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Xiao Huo,1 Xiaoshuang Zhou,2,3 Peng Peng,2 Mei Yu,2 Ying Zhang,2 Jiaxin Yang,2 Dongyan Cao,2 Hengzi Sun,4 Keng Shen2 1Medical Research Center, Peking University Third Hospital, Beijing,, People’s Republic of China; 2Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 3Department of Ultrasound, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Beijing, People’s Republic of China; 4Department of Obstetrics and Gynecology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Keng ShenDepartment of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan, Dongcheng District, Beijing, 100730, People’s Republic of ChinaTel +86-10-69155200Email shenkengpumc@163.comCorrespondence: Hengzi SunDepartment of Obstetrics and Gynecology, Beijing Chao-Yang Hospital, Capital Medical University, No. 8 GongTiNan Road, Chaoyang District, Beijing, 100020, People’s Republic of ChinaTel +86 010 85231760Email Summerel1990@163.comBackground: Although the incidence of cervical cancer has decreased in recent decades with the development of human papillomavirus vaccines and cancer screening, cervical cancer remains one of the leading causes of cancer-related death worldwide. Identifying potential biomarkers for cervical cancer treatment and prognosis prediction is necessary.Methods: Samples with mRNA sequencing, copy number variant, single nucleotide polymorphism and clinical follow-up data were downloaded from The Cancer Genome Atlas database and randomly divided into a training dataset (N=146) and a test dataset (N=147). We selected and identified a prognostic gene set and mutated gene set and then integrated the two gene sets with the random survival forest algorithm and constructed a prognostic signature. External validation and immunohistochemical staining were also performed.Results: We obtained 1416 differentially expressed prognosis-related genes, 624 genes with copy number amplification, 1038 genes with copy number deletion, and 163 significantly mutated genes. A total of 75 candidate genes were obtained after overlapping the differentially expressed genes and the genes with genomic variations. Subsequently, we obtained six characteristic genes through the random survival forest algorithm. The results showed that high expression of SLC19A3, FURIN, SLC22A3, and DPAGT1 and low expression of CCL17 and DES were associated with a poor prognosis in cervical cancer patients. We constructed a six-gene signature that can separate cervical cancer patients according to their different overall survival rates, and it showed robust performance for predicting survival (training set: p ˂ 0.001, AUC = 0.82; testing set: p ˂ 0.01, AUC = 0.59).Conclusion: Our study identified a novel six-gene signature and nomogram for predicting the overall survival of cervical cancer patients, which may be beneficial for clinical decision-making for individualized treatment.Keywords: cervical cancer, bioinformatics, prognostic signature, Gene Expression Omnibus, overall survival

Background and aim Nonattendance of outpatient colonoscopy leads to inefficient use of health-care resources. We aimed to study the effectiveness of using Short Message Service (SMS) reminder prior in patients scheduled for outpatient colonoscopy on their nonattendance rate. Methods Patients who scheduled for an outpatient colonoscopy and had access of SMS were recruited from three clinics in Hong Kong. Patients were randomized to SMS group and standard care (SC) group. All patients were given a written appointment slip on the booking date. In addition, patients in the SMS group received an SMS reminder 7-10 days before their colonoscopy appointment. Patients' demographics, attendance, colonoscopy completion, and bowel preparation quality were recorded. Logistic regression was performed to identify predictors of nonattendance. Results From November 2013 to October 2019, a total of 2225 eligible patients were recruited. A total of 1079 patients were allocated to the SMS group and 1146 to the SC group. The nonattendance rate of patients in the SMS group was significantly lower than that in the SC group (8.9% vs 11.9%, P = 0.022). There were no significant differences in their baseline characteristics and colonoscopy completion rate and bowel preparation quality. A trend towards a higher rate of adequate bowel preparation was observed in the SMS group when compared with the SC group (69.9% vs 65.8%, P = 0.053). Independent predictors for nonattendance included younger age, underprivilege, and existing diabetes. Conclusions An SMS reminder for outpatient colonoscopy is effective in reducing the nonattendance rate and may potentially improve the bowel preparation quality.

BACKGROUND Previously, a stretching regimen was designed for manual material handling (MMH) of gas cylinders as a potential ergonomic solution for reducing occupational injury. No studies have made use of objective process measures, such as muscle activation levels, for evaluation of effects of stretching programs. OBJECTIVE Examine acute effects of stretching on muscle activation levels and driver perceived level of exertion in gas cylinder handling during simulated delivery operations. METHODS A within-subject experiment was conducted with eight male participants being subjected randomly to two conditions over a two-day period: stretching before delivery trials and no stretching. Surface electromyography and the Borg CR-10 scale for perceived exertion were used. RESULTS Generally, results were variable among muscle responses. The extensor muscle bundle in the forearm was found to show a significant decrease (p = 0.0464) in activation level because of stretching. The anterior deltoid and trapezius significantly increased (p

We report the development of a theranostic collagen targeted cell penetrating drug delivery system toward treatment of cardiovascular disease. Caused by the action of matrix metalloproteinases (MMP...

Introduction: Youth with classical congenital adrenal hyperplasia (CAH) have higher prevalence of cardiometabolic risk factors such as obesity, abdominal adiposity, and hypertension. Patients with CAH also exhibit an earlier adiposity rebound (AR) compared to normative populations. However, the predictive relationship between AR and cardiometabolic risk factors needs to be better understood. Methods: We performed a retrospective cohort study at a US tertiary pediatric center in youth with classical CAH due to 21-hydroxylase deficiency. AR was determined by cubic polynomial modeling. A subset of participants had fasting analytes, whole-body dual-energy X-ray absorptiometry, and magnetic resonance imaging as adolescents. Results: In 42 youth with CAH (45.2% female, 54.8% Hispanic, and 90.5% salt-wasting form), the average age at AR was 3.4 ± 1.3 years. AR differed by BMI-z, with youth with obesity having an earlier AR (2.8 ± 1.0 years) compared to lean youth (4.1 ± 1.3 years, p = 0.001). However, AR did not differ by either CAH form or sex. Earlier AR predicted higher BMI-z at 7 and 12 years of age. In addition, earlier AR predicted increased central obesity (as measured by waist circumference, subcutaneous adipose tissue, and trunk fat) and total body fat in adolescence. AR was negatively correlated with bone age, and its relationships with HDL and hypertension were trending towards significance. Conclusions: AR in youth with classical CAH could serve as a useful clinical marker to identify those patients who are at higher risk for developing cardiometabolic risk factors during childhood and adolescence.

