ClpL is a functionally active tetradecameric AAA+ chaperone, distinct from hexameric/dodecameric ones.

AAA+ (ATPases associated with diverse cellular activities) chaperones are involved in a plethora of cellular activities to ensure protein homeostasis. The function of AAA+ chaperones is mostly modulated by their hexameric/dodecameric quaternary structures. Here we report the structural and biochemical characterizations of a tetradecameric AAA+ chaperone, ClpL from Streptococcus pneumoniae. ClpL exists as a tetradecamer in solution in the presence of ATP. The cryo-EM structure of ClpL at 4.5 Å resolution reveals a striking tetradecameric arrangement. Solution structures of ClpL derived from small-angle X-ray scattering data suggest that the tetradecameric ClpL could assume a spiral conformation found in active hexameric/dodecameric AAA+ chaperone structures. Vertical positioning of the middle domain accounts for the head-to-head arrangement of two heptameric rings. Biochemical activity assays with site-directed mutagenesis confirmed the critical roles of residues both in the integrity of the tetradecameric arrangement and activities of ClpL. Non-conserved Q321 and R670 are crucial in the heptameric ring assembly of ClpL. These results establish that ClpL is a functionally active tetradecamer, clearly distinct from hexameric/dodecameric AAA+ chaperones.

Ectopic Expression of OsPYL/RCAR7, an ABA Receptor Having Low Signaling Activity, Improves Drought Tolerance without Growth Defects in Rice.

Overexpression of abscisic acid (ABA) receptors has been reported to enhance drought tolerance, but also to cause stunted growth and decreased crop yield. Here, we constructed transgenic rice for all monomeric ABA receptors and observed that only transgenic rice over-expressing OsPYL/RCAR7 showed similar phenotype with wild type, without total yield loss when grown under normal growth condition in a paddy field. Even though transgenic rice over-expressing OsPYL/RCAR7 showed neither an ABA-sensitivity nor an osmotic stress tolerance in plate assay, it showed drought tolerance. We investigated the ABA-dependent interaction with OsPP2CAs and ABA signaling induction by OsPYL/RCAR7. In yeast two hybrid assay, OsPYL/RCAR7 required critically higher ABA concentrations to interact with OsPP2CAs than other ABA receptors, and co-immunoprecipitation assay showed strong interaction under ABA treatment. When ABA-responsive signaling activity was monitored using a transient expression system in rice protoplasts, OsPYL/RCAR7 had the lowest ABA-responsive signaling activity as compared with other ABA receptors. OsPYL/RCAR7 also showed weak suppression of phosphatase activity as compared with other ABA receptors in vitro. Transcriptome analysis of transgenic rice over-expressing OsPYL/RCAR7 suggested that only a few genes were induced similar to control under without exogenous ABA, but a large number of genes was induced under ABA treatment compared with control. We conclude that OsPYL/RCAR7 is a novel functional ABA receptor that has low ABA signaling activity and exhibits high ABA dependence. These results lay the foundation for a new strategy to improve drought stress tolerance without compromising crop growth.

Comprehensive survey of the VxGΦL motif of PP2Cs from Oryza sativa reveals the critical role of the fourth position in regulation of ABA responsiveness.

Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein phosphatase (PP2C), a co-receptor, form a complex upon binding to ABA. Previously we reported that the second and fourth positions in the VxGΦL motif of PP2Cs from Oryza sativa are critical in the interaction of PP2Cs with PYL/RCARs. Considering substantial effects of the VxGΦL motif on ABA signaling outputs, further comprehensive characterization of residues in the second and fourth positions are required. Here we surveyed the second and fourth positions of the VxGΦL motif by combination of biochemical, structural and physiological analyses. We found that the fourth position of the VxGΦL motif, highly conserved to small hydrophobic residues, was a key determinant of the OsPP2C50:OsPYL/RCAR interactions across subfamilies. Large hydrophobic or any hydrophilic residues in the fourth position abrogated ABA responsiveness. Analysis of crystal structures of OsPP2C50 mutants, S265L/I267V ("LV"), I267L ("SL") and I267W ("SW"), in complex with ABA and OsPYL/RCAR3, along with energy calculation of the complexes, uncovered that a bulky hydrophobic residue in the fourth position of the VxGΦL motif pushed away side chains of nearby residues, conferring side-chain rotameric energy stress. Hydrophilic residues in this position imposed solvation energy stress to the PP2C:PYL/RCAR complex. Germination and gene expression analyses corroborated that OsPP2C50 AS and AK mutants modulated ABA responsiveness in Arabidopsis. Our results suggest that ABA responsiveness could be fine-tuned by the fourth position of the VxGΦL motif on PP2Cs. KEY MESSAGE: We comprehensively surveyed the VxGΦL motif to find that the fourth position, highly conserved to small hydrophobic residues, was critical in regulating ABA responsiveness.

