Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. METHODOLOGY To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). FINDINGS Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. CONCLUSIONS Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

Insight into the strand exchange reaction promoted by the RecA protein of {\it Escherichia coli\/}

In vitro, RecA protein catalyses the exchange of single strands of DNA between different DNA molecules with sequence complementarity. In order to gain insight into this complex reaction and the roles of ATP binding and hydrolysis, two different approaches have been taken. The first is to use short single-stranded deoxyoligonucleotides as the ssDNA in strand exchange. These were used to determine the signal for hydrolysis and the structure of the RecA-DNA complex that hydrolyses ATP. I present a defined kinetic analysis of the nucleotide triphosphatase activity of RecA protein using short oligonucleotides as ssDNA cofactor. I compare the effects of both homopolymers and mixed base composition oligomers on the ATPase activity of RecA protein. I examine the steady state kinetic parameters of the ATPase reaction using these oligonucleotides as ssDNA cofactor, and show that although RecA can both bind to, and utilise, oligonucleotides 7 to 20 residues in length to support the repressor cleavage activity of RecA, these oligonucleotides are unable to efficiently stimulate the ATPase activity of RecA protein. I show that the K$\sb{\rm m}\sp{\rm ATP}$, the Hill coefficient for ATP binding, the extent of reaction, and k$\sb{\rm cat}$ are all a function of ssDNA chain length and that secondary structure may also play a role in determining the effects of a particular chain length on the ATPase activity of RecA protein.^ The second approach is to utilise one of the many mutants of RecA to gain insight into this complex reaction. The mutant selected was RecA1332. Surprisingly, in vitro, this mutant possesses a DNA-dependent ATPase activity. The K$\sb{\rm m}\sp{\rm ATP}$, Hill coefficient for ATP binding, and K$\sb{\rm m}\sp{\rm DNA}$ are similar to that of wild type. k$\sb{\rm cat}$ for the ATPase activity is reduced 3 to 12-fold, however. RecA1332 is unable to use deoxyoligonucleotides as DNA cofactors in the ATPase reaction, and demonstrates an increased sensitivity to inhibition by monovalent ions. It is able to perform strand exchange with ATP and ATP$\lbrack\gamma\rbrack$S but not with UTP, whereas the wild type protein is able to use all three nucleotide triphosphates. RecA1332 appears to be slowed in its ability to form intermediates and to convert these intermediates to products. (Abstract shortened by UMI.) ^

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.

Iron-Promoted Nucleophilic Additions to Diimine-Type Ligands: A Synthetic and Structural Study

We report here three examples of the reactivity of protic nucleophiles with diimine-type ligands in the presence of FeII salts. In the first case, the iron-promoted alcoholysis reaction of one nitrile group of the ligand 2,3-dicyano-5,6-bis(2-pyridyl)-pyrazine (L1) permitted the isolation of an stable E-imido−ester, [Fe(L1‘)2](CF3SO3)2 (1), which has been characterized by spectroscopic studies (IR, ES-MS, Mössbauer), elemental analysis, and crystallographically. Compound 1 consists of mononuclear octahedrally coordinated FeII complexes where the FeII ion is in its low-spin state. The iron-mediated nucleophilic attack of water to the asymmetric ligand 2,3-bis(2-pyridyl)pyrido[3,4-b]pyrazine (L2) has also been studied. In this context, the crystal structures of two hydration−oxidation FeIII products, [Fe(L2‘)2](ClO4)3·3CH3CN (2) and trans-[FeL2‘‘Cl2] (3), are described. Compounds 2 and 3 are both mononuclear FeIII complexes where the metals occupy octahedral positions. In principle, L2 is expected to coordinate to metal ions through its bipyridine-type units to form a five-membered ring; however, this is not the case in compounds 2 and 3. In 2, the ligand coordinates through its pyridines and through the hydroxyl group attached to the pyrazine imino carbon after hydration, that is, in an N,O,N tridentate manner. In compound 3, the ligand has suffered further transformations leading to a very stable diamido complex. In this case, the metal ion achieves its octahedral geometry by means of two pyridines, two amido N atoms, and two axial chlorine atoms. Magnetic susceptibility measurements confirmed the spin state of these two FeIII species:  compounds 2 and 3 are low-spin and high-spin, respectively.

