Immunohistochemical detection and quantification of T cells in the small intestine of Isospora suis-infected piglets--influence of fixation technique and intestinal segment

Quantification of immunohistochemical results constitutes an important tool in the analysis of cells and tissue that is not readily replaced by other techniques. For reliable quantification, it is essential to consider factors such as tissue fixation and tissue sampling. We report a study on the model of the intestine of Isospora suis‐infected piglets, in which we addressed (1) whether the quantity of detectable T cells in the intestinal mucosa is the same in formalin‐, HOPE®‐, and cryo‐conserved material or whether the amounts of T cells at least correlate with one another; and (2) whether single jejunal segments differ in regard to the quantity of mucosal T cells and variability of lymphocyte infiltration. Quantification of T cells in histological sections of different parts of the jejunum of 15‐22 day old piglets infected with I. suis was performed using an anti‐CD3‐antibody and stereological point counting. Area fractions of T‐cell profiles per intestinal mucosa profile were higher in cryo‐conserved samples than in HOPE®‐ and formalin‐conserved material but no correlation between different fixations could be found. The proximal part of the jejunum contained fewer T cells compared with mid‐ and end‐jejunum. Coefficients of variation did not differ between the intestinal segments. For quantification of T cells in the gut mucosa of piglets infected with I. suis, the cryo‐conserved mid jejunum seems most suitable in cases when unbiased sampling of the complete intestine is not feasible. It is generally not possible to compare quantitative results of immunostaining in samples conserved by different methods. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Broad-Band Measurements of Electromagnetic Properties of Film-Shaped Materials Using Striplines

Two broad-band techniques for measuring the electromagnetic properties of isotropic film-shaped materials are presented. The electromagnetic properties are computed from S-parameter measurements of coplanar and microstrip lines. These lines can be used as cells, propagating the quasi-TEM mode. The measurements are easy to be implemented. They are carried out with a network analyzer and on-wafer systems covering 0.05–40 GHz. The quasi-TEM dispersion is very low for a coplanar cell shape such as h>W+2S. Thus, an extraction method of the coplanar substrate properties ( _r , _r ) has been developed from analytical relationships. It is faster than the microstrip extraction method, which requires a numerical analysis method of the propagation in order to take into account the quasi-TEM dispersion. Moreover, a theoretical study of the electromagnetic propagation through these cells is presented. It has allowed us to determinate the best cell to be used according to the material properties to be characterized. Measured _r and _r data for several materials are presented in the 0.05–40 GHz frequency range. These techniques show good agreement between measured and predicted values. However, the loss tangent measurements of low-loss materials are not possible with these techniques.     

Proteome-wide screening for the analysis of protein targeting of <i>Chlamydia pneumoniae</i> in endoplasmic reticulum of host cells and their possible implication in lung cancer development

Available reports have confirmed a link between bacterial infection and the progression of different types of cancers, including colon, lungs, and prostate cancer. Here we report the Chlamydia pneumonia proteins targeting in endoplasmic reticulum (ER) using in-silico approaches and their possible role in lung cancer etiology. We predicted 48 proteins that target human ER, which may be associated with protein folding and protein-protein interactions during infection. The results showed C. pneumoniae proteins targeting human ER and their implications in lung cancer growth. These targeted proteins may be involved in competitive interactions between host and bacterial proteins, which may change the usual pathway functions and trigger the development of lung cancer. Moreover, C. pneumoniae unfolded protein accumulation in the human ER possibly induces ER stress, consequently activating the unfolded protein response (UPR), and providing a favorable microenvironment for cancer growth. The current study showed the C. pneumoniae protein targeting in ER of host cell and their implication in lung cancer growth. These results may help researchers better manage lung cancer and establish a molecular mechanism for C. pneumoniae lung cancer association.     

