Hyperglycaemia disproportionately affects African-Americans (AfAs).
We tested the transferability of 18 single-nucleotide polymorphisms (SNPs)
associated with glycaemic traits identified in European ancestry (EuA)
populations in 5,984 non-diabetic AfAs.
We meta-analysed SNP associations with fasting glucose (FG) or
insulin (FI) in AfAs from five cohorts in the Candidate Gene Association
Resource. We: (1) calculated allele frequency differences, variations in
linkage disequilibrium (LD), fixation indices (Fsts) and
integrated haplotype scores (iHSs); (2) tested EuA SNPs in AfAs; and (3)
interrogated within ±250 kb around each EuA SNP in AfAs.
Allele frequency differences ranged from 0.6% to 54%.
Fst exceeded 0.15 at 6/16 loci, indicating modest population
differentiation. All iHSs were <2, suggesting no recent positive
selection. For 18 SNPs, all directions of effect were the same and
95% CIs of association overlapped when comparing EuA with AfA. For
17 of 18 loci, at least one SNP was nominally associated with FG in AfAs.
Four loci were significantly associated with FG (GCK,
p=5.8 × 10-8;
MTNR1B, p=8.5 ×
10-9; and FADS1,
p=2.2 × 10-4) or FI
(GCKR, p=5.9 ×
10-4). At GCK and MTNR1B
the EuA and AfA SNPs represented the same signal, while at
FADS1, and GCKR, the EuA and best AfA
SNPs were weakly correlated (r2<0.2),
suggesting allelic heterogeneity for association with FG at these loci.
Few glycaemic SNPs showed strict evidence of transferability from EuA
to AfAs. Four loci were significantly associated in both AfAs and those with
EuA after accounting for varying LD across ancestral groups, with new
signals emerging to aid fine-mapping.
African ancestry; Genetics; Genome-wide association; LD mapping; Minorities; Type 2 diabetes
We describe the design and fabrication trials of x-ray absorption gratings of 200 nm period and up to 100:1 depth-to-period ratios for full-field hard x-ray imaging applications. Hard x-ray phase-contrast imaging relies on gratings of ultra-small periods and sufficient depth to achieve high sensitivity. Current grating designs utilize lithographic processes to produce periodic vertical structures, where grating periods below 2.0 μm are difficult due to the extreme aspect ratios of the structures. In our design, multiple bilayers of x-ray transparent and opaque materials are deposited on a staircase substrate, and mostly on the floor surfaces of the steps only. When illuminated by an x-ray beam horizontally, the multilayer stack on each step functions as a micro-grating whose grating period is the thickness of a bilayer. The array of micro-gratings over the length of the staircase works as a single grating over a large area when continuity conditions are met. Since the layers can be nanometers thick and many microns wide, this design allows sub-micron grating periods and sufficient grating depth to modulate hard x-rays. We present the details of the fabrication process and diffraction profiles and contact radiography images showing successful intensity modulation of a 25 keV x-ray beam.
To assess the value of contrast-enhanced ultrasound (CEUS) in differentiating hepatocellular carcinoma (HCC) from non-neoplastic lesion in cirrhotic liver in comparison with baseline ultrasound.
A total of 147 nodules (diameter ≤5.0 cm) in 133 cirrhotic patients (mean age±standard deviation: 52±13 years, range 20–82 years; gender: 111 males and 22 females) were examined with CEUS. There were 116 HCCs, 26 macroregenerative nodules and 5 high-grade dysplastic nodules. CEUS was performed with a real-time contrast-specific mode and a sulphur hexafluoride-filled microbubble contrast agent.
Hypervascularity was observed in 94.8% (110/116) HCCs, 3.8% (1/26) macroregenerative nodules and 60.0% (3/5) high-grade dysplastic nodules during arterial phase on CEUS. Detection rates of typical vascular pattern (i.e. hypervascularity during arterial phase and subsequent washout) in HCCs with a diameter of ≤2.0 cm, 2.1–3.0 cm and 3.1–5.0 cm were 69.2% (27/39), 97.1% (33/34) and 100.0% (43/43), respectively. CEUS significantly improved the sensitivity [88.8% (103/116) vs 37.1% (43/116), p<0.001], negative predictive value [70.5% (31/44) vs 31.5% (29/92), p<0.001], and accuracy [91.2% (134/147) vs 49.0% (72/147), p<0.001] in differentiating HCCs from non-neoplastic lesions when compared with baseline ultrasound. However, the sensitivity and accuracy of CEUS for HCCs ≤2.0 cm in diameter were significantly lower than those for HCCs of 2.1–3.0 cm and 3.1–5.0 cm in diameter.
