Neutrophil Gelatinase-Associated Lipocalin (NGAL) Predicts Response to Neoadjuvant Chemotherapy and Clinical Outcome in Primary Human Breast Cancer

In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.     

Precise Mapping of the CD95 Pre-Ligand Assembly Domain

Pre-association of CD95 at the plasma membrane is mandatory for efficient death receptor signaling. This homotrimerization occurs through self-association of an extracellular domain called the pre-ligand assembly domain (PLAD). Using novel molecular and cellular tools, we confirmed that CD95-PLAD is necessary to promote CD95 multimerization and plays a pivotal role in the transmission of apoptotic signals. However, while a human CD95 mutant deleted of the previously described PLAD domain (amino acids 1 to 66) fails to interact with its wild-type counterpart and trigger autonomous cell death, deletion of amino acids 1 to 42 does not prevent homo- or hetero (human/mouse)-oligomerization of CD95, and thus does not alter transmission of the apoptotic signal. Overall, these findings indicate that the region between amino acids 43 to 66 corresponds to the minimal motif involved in CD95 homotypic interaction and is necessary to convey an efficient apoptotic signal. Interfering with this PLAD may represent a new therapeutic strategy for altering CD95-induced apoptotic and non-apoptotic signals.     

HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 “Aging” Study

Background

The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes.

Methods

35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, {"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999). In more than 88% of patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm³. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L).

Results

Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60–80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration.

Conclusions

Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999 http://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT01038999","term_id":"NCT01038999"}}NCT01038999.     

Conveniently Pre-Tagged and Pre-Packaged: Extended Molecular Identification and Metagenomics Using Complete Metazoan Mitochondrial Genomes

Background

Researchers sorely need markers and approaches for biodiversity exploration (both specimen linked and metagenomics) using the full potential of next generation sequencing technologies (NGST). Currently, most studies rely on expensive multiple tagging, PCR primer universality and/or the use of few markers, sometimes with insufficient variability.

Methodology/Principal Findings

We propose a novel approach for the isolation and sequencing of a universal, useful and popular marker across distant, non-model metazoans: the complete mitochondrial genome. It relies on the properties of metazoan mitogenomes for enrichment, on careful choice of the organisms to multiplex, as well as on the wide collection of accumulated mitochondrial reference datasets for post-sequencing sorting and identification instead of individual tagging. Multiple divergent organisms can be sequenced simultaneously, and their complete mitogenome obtained at a very low cost. We provide in silico testing of dataset assembly for a selected set of example datasets.

Conclusions/Significance

This approach generates large mitogenome datasets. These sequences are useful for phylogenetics, molecular identification and molecular ecology studies, and are compatible with all existing projects or available datasets based on mitochondrial sequences, such as the Barcode of Life project. Our method can yield sequences both from identified samples and metagenomic samples. The use of the same datasets for both kinds of studies makes for a powerful approach, especially since the datasets have a high variability even at species level, and would be a useful complement to the less variable 18S rDNA currently prevailing in metagenomic studies.     

Concomitant targeting of EGF receptor, TGF-beta and SRC points to a novel therapeutic approach in pancreatic cancer

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways.     

The loss of metabolic control on alcohol drinking in heavy drinking alcohol-dependent subjects

Background

Most physiological studies interested in alcohol-dependence examined ethanol as a pharmacological agent rather than a nutrient. We conducted two studies, which assessed the metabolic and endocrine factors involved in the regulation of alcohol and nutrient intake in alcohol-dependent (AD) subjects. We also examined the potential role of a disruption in energy balance in alcohol-dependence.

Methods and Results

In Study-1, quantitative dietetic interviews of eating and drinking habits were conducted with 97 AD subjects. The population was split around a median alcohol intake value of 12.5 kcal/kg/day. The results showed that the “low alcohol” drinking AD subjects had high Body Mass Index (BMI) and Fat Mass (FM) and alcohol intake was compensated for by a decrease in non-alcoholic intakes. “High alcohol” drinking AD subjects, on the other hand, had low BMI and FM and the total caloric intakes were largely above norms. In Study-2, 24 AD inpatients were submitted to dietetic interviews, calorimetry and blood samplings for the measurement of biomarkers of the regulation of metabolism and satiety, on day 2, 5 and 16 of abstinence. These patients were compared with 20 controls matched for age and gender. We observed in AD patients an increase in cortisol, leptin and PYY plasma levels and a decrease in ghrelin, which might explain the observed decrease in non-alcoholic intakes. However, alcoholic and non-alcoholic intakes correlated positively with basal metabolism and negatively with leptin and leptin/BMI.

Conclusion

For individuals consuming below12.5 kcal/kg/day of alcohol, alcohol intake is compensated for by a decrease in non-alcoholic nutrient intakes, probably due to changes in metabolic and satiety factors. For individuals consuming above 12.5 kcal/kg/day of alcohol, alcohol accelerates metabolism and decreases fat mass and leptin levels, and the total caloric intake largely exceeds norms. A dual model for regulation of energy intake in AD subjects is proposed.     

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.     

Microarray analyses of inflammation response of human dermal fibroblasts to different strains of Borrelia burgdorferi sensu stricto

In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease. Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans). The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-κB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated. In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts. Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs.