Taxol, a microtubule stabilizer, prevents atrial fibrillation in in vitro atrial fibrillation models using rabbit hearts. Increased atrial oxidative stress plays an important role in the pathogenesis of atrial fibrillation. A predisposition for atrial fibrillation can be produced by altering the activities of enzymes related to reactive oxygen species metabolism. Microtubule integrity is necessary for cardio-protection; microtubules are involved in many cellular processes, including enzymatic activity. Taxol, a common microtubule stabilizer, can protect cardiac myocytes from oxidative stress. However, its effects on atrial fibrillation remain unknown. In vitro rabbit heart models of ischemia-, stretch-, and cholinergic agitation-induced atrial fibrillation were developed. Using either a fluorometric or spectrophotometric assay, we measured reactive oxygen species and the activities of oxidative enzymes including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, mitochondrial electron transport chain complexes I, mitochondrial electron transport chain complexes III, and superoxide dismutase. Taxol effectively reduced the incidence of ischemia-, stretch- and cholinergic agitation-induced atrial fibrillation in in vitro rabbit heart models by 50%, 60%, and 40%. Moreover, Taxol decreased the level of reactive oxygen species by 46%, 49%, and 70% in ischemia-, stretch- and cholinergic agitation-induced atrial fibrillation models. Additionally, the activities of NADPH oxidase and xanthine oxidase were increased, whereas those of mitochondrial electron transport chain complexes I and III and superoxide dismutase were not affected. Taxol, a microtubule stabilizer, prevents atrial fibrillation in in vitro atrial fibrillation model using rabbit hearts. This stabilizer most likely prevents atrial fibrillation by reducing the level of reactive oxygen species.


Plasma inhibin B and antimüllerian hormone concentrations in boys: discriminating between congenital hypogonadotropic hypogonadism and constitutional pubertal delay. Inhibin B and antimüllerian hormone (AMH) are secreted by the Sertoli cells, stimulated by gonadotropins. We evaluated their use for discriminating between congenital hypogonadotropic hypogonadism (HH) and constitutional pubertal delay. Group 1 (n=39) was diagnosed with constitutional pubertal delay; group 2 (n=15), isolated HH; and group 3, pituitary stalk interruption syndrome (3a with [n=5] and 3b [n=8] without HH). At pubertal age, mean plasma inhibin B concentrations were similar in the groups having the same gonadotropin status. Individual concentrations were normal in groups 1 and 3b, but low in 6 group 2, and 3 group 3a patients. Four group 2, and 1 group 3a, also had low AMH. Among these 9 with low inhibin B, 1 in each group evaluated before puberty had had normal inhibin B and AMH. In contrast, inhibin B increased (P<.03) and AMH decreased (P<.02) in 7 group 3b patients from before to after puberty. There were 20 patients with inhibin B below 100 pg/mL including 4 group 1, 13 group 2, and 3 group 3a. The basal plasma follicle stimulating (FSH) and luteinizing (LH) hormone concentrations were < 1 IU/L in 18%, 36% in group 1, and in 56% and 100% in group 2. Before puberty, plasma inhibin B and AMH concentrations may be normal despite HH. At pubertal age, both are low in some patients with HH. These hormone values may help discriminate between HH and constitutional pubertal delay together with plasma FSH and LH.


Development of a surgical algorithm and optimized management of complications - based on a review of 706 abdominal free flaps for breast reconstruction. Microsurgical free flaps are a common method of breast reconstruction. Our institutional experiences with 706 lower abdomen based free perforator flaps are reported with special interest in presenting a therapeutic algorithm for efficient decision-making. A retrospective chart review was performed. All patients undergoing free flap surgery for breast reconstruction between July 2004 and November 2009 were included. Seven hundred and six free flaps were performed in 635 patients: 451 DIEAP-flaps, 254 fasciasparing (fs) TRAM-flaps and one SIEA flaps were performed. Five hundred sixty four women had a unilateral and 71 a bilateral reconstruction (142 flaps). The complication rate was 0.84% (6 flaps) for total flap loss, 1.27% (9 flaps) for partial flap loss and 2.40% (17 flaps) for partial flap loss less than 20%. 7 patients (1.11%) underwent microsurgical revision, where venous problems occurred. 3 of them were successful, in two cases a partial flap loss less than 30% occurred after the revision. In 2 cases a complete flap loss appeared. Other minor complications included: 23 patients (3.65%) had breast hematomas, 18 patients (2.54%) presented with delayed wound healing (9 abdominal and 9 breast delayed wound healings), and 3 patients (0.42%) with abdominal hernias, 14 patients (1.98%) complained of weakness of abdominal wall. Abdominally based free flaps a safe and reliable method and should therefore be offered as a standard method in a breast cancer center. The protocol and algorithm presented here can reduce complications in microsurgical breast reconstructive surgery.


Pulsed perfusion in a venous human organ culture model with a Windkessel function (pulsed perfusion venous HOC-model). The effectiveness of human saphenous vein grafting is limited by hyperplasia of the vessel wall. The current paper reports on a pulsed perfused venous human organ culture model (pp-venous HOC-model) with a Windkessel function. Saphenous vein grafts from 21 patients undergoing coronary bypass grafting were cultured either in venous or arterial hemodynamic conditions. Up to four vein segments were fixed in parallel connection and attached to a closed loop pulsed perfusion system consisting of large and small elastic tubes, mimicking the Windkessel function. First, after exposure to arterial blood pressure first signs of reactive cell proliferation (n.s.) were detected at day 4. Second, media thickness of the venous segments in the pulsed pressure group was decreased at day 4 (n.s.) and day 7 (n.s.). Third, staining against smooth muscle alpha actin and v. Willebrand factor was always positive at day 1, 4, and 7. Pulsed perfusion in a human venous organ culture model with a Windkessel function is an approach to better understand the events taking place during early arterial-vein grafting. First signs of reactive cell proliferation were detected at day four. A period of seven days as described in the current model is probably too short to detect reactive cell proliferation and medial thickening. If the device might be activated for a longer period of time, it should be a suitable model to characterize the effects of intra- and extravascular drug administration as treatment strategies of vein graft disease.


Comparative molecular epidemiology of Leishmania major and Leishmania tropica by PCR-RFLP technique in hyper endemic cities of Isfahan and Bam, Iran. Leishmania is an obligate intracellular protozoa, and the sandfly, as a vector, transmits infectious forms of the parasite to the vertebrate host. The etiologic agents of cutaneous leishmaniasis (CL), Leishmania major and Leishmania tropica, are the most prevalent factor in Iran, especially in the Isfahan and Bam regions. Because of the importance of CL in endemic regions and the interaction of species diversity factors in developing control strategies, several isolated Leishmania species from 2 hyperendemic regions of Iran, Isfahan and Bam cities, were examined in this study by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In this study, 340 samples were taken from clinically suspected CL patients to prepare slides for direct microscopy and cultures for promastigotes by PCR-RFLP. The internal transcribed spacer 1 (ITS1) region of genomic DNA was extracted and amplified with LITSr and L5.8s primers. Amplification by PCR-RFLP was performed to determine 4 kinds of genotype pattern of the species in the 2 main cities of Isfahan and Bam. Some of the product samples were sequenced and analyzed. Two genotypic groups were detected from L. major isolates, LmA and LmB; also L. tropica showed 2 patterns, LtA and LtB, in comparison with standard species. The most prevalent genotypes related to isolates of Isfahan were LmA and of Bam were LtA. These 2 genotypes were recorded as major etiologic factors of CL in these 2 regions. Leishmania major and L. tropica, the causative agents of zoonotic CL and anthroponotic CL, respectively, in Isfahan and Bam, are genetically highly polymorphic species, and a correlation may exist between genetic heterogeneous and clinical manifestation and geographic regions of the disease in humans.


Serum asymmetric dimethylarginine levels in normotensive obese individuals. Obesity is associated with an increased risk of developing insulin resistance, hyperinsulinemia, glucose intolerance, dyslipidemia, hypertension, premature atherosclerosis, and coronary artery disease. This study is designed to compare serum asymmetric dimethylarginine (ADMA) levels between obese individuals and controls. Fifty volunteers, 30 obese (13 men; mean age, 40±11 years) and 20 healthy controls (13 men; mean age, 44±10 years) were enrolled to this study. Measurement of ADMA was accomplished by high performance liquid chromatography. The mean body mass index of the obese group was significantly higher than that of the control group (35±4 vs 26±3 kg/m2; P=.001). The mean waist circumference of the obese subjects was also significantly higher compared with controls (111±11 vs 93±10 cm; P=.001). No significant difference was found concerning age, sex, blood pressures, and biochemistry parameters. Serum ADMA levels were significantly higher in obese individuals compared with healthy controls (5.4±3.3 vs 3.1±1.8 µmol/L; P=.006). A weak but significant correlation was identified between serum ADMA concentration and the waist circumference (r=0.282, P=.047). The results of the present study demonstrated that serum ADMA levels of normotensive obese individuals were significantly higher than healthy controls. Increased ADMA concentrations observed only in the obese group were deemed to be important regarding the development future of cardiovascular disease in the future.


