG receptor complexes. The mechanistic basis of this perception has now been elucidated showing that the co-receptor LLG binds RALF23 to nucleate a FER receptor complex. This interaction likely occurs in other tissues where RALFs meet CrRLK1L receptors and LRE/LLG co-receptors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)