Evaluation and validation of Biolog OmniLog® system for antibacterial activity assays.

Minimal inhibitory concentration of antimicrobials, determined by the broth microdilution method, requires visual assessment or absorbance measurement using a spectrophotometer. Both procedures are usually performed manually, requiring the presence of an operator to assess the plates at specific time point. To increase the throughput of antimicrobial susceptibility testing, and concurrently convert into an automatic assay, the Biolog OmniLog® system was validated for a new, label-free application using standard 96-well microplates. OmniLog was evaluated for its signal strength to ensure that the signal intensity, detected and measured by the system's camera, was satisfactory. Variability due to the plate location inside the OmniLog incubator, as well as variation between wells, was investigated. Then the system was validated by determining the minimal inhibitory concentration of ciprofloxacin, piperacillin and linezolid against a selected Gram-negative and Gram-positive strains. No significant difference was observed in relation to position of the plates within the system. Plate edge effects were noticeable, thus the edge wells were not included in further experiments. Minimal inhibitory concentration results were comparable to those obtained by conventional protocol as well as to values defined by the Clinical Laboratory Standards Institute or published in the literature.

Non-motor symptoms burden, mood, and gait problems are the most significant factors contributing to a poor quality of life in non-demented Parkinson's disease patients: Results from the COPPADIS Study Cohort.

OBJECTIVE: To identify factors related to a poor health-related and global quality of life (QoL) in a cohort of non-demented Parkinson's disease (PD) patients and compare to a control group. METHODS: The data correspond to the baseline evaluation of the COPPADIS-2015 Study, an observational, 5-year follow-up, multicenter, evaluation study. Three instruments were used to assess QoL: (1) the 39-item Parkinson's disease Questionnaire (PDQ-39), (2) a subjective rating of global QoL (PQ-10), and (3) the EUROHIS-QOL 8-item index (EUROHIS-QOL8). Multiple linear regression methods were used to evaluate the direct impact of different variables on these QoL measures. RESULTS: QoL was worse in PD patients (n = 692; 62.6 ±â€¯8.9 years old, 60.3% males) than controls (n = 206; 61 ±â€¯8.3 years old, 49.5% males): PDQ-39, 17.1 ±â€¯13.5 vs 4.4 ±â€¯6.3 (p < 0.0001); PQ-10, 7.3 ±â€¯1.6 vs 8.1 ±â€¯1.2 (p < 0.0001); EUROHIS-QOL8, 3.8 ±â€¯0.6 vs 4.2 ±â€¯0.5 (p < 0.0001). A high correlation was observed between PDQ-39 and Non-Motor Symptoms Scale (NMSS) (r = 0.72; p < 0.0001), and PDQ-39 and Beck Depression Inventory-II (BDI-II) (r = 0.65; p < 0.0001). For health-related QoL (PDQ-39), non-motor symptoms burden (NMSS), mood (BDI-II), and gait problems (Freezing Of Gait Questionnaire [FOGQ]) provided the highest contribution to the model (ß = 0.32, 0.28, and 0.27, respectively; p < 0.0001); whereas mood and gait problems contributed the most to global QoL (PQ-10, ß = -0.46 and -0.21, respectively; EUROHIS-QOL8, ß = -0.44 and -0.23, respectively). CONCLUSIONS: QoL is worse in PD patients than in controls. Mood, non-motor symptoms burden, and gait problems seem to be the most relevant factors affecting health-related and global perceived QoL in non-demented PD patients.

Safety, efficacy and outcome-related factors of perampanel over 12 months in a real-world setting: The FYDATA study.

BACKGROUND: Real-world data of current antiepileptic drugs (AEDs) used to treat focal seizures is of importance to understand the efficacy and safety outside of the clinical trial setting. Here we report real-world data from a large series of patients treated with perampanel for 1year. METHODS: FYDATA was a multicentre, retrospective, 1-year observational study assessing the efficacy and safety of adjuvant perampanel in patients ≥12 years of age with focal epilepsy in a real-world setting. At 12 months, the proportion of patients who were seizure free, median percentage seizure reduction, proportion of responders, retention rate and proportion of patients with adverse events (AEs) were assessed. Analyses were also performed to identify any patient-, medication- and disease-related factors associated with a large clinical response or carry a risk for AEs. RESULTS: A total of 464 patients were included in the study with a retention rate of 60.6% at 1year. The mean number of prior AEDs was 7.8. The median percentage reduction in overall seizures was 33.3% (75% for secondary generalised seizures) after 1year, with 7.2% of patients achieving seizure freedom. Furthermore, patients on non-enzyme-inducing AEDs were more likely to achieve seizure freedom, and logistic regression revealed that patients aged ≥65 years, those with epilepsy due to a vascular aetiology and those who had received fewer prior AEDs showed a better clinical response to perampanel. A total of 62.9% of the patients experienced AEs at 12 months; dizziness, somnolence and irritability were the most frequent AEs. Patients with prior psychiatric comorbidities (hyperactivity and personality disorder) were more likely to experience psychiatric AEs with perampanel, and slower titration schedules were associated with less AEs overall. CONCLUSION: Perampanel, for the treatment of focal epilepsy in a real-world setting in a refractory population, over 1year, demonstrates a similar efficacy and safety profile to that observed in clinical trials. Our results have implications for the optimisation of perampanel use in a clinical setting.

[Correction: Incorrect spelling of last author name in Neuropsychological profile of frontotemporal lobar degeneration]. / Perfil neuropsicológico de la degeneración lobar frontotemporal.

This corrects the article on p. 79 in volume 38(1), PMID 25963461.

Automatic breast parenchymal density classification integrated into a CADe system.

PURPOSE: Breast parenchymal density is an important risk factor for breast cancer. It is known that mammogram interpretation is more difficult where dense tissue is involved. Therefore, automated breast density classification may aid in breast lesion detection and analysis. METHODS: Several image pattern classification techniques for screen-film (SFM) mammography datasets were tested and classified according to BIRADS categories using known cases. A hierarchical classification procedure based on k-NN, SVM and LBN combined with principal component analysis on texture features uses the breast density features. The classification techniques have been incorporated into a CADe system to drive the detection algorithms. RESULTS: The results obtained on 322 mammograms demonstrate that up to 84% of samples were correctly classified. The results of the lesion detection algorithms were obtained from modules integrated within the CADe system developed by the authors. CONCLUSIONS: The ability to detect suspicious lesions on dense and heterogeneous tissue has been tested. The tools enhance the detectability of lesions and they are able to distinguish their local attenuation without local tissue density constraints.

HIV-1 glycoprotein 120 induces the MMP-9 cytopathogenic factor production that is abolished by inhibition of the p38 mitogen-activated protein kinase signaling pathway.

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4(-)/CXCR4(+)/CCR5(+) T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 alpha (SDF-1alpha) (CXCL12) and macrophage inflammatory protein 1 beta (MIP-1beta) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) are activated on stimulation of these cells with either T- or M-tropic gp120, as well as with SDF-1alpha or MIP-1beta. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1beta but not by M-tropic gp120. Importantly, T- and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood. 2001;98:541-547)

Bends in human mitotic metaphase chromosomes revisited: 15q11-13 is the most frequent non-random autosomal bend in blood cultures.

We have investigated the preferential bending of some chromosome sites in blood cultures from normal and chromosomally abnormal subjects. A total of 2,262 centromeric and 2,718 non-centromeric bends were recorded, and 69 non-centromeric sites were found not to bend at random. 15q11-13 bending was found to be the most frequent non-random autosomal bend. Bends on chromosomes may be remnants of a folded chromosome state in the nucleus, and may facilitate the preferential involvement of some chromosomal bands in structural reorganizations such as the isoacentric fragments, or contribute to the high frequency of interstitial deletions and isodicentric inversion duplications involving the 15q11-13 region.

Bmp4 mediates apoptotic cell death in the developing chick eye.

