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Aims: Cold temperatures are known to affect heart failure (HF) hospitalizations, but the dynamic status of multi-morbidity of HF was rarely incorporated. We investigated the relationship between temperature and new-onset HF by risk strata., Methods and Results: This nationwide cohort study analysed daily data on ambient temperature, the dynamic status of risk factors (age, diabetes, chronic obstructive pulmonary disease, coronary artery disease, chronic kidney disease, hypertension, myocardial infarction, and atrial fibrillation), and new-onset HF among the Taiwan population from 2012 to 2019. Poisson regression, Austin's algorithm, and classification and regression tree (CART) were used to determine risk strata and obtain the predicted HF rate. 148 708 patients developed new-onset HF over 152.52 million person-years. Three risk strata for HF were identified: Stratum 1 was predominantly those without any comorbidity (89.9%); Stratum 2 was those aged 60-69 with 2-3 comorbidities or aged 70+ with 1-2 comorbidities (9.0%), and Stratum 3 was those aged 70+ and had four or more comorbidity (1.1%). The HF incidence rates for these three strata were 25.54, 555.27, and 2315.52 per 100 000 person-years, respectively. The R2 of the Poisson regression with the three risk strata and the daily minimum temperature on the ln HF incidence rates was 77.99%. The risk of HF increased as temperatures decreased, and the slopes were 1.032, 1.040, and 1.034 for Strata 1-3, respectively. The rate ratios of HF at the winter median temperature of 17°C vs. the summer median temperature of 29°C were 1.45, 1.58, and 1.49 for Strata 1-3, respectively. Cross-validation reveals a good fit and predicted HF rates by ambient temperature for the three strata were provided., Conclusion: Cold temperatures are associated with an increased risk of new-onset HF. Stratum 2 (aged 60-69 with 2-3 comorbidities or aged 70+ with 1-2 comorbidities) are particularly susceptible to cold-related new-onset HF., Competing Interests: Conflict of interest: The remaining authors have nothing to disclose., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objective: This study aimed to assess the incidence and risk factors surrounding mental illnesses in patients diagnosed with systemic autoimmune rheumatic diseases (SARDs)., Methods: This retrospective cohort study used nationwide, population-based claim data taken from Taiwan's National Health Insurance Research Database (NHIRD) to identify patients certified as having a catastrophic illness for Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), Systemic sclerosis (SSc), Dermatomyositis (DM), Polymyositis (PM) or Sjogren's syndrome (SS) from the years 2002-2020. We furthermore calculated the incidence of mental illness in patients diagnosed with SARDs while exploring factors associated with the development of mental illness using multivariable Cox regression analysis shown as adjusted hazard ratios (HRs) with 95% confidence intervals (CIs)., Results: Among the 28 588 participants, the average age was 47.4 (SD 14.9) years, with most participants being female (76.4%). When compared with patients with rheumatoid arthritis, patients with SLE (HR: 1.20, 95% CI: 1.10-1.32), SS (HR: 1.29, 95% CI: 1.19-1.39), and DM (HR: 1.28, 95% CI: 1.04-1.32) showed a significantly increased risk of developing mental illness. Additionally, when compared with patients with rheumatoid arthritis, patients with SLE (HR: 1.32, 95% CI: 1.21-1.44), SSc (HR: 1.20, 95% CI: 1.02-1.41), SS (HR: 1.17, 95% CI: 1.08-1.26), DM (HR: 1.73, 95% CI: 1.44-2.07), and PM (HR: 1.64, 95% CI: 1.32-2.03) showed a significantly increased risk of antidepressant use., Conclusions: This population-based cohort study revealed that patients diagnosed with SLE, SS and DM had significantly higher risks of developing mental illness when compared with patients with RA., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Numerous cases of long coronavirus disease (long COVID) have been reported in patients with autoimmune rheumatic diseases (ARDs). Despite the reviews on clinical manifestations of long COVID in the general population, systematic reviews on ARD patients are scarce. Herein, we conducted a systematic review and meta-analysis on the prevalence and characteristics of long COVID in ARD patients., Methods: We searched the literature in PubMed and Embase as of 27 December 