Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine with close structural analogy to substituted amphetamines and cathinone derivatives. Abuse of mephedrone has increased dramatically in recent years and has become a significant public health problem in the US and Europe. Unfortunately, very little information is available on the pharmacological and neurochemical actions of mephedrone. In light of the proven abuse potential of mephedrone and considering its similarity to methamphetamine and methcathinone, it is particularly important to know if mephedrone shares with these agents an ability to cause damage to dopamine nerve endings of the striatum. Accordingly, we treated mice with a binge-like regimen of mephedrone (4X 20 or 40 mg/kg) and examined the striatum for evidence of neurotoxicity 2 or 7 days after treatment. While mephedrone caused hyperthermia and locomotor stimulation, it did not lower striatal levels of dopamine, tyrosine hydroxylase or the dopamine transporter under any of the treatment conditions used presently. Furthermore, mephedrone did not cause microglial activation in striatum nor did it increase glial fibrillary acidic protein levels. Taken together, these surprising results suggest that mephedrone, despite its numerous mechanistic overlaps with methamphetamine and the cathinone derivatives, does not cause neurotoxicity to dopamine nerve endings of the striatum.
mephedrone; dopamine; neurotoxicity; β-ketoamphetamines; microglial activation; methamphetamine
Dystrophin is an actin-binding protein thought to stabilize cardiac and skeletal muscle cell membranes during contraction. Here, we investigated the contributions of each dystrophin domain to actin binding function. Cosedimentation assays and pyrene-actin fluorescence experiments confirmed that a fragment spanning two-thirds of the dystrophin molecule (from N-terminal ABD1 through ABD2) bound actin filaments with high affinity and protected filaments from forced depolymerization, but was less effective in both assays compared to full-length dystrophin. While a construct encoding the C-terminal third of dystrophin displayed no specific actin binding activity or competition with full-length dystrophin, our data show that it confers an unexpected regulation of actin binding by the N-terminal two-thirds of dystrophin when present in cis. Time-resolved phosphorescence anisotropy experiments demonstrated that the presence of the C-terminal third of dystrophin in cis also influences actin interaction in terms of restricting actin’s rotational amplitude. We propose that the C-terminal region of dystrophin allosterically stabilizes an optimal actin binding conformation of dystrophin.
Dystrophin; actin binding protein; actin dynamics; cooperative binding; and muscular dystrophy
The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T′ state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.
phospholamban; excited states; NMR; Ca-ATPase; membrane proteins
The effects of short-term (5-week) exposure to wet or dry diets on fecal bacterial populations in the cat were investigated. Sixteen mixed-sex, neutered, domestic short-haired cats (mean age = 6 years; mean bodyweight = 3.4 kg) were randomly allocated to wet or dry diets in a crossover design. Fecal bacterial DNA was isolated and bacterial 16S rRNA gene amplicons generated and analyzed by 454 Titanium pyrosequencing. Cats fed dry diets had higher abundances (P < 0.05) of Actinobacteria (16.5% vs. 0.1%) and lower abundances of Fusobacteria (0.3% vs. 23.1%) and Proteobacteria (0.4% vs. 1.1%) compared with cats fed the wet diet. Of the 46 genera identified, 30 were affected (P < 0.05) by diet, with higher abundances of Lactobacillus (31.8% vs. 0.1%), Megasphaera (23.0% vs. 0.0%), and Olsenella (16.4% vs. 0.0%), and lower abundances of Bacteroides (0.6% vs. 5.7%) and Blautia (0.3% vs. 2.3%) in cats fed the dry diet compared with cats fed the wet diet. These results demonstrate that short-term dietary exposure to diet leads to large shifts in fecal bacterial populations that have the potential to affect the ability of the cat to process macronutrients in the diet.
