Inflammatory chemokine (C-C motif) ligand 8 inhibition ameliorates peritoneal fibrosis.

Peritoneal fibrosis (PF) is an irreversible complication of peritoneal dialysis (PD) that leads to loss of peritoneal membrane function. We investigated PD effluent and serum levels and the tissue expression of chemokine (C-C motif) ligand 8 (CCL8) in patients with PD. Additionally, we investigated their association with PF in a mouse model. Eighty-two end-stage renal disease (ESRD) patients with PD were examined. CCL8 levels were measured via enzyme-linked immunosorbent assays in PD effluents and serum and analyzed with peritoneal transport parameters. Human peritoneal mesothelial cells (hPMCs) were obtained from the PD effluents of 20 patients. Primary cultured hPMCs were treated with recombinant (r) transforming growth factor (TGF)-ß, and CCL8 expression was assessed via western blotting. As the duration of PD increased, the concentration of CCL8 in PD effluents significantly increased. Correlations between peritoneal transport parameters and dialysate CCL8 levels were observed. Western blotting analysis showed that CCL8 was upregulated via rTGF-ß treatment, accompanied by increases in markers of inflammation, fibrosis, senescence, and apoptosis in hPMCs after induction of fibrosis with rTGF-ß. Anti-CCL8 monoclonal antibody (mAb) treatment suppressed the rTGF-ß-induced increase in all analyzed markers. Immunohistochemical analysis revealed that CCL8 along with fibrosis- and inflammation-related markers were significantly increased in the PF mouse model. Functional blockade of CCL8 using a CCR8 inhibitor (R243) abrogated peritoneal inflammation and fibrosis in vivo. In conclusion, high CCL8 levels in PD effluents may be associated with an increased risk of PD failure, and the CCL8 pathway is associated with PF. CCL8 blockade can ameliorate peritoneal inflammation and fibrosis.

Determination of honey adulterated with corn syrup by quantitative amplification of maize residual DNA using ultra-rapid real-time PCR.

BACKGROUND: Honey is a naturally sweet syrup made by honeybees from floral nectar. However, high-fructose corn syrup has been prevalently used for the adulteration of honey. A novel molecular method was developed for the characterization of corn syrup-adulterated honey by specific amplification and quantification of maize residual DNA in honey. An ultra-rapid real-time polymerase chain reaction (UR-qPCR) system for rapid amplification and protocol for direct purification of residual DNA from honey were described. RESULTS: Rapidity of maize DNA amplification was acquired within 20 min for a limit of detection of around three copies of targeted DNAs. The amplification of maize residual DNA in honeys adulterated with corn syrup from 5% to 80% (v/v) showed that a minimum rate of 10% adulteration can be identified, and Maize genomic DNA in 5 mL of adulterated honeys was from 13 ± 9 copies to 2478 ± 827 copies, respectively. However, the residual DNA of maize was also detected in natural honey produced in the region where pollen and nectar of maize were collected, and the quantity of maize genomic DNA in these natural honeys was in the range of 10% adulteration with corn syrup. Therefore, detection of both pollen and residual DNA of maize in honey is important in identifying the source of maize residual DNA present in honey. CONCLUSION: A rapid PCR assay was first developed for the accurate detection and quantification of maize residual DNA in honey. It is a useful tool for specific identification of the corn syrup used for honey adulteration. Further studies on residual DNA in various types of corn syrup and specificity of primer are recommended. © 2021 Society of Chemical Industry.

Mineral trioxide aggregate-induced AMPK activation stimulates odontoblastic differentiation of human dental pulp cells.

AIM: To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). METHODOLOGY: In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. RESULTS: Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P < 0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P < 0.05) and DMP1 (P < 0.05) as well as formation of mineralized nodules (P < 0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P < 0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P < 0.01) and DMP1 (P < 0.05), but increased the phosphorylation of mTOR (P < 0.05), p70S6 (P < 0.01) and Unc-51-like kinases 1 (ULK1) (ser757) (P < 0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. CONCLUSION: MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.