CEUS improves diagnostic performance in differentiating HCCs from non-neoplastic nodules in cirrhotic patients compared with baseline ultrasound. Diagnosis of HCCs ≤2.0 cm diameter by CEUS is still a clinical concern, and thus needs further investigation.
We conducted a systematic study of top susceptibility variants from a genome-wide association (GWA) study of Bipolar Disorder to gain insight into the functional consequences of genetic variation influencing disease risk. We report here the results of experiments to explore the effects of these susceptibility variants on DNA methylation and mRNA expression in human cerebellum samples. Among the top susceptibility variants, we identified an enrichment of cis regulatory loci on mRNA expression (eQTLs), and a significant excess of quantitative trait loci for DNA CpG methylation, hereafter referred to as mQTLs. Bipolar Disorder susceptibility variants that cis-regulate both cerebellar expression and methylation of the same gene are a very small proportion of Bipolar Disorder susceptibility variants. This finding suggests that mQTLs and eQTLs provide orthogonal ways of functionally annotating genetic variation within the context of studies of pathophysiology in brain. No lymphocyte mQTL enrichment was found, suggesting that mQTL enrichment was specific to the cerebellum, in contrast to eQTLs. Separately, we found that using mQTL information to restrict the number of SNPs studied enhances our ability to detect a significant association. With this restriction a priori informed by the observed functional enrichment, we identified a significant association (rs12618769, Pbonferroni<0.05) from two other GWA studies (TGen+GAIN; 2,191 cases and 1,434 controls) of Bipolar Disorder, which we replicated in an independent GWA study (WTCCC). Collectively, our findings highlight the importance of integrating functional annotation of genetic variants for gene expression and DNA methylation to advance biological understanding of Bipolar Disorder.
Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development
of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9),
interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD.
Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B
(NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal
degeneration; therefore, we investigated whether this action was mediated via
activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells
were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to
investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We
found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9
were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in
SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720
inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our
findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal
degeneration and inflammation in AMD.
Amyloid-beta; SRT1720; Age-related macular degeneration; Barrier integrity; Tight junction; Matrix metalloproteinase-9
To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)–mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES–mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES–mediated translation.
HCV IRES; hepatitis C virus; IFITM3 (1-8U); mouse oestrogen receptor; STAT1 dimerization
To investigate the correlation between enhancement patterns of intrahepatic cholangiocarcinoma (ICC) on contrast-enhanced ultrasound (CEUS) and pathological findings.
The CEUS enhancement patterns of 40 pathologically proven ICC lesions were retrospectively analysed. Pathologically, the degree of tumour cell and fibrosis distribution in the lesion was semi-quantitatively evaluated.
4 enhancement patterns were observed in the arterial phase for 32 mass-forming ICCs: peripheral rim-like hyperenhancement (n=19); heterogeneous hyperenhancement (n=6); homogeneous hyperenhancement (n=3); and heterogeneous hypo-enhancement (n=4). Among the four enhancement patterns, the differences in tumour cell distribution were statistically significant (p<0.05). The hyperenhancing area on CEUS corresponded to more tumour cells for mass-forming ICCs. Heterogeneous hyperenhancement (n=2) and heterogeneous hypo-enhancement (n=2) were observed in the arterial phase for four periductal infiltrating ICCs. In this subtype, fibrosis was more commonly found in the lesions. Heterogeneous hyperenhancement (n=1) and homogeneous hyperenhancement (n=3) were observed in the arterial phase for four intraductal growing ICCs. This subtype tended to have abundant tumour cells.
The CEUS findings of ICC relate to the degree of carcinoma cell proliferation at pathological examination. Hyperenhancing areas in the tumour always indicated increased density of cancer cells.
In the field of metallic materials with amorphous structures, it is vitally important to understand the glass formation and to predict glass-forming ability (GFA) in terms of constituent elements and alloy compositions. In this study, an expression has been formulated from first-principles calculations to predict the trend of GFA by hybridizing both internal energies and atomic-scale defect structures. The prediction of GFA from compositions has been verified successfully by available experimental data in the model Zr-Cu alloy system. The physical scenario revealed here has extensive implications for the design of bulk metallic glasses with superior GFA.
