Merlin has broad tumor suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In Neurofibromatosis type 2, a dominantly inherited tumor disease due to the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/MEK/ERK scaffold Kinase Suppressor of Ras 1 (KSR1) plays a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell-matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1’s function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 Ubiquitin Ligase CRL4DCAF1. Further functional study suggests that KSR1 and DCAF1 might co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.
KSR1; Merlin; DCAF1; schwannoma and MEK1/2
The present study tested the hypothesis that the amount and severity of preoperative pain will affect the anesthetic efficacy of inferior alveolar nerve block (IANB) in patients with symptomatic irreversible pulpitis. One-hundred seventy-seven adult volunteer subjects, actively experiencing pain in a mandibular molar, participated in this prospective double-blind study carried out at 2 different centers. The patients were classified into 3 groups on the basis of severity of preoperative pain: mild, 1–54 mm on the Heft-Parker visual analog scale (HP VAS); moderate, 55–114 mm; and severe, greater than 114 mm. After IANB with 1.8 mL of 2% lidocaine, endodontic access preparation was initiated. Pain during treatment was recorded using the HP VAS. The primary outcome measure was the ability to undertake pulp access and canal instrumentation with no or mild pain. The success rates were statistically analyzed by multiple logistic regression test. There was a significant difference between the mild and severe preoperative pain group (P = .03). There was a positive correlation between the values of preoperative and intraoperative pain (r = .2 and .4 at 2 centers). The amount of preoperative pain can affect the anesthetic success rates of IANB in patients with symptomatic irreversible pulpitis.
Inferior alveolar nerve block; Preoperative pain; Central sensitization; Hyperalgesia; Irreversible pulpitis
Remote Ischemic Preconditioning (RIPC) is a non-invasive cardioprotective intervention that involves brief cycles of limb ischemia and reperfusion. This is typically delivered by inflating and deflating a blood pressure cuff on one or more limb(s) for several cycles, each inflation-deflation being 3–5 min in duration. RIPC has shown potential for protecting the heart and other organs from injury due to lethal ischemia and reperfusion injury, in a variety of clinical settings. The mechanisms underlying RIPC are under intense investigation but are just beginning to be deciphered. Emerging evidence suggests that RIPC has the potential to improve exercise performance, via both local and remote mechanisms. This review discusses the clinical studies that have investigated the role of RIPC in cardioprotection as well as those studying its applicability in improving athletic performance, while examining the potential mechanisms involved.
Remote ischemic preconditioning; Exercise performance; Sports; Cardioprotection; Ischemia-reperfusion injury; CABG; PCI; Perconditioning; Postconditioning; Acute kidney injury
Background and Aims
Portal hypertension is characterized by reduced hepatic eNOS activity. Asymmetric-dimethylarginine (ADMA), an eNOS inhibitor, is elevated in cirrhosis and correlates with severity of portal hypertension. Dimethylargininedimethylaminohydrolase-1 (DDAH-1) is the key enzyme metabolizing hepatic ADMA. This study characterized DDAH-1 in cirrhosis, and explored hepatic DDAH-1 reconstitution through FXR agonism and DDAH-1 gene therapy.
DDAH-1 Immunohistochemistry was conducted on human cirrhosis and healthy liver tissue. Subsequently, sham-operated or bile-duct-ligated (BDL) cirrhosis rats were treated with FXR agonist Obeticholic acid (OA, 5mg/kg) or vehicle for 5 days. Further animals underwent hydrodynamic injection with DDAH-1-expressing plasmid or saline control. Groups: Sham+saline, BDL+saline, BDL+DDAH-1-plasmid. Portal pressure (PP) measurements were performed. Plasma ALT was measured by Cobas-Integra; DDAH-1 expression by qPCR and Western blot; eNOS activity by radiometric assay.
Immunohistochemistry and Western-blotting confirmed hepatic DDAH-1 was restricted to hepatocytes, and expression decreased significantly in cirrhosis. In BDL rats, reduced DDAH-1 expression was associated with elevated hepatic ADMA, reduced eNOS activity and high PP. OA treatment significantly increased DDAH-1 expression, reduced hepatic tissue ADMA, and increased liver NO generation. PP was significantly reduced in BDL+OA vs. BDL+vehicle (8±1 vs. 13.5±0.6 mmHg; p<0.01) with no change in MAP. Similarly, DDAH-1 hydrodynamic injection significantly increased hepatic DDAH-1 gene and protein expression, and significantly reduced PP in BDL+DDAH-1 vs. BDL+ saline (p<0.01).
This study demonstrates DDAH-1 is a specific molecular target for portal pressure reduction, through actions on ADMA-mediated regulation of eNOS activity. Our data support translational studies targeting DDAH-1 in cirrhosis and portal hypertension.