Structural determinants for pyrabactin recognition in ABA receptors in Oryza sativa.

KEY MESSAGE: We determined the structure of OsPYL/RCAR3:OsPP2C50 complex with pyrabactin. Our results suggest that a less-conserved phenylalanine of OsPYL/RCAR subfamily I is one of considerations of ABA agonist development for Oryza sativa. Pyrabactin is a synthetic chemical mimicking abscisic acid (ABA), a naturally occurring phytohormone orchestrating abiotic stress responses. ABA and pyrabactin share the same pocket in the ABA receptors but pyrabactin modulates ABA signaling differently, exhibiting both agonistic and antagonistic effects. To explore structural determinants of differential functionality of pyrabactin, we determined the crystal structure of OsPYL/RCAR3:pyrabactin:OsPP2C50, the first rice ABA receptor:co-receptor complex structure with a synthetic ABA mimicry. The water-mediated interaction between the wedging Trp-259 of OsPP2C50 and pyrabactin is lost, undermining the structural integrity of the ABA receptor:co-receptor. The loss of the interaction of the wedging tryptophan of OsPP2C with pyrabactin appears to contribute to the weaker functionality of pyrabactin. Pyrabactin in the OsPYL/RCAR3:OsPP2C50 complex adopts a conformation different from that in ABA receptors from Arabidopsis. Phe125, specific to the subfamily I of OsPYL/RCARs in the ABA binding pocket, appears to be the culprit for the differential conformation of pyrabactin. Although the gate closure essential for the integrity of ABA receptor:co-receptor is preserved in the presence of pyrabactin, Phe125 apparently restricts accessibility of pyrabactin, leading to decreased affinity for OsPYL/RCAR3 evidenced by phosphatase assay. However, Phe125 does not affect conformation and accessibility of ABA. Yeast two-hybrid, germination and gene transcription analyses in rice also support that pyrabactin imposes a weak effect on the control of ABA signaling. Taken together, our results suggest that phenylalanine substitution of OsPYL/RCARs subfamily I may be one of considerations for ABA synthetic agonist development.

Molecular characterization of single-chain antibody variable fragments (scFv) specific to Pep27 from Streptococcus pneumoniae.

Pep27 from Streptococcus pneumoniae is reported to initiate pneumococcal autolysis, thereby constituting a major virulence factor. Although a few antisera recognizing Pep27 have been reported, no monoclonal, well-characterized antibody for Pep27 has been developed. Here we screened two single-chain antibody variable fragments (scFv) using a phage display from a large human synthetic scFv library to select clones E2 and F9. Dissociation constants (Kd) of E2 and F9 were 1.1 µM and 0.50 µM, respectively. E2 and F9 did not cross-react with other pneumococcal and unrelated proteins. The epitopes of Pep27 were localized to residues 24, 26 and 27 by alanine scanning. Molecular docking analysis supported the experimentally investigated epitope. The E2 and F9 clones specifically detected Pep27 in an environment mimicking in vivo conditions, demonstrated in human serum. The scFv clones characterized here represent molecular tools for the detection of pneumococcal diseases with potential for further improvement in affinity.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

Primary cervical choriocarcinoma during viable intrauterine pregnancy.

A 31-year-old multigravida woman at 27 weeks' gestation was admitted with vaginal bleeding and a hypervascular mass near the cervix on ultrasonography. After discharge with improvement, she was readmitted the next day for uncontrolled, heavy vaginal bleeding and underwent emergency cesarean section at 29 weeks' gestation. A 3-cm friable mass found near the cervix was removed surgically; this lesion was shown to be primary cervical choriocarcinoma. On the 17th postoperative day the patient underwent total abdominal hysterectomy with preservation of both ovaries and biopsy was performed on the right ovary. The International Federation of Gynecology and Obstetrics (FIGO) stage was I and her World Health Organization prognostic score was 9, representing high risk. The patient received three rounds of chemotherapy until achieving three consecutive normal human chorionic gonadotropin levels with two additional courses to address risk of relapse. DNA genotyping on short tandem repeat polymorphism confirmed the gestational choriocarcinoma.

Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting.

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 µM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.

Structure-based elucidation of the regulatory mechanism for aminopeptidase activity.