Since the introduction of bone-anchored hearing aids (BAHAs) in the 1980s, the practices of surgeons who implant these hearing aids have become varied; different indications and surgical techniques are utilized depending on the surgeon and institution. The objective of the current study is to describe the clinical and surgical practices of otolaryngologists in Canada who perform pediatric BAHA operations.
A detailed practice questionnaire was devised and sent to all members of the Canadian Society of Otolaryngology-Head and Neck Surgery. Those who performed pediatric BAHA surgeries were asked to participate.
Twelve responses were received (response rate of 80%). All of the respondents identified congenital aural atresia to be an indication for pediatric BAHAs. Other indications were chronic otitis externa or media with hearing loss (92%), allergic reactions to conventional hearing aids (75%), congenital fixation or anomaly of ossicular chain (67%), and unilateral deafness (25%). Minor complications, such as skin reactions, were reported in 25% of cases, while major complications were very rare. There was great variability with regards to surgical techinque and post-operative management. The extent of financial support for the BAHA hardware and device also varied between provinces, and even within the same province.
There is a lack of general consensus regarding pediatric BAHA surgeries in Canada. With such a small community of otolaryngologists performing this procedure, we are hopeful that this survey can serve as an impetus for a national collaboration to establish a set of general management principles and inspire multi-site research ventures.
Bone-anchored hearing aid; BAHA; Surgical practice; Clinical practice; Practice survey; Pediatrics
Cardiomyocyte death is an important reason for the cardiac syndromes, such as heart failure (HF) and myocardial infarction (MI). In the heart diseases, necrosis is one of the main forms of cell death. MicroRNAs (miRNAs) are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. Hitherto, it is not yet clear whether miRNA can regulate necrosis in cardiomyocyte. In this work, we performed a microarray to detect miRNAs in response to H2O2 treatment, and the results showed that miR-874 was substantially increased. We further studied the function of miR-874, and observed that knockdown of miR-874 attenuated necrosis in the cellular model and also MI in the animal model. We searched for the downstream mediator of miR-874 and identified that caspase-8 was a target of miR-874. Caspase-8 was able to antagonize necrosis. When suppressed by miR-874, caspase-8 lost the ability to repress necrotic program. In exploring the molecular mechanism by which miR-874 expression is regulated, we identified that Foxo3a could transcriptionally repress miR-874 expression. Foxo3a transgenic or knockout mice exhibited a low or high expression level of miR-874, and a reduced or enhanced necrosis and MI. Our present study reveals a novel myocardial necrotic regulating model, which is composed of Foxo3a, miR-874 and caspase-8. Modulation of their levels may provide a new approach for tackling myocardial necrosis.
myocardial necrosis; miR-874; caspase-8
Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.
A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the allo-response and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.
Dendritic cells; Dendritic cell therapies; Transplantation; Immunosuppression; Tolerance
The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis, which is regulated by multiple signaling pathways. The Wnt/β-catenin pathway has a critical role in this process. Previously, we have shown that the calcineurin-dependent nuclear factor of activated T cell (NFAT) is involved in the regulation of intestinal cell differentiation, as noted by the alteration of brush-border enzyme intestinal alkaline phosphatase (IAP) activity. Here, we show that calcineurin-independent NFAT5 interacts with β-catenin to repress Wnt signaling. We found that overexpression of NFAT5 inhibits, whereas knockdown of NFAT5 increases, TOPflash reporter activity and the expression of Wnt/β-catenin target genes, suggesting that NFAT5 inhibits Wnt signaling. In addition, we demonstrated that NFAT5 directly interacts with the C-terminal transactivation domain (TAD) of β-catenin, inhibits CBP interaction with β-catenin, and inhibits CBP-mediated β-catenin acetylation. Moreover, NFAT5 is expressed in the mucosa of human intestine, with the most pronounced staining in the most differentiated region near the epithelial surface. Knockdown of NFAT5 attenuated sodium butyrate (NaBT)-mediated induction of IAP and sucrase activities; overexpression of NFAT5 induced IAP promoter activity. In summary, we provide evidence showing that NFAT5 is a regulator of Wnt signaling. Importantly, our results suggest that NFAT5 regulation of intestinal cell differentiation may be through inhibition of Wnt/β-catenin signaling.