2022. Cohort, cross-sectional and case-control studies relevant to long COVID in ARD patients were collected. Stratification based on the severity of COVID infection and subtypes of rheumatic diseases [systemic autoimmune rheumatic disease (SARD) vs non-autoimmune rheumatic disease (NARD)] was also undertaken. A random-effects model was used in the meta-analysis., Results: A total of 15 relevant studies were identified from the literature. The prevalence of long COVID was 56% (95% CI 34, 76) in 2995 patients. Hospitalized COVID patients had a higher proportion of long COVID than non-hospitalized patients. The prevalence of long COVID was similar between SARD and NARD patients. In terms of symptoms, fatigue, arthralgia and pain were commonly reported in long COVID patients with ARDs., Conclusion: The characteristics of long COVID in ARD patients are generally similar to those in the general population despite a higher prevalence and a higher proportion of arthralgia and pain., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Functional MRI measures the blood-oxygen-level dependent signals, which provide an indirect measure of neural activity mediated by neurovascular responses. Cerebrovascular reactivity affects both task-induced and resting-state blood-oxygen-level dependent activity and may confound inter-individual effects, such as those related to aging and biological sex. We examined a large dataset containing breath-holding, checkerboard, and resting-state tasks. We used the breath-holding task to measure cerebrovascular reactivity, used the checkerboard task to obtain task-based activations, and quantified resting-state activity with amplitude of low-frequency fluctuations and regional homogeneity. We hypothesized that cerebrovascular reactivity would be correlated with blood-oxygen-level dependent measures and that accounting for these correlations would result in better estimates of age and sex effects. We found that cerebrovascular reactivity was correlated with checkerboard task activations in the visual cortex and with amplitude of low-frequency fluctuations and regional homogeneity in widespread fronto-parietal regions, as well as regions with large vessels. We also found significant age and sex effects in cerebrovascular reactivity, some of which overlapped with those observed in amplitude of low-frequency fluctuations and regional homogeneity. However, correcting for the effects of cerebrovascular reactivity had very limited influence on the estimates of age and sex. Our results highlight the limitations of accounting for cerebrovascular reactivity with the current breath-holding task., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Long-range dependence is a prevalent phenomenon in various biological systems that characterizes the long-memory effect of temporal fluctuations. While recent research suggests that functional magnetic resonance imaging signal has fractal property, it remains unknown about the multifractal long-range dependence pattern of resting-state functional magnetic resonance imaging signals. The current study adopted the multifractal detrended fluctuation analysis on highly sampled resting-state functional magnetic resonance imaging scans to investigate long-range dependence profile associated with the whole-brain voxels as specific functional networks. Our findings revealed the long-range dependence's multifractal properties. Moreover, long-term persistent fluctuations are found for all stations with stronger persistency in whole-brain regions. Subsets with large fluctuations contribute more to the multifractal spectrum in the whole brain. Additionally, we found that the preprocessing with band-pass filtering provided significantly higher reliability for estimating long-range dependence. Our validation analysis confirmed that the optimal pipeline of long-range dependence analysis should include band-pass filtering and removal of daily temporal dependence. Furthermore, multifractal long-range dependence characteristics in healthy control and schizophrenia are different significantly. This work has provided an analytical pipeline for the multifractal long-range dependence in the resting-state functional magnetic resonance imaging signal. The findings suggest differential long-memory effects in the intrinsic functional networks, which may offer a neural marker finding for understanding brain function and pathology., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