Characterization of multidrug-resistant <i>Klebsiella pneumoniae</i> isolated from the Chinese cobra <i>Naja atra</i> in a Beijing suburb

The emergence and spread of antibiotic resistance genes among Bacteria are a serious threat to global health. Their occurrence in animals which are in contact with humans is also important. The Chinese cobra (Naja atra, Elapidae), though a highly venomous species, is appreciated as food and as a source of materials used in traditional Chinese medicine. We are here reporting the isolation of multidrug-resistant Klebsiella pneumoniae (Enterobacteriaceae) from the lung of Naja atra, obtained from a snake farm in a Beijing suburb. Our study analyzed, using gene sequencing, the occurrence of antibiotic resistance genes (ARGs) in three K. pneumoniae isolates from two snakes. In addition, bacterial clones were identified by biochemical tests and phylogenetic analysis. Tests of antimicrobial susceptibility showed that all K. pneumoniae isolates were resistant to a host of antibiotics (piperacillin, cefazolin, gentamicin, tetracycline, doxycyclin, ciprofloxacin, levofloxacin, lomefloxacin, ofloxacin, norfloxacin, nalidixic acid, chloramphenicol, nitrofurantoin, sulfamethoxazole, and sulfamethoxazole/trimethoprim) but were susceptible to cefotaxime, cefixime, aztreonam, bramycin, amikacin, kanamycin, netilmicin, and streptomycin. Eighteen ARGs were detected in total DNA extracted from the isolates. Results showed three quinolone resistance genes (oqxA, oqxB, qnrB), the gyrA gene that confers resistance to beta-lactam antibiotics, and the emerging aac(3)-II gene that confers resistance to aminoglycosides. K. pneumoniae is an important opportunistic human pathogen and the emergence of multidrug-resistant K. pneumoniae in N. atra suggests the increasing risk of pathogen transmission between humans, livestock, and wildlife. Given the close association between foodborne pathogenic microorganisms and humans, it is key factor to identify these antibiotic resistance genes profile thereby minimize the risk of K. pneumoniae transmission.     

Design and testing of a 1.5 Tesla double-tuned (H/P) RF surface coil with intrinsic geometric isolation

We analyse the isolation characteristics of axial and transverse field surface radio frequency coils, used to design a double-tuned surface coil composed by two coils, that combine proton (¹H) detection and localized spectra of phosphorous (³¹P). Several geometrical configurations were analysed, including circular loop and figure-of-eight coils, with the aim to optimize the isolation between the two channels. Our analysis shows that by using at least one transverse coil for the design of the double-tuned probe, it is possible to achieve a good intrinsic geometrical decoupling, without the need of additional decoupling circuits. On the basis of the experimental results, we have designed and built a 1.5 T double-tuned probe composed by a circular loop coil and a transverse field coil, with the external coil tuned at the ¹H frequency and the internal coil tuned at the ³¹P frequency, that shows a good intrinsic decoupling and signal-to-noise ratio.     

Brief Note: A glass bead protocol for recovery of host cell free <i>Ehrlichia canis</i> and quantification by Sybr-green real-time PCR

E. canis infection of the canine cell line DH82 is a routine in studies with this bacteria. A protocol for isolation of host cell free bacteria was developed based on the use of glass beads. Improvement of infection with E. canis isolated by this method was detected by real-time PCR.     

Apertureless scanning near-field optical microscopy for ion exchange channel waveguide characterization

We report the characterization of an integrated Ag⁺/Na⁺ ion exchange waveguide realized in a silicate glass substrate using apertureless scanning near‐field optical microscopy. Our experimental set‐up is based on the combination of a commercial atomic force microscope with an optical confocal detection system. Thanks to this system, the topography and evanescent optical field at the waveguide top surface are mapped simultaneously. Also, the process of apertureless scanning near‐field optical microscopy image formation is analysed. In particular, fringe patterns appearing in the image reveal the intrinsic interferometric nature of the collected signal, due to interference between the field scattered by the tip end and background fields related to guide losses. We give a quantitative interpretation of these fringes. Evanescent intensity mapping on the sample surface allowed us to extract physical waveguide parameters. In particular, it shows an unambiguous multimode beat along the waveguide propagation axis. Furthermore, we show that analysis of this intensity profile reveals back‐reflection effects from the waveguide exit facet. The resulting standing waves pattern allows us to evaluate the eigenmode propagation constants.     

