Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.     

细菌双杂交系统的改进

解析蛋白质之间的相互作用,对理解调控和代谢等生物学过程具有重要意义。其中基于互补腺苷酸环化酶功能的细菌双杂交系统是一种研究蛋白质之间相互作用的有效手段。本文在利用该系统时发现存在假阳性高的缺陷。进一步报告基因活性分析表明产生假阳性的原因是不同克隆的生理状态影响了LacZ的输出,即lacZ启动子除了受cAMP信号分子的控制,还受到其他调控蛋白的直接或间接的调控。为了降低生理因素对报告基因的影响,我们向该系统引入用多拷贝lacZ启动子控制的gfp报告基因。这不仅通过增加lacZ启动子的数量,弱化了生理因素的影响;还实现了第二个报告基因gfp与lacZ同时报告蛋白质之间的相互作用情况,提高了输出的准确性。系统的实验验证表明,我们改进的细菌双杂交系统的灵敏性确实得到大幅提高。     

Mapping inhibitor response to the in-frame deletions,insertions and duplications of epidermal growth factor receptor (EGFR) in non-small cell lung cancer

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of non-small cell lung cancer (NSCLC). However, a large number of in-frame deletion, insertion and duplication mutations in the EGFR tyrosine kinase (TK) domain have been observed to alter drug response to such a kinase target. Thus, a systematic investigation of the intermolecular interactions between the clinical small-molecule agents and various EGFR in-frame mutants would help to establish a complete picture of drug response to kinase mutations in lung cancer, and to design new EGFR inhibitors with high potency and selectivity to target drug-resistant mutants. Here, we describe a combined pipeline to explore the drug response of five representative EGFR inhibitors, including three FDA-approved agents (gefitinib, erlotinib and lapatinib) and two compounds under clinical development (AEE788 and TAK-285) to a number of clinically relevant EGFR in-frame mutations, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity to EGFR in-frame mutations. It was found that the insertion and duplication mutations in exon 20 can generally cause drug resistance to EGFR due to the reduced size of kinase’s active pocket, while deletion mutations in exon 19 associate closely with increased inhibitor sensitivity to EGFR by establishing additional non-bonded interactions across complex interface, including hydrogen bonds, cation–π interactions and hydrophobic contacts.     

Vibrational sensitivity of the wing of the pigeon (Columba livia) — a study using heart rate conditioning

Summary The vibrational sensitivity of awake pigeons was tested with the heart rate conditioning method. This method proved to be a very sensitive instrument for the behavioural measurement of the pigeon's sensitivity to mechanical stimuli.Sine wave vibrational stimuli between 50 and 2000 Hz were applied to the 1st, 7th and 16th primary feathers of the wing. The resulting threshold curves were U- or V-shaped with 3 characteristics: (i) The frequency of best response was either 800 or 900 Hz. (ii) Within the broad frequency range of vibrational sensitivity (50 to 2000 Hz) the sensitivity was extremely high at the best frequency: threshold amplitudes lay between 0.5 and 0.09 m. (iii) The threshold curves showed very sharp tuning at best frequency with bandwidths between 0.3 and 0.7 octaves (measured at best frequency 10fold above threshold). The functional meaning of the characteristic vibrational sensitivity of the wing for flight control is discussed; it is interpreted as an adaptation to special situations occurring during flight.Abbreviations CR conditioned response - CS conditioned stimulus - ECG electrocardiogram - HC(s) Herbst corpuscle(s) - UCS unconditioned stimulus All experiments were carried out at the Ruhr-Universität Bochum, Lehrstuhl für Allgemeine Zoologie     

Spatial orientation of rainbow trout to plane-polarized light: The ontogeny of E-vector discrimination and spectral sensitivity characteristics

Summary The results of this study demonstrate that trout (Salmo gairdneri) are capable of orienting to polarized light fields. The spectral composition of the polarized light fields can significantly influence the orientation of trout. Rainbow trout exhibit ontogenetic losses in orientation to polarized light fields which appears coincident with the ontogenetic loss of the UV-sensitive cones. Trout were trained to swim to a refuge located at one end of the training tank under a polarized light field. The E-vector of the polarized light field was oriented parallel or perpendicular to the long axis of the training tank. Trained fish were released in a circular test tank and their angular response scored. Under a white plus ultraviolet polarized light field, trout oriented in the trained E-vector orientation. For instance, fish trained under a parallel E-vector orientation exhibited angular responses close to parallel in the test tank. However, when the spectral composition of the polarized light field was manipulated, the accuracy of spatial orientation of the trout varied. Trout weighing about 30 g exhibited accurate orientation to the white plus UV polarized light field. The trout were incapable of orientation at a body weight of 50 to 60 g.     

Different modes of action of substance P in the motor control of the feline stomach and pylorus

In an experimental in vivo model to study gastropyloric motility in the cat a contraction of the stomach and the pyloric sphincter was regularly obtained in animals subjected to electrical vagal nerve stimulation or local intraarterial (i.a.) injection of substance P (SP). Much more infrequently contractile motor responses were recorded at splanchnic nerve stimulation. The contractile effects of SP were sensitive to atropine or local infusion of a SP analogue, (d-Pro²,d-Trp^7,9)-SP, indicating that SP activated a final common cholinergic neuron in both stomach and pylorus. However, there seemed to be separate transmission mechanisms in these two regions based on the results of the physiological studies. The vagally induced pyloric contraction was noncholinergic, nonadrenergic, but sensitive to ganglionic blockade (hexamethonium) or the SP analogue, indicating involvement of SP in a peptidergic pathway to the sphincter. The infrequent splanchnically induced pyloric contraction was sensitive to atropine, the SP analogue or ganglionic blockade (hexamethonium) in favour of SP acting on a final cholinergic neuron in this system. On the other hand the gastric contraction, obtained at either extrinsic nerve stimulations or local i.a. injection of SP, was sensitive to atropine or the SP analogue but hexamethonium resistant. These findings suggest antidromic activation of SP-containing axon collaterals of the extrinsic nerves terminating on cholinergic neurons of the gastric wall. When afferent C-fibres of the vagal nerve were selectively activated by local heating, pyloric contraction and gastric relaxation were obtained via vago-vagal reflexes. After cervical vagotomy heating of the distal end of the vagal nerve elicited a gastric contraction, previously demonstrated to be atropine sensitive and hexamethonium resistant, but no pyloric motor response. This suggests that the antidromic activation mechanism was present only in the stomach, not in the pylorus.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.