This study features an functional magnetic resonance imaging (fMRI) hyperscanning experiment from 2 sites, 305 km apart. The experiment contains 2 conditions: the dyad collaborated to win and then split the reward in the cooperation condition, whereas the winner took all the reward in the competition condition, thereby resulting in dynamic strategic interactions. To calculate the cerebral coherence in such jittered event-related fMRI tasks, we first iteratively estimated the feedback-related blood oxygenation level-dependent responses of each trial, using 8 finite impulse response functions (16 s) and then concatenated the beta volume series. With the right temporal-parietal junction (rTPJ) as the seed, the interpersonal connected brain areas were separately identified: the right superior temporal gyrus (rSTG) (cooperation) and the left precuneus (lPrecuneus) (competition), both peaking at the designated frequency bin (1/16 s = 0.0625 Hz), but not in permuted pairs. In addition, the extended coherence analyses on shorter and longer concatenated volumes verified that only in the optimal trial frequency did the rTPJ-rSTG and rTPJ-lPrecuneus couplings peak. In sum, our approach both showcases a flexible analysis method that widens the applicability of interpersonal coherence in the rapid event-related fMRI hyperscanning and reveals a context-based inter-brain coupling between interacting pairs during cooperation and during competition., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To determine the bidirectional relationship between macrophage activation syndrome (MAS) and SLE., Methods: Using the 1997-2013 Taiwan National Health Insurance Research Database, we identified patients with newly diagnosed SLE from 2001 to 2013 and selected individuals without SLE from a 1 million representative population. Propensity score (PS) matching was performed to balance incident SLE patients and individuals without SLE according to age, sex, comorbidities and medical utilization. The association between a history of MAS and SLE was studied using conditional logistic regression analysis shown as an adjusted odds ratio (aOR). The risk of MAS associated with SLE was analysed using Cox proportional regression analysis, shown as an adjusted hazard ratio (aHR), and we conducted a sensitivity analysis using various definitions of MAS., Results: We included 10 481 SLE patients and 20 962 PS-matched (1:2) non-SLE individuals. The correlation between a history of MAS and SLE did not reach statistical significance after adjustment for potential confounders [aOR 1.18 (95% CI, 0.80, 1.75)] in the age-/sex-matched populations. In the 1:2 PS-matched populations, the risk of MAS markedly increased in patients with SLE [aHR 7.18 (95% CI 4.97, 10.36)]. Other risk factors for MAS included female gender, age ≥65 years, low income, a history of inflammatory bowel disease and a history of MAS., Conclusion: This nationwide, population-based study revealed that a history of MAS was not significantly associated with SLE risk. However, the risk of MAS was markedly associated with SLE and a history of MAS., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To assess the association of severe pulmonary arterial hypertension (PAH) with particulate matter <2.5 μm (p.m.2.5) and clinical data in patients with systemic autoimmune rheumatic diseases (SARDs)., Methods: We used the 2003-2017 nationwide data in Taiwan to identify patients with SARDs, including systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, dermatomyositis/polymyositis and primary Sjögren's syndrome. We identified 479 cases with severe PAH and selected controls matched (1:4) for age, sex, and index year. We used conditional logistic regression analysis to determine factors associated with risks for severe PAH shown as odds ratios (ORs) with 95% confidence intervals (CIs)., Results: We found that severe PAH was highly associated with interstitial lung disease (OR, 8.57; 95% CI: 5.52, 13.32), congestive heart failure (OR, 7.62; 95% CI: 5.02, 11.55), valvular heart disease (OR, 3.34; 95% CI: 2.03, 5.50) and slightly associated with thyroid diseases (OR, 1.88; 95% CI: 1.18, 3.00), but not the level of exposure to p.m.2.5. Increased risk for PAH was found in patients receiving corticosteroid (prednisolone equivalent dosage, mg/day, OR, 1.03; 95% CI: 1.01, 1.05), biologics (OR, 2.18; 95% CI: 1.15, 4.12) as well as immunosuppressants, including ciclosporin (OR, 2.17; 95% CI: 1.31, 3.59), azathioprine (OR, 1.96; 95% CI: 1.48, 2.61), cyclophosphamide (OR, 2.01; 95% CI: 1.30, 3.11) and mycophenolate mofetil/mycophenolic acid (OR, 2.42; 95% CI: 1.37, 4.27), and those with the highest level of insured amount (reference, lowest level; OR, 0.53; 95% CI: 0.34, 0.83)., Conclusion: The population-based study identified risks for severe PAH in patients with SARDs, and these findings provide evidence for PAH risk stratification in patients with SARDs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Extracorporeal membrane oxygenation (ECMO) is used for life support in patients with liver cirrhosis (LC). However, there have been no studies evaluating the outcome of ECMO support in patients with LC., Methods: Using Taiwan's National Health Insurance (NHI) database, we retrospectively evaluated patients with LC who received veno-venous or veno-arterial ECMO between 1 January 2000 and 31 December 2013. The outcomes included ECMO-related complications, in-hospital mortality, all-cause mortality and long-term outcomes in patients with and without LC., Results: A total of 7003 patients who received ECMO, of whom 233 (3.3%) had LC, were eligible for analysis. The LC patients who received ECMO support had a significantly higher risk of in-hospital mortality than the non-LC group (76.4% vs 60.7%; odds ratio 1.97; 95% confidence interval 1.44-2.70). The LC group also had a higher risk of complications, including de novo dialysis and massive blood transfusion with >10 units of red blood cells. Patients ≥65 years of age, patients with respiratory disease, patients with hypoalbuminaemia and liver transplant patients had higher in-hospital and 1-year mortality. The mortality rates for patients with 2 or more risk factors were 90.3% and 95.8%, respectively. LC was associated with a higher incidence of all-cause mortality and liver-related outcomes during follow-up., Conclusions: Our findings raise questions regarding the utility of ECMO for LC patients, especially when >2 risk factors have been identified. ECMO support for LC patients should be used with caution and with careful patient selection., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background: This study investigated the predictors of subsequent cardiovascular events in stable post-myocardial infarction patients in Taiwan., Methods: A total of 11,183 patients were recruited who had survived one year post-myocardial infarction without subsequent events of recurrent myocardial infarction or stroke from the Taiwan National Health Insurance Research Database. Their composite cardiovascular event rates were identified., Results: The composite cardiovascular events rate in three year follow-up in the post-myocardial infarction population was 13.8%. Corresponding event rates were 5.8% recurrent myocardial infarction, 5.0% stroke, and 5.2% death. Independent factors associated with a higher risk of ischemic events or death included heart failure (hazard ratio (HR)=1.19), hypertension (HR=1.16), age (65-75 vs <65 years: HR=1.29; 75-85 vs <65 years: HR=1.50; >85 vs <65 years: HR=1.70), diabetes (HR=1.33), prior stroke (HR=1.24), chronic kidney disease (HR=1.4), atrial fibrillation (HR=1.27), and underutilization of guideline-based medication (HR=1.73). Composite risk for myocardial infarction, stroke and death increased progressively from 4.9% in patients with zero risk factor to 100.0% in patients with eight risk factors., Conclusions: For acute myocardial infarction patients surviving one year without subsequent events of recurrent myocardial infarction or stroke, the risk of cardiovascular events remained high. Eight predictors identified patients at increased risk for subsequent cardiovascular events within the next three years. These results suggest an unmet need, particularly in patients with additional risk factors.