Integrating lung and plasma expression of pneumo-proteins in developing biomarkers in COPD: a case study of surfactant protein D. Surfactant protein D (SP-D) is a promising blood biomarker in patients with chronic obstructive pulmonary disease (COPD). Nevertheless, circulating levels of SP-D are not related to pulmonary functions. In the present exploratory study, we created a simple index of plasma to bronchoalveolar lavage (BAL) fluid ratio of SP-D (pSP-D/bSP-D), and determined whether this index would relate to the severity of airflow limitation and hence represent a superior biomarker than pSP-D alone. In 50 ex and current smokers (mean age 57.6±7.8 years, 74% men), SP-D was measured in BAL fluid and plasma samples, and the relationships between spirometric variables and a composite parameter - the pSP-D/bSP-D ratio were determined. There was a significant inverse correlation between the pSP-D/bSP-D ratio and the severity of airflow obstruction, as measured by FEV1/FVC (p=0.012). In contrast, no relationship was observed between FEV1/FVC and pSP-D alone. We suggest that integrating both lung and plasma expression of pneumo-proteins may be more useful than plasma expression alone in developing pneumo-proteins as potential biomarkers in COPD.


Association between documented coronary artery disease and urinary albumin, albumin to creatinine ratio. The aim of this study was to investigate whether the amount of urinary albumin concentration (UAC) or urinary albumin to creatinine ratio (UACR) is more strongly associated with angiographically documented coronary artery disease (CAD). A total of 199 consecutive patients [11 9(60%) male, 80 (40%) female, mean age =57±10] undergoing diagnostic coronary angiography were included in the study. Significant coronary artery disease was defined as a stenosis equal to or above 50% in the main coronary artery or in one of the other branches. UAC and UACR were calculated from the urine. Baseline clinical parameters, UAC and UACR were compared between subjects with and without CAD. Factors predicting CAD were evaluated by multivariate analysis. Baseline clinical and laboratory characteristics of patients with and without CAD were not different except for a slightly male predominance in patients with CAD. Patients with CAD had significantly higher UACs and UACRs than patients without CAD (32.14±31.27 mg/day vs. 15.61±16.70 mg/day, p=0.01; 9.11±7.42 mg/g vs. 4.80±3.28 mg/g, p=0.009). A positive correlation was found between Gensini score and UACR (p=0.01), whereas no correlation was found between Gensini and UAC. UACR was the only significant parameter for the presence of CAD in the multivariate analysis adjusted for age, sex, other well known CAD risk factors, UAC and UACR. Our preliminary results suggest that UACR is more closely associated with angiography documented CAD than is the level of UAC itself, but UACR maybe more significantly associated with angiography documented CAD than with the levels of UAC.


Platinum - sensitive relapsed epithelial ovarian cancer: not all the patients benefit from reinduction with carboplatin and paclitaxel. It is widely accepted that patients with ovarian cancer relapsing 6 to 12 months after completion of a platinum-based regimen are considered to be partially platinum-sensitive. The aim of this study was to evaluate and correlate the efficacy and toxicity of reinduction with paclitaxel-carboplatin in a platinum-sensitive epithelial ovarian cancer patient cohort with previous platinum-free interval (PFI). We studied retrospectively 39 patients with platinum-sensitive epithelial ovarian cancer, who received primary chemotherapy at the Institute for Oncology and Radiology of Serbia, between January 2002 and May 2008. All patients were treated with paclitaxel and carboplatin for metastatic disease. Subsequent to progression, patients were re-treated with the same chemotherapy in group A (PFI 6-12 months) or group B (PFI ≥12 months). The number of patients in group A was 14, and in group B it was 25. Response rate to reinduction in group A was 36% (partial response, 36%; stable disease, 0%; progressive disease, 64%) and in group B was 68% (complete response, 60%; partial response, 8%; stable disease, 4%; progressive disease, 28%; P=.05). The median response duration of the patients in group A arm was 20 months, whereas it was 17 months for those in group B (P=.721). There were no significant differences in the toxicity profile between the 2 groups. In terms of objective response rate this study supports the reinduction of carboplatin and paclitaxel in patients with a prior PFI of at least 12 months only. To define the best approach and the optimal treatment of patients with partially platinum-sensitive disease more precisely, future studies should apply treatment-free interval as a stratification criterion.


Ruptured sinus of Valsalva aneurysm in a child with Down syndrome: a rare cardiac anomaly. As many as forty to fifty per cent of children with Down syndrome are born with a cardiac anomaly. Sinus of Valsalva aneurysm in this syndrome is extremely rare, only three previous reports, and of adult onset. An asymptomatic 9 and a half year boy with Down syndrome presented with a cardiac murmur at routine examination. Clinical examination, transthoracic and transesopahageal echocardiography revealed the presence of a ruptured sinus of Valsalva aneurysm from the non - coronary sinus protruding into the right atrium. The child underwent successful surgical repair with full recovery. This fourth case report of sinus of Valsalva aneurysm occurring in association with Down syndrome is the youngest to date. Physicians caring for Down syndrome patients ought to be aware of this late onset cardiac malformation. Serial echocardiographic assessment of the aortic sinus of Valsalva in these patients may offer early detection.


Hearing preservation in partial deafness treatment. Partial deafness (PD) is a condition in which hearing loss occurs in at least 1 frequency critical to speech understanding. Current options for partial deafness treatment (PDT) rely on preoperative hearing preservation, which, along with the use of different means of acoustic and electric stimulation, enable extending the indications for various assistive hearing devices. Possible solutions include acoustic methods only, the use of hearing aids or middle ear implants, electric complementation, and a combination of electric and acoustic stimulation. A total of 95 patients (63 adults, 32 children) with different types of PD were treated using selected types of electrodes and the optimal "round window" approach to the inner ear, with at least 36 months of observation. The extension of PDT indications created an opportunity for patients with different hearing impairments who obtained no benefit from a hearing aid and did not qualify for standard cochlear implant application. The authors' observations are based on the findings that preservation of preoperative hearing had been achieved in 97.1% of adult patients (8 years' observation) and in 100% of children (6 years' observation). Those results send the important message that PDT is feasible and effective. To allow comparison of PDT results from different studies, the authors developed the Skarzynski PDT classification system, which permits the comparison of postoperative results, including the degree of hearing preservation and, more importantly, the patient's understanding of speech after treatment.


Immunosuppressive regimen and prevalence of chronic kidney disease in orthotopic heart transplant recipients. Heart transplantation is an established treatment for advanced heart failure. Heart transplant recipients experience a number of complications related to pharmacological immunosuppression and infections. Chronic kidney disease (CKD) is an important long-term complication of all forms of organ transplantation other than renal. It has been shown that a GFR <60ml/min is predictive of premature cardiovascular death. The aim of our study was to assess kidney function in relation to the type of immunosuppression regimen used. We analyzed 169 patients who underwent their first orthotopic heart transplant. The immunosuppressive regimen of prevalent patients consisted of tacrolimus (n=60), cyclosporine (n=109), in combination with mycophenolate mofetil (n=134), azathioprine (n=4), everolimus (n=28) or sirolimus (n=8). GFR was estimated using 3 formulae: CKD-EPI, MDRD and the Cockcroft-Gault. Complete blood count, urea, serum lipids, fasting glucose, creatinine, and NT-proBNP were also analyzed. In patients treated with tacrolimus, we observed a higher eGFR (all formulae), a greater 24-hour creatinine clearance, and lower serum cholesterol, LDL-cholesterol and triglyceride values than in patients treated with cyclosporine. Patients treated with tacrolimus were younger than those treated with cyclosporine. However, the time elapsed after OHT was similar in both groups. The prevalence of CKD (eGFR <60 ml/min) in the tacrolimus-treated patients was 45%, whereas in the cyclosporine treated group it was 87% (p<0.01). The prevalence of CKD is high in heart transplant recipients. Tacrolimus-based immunosuppression was associated with better kidney function and more favorable lipid profile. Evaluation of renal function is important in order to select the appropriate strategy by individually tailored therapy to achieve the best possible outcomes.


Application of manuka honey and GENADYNE A4 negative pressure wound therapy system in a 55-year-old woman with extensive phlegmonous and necrotic lesions in the abdominal integuments and lumbar region after traumatic rupture of the colon. Antibiotic resistance of bacteria is on the rise and thus, the discovery of alternative therapeutic agents is urgently needed. Honey possesses good therapeutic potential, including wound healing properties and antimicrobial activity. The authors report on the case of a 55-year-old woman with extensive phlegmonous and necrotic lesions of the abdominal integuments and the lumbar area following traumatic colonic rupture, treated with Manuka honey wound dressings and the GENADYNE A4 negative pressure wound healing system. The application of the Manuka honey and the GENADYNE A4 negative pressure wound healing system in treating phlegmonous lesions of the abdominal integuments after rupture of the colon brought good effects, ultimately enabling skin autografting on the wound site and complete wound healing.


Stabilizing severely open fractures of the tibia with a surgical suture: an alternative and feasible method for osteosynthesis. Outcomes concerning treatment of severely open fractures of the tibia have achieved great success with the advent of powerful antibiotics, superb microsurgery techniques, and advanced stabilization instruments. However, severe pollution in the wound and high comminution of the fragments may restrict application of internal or external fixators. The condition could appear more complicated if osteoporosis and polytrauma are present or if those special instruments are unavailable. We tried using a surgical suture to stabilize severely open fractures of the tibia in the elderly with osteoporosis. A 65-year-old woman with a severely open fracture of the tibia was treated with a surgical suture, and the result was satisfactory in early period. We discuss the new but traditional osteosynthesis method. A surgical suture osteosynthesis could act as an additional alternative when special fractures or conditions occur. Postoperative results in early period are satisfactory. Long-term results and clinical experience should be analyzed and discussed.