Portal hypertension; ADMA; DDAH-1; Nitric oxide
Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Student’s t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.
Remote Ischemic Preconditioning (RIPC) is emerging as a new noninvasive intervention that has the potential to protect a number of organs against ischemia–reperfusion (IR) injury. The standard protocols normally used to deliver RIPC involve a number of cycles of inflation of a blood pressure (BP) cuff on the arm and/or leg to an inflation pressure of 200 mmHg followed by cuff deflation for a short period of time. There is little evidence to support what limb (upper or lower) or cuff inflation pressures are most effective to deliver this intervention without causing undue discomfort/pain in nonanesthetized humans. In this preliminary study, a dose–response assessment was performed using a range of cuff inflation pressures (140, 160, and 180 mmHg) to induce limb ischemia in upper and lower limbs. Physiological changes in the occluded limb and any pain/discomfort associated with RIPC with each cuff inflation pressure were determined. Results showed that ischemia can be induced in the upper limb at much lower cuff inflation pressures compared with the standard 200 mmHg pressure generally used for RIPC, provided the cuff inflation pressure is ~30 mmHg higher than the resting systolic BP. In the lower limb, a higher inflation pressure, (~55 mmHg > resting systolic BP), is required to induce ischemia. Cyclical changes in capillary blood O2, CO2, and lactate levels during the RIPC stimulus were observed. RIPC at higher cuff inflation pressures of 160 and 180 mmHg was better tolerated in the upper limb. In summary, limb ischemia for RIPC can be more easily induced at lower pressures and is much better tolerated in the upper limb in young healthy individuals. However, whether benefits of RIPC can also be derived with protocols delivered to the upper limb using lower cuff inflation pressures and with lesser discomfort compared to the lower limb, remains to be investigated.
Remote Ischemic Preconditioning (RIPC) is emerging as a new noninvasive intervention that has the potential to protect a number of organs against ischemia‐reperfusion (IR) injury. Currently, there is little evidence to support what limb (upper or lower) or cuff inflation pressures are most effective to deliver this intervention without causing undue discomfort/pain in nonanesthetized humans. In this study we have demonstrated that limb ischemia for RIPC can be more easily induced at lower pressures and is much better tolerated in the upper limb compared with the lower limb, in young healthy individuals.
Characterization; cuff inflation pressure; remote ischemic preconditioning; tolerability
Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70–80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints.
The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC).
IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD.
TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith–Magenis and Potocki–Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD.
Genetics; Molecular genetics; Chromosomal; Clinical genetics; Psychiatry
Interleukin-1β (IL-1β) is an inflammatory cytokine that plays a prominent role in stress-induced behavioral changes. In a model of repeated social defeat (RSD), elevated IL-1β expression in the brain was associated with recruitment of primed macrophages that were necessary for development of anxiety-like behavior. Moreover, microglia activation and anxiety-like behavior associated with RSD did not occur in IL-1 receptor type-1 knock-out (IL-1R1KO) mice. Therefore, the objective of this study was to examine the role of IL-1 signaling in RSD-induced macrophage trafficking to the brain and anxiety-like behavior. Initial studies revealed that RSD did not increase circulating myeloid cells in IL-1R1KO mice, resulting in limited macrophage trafficking to the brain. In addition, IL-1R1KO bone marrow-chimera mice showed that IL-1R1 expression was essential for macrophage trafficking into the brain. To differentiate cellular mediators of stress-induced IL-1 signaling, endothelial-specific IL-1R1 knock-down (eIL-1R1kd) mice were used. Both wild-type (WT) and eIL-1R1kd mice had increased circulating monocytes, recruitment of macrophages to the brain, and altered microglia activation after RSD. Nonetheless, RSD-induced expression of IL-1β, TNF-α, and IL-6 mRNA in brain CD11b+ cells was attenuated in eIL-1R1kd mice compared with WT. Moreover, anxiety-like behavior did not develop in eIL-1R1kd mice. Collectively, these findings demonstrated that there was limited RSD-induced priming of myeloid cells in IL-1R1KO mice and disrupted propagation of neuroinflammatory signals in the brain of eIL-1R1kd mice. Furthermore, these data showed that transduction of IL-1 signaling by endothelial cells potentiates stress-induced neuroinflammation and promotes anxiety-like behavior.
anxiety; blood-brain barrier; interleukin-1; microglia; neuroinflammation; stress
The purpose of the present study was to comparatively evaluate the effect of presence of a 2 mm ferrule and different type of dowels on fracture resistance of mandibular premolars.