NFAT5; Wnt; β-catenin; intestinal cell differentiation
The effect of bisphenol A (BPA) on the reproductive system is highly debated but has been associated with meiotic abnormalities. However, evidence is lacking with regard to the mechanisms involved. In order to explore the underlying mechanisms of BPA-induced meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 0, 2, 20 or 200 μg/kg body weight (bw)/day for 60 consecutive days. 17β-Estradiol (E2) was administered at 10 μg/kg bw/day as the estrogenic positive control. Treatments with 200 μg/kg bw/day of BPA and E2 significantly decreased sperm counts and inhibited spermiation, characterized by an increase in stage VII and decrease in stage VIII in the seminiferous epithelium. This was concomitant with a disruption in the progression of meiosis I and the persistence of meiotic DNA strand breaks in pachytene spermatocytes,and the ataxia–telangiectasia-mutated and checkpoint kinase 2 signal pathway was also activated; Eventually, germ cell apoptosis was triggered as evaluated by terminal dUTP nick-end labeling assay and western blot for caspase 3. Using the estrogen receptor (ER) antagonist ICI 182780, we determined that ER signaling mediated BPA-induced meiotic disruption and reproductive impairment. Our results suggest that ER signaling-mediated meiotic disruption may be a major contributor to the molecular events leading to BPA-related male reproductive disorders. These rodent data support the growing association between BPA exposure and the rapid increase in the incidence of male reproductive disorders.
bisphenol A; meiocyte spreading; meiosis; spermatogenesis; stage of seminiferous epithelium
One of the most important steps in accurate determination of copy number variants using a case-control comparison is normalizing the coverage level between the different projects. NextGENe's CNV tool uses unique methods for this global normalization and also for log2 ratio calculation. The global normalization method is based on the median allele coverage of selected heterozygous positions. After normalization, log2 ratios are calculated by selecting one position in each specified region (based on annotation or amplicon location). The positions are selected if they are adequately representative of the region, and (ideally) if they are heterozygous, so that allele frequency changes can be noted. In this analysis, the tool is used to analyze data from multiple targeted sequencing technologies. A known variant (deletion in the KCNH2 gene) was detected using HaloPlex™ Target Enrichment System Panels. Large chromosomal abnormalities (such as trisomy 21) were detected using Ion AmpliSeq™ Panels.
Toll-like receptor-4 (TLR4) plays a critical role in innate and acquired immunity, but its role in radio-resistance is unknown. We used TLR4 knockout (KO,−/−) mice and gut commensal depletion methods, to test the influence of TLR4 and its' in vivo agonist on basal radio-resistance. We found that mice deficient in TLR4 were more susceptible to IR-induced mortality and morbidity. Mortality of TLR4-deficient mice after IR was associated with a severe and persistent bone marrow cell loss. Injection of lipopolysaccharide into normal mice, which is known to activate TLR4 in vivo, induced radio-resistance. Moreover, TLR4 in vivo ligands are required for basal radio-resistance. We found that exposure to radiation leads to significant endotoxemia that also confers endogenous protection from irradiation. The circulating endotoxins appear to originate from the gut, as sterilization of the gut with antibiotics lead to increased mortality from radiation. Further data indicated that Myd88, but not TRIF, may be the critical adaptor in TLR4-induced radio-resistance. Taken together, these data strongly suggest that TLR4 plays a critical role in basal radio-resistance. Our data suggest, it is important not to give antibiotics that may sterilize the gut before the whole body irradiation. Further, these data also suggest that management of gut flora through antibiotic or possibly probiotic therapy may alter the innate response to the total body irradiation.
