Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes.

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on the distance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.

AGAMOUS-LIKE13, a putative ancestor for the E functional genes, specifies male and female gametophyte morphogenesis.

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant-negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet-like complexes (AGL13-AG)2 and interact with AG-AP3-PI to form a higher-order heterotetrameric complex (AGL13-AG-AP3-PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.

The C-Terminal Sequence and PI motif of the Orchid (Oncidium Gower Ramsey) PISTILLATA (PI) Ortholog Determine its Ability to Bind AP3 Orthologs and Enter the Nucleus to Regulate Downstream Genes Controlling Petal and Stamen Formation.

This study focused on the investigation of the effects of the PI motif and C-terminus of the Oncidium Gower Ramsey MADS box gene 8 (OMADS8), a PISTILLATA (PI) ortholog, on floral organ formation. 35S::OMADS8 completely rescued and 35S::OMADS8-PI (with the PI motif deleted) partially rescued petal/stamen formation, whereas these deficiencies were not rescued by 35S::OMADS8-C (C-terminal 29 amino acids deleted) in pi-1 mutants. OMADS8 could interact with Arabidopsis APETALA3 (AP3) and enter the nucleus. The nuclear entry efficiency was reduced for OMADS8-PI/AP3 and OMADS8-C/AP3. OMADS8 could also interact with OMADS5/OMADS9 (the Oncidium AP3 ortholog) and enter the nucleus with an efficiency only slightly affected by the deletion of the C-terminal sequence or PI motif. However, the stability of the OMADS8/OMADS5 and OMADS8/OMADS9 complexes was significantly reduced by deletion of the C-terminal sequence or PI motif. Further analysis indicated that the expression of genes downstream of AP3/PI (BNQ1/BNQ2/GNC/At4g30270) was compensated by 35S::OMADS8 and 35S::OMADS8-PI to a level similar to wild-type plants but was not affected by 35S::OMADS8-C in the pi-1 mutants. A similar FRET (fluorescence resonance energy transfer) efficiency was observed for Arabidopsis AGAMOUS (AG) and the Oncidium AG ortholog OMADS4 for OMADS8, OMADS8-PI and OMADS8-C. These results indicated that the OMADS8 PI motif and C-terminus were valuable for the interaction of OMADS8 with the AP3 orthologs to form higher order heterotetrameric complexes that regulated petal/stamen formation in both Oncidium orchids and transgenic Arabidopsis. However, the C-terminal sequence and PI motif were dispensable for the interaction of OMADS8 with the AG orthologs.

Confirmed False Positive Proteinuria in Patients with Systemic Lupus Erythematosus Taking Hydroxychloroquine: a Spot Sample Measurement.

BACKGROUND: A false-positive screening result is associated with harmful treatment or follow-up costs. This study aimed to estimate the rate of false positive proteinuria with the dipstick in patients with systemic lupus erythematosus (SLE) taking hydroxychloroquine. METHODS: A total of 334 patients with a positive dipstick and confirmed by total urine protein with quantification assay were enrolled. The experimental group included those with SLE taking hydroxychloroquine, and the rest was the control group. The difference of the rate of false positive in the dipstick was analyzed using the chi-square test and odds ratio (OR) between groups. Qualitative tracking of potential interference in the dipstick was performed. RESULTS: The results revealed that the rate of false positive with a dipstick for the experimental and control groups were 29.5% and 5.0% (p = 0.000), respectively. The OR with 95% confidence interval (CI) of the rate of false positive for the experimental group with respect to the control group was 5.95 (95% CI: 2.80 - 12.65). Qualitative tracking showed that the dipstick was influenced to become false-positive when hydroxychloroquine concentration was ≥ 30 mg/dL. CONCLUSIONS: Hydroxychloroquines like plaquenil or geniquin may lead to a high rate of false positive with the dipstick method. A quantification assay is recommended for proteinuria measurement in patients with SLE taking hydroxychloroquines.