Comparison of hemodialysis and peritoneal dialysis patients' dietary behaviors.

BACKGROUND: Nutritional factors are associated with high mortality and morbidity in dialysis patients, and protein-energy wasting is regarded as an important one. The modality of dialysis may affect patients' dietary behavior and nutritional status, but no study has compared the dietary behavior, nutrient intake, and nutritional adequacy of hemodialysis (HD) and peritoneal dialysis (PD) patients. METHODS: From December 2016 to May 2017, a dietary behavior survey and Semi-quantitative Food Frequency Questionnaire (Semi-FFQ) were conducted on 30 HD patients and 30 PD patients in Ewha Womans University Mokdong Hospital, and laboratory parameters were obtained. The results of prevalent HD and PD patients were then compared. RESULTS: The mean age of HD patients was higher than that of PD patients; HD: 58.5 ± 9.1 years, PD: 49.3 ± 9.7 years (p = 0.001). In the dietary behavior survey, HD patients showed more appropriate dietary behavior patterns overall than PD patients. In the dietary intake analysis with the Semi-FFQ, energy intake was significantly lower in the PD group than in the HD group due to the lower intake of carbohydrates, fat, and protein. A comparison of nutrient intake-to-recommended allowance ratio between the HD and PD groups revealed that the HD group showed higher nutrient intake than the PD group. Serum albumin and potassium levels were significantly higher in HD than in PD patients. CONCLUSION: According to this study, the dietary behavior and nutritional intake of prevalent PD patients were worse than those of HD patients.

The interactive effects of input and output on managing fluid balance in patients with acute kidney injury requiring continuous renal replacement therapy.

BACKGROUND: The interactive effect of cumulative input and output on achieving optimal fluid balance has not been well elucidated in patients with acute kidney injury (AKI) requiring continuous renal replacement therapy (CRRT). This study evaluated the interrelation of fluid components with mortality in patients with AKI requiring CRRT. METHODS: This is a retrospective observational study conducted with a total of 258 patients who were treated with CRRT due to AKI between 2016 and 2018 in the intensive care unit of Ewha Womans University Mokdong Hospital. The amounts of fluid input and output were assessed at 24-h and 72-h from the initiation of CRRT. The study endpoints were 7- and 28-day all-cause mortality. RESULTS: The mean patient age was 64.7 ± 15.8 years, and 165 (64.0%) patients were male. During the follow-up, 7- and 28-day mortalities were observed in 120 (46.5%) and 157 (60.9%) cases. The patients were stratified into two groups (28-day survivors vs. non-survivors), and the cumulative fluid balances (CFBs) at 24 h and 72 h were significantly higher in the 28-day non-survivors compared with the survivors. The increase in 24-h and 72-h CFB was significantly associated with an increase in 7- and 28-day mortality risks. To examine the interactive effect of cumulative input or output on the impact of CFB on mortality, we also stratified patients into three groups based on the tertile of 24-h and 72-h cumulative input or output. The increases in 24-h and 72-h CFBs were still significantly related to the increases in 7-day and 28-day mortality, irrespective of the cumulative input. However, we did not find significant associations between increase in 24-h and 72-h CFB and increase in mortality risk in the groups according to cumulative output tertile. CONCLUSIONS: The impact of cumulative fluid balance on mortality might be more dependent on cumulative output. The physicians need to decrease the cumulative fluid balance of CRRT patients as much as possible and consider increasing patient removal.

Synthesis and Characterization of Self-Polishing Polyurethane Copolymers.

Synthesizing copolymers from Zn acrylate monomers is an effective strategy employed in the self-polishing materials industry. In this study, we designed and synthesized polyurethane-based self-polishing copolymers with improved surface adhesion properties. The synthesized polyurethane copolymers were characterized using Fourier transform infrared spectroscopy and inductively coupled plasma optical emission spectrometry. The properties of Zn-based polyurethane copolymers were compared with those of Zn-free polyurethane as reference. The erosion rates of the Zn-based polyurethane SPC films were determined by measuring changes in the film thickness after dynamic immersion tests. In comparison to Zn-free polyurethane, the Zn-based polyurethane self-polishing copolymers demonstrated effective self-polishing and surface adhesion properties.