Feline; fecal bacterial community; high-throughput nucleotide sequencing
We used immunoblots to determine whether inotropic and lusitropic effects of high-dose insulin (HDI) in cardiogenic shock, induced by a β-blocker (BB) or a calcium channel blocker (CCB), are mediated by phosphorylation of phospholamban (PLB). PLB is a membrane protein that regulates calcium uptake into the sarcoplasmic reticulum (SR) by inhibition of the cardiac calcium pump (SERCA2a). Phosphorylation of PLB relieves SERCA inhibition, thus enhancing diastolic relaxation and preload.
Our Institutional Animal Care and Use Committee approved this research. Swine myocardia from six groups were flash frozen immediately upon death or sacrifice. Groups 1–6 received: (1) no medications, (2) HDI and glucose only, (3) toxic propranolol infusions and saline resuscitation, (4) toxic propranolol infusions and HDI resuscitation, (5) toxic verapamil infusions and saline resuscitation, and (6) toxic verapamil infusions and HDI resuscitation. Groups 3–6 were resuscitated for 4 h. Tissue samples from all six groups were analyzed by quantitative immunoblots, using antibodies to both unphosphorylated PLB (uPLB) and phosphorylated PLB (pPLB), to determine the total PLB content and the fraction of PLB phosphorylated.
There were no differences in either pPLB or total PLB in cardiac tissue among any of the six groups. However, infusion of a pig with the β-adrenergic agonist, isoproterenol, produced enhanced PLB phosphorylation.
The mechanism by which HDI produces its inotropic and lusitropic effects in CCB- and BB-induced cardiovascular toxicity, resulting in resuscitation, is not due to changes in phosphorylation of PLB or a change in the total PLB in the SR.
Phospholamban; High-dose insulin; Cardiogenic shock; Verapamil; Propranolol
Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases.
To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05).
Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested.
Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases.
Protein trafficking; Corrector; Proteostasis; CFTR; hERG; KCNH2; AVPR2; V2R; ABCC8; SUR1
Aortic coarctation (AC) is a significant cause of secondary hypertension and is diagnosed in childhood in the vast majority of patients. Mild or moderate coarctation may exist undetected into adult life, when it usually presents due to its sequelae. The authors present the case of a 20-year-old woman, previously extensively investigated for severe hypertension, who was admitted following sever, sudden-onset headache. CT scanning of the head showed the presence of subarachnoid blood (SAH), with subsequent CT angiography revealing two intracerebral aneurysms as the source. On attempting to catheterise the femoral artery her pulses were noted to be weak and during passage of the catheter she was found to have significant AC. The aneurysms were duly treated with detachable coils and the clinical course with regard to the SAH was unremarkably safe for high-pressure headache.
To evaluate the validity of fruit and vegetable intake, using three classification schemes, as it relates to plasma carotenoid and vitamin C concentrations among Chinese women.
Intakes were calculated from an interviewer-administered food frequency questionnaire. Fruits and vegetables, botanical groups, and high-nutrient groups were evaluated. These three classification schemes were compared with plasma carotenoid and vitamin C concentrations from blood drawn within one week of questionnaire completion.
Participants (n=2031) were drawn from women who participated in a case-control study of diet and breast diseases nested within a randomized trial of breast self-examination among textile workers (n=266,064)
Fruit intake was significantly (p<0.05) and positively associated with plasma concentrations of α-tocopherol, β-cryptoxanthin, lycopene, α-carotene, β-carotene, retinyl palmitate, and vitamin C. Fruit intake was inversely associated with γ-tocopherol and lutein+zeaxanthin concentrations. Vegetable consumption was significantly and positively associated with γ-tocopherol, and β-cryptoxanthin concentrations. Each botanical and high-nutrient group was also significantly associated with particular plasma nutrient concentrations. Fruit and vegetable intake and most plasma nutrient concentrations were significantly associated with season of interview.