Materials and Methods:
Fifty uniradicular mandibular premolars were divided into five groups (n = 10). Ten teeth received no treatment (group I). Samples in group II & III were decoronated 2 mm above cemento-enamel junction and received custom cast dowel-core and fiber dowel-composite core respectively, with 2 mm ferrule. Samples in group IV & V were decoronated at CEJ and were restored using cast dowels and fiber dowel-composite cores, without any ferrule. The restored teeth received metal ceramic crowns and were mechanically loaded. The specimens were subjected to a static load, until fracture, to determine the fracture resistance and fracture mode.
The samples with 2 mm ferrule had a higher fracture resistance than non ferrule groups. Within non ferrule groups, there were no significant differences in the fracture resistance. Specimen restored with cast dowel had more incidence of non-repairable fracture.
Presence of ferrule increased the fracture resistance of endodontically treated teeth. In case of absence of ferrule, fiber dowels had similar fracture resistance as that of cast dowels and showed increased incidence of repairable fracture.
Cast dowel; Fiber dowels; Fracture resistance; Fracture mode
The aim of this study was to assess the prevalence of alcohol consumption, tobacco use and risky sexual behaviour among adolescents, and to evaluate the socioeconomic factors potentially influencing these behaviours.
This cross-sectional study was conducted from January to April 2011 among 376 adolescents (15–19 years old) studying in different schools and colleges in Udupi, India. The Youth Risk Behavior Survey questionnaire and guidelines were followed for data collection. Participants’ alcohol consumption, smoking habits and sexual behaviour patterns were explored. Univariate analysis followed by multivariate logistic regression was done.
The prevalence of alcohol consumption, tobacco use and sexual activity was found to occur in 5.7%, 7.2% and 5.5% of participants, respectively. The mean age of the participants’ first sexual activity, consumption of alcohol and tobacco use was reported to be approximately 16.8 years. Multivariate analysis showed that males were more likely to have used alcohol and tobacco. Other factors, such as religion and tobacco use among family members, were found to be influential.
The potential coexistence of multiple risk behaviours in a student demands an integrated approach. Emphasis should be placed on health education in schools and an increased awareness among parents in order to prevent adolescents’ behaviours from becoming a risk to their health.
Adolescents; Risk Behaviors; Tobacco; Alcoholic Beverages; Sexual Behavior; India
HIV–TB (tuberculosis) coinfection has emerged as a major public health threat. Given the multifactorial enabling environment in a resource-constrained setting like India, the consequences are of epidemic proportions.
This study was aimed at identifying the clinical and epidemiological determinants underlying HIV–TB coinfection.
Settings and Design:
A retrospective review of patient records was done from the antiretroviral therapy center (ART) center at a district hospital in southern India between May and August 2012.
Materials and Methods:
Secondary data of 684 patients on ART as well as pre-ART were collected between July 2008 and June 2012 and were analyzed.
Descriptive analysis, χ2, and Wilcoxon signed rank tests were used with SPSS version 15.0 to draw significant statistical inferences.
HIV–TB coinfection was diagnosed in 18.9% with higher prevalence among males (75.3%), in the sexually active age group 31-45 years (61.3%), with less than primary education (44.15%), who were married (56.1%), laborers (42.4%), from rural backgrounds (88.2%), and having low income-earning capacity (94.4%). Transmission was predominantly through the heterosexual route. The key entry point was the integrated counseling and testing center (ICTC) (47.4%). Pulmonary tuberculosis (58.8%) was predominantly found followed by extrapulmonary tuberculosis (38.2%) and both in 3.1%. A favorable outcome was observed in 69.3% of coinfected patients with 89.2% on ART and 97.2% currently on DOTS therapy. The Wilcoxon signed-rank test found significant association between rises in CD4 counts after the 6th-month follow up (P < 0.05). Coinfected patients had a case fatality rate of 25%.
The prevalence of HIV–TB coinfection recorded in this sample was 18.86%. ICTC implemented by NACO emerged as an effective entry point, while Revised National Tuberculosis Control Program referred 1.6% (n = 11) of the patients to the ART center. Coinfection is associated with lower CD4 counts than those with HIV alone, which could translate into increased morbidity and progression of HIV to AIDS.
ART; CD4; DOTS; HIV–TB coinfection; ICTC
Craniosynostosis, the premature fusion of the cranial sutures, is a heterogeneous disorder with a prevalence of ~1 in 2,200 (refs. 1,2). A specific genetic etiology can be identified in ~21% of cases3, including mutations of TWIST1, which encodes a class II basic helix-loop-helix (bHLH) transcription factor, and causes Saethre-Chotzen syndrome, typically associated with coronal synostosis4-6. Starting with an exome sequencing screen, we identified 38 heterozygous TCF12 mutations in 347 samples from unrelated individuals with craniosynostosis. The mutations predominantly occurred in patients with coronal synostosis and accounted for 32% and 10% of subjects with bilateral and unilateral pathology, respectively. TCF12 encodes one of three class I E-proteins that heterodimerize with class II bHLH proteins such as TWIST1. We show that TCF12 and TWIST1 act synergistically in a transactivation assay, and that mice doubly heterozygous for loss-of-function mutations in Tcf12 and Twist1 exhibit severe coronal synostosis. Hence, the dosage of TCF12/TWIST1 heterodimers is critical for coronal suture development.