The biopsychosocial model, religion and a unified theory of diseases.


The pulse arrival time approach for non-invasive hemodynamic monitoring in low-acuity settings. Hemodynamic monitoring in hospitalized patients is crucial since in clinical practice unexpected deterioration of cardiovascular function (e.g. pulmonary embolism resulting in syncope) remains a serious problem and an important cause of death. Standard pulse oximetrie in low acuity settings is nowadays predominately used to monitor peripheral oxygen saturation. Of note, there is evidence that additional analyses of pulse wave characteristics might be a valuable source of information to generate additional insights into the cardiorespiratory status of the patient. Herein, we present our initial experience using a hemodynamic surrogate measure called pulse arrival time (PAT) derived from standard photoplethysmography (PPG), single-lead electrocardiogram (ECG), and context information based on a small body-worn monitoring system for continuous unsupervised monitoring during short-term physical exercise and posture changes. This paper presents examples of using PPG signals for continuous non-invasive monitoring and discusses PAT and heart rate responses observed during short-term physical exercise, posture changes, and tilt table testing resulting in syncope. Our results indicate that hemodynamic adaptatations being detected by PPG and ECG analysis might be useful to monitor high-risk patients in low acuity settings. Context information on posture and physical activity is imperatively needed to interpret PAT monitoring.


Prevalence of psychoactive substances use in a Lithuanian women's prison revisited after 5 years. This paper re-examines the prevalence of psychoactive substances (tobacco, alcohol, narcotic drugs) use among inmates in a Lithuanian women's prison. The main goal of this study was to determinate the changes in the use of the psychoactive substances in a women's prison in Lithuania. We accomplished the retesting of the first ever investigation of this kind, carried out in 2004, using the same questionnaire, in the only women's prison in Lithuania. In June 2009, 71 (27.8%) women of 255 inmates of the prison were given questionnaires with information about the aim of the study, stating that the study was voluntary and anonymous, and obtaining permission for release of information. The results were compared with the previous investigation. A statistical analysis was carried out using SPSS 17.0. Tobacco smokers comprised 85.3% of respondents.; the average age at which respondents started to smoke was 14±7.3 years; 57.7% of respondents had tried narcotic drugs at least once; 22.5% of respondents used drugs (in 2004 we had found no drug use in this women's prison); 18.3% of respondents indicated that they narcotic drugs were tried for the first time away, 4.2% - in a custodial establishment. Psychoactive substances are often used due to their psychological effect. inmates constitute a high-risk group of drug users and distributors of narcotic drugs. Intravenous narcotics stimulating dangerous behavior prevail in Lithuanian prisons. Women in prison are especially prone to smoking.


In utero surgery - current state of the art: part I. Fetal surgery, also referred to as in utero, prenatal or intrauterine surgery, consists of treatment of congenital malformations during fetal period. The idea of treating malformations diagnosed in the course of intrauterine life dates back to 1963, when Lilly performed first blood transfusion in the fetus. Ever since then, it has been introduced as treatment option in series of lethal malformations. Efforts are being made to treat nonlethal malformations by means of fetal surgery. A comprehensive search of the literature using MEDLINE and PubMed between 1925 and February 2009 was performed. Search terms for MEDLINE and PubMed were: fetal surgery, foetal surgery, in utero surgery, prenatal surgery, and in utero treatment. In addition, information was obtained at Web sites of International Medicine and Surgery Society and the University of California Fetal Treatment Centre. Our attention focused on the overall view on fetal surgery history, its development and perspectives. Moreover, owing to the authors' interest in plastic surgery, some remarks on the role of intrauterine medicine and surgery in this branch of medicine were made. In utero surgery may be regarded as an efficient tool of preventive medicine. It offers some advantages that no other branch of medicine may offer. However, its implementation is more troublesome than it used to be regarded as before. Therefore only selected cases may benefit from its advantages. Nevertheless, current tendencies are to include, after thorough evaluation of benefit - risk ratio, some new indications for fetal surgery.


Influence of statins on effector and regulatory immune mechanisms and their potential clinical relevance in treating autoimmune disorders. Studies have shown that HMG-CoA reductase inhibitors possess anti-inflammatory properties, for example, decreasing serum levels of proinflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) or decreasing the activity of numerous effector mechanisms. However, they also may influence the immune system by affecting a wide range of regulatory actions, for example, Treg- and Th2-type response activation as well as Th1 and Th17 response diminishing. These features provide the rationale for potential application of statins in treating chronic inflammatory diseases and other disorders with the involvement of the immune system. This article seeks to review the immunomodulatory actions of statins and their possible implementation in treating rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders in humans.


Future possibilities in glaucoma therapy. Glaucoma is a group of eye diseases causing irreversible optic nerve damage. This review presents 4 elements of future glaucoma treatment strategies: baroprotection, vasoprotection, neuroprotection and gene therapy. New baroprotection includes compounds that alter the actin cytoskeleton (rho-kinase inhibitors, latrunculin, cytochalasin), new drugs that enhance aqueous outflow via the trabecular meshwork (statins, steroid antagonists) and via the uveoscleral route (EP2 agonists, 5-HT2 agonists), as well as new classes of components that suppress aqueous humor formation (cannabinoids). Vasoprotection includes blocking reperfusion injury (NOS-2 inhibitors, endothelin blockers, MMP-9 inhibitors). Concerning neuroprotection antiamyloids antibodies, erythropoietin and caspase inhibitors are discussed. Gene therapy may target various effectors: the trabecular meshwork (cytoskeleton regulatory proteins, miocyllin, MMPs), the ciliary body epithelium (genes modifying aqueous humor production, neuropeptides), the ciliary body cells (MMPs, genes of local PGs biosynthesis, ciliary muscle relaxants), the retinal ganglion cells (neurotrophin genes, anti-apoptotic genes), Müller cells (neurotrophins, GLAST) and conjunctiva (gene of chloramphenicol acetyltransferase, inhibitor p21). Experimental studies on the graft mesenchymal stem cells and mature retinal cells to replace the dead retinal ganglion cells are advanced. Immunotherapy, offering a vaccination, providing protection against loss of retinal ganglion cells, has been investigated.


Targeting transcription factor Stat4 uncovers a role for interleukin-18 in the pathogenesis of severe lupus nephritis in mice. Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.


De novo development of circulating anti-endothelial cell antibodies rather than pre-existing antibodies is associated with post-transplant allograft rejection. Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.


Predicting idiopathic toxicity of cisplatin by a pharmacometabonomic approach. Cisplatin has been one of the most widely used anticancer agents, but its nephrotoxicity remains a dose-limiting complication. Here, we evaluated the idiopathic nature and the predose prediction of cisplatin-induced nephrotoxicity using a nuclear magnetic resonance (NMR)-based pharmacometabonomic approach. Cisplatin produced serious toxic responses in some animals (toxic group), but had little effect in others (nontoxic group), as judged by hematological and histological results. The individual metabolic profiles, assessed by urine NMR spectra, showed large differences between the post-administration profiles of the two groups, indicating the relevance of the NMR approach. Importantly, multivariate analysis of the NMR data showed that the toxic and nontoxic groups can be differentiated based on the pretreatment metabolite profiles. Leave-one-out analysis, performed to evaluate the practical performance of our approach, gave a 66% accuracy rate in predicting toxic responses based on the pretreatment metabolite profiles. Hence, we provide a working model that can explain the idiopathic toxicity mechanism based on marker metabolites found by NMR analysis consistent with tissue NADH measurements. Thus, a pharmacometabonomic approach using pretreatment metabolite profiles may help expedite personalized chemotherapy of anticancer drugs.


Urinary levels of regenerating islet-derived protein III β and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats. A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicin's nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine.


Changes in dietary protein intake has no effect on serum cystatin C levels independent of the glomerular filtration rate. Cystatin C is being considered as a replacement for serum creatinine in the estimation of the glomerular filtration rate (GFR); however, its plasma levels might be affected by factors other than the GFR, such as protein intake. We performed a post hoc analysis of the data in the Modification of Diet in Renal Disease study, in which we compared serum creatinine and cystatin C levels in 741 patients with available estimates of protein intake at baseline prior to their randomization to diets containing various amounts of protein, and at 2 years of follow-up in 426 of these patients in whom a cystatin C measurement was available. The 503 patients in study A (GFR 25-55 ml/min per 1.73 m(2)) had been assigned a low (0.58 g/kg per day) or a usual (1.3 g/kg per day) protein intake, and the 238 participants in study B (GFR 13-24 ml/min per 1.73 m(2)) were assigned a very low (0.28 g/kg per day) or the low protein intake. In either study group, lowering the dietary protein intake reduced the change in creatinine, but did not have a significant change in cystatin C. Thus, in patients with moderate-to-severe chronic kidney disease, serum cystatin C unlike serum creatinine was not affected by dietary protein intake independent of changes in GFR. Hence, cystatin C may allow more accurate estimates of GFR than creatinine for patients with reduced protein intake. Further study of other non-GFR determinants of cystatin C is needed before the widespread adoption.


Discussing standards.

Discussing standards.


The Archon Genomics X PRIZE for whole human genome sequencing.