The specificity of proteases for the residues in and length of substrates is key to understanding their regulatory mechanism, but little is known about length selectivity. Crystal structure analyses of the bacterial aminopeptidase PepS, combined with functional and single-molecule FRET assays, have elucidated a molecular basis for length selectivity. PepS exists in open and closed conformations. Substrates can access the binding hole in the open conformation, but catalytic competency is only achieved in the closed conformation by formation of the S1 binding pocket and proximal movement of Glu343, a general base, to the cleavage site. Hence, peptides longer than the depth of the binding hole block the transition from the open to the closed conformation, and thus length selection is a prerequisite for catalytic activation. A triple-sieve interlock mechanism is proposed featuring the coupling of length selectivity with residue specificity and active-site positioning.

A Complex of Cirsium japonicum var. maackii (Maxim.) Matisum. and Thymus vulgaris L. Improves Menopausal Symptoms and Supports Healthy Aging in Women.

We evaluated the efficacy and safety of MS-10® for the treatment of menopausal symptoms. A double-blind randomized placebo-controlled clinical trial was performed in 71 premenopausal women for 4 and 12 weeks. A total of 12 individual menopausal symptom scores were assessed using the Kupperman index. MS-10 treatment effectively improved the symptoms by ∼48%. In addition, the quality of life of the women improved by 36% from four perspectives: vasomotor, psychosocial, physical, and sexual symptoms as evaluated using the menopause-specific quality of life (MenQoL) questionnaire. Our results show that MS-10 improves insulin-like growth factor-1 (IGF-1) and estrogen utilization through receptor activation, which are thought to have causative therapeutic effects on menopause and aging inhibition in women. Improvement of Enthotheline-1 (ET-1) in the blood after MS-10 intake led to an improvement in menopausal vascular symptoms. Improvements in bone formation and absorption markers such as osteocalcin, bone-specific alkaline phosphatase (BSALP), C-telopeptides of type I collagen (CTx), deoxypyridinoline (deoxyPYD), and N-telopeptides of type I collagen (NTx) in blood or urine indicate that MS-10 fundamentally improves bone health in women. By confirming the improvement of the psychological well-being index based on the improvement of stress hormone cortisol, MS-10 can solve causative psychological and physical stress-related symptoms. Moreover, various safety tests, such as those for female hormones, were confirmed. Therefore, it can be confirmed that MS-10 is a natural pharmaconutraceutical that causatively and safely improves health of women and aids in antiaging processes.

Structural mechanism for regulation of Rab7 by site-specific monoubiquitination.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

Underuse of ovarian transposition in reproductive-aged cancer patients treated by primary or adjuvant pelvic irradiation.

AIM: To investigate the application status of ovarian transposition (OT) in reproductive-aged cancer patients undergoing radiation therapy. MATERIAL & METHODS: Between November 1999 and December 2008, 2524 patients had received pelvic irradiation at Seoul National University Hospital. We filtered the patients with the indications of (i) within 12 to 40 years of age, and (ii) receiving primary or adjuvant pelvic irradiation. There were 241 patients within 12 to 40 years of age. After excluding 133 patients with metastatic disease or under palliative radiation treatment, 108 patients were discovered appropriate for OT. We analyzed the application status of OT, surgical types of OT, cancer types and radiation types in those 108 patients. RESULTS: Cervical cancer was the major indication (n = 68, 62.9%). Another 37.1% of indicated disease were composed of rectal cancer (n = 19), vulvo-vaginal cancer (n = 4), non-Hodgkin's lymphoma (n = 3), and other pelvic tumors (n = 14). Among the 108 patients, only 31 (28.7%) patients had received OT before pelvic irradiation. Most of the operations were applied on cervical cancer patients (n = 29) and only two procedures on rectal and endometrial cancer, respectively. OT had been mostly performed during laparotomy. Laparoscopic procedure was applied in only one case with advanced cervical cancer. CONCLUSIONS: Although OT could be a preventive measure of premature ovarian failure from radiation therapy, this procedure has been considerably underused at our institution. This procedure should be applied more widely to preserve the fertility and improve the quality of life in reproductive-aged cancer patients.

Study of the process-induced cell damage in forced extrusion bioprinting.

