Complete loss of RelA and SpoT homologs in Arabidopsis reveals the importance of the plastidial stringent response in the interplay between chloroplast metabolism and plant defense response.

The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used the CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases, and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type (WT) under normal growth conditions, and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense.

Efficacy of Nostril Supportive Splint Treatment for Nasal Septum Deformities after Le Fort I Osteotomy.

This retrospective study aimed to evaluate the efficacy of support splint treatment for deformities and deviations of the nasal septum after Le Fort I osteotomy (LFI). Patients were divided into two groups: the retainer group wore a nasal support splint immediately after LFI for 7 days, and the no retainer group did not wear a nasal support splint. Evaluation was performed by measuring the ratio of the difference between the left and right sides of the nasal cavity area (ratio of nasal cavity) and the angle of the nasal septum using three computed tomography frontal images (anterior, middle, and posterior) before and one year postoperatively. Sixty patients were included and divided into two groups, the retainer and no retainer group (n=30 each). Regarding the ratio of nasal cavity on middle images at one year postoperatively, the retainer and no retainer groups differed significantly (0.79±0.13 and 0.67±0.24, respectively; P =0.012). The angle of the nasal septum on anterior images at one year postoperatively was 164.8±11.7° in the retainer group and 156.9±13.5° in the no retainer group, showing a significant difference ( P =0.019). This study suggests that support splint treatment after LFI is effective in preventing post-LFI nasal septal deformation or deviation.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

Significance of accumulation of the alarmone (p)ppGpp in chloroplasts for controlling photosynthesis and metabolite balance during nitrogen starvation in Arabidopsis.

The regulatory nucleotides, guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon-nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation.

Maxillary bone healing and CT value after Le Fort I osteotomy using absorbable plate system: A retrospective study.

This study aimed to evaluate maxillary bone healing and computed tomography (CT) values after Le Fort I osteotomy with sagittal split ramus osteotomy in patients with class II and III malocclusion. Four absorbable plates and screws were used to fix the maxillary segments in all patients. For 112 sides (58 patients), the bone defect areas at the anterior and posterior sites between the maxillary segments were measured using 3-dimensional CT views reconstructed over a constant CT value at 1 week and 1 year postoperatively. Subsequently, CT values at the upper, middle, and lower sites around the osteotomy line in the medial, middle, and lateral regions were measured. The bone defect area after 1 year increased at the anterior site in class III and at both the anterior and posterior sites in class II (P < 0.05). This study suggests that the increase in bone defect area was affected by lower CT values at the middle site of the middle and lateral regions in class II, and that bony defects between fragments in the maxilla could partially remain in both classes II and III within 1 year after Le Fort I osteotomy.

Bone healing and stability after advancement genioplasty using a pre-bent absorbable plate and screws.

This study aimed to compare the bone healing and stability of the chin with a pre-bent absorbable plate and a manually bent absorbable plate after advancement genioplasty. Patients with class II malocclusion who underwent genioplasty with bimaxillary surgery were included. After genioplasty, two absorbable bicortical screws were fixed on both sides. Then, a pre-bent absorbable plate and screws were used in the center area to fix the segment in advancement genioplasty (PB group). A manually bent absorbable plate was used for the remaining patients (MB group). Computed tomography (CT) was performed before surgery and 1 week and 1 year after surgery. Changes in the pogonion (Pog) and menton (Me) points, soft tissue points, and the ratio of bone squares under the plate were evaluated using lateral cephalometric images reconstructed with 3-dimensional CT data. 32 patients were included in the study. There were no significant differences in the cephalometric measurements in the time interval from 1 week to 1 year. However, the bone square ratio in the PB group showed a significant increase after 1 year (P = 0.0021). Within the limitations of the study it seems that the use of a pre-bent absorbable plate is effective in promoting bone healing after advancement genioplasty.

Assessment of lateral pterygoid muscle and temporomandibular joint disc after Le Fort I osteotomy with and without intentional pterygoid plate fracture and sagittal split ramus osteotomy in class II and class III patients.

The aim of the study was to examine lateral pterygoid muscle (LPM) and temporomandibular joint (TMJ) disc before and after Le Fort I osteotomy with and without intentional pterygoid plate fracture and sagittal split ramus osteotomy (SSRO) in class II and class III patients. Le Fort I osteotomy and SSRO were performed in class II and class III patients. LPM measurements using oblique sagittal computed tomography (CT) images and TMJ disc position using magnetic resonance imaging (MRI) were examined. Statistical comparisons were performed for the LPM and TMJ between class II and class III patients and between those with and without intentional pterygoid plate fracture in Le Fort I osteotomy. The subjects comprised 60 female patients (120 sides), with 30 diagnosed as class II and 30 as class III. Preoperatively, the width of the condylar attachment, width at eminence, length of the LPM, angle of the LPM, and square of the LPM were significantly smaller in the class II group than in the class III group (p < 0.05). After 1 year, the width of the condylar attachment, width at eminence, and angle of the LPM remained significantly smaller in the class II group than in the class III group (p < 0.0001). TMJ disc position was significantly related to the width of the condylar attachment of the LPM, both pre- and postoperatively (p < 0.0001). However, postoperative disc position did not change in all patients. Next, the class II patients (60 sides) were divided into two groups who underwent Le Fort I osteotomy with or without intentional pterygoid plate fracture. Changes in all measurements of the LPM showed no significant differences between these two groups. Our study suggested that TMJ disc position classification could be associated with the width of condylar attachment of the LPM before and after surgery, while the surgical procedure, including Le Fort I osteotomy with intentional pterygoid plate fracture, might not affect postoperative LMP or disc position in class II patients.

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice.

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

Toward an understanding of the pathophysiology of clear cell carcinoma of the ovary (Review).

Endometriosis-associated ovarian cancers demonstrate substantial morphological and genetic diversity. The transcription factor, hepatocyte nuclear factor (HNF)-1ß, may be one of several key genes involved in the identity of ovarian clear cell carcinoma (CCC). The present study reviews a considerably expanded set of HNF-1ß-associated genes and proteins that determine the pathophysiology of CCC. The current literature was reviewed by searching MEDLINE/PubMed. Functional interpretations of gene expression profiling in CCC are provided. Several important CCC-related genes overlap with those known to be regulated by the upregulation of HNF-1ß expression, along with a lack of estrogen receptor (ER) expression. Furthermore, the genetic expression pattern in CCC resembles that of the Arias-Stella reaction, decidualization and placentation. HNF-1ß regulates a subset of progesterone target genes. HNF-1ß may also act as a modulator of female reproduction, playing a role in endometrial regeneration, differentiation, decidualization, glycogen synthesis, detoxification, cell cycle regulation, implantation, uterine receptivity and a successful pregnancy. In conclusion, the present study focused on reviewing the aberrant expression of CCC-specific genes and provided an update on the pathological implications and molecular functions of well-characterized CCC-specific genes.