Regression of microalbuminuria in type 1 diabetes is associated with lower levels of urinary tubular injury biomarkers, kidney injury molecule-1, and N-acetyl-β-D-glucosaminidase. Elevated urinary albumin excretion in patients with type 1 diabetes reverts to normoalbuminuria in a majority of patients but advances toward proteinuria in some. In order to gain valuable insights into the early pathophysiology of diabetic nephropathy we evaluated the association of kidney tubular injury biomarkers with changes in albuminuria in patients with type 1 diabetes mellitus. Urine levels of kidney injury molecule-1 (KIM-1), N-acetyl-β-D-glucosaminidase (NAG), and some inflammatory markers were determined in 38 healthy individuals and 659 patients with type 1 diabetes mellitus having varying degrees of albuminuria. Urinary interleukin-6, CXCL10/IP-10, NAG, and KIM-1 levels were very low in healthy individuals, increased in type 1 patients with normoalbuminuria, and were highest in diabetic patients that had microalbuminuria. Low baseline concentrations of urinary KIM-1 and NAG both individually and collectively were significantly associated with the regression of microalbuminuria over the subsequent 2 years; an effect independent of clinical characteristics. Progression and regression of microalbuminuria were unrelated to urinary levels of interleukins 6 and 8, CXCL10/IP-10, and monocyte chemoattractant protein-1. Thus our results show that lower urinary KIM-1 and NAG levels were associated with the regression of microalbuminuria in type 1 diabetes mellitus. Hence, tubular dysfunction is a critical component of the early course of diabetic nephropathy.


No evidence for lateral gene transfer between salmonids and schistosomes.


Marker papers and data citation.

Marker papers and data citation.


Lack of evidence for DNA methylation of Invader4 retroelements in Drosophila and implications for Dnmt2-mediated epigenetic regulation.


A common mechanism for microcephaly.

A common mechanism for microcephaly.


Glutamate receptors and learning and memory.


Genome-wide association studies coming of age in rice.


High-novelty-preference rats are predisposed to compulsive cocaine self-administration. Sensation/novelty-seeking is amongst the best markers of cocaine addiction in humans. However, its implication in the vulnerability to cocaine addiction is still a matter of debate, as it is unclear whether this trait precedes or follows the development of addiction. Sensation/novelty-seeking trait has been identified in rats on the basis of either novelty-induced locomotor activity (high-responder (HR) trait) or novelty-induced place preference (high-novelty-preference trait (HNP)). HR and HNP traits have been associated with differential sensitivity to psychostimulants. However, it has recently been demonstrated that HR rats do not develop compulsive cocaine self-administration (SA) after protracted exposure to the drug, thereby suggesting that at least one dimension of sensation/novelty seeking in the rat is dissociable from the vulnerability to switch from controlled to compulsive cocaine SA. We therefore investigated whether HNP, as measured as the propensity to choose a new environment in a free choice procedure, as opposed to novelty-induced locomotor activity, predicts the vulnerability to, and the severity of, addiction-like behavior for cocaine. For this, we identified HR/LR rats and HNP/LNP rats before any exposure to cocaine. After 60 days of cocaine SA, each rat was given an addiction score based on three addiction-like behaviors (persistence of responding when the drug is signaled as not available, high breakpoint under progressive ratio schedule and resistance to punishment) that resemble the clinical features of drug addiction, namely inability to refrain from drug seeking, high motivation for the drug and compulsive drug use despite adverse consequences. We show that, as opposed to HR rats, HNP rats represent a sub-population predisposed to compulsive cocaine intake, displaying higher addiction scores than LNP rats. This study thereby provides new insights into the factors predisposing to cocaine addiction, supporting the hypothesis that addiction is sustained by two vulnerable phenotypes: a 'drug use prone' phenotype such as HR which brings an individual to develop drug use and an 'addiction prone' phenotype, such as HNP, which facilitates the shift from sustained to compulsive drug intake and addiction.


Efficacy of antimanic treatments: meta-analysis of randomized, controlled trials. We conducted meta-analyses of findings from randomized, placebo-controlled, short-term trials for acute mania in manic or mixed states of DSM (III-IV) bipolar I disorder in 56 drug-placebo comparisons of 17 agents from 38 studies involving 10,800 patients. Of drugs tested, 13 (76%) were more effective than placebo: aripiprazole, asenapine, carbamazepine, cariprazine, haloperidol, lithium, olanzapine, paliperdone, quetiapine, risperidone, tamoxifen, valproate, and ziprasidone. Their pooled effect size for mania improvement (Hedges' g in 48 trials) was 0.42 (confidence interval (CI): 0.36-0.48); pooled responder risk ratio (46 trials) was 1.52 (CI: 1.42-1.62); responder rate difference (RD) was 17% (drug: 48%, placebo: 31%), yielding an estimated number-needed-to-treat of 6 (all p<0.0001). In several direct comparisons, responses to various antipsychotics were somewhat greater or more rapid than lithium, valproate, or carbamazepine; lithium did not differ from valproate, nor did second generation antipsychotics differ from haloperidol. Meta-regression associated higher study site counts, as well as subject number with greater placebo (not drug) response; and higher baseline mania score with greater drug (not placebo) response. Most effective agents had moderate effect-sizes (Hedges' g=0.26-0.46); limited data indicated large effect sizes (Hedges' g=0.51-2.32) for: carbamazepine, cariprazine, haloperidol, risperidone, and tamoxifen. The findings support the efficacy of most clinically used antimanic treatments, but encourage more head-to-head studies and development of agents with even greater efficacy.


The subjective, reinforcing, and analgesic effects of oxycodone in patients with chronic, non-malignant pain who are maintained on sublingual buprenorphine/naloxone. Some sources suggest that significant misuse of opioid drugs exists among patients with chronic pain. However, the risk factors and motivation behind their abuse may differ from those of other opioid abusers. This study sought to examine the abuse liability of oxycodone among patients with chronic, non-malignant pain who met the DSM-IV criteria for opioid abuse. Eighteen opioid-dependent patients with chronic pain lived on an in-patient unit of the New York State Psychiatric Institute during the 7-week study. Participants were given oral oxycodone (0, 10, 20, 40, and 60 mg/70 kg) while maintained on various doses of sublingual buprenorphine/naloxone (Bup/Nx; 2/0.5, 8/2, and 16/4 mg/day). Doses of both medications were administered under double-blind conditions. Oxycodone produced an overall positive, but less robust, subjective profile than previously reported in recreational opioid users without pain. Furthermore, unlike our findings in recreational opioid users and more similar to effects in non-drug-abusing individuals, oxycodone failed to serve as a reinforcer. As for the maintenance drug, Bup/Nx produced a dose-related reduction in some of the effects of acutely administered oxycodone. These data suggest that sublingual Bup/Nx has the potential as an analgesic medication and further research should investigate its use in treating patients with chronic pain who abuse opioids.


Serotonin and dopamine play complementary roles in gambling to recover losses. Continued gambling to recover losses--'loss chasing'--is a prominent feature of social and pathological gambling. However, little is known about the neuromodulators that influence this behavior. In three separate experiments, we investigated the role of serotonin activity, D(2)/D(3) receptor activity, and beta-adrenoceptor activity on the loss chasing of age and IQ-matched healthy adults randomized to treatment or an appropriate control/placebo. In Experiment 1, participants consumed amino-acid drinks that did or did not contain the serotonin precursor, tryptophan. In Experiment 2, participants received a single 176 μg dose of the D(2)/D(3) receptor agonist, pramipexole, or placebo. In Experiment 3, participants received a single 80 mg dose of the beta-adrenoceptor blocker, propranolol, or placebo. Following treatment, participants completed a computerized loss-chasing game. Mood and heart rate were measured at baseline and following treatment. Tryptophan depletion significantly reduced the number of decisions made to chase losses, and the number of consecutive decisions to chase, in the absence of marked changes in mood. By contrast, pramipexole significantly increased the value of losses chased and diminished the value of losses surrendered. Propranolol markedly reduced heart rate, but produced no significant changes in loss-chasing behavior. Loss chasing can be thought of as an aversively motivated escape behavior controlled, in part, by the marginal value of continued gambling relative to the value of already accumulated losses. Serotonin and dopamine appear to play dissociable roles in the tendency of individuals to gamble to recover, or to seek to 'escape' from, previous losses. Serotonergic activity seems to promote the availability of loss chasing as a behavioral option, whereas D(2)/D(3) receptor activity produces complex changes in the value of losses judged worth chasing. Sympathetic arousal, at least as mediated by beta-adrenoceptors, does not play a major role in laboratory-based loss-chasing choices.


A dopamine D1 receptor-dependent β-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice. Morphine is a widely used analgesic in humans that is associated with multiple untoward effects, such as addiction and physical dependence. In rodent models, morphine also induces locomotor activity. These effects likely involve functionally selective mechanisms. Indeed, G protein-coupled receptor desensitization and adaptor protein β-arrestin 2 (βarr2) through its interaction with the μ-opioid receptor regulates the analgesic but not the rewarding properties of morphine. However, βarr2 is also required for morphine-induced locomotor activity in mice, but the exact cellular and molecular mechanisms that mediate this arrestin-dependent behavior are not understood. In this study, we show that βarr2 is required for morphine-induced locomotor activity in a dopamine D1 receptor (D1R)-dependent manner and that a βarr2/phospho-ERK (βarr2/pERK) signaling complex may mediate this behavior. Systemic administration of SL327, an MEK inhibitor, inhibits morphine-induced locomotion in wild-type mice in a dose-dependent manner. Acute morphine administration to mice promotes the formation of a βarr2/pERK signaling complex. Morphine-induced locomotor activity and formation of the βarr2/pERK signaling complex is blunted in D1R knockout (D1-KO) mice and is presumably independent of D2 dopamine receptors. However, D1Rs are not required for morphine-induced reward as D1-KO mice show the same conditioned place preference for morphine as do control mice. Taken together, these results suggest a potential role for a D1R-dependent βarr2/pERK signaling complex in selectively mediating the locomotor-stimulating but not the rewarding properties of morphine.