New genes are of recent origin and only present in a subset of species in a phylogeny. Accumulated evidence suggests that new genes, like old genes that are conserved across species, can also take on important functions and be essential for the survival and reproductive success of organisms. Although there are detailed analyses of the mechanisms underlying new genes' gaining fertility functions, how new genes rapidly become essential for viability remains unclear. We focused on a young retro-duplicated gene (CG7804, which we named Cocoon) in Drosophila that originated between 4 and 10 Ma. We found that, unlike its evolutionarily conserved parental gene, Cocoon has evolved under positive selection and accumulated many amino acid differences at functional sites from the parental gene. Despite its young age, Cocoon is essential for the survival of Drosophila melanogaster at multiple developmental stages, including the critical embryonic stage, and its expression is essential in different tissues from those of its parental gene. Functional genomic analyses found that Cocoon acquired unique DNA-binding sites and has a contrasting effect on gene expression to that of its parental gene. Importantly, Cocoon binding predominantly locates at genes that have other essential functions and/or have multiple gene-gene interactions, suggesting that Cocoon acquired novel essential function to survival through forming interactions that have large impacts on the gene interaction network. Our study is an important step toward deciphering the evolutionary trajectory by which new genes functionally diverge from parental genes and become essential., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An efficient method was developed for the simultaneous determination of 10 industrial dyes (basic orange 2, basic orange 21, basic orange 22, acid orange II, auramine, basic rhodamine B and Sudan I-IV) in the foodstuffs using high-performance liquid chromatography coupled with diode array detector. Samples were extracted with acetonitrile and cleaned up on a solid phase extraction cartridge using HLB. The chromatographic separation was achieved on a C18 column using a mobile phase consisting of methanol and 10 mmol/L ammonium acetate with 0.1% formic acid by gradient elution. Good linearity (r ≥ 0.9993) was observed between 0.050 and 5.0 μg/mL. The limits of detection were in the range of 0.007-0.01 mg/kg, high recoveries (80.6-104%) and good reproducibility (1.1-5.7%) were obtained. Such method is simple, feasible and accurate, which can be applied to the quantification of 10 dyes in food samples., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Gout is characterized by recurrent attacks of arthritis with hyperuricaemia and urate crystal-induced inflammation. Although urate transporters are known as risk factors, the immunogenetics of gouty inflammation remains unclear. This study aimed to investigate the genetic association between immune/metabolism regulators and gout., Methods: We enrolled 448 gout patients and 943 population controls from Taiwan; all were Han Chinese. We screened association between gout and 22 variants of candidate genes, including NLRP3 , caspase 1, peroxisome proliferator-activated receptor-γ, proliferator-activated receptor-γ coactivator 1α ( PPARGC1A ) and 1β ( PPARGC1B ). The association was validated by replication and combined-sample analyses. Functional assays were performed by quantitative PCR, ELISA, siRNA knockdown and transfection using THP-1 cells, peripheral blood mononuclear cells and synovial cells from patients., Results: Gouty arthritis exhibited significant association with variants of peroxisome PPARGC1B , which included a missense single nucleotide polymorphism, rs45520937 [P = 6.66 × 10 -9 ; odds ratio (95% CI): 1.85 (1.51, 2.28)]. Expression of PPARGC1B and NLRP3 was induced in urate crystal-activated THP-1, peripheral blood mononuclear cells and synovial cells from gout patients in acute stage. siRNA knockdown of PPARGC1B upregulated NLRP3 in urate crystal-activated macrophages. Compared with the wild-type carriers, patients with the risk A allele of rs45520937 showed statistically increased NLRP3 (P = 0.044) and plasma IL-1β (P = 0.006). Transfection of PPARGC1B cDNA with rs45520937 A allele to macrophages significantly augmented the expression of NLRP3 and IL-1β., Conclusion: Genetic variants of PPARGC1B are significantly associated with gout, and a missense single nucleotide polymorphism, rs45520937, augments NLRP3 and IL-1β expression. These data suggest that variants of PPARGC1B , a regulator of metabolism and inflammation, contribute to the pathogenesis of gouty arthritis., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Objective: To evaluate the impact of adalimumab (ADA) dose-halving on therapeutic responses and drug levels, the differences in drug levels among patients with different therapeutic responses and the optimal baseline cut-off ADA levels for predicting persistent remission or low disease activity (LDA) at week 24 of dose-halving therapy in 64 RA patients who had already achieved LDA or remission at baseline., Methods: Anti-ADA antibody levels were determined by bridging ELISA, ADA levels were evaluated using sandwich ELISA and therapeutic responses were assessed by the 28-joint DAS change. The optimal cut-off drug levels for predicting persistent remission were determined by receiver operating characteristic curve analysis., Results: At baseline, 25 (39.1%) and 39 (60.9%) patients had achieved remission and LDA, respectively. After 24 week ADA dose-halving, persistent remission was observed in 23 patients, remission turned LDA in 2 patients, persistent LDA in 24 patients and disease flare in 15 (23.5%) patients. Three patients who developed anti-ADA antibodies at week 24 of dose-halving had very low drug levels and disease flare. Among 61 anti-ADA antibody-negative patients, ADA levels declined by half after 24 weeks of dose-halving (median 5.5 vs 2.6 μg/ml). Baseline ADA levels were significantly higher in patients with persistent remission (median 10.5 μg/ml) or LDA (4.5 μg/ml) than in those with disease flare (0.9 μg/ml), indicating associations of ADA levels with therapeutic responses. An ADA level above the cut-off value of 6.4 μg/ml might predict persistent remission after dose-halving with high sensitivity and specificity., Conclusion: ADA dose-halving is feasible for patients who have achieved remission and sufficient drug levels. Drug level monitoring may help clinicians optimize the dosing regimen and prevent overtreatment for patients receiving anti-TNF-α therapy., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: We investigated factors associated with discontinuation of anti-tumor necrosis factor (TNF) therapy in patients with ankylosing spondylitis (AS), who were anti-TNF-naïve and were given etanercept (ETN) or adalimumab (ADA)., Methods: This is a retrospective nationwide population-based cohort study. We identified 1401 anti-TNF-naïve patients with AS who initiated ETN (n = 441) or ADA (n = 960) and measured the duration of anti-TNF drug use. We recorded demographic and clinical data of all patients, and calculated adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) using Cox proportional hazard regression analyses., Results: Overall, the ADA and ETN groups had similar risk for drug discontinuation (HR: 0.83; 95% CI: 0.63-1.08). In each group, concomitant use of methotrexate (MTX) or a non-steroidal anti-inflammatory drug was associated with a lower risk of discontinuation. Subgroup analysis indicated that concomitant MTX use reduced risk of discontinuation of ADA (HR: 0.54; 95% CI: 0.40-0.74), but not ETN (HR: 1.03; 95% CI: 0.65-1.63)., Conclusions: This study of anti-TNF-naïve patients with AS indicated that users of ADA and ETN had similar overall risk of drug discontinuation. However, patients taking ADA with MTX had a lower risk of discontinuation than those taking ADA alone.