Background: Calcific aortic valve disease is common in the aging population and is characterized by the histological changes of the aortic valves including extracellular matrix remodeling, osteochondrogenic differentiation, and calcification. Combined, these changes lead to aortic sclerosis, aortic stenosis (AS), and eventually to heart failure. Runt-related transcription factor 2 (Runx2) is a transcription factor highly expressed in the calcified aortic valves. However, its definitive role in the progression of calcific aortic valve disease (CAVD) has not been determined. In this study, we utilized constitutive and transient conditional knockout mouse models to assess the molecular, histological, and functional changes in the aortic valve due to Runx2 depletion.Methods: Lineage tracing studies were performed to determine the provenance of the cells giving rise to Runx2+ osteochondrogenic cells in the aortic valves of LDLr−/− mice. Hyperlipidemic mice with a constitutive or temporal depletion of Runx2 in the activated valvular interstitial cells (aVICs) and sinus wall cells were further investigated. Following feeding with a diabetogenic diet, the mice were examined for changes in gene expression, blood flow dynamics, calcification, and histology.Results: The aVICs and sinus wall cells gave rise to Runx2+ osteochondrogenic cells in diseased mouse aortic valves. The conditional depletion of Runx2 in the SM22α+ aVICs and sinus wall cells led to the decreased osteochondrogenic gene expression in diabetic LDLr−/− mice. The transient conditional depletion of Runx2 in the aVICs and sinus wall cells of LDLr−/−ApoB100 CAVD mice early in disease led to a significant reduction in the aortic peak velocity, mean velocity, and mean gradient, suggesting the causal role of Runx2 on the progression of AS. Finally, the leaflet hinge and sinus wall calcification were significantly decreased in the aortic valve following the conditional and temporal Runx2 depletion, but no significant effect on the valve cusp calcification or thickness was observed.Conclusions: In the aortic valve disease, Runx2 was expressed early and was required for the osteochondrogenic differentiation of the aVICs and sinus wall cells. The transient depletion of Runx2 in the aVICs and sinus wall cells in a mouse model of CAVD with a high prevalence of hemodynamic valve dysfunction led to an improved aortic valve function. Our studies also suggest that leaflet hinge and sinus wall calcification, even in the absence of significant leaflet cusp calcification, may be sufficient to cause significant valve dysfunctions in mice.

We explored the severity and risk factors for cardiac and liver iron overload (IOL) in 69 thalassemia patients who underwent T2* magnetic resonance imaging (T2* MRI) in a Malaysian tertiary hospital from 2011 to 2015. Fifty-three patients (76.8%) had transfusion-dependent thalassemia (TDT) and 16 (23.2%) had non transfusion-dependent thalassemia (NTDT). Median serum ferritin prior to T2* MRI was 3848.0 μg/L (TDT) and 3971.0 μg/L (NTDT). Cardiac IOL was present in 16 (30.2%) TDT patients and two (12.5%) NTDT patients, in whom severe cardiac IOL defined as T2* 10 ms affected six (11.3%) TDT patients. Liver IOL was present in 51 (96.2%) TDT and 16 (100%) NTDT patients, 37 (69.8%) TDT and 13 (81.3%) NTDT patients were in the most severe category (15 mgFe/gm dry weight). Serum ferritin showed a significantly strong negative correlation with liver T2* in both TDT (

Background: Distant metastasis is the main reason for treatment failure in patients with nasopharyngeal carcinoma (NPC). In this study, we evaluated the effect of ulinastatin (UTI) on NPC metastasis and investigated its underlying mechanism.Methods: Highly-metastatic NPC cell lines S18 and 5-8F were treated with UTI and the effect on cellular growth, migration, and invasion of NPC cells was determined using MTS and transwell assays. The luciferase-expressing S18 cells (S18-1C3) were injected into the left hind footpad of nude mice to establish a spontaneous footpad to popliteal lymph node (LN) metastasis model. The luciferase mRNA was measured by qPCR to calculate the metastatic inhibition rate. The UTI-related uPA, uPAR and the main molecular members of the JAT/STAT3 pathway were detected by qPCR and immunoblotting.Results: UTI inhibited the migration and invasion of S18 and 5-8F cells with no effect on cellular growth in vitro, and suppressed metastasis of S18 cells in vivo. uPAR expression was reduced from 24 to 48 hours after UTI treatment. The anti-metastasis effect of UTI partially relies on the suppression of uPA and uPAR.Conclusion: UTI partially inhibits NPC metastasis via downregulating the expression of uPA and uPAR.

Improvement in outcomes of children with acute myeloid leukemia (AML) is attributed to several refinements in clinical management. We evaluated treatment outcomes of Taiwanese pediatric AML patients in the past 20 years. Overall, 860 de novo AML patients aged 0–18 years and registered in the Childhood Cancer Foundation of R.O.C during January 1996–December 2019 were included. Survival analysis was performed to identify factors that improved treatment outcomes. Regardless of treatment modalities used, patients during 2008–2019 had better 5-year event-free survival (EFS) and overall survival (OS) rates than patients during 1996–2007. For patients received the TPOG-AML-97A treatment, only 5-year OS rates were significantly different between patients diagnosed before and after 2008. Patients with RUNX1–RUNX1T1 had similar relapse-free survival rates, but 5-year OS rates were better during 2008–2019. However, the survival of patients who received hematopoietic stem-cell transplantations (HSCT) did not differ significantly before and after 2008. For patients without relapse, the 5-year OS improved during 2008–2019. Non-relapse mortality decreased annually, and cumulative relapse rates were similar. In conclusion, 5-year EFS and OS rates improved during 2008–2019, though intensities of chemotherapy treatments were similar before and after 2008. Non-relapse mortality decreased gradually. Further treatment strategies including more intensive chemotherapy, novel agents’ use, identification of high-risk patients using genotyping and minimal residual disease, early intervention of HSCT, and antibiotic prophylaxis can be considered for future clinical protocol designs in Taiwan.