Correlation between imatinib pharmacokinetics and clinical response in Japanese patients with chronic-phase chronic myeloid leukemia. Despite the outstanding results generally obtained with imatinib mesylate (IM) in the treatment of chronic myeloid leukemia (CML), some patients show a poor molecular response. To evaluate the relationship between steady-state trough plasma IM concentration (IM-C(min)) and clinical response in CML patients, we integrated data from six independent Japanese studies. Among 254 CML patients, the mean IM-C(min) was 1,010.5 ng/ml. Importantly, IM-C(min) was significantly higher in patients who achieved a major molecular response (MMR) than in those who did not (P = 0.002). Multivariate analysis showed that an MMR was associated with both age (odds ratio (OR) = 0.97 (0.958-0.995); P = 0.0153) and with IM-C(min) (OR = 1.0008 (1.0003-1.0015); P = 0.0044). Given that patients with IM-C(min) values >1,002 ng/ml had a higher probability of achieving an MMR in our large cohort (P = 0.0120), the data suggest that monitoring of IM levels in plasma may improve the efficacy of IM therapy for CML patients.


Control of intestinal Nod2-mediated peptidoglycan recognition by epithelium-associated lymphocytes. Innate immune recognition of the bacterial cell wall constituent peptidoglycan by the cytosolic nucleotide-binding oligomerization domain 2 (Nod2) receptor has a pivotal role in the maintenance of intestinal mucosal homeostasis. Whereas peptidoglycan cleavage by gut-derived lysozyme preserves the recognition motif, the N-acetylmuramoyl-L-alanine amidase activity of the peptidoglycan recognition protein 2 (PGLYRP-2) destroys the Nod2-detected muramyl dipeptide structure. PGLYRP-2 green fluorescent protein (GFP) reporter and wild-type mice were studied by flow cytometry and quantitative RT-PCR to identify Pglyrp-2 expression in cells of the intestinal mucosa and reveal a potential regulatory function on epithelial peptidoglycan recognition. CD3(+)/CD11c(+) T lymphocytes revealed significant Pglyrp-2 expression, whereas epithelial cells and intestinal myeloid cells were negative. The mucosal Pglyrp-2-expressing lymphocyte population demonstrated a mixed T-cell receptor (TCR) αβ or γδ phenotype with predominant CD8α and less so CD8β expression, as well as significant staining for the activation markers B220 and CD69, presenting a typical intraepithelial lymphocyte phenotype. Importantly, exposure of peptidoglycan to PGLYRP-2 significantly reduced Nod2/Rip2-mediated epithelial activation. Also, moderate but significant alterations of the intestinal microbiota composition were noted in Pglyrp-2-deficient animals. PGLYRP-2 might thus have a significant role in regulation of the enteric host-microbe homeostasis.


Twice-weekly administration of kisspeptin-54 for 8 weeks stimulates release of reproductive hormones in women with hypothalamic amenorrhea. Kisspeptin is a novel therapeutic target for infertility. A single kisspeptin-54 (KP-54) injection acutely stimulates the release of reproductive hormones in women with hypothalamic amenorrhea (HA), a commonly occurring condition characterized by absence of menstruation; however, twice-daily administration of KP-54 results in tachyphylaxis. We determined the time course of desensitization to twice-daily KP-54 injections, compared the effects of twice-daily and twice-weekly administration regimens of KP-54, and studied the effects of long-term twice-weekly administration of KP-54 on the release of reproductive hormones in women with HA. When KP-54 was administered twice daily, responsiveness to luteinizing hormone (LH) diminished gradually, whereas responsiveness to follicle-stimulating hormone (FSH) was nearly abolished by day 2. Twice-weekly KP-54 administration resulted in only partial desensitization, in contrast to the complete tolerance achieved with twice-daily administration. Women with HA who were treated with twice-weekly KP-54 injections had significantly elevated levels of reproductive hormones after 8 weeks as compared with treatment with saline. No adverse effects were observed. This study provides novel pharmacological data on the effects of KP-54 on the release of reproductive hormones in women with HA.


A phase I study of the pharmacokinetic and safety profiles of oral pazopanib with a high-fat or low-fat meal in patients with advanced solid tumors. Pazopanib is an oral angiogenesis inhibitor of vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor receptor, and cytokine receptor. This open-label, randomized, crossover, phase I study evaluated the effect of low- and high-fat meals on the pharmacokinetics (PK) of pazopanib in patients with advanced solid tumors. Patients participated in either the lead-in cohort or randomized food-effect cohort. Patients in the lead-in cohort were administered a single dose of pazopanib 400 mg with a high-fat meal. Patients in the food-effect cohort were randomized to receive single doses of pazopanib 800 mg in fed condition (high- or low-fat meal) or fasting condition, in random sequence 14 days apart. After completion of the study, patients were given the opportunity to continue treatment with daily pazopanib 800 mg. Administration of pazopanib with both low- and high-fat meals increased maximum observed plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) by approximately twofold as compared with the corresponding values when administered to patients in the fasted condition. Therefore, pazopanib should be administered to patients in the fasted state so as to minimize within- and between-day variability in the systemic exposure to pazopanib in patients with cancer.


The TNF-family cytokine TL1A drives IL-13-dependent small intestinal inflammation. The tumor necrosis factor (TNF)-family cytokine TL1A (TNFSF15) costimulates T cells through its receptor DR3 (TNFRSF25) and is required for autoimmune pathology driven by diverse T-cell subsets. TL1A has been linked to human inflammatory bowel disease (IBD), but its pathogenic role is not known. We generated transgenic mice that constitutively express TL1A in T cells or dendritic cells. These mice spontaneously develop IL-13-dependent inflammatory small bowel pathology that strikingly resembles the intestinal response to nematode infections. These changes were dependent on the presence of a polyclonal T-cell receptor (TCR) repertoire, suggesting that they are driven by components in the intestinal flora. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) were present in increased numbers despite the fact that TL1A suppresses the generation of inducible Tregs. Finally, blocking TL1A-DR3 interactions abrogates 2,4,6 trinitrobenzenesulfonic acid (TNBS) colitis, indicating that these interactions influence other causes of intestinal inflammation as well. These results establish a novel link between TL1A and interleukin 13 (IL-13) responses that results in small intestinal inflammation, and also establish that TL1A-DR3 interactions are necessary and sufficient for T cell-dependent IBD.


Acute alertness-promoting effects of a novel histamine subtype-3 receptor inverse agonist in healthy sleep-deprived male volunteers. The alertness-promoting effect of MK-0249 (10 or 50 mg), a histamine subtype-3 receptor (HRH3) inverse agonist (IA), was evaluated in the stimulant reference sleep deprivation model (SRSDM) using a double-blind, double-dummy, placebo- and modafinil- (200 mg) controlled, four-period crossover design in 24 healthy young men. The two primary hypotheses were related to sleep latency (first appearance of one epoch of stage 2, 3, or 4 or REM sleep, as detected using polysomnography (PSG)) at 8:00 AM on day 2. Statistically significant increases in sleep latency were observed in association with the use of modafinil 200 mg (9.07 min; P < 0.0001), MK-0249 50 mg (5.17 min; P = 0.008), and MK-0249 10 mg (5.45 min; P = 0.005) at the maintenance of wakefulness test (MWT) at 8:00 AM. Sleep latency was higher when averaged over all MWT time points (P < 0.0001 for modafinil and for both doses of MK-0249). The alertness-promoting effect with the use of MK-0249 in the SRSDM suggests that HRH3 IAs may be effective in disorders involving excessive somnolence.


Short-term adaptation of conditioned fear responses through endocannabinoid signaling in the central amygdala. The cannabinoid receptor type 1 (CB1) and the central nucleus of the amygdala (CeA) are both known to have crucial roles in the processing of fear and anxiety, whereby they appear to be especially involved in the control of fear states. However, in contrast to many other brain regions including the cortical subregions of the amygdala, the existence of CB1 in the CeA remains enigmatic. In this study we show that CB1 is expressed in the CeA of mice and that CB1 in the CeA mediates short-term synaptic plasticity, namely depolarization-induced suppression of excitation (DSE) and inhibition (DSI). Moreover, the CB1 antagonist AM251 increased both excitatory and inhibitory postsynaptic responses in CeA neurons. Local application of AM251 in the CeA in vivo resulted in an acutely increased fear response in an auditory fear conditioning paradigm. Upon application of AM251 in the basolateral nucleus of the amygdala (BLA) in an otherwise identical protocol, no such acute behavioral effects were detected, but CB1 blockade resulted in increased fear responses during tone exposures on the subsequent days. Moreover, we observed that the efficacy of DSE and DSI in the CeA was increased on the day following fear conditioning, indicating that a single tone-shock pairing resulted in changes in endocannabinoid signaling in the CeA. Taken together, our data show the existence of CB1 proteins in the CeA, and their critical role for ensuring short-term adaptation of responses to fearful events, thereby suggesting a potential therapeutic target to accompany habituation-based therapies of post-traumatic symptoms.