With remarkable developments in technologies, the possibility of replacing injured tissue or organs with artificial ones via three-dimensional bioprinting is being improved. The basic prerequisite for successful application of bioprinting is high cell survival following printing. In this study, numerical calculations and experiments were performed to understand cell damage process incurred by forced extrusion bioprinters. Compressible and shear stresses were presumed to play a pivotal role within the syringe and needle, respectively, based on numerical calculation. To verify the numerical results, two experiments-pressurization in a clogged syringe and extrusion through syringe-needle-were conducted, and the damaged cell ratio (DCR) were measured by live/dead assays. Shear stress of needle flow had a great influence on DCR of discharged bioink, whereas effect of compressible stress in clogged syringe was relatively small. Cell damage in the needle flow is affected by moving distance under load as well as magnitude of shear stress. Applying this concept the differential equation of DCR growing was established, similar to the historied logistic equation for population dynamics, and the mathematical formula to predict DCR was explicitly represented splendidly as a function of only one independent variable, pressure work. The proposed formula was able to effectively predict DCR measurements for 43 bioprinting conditions, and the exactness confirmed the hypothesis for the theory. The presence of safe core zone, which may be related to the critical shear stress and stressed duration on cells, was theoretically conjectured from the DCR measurements, and further studies are necessary for an extensive and profound understanding. Fast printing is required for efficiency of a bio-structure fabrication; however, the higher shear stress accompanying increased operating pressure to speed up bioink discharge rate causes more cell damage. Employing the accurate formula presented, the optimal bioprinting conditions can be designed with ensuring targeted cell viability.

Bioorthogonally surface-edited extracellular vesicles based on metabolic glycoengineering for CD44-mediated targeting of inflammatory diseases.

Extracellular vesicles (EVs) are essential mediators in intercellular communication that have emerged as natural therapeutic nanomedicines for the treatment of intractable diseases. Their therapeutic applications, however, have been limited by unpredictable in vivo biodistribution after systemic administration. To control the in vivo fate of EVs, their surfaces should be properly edited, depending on the target site of action. Herein, based on bioorthogonal copper-free click chemistry (BCC), surface-edited EVs were prepared by using metabolically glycoengineered cells. First, the exogenous azide group was generated on the cellular surface through metabolic glycoengineering (MGE) using the precursor. Next, PEGylated hyaluronic acid, capable of binding specifically to the CD44-expressing cells, was labelled as the representative targeting moiety onto the cell surface by BCC. The surface-edited EVs effectively accumulated into the target tissues of the animal models with rheumatoid arthritis and tumour, primarily owing to prolonged circulation in the bloodstream and the active targeting mechanism. Overall, these results suggest that BCC combined with MGE is highly useful as a simple and safe approach for the surface modification of EVs to modulate their in vivo fate.

Evaluation of preoperative criteria used to predict lymph node metastasis in endometrial cancer.

OBJECTIVE: To evaluate whether we could accurately predict lymph node (LN) metastasis with preoperative tests in endometrial cancer. Design. Retrospective study. SETTING: Seoul National University Hospital, South Korea. Population. Three hundred patients with endometrial cancer who underwent surgical staging including lymphadenectomy between January 1999 and July 2007. METHODS: We reviewed the medical records of 300 patients with endometrial cancer. The preoperative factors used to predict LN metastasis were as follows: old age (> or = 55 years), serum CA-125 level [level > or = 20 U/mL (if age < 50 years), level > or = 28 U/mL (if age > or = 50 years)], non-endometrioid histologic type and Grade 3, metastatic LN assessed by pelvic MRI or CT, and deep myometrial invasion assessed by pelvic MRI only. Logistic regression analysis was used to determine the significant predictive factors. MAIN OUTCOME MEASURES: Sensitivity/specificity and false positive/negative rates. RESULTS: Thirty patients had LN metastasis. Although LN evaluation by pelvic MRI or CT and high CA-125 level were the significant independent predictors for LN metastasis, the sensitivity/specificity and false positive/negative rates for LN metastasis by these two combined preoperative tests were 86.7%/71.4% and 68.7%/2.7%, respectively. However, the sensitivity/specificity and false positive/negative rates for LN metastasis by six combined preoperative tests were 100%/28.9% and 84.6%/0%, respectively. CONCLUSIONS: The six combined preoperative tests are useful in selecting patients without LN metastasis in endometrial cancer. Lymphadenectomy could be avoided in about 29% of patients with endometrial cancer who have no LN metastasis by using six combined preoperative tests.

Selection and functional identification of a synthetic partial ABA agonist, S7.

The stress hormone abscisic acid (ABA) helps plants to survive under abiotic stresses; however, its use as an agrochemical is limited by its chemical instability and expense. Here, we report the development of an in vivo screening system to isolate chemicals able to induce ABA signalling responses in rice (Oryza sativa) protoplasts. This system consists of an ABA-hypersensitive synthetic promoter containing ABRE and DRE motifs driving a luciferase reporter gene. After efficiently transfecting rice protoplasts with this construct, we screened chemicals library with a similar molecular weight and chemical structure to ABA and identified one chemical, S7, that induced ABA signalling by mediating interactions between the group I and II OsPYL receptors and certain OsPP2CAs in a yeast two-hybrid assay. In an in vitro pulldown assay, S7 was found to mediate a weak interaction between OsPYL5/8 and various OsPP2CAs. S7 treatments did not affect seedling growth or seed germination, but could reduce water loss. Rice seedlings treated with S7 exhibited transcriptome profiles that partially overlapped those treated with ABA. Taken together, we concluded that S7 is a new partial ABA agonist, which has potential use in future dissections of ABA signalling and as an agrochemical.

