Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis.

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal (distal-proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP-MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell-like gene expression profiles. Although in myb98, egg cell-specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell-specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

Exploring Novel Polytubey Reproduction Pathways Utilizing Cumulative Genetic Tools.

In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants.

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

Optic nerve sheath diameter as a quantitative parameter associated with the outcome in patients with traumatic brain injury undergoing hematoma removal.

PURPOSE: To determine the association between optic nerve sheath diameter (ONSD) and outcome in patients with traumatic brain injury (TBI) who undergo hematoma removal (HR). METHODS: This study was a retrospective analysis of data from a single center between 2016 and 2021. Adult patients with TBI who underwent HR within 24 h after admission were included in this study. Preoperative and postoperative ONSD of the surgical side and the mean ONSD of both sides were measured for analysis. The primary outcome was mortality at 30 days. Receiver operating characteristic curve analysis was performed to calculate the area under the curve (AUC) and 95% confidence interval (CI) for 30 days mortality. RESULTS: Sixty-one patients were enrolled in the study. Among them, 48 (78.7%) survived for 30 days after admission. The AUC and 95% CI of the postoperative mean ONSD on both sides and postoperative/preoperative mean of the ONSD ratio on both sides were 0.884 [0.734-0.955] and 0.875 [0.751-0.942], respectively. The postoperative mean of both ONSDs of 6.0 mm had high accuracy as a cut-off value with a sensitivity of 85%, specificity of 83%, positive likelihood ratio (LR) of 5.0, and negative LR- of 0.18. CONCLUSION: This study demonstrated that postoperative ONSD and the postoperative/preoperative ONSD ratio were associated with postoperative outcome in patients with TBI who underwent HR.

Effectiveness of Administration of Fibrinogen Concentrate as Prevention of Hypofibrinogenemia in Patients with Traumatic Brain Injury with a Higher Risk for Severe Hyperfibrinolysis: Single Center Before-and-After Study.

BACKGROUND: Coagulopathy is often observed in severe traumatic brain injury (sTBI), and hyperfibrinolysis (HF) is associated with a poor prognosis. Although the efficacy of fibrinogen concentrate (FC) in multiple trauma has been reported, its efficacy in sTBI is unclear. Therefore, we delineated severe HF risk factors despite fresh frozen plasma transfusion. Using these risk factors, we defined high-risk patients and determined whether FC administration to this group improved fibrinogen level. METHODS: In the first part of this study, successive adults with sTBI treated at our hospital between April 2016 and March 2019 were reviewed. Patients underwent transfusion as per our conventional protocol and were divided into two groups based on whether fibrinogen levels of ≥ 150 mg/dL were maintained 3-6 h after arrival to delineate the risk factors of severe HF. In the second part of the study, we conducted a before-and-after study in patients with sTBI who were at a higher risk for severe HF (presence of at least one of the risk factors identified in the first part of the study), comparing those treated with FC between April 2019 and March 2021 (FC group) with those treated with conventional transfusion before FC between April 2016 and March 2019. The primary outcome was maintenance of fibrinogen levels, and the secondary outcome was 30-day mortality. RESULTS: In the first part of the study, 78 patients were included. Twenty-three patients did not maintain fibrinogen levels ≥ 150 mg/dL. A D-dimer level on arrival > 50 µg/mL, a fibrinogen level on arrival < 200 mg/dL, depressed skull fracture, and multiple trauma were severe HF risk factors. In the second part, compared with 46 patients who were identified as being at high risk for severe HF but were not administered FC (non-FC group), fibrinogen levels ≥ 150 mg/dL 3-6 h after arrival were maintained in 14 of 15 patients in the FC group (odds ratio: 0.07; 95% confidence interval: 0.01-0.59). Although there were significant differences in fibrinogen levels, no significant differences were observed in terms of 30-day mortality between the groups. CONCLUSIONS: Coagulation abnormalities on arrival, severe skull fracture, and multiple trauma are severe HF risk factors. FC administration may contribute to rapid correction of developing hypofibrinogenemia.

Migration of lipiodol into lateral ventricles after embolization of cerebral arteriovenous malformation: a case report.

N-butyl cyanoacrylate (NBCA) has been used to embolise brain arteriovenous malformations (AVMs) for over 30 years. It is a mixed with lipiodol in varying proportions. We report a 22-year-old male with intraventricular hemorrhage from a ruptured intranidal AVM aneurysm in the left temporal lobe. The intranidal aneurysm and the nidus were successfully embolized using a 20% NBCA and lipiodol mixture without any complications according to computed tomography (CT) immediately after treatment. Scattered high-density spots were observed in both lateral ventricles on CT 5 days after embolization, suggesting migration of lipiodol. We speculated that the aneurysm was a pseudoaneurysm whose wall protruded into the inferior horn of the left lateral ventricle, and the lipiodol in the NBCA migrated into the ventricles after the thin part of the wall ruptured. The patient developed pyrexia due to chemical meningitis, which responded to steroid treatment for one month.

Super-selective balloon test occlusion with electrophysiological monitoring to occlude angiographically invisible posterior communicating artery perforators with unruptured aneurysm.

Balloon test occlusion (BTO) can predict the ischemic complication risk associated with arterial occlusion. We present a case of an unruptured, broad-necked internal carotid artery-posterior communicating artery (PcomA) aneurysm that was successfully embolized after super-selective BTO of fetal PcomA with electrophysiological monitoring. The proximal portion of the PcomA was internally occluded without causing major neurological deficits, although we observed a small new infarction in the ipsilateral anterior thalamus postoperatively. We recognized small perforators arising from the proximal PcomA during a previous clipping surgery. Super-selective BTO with electrophysiological monitoring could be useful for functional preservation after infarction from angiographically invisible perforators.