The extracellular signal-related kinases (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets1. Here we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs 2-7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf reduced to ~30% of normal exhibit postnatal multisuture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to distal regulatory elements containing RUNX or AP1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.
This open-labeled, post-marketing study was conducted to assess the efficacy and tolerability of fixed dose combination of amlodipine and metoprolol extended release capsules in mild to moderate hypertension in adult Indian patients.
Materials and Methods:
Of 101 enrolled patients, 64 drug naïve patients were treated with regimen A (amlodipine 5 mg + metoprolol 25 mg) and those with prior history of hypertension (n = 37) were treated with regimen B (amlodipine 5 mg + metoprolol 50 mg) for 8 weeks. Treatment response was assessed at week 4 and 8. Dose up titration to regimen B was carried out for those who failed to achieve the target blood pressure (BP) at week 4 in regimen A and additional antihypertensives were added to those in regimen B. Safety laboratory tests were performed at baseline and end of study.
Mean age (±SD) of patients was 53.36 (±11.26) years and body weight (±SD) 63.40 (10.03) kg. Ninety five patients (94.06%) were only hypertensive and 6 (5.94%) had hypertension with history of coronary artery disease; mean duration (±SD) of hypertension was 42.50 (48.07) months. At baseline, patients had a mean (±SD) systolic blood pressure (SBP) and diastolic blood pressure (DBP) of 154.98 (±7.76) mmHg and 95.55 (±5.70) mmHg respectively. There was a statistically significant (P < 0.001) reduction of 12.16% and 14.69% in SBP, 11.49% and 14.65% in DBP at week 4 and week 8 respectively, compared to baseline. Normalization of overall BP was achieved in 49.49% and 70.71% patients at week 4 and 8, respectively. Peripheral edema was reported in 2.97% (3/101) patients.
This combination was safe, efficacious, and well-tolerated in study population.
Amlodipine and metoprolol; essential hypertension; fixed dose combination; peripheral edema
The human tumour antigen PRAME (preferentially expressed antigen in melanoma) is frequently overexpressed during oncogenesis, and high PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.
Nucleosome translocation along DNA is catalyzed by eukaryotic SNF2-type ATPases. One class of SNF2-ATPases is distinguished by the presence of a C-terminal bromodomain and is conserved from yeast to man and plants. This class of SNF2 enzymes forms rather large protein complexes that are collectively called SWI/SNF complexes. They are involved in transcription and DNA repair. Two broad types of SWI/SNF complexes have been reported in the literature; PBAF and BAF. These are distinguished by the inclusion or not of polybromo and several ARID subunits. Here we investigated human SS18, a protein that is conserved in plants and animals. SS18 is a putative SWI/SNF subunit which has been implicated in the etiology of synovial sarcomas by virtue of being a target for oncogenic chromosomal translocations that underlie synovial sarcomas.
We pursued a proteomic approach whereby the SS18 open reading frame was fused to a tandem affinity purification tag and expressed in amenable human cells. The fusion permitted efficient and exclusive purification of so-called BAF-type SWI/SNF complexes which bear ARID1A/BAF250a or ARID1B/BAF250b subunits. This demonstrates that SS18 is a BAF subtype-specific SWI/SNF complex subunit. The same result was obtained when using the SS18-SSX1 oncogenic translocation product. Furthermore, SS18L1, DPF1, DPF2, DPF3, BRD9, BCL7A, BCL7B and BCL7C were identified. ‘Complex walking’ showed that they all co-purify with each other, defining human BAF-type complexes. By contrast,we demonstrate that human PHF10 is part of the PBAF complex, which harbors both ARID2/BAF200 and polybromo/BAF180 subunits, but not SS18 and nor the above BAF-specific subunits.
SWI/SNF complexes are found in most eukaryotes and in the course of evolution new SWI/SNF subunits appeared. SS18 is found in plants as well as animals. Our results suggest that in both protostome and deuterostome animals, a class of BAF-type SWI/SNF complexes will be found that harbor SS18 or its paralogs, along with ARID1, DPF and BCL7 paralogs. Those BAF complexes are proteomically distinct from the eukaryote-wide PBAF-type SWI/SNF complexes. Finally, our results suggests that the human bromodomain factors BRD7 and BRD9 associate with PBAF and BAF, respectively.