These results suggest that the manner in which fruits and vegetables are grouped provides different plasma nutrient exposure information, which may be an important consideration when testing and generating hypotheses regarding disease risk in relation to diet. Interview season should be considered when evaluating associations of reported intake and plasma nutrients with disease outcomes.
fruit; vegetable; botanical; China
A modification to the 3D modified driven equilibrium Fourier transform
(MDEFT) imaging technique is proposed that reduces its sensitivity to RF
inhomogeneity. This is especially important at high field strengths where RF
focusing effects exacerbate B1 inhomogeneity,
causing significant signal nonuniformity in the images. The adiabatic inversion
pulse used during the preparation period of the MDEFT sequence is replaced by a
hard (nonadiabatic) pulse with a nominal flip angle of 130°. The spatial
inhomogeneity of the hard pulse preparation compensates for the inhomogeneity of
the excitation pulses. Uniform signal intensity is obtained for a wide range of
B1 amplitudes and the high CNR characteristic of
MDEFT is retained. The new approach was validated by numerical simulations and
successfully applied to human brain imaging at 4.7 T, resulting in highquality
T1-weighted images of the whole human brain at
high field strength with uniform signal intensity and contrast, despite the
presence of significant RF inhomogeneity.
high field MRI; T1-weighted imaging; RF inhomogeneity; human brain imaging; structural brain imaging
Background. Proinflammatory cytokines play a critical role in antiviral immune responses. Large-scale genome studies have found correlations between single-nucleotide polymorphisms (SNPs) in the interleukin (IL) 18 promoter and spontaneous control of hepatitis C virus (HCV), suggesting a role in clearance.
Methods. Plasma IL-18, IL-1β, IL-6, IL-8, IL-12, interferon-γ, tumor necrosis factor–α, alanine aminotransferase (ALT), and HCV RNA levels were assessed longitudinally in subjects with known dates of HCV acquisition and analyzed according to IL-18 SNPs and outcome, either spontaneous clearance (SC) (n = 13) or persistent infection (PI) (n = 25).
Results. No significant change in plasma proinflammatory cytokine expression was observed with the exception of IL-18, which increased in every subject with initial detection of HCV RNA. In every SC subject, IL-18 returned to the preinfection baseline concomitant with HCV control. In PI subjects, IL-18 declined following the acute phase of infection but remained above the preinfection baseline throughout chronic infection and did not correlate with HCV RNA or ALT levels.
Conclusions. Plasma IL-18 was an early and the most reliably detected host response to HCV infection measured in blood. Reduced IL-18 production with transition to chronic infection without correlation with HCV RNA or ALT levels suggests modulation of the innate response with persistent infection.
Background Few studies have examined the possible effects of reproductive factors on cardiovascular disease (CVD) risks in Asian women.
Methods A cohort of 267 400 female textile workers in Shanghai, China, was administered a questionnaire at enrolment (1989–91) and followed for mortality through 2000. Relative risks (hazard ratios) for ischaemic heart disease (IHD), ischaemic stroke and haemorrhagic stroke were calculated using Cox proportional hazards modelling, adjusting for relevant co-variates.
Results Risks were not consistently associated with age at menopause, parity, stillbirths, miscarriages or duration of lactation. An increasing trend in IHD mortality risk, but not stroke, was observed with decreasing age at menarche. There was no evidence of increased CVD mortality risk by oral or injectable contraceptive use or induced abortions. As expected, greater mortality rates from CVD and increased CVD risks were also observed with smoking.
Conclusions Use of steroid contraceptives, induced abortions and reduced parity from China's one-child-per-family policy has not had an adverse effect on risk of CVD mortality in this cohort.
Cohort; cardiovascular diseases; reproductive history; mortality; China
To investigate chronic obstructive pulmonary disease (COPD) mortality among textile workers.
A total of 267 400 Chinese female textile employees were monitored for COPD mortality from 1989 to 2000. Textile factories in the cohort were classified into 10 industrial sectors. Age-adjusted mortality, standardized mortality ratios (SMRs) and 95% CIs were calculated by sector. In addition, RRs (HRs) adjusted for smoking and age were calculated for exposure to cotton and silk textile work compared with the other sectors in the cohort.