Relationship of multidrug-resistant gene and extended-spectrum carbapenem-resistance in <i>Staphylococcus aureus</i>

The aim of this study was to determine the relationship between phenotypic antimicrobial susceptibility patterns and extended-spectrum, carbapenem-resistance genes. A total of 109 clinical Staphilococcus aureus strains were subjected to 19 antimicrobial susceptibility tests. Resistance to methicillin (mecA), penicillin (blaTEM), and tetracycline (tetM) was detected. We compared the presence of the blaTEM genes with extended-spectrum, carbapenem-related genes and identified the types of SCCmec genes. Of 109 clinical S. aureus strains, 62 (56.88%) had methicillin resistance and 60 strains carried mecA. The prevalence of blaTEM and tetM genes was 81.65% and 37.61%, respectively. The most predominant SCCmec type was SCCmec type II 28/60 (46.67%), in 60 mecA-positive methicillin-resistant S. aureus (MRSA) isolates. The SCCmec prevalence rates were type IVA 30.00% (18/60), type IVb 8.33% (5/60), type IVd 6.67% (4/60), and non-typable 8.33% (5/60). Sixty of the 109 (55.05%) MRSA isolates were positive for extended-spectrum carbapenems (31/60) (51.67%), cephalosporins 40/60 (66.67%) and carbapenems 31/60 (51.67%). The predominant SCCmec type II demonstrated more carbapenem-resistance than the IVA, IVb and IVd types.     

Identification of key long noncoding RNAs and their biological functions in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) are vital regulators in tumorigenesis and metastasis. However, the pathological role of lncRNAs in hepatocellular carcinoma (HCC) is still unclear. In this study, we filtered out three lncRNAs from The Cancer Genome Atlas (TCGA) data that were screened for basic expression and clinical research. We selected lncRNA-NEAT1 for further study to explore its function in HCC progression and its regulatory mechanism. We identified three differentially expressed lncRNAs (DElncRNAs) in tumor and adjacent normal tissues from the TCGA library using data mining methods: lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11. Their basic expression levels were detected by qPCR. Then, we selected lncRNA-NEAT1 as a potentially important lncRNA to verity its biological function and mechanism in HCC cell lines. lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11 were overexpressed in liver cancer tissues and cell lines. We found that silencing NEAT1 in vitro can inhibit the proliferation of HuH-7 and Li-7 cells, inhibit cell migration, and induce apoptosis as well as significantly increase the level of miR-16-5p. We also confirmed that miR-16-5p has a significant correlation with Bcl-2. When NEAT1 is silenced, the expression of Bcl-2 decreases. Inhibiting miR-16-5p can restore Bcl-2 to its original level. We conclude that miR-16-5p1/lncRNA NEAT1 plays a crucial role in regulating the delivery of Bcl-2 in HCC. Overall, the miR-16-5p/lncRNA-NEAT1/Bcl-2 signaling axis may be a promising target for HCC treatment.     

Characterisation of DLC coating adherence by scratch testing

In order to characterize the adherence of DLC coatings (Diamond Like Carbon), scratch testing was performed on a unit equipped with sensors for normal and tangential forces, and an acoustic detector to detect the nucleation and the propagation of cracks. The system is also equipped with a microscope permitting observation of each event on the scratch according to the friction tangential force signal or the acoustic signal. The local microscopic observation allows identification of the damage with respect to the normal load. The test was performed with a Rockwell C indenter at the relative displacement speed, v=10 mm/min under a progressive normal load from 5 to 55 N.Coating failure appears in various modes, particularly the following: propagation of the cracks along the longitudinal edges of the scratch; propagation in front of the indenter; rupture along the maximum principal stress lines; and, detachment in the subsurface by shearing of the coating. The microscopic analysis of the evolution of the scratch under a progressive normal load permits identification of the various traces and the damage mechanisms of the coating.In this study, experimental results are shown for the scratch tests on bulk glass and DLC coating. Various modes of crack initiation, damage and rupture of these materials according to the critical normal load are presented. The analysis of the contact stress field distribution in bulk glass enables identification of the crack initiation and its propagation in the coating.     

Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

Theory of Friction with Applied Load

We present a molecular dynamics simulation for the static friction under the effect of load. We chose a system formed by slabs of molecules of N ₂ deposited on a (111) surface of Pb. In contrary to many calculations, we assume that the Pb atoms are not kept fixed in the lattice positions, but can vibrate in their own phonon’s field. This has the important consequence that the upper and lower block can exchange energy and momentum. During the molecular dynamics simulation, the two systems can reach a thermodynamical equilibrium. When in our molecular dynamics simulation the equilibrium is reached the N ₂ plane at the interface reconstructs. The unit cell is still hexagonal but it contains 16 molecules in disordered positions. These positions of the N ₂ molecules are strongly modified by the presence of load. For small load there is a small increase of the disorder that produces a small reduction in the static friction. For larger loads the formation of clusters begins, and for high loads the size of the clusters increases and there is a tendency to the formation of vacancies. These effects are producing a large increase in the force friction. We can then distinguish different regions that characterize the behaviour of the static friction as a function of the load.     

MicroRNA-181a is elevated by 10-hydroxycamptothecin and represses lung carcinoma progression by downregulating FOXP1

Tumor progression is usually characterized by proliferation, migration, and angiogenesis, which is essential for supplying both nutrients and oxygen to the tumor cells. Therefore, targeting angiogenesis has been considered a promising therapeutic strategy for cancer prevention and treatment. In the present study, we demonstrated that in addition to suppressing lung cancer cell proliferation and migration in vitro, 10-hydroxycamptothecin (10-HCPT) is also capable of inhibiting angiogenesis in vivo with a miR-181a-dependent manner. Mechanistically, by upregulating miR-181a, which in turn downregulating FOXP1, 10-HCPT can inhibit the PI3K/Akt/ERK signaling pathwaymediated angiogenesis. Furthermore, reduced levels of miR-181a have been found in both lung cancer cell lines and xenograft with concurrently elevated levels of FOXP1, VEGF, bFGF, and HDGF. Consistent with the findings from the in vitro experiments, miR-181a impairs neovascularization in our xenograft model. In summary, our findings have not only established the anti-oncogenic role of miR-181a in lung cancer angiogenesis but also suggest that 10-HCPT could be a potential therapeutic reagent for lung cancer treatment.     

Nondestructive control of objects made of electroconductive materials in pulsed magnetic fields

We have measured the values of the maximum tagential component of magnetic field strength H _τm on the surfaces of metal objects as a function of the time of increase in charge pulses and as a function of sample thickeness. We have found the empirical formulas of dependence H _τSm of the secondary field on sample thicknesses and on the time of increase in pulses, for which we give a theoretical explanation. We expound on methods for controlling the electric and magnetic properties of objects, as well as their geometric parameters and integrity defects in them.     

Synergistic combination of colistin with imipenem,amikacine or ciprofloxacin against <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i> carbapenem-resistant isolated in Annaba hospital Algeria

Objective: The aim of this study is to detect in vitro the synergetic activity of colistin in combination with imipenem, amikacin or ciprofloxacin, at sub-inhibitory concentrations, against carbapenems-resistant (CR) Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.
Materials and Methods: The minimal inhibitory concentrations (MIC) were determined by broth macrodilution (BMD). Carbapenemase encoding genes were screened using polymerase chain reaction (PCR). The activity of colistin in combination with second antibiotic was evaluated by the Checkerboard Technique.
Results: 39 CR P. aeruginosa and 21 CR A. baumanni strains where collected. The MIC values ranging from (0.25 to 4 µg/ml) to colistin, ≥16 µg/ml for imipenem, ≥4 µg/ml to amikacin and ≥8 µg/ml ciprofloxacin. The PCR reveals the presence of the genes bla_OXA23 (n = 12), blaOXA24 (n = 6), blaNDM1 (n = 3) in A. baumannii and blaVIM2 (n = 12) in P. aeruginosa. The combination of colistin with imipenem showed synergistic effect on 57.14% and 46.15% of A. baumannii and P. aeruginosa isolates, respectively. For colistin and amikacin, the synergistic effect is detected in 28.6% of A. baumannii and 30.8% of P. aeruginosa. While colistin and ciprofloxacin showed synergy on 14.29% and 15.38% of A. baumannii and P. aeruginosa isolates, respectively.
Conclusion: CR A. baumannii and P. aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital. Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.     