The bone morphogenetic protein (BMP) expression in vertebrates suggests a reiterative function of these molecules during eye development. However, genetic analysis in mice has provided only partial information. Using the chick embryo as a model system, we have analyzed possible additional functions of BMP4 during optic cup formation. Here we describe the expression pattern of Bmp4 and Bmp7 and we show that, in contrast to the mouse, the prospective lens placode ectoderm expresses high levels of Bmp4 but no Bmp7. After optic vesicle invagination, Bmp4 is expressed in the prospective dorsal neural retina, where BmprIA, BmprII, and Smad1, components of the BMP4 signal transduction pathway, are also expressed. In toto terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling analysis shows that the dorsal optic cup is the site of a spatiotemporally restricted apoptosis, which parallels the expression not only of Bmp4 but also of Msx1 and Msx2, genes implicated in BMP4-mediated apoptosis. The use of optic vesicle cultures as well as in ovo local addition of BMP4 and its antagonist Noggin proves that the local activity of BMP4 is responsible for programmed cell death in the dorsal optic cup. In addition, we show that Noggin is able to reduce the rate of cell proliferation in the dorsal part of the optic cup whereas BMP4 increases the number of BrdU-positive cells in retina cultures. These results provide evidence that BMP4 contributes to eye development by promoting cell proliferation and programmed cell death.

Early and dynamic expression of cSfrp1 during chick embryo development.

Secreted frizzled related proteins (SFRPs) are a new class of signalling molecules that appear to antagonise the activity of the Wnt proteins. Here we report the dynamic expression pattern of cSfrp1, a new member of this family, at early stages of chick embryo development. cSfrp1 transcripts are first detected at pre-streak stages throughout the chick blastula but, during early primitive streak formation, expression is restricted to the anterior primitive streak and later to the blastoderm anterior to the Hensen' s node. This pattern of expression overlaps with that of Otx2 and is complementary to that of cWnt8c. During neural plate formation cSfrp1 mRNAs are abundantly localized only to the anterior domain of the embryo but, as neural tube closes, the expression extends caudally. Later, the main sites of expression in the neural tissue are the telencephalic vesicles, the epiphysis, the developing eyes and the ventral hindbrain and neural tube. Additionally, cSfrp1 transcripts were found in the axial and lateral mesoderm, the otic placode, the trigeminal ganglia, the mesoderm of the branchial arches, the developing limb buds, as well as in the mesodermal component of the developing kidney.

cSix4, a member of the six gene family of transcription factors, is expressed during placode and somite development.

We describe the expression pattern of cSix4, a chick homologue of the murine Six4/AREC3 gene. cSix4 transcripts are detected at gastrula stages in the blastoderm surrounding the developing axial midline. As the neural plate begins to form cSix4 mRNA is detected in a crescent-shaped band, which surrounds the anterior developing neural plate and corresponds to the presumptive placode region. This expression is maintained in all the placodes (olfactory, optic, neural and otic) as they develop but with different characteristics. Further, abundant expression of cSix4 was localised to the paraxial mesoderm and the entire developing somites, becoming restricted first to their dorsal portion, then to the dermomyotome and finally to the myotome. cSix4 expression is maintained in the developing and adult muscular tissue. Additional sites of cSix4 expression are the presumptive and developing limb buds, the notochord, trigeminal ganglia, cells of the spinal cord, particularly the motor neurones, the dorsal root ganglia, the neural retina, as well as the epithelial component of the developing kidney.

Rho-regulated signals induce apoptosis in vitro and in vivo by a p53-independent, but Bcl2 dependent pathway.

Rho proteins are a branch of GTPases that belongs to the Ras superfamily which are critical elements of signal transduction pathways leading to a variety of cellular responses. This family of small GTPases has been involved in diverse biological functions such as cytoskeleton organization, cell growth and transformation, cell motility, migration, metastasis, and responses to stress. We report that several human Rho proteins including Rho A, Rho C and Rac 1, are capable of inducing apoptosis in different cell systems like murine NIH3T3 fibroblasts and the human erythroleukemia K562 cell line. Since K562 cells are devoid of p53, apoptosis induced by Rho in this system is independent of p53. Rho-dependent apoptosis is mediated by the generation of ceramides, and it is drastically inhibited by ectopic expression of Bcl2, both under in vitro and in vivo conditions. Furthermore, the human oncogenes vav and ost that have been shown to function as guanine exchange factors for Rho proteins, were also able to induce apoptosis under similar conditions. Finally, we also report that the levels of endogenous Rho proteins are increased when U937 myeloid leukemia cells are exposed to apoptosis-inducing conditions such as TNF alpha treatment. Furthermore, TNF alpha-induced apoptosis in these cells is inhibited by expression of a dominant negative mutant of Rac 1 but it is not affected by a similar mutant of Rho A. These results suggest that Rho proteins play an important role in the physiological regulation of the apoptotic response to stress-inducing agents.