Breakdown of chlorophyll: A nonenzymatic reaction accounts for the formation of the colorless "nonfluorescent" chlorophyll catabolites

Senescent higher plants degrade their chlorophylls (Chls) to polar colorless tetrapyrrolic Chl catabolites, which accumulate in the vacuoles. In extracts from degreened leaves of the tree Cercidiphyllum japonicum an unpolar catabolite of this type was discovered. This tetrapyrrole was named Cj-NCC-2 and was found to be identical with the product of a stereoselective nonenzymatic isomerization of a “fluorescent” Chl catabolite. This (bio-mimetic) formation of the “nonfluorescent” catabolite Cj-NCC-2 took place readily at ambient temperature and at pH 4.9 in aqueous solution. The indicated nonenzymatic process is able to account for a crucial step during Chl breakdown in senescent higher plants. Once delivered to the acidic vacuoles, the fluorescent Chl catabolites are due to undergo a rapid, stereoselective isomerization to the ubiquitous nonfluorescent catabolites. The degradation of the Chl macrocycle is thus indicated to rely on just two known enzymes, one of which is senescence specific and cuts open the chlorin macroring. The two enzymes supply the fluorescent Chl catabolites, which are “programmed” to isomerize further rapidly in an acidic medium, as shown here. Indeed, only small amounts of the latter are temporarily observable during senescence in higher plants.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^

Thermal reaction norms of growth in Atlantic and Pacific silverside fishes

How organisms may adapt to rising global temperatures is uncertain, but concepts can emerge from studying adaptive physiological trait variations across existing spatial climate gradients. Many ectotherms, particularly fish, have evolved increasing genetic growth capacities with latitude (i.e. countergradient variation (CnGV) in growth), which are thought to be an adaptation primarily to strong gradients in seasonality. In contrast, evolutionary responses to gradients in mean temperature are often assumed to involve an alternative mode, 'thermal adaptation'. We measured thermal growth reaction norms in Pacific silverside populations (Atherinops affinis) occurring across a weak latitudinal temperature gradient with invariant seasonality along the North American Pacific coast. Instead of thermal adaptation, we found novel evidence for CnGV in growth, suggesting that CnGV is a ubiquitous mode of reaction-norm evolution in ectotherms even in response to weak spatial and, by inference, temporal climate gradients. A novel, large-scale comparison between ecologically equivalent Pacific versus Atlantic silversides (Menidia menidia) revealed how closely growth CnGV patterns reflect their respective climate gradients. While steep growth reaction norms and increasing growth plasticity with latitude in M. menidia mimicked the strong, highly seasonal Atlantic coastal gradient, shallow reaction norms and much smaller, latitude-independent growth plasticity in A. affinis resembled the weak Pacific latitudinal temperature gradient.

An Empirical Analysis of the Monetary Policy Reaction Function in India

This paper empirically analyzes India’s monetary policy reaction function by applying the Taylor (1993) rule and its open-economy version which employs dynamic OLS. The analysis uses monthly data from the period of April 1998 to December 2007. When the simple Taylor rule was estimated for India, the output gap coefficient was statistically significant, and its sign condition was found to be consistent with theoretical rationale; however, the same was not true of the inflation coefficient. When the Taylor rule with exchange rate was estimated, the coefficients of output gap and exchange rate had statistical significance with the expected signs, whereas the results of inflation remained the same as before. Therefore, the inflation rate has not played a role in the conduct of India’s monetary policy, and it is inappropriate for India to adopt an inflation-target type policy framework.

Influence of cement properties in the reaction rate and mechanical behavior of concrete with high fl y ash content

The use of fly ash (FA) as an admixture to concrete is broadly extended for two main reasons: the reduction of costs that supposes the substitution of cement and the micro structural changes motivated by the mineral admixture. Regarding this second point, there is a consensus that considers that the ash generates a more compact concrete and a reduction in the size of the pore. However, the measure in which this contributes to the pozzolanic activity or as filler is not well defined. There is also no justification to the influence of the physical parameters, fineness of the grain and free water, in its behavior. This work studies the use of FA as a partial substitute of the cement in concretes of different workability (dry and wet) and the influence in the reactivity of the ash. The concrete of dry consistency which serves as reference uses a cement dose of 250 Kg/m 3 and the concrete of fluid consistency utilized a dose of cement of 350 Kg/m 3 . Two trademark of Portland Cement Type 1 were used. The first reached the resistant class for its fineness of grain and the second one for its composition. Moreover, three doses of FA have been used, and the water/binder ratio was constant in all the mixtures. We have studied the mechanical properties and the micro-structure of the concretes by means of compressive strength tests, mercury intrusion porosimetry (MIP) and thermal analysis (TA). The results of compressive strength tests allow us to observe that concrete mixtures with cements of the same classification and similar dosage of binder do not present the same mechanical behavior. These results show that the effective water/binder ratio has a major role in the development of the mechanical properties of concrete. The study of different dosages using TA, thermo-gravimetry and differential thermal analysis, revealed that the portlandite content is not restrictive in any of the dosages studied. Again, this proves that the rheology of the material influences the reaction rate and content of hydrated cement products. We conclude that the available free water is determinant in the efficiency of pozzolanic reaction. It is so that in accordance to the availability of free water, the ashes can react as an active admixture or simply change the porous distribution. The MIP shows concretes that do not exhibit significant changes in their mechanical behavior, but have suffered significant variation in their porous structure