Synthesis of Novel Polyacrylates Containing Cyclotetrasiloxane for Fouling-Release Coating Applications.

An acrylate monomer containing cyclotetrasiloxane (CTS) were designed and synthesized for anti-fouling coating applications. New acryl-based copolymers consisting of styrene and CTS, poly(styrene-co-CTS)s, were synthesized by changing molar ratios via free radical polymerization. The properties of poly(styrene-co-CTS)s were compared with those of poly(styrene) (PS) as a reference. The content of CTS in the copolymer increased its hydrophobicity also decreased whereas its surface decreased. Protein adsorption studies were conducted to evaluate their fouling-release properties.

A Retrospective Study of Association between Peg-shaped Maxillary Lateral Incisors and Dental Anomalies.

OBJECTIVE: The purpose of this study was to investigate the prevalence of peg-shaped maxillary lateral incisors and the incidence of associated dental anomalies in children. STUDY DESIGN: We investigated the prevalence of peg-laterals and incidence of associated dental anomalies in 3,834 children aged 7-15 who visited the Department of Pediatric Dentistry from January 2010 to December 2015 and underwent panoramic radiographs. RESULTS: The prevalence of peg-laterals was 1.69% in boys, 1.75% in girls, and 1.72% overall. Among children with peg-laterals, the frequencies of associated dental anomalies were as follows: congenitally missing teeth, 31.8%; dens invaginatus, 19.7%; palatally displaced canines, 12.1%; supernumerary teeth, 7.6%; and transposition, 7.6%. CONCLUSION: As children with peg-laterals have a higher incidence of other dental anomalies, careful consideration is needed when planning diagnosis and treatment.

Anti-Biofouling Effect of PEG-Grafted Block Copolymer Synthesized by RAFT Polymerization.

Poly(glycidyl methadrylate-block-styrene) (PGMA-b-PS), a block copolymer consisting of glycidyl methacrylate and styrene, was synthesized via reversible addition-fragmentation chain transfer living polymerization. The synthesized PGMA-b-PS was then grafted with low-molecular-weight polyethylene glycol (PEG) via epoxy ring opening to give PGMA-g-PEG-b-PS, which was evaluated as an anti-biofouling coating material. As a preliminary test for the anti-biofouling effect, a protein adsorption experiment was performed on the synthesized block copolymer surface. The block copolymers were spin-coated onto silicon wafers, and protein adsorption experiments were carried out using fluorescein isothiocyanate conjugate-labeled bovine serum albumin. The fluorescence intensity of the protein adsorbed on the block copolymer surface was compared with that of a polystyrene film as a reference. The synthesized PGMA-g-PEG-b-PS film showed much lower fluorescence intensity than that of the PS film.

Characteristics of patients and facility of peritoneal dialysis in Korea: Results from the Peritoneal Dialysis Outcomes and Practice Patterns Study (PDOPPS) Korea.

BACKGROUND: Varying peritoneal dialysis (PD)-related clinical outcomes have been reported in different countries. As a participant of the Peritoneal Dialysis Outcomes and Practice Patterns Study (PDOPPS), this study investigated the characteristics of Korean PD patients, PD facilities and the incidence rates of clinical outcomes including mortality and PD-related outcomes. METHODS: From July 2019 to December 2021, a total of 766 Korean PD patients were included for analysis. Poisson regression analysis was used to explore the incidence rates of various clinical events including mortality, modality transfer, exit site or catheter tunnel infection and peritonitis. RESULTS: Among the 766 patients (median age 55.5 years, males 59.5%), 276 were incident and 490 were prevalent PD patients. The incidence rates of events were as follows: all-cause mortality (0.048), modality transfer (0.051), exit site or catheter tunnel infection (0.054) and peritonitis (0.136) events per person year. The most common causative organism for exit site or tunnel infection was staphylococcus species (47%) and that for peritonitis was streptococcus (28%) followed by staphylococcus (27%) species. CONCLUSIONS: Up to now, PDOPPS Korea has recruited 766 Korean PD patients and started documentation of major PD-related outcomes which occurred during the follow-up period. The overall incidence rates of clinical outcomes in Korean PD patients were relatively favourable. There was no statistically significant difference in the incidence rates of clinical outcomes according to both facility and patient factors.