Objective: SLE is associated with increased risk of diabetes mellitus. Treatment for SLE requires high-dose glucocorticoids that may worsen glucose homoeostasis. HCQ can reduce diabetes risk in RA. This study aimed to investigate the association of HCQ use and diabetes mellitus risk in SLE patients., Methods: This nationwide, population-based cohort study was conducted using the Taiwan National Health Insurance Research Database. In the period 2001-10, 8628 newly diagnosed SLE patients were identified after excluding those with a previous diagnosis of RA, psoriasis or diabetes mellitus. Incidence of diabetes mellitus was identified as a new diagnostic code using a diabetes mellitus-specific medication., Results: Two hundred and twenty-one newly diagnosed diabetes mellitus patients were identified among SLE patients (6795 had taken HCQ and 1833 had never taken HCQ), with an average follow-up period of 5.6 years. Compared with patients without HCQ treatment, the hazard ratio (HR) of diabetes mellitus in patients taking HCQ at a cumulative dose ≥129 g was reduced [HR 0.26 (95% CI 0.18, 0.37), P < 0.001]. Daily glucocorticoid ≥10 mg prednisolone-equivalent dose was associated with increased risk of developing diabetes mellitus [HR 2.47 (95% CI 1.44, 4.23), P = 0.001], which was minimized by concomitant HCQ use at a cumulative dose ≥129 g., Conclusion: In SLE patients, the use of HCQ is associated with reduced risk of incident diabetes mellitus in a dose-dependent manner. High-dose glucocorticoids increase the risk of diabetes, which can be decreased by concomitant HCQ use., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: The objective of this study was to investigate the impact of disease onset age on mortality and renal survival in female SLE patients., Methods: This nationwide, population-based, retrospective cohort study used data from the National Health Insurance Research Database of Taiwan. Female patients newly diagnosed with SLE from 2001 to 2004 were identified as the study cohort. A non-SLE group was matched for age, sex and initial diagnosis date (index date) as the comparison cohort. Co-morbidities, mortality rates and end-stage renal disease (ESRD) incidences were compared among SLE patients of different onset age. Hazard ratios with a 95% CI were determined by the Cox proportional hazard model to quantify the mortality rates and ESRD incidences. Juvenile-onset, adult-onset and late-onset SLE patients were categorized according to disease onset age: <18, 18-50 and >50 years old., Results: In total, 513 juvenile-onset, 3076 adult-onset and 764 late-onset SLE patients were identified. Compared with non-SLE controls, the hazard ratios of mortality were 6.49 (95% CI 3.73, 11.32, P < 0.001) for juvenile-onset, 1.75 (95% CI 1.47, 2.08, P < 0.001) for adult-onset and 3.44 (95% CI 2.76, 4.28, P < 0.001) for late-onset SLE patients. The hazard ratios of incident ESRD were 20.28 (95% CI 12.79, 32.15, P < 0.001) for adult-onset lupus patients and 1.99 (95% CI 1.36, 2.93, P < 0.001) for late-onset patients., Conclusion: Female patients with late-onset SLE carried a higher risk of mortality than those with adult-onset disease in the presence of co-morbidities. Juvenile-onset SLE patients were at greatest risk of mortality, which is probably due to disease severity.

