Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] 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)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC 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)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Parathyroid carcinoma: impact of preoperative diagnosis on the choice of surgical procedure.

The operative procedure in the surgical treatment of parathyroid carcinoma differs from that of benign hyperparathyroidism. However, preoperative differentiation is often difficult. This study elucidated how clinicians diagnose parathyroid carcinoma and the relationship between preoperative diagnosis and the operative course. Using a retrospective chart review, twenty cases of parathyroid carcinoma from nine participating centers were examined. In 11 cases with preoperative suspicion of malignancy, at least one of these three features was found: elevated serum calcium level (>14 mg/dL), palpable mass, and irregular margin on ultrasonography. Although an intact parathyroid hormone (iPTH) threshold to suspect malignancy has not been established, six cases showed marked iPTH elevation exceeding 8.0 times the upper limit of normal. One case was excluded from analysis due to hemodialysis. Compared with the four cases that showed calcium elevation, the iPTH threshold might represent better sensitivity. Among 9 cases of benign preoperative diagnosis, six cases were performed with pericapsular resection. In three cases where malignancy was suspected in the middle of the operation, the recommended en bloc resection with ipsilateral thyroid lobectomy was not performed but a parathyroidectomy with surrounding soft tissue. In contrast, 10 preoperatively suspected cases underwent en bloc resection, and one case underwent pericapsular resection followed by supplementary ipsilateral hemithyroidectomy due to the uncertain pre- and intraoperative findings to determine the diagnosis. In conclusion, the surgical procedure for parathyroid carcinoma strongly depends on the preoperative diagnosis. The presence of excessive iPTH levels might contribute to improved preoperative diagnostic sensitivity for parathyroid carcinoma.

Is surgical resection without radioactive iodine treatment a safe alternative treatment for T1-2N1bM0 papillary thyroid carcinoma?

OBJECTIVE: Postoperative radioactive iodine (RAI) adjuvant therapy improves the prognosis in patients with advanced papillary thyroid carcinoma (PTC), although the role of RAI adjuvant therapy remains unclear in intermediate-risk patients, as defined by the American Thyroid Association. The ATA cautiously recommended RAI adjuvant therapy in patients with T1-3N1b, but the Japanese Society of Thyroid Surgery suggests lobectomy without RAI adjuvant therapy in these patients. This study assessed the role and efficacy of RAI adjuvant therapy in patients with T1-3N1b PTC. METHODS: A single-center retrospective observational study was performed. We included patients with T1-3N1bM0 PTC who underwent complete resection between January 2003 and December 2017. Patients with bilateral PTC were excluded. We compared recurrence rates after surgery with RAI adjuvant therapy and surgery alone. RESULTS: A total of 61 patients (male:female ratio, 18:43; mean age, 57.1 ± 16.7 years) were included, and the median follow-up period was 6.8 years. Of the included patients, 33 were treated with surgery with RAI adjuvant therapy and 28 were treated with surgery alone. The RAI treatment group that underwent surgery had larger tumors, more lymph node metastases, and were younger. There were no disease-specific deaths, and 10 patients experienced local recurrence. The recurrence rate was 24.2% in patients who underwent surgery with RAI adjuvant therapy and 7.1% in patients who underwent surgery alone. Compared to T1-2 stage patients, the T3 stage patients tended to have a higher recurrence rate (odds ratio, 2.38; 95% confidence interval, 0.51-10.7). CONCLUSIONS: The recurrence rate was higher in patients who underwent surgery with RAI adjuvant therapy than in patients who underwent surgery alone. Patients in the former group had larger tumors and more lymph node metastases, and this difference in baseline characteristics could explain their higher recurrence rate. The recurrence rate was lower in patients with small tumors, and RAI adjuvant therapy would likely not play a major role in T1-2N1bM0 patients.

Perioperative local anaesthesia for reducing pain following septal surgery.