Association of Chronic Kidney Disease With Atrial Fibrillation in the General Adult Population: A Nationwide Population-Based Study.

Background The incidences of atrial fibrillation (AF) and chronic kidney disease (CKD) are increasing, and AF is prevalent in patients with CKD. However, few studies have investigated the incidence or association of AF in a large CKD population from a longitudinal study. Methods and Results From a nationwide cohort, a total of 4 827 987 Korean individuals without prior AF, who received biennial health checkups provided by the National Health Insurance Service between 2009 and 2012 in Korea, were analyzed. Incidence of AF was ascertained through the end of 2018. During a median follow-up of 8.1 years, the annual incidence rate of AF was 1.17 per 1000 person-years among subjects without CKD, 1.55 for stage 1 CKD, 1.86 for stage 2 CKD, 2.1 for stage 3 CKD, and 4.33 for stage 4 CKD. In Fine-Gray regression models, CKD was associated with an increased risk of AF; the adjusted hazard ratios and 95% CIs of AF occurrence were 1.77 (1.69-1.85), 1.85 (1.80-1.91), 1.99 (1.95-2.04), and 4.04 (3.07-5.33) in individuals with CKD stages 1, 2, 3, and 4, respectively, compared with non-CKD. The association between CKD and incident AF remained statistically significant after adjustment for multiple confounding factors and was consistent across subgroups stratified by sex and age. Conclusions CKD is associated with an increased incidence of AF. Even mild CKD is associated with incident AF, and there was a stepwise increase in the risk of incident AF with a decrease in renal function.

Measured sodium excretion is associated with cardiovascular outcomes in non-dialysis CKD patients: results from the KNOW-CKD study.

Background: There are insufficient studies on the effect of dietary salt intake on cardiovascular (CV) outcomes in chronic kidney disease (CKD) patients, and there is no consensus on the sodium (Na) intake level that increases the risk of CV disease in CKD patients. Therefore, we investigated the association between dietary salt intake and CV outcomes in CKD patients. Methods: In the Korean cohort study for Outcome in patients with CKD (KNOW-CKD), 1,937 patients were eligible for the study, and their dietary Na intake was estimated using measured 24h urinary Na excretion. The primary outcome was a composite of CV events and/or all-cause death. The secondary outcome was a major adverse cardiac event (MACE). Results: Among 1,937 subjects, there were 205 (10.5%) events for the composite outcome and 110 (5.6%) events for MACE. Compared to the reference group (urinary Na excretion< 2.0g/day), the group with the highest measured 24h urinary Na excretion (urinary Na excretion ≥ 8.0g/day) was associated with increased risk of both the composite outcome (hazard ratio 3.29 [95% confidence interval 1.00-10.81]; P = 0.049) and MACE (hazard ratio 6.28 [95% confidence interval 1.45-27.20]; P = 0.013) in a cause-specific hazard model. Subgroup analysis also showed a pronounced association between dietary salt intake and the composite outcome in subgroups of patients with abdominal obesity, female, lower estimated glomerular filtration rate (< 60 ml/min per 1.73m2), no overt proteinuria, or a lower urinary potassium-to-creatinine ratio (< 46 mmol/g). Conclusion: A high-salt diet is associated with CV outcomes in non-dialysis CKD patients.

Aminoindanol-based chiral derivatizing agents for the determination of the absolute configuration of carboxylic acids.