Objective: To investigate the influence of rituximab therapy on released IFN-γ levels in the QuantiFERON-TB-Gold In-Tube (QFT-GIT) assay among RA patients of different Mycobacterium tuberculosis infection status., Methods: Change in levels of released IFN-γ in the QFT-GIT assay was evaluated in RA patients who had received 1 year of rituximab treatment. A tuberculin skin test was performed using the Mantoux method and the QFT-GIT assay was performed by measuring IFN-γ levels in whole blood treated with tuberculosis (TB)-specific antigens., Results: Among 56 patients, 43 patients were presumed to be without latent TB infection (LTBI), 7 patients had LTBI and 6 patients had anti-TNF-associated TB. During the 1-year period of rituximab therapy, no patient developed active TB or had QFT-GIT conversion. No significant change in released IFN-γ levels on QFT-GIT assay after 1-year rituximab therapy was observed in patients with LTBI [3.39 (1.21) vs 2.47 (0.82) IU/ml] or in those with anti-TNF-associated TB [1.06 (0.22) vs 0.87 (0.39) IU/ml]. Rituximab did not inhibit TB antigen-stimulated IFN-γ production ex vivo. The frequency of circulating CD19(+) B cells was significantly decreased [8.56 (0.84) vs 1.52 (0.44)%, P < 0.001], paralleling the decrease in RF titre [318.5 (76.0) vs 115.1 (32.1) IU/ml, P < 0.001] and DAS28 [6.49 (0.13) vs 4.59 (0.14), P < 0.001] after 1 year of rituximab therapy. However, there was no significant change in the frequency of CD3(+) T cells after rituximab therapy., Conclusion: No occurrence of active TB or QFT-GIT conversion was observed in patients receiving 1 year of rituximab therapy. No significant effect of rituximab therapy on IFN-γ release levels was observed in patients with LTBI and with anti-TNF-α-associated TB. Rituximab may be an alternative therapeutic agent for these patients.