Evaluating the performance of urine conductivity as screening for early stage chronic kidney disease.

BACKGROUND: This study aimed to determine if urine conductivity (Cond) is better for screening early stage chronic kidney disease (CKD) instead of the currently routinely used parameters of urine creatinine (UCr), urine osmolality (Osmo), urine specific gravity (SpGr), and urine protein (UP). METHODS: One hundred and forty participants (86 male, 54 female) with eGFR > 60 were grouped as either early stage CKD (kidney damage longer than 3 months with either structural or functional abnormalities [n = 72]) or the control group (without CKD and without kidney damage or functional abnormalities [n = 681]). Sensitivty (Sn) and specificity (Sp) of UP and the ROC curves were calculated. The area under the curve (AUC) with 95% confidence interval (CI) was used to compare Cond, UCr, Osmo, and SpGr. Pearson's correlation was used to analyze the correlation between Cond and UCr, Osmo, and SpGr in the early stage CKD group. RESULTS: The Sn and Sp of UP were 22.2% and 92.6%, respectively. By ROC analysis, Cond had the largest AUC (0.752, 95% CI: 0.672-0.832), with 52.9% Sn and 86.1% Sp. Pearson's correlation showed that the coefficient (p < 0.01) of Cond to UCr, Osmo, and SpG were 0.696, 0.907, and 0.820, respectively. CONCLUSIONS: Cond has better screening ability than UP for early stage CKD and may be a potential surrogate parameter for Osmo, SpGr and UCr.

Factors Associated with Postoperative Lipiduria and Hypoxemia in Patients Undergoing Surgery for Orthopedic Fractures.

We investigated factors associated with postoperative lipiduria and hypoxemia in patients undergoing surgery for orthopedic fractures. We enrolled patients who presented to our emergency department due to traumatic fractures between 2016 and 2017. We collected urine samples within 24 h after the patients had undergone surgery to determine the presence of lipiduria. Hypoxemia was defined as an SpO2 <95% determined with a pulse oximeter during the hospitalization. Patients' anthropometric data, medical history, and laboratory test results were collected from the electronic medical record. Logistic regression analyses were used to determine the associations of clinical factors with postoperative lipiduria and hypoxemia with multivariate adjustments. A total of 144 patients were analyzed (mean age 51.3 ± 22.9 years, male 50.7%). Diabetes (odd ratio 3.684, 95% CI, 1.256-10.810, p = 0.018) and operation time (odd ratio 1.005, 95% CI, 1.000-1.009, p = 0.029) were independently associated with postoperative lipiduria, while age (odd ratio 1.034, 95% CI, 1.003-1.066, p = 0.029), body mass index (odd ratio 1.100, 95% CI, 1.007-1.203, p = 0.035), and operation time (odd ratio 1.005, 95% CI, 1.000-1.010, p = 0.033) were independently associated with postoperative hypoxemia. We identified several factors independently associated with postoperative lipiduria and hypoxemia in patients with fracture undergoing surgical intervention. Operation time was associated with both postoperative lipiduria and hypoxemia, and we recommend that patients with prolonged operation for fractures should be carefully monitored for clinical signs related to fat embolism syndrome.

Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid.

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.

Effects of Water Extract of Cynanchum paniculatum (Bge.) Kitag. on Different Breast Cancer Cell Lines.