Endoxifen, a new cornerstone of breast cancer therapy: demonstration of safety, tolerability, and systemic bioavailability in healthy human subjects. Endoxifen is an active metabolite of tamoxifen, a drug used in the treatment of breast cancer. In order to be clinically effective, tamoxifen must be converted to endoxifen by cytochrome P450 2D6 (CYP2D6). A study involving single escalating oral doses was conducted in humans to evaluate the safety, tolerability, and pharmacokinetics (PK) of endoxifen. This is the first study demonstrating that single oral doses of endoxifen are safe and well tolerated and have sufficient bioavailability to reach systemically effective levels in human subjects. Furthermore, it was found that endoxifen is rapidly absorbed and systemically available and that it displays dose proportionality in peak drug concentrations in plasma (C(max)) and area under the concentration-time curve extrapolated from 0 to ∞ (AUC(0-∞)) over the dose range 0.5-4.0 mg.


Accelerator mass spectrometry measurement of intracellular concentrations of active drug metabolites in human target cells in vivo. Accelerator mass spectrometry (AMS) is an ultrasensitive technique to detect radiolabeled compounds. We administered a microdose (100 µg) of (14)C-labeled zidovudine (ZDV) with or without a standard unlabeled dose (300 mg) to healthy volunteers. Intracellular ZDV-triphosphate (ZDV-TP) concentration was measured using AMS and liquid chromatography-tandem mass spectrometry (LC/MS/MS). AMS analysis yielded excellent concordance with LC/MS/MS and was 30,000-fold more sensitive. The kinetics of intracellular ZDV-TP formation changed several-fold over the dose range studied (100 µg-300 mg). AMS holds promise as a tool for quantifying intracellular drug metabolites and other biomediators in vivo.


Breaking barriers in the genomics and pharmacogenetics of drug addiction. Drug addiction remains a substantial health issue with limited treatment options currently available. Despite considerable advances in the understanding of human genetic architecture, the genetic underpinning of complex disorders remains elusive. On the basis of our current understanding of neurobiology, numerous candidate genes have been implicated in the etiology and response to treatment for different addictions. Genome-wide association (GWA) studies have also identified novel targets. However, replication of these studies is often lacking, and this complicates interpretation. The situation is expected to improve as issues such as phenotypic characterization, the apparent "missing heritability," the identification of functional variants, and possible gene-environment (G × E) interactions are addressed. In addition, there is growing evidence that genetic information can be useful in refining the choice of addiction treatment. As genetic testing becomes more common in the practice of medicine, a variety of ethical and practical challenges, some of which are unique to drug addiction, will also need to be considered.


Developing pharmacogenetic evidence throughout clinical development. Genetics as a discipline is fundamental for the pharmaceutical industry; it contributes to all therapeutic areas and has an impact throughout the research and development continuum, right up to and including clinical practice. Pharmacogenetics is seen as a significant contributor to increasing the efficiency and effectiveness of pharmaceutical R&D, and it enhances the growing interest in personalized medicine. This article discusses some contemporary issues that influence drug development and examines the potential of pharmacogenetics to reduce the risk and uncertainty that are inherent in the drug development process.


Assessing the clinical utility of diagnostics used in drug therapy. There is an ongoing debate over the evidentiary standards that should be applied for introduction of new diagnostics into routine clinical practice. Many call for evidence of "clinical utility," i.e., a positive impact on patient outcomes. A diagnostic, when used with a medicine, has clinical utility if it improves the outcomes of drug therapy. Improved outcomes may be defined broadly, including benefits, harm reduction, and patient-reported outcomes. Much of the controversy centers around the methods of demonstrating clinical utility. For instance, are randomized prospective trials the only acceptable source of data? Practically speaking, many sources of evidence-mechanistic, pharmacologic, and observational-can contribute to a finding of clinical utility, depending on the circumstances. Clinical utility is highly indication specific, and achieving it is dependent on good analytical and diagnostic test performance.


Acceptance of biomarker-based tests for application in clinical practice: criteria and obstacles. The journey from the discovery of a biomarker to its implementation in clinical practice is long and complex. There are many obstacles along the way, including lack of consistent evidence, inadequate validation of the biomarker, inadequate evidence of clinical utility, operational barriers to clinical implementation, and inadequate evidence of operational effectiveness or impact in clinical care. Multipronged approaches, incorporating input from many disciplines working together, will be required to overcome the obstacles to the clinical implementation and to allow the progression of clinical practice from the current paradigm of a one-size-fits-all strategy to more personalized treatment pathways that are more predictable in terms of health gains.


Quantum tunnelling of the magnetization in a monolayer of oriented single-molecule magnets. A fundamental step towards atomic- or molecular-scale spintronic devices has recently been made by demonstrating that the spin of an individual atom deposited on a surface, or of a small paramagnetic molecule embedded in a nanojunction, can be externally controlled. An appealing next step is the extension of such a capability to the field of information storage, by taking advantage of the magnetic bistability and rich quantum behaviour of single-molecule magnets (SMMs). Recently, a proof of concept that the magnetic memory effect is retained when SMMs are chemically anchored to a metallic surface was provided. However, control of the nanoscale organization of these complex systems is required for SMMs to be integrated into molecular spintronic devices. Here we show that a preferential orientation of Fe(4) complexes on a gold surface can be achieved by chemical tailoring. As a result, the most striking quantum feature of SMMs-their stepped hysteresis loop, which results from resonant quantum tunnelling of the magnetization-can be clearly detected using synchrotron-based spectroscopic techniques. With the aid of multiple theoretical approaches, we relate the angular dependence of the quantum tunnelling resonances to the adsorption geometry, and demonstrate that molecules predominantly lie with their easy axes close to the surface normal. Our findings prove that the quantum spin dynamics can be observed in SMMs chemically grafted to surfaces, and offer a tool to reveal the organization of matter at the nanoscale.


Climate-driven population divergence in sex-determining systems. Sex determination is a fundamental biological process, yet its mechanisms are remarkably diverse. In vertebrates, sex can be determined by inherited genetic factors or by the temperature experienced during embryonic development. However, the evolutionary causes of this diversity remain unknown. Here we show that live-bearing lizards at different climatic extremes of the species' distribution differ in their sex-determining mechanisms, with temperature-dependent sex determination in lowlands and genotypic sex determination in highlands. A theoretical model parameterized with field data accurately predicts this divergence in sex-determining systems and the consequence thereof for variation in cohort sex ratios among years. Furthermore, we show that divergent natural selection on sex determination across altitudes is caused by climatic effects on lizard life history and variation in the magnitude of between-year temperature fluctuations. Our results establish an adaptive explanation for intra-specific divergence in sex-determining systems driven by phenotypic plasticity and ecological selection, thereby providing a unifying framework for integrating the developmental, ecological and evolutionary basis for variation in vertebrate sex determination.


Promoter polymorphisms in the β-2 adrenergic receptor are associated with drug-induced gene expression changes and response in acute lymphoblastic leukemia. We investigated whether genetic polymorphisms in the promoter region of the proapoptotic β-2 adrenergic receptor gene (ADRB2) influence treatment-induced changes in ADRB2 expression in leukemia cells and response to chemotherapy. The ADRB2 promoter region was genotyped in germline DNA from 369 children with acute lymphoblastic leukemia (ALL). For 95 of the patients, sufficient RNA was available before and after in vivo treatment to assess treatment-induced gene expression changes in ALL cells. After treatment, the median ADRB2 mRNA expression was ninefold lower in leukemia cells of patients who ultimately relapsed as compared with patients who remained in continuous complete remission (CCR). Polymorphisms in the ADRB2 promoter were significantly linked to methotrexate (MTX)-induced upregulation in ADRB2 gene expression in ALL cells. Moreover, the ADRB2 promoter haplotype was significantly related to early treatment response in 245 children with ALL who received uniform treatment. We conclude that germline polymorphisms in ADRB2 are linked to the antileukemic effects of ALL chemotherapy.


Unveiling thermal transitions of polymers in subnanometre pores. The thermal transitions of confined polymers are important for the application of polymers in molecular scale devices and advanced nanotechnology. However, thermal transitions of ultrathin polymer assemblies confined in subnanometre spaces are poorly understood. In this study, we show that incorporation of polyethylene glycol (PEG) into nanochannels of porous coordination polymers (PCPs) enabled observation of thermal transitions of the chain assemblies by differential scanning calorimetry. The pore size and surface functionality of PCPs can be tailored to study the transition behaviour of confined polymers. The transition temperature of PEG in PCPs was determined by manipulating the pore size and the pore-polymer interactions. It is also striking that the transition temperature of the confined PEG decreased as the molecular weight of PEG increased.