BACKGROUND: Septal surgery is a well-established procedure used to treat nasal obstruction due to deviation of the nasal septum, which is carried out under local or general anaesthesia. Local anaesthesia is used for postoperative pain control, but its effectiveness and safety are unclear. OBJECTIVES: To assess the effectiveness of perioperative local anaesthesia for reducing pain in septal surgery and to evaluate the risk of associated complications. SEARCH METHODS: The Cochrane ENT Information Specialist searched the Cochrane ENT Trial Register; Central Register of Controlled Trials; Ovid MEDLINE; Ovid Embase; CINAHL; Web of Science; ClinicalTrials.gov; ICTRP and additional sources for published and unpublished trials. The date of the search was 9 January 2018. SELECTION CRITERIA: Randomised controlled trials and cluster-randomised controlled trials involving adults or children (or both) who underwent septal surgery. We included studies comparing local anaesthesia versus no treatment/placebo. We also included studies comparing different types of local anaesthesia to each other (i.e. local injection, the addition of an anaesthetic agent to nasal packing, where used, and sphenopalatine ganglion block). DATA COLLECTION AND ANALYSIS: We used the standard methodological procedures expected by Cochrane. The primary outcome was postoperative pain intensity at 12, 24 and 48 hours measured by visual analogue scale (VAS) or another pain outcome tool including numerical or verbal rating scales. Secondary outcomes were requirement for additional analgesia, duration of hospitalisation and adverse effects (postoperative bleeding and postoperative vomiting). We used GRADE to assess the quality of the evidence for each outcome; this is indicated in italics. MAIN RESULTS: We included seven randomised controlled trials involving 493 participants. In all studies the participants were adults undergoing septoplasty. These studies were heterogeneous and the quality of the body of evidence ranged from low to very low. Few of the studies provided reliable data for the primary outcome in this review.Local anaesthetic injection versus no treatment/placeboTwo studies (142 participants) compared local anaesthetic injection versus placebo but these studies did not report postoperative pain at 12, 24 or 48 hours. It is unclear whether local anaesthetic injection changed the risk of vomiting (odds ratio (OR) 3.10, 95% confidence interval (CI) 0.12 to 79.23; 60 participants; one study) (low-quality evidence). Neither study reported the requirement for additional analgesia, duration of hospitalisation or uncontrollable postoperative bleeding.Local anaesthetic application via nasal packing versus no packing/packing with placeboFour studies (301 participants) used nasal packing postoperatively and compared the addition of local anaesthetic to the pack versus packing with a placebo added. Compared with packing with placebo, the addition of local anaesthetic to nasal packing reduced the pain score on a VAS (ranging from 0 to 100) at 12 hours (mean difference (MD) -16.95, 95% CI -22.27 to -11.62; 151 participants; two studies; I2 = 49%) (low-quality evidence) and at 24 hours postoperatively (MD -7.53, 95% CI -9.76 to -5.29; 268 participants; four studies; I2 = 83%) (very low-quality evidence). These studies did not report postoperative pain at 48 hours. The addition of local anaesthetic to nasal packing decreased the requirement for additional analgesia (OR 0.15, 95% CI 0.07 to 0.34; 151 participants; two studies; I2 = 15%) (moderate-quality evidence). No studies reported duration of hospitalisation, postoperative vomiting or uncontrollable postoperative bleeding.No studies compared the addition of local anaesthetic to nasal packing versus no packing.Sphenopalatine ganglion block versus no treatment/placeboOne study (50 participants) compared sphenopalatine ganglion block versus no treatment but this study did not report postoperative pain, requirement for additional analgesia, duration of hospitalisation, vomiting or uncontrollable postoperative bleeding. AUTHORS' CONCLUSIONS: The addition of local anaesthesia to nasal packs (if these are being used) following septal surgery may reduce postoperative pain within the first 12 hours, compared to nasal packing with a placebo added. The effect is uncertain at 24 hours because the quality of the evidence is very low. Evidence was lacking for other outcomes, including adverse effects. There is a lack of evidence about the effects of local anaesthesia added to nasal packing compared to no nasal packing. There is also a lack of evidence about the effects of local anaesthesia given by injection and the effects of sphenopalatine ganglion block.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

Efficacy of chlorophyll c2 for seasonal allergic rhinitis: single-center double-blind randomized control trial.

Chlorophyll c2 extracted from Sargassum horneri improved allergic symptoms in an animal model of allergic rhinitis. In the present study, we explored the efficacy of chlorophyll c2 in patients with seasonal allergic rhinitis. This was a single-center, randomized, double-blind placebo-controlled trial. Sixty-six patients aged 20-43 years, each with a 2-year history of seasonal allergic rhinitis, were randomly assigned to receive either a single daily dose (0.7 mg) of chlorophyll c2 or placebo for 12 weeks. The use of medications including H1-antihistamines and topical nasal steroids was recorded by rescue medication scores (RMSs) noted after 4, 8, and 12 weeks of treatment. Disease-specific quality of life was measured using the Japan Rhinitis Quality of Life Questionnaire (JRQLQ) both before and after 4, 8, and 12 weeks of treatment. The RMS at 8 weeks was significantly better in the chlorophyll c2 than the placebo group (mean RMS difference = -3.09; 95 % confidence interval = -5.96 to -0.22); the mean RMS at 4 weeks was only slightly better in the chlorophyll c2 group. The JRQLQ scores did not differ significantly between the two groups. Chlorophyll c2 would have a potential to be an alternative treatment for allergic rhinitis.