Objective Calcific aortic valve disease (CAVD) is a major cause of aortic stenosis (AS) and cardiac insufficiency. Patients with type II diabetes mellitus (T2DM) are at heightened risk for CAVD, and their valves have greater calcification than nondiabetic valves. No drugs to prevent or treat CAVD exist, and animal models that might help identify therapeutic targets are sorely lacking. To develop an animal model mimicking the structural and functional features of CAVD in people with T2DM, we tested a diabetogenic, procalcific diet and its effect on the incidence and severity of CAVD and AS in the, LDLr -/- ApoB 100/100 mouse model. Results LDLr -/- ApoB 100/100 mice fed a customized diabetogenic, procalcific diet (DB diet) developed hyperglycemia, hyperlipidemia, increased atherosclerosis, and obesity when compared with normal chow fed LDLr -/- ApoB 100/100 mice, indicating the development of T2DM and metabolic syndrome. Transthoracic echocardiography revealed that LDLr -/- ApoB 100/100 mice fed the DB diet had 77% incidence of hemodynamically significant AS, and developed thickened aortic valve leaflets and calcification in both valve leaflets and hinge regions. In comparison, normal chow (NC) fed LDLr -/- ApoB 100/100 mice had 38% incidence of AS, thinner valve leaflets and very little valve and hinge calcification. Further, the DB diet fed mice with AS showed significantly impaired cardiac function as determined by reduced ejection fraction and fractional shortening. In vitro mineralization experiments demonstrated that elevated glucose in culture medium enhanced valve interstitial cell (VIC) matrix calcium deposition. Conclusions By manipulating the diet we developed a new model of CAVD in T2DM, hyperlipidemic LDLr -/- ApoB 100/100 that shows several important functional, and structural features similar to CAVD found in people with T2DM and atherosclerosis including AS, cardiac dysfunction, and inflamed and calcified thickened valve cusps. Importantly, the high AS incidence of this diabetic model may be useful for mechanistic and translational studies aimed at development of novel treatments for CAVD.

The aim of this study was to investigate in vitro effects of fenitrothion (FNT) on mouse splenic lymphocytes. Here, naïve mice had their spleens harvested and splenocytes isolated. After exposure to FNT for 48 hr: splenocyte viability was measured using a tetrazolium dye assay; cell phenotypes, i.e., B-cells (CD19

Aims Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro. A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE −/− mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size. Methods and results We used an improved targeting construct to generate LDLr −/− mice with floxed-Runx2 alleles ( LDLr −/− :Runx2f/f ) such that Cre-mediated recombination ( LDLr −/− :Runx2ΔSM ) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both LDLr −/− :Runx2f/f and LDLr −/− :Runx2ΔSM mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of LDLr −/− :Runx2ΔSM mice compared to LDLr −/− :Runx2f/f counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced. Conclusions SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression.

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/- mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification.

In Arabidopsis, CONSTANS (CO) integrates light and circadian clock signals to promote flowering under long days (LD). In the grasses, a duplication generated two paralogs designated as CONSTANS1 (CO1) and CONSTANS2 (CO2). Here we show that in tetraploid wheat plants grown under LD, combined loss-of-function mutations in the A and B-genome homeologs of CO1 and CO2 (co1 co2) result in a small (3 d) but significant (P60 d relative to the co1 mutant allele under LD. We detected significant genetic interactions among CO1, CO2 and PPD1 genes on heading time, which were reflected in complex interactions at the transcriptional and protein levels. Loss-of-function mutations in PPD1 delayed heading more than combined co1 co2 mutations and, more importantly, PPD1 was able to perceive and respond to differences in photoperiod in the absence of functional CO1 and CO2 genes. Similarly, CO1 was able to accelerate heading time in response to LD in the absence of a functional PPD1. Taken together, these results indicate that PPD1 and CO1 are able to respond to photoperiod in the absence of each other, and that interactions between these two photoperiod pathways at the transcriptional and protein levels are important to fine-tune the flowering response in wheat., Author summary An understanding of the mechanisms involved in the regulation of wheat heading time is required to engineer more productive varieties better adapted to new or changing environments. A large proportion of wheat’s natural variation in heading time is determined by differences in genes controlling the photoperiodic response. In this study, we show that the wheat PHOTOPERIOD1 (PPD1) gene has a stronger effect on heading time than CONSTANS1 (CO1) and CO2 in the regulation of the photoperiodic response, and that complex genetic interactions among these genes are important to fine-tune heading time. Using loss-of-function mutants for both CO1 and CO2, we demonstrate that these genes are not required for PPD1 to perceive differences in photoperiod and regulate heading time. Similarly, we show that in the absence of PPD1, CO1 can accelerate heading time more than 60 days in response to long days. Our results indicate that each of these two wheat photoperiod pathways can respond to differences in photoperiod even in the absence of the other one. Differences in the relative importance of these two pathways and in their epistatic interactions have contributed to the diversity of photoperiodic responses observed in different grass species.

Background To eliminate cranial irradiation (CrRT)-related sequelae and to minimize the adverse impact of traumatic lumbar puncture (TLP) with blasts, the Taiwan Pediatric Oncology Group (TPOG) introduced a modified central nervous system (CNS)-directed regimen characterized by delayed triple intrathecal therapy (TIT) and the omission of CrRT for all children with newly diagnosed acute lymphoblastic leukemia (ALL). Methods This study compared the treatment outcomes of patients overall and patients with a non-CNS-1 status (CNS-2, CNS-3, or TLP with blasts) in 2 treatment eras, one before and another after the revision of the TPOG-ALL-2002 protocol by the introduction of the modification (era 1 [2002-2008] with CrRT and era 2 [2009-2012] with delayed first TIT and no CrRT). Results There were no statistically significant differences in major outcomes between the 903 patients treated in era 1 and the 444 patients treated in era 2: the 5-year event-free survival (EFS) rates were 75.7% ± 1.4% and 72.1% ± 2.4%, respectively (P = .260), and the cumulative risks of isolated CNS relapse were 4.0% ± 0.7% and 4.1% ± 1.0%, respectively (P = .960). There were also no differences between non-CNS-1 patients treated in era 1 (n = 76) and era 2 (n =28): the 5-year EFS rates were 52.3% ± 5.8% and 62.9% ± 9.4%, respectively (P = .199), and the cumulative risks of isolated CNS relapse were 6.3% ± 3.1% and 3.6% ± 3.5%, respectively (P = .639). Notably, TLP with blasts was completely eliminated after the first TIT was delayed in era 2. Conclusions The delay of the first TIT until the clearance of circulating blasts and the total omission of CrRT did not compromise survival or CNS control in patients with childhood ALL, including those with a non-CNS-1 status.

