Aluminium foil as an alternative substrate for the spectroscopic interrogation of endometrial cancer

Biospectroscopy has the potential to investigate and characterize biological samples and could, therefore, be utilized to diagnose various diseases in a clinical environment. An important consideration in spectrochemical studies is the cost‐effectiveness of the substrate used to support the sample, as high expense would limit their translation into clinic. In this paper, the performance of low‐cost aluminium (Al) foil substrates was compared with the commonly used low‐emissivity (low‐E) slides. Attenuated total reflection‐Fourier transform infrared spectroscopy was used to analyse blood plasma and serum samples from women with endometrial cancer and healthy controls. The 2 populations were differentiated using principal component analysis with support vector machines with 100% sensitivity in plasma samples (endometrial cancer = 70; healthy controls = 15) using both Al foil and low‐E slides as substrates. The same sensitivity results (100%) were achieved for serum samples (endometrial cancer = 60; healthy controls = 15). Specificity was found higher using Al foil (90%) in comparison to low‐E slides (85%) and lower using Al foil (70%) in comparison to low‐E slides in serum samples. The establishment of Al foil as low‐cost and highly performing substrate would pave the way for large‐scale, multicentre studies and potentially for routine clinical use.

     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

The 1- to 2-Hz oscillations in muscle force are exacerbated by stress, especially in older adults.

Although force fluctuations during a steady contraction are often heightened in old adults compared with young adults and are enhanced in young adults during the stress response, the mechanisms underlying the augmentation are uncertain. The purpose of the study was to compare the effect of a stressor on the plasma concentrations of selected stress hormones and on the force fluctuations experienced by young and old adults during the performance of a precision grip. Thirty-six men and women (19-86 yr) participated in a protocol that comprised anticipatory (30 min), stressor (15 min), and recovery periods (25 min). The stressor was a series of noxious electrical stimuli applied to the dorsal surface of the left hand. Subjects sustained a pinch-grip force with the right hand at 2% of the maximal voluntary contraction force. The fluctuations in pinch-grip force, the interference electromyogram (EMG) of six muscles, and the spectra for the force and EMG were quantified across the 70-min protocol. The stressor increased the force fluctuations, largely due to an enhancement of the power at 1-2 Hz in the force spectrum (r(2) = 0.46). The effect was greatest for the old adults compared with young and middle-aged adults. The plasma concentrations of the stress hormones (adrenocorticotropin, epinephrine, and norepinephrine) were elevated to similar levels for all three age groups, and the changes were not associated with modulation of the force fluctuations. Furthermore, the heightened EMG activity exhibited by the old adults during all periods was not related to the changes in the force fluctuations or the 1- to 2-Hz force oscillations. The absence of a change in the mean pinch-grip force during the protocol and the lack of an association between elevation of the plasma concentrations for the stress hormones and modulation of the force fluctuations suggest that the enhanced force fluctuations caused by the stressor was due to an increase in the low-frequency output of the spinal motor neurons.     

Structure of PICK1 and other PDZ domains obtained with the help of self-binding C-terminal extensions

PDZ domains are protein-protein interaction modules that generally bind to the C termini of their target proteins. The C-terminal four amino acids of a prospective binding partner of a PDZ domain are typically the determinants of binding specificity. In an effort to determine the structures of a number of PDZ domains we have included appropriate four residue extensions on the C termini of PDZ domain truncation mutants, designed for self-binding. Multiple truncations of each PDZ domain were generated. The four residue extensions, which represent known specificity sequences of the target PDZ domains and cover both class I and II motifs, form intermolecular contacts in the expected manner for the interactions of PDZ domains with protein C termini for both classes. We present the structures of eight unique PDZ domains crystallized using this approach and focus on four which provide information on selectivity (PICK1 and the third PDZ domain of DLG2), binding site flexibility (the third PDZ domain of MPDZ), and peptide-domain interactions (MPDZ 12th PDZ domain). Analysis of our results shows a clear improvement in the chances of obtaining PDZ domain crystals by using this approach compared to similar truncations of the PDZ domains without the C-terminal four residue extensions.     