In vitro expression of MMP-2 and MMP-9 in glioma cells following exposure to inflammatory mediators.

Progression of glioma is associated with local degenerative processes which are attributed to the activity of gelatinases. As glioma cells are candidate for secretion of these enzymes, we have studied in vitro the potential of cytokines (interleukin-1alpha (IL-1), tumor necrosis factor-alpha (TNFalpha) and transforming growth factor-beta (TGFbeta2)) to regulate the expression of gelatinase A and B (Gels A and B, respectively) in two glioma cells of human (A172) and rat origin (C6). We showed that IL-1 and TNFalpha both induced gene expression and protein secretion of Gel B in both cell lines, as revealed by RT-PCR and gelatin zymography, respectively. In C6 cells, TNFalpha had no effect on Gel A constitutive expression while IL-1 increased its production, but only at high doses. We have also demonstrated that TGFbeta2 inhibited both IL-1- or TNFalpha-induced gene expression and Gel B production in a dose-dependent manner but had no effect on Gel A secretion. The effect of TGFbeta2 on Gel B secretion was reversed by phorbol myristate acetate (PMA). Taken together, these data suggest that IL-1, TNFalpha and TGFbeta2 tightly regulate Gel B secretion in glioma cells, an enzyme which is believed to play an important role in the local invasion of brain tissue by tumor cells.

Multidrug-resistance drug-binding peptides generated by using a phage display library.

A phage display library of random decapeptides was used to generate peptide ligands that can bind multidrug-resistance (MDR) drugs mimicking, in this respect, the drug-binding activity of P-glycoprotein. Seven peptide sequences were identified that specifically bound doxorubicin. Five of these sequences expressed the core consensus motif WXXW. The displacement assay showed that the phages expressing these peptides bound MDR type drugs (vinblastine, doxorubicin, verapamil, and genistein) with the same selectivity as P-glycoprotein and did not interact with non-MDR type drugs, such as arabinosylcytosine (Ara-C) and melphalan. One of the selected peptides that showed a highest capacity for the binding (VCDWWGWGIC) was synthesized and displayed competition with the phage for doxorubicin binding. The structure modeling suggested that all the selected sequences contained a hydrophobic envelope in which MDR drugs could be docked with substantial energy minimization. Western blot analysis showed that monospecific antibody obtained against the phage expressing VCDWWGWGIC peptide could specifically recognize P-glycoprotein in the membrane fraction of MDR phenotype MCF-7ADR cells. The MDR drug-binding sequences generated during this work could provide an important tool for design and screening of new chemotherapeutic agents.

Gelatinase B (MMP-9) production and expression by stromal cells in the normal and adult thymus and experimental thymic lymphoma.

Correlative and functional evidence support a crucial role for metalloproteinase (MMP) activity in tumor progression. Dysregulation of MMP production at local tumor sites is thought to participate in the remodeling of the local stromal tissue necessary for tumor growth. The extent of damages in local tissues is often reflected by the high concentration of MMP released in the bloodstream of cancer patients. The integrity of the thymic architecture plays a crucial role in the development of mature T cells, but it is compromised by extensive remodeling occurring during the development of thymic lymphomas. In the present work, we have used an experimental thymic lymphoma model to investigate the regulation of MMP-9 (gelatinase B) production in animals bearing large thymic lymphomas. We show a 3-fold increase in serum gelatinase B (Gel B) levels in animals bearing thymic lymphoma compared with those found in normal animals and a correlation between these levels and the size of the tumor. Although Gel B was found within the thymic tumor, lymphoma cells did not express it in vivo, indicating that Gel B expression was associated with thymic stromal cells rather than lymphoma cells. This was corroborated by evidence that lymphoma cells have the capacity to stimulate Gel B gene expression in stromal cells. Our results suggest that lymphoma cells can exert a significant control over Gel B expression by local stromal cells, thereby inducing the extensive remodeling necessary for tumor growth.