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2(f/f)) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22-recombinase transgenic allele mice (Runx2(ΔSM)) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2(f/f) mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D-treated Runx2(ΔSM) mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2(f/f) SMCs calcified to a much greater extent than those derived from Runx2(ΔSM) mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

PiT-2, a type III sodium-dependent phosphate transporter, is a causative gene for the brain arteriolar calcification in people with familial basal ganglion calcification. Here we examined the effect of PiT-2 haploinsufficiency on vascular calcification in uremic mice using wild-type and global PiT-2 heterozygous knockout mice. PiT-2 haploinsufficiency enhanced the development of vascular calcification in mice with chronic kidney disease fed a high-phosphate diet. No differences were observed in the serum mineral biomarkers and kidney function between the wild-type and PiT-2 heterozygous knockout groups. Micro computed tomography analyses of femurs showed that haploinsufficiency of PiT-2 decreased trabecular bone mineral density in uremia. In vitro, sodium-dependent phosphate uptake was decreased in cultured vascular smooth muscle cells isolated from PiT-2 heterozygous knockout mice compared with those from wild-type mice. PiT-2 haploinsufficiency increased phosphate-induced calcification of cultured vascular smooth muscle cells compared to the wild-type. Furthermore, compared to wild-type vascular smooth muscle cells, PiT-2 deficient vascular smooth muscle cells had lower osteoprotegerin levels and increased matrix calcification, which was attenuated by osteoprotegerin supplementation. Thus, PiT-2 in vascular smooth muscle cells protects against phosphate-induced vascular calcification and may be a therapeutic target in the chronic kidney disease population.

Calcific aortic valve disease (CAVD), the progressive accumulation of fibrocalcific matrices and calcified, bony nodules in aortic valves that results in aortic stenosis, accounts for ≈50% of cardiac valve disease.1,2 In developed countries, CAVD is the third most common cardiovascular disease behind coronary artery disease and hypertension.3 Although CAVD is often asymptomatic during the first several decades of life, notably, approximately one third of our elderly have early valve disease as indicated by echocardiographic or radiological evidence of aortic sclerosis.2,4,5 By age 65, ≈2% of individuals develop symptomatic aortic stenosis, characterized by severe valve calcification, impaired leaflet motion, and cardiac outflow, which, if untreated, leads to life-threatening left ventricular dysfunction, angina, syncope, and heart failure.6,7 See accompanying article on page 543 CAVD risk factors include congenital malformation, age, male sex, smoking, hypercholesterolemia, …

The relationship between body mass index (BMI) and fracture risk is controversial. We sought to investigate the effect of collinearity between BMI and bone mineral density (BMD) on fracture risk, and to estimate the direct and indirect effect of BMI on fracture with BMD being the mediator. The study involved 2199 women and 1351 men aged 60 years or older. BMI was derived from baseline weight and height. Femoral neck BMD was measured by dual-energy X-ray absorptiometry (DXA; GE-LUNAR, Madison, WI, USA). The incidence of fragility fracture was ascertained by X-ray reports from 1991 through 2012. Causal mediation analysis was used to assess the mediated effect of BMD on the BMI-fracture relationship. Overall, 774 women (35% of total women) and 258 men (19%) had sustained a fracture. Approximately 21% of women and 20% of men were considered obese (BMI ≥ 30). In univariate analysis, greater BMI was associated with reduced fracture risk in women (hazard ratio [HR] 0.92; 95% confidence interval [CI], 0.85 to 0.99) and in men (HR 0.77; 95% CI, 0.67 to 0.88). After adjusting for femoral neck BMD, higher BMI was associated with greater risk of fracture in women (HR 1.21; 95% CI, 1.11 to 1.31) but not in men (HR 0.96; 95% CI, 0.83 to 1.11). Collinearity had minimal impact on the BMD-adjusted results (variance inflation factor [VIF] = 1.2 for men and women). However, in mediation analysis, it was found that the majority of BMI effect on fracture risk was mediated by femoral neck BMD. The overall mediated effect estimates were −0.048 (95% CI, −0.059 to −0.036; p

Review objectives The objective of this review is to synthesize the best available evidence regarding the effectiveness of patient centered self-care education for adult patients with heart failure relating to heart failure knowledge, self-care behaviors centered on heart failure, heart failure-related quality of life, and heart failure readmissions within one year of an index hospitalization. A secondary objective of this review is to determine the most effective education approach for interventions included in the review to deliver patient-centered self-care education to adult patients with heart failure. Inclusion criteria Types of participants This review will consider studies that include all adult patients, 18 years and older, of any race, ethnicity, or gender with a diagnosis of HF regardless of etiology, severity, duration of HF, or presence of comorbid conditions. Type of intervention This review will consider all types of patient-centered, self-care education interventions for adult patients with HF provided by any health care provider, regardless of frequency, duration and intensity. For this review, patient-centered, self-care education interventions are defined as interventions designed towards the patient as a unique individual, taking into consideration a patient’s individual needs, preferences and values. The patient is the central focus of the planned intervention with a goal of meeting patient specific self-care educational goals and desired outcomes. Comparator intervention This review will consider as comparators standard care or non-patient-centered education programs such as written or video taped education materials that have not been individualized to a patient’s specific needs, preferences or values. Types of outcomes This review will consider studies that include the following outcomes as measured by valid and reliable instruments within one year post-intervention: Heart failure knowledge such as knowledge related to the general pathophysiology of HF, treatment strategies, and signs/symptoms of HF as measured by valid and reliable instruments such as the Atlanta Heart Failure Knowledge Test (A-HFKT). Heart failure self-care behaviors including, but not limited to, HF management, symptom monitoring, and implementation of treatment strategies as measured by valid and reliable instruments such as the Self-Care Heart Failure Index (SCHFI) or the European Heart Failure Self-Care Behavior Scale (EHFScBS). Heart failure-related quality of life related to areas such as maintaining physical activity and exercise, symptom burden, self-efficacy, and maintaining social interaction and support, as measured by valid and reliable instruments such as the generic Short Form 36 (SF-36), or HF specific instruments such as the Minnesota Living with Heart Failure Questionnaire (MLHFQ) or the Kansas City Cardiomyopathy Questionnaire (KCCQ). Heart failure admissions or readmissions within 1 year post intervention. For this review, hospital readmission is the consecutive admission to the same hospital, a different hospital, or another acute care facility related to a specific condition for which treatment was rendered on a previous hospitalization.