Cynanchum paniculatum (Bge.) Kitag. (CP) is an important medicinal herb used in Chinese herbal medicine, with a variety of biological activities including anticancer property. In this study, we explored the water extract of CP, for its anticancer effects against breast cancer cells with different mutation types. Cells were grouped as untreated (Control); CP direct treatment (dir-CP); Conditioned medium from CP treated (sup-CP), and untreated cells (sup-Control). Effects of dir-CP and sup-CP were compared to corresponding untreated cells on cytotoxicity, cell migration, and protein expression (cleaved caspase-3, caspase-9, and MMP-2 and 9). CP treatment showed time-dependent decrease in cell number of MDA-MB-231 and SK-Br-3 (both ER(-) PR(-)), while the decrease in cell number was not as significant in MCF-7 and ZR-75-1 cells (both ER(+) PR(+)). sup-CP treatment inhibited the cell migration of MDA-MB-231 and MCF-7 (Her2(-)) in a 24 h scratch assay. Our data suggested that ER(-) PR(-) cells are more sensitive to the CP in terms of direct cytotoxicity, which is not regulated by caspase-3. CP inhibited the migration of the two Her2(-) cells, and this correlated with MMP-2 regulation. The migration of ER(-) PR(-) cells was more sensitive to conditioned medium with CP treatment than to direct CP, and this is not regulated by MMP-2. Our data suggested that CP has anticancer potential on various breast cancer cells through different mechanisms and is specifically effective in inhibiting the migration of the triple negative MDA-MB-231. Our data provide insight into the mechanism of CP against breast cancer progression and would benefit the medical practitioners in better management with CP usage.

Processing Cycle Efficiency to Monitor the Performance of an Intelligent Tube Preparation System for Phlebotomy Services.

BACKGROUND: The waiting time (WT) for a phlebotomy is directly related to patient satisfaction with a health service. However, the processing time varies widely depending on the type of patients. Monitoring of the WT alone may not enable an effective evaluation of the lean performance of the medical staff for patients with different characteristics. The objective of this study was to use process cycle efficiency (PCE) to assess the performance of an intelligent tube preparation system (ITPS) which automatically labeled test tubes and conducted patient rerouting for phlebotomy services, and to interpret the WT during peak hours. METHODS: Three time periods were used. The baseline period was from 1 July to 31 July 2014. Phase 1 was after the establishment of the ITPS, with patients ≥80 years old being rerouted. In phase 2, patients ≥78 years old were rerouted. Those data were recorded with a calling system and ITPS, respectively. RESULTS: PCE was significantly improved from 12.9% at baseline to 51.1% (p < 0.001) in phase 1 and 53.0% (p < 0.001) in phase 2. The WT of 16.9 min at baseline was reduced to 3.8 min in phase 1 (p < 0.001), and 3.6 min in phase 2 (p < 0.001). Moreover, the results showed that a WT < 10 min was consistent with a PCE ≥ 25%. CONCLUSIONS: Establishing an ITPS for phlebotomy can significantly increase PCE and shorten the WT. Furthermore, the PCE ≥ 25% could be a good assessment reference for the management of appropriate human resources for phlebotomy services, although it is a complex parameter.

Kaempferitrin-Treated HepG2 Differentially Expressed Exosomal Markers and Affect Extracellular Vesicle Sizes in the Secretome.

Kaempferitrin is extracted in significantly high quantities from the leaves of Cinnamomum osmophloeum, which belongs to a group of plant species that comes under the genus Cinnamomum, well-known for its established anti-diabetic property in Chinese medicine. Oral administration of kaempferitrin and Cinnamomum osmophloeum extract reduced blood sugar in alloxan-induced diabetic rats and improved the lipid profile in hamsters respectively. In this paper we studied the differential protein expression profile using mass spectrometry approach in the kaempferitrin-treated conditioned medium of liver cancer cell line HepG2. We discovered that 33 genes were up/down-regulated consistently between two biological samples. A slightly different version of the analysis software selected 28 genes, and the final 18 genes that appeared in both lists were selected. Interestingly, 5 proteins out of 18 were either exosomal markers or reported in high frequency of occurrence in exosome/secreted vesicles. We also examined the extracellular particles with atomic force microscopy (AFM), which showed that the conditioned medium of kaempferitrin treated had larger vesicles and fewer small vesicles. Expression of some lipid-regulating genes were also altered. Our data suggested that extracellular vesicle secretions may be regulated by kaempferitrin, and regulation of lipid profile by kampeferitrin involves multiple mechanisms.