Activation of phospholipase D by growth factors and oncogenes in murine fibroblasts follow alternative but cross-talking pathways.

Phospholipase D (PLD) is activated by a variety of stimuli, including mitogenic stimulation by growth factors and oncogene transformation. Activation of PLD by growth factors requires protein kinase C (PKC) since depletion of the enzyme by down-regulation or direct inhibition by specific drugs completely abrogates this effect. Transformation by the ras and src oncogenes is also associated with an increase in basal PLD activity. However, this effect is not dependent on PKC, suggesting that growth factors and oncogenes may activate PLD by two independent mechanisms. Here we demonstrate that activation of PLD by phorbol esters is greatly enhanced in ras-transformed cells, suggesting synergistic activation of PLD by ras oncogenes and PKC. Also, ras-transformed cells showed a dramatic attenuation of the PLD activation induced by growth factors, although receptor function was still detectable. This attenuation paralleled the specific uncoupling of the phosphatidylinositol-specific phospholipase C (PI-PLC) pathway, indicating that activation of PLD by growth factors may be mediated by PI-PLC and PKC activation. Attenuation of PLD activation by platelet-derived growth factor was also observed in several oncogene-transformed cells, as well as the uncoupling of the PI-PLC pathway. Neither the co-operation with PKC activation nor the attenuation of the PLD response to growth factors in ras-transformed cells was a general consequence of cell transformation, since cells transformed by other oncogenes showed a normal response to either treatment. These results support the existence of at least two alternative signalling routes for the activation of PLD, one mediated by the PI-PLC/diacylglycerol/PKC pathway and a second one mediated by several oncogenes, independent of the PKC pathway, which synergizes with the PI-PLC/PKC-dependent pathway.

Rho proteins induce metastatic properties in vivo.

Rho proteins have been implicated in the regulation of multiple signal transduction processes. Some of the members of this family, including the rho gene from Aplysia californica and the human genes (rhoA, rhoB and rac-1), are proto-oncogenes since when properly mutated they can induce cell transformation, and the generated rho-transformed cells are tumorigenic when inoculated into mice. In addition to their tumorigenic activity, there is evidence suggesting that Rho proteins may contribute to the metastatic phenotype. However, all the experiments implicating Rho proteins or Rho-regulating proteins in the induction of metastatic potential are either indirect or have been performed in vitro. In this study we investigated whether cells transformed by rho oncogenes do have metastatic potential in vivo. We present evidence that cells transformed by the Aplysia californica rho gene, when injected directly into the blood stream are able to efficiently colonize lungs and secondary organs, consistent with the acquisition of the metastatic potential. Moreover, tumors derived from subcutaneous injections of these rho-transformed cells are also able to metastasize in distant organs, a strong support to the hypothesis that Rho proteins play a role in the metastatic phenotype. Finally, cells transformed by the human oncogenes dbl, vav and ost, three well-known guanine exchange factors for members of the Rho family, or cells transformed by the activated human rac-1 or rhoA genes do also have metastatic potential when injected into the blood stream. These results demonstrate that signaling pathways regulated by Rho proteins play an important role in the acquisition of the metastatic phenotype in vivo.

Flow cytometric analysis of gelatinase B (MMP-9) activity using immobilized fluorescent substrate on microspheres.

Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes.

Activation of phospholipase D by ras proteins is independent of protein kinase C.

Growth factors activate phospholipases, causing the generation of diverse lipid metabolites with second messenger function. Among them, the phosphatidylcholine-preferring phospholipase D (PLD) has attracted great interest, since in addition to the transient activation by growth factors stimulation, it is constitutively activated in some of the src- and ras-transformed cells investigated. To establish further the functional relationship of ras oncogenes with PLD, we have investigated its mechanism of regulation. Growth factors such as PDGF or FGF activate the PC-PLD enzyme by a common, PKC-dependent mechanism. By contrast, ras oncogenes activate the PC-PLD enzyme by a PKC-independent mechanism. These results suggest that existence of at least two mechanisms for PLD activation, and ras oncogenes contribute to one of them.