Objective Many studies on integration have reported conflicting results regarding the role of HPV integration in cervical cancer. We hypothesized that high viral load and disruption of E2 gene associated with integration of HPV were not the only pathway leading to cancer development. Methods This study analysed the viral load and integration status of HPV16, measured the HPV16 E6/E7 mRNA transcript levels, delineated the E2 and LCR sequence variation, and determined the methylation status of two E2 binding sites. Results The results showed that viral load was not associated with the physical status of HPV genome. Levels of the three E6/E7 mRNA transcripts in invasive cervical cancers containing purely episomal viral genome were found to be similar to those containing integrated viral genome, suggesting that cancers containing episomal viral genome were also mediated by an up-regulated E6/E7 mRNA expression, and more importantly, did not depend on integration and disruption of the E2 gene. Conclusions The alternative mechanism that up-regulated the expression of E6 and E7 in invasive cancers harbouring episomal viral genome was likely to be a consequence of methylation of the two E2 binding sites located at the promoter region of HPV16. These observations are in line with the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer.

Blood-brain barrier (BBB) dysfunction in acute liver failure (ALF) results in increased BBB permeability that often precludes the patients from obtaining a life-saving liver transplantation. It remains controversial whether matrix metalloproteinase-9 (MMP-9) from the injured liver contributes to the deregulation of BBB function in ALF. We selectively upregulated a physiologic inhibitor of MMP-9 (TIMP-1) with a single intracerebroventricular injection of TIMP-1 cDNA plasmids at 48 and 72 hours, or with pegylated-TIMP-1 protein. Acute liver failure was induced with tumor necrosis factor-α and D-(+)-galactosamine in mice. Permeability of BBB was assessed with sodium fluorescein (NaF) extravasation. We found a significant increase in TIMP-1 within the central nervous system (CNS) after the administration of TIMP-1 cDNA plasmids and that increased TIMP-1 within the CNS resulted in an attenuation of BBB permeability, a reduction in activation of epidermal growth factor receptor and p38 mitogen-activated protein kinase signals, and a restoration of the tight junction protein occludin in mice with experimental ALF. Pegylated TIMP-1 provided similar protection against BBB permeability in mice with ALF. Our results provided a proof of principle that MMP-9 contributes to the BBB dysfunction in ALF and suggests a potential therapeutic role of TIMP-1 in ALF.

Background Reinduction therapy has improved the outcomes in children with acute lymphoblastic leukemia (ALL). We sought to determine the optimal course(s) of reinduction therapy for standard-risk (SR, or “low-risk” in other groups) patients. Also, we evaluated outcomes using triple intrathecal therapy without cranial radiation (CrRT) for central nervous system (CNS) preventive therapy. Procedure From 2002 to 2012, all newly diagnosed children with ALL in Taiwan were enrolled in Taiwan Pediatric Oncology Group ALL-2002 protocol. SR patients were randomized to receive single or double reinduction courses. The patients enrolled before 2009 received CrRT, while those enrolled later did not. The Kaplan–Meier method was used to estimate survival rates and the difference between two groups was compared by the two-sided log-rank test. Results In 1,366 eligible patients, the 5-year overall survival (OS) was 81.6 ± 1.1% (standard error) and 5-year event-free survival (EFS) was 74.3 ± 1.2%. In SR patients, the 5-year OS for one and two reinduction courses was 91.6 ± 2.1% and 93.7 ± 1.8%, respectively, and the 5-year EFS was 85.2 ± 2.7% and 89.8 ± 2.3%, respectively. There were no significant differences in survival between these two groups. Patients with MLL or BCR-ABL1 had the worst outcomes: 5-year EFS was 23.4 and 31.8% and 5-year OS was 28.6 and 44.7%, respectively. There was no significant difference in CNS relapse or survival between the era with or without CrRT. Conclusions For SR patients, one-course reinduction was adequate. Triple intrathecal therapy alone successfully prevented CNS relapse.

The maxillary first molar usually has single palatal root. Incidence rate of maxillary first molar with two palatal roots is especially rare. We reported a case of young maxillary first molar with two palatal roots in this article. Anatomic characteristic of permanent maxillary molar is generally described as a group of teeth with three roots, one palatal and two buccalbut the root canal system is complexed. The second mesiobuccal root was common finding of the teeth studied, the second distobuccal and palatal root was rare. We reported the unusual variation in root and canal morphology of one four-rooted maxillary first molar with two palatal roots.

Background The episiotomy rate has declined worldwide but remains high in several countries such as Taiwan. The effects of episiotomy on women's health should be a constant concern. Few data are available on the effect of episiotomy by validated measures. Objective The present study examined the effect of episiotomy on pain, urinary incontinence, and sexual function up to 3 months postpartum. Design, setting and participants A prospective follow-up study of 243 women who completed the Taiwanese versions of the Short Form McGill Pain Questionnaire, International Consultation on Incontinence Questionnaire – Urinary Incontinence Short Form, Female Sexual Function Index, and a demographic questionnaire after vaginal delivery in a Taiwanese medical center. Methods Differences between those who did and did not have an episiotomy were tested using ANCOVA, adjusting for age, parity, newborn weight, and vacuum delivery. The reliability and validity of the measuring instruments were assessed using Cronbach's α coefficient and factor analysis. Results Women who delivered without an episiotomy had significantly lower perineal pain scores at weeks 1, 2 and 6 postpartum compared to women who had an episiotomy ( p =0.0065, 0.0391, 0.0497, respectively). Women in the no-episiotomy group had significantly lower non-localized pain scores at week 2 postpartum compared to women in the episiotomy group ( p =0.0438). The mean urinary incontinence score was significantly higher in the episiotomy group 3 months postpartum ( p =0.0293). No significant difference in sexual function score was found between groups. Conclusions The results indicate that episiotomy increased pain at weeks 1, 2 and 6 postpartum, and urinary incontinence at 3 months postpartum. Awareness of the relationship between episiotomy and women's health will help health care professionals develop policy and promote the application of restrictive episiotomy.