Statin therapy, alone or with rapamycin, does not reverse monocrotaline pulmonary arterial hypertension: the rapamcyin-atorvastatin-simvastatin study

Pulmonary arterial hypertension (PAH) is characterized by excessive pulmonary artery smooth muscle cell proliferation and impaired apoptosis leading to obstruction of resistance pulmonary arteries. We hypothesized that antiproliferative (rapamycin) and proapoptotic (statins) agents, already used clinically for other indications, would decrease experimental PAH, facilitating translation to human therapies. Prior studies in the rat monocrotaline-PAH model have indicated that simvastatin regresses and rapamycin prevents, but cannot reverse, PAH. Two PAH regression strategies (rapamycin monotherapy vs. rapamycin + atorvastatin) and one prevention strategy (simvastatin) were tested in a rat monocrotaline-PAH model. Adult male Sprague-Dawley rats were randomized to saline (n = 6) or monocrotaline (60 mg/kg ip, n = 36) treatment groups. Monocrotaline rats were randomized to gavage with vehicle, rapamycin (2.5 mgxkg(-1)xday(-1)), or rapamycin + atorvastatin (10 mgxkg(-1)xday(-1)) treatment groups, beginning 12 days post-monocrotaline. Echocardiographic and hemodynamic end points were assessed 2 wk later. Additional monocrotaline-PAH rats (n = 20) were randomized to vehicle or simvastatin (2 mgxkg(-1)xday(-1)) treatment groups and followed echocardiographically for 4 wk. Monocrotaline-PAH increased lung p70 S6 kinase phosphorylation, and this was reversed by rapamycin, confirming the biological activity of rapamycin. Despite the use of high doses, neither rapamcyin nor rapamycin + atorvastatin improved survival nor reduced PAH, vascular remodeling, and right ventricular hypertrophy. Although prophylactic simvastatin slowed PAH progression, by 4 wk PAH severity and mortality were not different from placebo. Apart from the new finding of p70 S6 kinase phosphorylation in monocrotaline-PAH, this is a negative therapeutic trial (none of these promising therapies improved monocrotaline-PAH). These negative results should be considered as human trials with these agents are underway (simvastatin) or proposed (rapamycin).     

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background

Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results

In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion

A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.     

Human Schlafen 5 (SLFN5) Is a Regulator of Motility and Invasiveness of Renal Cell Carcinoma Cells

We provide evidence that human SLFN5, an interferon (IFN)-inducible member of the Schlafen (SLFN) family of proteins, exhibits key roles in controlling motility and invasiveness of renal cell carcinoma (RCC) cells. Our studies define the mechanism by which this occurs, demonstrating that SLFN5 negatively controls expression of the matrix metalloproteinase 1 gene (MMP-1), MMP-13, and several other genes involved in the control of malignant cell motility. Importantly, our data establish that SLFN5 expression correlates with a better overall survival in a large cohort of patients with RCC. The inverse relationship between SLFN5 expression and RCC aggressiveness raises the possibility of developing unique therapeutic approaches in the treatment of RCC, by modulating SLFN5 expression.     

Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar-specific virulence

The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola ( Psph ), is differentiated into nine races based on the presence or absence of five avirulence ( avr ) genes in the bacterium, which interact with corresponding resistance genes, R1–R5 , in Phaseolus vulgaris . The resistance gene R2 is matched by avrPphE , which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph . Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph . DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104 bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens -directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene-specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph , elicited a weak HR if expressed in planta , whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB , which matches the R3 resistance gene, also caused a gene-specific HR following expression in the plant after delivery by A. tumefaciens .     

The Network RamDisk: Using remote memory on heterogeneous NOWs

Efficient data storage, a major concern in the modern computer industry, is mostly provided today by traditional magnetic disks. However, the cost of a disk transfer (measured in processor cycles) continues to increase with time, making disk accesses increasingly expensive. In this paper we describe the design, implementation and evaluation of a Network RamDisk device that uses main memory of remote workstations as a fasterthandisk storage device. In our study we propose various reliability policies, making the device tolerant to single workstation crashes. We show that the Network RamDisk is portable, flexible, and can operate under any of the existing Unix file systems. The Network RamDisk has been implemented both on the Linux and the Digital Unix operating systems, as a block device driver without any modifications to the kernel code. Using several real applications and benchmarks, we measure the performance of the Network RamDisk over an Ethernet and an ATM network, and find it to be usually four to eight times better than the magnetic disk. In one benchmark, our system was two orders of magnitude faster than the disk. We believe that a Network RamDisk can be efficiently used to provide reliable lowlatency access to files that would otherwise be stored on magnetic disks.