A systems approach towards the stoichiometry-controlled hetero-assembly of nanoparticles. A central theme in nanotechnology is to advance the fundamental understanding of nanoscale component assembly, thereby allowing rational structural design that may lead to materials with novel properties and functions. Nanoparticles (NPs) are often regarded as 'artificial atoms', but their 'reactions' are not readily controllable. Here, we demonstrate a complete nanoreaction system whereby colloidal NPs are rationally assembled and purified. Two types of functionalized gold NPs (A and B) are bonded to give specific products AB, AB(2), AB(3) and AB(4). The stoichiometry control is realized by fine-tuning the charge repulsion among the B-NPs. The products are protected by a polymer, which allows their isolation in high purity. The integration of hetero-assembly, stoichiometry control, protection scheme and separation method may provide a scalable way to fabricate sophisticated nanostructures.


Two-dimensional superconductivity at a Mott insulator/band insulator interface LaTiO3/SrTiO3. Transition metal oxides show a great variety of quantum electronic behaviours where correlations often have an important role. The achievement of high-quality epitaxial interfaces involving such materials gives a unique opportunity to engineer artificial structures where new electronic orders take place. One of the most striking result in this area is the recent observation of a two-dimensional electron gas at the interface between a strongly correlated Mott insulator LaTiO(3) and a band insulator SrTiO(3). The mechanism responsible for such a behaviour is still under debate. In particular, the influence of the nature of the insulator has to be clarified. In this article, we show that despite the expected electronic correlations, LaTiO(3)/SrTiO(3) heterostructures undergo a superconducting transition at a critical temperature T(c)(onset)~300 mK. We have found that the superconducting electron gas is confined over a typical thickness of 12 nm and is located mostly on the SrTiO(3) substrate.


Bottom-up effects of plant diversity on multitrophic interactions in a biodiversity experiment. Biodiversity is rapidly declining, and this may negatively affect ecosystem processes, including economically important ecosystem services. Previous studies have shown that biodiversity has positive effects on organisms and processes across trophic levels. However, only a few studies have so far incorporated an explicit food-web perspective. In an eight-year biodiversity experiment, we studied an unprecedented range of above- and below-ground organisms and multitrophic interactions. A multitrophic data set originating from a single long-term experiment allows mechanistic insights that would not be gained from meta-analysis of different experiments. Here we show that plant diversity effects dampen with increasing trophic level and degree of omnivory. This was true both for abundance and species richness of organisms. Furthermore, we present comprehensive above-ground/below-ground biodiversity food webs. Both above ground and below ground, herbivores responded more strongly to changes in plant diversity than did carnivores or omnivores. Density and richness of carnivorous taxa was independent of vegetation structure. Below-ground responses to plant diversity were consistently weaker than above-ground responses. Responses to increasing plant diversity were generally positive, but were negative for biological invasion, pathogen infestation and hyperparasitism. Our results suggest that plant diversity has strong bottom-up effects on multitrophic interaction networks, with particularly strong effects on lower trophic levels. Effects on higher trophic levels are indirectly mediated through bottom-up trophic cascades.


Evidence for infragravity wave-tide resonance in deep oceans. Ocean tides are the oscillatory motions of seawater forced by the gravitational attraction of the Moon and Sun with periods of a half to a day and wavelengths of the semi-Pacific to Pacific scale. Ocean infragravity (IG) waves are sea-surface gravity waves with periods of several minutes and wavelengths of several dozen kilometres. Here we report the first evidence of the resonance between these two ubiquitous phenomena, mutually very different in period and wavelength, in deep oceans. The evidence comes from long-term, large-scale observations with arrays of broadband ocean-bottom seismometers located at depths of more than 4,000 m in the Pacific Ocean. This observational evidence is substantiated by a theoretical argument that IG waves and the tide can resonantly couple and that such coupling occurs over unexpectedly wide areas of the Pacific Ocean. Through this resonant coupling, some of ocean tidal energy is transferred in deep oceans to IG wave energy.


A subunit-selective potentiator of NR2C- and NR2D-containing NMDA receptors. NMDA receptors are tetrameric complexes of NR1 and NR2A-D subunits that mediate excitatory synaptic transmission and have a role in neurological disorders. In this article, we identify a novel subunit-selective potentiator of NMDA receptors containing the NR2C or NR2D subunit, which could allow selective modification of circuit function in regions expressing NR2C/D subunits. The substituted tetrahydroisoquinoline CIQ (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone) enhances receptor responses two-fold with an EC(50) of 3 μM by increasing channel opening frequency without altering mean open time or EC(50) values for glutamate or glycine. The actions of CIQ depend on a single residue in the M1 region (NR2D Thr592) and on the linker between the N-terminal domain and agonist binding domain. CIQ potentiates native NR2D-containing NMDA receptor currents from subthalamic neurons. Our identification of a subunit-selective NMDA receptor modulator reveals a new class of pharmacological tools with which to probe the role of NR2C- and NR2D-containing NMDA receptors in brain function and disease.


Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A. Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, INDY, a benzothiazole derivative showing a potent ATP-competitive inhibitory effect with IC(50) and K(i) values of 0.24 and 0.18 μM, respectively. X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme. INDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed NFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo.


'Quantized' states of the charge-density wave in microcrystals of K(0.3)MoO3. Quantization of electrons in solids can typically be observed in microscopic samples if the mean free path of the electrons exceeds the dimensions of the sample. A special case is a quasi one-dimensional metal, in which electrons condense into a collective state. This state, a charge-density wave (CDW), is a periodic modulation of both the lattice and electron density. Here, we demonstrate that samples of K(0.3)MoO(3), a typical CDW conductor, show jumps in conduction, regular in temperature. The jumps correspond to transitions between discrete states of the CDW and reveal the quantization of the wave vector of electrons near the Fermi vector. The effect involves both quantum and classical features of the CDW: the quantum condensate demonstrates modes, resembling those of a classical wave in a resonator. The analysis of the steps allows extremely precise studies of the CDW wave-vector variations and reveals new prospects for structural studies of electronic crystals and fine effects in their electronic states and lattice motions.


Enhanced proton transport in nanostructured polymer electrolyte/ionic liquid membranes under water-free conditions. Proton exchange fuel cells (PEFCs) have the potential to provide power for a variety of applications ranging from electronic devices to transportation vehicles. A major challenge towards economically viable PEFCs is finding an electrolyte that is both durable and easily passes protons. In this article, we study novel anhydrous proton-conducting membranes, formed by incorporating ionic liquids into synthetic block co-polymer electrolytes, poly(styrenesulphonate-b-methylbutylene) (S(n)MB(m)), as high-temperature PEFCs. The resulting membranes are transparent, flexible and thermally stable up to 180 °C. The increases in the sulphonation level of S(n)MB(m) co-polymers (proton supplier) and the concentration of the ionic liquid (proton mediator) produce an overall increase in conductivity. Morphology effects were studied by X-ray scattering and electron microscopy. Compared with membranes having discrete ionic domains (including Nafion 117), the nanostructured membranes revealed over an order of magnitude increase in conductivity with the highest conductivity of 0.045 S cm(-1) obtained at 165 °C.


One-by-one trap activation in silicon nanowire transistors. Flicker or 1/f noise in metal-oxide-semiconductor field-effect transistors (MOSFETs) has been identified as the main source of noise at low frequency. It often originates from an ensemble of a huge number of charges becoming trapped and de-trapped. However, as a deviation from the well-known model of 1/f noise is observed for nanoscale MOSFETs, a new model is required. Here, we report the observation of one-by-one trap activation controlled by the gate voltage in a nanowire MOSFET and propose a new low-frequency-noise theory for nanoscale FETs. We show that the Coulomb repulsion between electronically charged trap sites prevents the activation of several traps simultaneously. This effect induces a noise reduction of more than one order of magnitude. It decreases when the electron density in the channel is increased due to the electrical screening of traps. These findings are technologically useful for any FET with a short and narrow channel.


Effects of electronegative substitution on the optical and electronic properties of acenes and diazaacenes. Large acenes, particularly pentacenes, are important in organic electronics applications such as thin-film transistors. Derivatives where CH units are substituted by sp(2) nitrogen atoms are rare but of potential interest as charge-transport materials. In this article, we show that pyrazine units embedded in tetracenes and pentacenes allow for additional electronegative substituents to induce unexpected redshifts in the optical transitions of diazaacenes. The presence of the pyrazine group is critical for this effect. The decrease in transition energy in the halogenated diazaacenes is due to a disproportionate lack of stabilization of the HOMO on halogen substitution. The effect results from the unsymmetrical distribution of the HOMO, which shows decreased orbital coefficients on the ring bearing chlorine substituents. The more strongly electron-accepting cyano group is predicted to shift the transitions of diazaacenes even further to the red. Electronegative substitution impacts the electronic properties of diazaacenes to a much greater degree than expected.


Piezoelectric-nanowire-enabled power source for driving wireless microelectronics. Harvesting energy from irregular/random mechanical actions in variable and uncontrollable environments is an effective approach for powering wireless mobile electronics to meet a wide range of applications in our daily life. Piezoelectric nanowires are robust and can be stimulated by tiny physical motions/disturbances over a range of frequencies. Here, we demonstrate the first chemical epitaxial growth of PbZr(x)Ti(1-x)O(3) (PZT) nanowire arrays at 230 °C and their application as high-output energy converters. The nanogenerators fabricated using a single array of PZT nanowires produce a peak output voltage of ~0.7 V, current density of 4 μA cm(-2) and an average power density of 2.8 mW cm(-3). The alternating current output of the nanogenerator is rectified, and the harvested energy is stored and later used to light up a commercial laser diode. This work demonstrates the feasibility of using nanogenerators for powering mobile and even personal microelectronics.