A majority of textile sectors had lower or similar COPD mortality (age-adjusted SMRs=0.58–1.15) compared with the general female population in the city of Nanjing, China. SMRs for cotton and silk workers were, respectively, 1.02 (95% CI: 0.81 to 1.28) and 2.03 (95% CI: 1.13 to 3.34). Compared with all other textile sectors in the cohort, there was greater COPD mortality among cotton workers (HR=1.40, 95% CI: 1.03 to 1.89) and silk workers (HR=2.54, 95% CI: 1.47 to 4.39).
Elevated COPD mortality among cotton workers is consistent with previous reports of adverse respiratory effects of cotton dust. The higher rate of COPD deaths among silk workers was unexpected.
We have examined the structural and functional effects of site-directed methionine oxidation in Dictyostelium (Dicty) myosin II using mutagenesis, in vitro oxidation, and site-directed spin-labeling for electron paramagnetic resonance (EPR). Protein oxidation by reactive oxygen and nitrogen species is critical for normal cellular function, but oxidative stress has been implicated in disease progression and biological aging. Our goal is to bridge understanding of protein oxidation and muscle dysfunction with molecular-level insights into actomyosin interaction. In order to focus on methionine oxidation and to facilitate site-directed spectroscopy, we started with a Cys-lite version of Dicty myosin II. For Dicty myosin containing native methionines, peroxide-treatment decreased actin-activated myosin ATPase activity, consistent with the decline in actomyosin function previously observed in biologically aged or peroxide-treated muscle. Methionine-to-leucine mutations, used to protect specific sites from oxidation, identified a single methionine that is functionally sensitive to oxidation: M394, near the myosin cardiomyopathy loop in the actin-binding interface. Previously characterized myosin labeling sites for spectroscopy in the force-producing region and actin-binding cleft were examined; spin label mobility and distance measurements in the actin-binding cleft were sensitive to oxidation, but particularly in the presence of actin. Overall secondary structure and thermal stability were unaffected by oxidation. We conclude that the oxidation-induced structural change in myosin includes a redistribution of existing structural states of the actin-binding cleft. These results will be applicable to the many biological and therapeutic contexts in which a detailed understanding of protein oxidation, function and structure relationships are sought.
Traditional herbal medicines are commonly used in sub-Saharan Africa and some herbs are known to be hepatotoxic. However little is known about the effect of herbal medicines on liver disease in sub-Saharan Africa.
500 HIV-infected participants in a rural HIV care program in Rakai, Uganda, were frequency matched to 500 HIV-uninfected participants. Participants were asked about traditional herbal medicine use and assessed for other potential risk factors for liver disease. All participants underwent transient elastography (FibroScan®) to quantify liver fibrosis. The association between herb use and significant liver fibrosis was measured with adjusted prevalence risk ratios (adjPRR) and 95% confidence intervals (CI) using modified Poisson multivariable logistic regression.
19 unique herbs from 13 plant families were used by 42/1000 of all participants, including 9/500 HIV-infected participants. The three most-used plant families were Asteraceae, Fabaceae, and Lamiaceae. Among all participants, use of any herb (adjPRR = 2.2, 95% CI 1.3–3.5, p = 0.002), herbs from the Asteraceae family (adjPRR = 5.0, 95% CI 2.9–8.7, p<0.001), and herbs from the Lamiaceae family (adjPRR = 3.4, 95% CI 1.2–9.2, p = 0.017) were associated with significant liver fibrosis. Among HIV infected participants, use of any herb (adjPRR = 2.3, 95% CI 1.0–5.0, p = 0.044) and use of herbs from the Asteraceae family (adjPRR = 5.0, 95% CI 1.7–14.7, p = 0.004) were associated with increased liver fibrosis.