Objectives— Vascular calcification is highly correlated with morbidity and mortality, and it is often associated with inflammation. Vitamin D may regulate vascular calcification and has been associated with cardiovascular survival benefits. Methods and Results— We developed a macrophage/smooth muscle cell (SMC) coculture system and examined the effects of vitamin D receptor activators (VDRA), calcitriol and paricalcitol, on SMC matrix calcification. We found that treatment of SMC alone with VDRA had little effect on phosphate-induced SMC calcification in vitro. However, coculture with macrophages promoted SMC calcification, and this was strikingly inhibited by VDRA treatment. Several VDRA-induced genes, including bone morphogenetic protein-2 (BMP2), tumor necrosis factor-α, and osteopontin, were identified as candidate paracrine factors for the protective effect of VDRA. Of these, osteopontin was further investigated and found to contribute significantly to the inhibitory actions of VDRA on calcification in macrophage/SMC cocultures. Conclusion— The ability of VDRA to direct a switch in the paracrine phenotype of macrophages from procalcific to anticalcific may contribute to their observed cardiovascular survival benefits.

SEDT (spondyloepiphyseal dysplasia tarda) is a late-onset X-linked recessive skeletal dysplasia caused by mutations in the gene SEDL coding for sedlin. In the present paper, we investigated four missense mutations observed in SEDT and compare biochemical and cellular characteristics relative to the wild-type protein to address the mechanism of disease and to gain insight into the function of the sedlin protein. In situ hybridization and immunohistochemical experiments in mouse growth plates revealed sedlin to be predominantly expressed in proliferating and hypertrophic chondrocytes. Cell culture studies showed that the wild-type protein localized predominantly in the vicinity of the nucleus and the Golgi, with further localization around the cytoplasm, whereas mutation resulted in mislocalization. The D47Y mutant was expressed similarly to the wild-type, but the S73L, F83S and V130D mutants showed particularly low levels of expression that were rescued in the presence of the proteasome inhibitor MG132 (benzyloxycarbonyl-leucylleucylleucinal). Furthermore, whereas the D47Y mutant folded similarly and had similar stability to the wild-type sedlin as shown by CD and fluorescence, the S73L, F83S and V130D mutants all misfolded during expression. Two independent assays showed that the D47Y mutation resulted in an increased affinity for the transport protein particle component Bet3 compared with the wild-type sedlin. Our results suggest that the sedlin mutations S73L, F83S and V130D cause SEDT by sedlin misfolding, whereas the D47Y mutation may influence normal TRAPP (transport protein particle) dynamics.

Title. Childbirth-related fatigue trajectories during labour. Aim. This paper is a report on a study identifying trajectories of childbirth-related fatigue intensity changes over time and the influencing factors related to specific trajectory patterns. Background. Childbirth is a period of time that encompasses considerable physiological and psychological fatigue, often having an adverse impact on women in labour. Empirical studies on this issue are scarce. How childbirth-related fatigue changes over time, and factors influencing fatigue development, remain unclear. Method. A prospective, correlational design with repeated measures was used. Data were collected by self-reported measures and laboratory analysis of blood specimens. From December 2004 to November 2005 a convenience sample of 209 low-risk pregnant Taiwanese women was followed throughout the labour process. Repeated measures of fatigue were analysed by using a semiparametric mixture model. Variables explaining trajectory class membership were identified by means of logistic regression. Results. Two distinct trajectories of childbirth-related fatigue were identified: low intensity (30·8% of women) and high intensity (69·2% of women). Fatigue level of both classes intensified following labour. The fastest period of fatigue-increasing rate was in the active phase. After birth, fatigue levels in the high-fatigue intensity class remained high. Primiparas in the high-fatigue intensity class experienced significantly more anxiety and higher lactate concentration at admission than the low-intensity class. Conclusion. Caregivers should endeavour to prevent high levels of fatigue once women enter the labour phase. Women who present with high fatigue at onset of labour should be targeted for early intervention, especially in the period of rapid fatigue increase.

Background. Much has been written about the anxiety of pregnant women undergoing maternal serum screening, but little has focused on the maternal self. Aims. The purpose of this study was to explore how the maternal self was affected by abnormal results of prenatal screening. Design. A qualitative design was used, based upon a grounded theory method. Methods. Twenty-seven women undergoing maternal serum screening engaged in qualitative interviews about how abnormal results affected them and their family life. Results. This study identified three forms of maternal self in women who had been informed of abnormal results of maternal serum screening. The three forms of maternal self could be described as self-stigmatizing, self-conflicting and self-knowledgeable. Conclusion. The self-stigmatizing and self-conflicting resulted from painful body image. The pregnant women responded to this vulnerability by reflecting on their experiences and constituting self-knowledgeable. Relevance to clinical practice. This study demonstrated that ethical issues were implicit in prenatal counselling in terms of various forms of maternal self. Understanding the self-perceptions of pregnant women coping with an increased risk of Down Syndrome could be very significant in delivering prenatal genetic counselling.

Background Cryptococcus gattii (Cg) infection emerged in British Columbia in 1999. A longitudinal, clinical description of patients has not been reported. Methods Medical records were reviewed for Cg patients identified through surveillance (1999-2007). Risk factors for Cg mortality were explored using multivariate Cox regression; longitudinal patterns in serum cryptococcal antigen (SCrAg) titers and the probability of chest cryptococcomas over time were estimated using cubic B-splines in mixed-effects regression models. Results Among 152 patients, 111 (73.0%) were culture confirmed. Isolated lung infection was present in 105 (69.1%) patients; 47 (30.9%) had central nervous system infection, with or without lung involvement. Malignancy was the provisional diagnosis in 64 (42.1%) patients. Underlying diseases were present in 91 (59.9%) patients; 23 (15.1%) were immunocompromised, and 23 (15.1%) had asymptomatic disease. There were only 2 (1.8%) culture positive relapses, both within 12 months of follow-up. The estimated median time to resolution of lung cryptococcomas and decline in SCrAg titer to 50 years (hazard ratio [HR], 15.6; 95% confidence interval [CI], 1.9-130.5) and immunocompromise (HR, 5.8; CI, 1.5-21.6). All Cg-related mortality occurred among culture-positive cases within 1 year of diagnosis. Conclusions Cryptococcomas and serum antigenemia were slow to resolve. However, late onset of failed therapy or relapse was uncommon, suggesting that delayed resolution of these findings does not require prolongation of treatment beyond that recommended by guidelines.