Infection of nonhuman primates (NHPs) with monkeypox virus (MPXV) is currently being developed as an animal model of variola infection in humans. We used positron emission tomography and computed tomography (PET/CT) to identify inflammatory patterns as predictors for the outcome of MPXV disease in NHPs. Two NHPs were sublethally inoculated by the intravenous (IV) or intrabronchial (IB) routes and imaged sequentially using fluorine-18 fluorodeoxyglucose ((18)FDG) uptake as a nonspecific marker of inflammation/immune activation. Inflammation was observed in the lungs of IB-infected NHPs, and bilobular involvement was associated with morbidity. Lymphadenopathy and immune activation in the axillary lymph nodes were evident in IV- and IB-infected NHPs. Interestingly, the surviving NHPs had significant (18)FDG uptake in the axillary lymph nodes at the time of MPXV challenge with no clinical signs of illness, suggesting an association between preexisting immune activation and survival. Molecular imaging identified patterns of inflammation/immune activation that may allow risk assessment of monkeypox disease.

Objective: To investigate the potential role of Th type 17 (Th17) cells and Th17-related cytokines in the pathogenesis of adult-onset Still's disease (AOSD)., Methods: The frequencies of circulating Th17 cells in 24 patients with active untreated AOSD, 16 patients with active SLE and 12 healthy volunteers were determined using intracellular cytokine staining and flow cytometry. Serum levels of Th17-related cytokines, including IL-1β, IL-6, IL-17, IL-18, IL-21 and IL-23 were measured by ELISA., Results: Significantly higher median frequencies of circulating Th17 cells were found in active untreated AOSD patients (1.01%) and active SLE patients (1.26%) than in healthy volunteers (0.12%, both P<0.001). The frequencies of circulating Th17 cells were positively correlated with activity score (r=0.527, P<0.01) and serum ferritin levels (r=0.724, P<0.001) in AOSD patients, and correlated with SLEDAI (r=0.663, P<0.01) in SLE patients. Additionally, the frequencies of circulating Th17 cells were positively and significantly correlated with serum levels of IL-1β, IL-6, IL-17, IL-18, IL-21 and IL-23 in both AOSD and SLE patients. The frequencies of circulating Th17 cells and serum IL-17 levels significantly decreased after effective therapy in AOSD patients (both P<0.001)., Conclusion: Elevated frequencies of circulating Th17 cells and a positive correlation with disease activity in our AOSD patients suggest that Th17 cells contribute to the pathogenesis of this disease. Dysregulation of Th17 cells may be a common pathogenic mechanism that underlies the development of both AOSD and SLE.

Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos.

Objective: Geriatric syndromes (GSs), i.e. cognitive impairment, depression, fall, incontinence and malnutrition, are extensively used to highlight the unique features of common health problems in the frail elderly. Although GS is common in older RA patients, it is rarely reported earlier. We evaluate the prevalence of GS in elderly RA patients and explore the interrelationship between GS and RA., Methods: All enrolled RA patients were categorized into elderly RA (aged > or =65 years) and younger RA (aged <65 years). A comprehensive geriatric assessment was done to determine the presence of GS. HAQ score and disease activity of RA, including 28-joint disease activity score (DAS28), were assessed., Results: In total, 65 elderly and 25 younger RA patients were enrolled. The prevalence of GS in the elderly participants, especially cognitive impairment and fall, was significantly higher than that in the young. Older RA patients were more physically dependent than the young. Compared with subjects without GS, older RA patients with GS had longer disease duration, higher DAS28 scores, lower haemoglobin levels and more physical dependence. By using binary logistic regression, we found that a higher DAS28 score and a lower haemoglobin level were independent risk factors for GS., Conclusions: The prevalence of GS was higher in the elderly RA patients than that in the young. A higher DAS28 score and a lower haemoglobin level were independent risk factors for GS in older RA patients. Further study is needed to evaluate the prognostic role of GS.

The role of androgen and androgen receptors (ARs) in males has been well established. This steroid and its receptor also exist in follicles, but their functions are still unclear. In this study, using a culture system containing a low dose of hypoxanthine, we revealed the positive contribution of testosterone to oocyte meiotic resumption. By performing ultracentrifugation to allow clear visualization of porcine germinal vesicles, our results provide evidence that mitogen-activated protein kinase (MAPK) in the oocyte itself but not in cumulus cells was activated before germinal vesicle breakdown (GVBD) after testosterone treatment. We further explored the signal cascade of testosterone-triggered GVBD and showed significant contributions of AR to testosterone-induced MAPK activation and GVBD. By using a potent and selective inhibitor of SRC and detecting activation of the kinase, we found that testosterone activated SRC in oocytes but not in cumulus cells and that SRC (as an essential upstream molecule of MAPK) mediated this testosterone- and AR-promoted reinitiation of meiosis. The present findings propose an undefined signaling pathway and suggest the potential competence of testosterone for meiotic resumption in mammalian oocytes.

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.

Spindle movement, including spindle migration during first meiosis and spindle rotation during second meiosis, is essential for asymmetric divisions in mouse oocytes. Previous studies by others and us have shown that microfilaments are required for both spindle migration and rotation. In the present study, we aimed to further investigate the mechanism controlling spindle movement during mouse oocyte meiosis. By employing drug treatment and immunofluorescence microscopy, we showed that dynamic microtubule assembly was involved in both spindle migration and rotation. Furthermore, we found that the calcium/CaM/CaMKII pathway was important for regulating spindle rotation.

Because some animals and human beings potentially engage in sexual activity at any day of the menstrual cycle, this may cause fertilization of postovulatory aged oocytes, which result in decreased potential of embryo development and longevity of offspring. To investigate the involvement of histone acetylation in the function of postovulatory aging, we examined the changes of histone acetylation by immunostaining with specific antibodies against various acetylated lysines on histones H3 and H4. We found that the acetylation levels of lysine 14 on histone H3 and lysines 8 and 12 on histone H4 in mouse oocytes were gradually increased during in vivo and in vitro postovulatory aging. Furthermore, the acetylation levels on these sites were markedly decreased or increased when the process of postovulatory aging was artificially delayed or accelerated, respectively. These results indicated that the gradual acetylation on some lysines of histones H3 and H4 is one of the phenomena in the process of postovulatory aging. Moreover, raising the level of histone acetylation by trichostatin A can accelerate the progression of postovulatory aging, suggesting that alteration of the acetylation on histones H3 and H4 can affect the progression of postovulatory aging in mouse oocytes.