Traditional herbal medicine use was independently associated with a substantial increase in significant liver fibrosis in both HIV-infected and HIV-uninfected study participants. Pharmacokinetic and prospective clinical studies are needed to inform herb safety recommendations in sub-Saharan Africa. Counseling about herb use should be part of routine health counseling and counseling of HIV-infected persons in Uganda.
Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (∼8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.
arsenic; carcinogenesis; life-stage; age susceptibility; urinary bladder; C3H mice
Adult smoking has its roots in adolescence. If individuals do not initiate smoking during this period it is unlikely they ever will. In high income countries, smoking rates among Indigenous youth are disproportionately high. However, despite a wealth of literature in other populations, there is less evidence on the determinants of smoking initiation among Indigenous youth. The aim of this study was to explore the determinants of smoking among Australian Indigenous young people with a particular emphasis on the social and cultural processes that underlie tobacco use patterns among this group.
This project was undertaken in northern Australia. We undertook group interviews with 65 participants and individual in-depth interviews with 11 youth aged 13–20 years led by trained youth ‘peer researchers.’ We also used visual methods (photo-elicitation) with individual interviewees to investigate the social context in which young people do or do not smoke. Included in the sample were a smaller number of non-Indigenous youth to explore any significant differences between ethnic groups in determinants of early smoking experiences. The theory of triadic influence, an ecological model of health behaviour, was used as an organising theory for analysis.
Family and peer influences play a central role in smoking uptake among Indigenous youth. Social influences to smoke are similar between Indigenous and non-Indigenous youth but are more pervasive (especially in the family domain) among Indigenous youth. While Indigenous youth report high levels of exposure to smoking role models and smoking socialisation practices among their family and social networks, this study provides some indication of a progressive denormalisation of smoking among some Indigenous youth.
Future initiatives aimed at preventing smoking uptake in this population need to focus on changing social normative beliefs around smoking, both at a population level and within young peoples’ immediate social environment. Such interventions could be effectively delivered in both the school and family environments. Specifically, health practitioners in contact with Indigenous families should be promoting smoke free homes and other anti-smoking socialisation behaviours.
Aboriginal; Youth; Smoking; Tobacco; Australia
In 2006, we were funded by the US National Institutes of Health to implement a study of tuberculosis epidemiology in Peru. The study required a secure information system to manage data from a target goal of 16,000 subjects who needed to be followed for at least one year. With previous experience in the development and deployment of web-based medical record systems for TB treatment in Peru, we chose to use the OpenMRS open source electronic medical record system platform to develop the study information system. Supported by a core technical and management team and a large and growing worldwide community, OpenMRS is now being used in more than 40 developing countries. We adapted the OpenMRS platform to better support foreign languages. We added a new module to support double data entry, linkage to an existing laboratory information system, automatic upload of GPS data from handheld devices, and better security and auditing of data changes. We added new reports for study managers, and developed data extraction tools for research staff and statisticians. Further adaptation to handle direct entry of laboratory data occurred after the study was launched.
Data collection in the OpenMRS system began in September 2009. By August 2011 a total of 9,256 participants had been enrolled, 102,274 forms and 13,829 laboratory results had been entered, and there were 208 users. The system is now entirely supported by the Peruvian study staff and programmers.
The information system served the study objectives well despite requiring some significant adaptations mid-stream. OpenMRS has more tools and capabilities than it did in 2008, and requires less adaptations for future projects. OpenMRS can be an effective research data system in resource poor environments, especially for organizations using or considering it for clinical care as well as research.