Objective— Advanced atherosclerotic lesions in the innominate arteries of chow-fed apolipoprotein E–deficient mice become highly calcified with 100% frequency by 75 weeks of age. The time course, cell types, and mechanism(s) associated with calcification were investigated. Methods and Results— The deposition of hydroxyapatite is preceded by the formation of fibro-fatty nodules that are populated by cells that morphologically resemble chondrocytes. These cells are spatially associated with small deposits of hydroxyapatite in animals between 45 and 60 weeks of age. Immunocytochemical analyses with antibodies recognizing known chondrocyte proteins show that these cells express the same proteins as chondrocytes within developing bone. Histological and electron microscopic analyses of lesions from animals between 45 and 60 weeks of age show that the chondrocyte-like cells are surrounded by dense connective tissue that stains positive for type II collagen. Nanocrystals of hydroxyapatite can be seen within matrix vesicles derived from the chondrocyte-like cells. In mice between 75 and 104 weeks of age, the lesions have significantly reduced cellularity and contain large calcium deposits. The few remaining chondrocyte-like cells are located adjacent to or within the large areas of calcification. Conclusions— Calcification of advanced lesions in chow-fed apolipoprotein E–deficient mice occurs reproducibly in mice between 45 and 75 weeks of age. The deposition of hydroxyapatite is mediated by chondrocytes, which suggests that the mechanism of calcification may in part recapitulate the process of endochondral bone formation.

The objective of this study was to elucidate if chronic and acute ammonia intoxication in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti, were associated with high levels of ammonia and/or glutamine in their brains, and if acute ammonia intoxication could be prevented by the administration of methionine sulfoximine [MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of N-methyl D-aspartate type glutamate (NMDA) receptors]. For P. schlosseri and B. boddaerti exposed to sublethal concentrations (100 and 8 mmol l(-1) NH4Cl, respectively, at pH 7.0) of environmental ammonia for 4 days, brain ammonia contents increased drastically during the first 24 h, and they reached 18 and 14.5 micromol g(-1), respectively, at hour 96. Simultaneously, there were increases in brain glutamine contents, but brain glutamate contents were unchanged. Because glutamine accumulated to exceptionally high levels in brains of P. schlosseri (29.8 micromol g(-1)) and B. boddaerti (12.1 micromol g(-1)) without causing death, it can be concluded that these two mudskippers could ameliorate those problems associated with glutamine synthesis and accumulation as observed in patients suffering from hyperammonemia. P. schlosseri and B. boddaerti could tolerate high doses of ammonium acetate (CH3COONH4) injected into their peritoneal cavities, with 24 h LC50 of 15.6 and 12.3 micromol g(-1) fish, respectively. After the injection with a sublethal dose of CH3COONH4 (8 micromol g(-1) fish), there were significant increases in ammonia (5.11 and 8.36 micromol g(-1), respectively) and glutamine (4.22 and 3.54 micromol g(-1), respectively) levels in their brains at hour 0.5, but these levels returned to normal at hour 24. By contrast, for P. schlosseri and B. boddaerti that succumbed within 15-50 min to a dose of CH3COONH4 (15 and 12 micromol g(-1) fish, respectively) close to the LC50 values, the ammonia contents in the brains reached much higher levels (12.8 and 14.9 micromol g(-1), respectively), while the glutamine level remained relatively low (3.93 and 2.67 micromol g(-1), respectively). Thus, glutamine synthesis and accumulation in the brain was not the major cause of death in these two mudskippers confronted with acute ammonia toxicity. Indeed, MSO, at a dosage (100 microg g(-1) fish) protective for rats, did not protect B. boddaerti against acute ammonia toxicity, although it was an inhibitor of GS activities from the brains of both mudskippers. In the case of P. schlosseri, MSO only prolonged the time to death but did not reduce the mortality rate (100%). In addition, MK801 (2 microg g(-1) fish) had no protective effect on P. schlosseri and B. boddaerti injected with a lethal dose of CH3COONH4, indicating that activation of NMDA receptors was not the major cause of death during acute ammonia intoxication. Thus, it can be concluded that there are major differences in mechanisms of chronic and acute ammonia toxicity between brains of these two mudskippers and mammalian brains.

This study investigated the expression profile of placental leptin and leptin receptor isoforms in preeclampsia, using placental tissue from normal pregnancies that were matched in gestational age and birth weight as controls. A total of 29 cases of preeclampsia were studied by immunohistochemistry, including 16 severe and 13 mild preeclampsia cases. Reverse transcriptase-polymerase chain reaction (RT-PCR) was further performed using RNA extracted from frozen tissue (10 severe preeclampsia, 10 mild preeclampsia, and 20 normal third trimester placentas). In all tissue sections, immunostaining signal was shown in the cytoplasmic compartment of the trophoblastic cells. Both the severe and mild preeclampsia groups showed significantly higher immunostaining for leptin compared with normal controls (p0.05), but there was no significant difference between the severe and mild preeclampsia groups (p0.05). There was no significant difference in immunostaining for leptin receptor between both severe and mild preeclampsia compared with controls (p0.05). RT-PCR showed significantly higher levels of mRNA transcripts of leptin in severe preeclampsia (p0.05), but not mild preeclampsia (p0.05), compared with normal controls. No significant difference in expression of all the receptor isoforms was demonstrated between both severe and mild preeclampsia groups compared with controls (p0.05). In conclusion, we confirmed an up-regulated expression of leptin in placental tissue in preeclampsia. However, there was no difference in the expression of all leptin receptor isoforms in placental tissue between preeclamptic and normal pregnancies. The leptin signal probably does not play a major primary role in the pathogenesis of preeclampsia.

Vascular calcification is highly correlated with cardiovascular disease (CVD) and is a significant predictor of cardiovascular events, especially in high risk patients such as the end stage renal disease (ESRD) population. Vascular calcification can lead to serious problems including valve stenosis, decreased vascular compliance, calciphylaxis, and even sudden death. However, the contribution of vascular calcification to progression of atherosclerosis is unknown and needs more study. Biochemical, histological, and genetic studies indicate that vascular calcification is actively regulated and involves both positive and negative modulators. Several nonmutually exclusive theories to account for vascular calcification based on current studies are discussed.

Intravenous leiomyomatosis is a rare smooth muscle tumor. We report two cases of intravenous leiomyomatosis that grew along different routes of the venous system into the inferior vena cava and the right atrium. The different route of extension makes a difference in the ease of excision of tumor masses. Using MEDLINE together with the references in each publication, we identified all cases of intracardiac leiomyomatosis reported in the English literature in the period between 1980 and 2003 and performed a brief review on this potentially lethal disease entity.