New chiral derivatizing agents have been prepared through a simple, short-step synthesis. The absolute configuration of α-chiral carboxylic acids can be assigned on the basis of the NMR chemical shift difference between diastereomeric esters. Because of the modular structures of the agents, the anisotropic effect could be easily manipulated to afford large chemical shift differences even in polar solvents.

Left Ventricular Diastolic Dysfunction and Progression of Chronic Kidney Disease: Analysis of KNOW-CKD Data.

Background Few studies have examined the association between the early diastolic mitral inflow velocity/early diastolic mitral annulus velocity ratio (E/e') and chronic kidney disease progression. Methods and Results We reviewed data from 2238 patients with nondialysis chronic kidney disease from the KNOW-CKD (Korean Cohort Study for Outcome in Patients With Chronic Kidney Disease); data from 163 patients were excluded because of missing content. A >50% decrease in estimated glomerular filtration rate from baseline, doubling of serum creatinine, or dialysis initiation and/or kidney transplantation were considered renal events. At baseline, median (interquartile range) ejection fraction and E/e' were 64.0% (60.0%-68.0%) and 9.1 (7.4-11.9), respectively. Proportions of ejection fraction <50% and E/e' ≥15 were 1.3% and 9.6%, respectively. More than one quarter of patients (27.2%) had an estimated glomerular filtration rate <30 mL/min per 1.73 m2. During the mean 59.1-month follow-up period, 724 patients (34.9%) experienced renal events. In multivariable Cox proportional hazard regression analysis, the hazard ratio with 95% CI per 1-unit increase in E/e' was 1.027 (1.005-1.050; P=0.016). Penalized spline curve analysis yielded a suggested threshold of E/e' for renal events of 12; in our data set, the proportion of E/e' ≥12 was 4.1%. Conclusions Increased E/e' was associated with an increased hazard of renal events, suggesting that diastolic heart dysfunction is a novel risk factor for chronic kidney disease progression.

Low Klotho/Fibroblast Growth Factor 23 Ratio Is an Independent Risk Factor for Renal Progression in Chronic Kidney Disease: Finding From KNOW-CKD.

Background: We aimed to evaluate soluble Klotho and circulating fibroblast growth factor 23 (FGF23) ratio as a risk factor for renal progression, cardiovascular (CV) events, and mortality in chronic kidney disease (CKD). Methods: We analyzed 2,099 subjects from a CKD cohort whose soluble Klotho and C-terminal FGF23 levels were measured at enrollment. The Klotho to FGF23 ratio was calculated as Klotho values divided by FGF23 values + 1 (hereinafter called the Klotho/FGF23 ratio). Participants were categorized into quartiles according to Klotho/FGF23 ratio. The primary outcome was renal events, defined as the doubling of serum creatinine, 50% reduction of estimated glomerular filtration rate from the baseline values, or development of end-stage kidney disease. The secondary outcomes consisted of CV events and death. Changes in CV parameters at the time of enrollment and during follow-up according to the Klotho/FGF23 ratio were also examined. Results: During the follow-up period of 64.0 ± 28.2 months, 735 (35.1%) and 273 (13.0%) subjects developed renal events and composite outcomes of CV events and death, respectively. After adjustment, the first (HR: 1.36; 95% CI: 1.08-1.72, P = 0.010) and second (HR: 1.45; 95% CI: 1.15-1.83, P = 0.002) quartiles with regard to the Klotho/FGF23 ratio showed elevated risk of renal events as compared to the fourth quartile group. There was no significant association between Klotho/FGF23 ratio and the composite outcome of CV events and death. The prevalence of left ventricular hypertrophy and vascular calcification was higher in the low Klotho/FGF23 ratio quartiles at baseline and at the fourth-year follow-up. Conclusions: Low Klotho/FGF23 ratio was significantly associated with increased renal events in the cohort of Korean predialysis CKD patients.