Electronic medical record; Developing country; Multidrug-resistant tuberculosis; MDR-TB
Single-nucleotide polymorphisms (SNPs) around IL28B are associated with spontaneous hepatitis C virus (HCV) clearance of genotypes 1 and 3 in white and African-American populations. This study investigated whether the IL28B SNP (rs12979860) is associated with spontaneous clearance of HCV, principally genotype 4, in 162 Egyptians (80 with clearance). The protective C allele was more common in those with spontaneous clearance (76.3% vs 57.9%; P = .0006). Individuals with clearance were 3.4 (95% confidence interval, 1.8–6.5) times more likely to have C/C genotype. Thus, IL28B plays a role in spontaneous clearance of HCV genotype 4 in North Africa.
To develop a robust sterile, fully demucosalized and vascularized seromuscular patch for use as an adjunct to novel bioengineering techniques aimed at augmenting, reconstructing, or replacing the bladder because of endstage disease. To eliminate deep colonic epithelial crypts to prevent the possibility of colonocyte regrowth. To maintain sterility by excluding the possibility of contamination from the bowel contents.
Pilot studies were performed on euthanized pigs to optimize the technique, with tissue samples examined by immunohistochemistry. In vivo, vascularized seromuscular colonic flaps were created from the bowel exterior in 7 large white hybrid pigs. The dissection was facilitated by placing an inflated Foley catheter within the colonic lumen. The seromuscular ends were approximated with 5/0 Vicryl sutures and excess mucosa intussuscepted within the lumen. Demucosalized flaps were used to augment the bladder by composite cystoplasty and were examined immunohistochemically at 3 months.
Pilot studies showed that the technique was successful in creating seromuscular segments with no epithelial remnants. When applied surgically, the seromuscular flaps survived and showed no evidence of colonocyte regrowth at 3 months.
Extraluminal dissection creates robust seromuscular flaps and prevents both regrowth by colonic epithelial cells and contamination of the tissue by exposure to the bowel contents. This technique should find application in a range of bladder reconstruction techniques, including composite cystoplasty and autoaugmentation.
Recent evidence demonstrates that the efficacy of conventional anticancer therapies including chemotherapy requires a functional immune system. Here, we addressed the possibility that the antitumor effect of a selective Smoothened antagonist and Hedgehog (Hh) pathway inhibitor (LDE225), a promising anticancer drug, might at least partially depend on the immune system. To this aim, we used tumor cell lines derived from a murine model of radiation-induced osteosarcoma. In vitro treatment of osteosarcoma cells with LDE225 resulted in a decreased ability of tumor cells to proliferate, but had no effect on their viability. Flow cytometry analysis demonstrated that LDE225-treatment did not detectably modulate the immunogenicity of tumor cells. Moreover, LDE225 did not display any pro-apoptotic properties on osteosarcoma cells, highlighting that its antitumor profile mainly derives from a cytostatic effect. Furthermore, calreticulin exposure, a key feature of immunogenic cell death, was not provoked by LDE225, neither alone nor combined with recognized immunogenic drugs. Finally, the oral administration of LDE225 to osteosarcoma-bearing mice did significantly delay the tumor growth even in an immunocompromised setting. These data suggest that inhibiting Hh signaling can control osteosarcoma cell proliferation but does not modulate the immunogenic profile of these cells.
apoptosis; Hedgehog signaling pathway; immune response; immunogenicity; mouse; osteosarcoma; proliferation
The HCV council 2011 convened 11 leading clinicians and researchers in hepatitis C virus from academic medical centers in the United States to provide a forum for the practical and comprehensive evaluation of current data regarding best practices for integrating new direct-acting antiviral agents into existing treatment paradigms. The council investigated 10 clinical practice statements related to HCV treatment that reflect key topical areas. Faculty members reviewed and discussed the data related to each statement, and voted on the nature of the evidence and their level of support for each statement. In this new era of DAAs, a comprehensive and critical analysis of the literature is needed to equip clinicians with the knowledge necessary to design, monitor, and modify treatment regimens in order to optimize patient outcomes.