Creation of a two-dimensional electron gas at an oxide interface on silicon. In recent years, reversible control over metal-insulator transition has been shown, at the nanoscale, in a two-dimensional electron gas (2DEG) formed at the interface between two complex oxides. These materials have thus been suggested as possible platforms for developing ultrahigh-density oxide nanoelectronics. A prerequisite for the development of these new technologies is the integration with existing semiconductor electronics platforms. Here, we demonstrate room-temperature conductivity switching of 2DEG nanowires formed at atomically sharp LaAlO(3)/SrTiO(3) (LAO/STO) heterointerfaces grown directly on (001) Silicon (Si) substrates. The room-temperature electrical transport properties of LAO/STO heterointerfaces on Si are comparable with those formed from a SrTiO(3) bulk single crystal. The ability to form reversible conducting nanostructures directly on Si wafers opens new opportunities to incorporate ultrahigh-density oxide nanoelectronic memory and logic elements into well-established Si-based platforms.


Structural bases for the interaction of frataxin with the central components of iron-sulphur cluster assembly. Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron-sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions.


Tectorial membrane travelling waves underlie abnormal hearing in Tectb mutant mice. Remarkable sensitivity and exquisite frequency selectivity are hallmarks of mammalian hearing, but their underlying mechanisms remain unclear. Cochlear insults and hearing disorders that decrease sensitivity also tend to broaden tuning, suggesting that these properties are linked. However, a recently developed mouse model of genetically altered hearing (Tectb(-/-)) shows decreased sensitivity and sharper frequency selectivity. In this paper, we show that the Tectb mutation reduces the spatial extent and propagation velocity of tectorial membrane (TM) travelling waves and that these changes in wave propagation are likely to account for all of the hearing abnormalities associated with the mutation. By reducing the spatial extent of TM waves, the Tectb mutation decreases the spread of excitation and thereby increases frequency selectivity. Furthermore, the change in TM wave velocity reduces the number of hair cells that effectively couple energy to the basilar membrane, which reduces sensitivity. These results highlight the importance of TM waves in hearing.


A role for TREX components in the release of spliced mRNA from nuclear speckle domains. The TREX complex, which functions in mRNA export, is recruited to mRNA during splicing. Both the splicing machinery and the TREX complex are concentrated in 20-50 discrete foci known as nuclear speckle domains. In this study, we use a model system where DNA constructs are microinjected into HeLa cell nuclei, to follow the fates of the transcripts. We show that transcripts lacking functional splice sites, which are inefficiently exported, do not associate with nuclear speckle domains but are instead distributed throughout the nucleoplasm. In contrast, pre-mRNAs containing functional splice sites accumulate in nuclear speckles, and our data suggest that splicing occurs in these domains. When the TREX components UAP56 or Aly are knocked down, spliced mRNA, as well as total polyA+ RNA, accumulates in nuclear speckle domains. Together, our data raise the possibility that pre-mRNA undergoes splicing in nuclear speckle domains, before their release by TREX components for efficient export to the cytoplasm.


Second-generation environmental sequencing unmasks marine metazoan biodiversity. Biodiversity is of crucial importance for ecosystem functioning, sustainability and resilience, but the magnitude and organization of marine diversity at a range of spatial and taxonomic scales are undefined. In this paper, we use second-generation sequencing to unmask putatively diverse marine metazoan biodiversity in a Scottish temperate benthic ecosystem. We show that remarkable differences in diversity occurred at microgeographical scales and refute currently accepted ecological and taxonomic paradigms of meiofaunal identity, rank abundance and concomitant understanding of trophic dynamics. Richness estimates from the current benchmarked Operational Clustering of Taxonomic Units from Parallel UltraSequencing analyses are broadly aligned with those derived from morphological assessments. However, the slope of taxon rarefaction curves for many phyla remains incomplete, suggesting that the true alpha diversity is likely to exceed current perceptions. The approaches provide a rapid, objective and cost-effective taxonomic framework for exploring links between ecosystem structure and function of all hitherto intractable, but ecologically important, communities.


Protein-binding assays in biological liquids using microscale thermophoresis. Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.


Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers. Concentration gradients of morphogenic proteins pattern the embryonic axes of Drosophila by activating different genes at different concentrations. The neurogenic ectoderm enhancers (NEEs) activate different genes at different threshold levels of the Dorsal (Dl) morphogen, which patterns the dorsal/ventral axis. NEEs share a unique arrangement of highly constrained DNA-binding sites for Dl, Twist (Twi), Snail (Sna) and Suppressor of Hairless (Su(H)), and encode the threshold variable in the precise length of DNA that separates one well-defined Dl element from a Twi element. However, NEEs also possess dense clusters of variant Dl sites. Here, we show that these increasingly variant sites are eclipsed relic elements, which were superseded by more recently evolved threshold encodings. Given the divergence in egg size during Drosophila lineage evolution, the observed characteristic clusters of divergent sites indicate a history of frequent selection for changes in threshold responses to the Dl morphogen gradient and confirm the NEE structure/function model.


G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion. Mitofusins (Mfns) mediate the fusion of mitochondrial membranes. However, little is known about how Mfns are regulated to control mitochondrial fusion, which is a multistep process requiring tethering and docking of the outer membranes of two mitochondria. In this study, we report that guanine nucleotide binding protein-β subunit 2 (Gβ2), a WD40 repeats protein and a member of the β-subunits of the heterotrimeric G proteins, has a crucial function in mitochondrial fusion. Gβ2 was found to be enriched on the surface of mitochondria and interacted with mitofusin 1 (Mfn1) specifically. Gβ2 also regulated the mobility of Mfn1 on the surface of the mitochondrial membrane and affected the mitochondrial fusion. Depletion of endogenous Gβ2 resulted in mitochondrial fragmentation, which could be rescued by exogenous Gβ2. These findings have thus uncovered a novel role of Gβ2 in regulating mitochondrial fusion through its interaction with Mfn1.


From casual blood pressure measurement to long-term blood pressure burden: better elucidation of the association between versatile blood pressures and cardiovascular events.


Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis. The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.


The risk of cold temperature: an important aspect of the determination of morning blood pressure surge.


Reactive oxygen species and vascular biology: implications in human hypertension. Increased vascular production of reactive oxygen species (ROS; termed oxidative stress) has been implicated in various chronic diseases, including hypertension. Oxidative stress is both a cause and a consequence of hypertension. Although oxidative injury may not be the sole etiology, it amplifies blood pressure elevation in the presence of other pro-hypertensive factors. Oxidative stress is a multisystem phenomenon in hypertension and involves the heart, kidneys, nervous system, vessels and possibly the immune system. Compelling experimental and clinical evidence indicates the importance of the vasculature in the pathophysiology of hypertension and as such much emphasis has been placed on the (patho)biology of ROS in the vascular system. A major source for cardiovascular, renal and neural ROS is a family of non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), including the prototypic Nox2 homolog-based NADPH oxidase, as well as other Noxes, such as Nox1 and Nox4. Nox-derived ROS is important in regulating endothelial function and vascular tone. Oxidative stress is implicated in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis and rarefaction, important processes involved in vascular remodeling in hypertension. Despite a plethora of data implicating oxidative stress as a causative factor in experimental hypertension, findings in human hypertension are less conclusive. This review highlights the importance of ROS in vascular biology and focuses on the potential role of oxidative stress in human hypertension.


B-cell depletion reveals a role for antibodies in the control of chronic HIV-1 infection. HIV can be partially contained by host immunity and understanding the basis of this may inform vaccine design. The importance of B-cell function in long-term control is poorly understood. One method of investigating this is in vivo cellular depletion. In this study, we take advantage of a unique opportunity to investigate the role of B cells in an HIV-infected patient. The HIV-1(+) patient studied here was not taking antiretroviral drugs and was treated for pre-existing low-grade lymphoplasmacytoid lymphoma by depletion of CD20+ B cells using rituximab. We demonstrate that B-cell depletion results in a decline in autologous neutralizing antibody (NAb) responses and a 1.7 log(10) rise in HIV-1 plasma viral load (pVL). The recovery of NAbs results in a decline in pVL. The HIV-1 sequences diversify and NAb-resistant mutants are subsequently selected. These data suggest that B-cell function can contribute to the long-term control of pVL, and that NAbs may be more important in controlling chronic HIV-1 infection than previously suspected.


The ENPP1 K121Q polymorphism is not associated with type 2 diabetes or obesity in the Chinese Han population. Type 2 diabetes (T2D) mellitus is a metabolic disorder characterized by chronic hyperglycemia and insulin resistance. It has been a worldwide public health problem, which is increasing rapidly, especially in developing countries such as China. The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, also known as plasma cell membrane glycoprotein 1, has been reported by genetic association studies as being associated with T2D and obesity in various populations, such as Caucasian and African American. However, there are also some controversial results in Asian populations. Our study tried to examine the associations between ENPP1 and T2D and obesity. Rs1044498 (K121Q) and rs7754561 were genotyped in 1912 patients and 2041 control subjects through TaqMan technology on the ABI7900 system. They showed no statistical association with T2D, obesity or any metabolic quantitative traits. Our meta-analysis result was consistent with it. Our study did not replicate the positive association found previously and suggested that K121Q of ENPP1 might not have a major role in the susceptibility to T2D or obesity in the Chinese Han population.


Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies. Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.