ionizing radiation; toll-like receptor 4; lipopolysaccharide; commensal microflora; Myd88
Coptidis Rhizoma (Coptis chinensis) has been reported to have antioxidative effect on hemolysis of erythrocytes induced by acetylphenylhydrazine in mice and rats. However, the ability of Coptidis Rhizoma to protect structure and function of erythrocytes membrane and morphology of erythrocytes against oxidative damage remains unknown. In this study, we undertook a characterization of antioxidative activity in erythrocytes membrane of Coptidis Rhizoma using an in vivo model of acetylphenylhydrazine-induced mice together with in vitro studies with 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced erythrocytes for further morphology characterization. Acetylphenylhydrazine-induced mice were treated intragastrically with Coptidis Rhizoma at doses of 0.3, 0.6, and 1.2 g/kg per day for 3 days and at the dose of 0.6 and 1.2 g/kg it showed that there was an increasing trend in membranes cytoskeletal proteins of band I-IV, especially a significant upregulation in band II. Significant increase in phosphatidylserine and phosphatidylcholine content at the dose of 1.2 g/kg Coptidis Rhizoma was obsereved. At all doses of Coptidis Rhizoma, the declined membrane fluidity of acetylphenylhydrazine-induced mice was significantly increased. In addition, at the dose of 1.2 g/kg Coptidis Rhizoma treatment showed a significant increase in Ca2+/Mg2+-ATPase activity and there was an increasing trend in the activity of Na+/K+-ATPase. In vitro, Coptidis Rhizoma protected erythrocytes from 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced hemolysis in a dose-dependent manner at concentrations of 0.25-1.5 mg/ml, and also significantly inhibited the 2,2-azo-bis (2-amidinopropane) dihydrochloride-induced morphological alterations in mice erythrocytes. These results demonstrate that Coptidis Rhizoma is capable of protecting erythrocytes against oxidative damage probably by acting as an antioxidant and maintaining membrane integrity.
Coptidis rhizoma; erythrocytes membranes; hemolysis; oxidative damage
A variety of aetiologies may cause third nerve palsy (TNP), and some manifestations may herald neurological emergencies. This article describes and illustrates various diseases that lead to TNP.
We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo
data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.
SC-59; sorafenib; STAT3; HCC
Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring V600EBRAF mutations. RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.
BRAF; PTEN; RB1; MEK inhibitor; BRAF inhibitor; CDKN2A
Transient hypoxia–ischemia (HI) leads to delayed neuronal death in both mature and immature neurons but the underlying mechanisms are not fully understood. To understand whether the pathogenesis of HI-induced neuronal death is different between mature and immature neurons, we used a rat HI model at postnatal days 7 (P7), 15 (P15), 26 (P26) and 60 (P60) in order to investigate ultrastructural changes and active caspase-3 distribution in HI-injured neurons as a function of developmental age. In P7 pups, despite more than 95% of HI-injured neurons highly expressing active caspase-3, most of these active caspase-3-positive neurons revealed mixed features of apoptosis and necrosis (a chimera type) under electron microscopy (EM). Classical apoptosis was observed only in small populations of HI-injured P7 neurons. Furthermore, in rats older than P7, most HI-injured neurons displayed features of necrotic cell death under EM and, concomitantly, active caspase-3-positive neurons after HI declined dramatically. Classical apoptosis after HI was rarely found in neurons older than P15. In P60 rats, virtually all HI-injured neurons showed the shrinkage necrotic morphology under EM and were negative for active caspase-3. These results strongly suggest that pathogenesis of HI-induced neuronal death is shifting from apoptosis to necrosis during brain development.
neonatal brain; hypoxia–ischemia; neuronal death; brain maturation; RNase protection assay; electron microscopy
Transient cerebral ischemia leads to protein aggregation mainly in neurons destined to undergo delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether ischemic preconditioning is able to alleviate neuronal protein aggregation, thereby protecting neurons from ischemic neuronal damage. Ischemic preconditioning was introduced by a sublethal 3 min period of ischemia followed by 48 h of recovery. Brains from rats with either ischemic preconditioning or sham-surgery were then subjected to a subsequent 7 min period of ischemia followed by 30 min, 4, 24, 48 and 72 h of reperfusion. Protein aggregation and neuronal death were studied by electron and confocal microscopy, as well as by biochemical analyses. Seven minutes of cerebral ischemia alone induced severe protein aggregation after 4 h of reperfusion mainly in CA1 neurons destined to undergo delayed neuronal death (which took place after 72 h of reperfusion). Ischemic preconditioning reduced significantly protein aggregation and virtually eliminated neuronal death in CA1 neurons. Biochemical analyses revealed that ischemic preconditioning decreased accumulation of ubiquitin-conjugated proteins (ubi-proteins) and reduced free ubiquitin depletion after brain ischemia. Furthermore, ischemic preconditioning also reduced redistribution of heat shock cognate protein 70 and Hdj1 from cytosolic fraction to protein aggregate-containing fraction after brain ischemia. These results suggest that ischemic preconditioning decreases protein aggregation after brain ischemia.
brain ischemia; preconditioning; protein aggregation; proteotoxicity; electron microscopy