Most cases of cystic fibrosis (CF) are caused by the deletion of a single phenylalanine residue at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutant F508del-CFTR is retained in the endoplasmic reticulum and degraded, but can be induced by low temperature incubation (29°C) to traffic to the plasma membrane where it functions as a chloride channel. Here we show that, cardiac glycosides, at nanomolar concentrations, can partially correct the trafficking of F508del-CFTR in human CF bronchial epithelial cells (CFBE41o-) and in an F508del-CFTR mouse model. Comparison of the transcriptional profiles obtained with polarized CFBE41o-cells after treatment with ouabain and by low temperature has revealed a striking similarity between the two corrector treatments that is not shared with other correctors. In summary, our study shows a novel function of ouabain and its analogs in the regulation of F508del-CFTR trafficking and suggests that compounds that mimic this low temperature correction of trafficking will provide new avenues for the development of therapeutics for CF.
cystic fibrosis; CFTR; trafficking; quabain; microarray; connectivity map; hierarchical clustering; CFBE cells
To evaluate the prevalence and risk factors for low bone mineral density (BMD) in persons co-infected with HIV and Hepatitis C.
HIV/HCV co-infected study participants (n=179) were recruited into a prospective cohort and underwent dual-energy X-ray absorptiometry (DXA) within 1 year of a liver biopsy. Fibrosis staging was evaluated according to the METAVIR system. Osteoporosis was defined as a T-score ≤ −2.5. Z-scores at the total hip, femoral neck, and lumbar spine were used as the primary outcome variables to assess the association between degree of liver disease, HIV-related variables, and BMD.
The population was 65% male, 85% Black with mean age 50.3 years. The prevalence of osteoporosis at either at the total hip, femoral neck, or lumbar spine was 28%, with 5% having osteoporosis of the total hip, 6% at the femoral neck, 25% at the spine. The mean Z-scores (standard deviation) were −0.42 (1.01) at the total hip, −0.16 (1.05) at the femoral neck, and −0.82 (1.55) at the lumbar spine. In multivariable models, controlled HIV replication (HIV RNA < 400 copies/mL vs ≥400 copies/mL) was associated with lower Z-scores (mean ± standard error) at the total hip (−0.44±0.17, p=0.01), femoral neck (−0.59±0.18, p=0.001), and the spine (−0.98±0.27, p=0.0005). There was no association between degree of liver fibrosis and Z-score.
Osteoporosis was very common in this population of predominately African-American HIV/HCV co-infected patients, particularly at the spine. Lower BMD was associated with controlled HIV replication, but not liver disease severity.
hepatitis C; bone mineral density; hepatic fibrosis; HIV
The metalloid arsenic is a natural environmental contaminant to which humans are routinely exposed in food, water, air, and soil. Arsenic has a long history of use as a homicidal agent, but in the past 100 years arsenic, has been used as a pesticide, a chemotherapeutic agent and a constituent of consumer products. In some areas of the world, high levels of arsenic are naturally present in drinking water and are a toxicological concern. There are several structural forms and oxidation states of arsenic because it forms alloys with metals and covalent bonds with hydrogen, oxygen, carbon, and other elements. Environmentally relevant forms of arsenic are inorganic and organic existing in the trivalent or pentavalent state. Metabolism of arsenic, catalyzed by arsenic (+3 oxidation state) methyltransferase, is a sequential process of reduction from pentavalency to trivalency followed by oxidative methylation back to pentavalency. Trivalent arsenic is generally more toxicologically potent than pentavalent arsenic. Acute effects of arsenic range from gastrointestinal distress to death. Depending on the dose, chronic arsenic exposure may affect several major organ systems. A major concern of ingested arsenic is cancer, primarily of skin, bladder, and lung. The mode of action of arsenic for its disease endpoints is currently under study. Two key areas are the interaction of trivalent arsenicals with sulfur in proteins and the ability of arsenic to generate oxidative stress. With advances in technology and the recent development of animal models for arsenic carcinogenicity, understanding of the toxicology of arsenic will continue to improve.
arsenic; cancer; exposure