(+)-Catechin protects dermal fibroblasts against oxidative stress-induced apoptosis.

BACKGROUND: Oxidative stress has been suggested as a mechanism underlying skin aging, as it triggers apoptosis in various cell types, including fibroblasts, which play important roles in the preservation of healthy, youthful skin. Catechins, which are antioxidants contained in green tea, exert various actions such as anti-inflammatory, anti-bacterial, and anti-cancer actions. In this study, we investigated the effect of (+)-catechin on apoptosis induced by oxidative stress in fibroblasts. METHODS: Fibroblasts (NIH3T3) under oxidative stress induced by hydrogen peroxide (0.1 mM) were treated with either vehicle or (+)-catechin (0-100 µM). The effect of (+)-catechin on cell viability, apoptosis, phosphorylation of c-Jun terminal kinases (JNK) and p38, and activation of caspase-3 in fibroblasts under oxidative stress were evaluated. RESULTS: Hydrogen peroxide induced apoptotic cell death in fibroblasts, accompanied by induction of phosphorylation of JNK and p38 and activation of caspase-3. Pretreatment of the fibroblasts with (+)-catechin inhibited hydrogen peroxide-induced apoptosis and reduced phosphorylation of JNK and p38 and activation of caspase-3. CONCLUSION: (+)-Catechin protects against oxidative stress-induced cell death in fibroblasts, possibly by inhibiting phosphorylation of p38 and JNK. These results suggest that (+)-catechin has potential as a therapeutic agent for the prevention of skin aging.

L-arginine stimulates fibroblast proliferation through the GPRC6A-ERK1/2 and PI3K/Akt pathway.

L-arginine is considered a conditionally essential amino acid and has been shown to enhance wound healing. However, the molecular mechanisms through which arginine stimulates cutaneous wound repair remain unknown. Here, we evaluated the effects of arginine supplementation on fibroblast proliferation, which is a key process required for new tissue formation. We also sought to elucidate the signaling pathways involved in mediating the effects of arginine on fibroblasts by evaluation of extracellular signal-related kinase (ERK) 1/2 activation, which is important for cell growth, survival, and differentiation. Our data demonstrated that addition of 6 mM arginine significantly enhanced fibroblast proliferation, while arginine deprivation increased apoptosis, as observed by enhanced DNA fragmentation. In vitro kinase assays demonstrated that arginine supplementation activated ERK1/2, Akt, PKA and its downstream target, cAMP response element binding protein (CREB). Moreover, knockdown of GPRC6A using siRNA blocked fibroblast proliferation and decreased phosphorylation of ERK1/2, Akt and CREB. The present experiments demonstrated a critical role for the GPRC6A-ERK1/2 and PI3K/Akt signaling pathway in arginine-mediated fibroblast survival. Our findings provide novel mechanistic insights into the positive effects of arginine on wound healing.

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley.

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline â†’ betaine aldehyde â†’ GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.

Characterization of a novel glycinebetaine/proline transporter gene expressed in the mestome sheath and lateral root cap cells in barley.

The accumulation of glycinebetaine (GB) is one of the adaptive strategies to adverse salt stress conditions. Although it has been demonstrated that barley plants accumulate GB in response to salt stress and various studies focused on GB synthesis were performed, its transport mechanism is still unclear. In this study, we identified a novel gene, HvProT2, encoding Hordeum vulgare GB/proline transporter from barley plants. Heterologous expression in yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of HvProT2 was highest for GB, intermediate for proline and lowest for gamma-aminobutyric acid. Transient expression of fusions of HvProT2 and green fluorescent protein in onion epidermal cells revealed that HvProT2 is localized at the plasma membrane. Relative quantification of mRNA level of HvProT2 using semi-quantitative reverse transcription-polymerase chain reaction analysis showed that HvProT2 is constitutively expressed in both leaves and roots, and the expression level was higher in old leaves than young leaves and roots. Moreover, we found that HvProT2 was expressed in the mestome sheath and lateral root cap cells. We discussed the possible involvement of HvProT2 for salt stress tolerance.