Aims Cysteamine is used to reduce tissue cystine content in patients suffering from nephropathic cystinosis. The objectives of the current study were to investigate pharmacokinetics and pharmacodynamics of cysteamine bitartrate in children and young adults with nephropathic cystinosis. Methods Cysteamine bitartrate was administered to 11 cystinosis patients at their regular dose level in a single-dose, open-label, steady-state study. Blood samples were collected and analysed for plasma cysteamine and white blood cell cystine content and pharmacokinetic and pharmacodynamic parameters estimated by NONMEM analysis using a linked pharmacokinetic–pharmacodynamic model. Results Cysteamine was rapidly cleared from the plasma (mean CL/F = 32.3 ml min−1 kg−1, range = 17.3–52.2), appeared to be extensively distributed (mean Vss/F = 15.1 l, range 2.7–32.3) and exhibited a mean Tmax of 1.4 h. White blood cell cystine content post-dosing was significantly decreased compared with pre- and post-dose values (average decrement approximately 47%). A counter-clockwise hysteresis was noted in all patients, suggestive of a lag time (mean Tlag = 0.44 h, range 0.22–0.92) between drug concentration and effect. Conclusions The results of this study establish that cysteamine is rapidly cleared from the plasma but that an every 6 h dosing interval adequately maintains white blood cell cystine content below the target of 1 nmol cystine per mg protein.

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions.

Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification.

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, β-glycerophosphate (βGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22α and smooth muscle α-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor α1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP −/− ) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5C1 demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5C1 and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5C1 and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

Objectives:To study collagen phagocytosis by human extravillous trophoblast.Methods:First-trimester extravillous trophoblastic cell lines and primary trophoblast cell preparations were cultured in vitro with collagen-coated fluorescent latex beads and fluorescent-labeled collagen. Confocal microscopy was used to demonstrate internalization of collagen and beads. The effect of cytochalasin B, temperature, metabolic inhibitors, and cytokines was studied by culturing trophoblast cells with tritated collagen. Acridine orange was used to stain for lysosomal compartments, and histochemical methods were used to demonstrate acid phosphatase in trophoblast cells.Results:Both cell lines and primary culture cells internalize collagen and beads. Confocal microscopy unequivocally localized the phagocytosed material to the intracellular compartment. Inhibition by cytochalasin B and culture at 4C of uptake of [3H] collagen suggested that the process was phagocytosis. Cytokines and growth factors did not affect phagocyto...

Objective To compare plasma catecholamine (noradrenaline and adrenaline) levels in pre-eclamptic to normotensive pregnancy, and to study the activity of synthetic enzymes for catecholamines in placental and trophoblastic cell cultures. We postulated that catecholamines might be an important signal secreted by the fetoplacental unit in pre-eclampsia. Methods We recruited 12 women with pre-eclampsia and 12 pregnant women with nonproteinuric hypertension undergoing delivery by caesarean section, 23 normotensive women undergoing elective caesarean section at term, and 26 normotensive primigravid women with ongoing pregnancies at gestations equivalent to those women with pre-eclampsia. We measured venous blood concentrations of catecholamines. Following delivery, we studied tyrosine hydroxylase (the rate limiting enzyme for catecholamine synthesis) activity in placental tissue of these women as well as from four eclamptic women not in the observer study. We used Northern blot analysis to quantify mRNA for tyrosine hydroxylase and dopamine-β-hydroxylase (D-P-H, a non-rate-limiting synthetic enzyme for catecholamine) in placental tissue, as well as in trophoblast cells in primary culture and trophoblast cell lines. Results Venous blood concentrations of noradrenaline were significantly higher in pre-eclamptic women compared with normotensive women. Tyrosine hydroxylase activity was greater in placental tissue from pre-eclamptic and eclamptic compared with normotensive pregnancies, as were mRNA levels for this enzyme. The mRNA levels for the non-rate-limiting D-β-H in women with pre-eclampsia were similar to those in normotensive pregnancies. First trimester trophoblast cells in primary culture and trophoblast cell lines transcript mRNA for tyrosine hydroxylase and D-β-H. Conclusions Trophoblasts have the capacity to secrete catecholamines, and we found increased activity of the rate-limiting synthetic enzyme in placental tissue from pre-eclamptic pregnancies. We postulate that the higher levels of catecholamines we found in the plasma of women with pre-eclampsia might be of placental origin. We hypothesise that in pre-eclampsia ischaemic trophoblast tissue secretes catecholamines as a physiological signal to increase maternal blood flow to the fetoplacental unit, which itself is spared the vasoconstrictor effects of catecholamines (placental vessels are known to be unresponsive to catecholamines). However, since the basic pathology—defective trophoblast invasion—is not corrected, the increased blood flow fails to resolve the ischaemia, and the secretion of catecholamines is therefore sustained or even enhanced. Noradrenaline is known to cause lipolysis. This results in breakdown of triglycerides to free fatty acids, which are oxidized to lipid peroxides. The latter are cytotoxic and cause widespread endothelial cell damage and dysfunction, culminating in the clinical syndrome of pre-eclampsia.

Background Computed tomography (CT), positron emission tomography CT (PET-CT) and magnetic resonance imaging (MRI) all play a role in the management of colorectal liver metastases (CRLM), but inappropriate over investigation can lead to delays in treatment and additional cost. This study aimed to determine the optimal sequence for pre-operative imaging pathway to minimise delays to treatment and healthcare costs. Methods All patients with colorectal liver metastases referred to a single tertiary liver specialist multidisciplinary team (MDT) between 2008 and 2011 were examined. Primary data of clinical and radiological outcomes of all patients were analysed. These data were used to construct and test 3 hypothetical imaging strategies – ‘Upfront’, ‘Sequential’ and ‘Hybrid’ models. Results Six hundred and forty four consecutive patients were included. One hundred and sixty five patients were excluded for curative resection following the initial CT review. Subsequently 167/433 patients did not proceed to hepatectomies. Eighty (47.9%) of these patients had extra-hepatic disease identified on PET-CT, and 29 were due to the exclusion by MRI liver. A resectable pattern of liver disease on initial CT did not exclude patients with occult disease on PET-CT. Based on cost analysis, assessment of initial CT, followed by MDT with subsequent PET-CT and MRI imaging thereafter (Hybrid model), was associated with the shortest time-to-decision and lowest cost. Conclusions Resectable pattern of liver metastases should not solely be used to determine the application of PET-CT for staging. Hybrid model is associated with the lowest cost and shortest time-to-treatment.