Rapid real-time fluorescent PCR gene dosage test for the diagnosis of DNA duplications and deletions

BACKGROUND: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. METHODS: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed. RESULTS: Area ratios were approximately 0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples. CONCLUSIONS: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.     

Neurólisis robótica del pudendo en el tratamiento del síndrome de atrapamiento del pudendo

Introduction

Pudendal nerve entrapment syndrome (PNE) is characterised by the presence of neuropathic pain in the pudendal nerve (PN) territory, associated or not with urinary, defecatory and sexual disorders. Surgical PN decompression is an effective and safe alternative for cases when conservative treatment fails. The aim of this study is to describe the first robot-assisted pudendal neurolysis procedure performed in our country.

Effect of Different Dialysis Modalities on Microinflammatory Status and Endothelial Damage

Background and objectives: We studied the relationship between microinflammation and endothelial damage in chronic kidney disease (CKD) patients on different dialysis modalities.Design, setting, participants, & measurements: Four groups of CKD stage 5 patients were studied: 1) 14 nondialysis CKD patients (CKD-NonD); 2) 15 hemodialysis patients (HD); 3) 12 peritoneal dialysis patients with residual renal function >1 ml/min (PD-RRF >1); and 4) 13 peritoneal dialysis patients with residual renal function ≤1 ml/min (PD-RRF ≤1). Ten healthy subjects served as controls. CD14⁺CD16⁺ cells and apoptotic endothelial microparticles (EMPs) were measured by flow cytometry. Serum vascular endothelial growth factor (VEGF) was measured by ELISA.Results: CKD-NonD and HD patients had a higher percentage of CD14⁺CD16⁺ monocytes than PD groups and controls. CD14⁺CD16⁺ was similar in the PD groups, regardless of their RRF, and controls. The four uremic groups displayed a marked increase in apoptotic EMPs and VEGF compared with controls. Apoptotic EMPs and VEGF were significantly higher in HD patients than in CKD-NonD and both PD groups. However, there were no significant differences between CKD-NonD and the two PD groups. There was a correlation between CD14⁺CD16⁺ and endothelial damage in CKD-NonD and HD patients, but not in PD and controls.Conclusions: There was an increase in CD14⁺CD16⁺ only in CKD-NonD and HD patients. In these patients, there was a relationship between increased CD14⁺CD16⁺ and endothelial damage. These results strongly suggest that other factors unrelated to the microinflammatory status mediated by CD14⁺CD16⁺ are promoting the endothelial damage in PD, regardless of their RRF.Chronic kidney disease (CKD) patients have a higher rate of mortality than the general population, which has been attributed to a higher rate of cardiovascular mortality (1–3). Several studies have also reported a strong association between endothelial damage and chronic inflammation in CKD patients (4–7).The percentage of proinflammatory blood monocytes with the CD14⁺CD16⁺ phenotype is higher in hemodialysis (HD) patients (8,9), suggesting that these cells play an important role in the chronic inflammatory disease associated with uremia. CD14⁺CD16⁺ cells have been described as a subset of human peripheral monocytes that are phenotypically more differentiated than CD14⁺⁺ and produce proinflammatory cytokines, such as TNF and IL-6 (10,11). In addition, a higher percentage of CD14⁺CD16⁺ monocytes is correlated with endothelial damage in CKD patients (12) and, interestingly, the high levels of CD14⁺CD16⁺ also predicted the appearance of cardiovascular disease.Recently, we reported that a decrease in the percentage of proinflammatory CD14⁺CD16⁺ cells in uremic patients was associated with a greater clearance of large uremic toxins. In fact, we have demonstrated that on-line hemodiafiltration with a high convective transport reduced the percentage of proinflammatory CD14⁺CD16⁺ cells in comparison with high-flux HD (13). Furthermore, we have also observed that higher levels of CD14⁺CD16⁺ in CKD patients were correlated with high plasma levels of endothelial microparticles, an early feature of endothelial damage (14).The aim of the present study was to evaluate the effects of different dialysis modalities on microinflammation as assessed by the percentage of CD14⁺CD16⁺ monocytes and endothelial damage associated with the CKD.     

Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer

Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. We found that tumor samples from patients with biopsy-accessible solid tumors of advanced disease treated with RAD001, a rapamycin derivative, showed an administration schedule–dependent increase in activation of the MAPK pathway. RAD001 treatment also led to MAPK activation in a mouse model of prostate cancer. We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway. Significantly, pharmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors, currently employed as single agents in the clinic, for the treatment of human cancers.     

Analyses of variants located in estrogen metabolism genes (ESR1, ESR2, COMT and APOE) and schizophrenia

Relationships between gender, age-of-onset of schizophrenia and reproductive age strongly suggest a key role for gonadal hormones, and more specifically for estrogens, in the etiology of the illness. Also, estrogens act as neural growth and trophic factors influencing neuron and glial cells in many areas of the central nervous system. Therefore, we investigated the association between schizophrenia and 4 genes related to estrogen metabolism. These genes are ESR1 (estrogen receptor 1), ESR2 (estrogen receptor 2), APOE (apolipoprotein E) and COMT (catechol-O-methyltransferase). The expression of APOE and COMT, which contain estrogen response elements, have been demonstrated to be regulated by the estrogen receptors. In this current association study, we examined 59 single nucleotide polymorphisms (SNPs) located in the ESR1 (26), ESR2 (14), APOE (7) and COMT (12) loci. Allele frequencies were evaluated in the schizophrenia (n = 585)-control (n = 615) sample and no association was found with any of the four genes. In conclusion, our data suggest that the four analyzed genes do not play an important role in susceptibility to schizophrenia.     

Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3–dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.     

Cost–effectiveness of initial antiretroviral treatment administered as single vs. multiple tablet regimens with the same or different components

Objective

To evaluate the efficiency of single-tablet regimens (STR) and multiple-tablet regimens (MTR) with exactly the same or different components.

The three most common CARD15 mutations associated with Crohn's disease and the chromosome 16 susceptibility locus for systemic lupus erythematosus

OBJECTIVE: To test if the three most common mutations contributing to Crohn's disease on the CARD15/NOD2 gene could contribute also to genetic susceptibility to systemic lupus erythematosus (SLE), which has been found to be linked to the region of chromosome 16q13 where the CARD15 gene is located. METHODS: We obtained DNA samples from the blood of 189 SLE patients (according to the American College of Rheumatology classification criteria) and 194 controls of Spanish ancestry. Genotypes for the three CARD15 mutations (3020insC, 2722G>C and 2104C>T) were determined by hybridization with fluorescence resonance energy transfer probes on a LightCycler real-time polymerase chain reaction system. RESULTS: CARD15 genotypes were similar in SLE patients and in controls from the general population (allelic frequencies for 3020insC 0.013 in SLE patients vs 0.013 in controls; for 2722G > C 0.011 vs 0.008; and for 2104C > T 0.032 vs 0.051). CONCLUSION: We did not find evidence that the Crohn's disease-associated mutations on CARD15 contributed to SLE susceptibility.