Detection of new antibiotic resistance gene profile in <i>Escherichia coli</i> associated with avian leukosis virus infection from broiler chickens

The Escherichia coli (E. coli) is prevailing worldwide, but the epidemiology of E. coli infections feature regional distribution characteristics to some extent. E. coli, as a zoonotic pathogen, can be transferred from animals to humans through food chain or via contact with wounds, causing a public health risk. We reported the swelling of proventriculus and tracheal bleeding following the death in two broiler chickens (Gallus gallus domesticus) from Beijing, China. To investigate whether a virus was involved in the infection, Madin Darby Bovine Kidney (MDCK) cells were co-cultured with supernatants of proventriculus, trachea and spleen homogenates. The avian leucosis virus was detected in the samples of proventriculus and trachea, but the avian influenza virus, the Newcastle disease virus and the avian infectious laryngotracheitis virus were not detected. E. coli isolates were resistant to almost all the antimicrobial as tested except for the combinations of amoxicillin/clavulanic acid and sulfamethoxazole/trimethoprim. PCR tests demonstrated the presence of antibiotic resistance genes in these E. coli isolates and further research revealed a novel gene profile with the presence of CTX-M-1, gyrA, gyrB, oqxA, oqxB, parC and Sul2 antibiotic resistance genes in a strain isolated from a proventriculus sample. These results demonstrated that the presence of antibiotic resistant E. coli would not necessarily cause outbreak of large-scale disease. However, when the bacteria carrying new antibiotic resistance genes enter the environment, it may result in the development of more virulent strains which will potentially impact human and animal health.     

Morphological characteristics and mycelial compatibilityof different Mycogone perniciosa isolates

The major disease of the cultivated mushroom Agaricus bisporus Lange (Imb) in Serbian mushroom farms is wet bubble caused by the fungus Mycogone perniciosa (Magnus) Delacr. In this study we report the morpho‐physiological characteristics and inter‐relationships between colonies of five isolates of M. perniciosa. The results suggest that mycelial compatibility could serve as an additional parameter for a more reliable determination of different pathotypes of M. perniciosa.     

Harmonic demodulation and minimum enhancement factors in field‐enhanced near‐field optical microscopy

Field‐enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near‐field information recorded in a field‐enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe–sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near‐field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near‐field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe–sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field‐enhanced scanning optical microscopy.     

Ultrastructural analysis of cell wall of drug resistant and sensitive Mycobacterium tuberculosis isolated from cerebrospinal fluid by transmission electron microscope

Drug‐resistant tuberculosis is being increasingly recognized and is one among the leading cause of death worldwide. Remarkable impermeability of cell wall to antituberculous drugs protects the mycobacteria from drug action. The present study analyzed the cell wall thickness among first‐line drug resistant and sensitive Mycobacterium tuberculosis (Mtb) isolated from cerebrospinal fluid by transmission electron microscopy (TEM). The average thickness of the cell wall of sensitive isolates was 13.60 ± 0.98 nm. The maximum difference (26.48%) in the cell wall thickness was seen among multi‐drug resistant (18.50 ± 1.71 nm) isolates and the least difference (4.14%) was shown by streptomycin‐resistant (14.18 ± 1.38 nm) isolates. The ultrastructural study showed evident differences in the cell wall thickness among sensitive and resistant isolates. Preliminary TEM examination of cells indicates that morphological changes occur in the cell wall which might be attributed to the drug resistance. The thickened wall of Mtb appears to help the bacilli to overcome the action of antituberculous drugs.