Both the infection of human cytomegalovirus (HCMV) and the immunization of its recombinant glycoprotein (gB) in mice have been known to induce autoimmunity, resulting in symptoms similar to those of human systemic lupus erythematosus (SLE). Research has also found that the murine cytomegalovirus (MCMV)-specific monoclonal antibody (mAb) is able to react with a human U1-70K-like autoantigen. To investigate HCMV involvement in autoimmunity, we analysed the humoral responses to HCMV by autoimmune patients and normal adults. Our studies show unambiguously that sera from SLE patients exhibited an elevated IgG titre to HCMV when compared with those observed in controls and other connective tissue disease (CTD) patients (P < 0.001). The IgM titres to HCMV and IgG to HBV were evaluated, and no significant differences were noted among all testing groups. In addition to initiating T cell activity, as reported by many investigators, we found that the HCMV pp65 antigen (also known as lower matrix protein) was able to induce humoral responses in SLE patients. Immunoblot assays showed that 82.56% of sera from SLE patients reacted with the HCMV pp65 antigen, but only 11.11%, 23.53% and 31.17% of patients from normal control, rheumatoid arthritis (RA) and CTD patients, respectively, reacted to it. Unlike HCMV pp65, HCMV pp150 induced B cell activity in most collected sera (92.22%-98.04%). Finally, female NZB/W F1 mice immunized with plasmids encoding HCMV pp65 open reading frame (pcDNApp65) developed an early onset of autoantibody activity and more severe glomerulonephritis. Thus, we conclude that the HCMV pp65 antigen triggers humoral immunity in SLE patients and autoimmune-prone mice and that it could very well exacerbate the autoimmune responses in susceptible animals.

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.

Summary: A web-based three-dimensional (3D) protein retrieval system is available for protein structure data including all PDB and FSSP dataset. In this system, we use a visual-based matching method to compare the protein structure from multiple viewpoints. It takes less than three seconds for each query with 90% accuracy on an average.

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.

Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.

Somatic cell nuclei of giant pandas can dedifferentiate in enucleated rabbit ooplasm, and the reconstructed eggs can develop to blastocysts. In order to observe whether these interspecies cloned embryos can implant in the uterus of an animal other than the panda, we transferred approximately 2300 panda-rabbit cloned embryos into 100 synchronized rabbit recipients, and none became pregnant. In another approach, we cotransferred both panda-rabbit and cat-rabbit interspecies cloned embryos into the oviducts of 21 cat recipients. Fourteen recipients exhibited estrus within 35 days; five recipients exhibited estrus 43-48 days after embryo transfer; and the other two recipients died of pneumonia, one of which was found to be pregnant with six early fetuses when an autopsy was performed. Microsatellite DNA analysis of these early fetuses confirmed that two were from giant panda-rabbit cloned embryos. The results demonstrated that panda-rabbit cloned embryos can implant in the uterus of a third species, the domestic cat. By using mitochondrial-specific probes of panda and rabbit, we found that mitochondria from both panda somatic cells and rabbit ooplasm coexisted in early blastocysts, but mitochondria from rabbit ooplasm decreased, and those from panda donor cells dominated in early fetuses after implantation. Our results reveal that mitochondria from donor cells may substitute those from recipient oocytes in postimplanted, interspecies cloned embryos.

Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization.

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.

Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intrinsic agonist activity, but both antagonized the growth-promoting activity of wild-type PRL, with the aspartate mutant proving to be a very effective antagonist. Two hundred picograms per ml of the aspartate mutant negated 75% of the growth response to 400 pg/ml wild-type PRL. When serine 179 was mutated to alanine or valine, mutant PRLs with 0% and 14% of the biological activity of wild-type PRL, respectively, were produced. These results demonstrate 1) that molecular mimicry of the phosphorylated hormone does produce a PRL antagonist, and 2) that the serine at position 179 is crucial to the growth-promoting activity of PRL. The aspartate mutant can now be used to study many aspects of the physiology of PRL.

Capillary electrophoresis provides very high efficiency separations of biological molecules. Laser-induced fluorescence produces very high sensitivity detection. The combination of the two techniques results in an analytical tool with unprecedented properties: separations with more than a million theoretical plates and detection limits of a few hundred analyte molecules. This paper considers the design of high-sensitivity laser-induced fluorescence detection for capillary zone electrophoresis separation of labeled amino acids and capillary gel electrophoresis separation of DNA sequencing fragments.