The effect of visceral fat on the hemodilution effect of serum carcinoembryonic antigen in Korean population.

OBJECTIVE: To investigate the relationship between visceral fat and the hemodilution effect of carcinoembryonic antigen in both sexes. METHODS: A total of 15,340 females and 20,024 males who visited the health promotion center at Chung-Ang University Hospital from 2011 to 2014 were retrospectively collected. Correlation analysis and chi-square test for linear by linear association were used to determine the correlation between carcinoembryonic antigen concentration, carcinoembryonic antigen mass and visceral fat. Multivariable linear regression analysis was used to calculate the mean of carcinoembryonic antigen concentration and the mean of carcinoembryonic antigen mass, reflecting age, aspartate aminotransferase, alanine aminotransferase, creatinine, body fat percentage, body mass index, lean body mass and waist circumference as confounding variables. RESULTS: Higher body mass index was related with lower carcinoembryonic antigen concentration in men (r = -0.019, P = 0.019), but higher carcinoembryonic antigen concentration in women (r = 0.084, P<0.001). Average of waist circumference for male is greater than that of female (P<0.01). Average of body fat percentage for male is lesser than that of female (P<0.01). Male lean body mass mean is larger than that of women (P<0.01). Increased waist circumference was significantly associated with higher carcinoembryonic antigen mass in both female and male (P<0.001 for trend). Postmenopausal women might be more likely to have increased carcinoembryonic antigen mass and carcinoembryonic antigen concentration (P<0.001 for trend). CONCLUSIONS: This study suggests that visceral fat may increase total amount of CEA in the body. Visceral fat should be taken into account when evaluating serum CEA levels in both sexes.

Two Clade A Phosphatase 2Cs Expressed in Guard Cells Physically Interact With Abscisic Acid Signaling Components to Induce Stomatal Closure in Rice.

BACKGROUND: The core ABA signaling components functioning in stomatal closure/opening, namely ABA receptors, phosphatases, SnRK2s and SLAC1, are well characterized in Arabidopsis, but their functions in guard cells of rice have not been extensively studied. RESULTS: In this study, we confirmed that OsSLAC1, the rice homolog of AtSLAC1, is specifically expressed in rice guard cells. Among the rice SAPKs, SAPK10 was specifically expressed in guard cells. In addition, SAPK10 phosphorylated OsSLAC1 in vitro and transgenic rice overexpressing SAPK10 or OsSLAC1 showed significantly less water loss than control. Thus, those might be major positive signaling components to close stomata in rice. We identified that only OsPP2C50 and OsPP2C53 among 9 OsPP2CAs might be related with stomatal closure/opening signaling based on guard cell specific expression and subcellular localization. Transgenic rice overexpressing OsPP2C50 and OsPP2C53 showed significantly higher water loss than control. We also characterized the interaction networks between OsPP2C50 and OsPP2C53, SAPK10 and OsSLAC1 and found two interaction pathways among those signaling components: a hierarchical interaction pathway that consisted of OsPP2C50 and OsPP2C53, SAPK10 and OsSLAC1; and a branched interaction pathway wherein OsPP2C50 and OsPP2C53 interacted directly with OsSLAC1. CONCLUSION: OsPP2C50 and OsPP2C53 is major negative regulators of ABA signaling regarding stomata closing in rice. Those can regulate the OsSLAC1 directly or indirectly thorough SAPK10.

Predictive value of individualized tumor response testing by ATP-based chemotherapy response assay in ovarian cancer.

The aim of this study is to investigate the correlation between ATP-based chemotherapy response assay (ATP-CRA) results and clinical outcomes in ovarian cancer. Twenty-nine fresh tumor specimens were collected. Tumor cells were isolated and cultured for 48 hrs in medium containing anticancer drugs. The median age of patients was 56 years. The sensitivity, positive predictive value, and accuracy of ATP-CRA were respectively 94.1%, 94.1%, and 90.0%. There was a significant relationship between ATP-CRA results and clinical responses (p = 0.046). This study suggests that ATP-CRA has high sensitivity, positive predictive value, and accuracy for predicting response to chemotherapy in ovarian cancer.