System of integrating biosignals during hemodialysis: the CONTINUAL (Continuous mOnitoriNg viTal sIgN dUring hemodiALysis) registry.

BACKGROUND: Appropriate monitoring of intradialytic biosignals is essential to minimize adverse outcomes because intradialytic hypotension and arrhythmia are associated with cardiovascular risk in hemodialysis patients. However, a continuous monitoring system for intradialytic biosignals has not yet been developed. METHODS: This study investigated a cloud system that hosted a prospective, open-source registry to monitor and collect intradialytic biosignals, which was named the CONTINUAL (Continuous mOnitoriNg viTal sIgN dUring hemodiALysis) registry. This registry was based on real-time multimodal data acquisition, such as blood pressure, heart rate, electrocardiogram, and photoplethysmogram results. RESULTS: We analyzed session information from this system for the initial 8 months, including data for some cases with hemodynamic complications such as intradialytic hypotension and arrhythmia. CONCLUSION: This biosignal registry provides valuable data that can be applied to conduct epidemiological surveys on hemodynamic complications during hemodialysis and develop artificial intelligence models that predict biosignal changes which can improve patient outcomes.

Associations among body composition parameters and quality of life in peritoneal dialysis patients.

Health-related quality of life (HRQOL) is an important issue among patients undergoing dialysis treatment. Peritoneal dialysis (PD) is associated with a number of adverse body composition changes. However, whether body composition is associated with HRQOL is uncertain. The purpose of this study was to analyze the effects of body composition on HRQOL in PD patients. We performed a cross-sectional observational study on the association between body composition and HRQOL in PD patients at a single center. Body composition was determined by multifrequency bioimpedance spectroscopy. HRQOL is summarized to three composite scores: kidney disease component summary (KDCS), physical component summary (PCS), and mental component summary (MCS). The relationships between HRQOL and the hydration index, lean tissue index (LTI), and fat tissue index (FTI) were analyzed by regression analysis. One hundred and ninety-seven PD patients were included in the present study. Patients with severe fluid overload showed a lower PCS. The hydration index and FTI showed statistically significant negative associations with PCS. In subgroup analysis, the associations between the hydration index and PCS remained robust after stratifying according to sex, age, and residual urine. Our results indicated that both the hydration index and FTI were negatively associated with HRQOL, especially PCS.

Optimal target blood pressure for major adverse cardiovascular and cerebrovascular events in hypertensive patients: a nationwide population-based study.

BACKGROUND: Generalizing an 'optimal' blood pressure (BP) level for individuals with hypertension remains controversial due to the implementation of different medical guidelines. This study investigated the association of BP with major adverse cardiovascular and cerebrovascular events (MACCE) and determined the optimal BP for patients with hypertension. METHOD: A total of 934 179 individuals who received antihypertensive medications were selected from the National Health Insurance Service Examination Database between 2003 and 2011 in Korea. Their BP was measured at the index date, which was the first health examination. The study outcomes were MACCE, including acute myocardial infarction, heart failure, stroke, and all-cause mortality. The participants were monitored until in December, 2017. The hazard ratios were calculated using Cox proportional hazard models. The cumulative incidence of MACCE for each BP group was estimated using the Kaplan-Meier method. RESULTS: A lower risk of MACCE was observed at a SBP of 120-129 mmHg and a DBP of 80-89 mmHg. The endpoint-specific incidence rates and hazard ratios for acute myocardial infarction, heart failure, stroke, and all-cause mortality were the lowest at a SBP of 120-129 mmHg and a DBP of 80-89 mmHg. CONCLUSION: Even though this observational study did not support inference of a causal relationship, a SBP of 120-129 mmHg and a DBP of 80-89 mmHg may be safely recommended considering the possibility of MACCE in Korean patients with hypertension. In addition, the target BP should be tailored individually according to age, sex, and comorbidities.

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein.

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8(AN015) or CSN5(null) mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR.

BACKGROUND: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. OBJECTIVES: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. METHODS: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. RESULTS: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. CONCLUSIONS: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.