A review of diagnostic tests for congenital syphilis in newborns

Congenital syphilis (CS) can occur when a mother is inadequately treated or not treated at all for an active Treponema pallidum infection. Symptoms of CS are often subtle and non-specific, and it is estimated that up to 60% of affected infants are asymptomatic at birth, making the diagnosis dependent on laboratory findings. Despite decades of experience with CS, problems still arise in its diagnosis because laboratory test results for children at risk for CS can be inconclusive and no single diagnostic test can be used to diagnose CS. The development of diagnostic tests such as enzyme immunoassays, immunoblotting and polymerase chain reaction (PCR) has increased the sensitivity and specificity of diagnoses, but the detection of specific IgM is currently the most sensitive serological method, and the presence of specific IgM should be considered as evidence of a congenital T. pallidum infection. Suspected cases can also be confirmed or excluded by serial post-partum tests of antibody kinetics. The authors note strongly that it is considered unethical not to treat a baby at risk of contracting CS, even without a definitive diagnosis. In this review, we describe the various microbiological methods—and their shortcomings—used in the laboratory diagnosis of CS.     

A simple PCR condition for detection of a single cyst of Acanthamoeba species

Acanthamoeba is a free-living protozoan with a worldwide distribution in a variety of natural and artificial habitats. It has even been found in contact lens solution. Acanthamoeba spp. can cause infections such as granulomatous amoebic encephalitis and amoebic keratitis. Specific and sensitive diagnosis of Acanthamoeba infections can prevent clinical symptoms from worsening. Recently, PCR technique has been used for Acanthamoeba diagnosis. Unfortunately the dormant cyst of Acanthamoeba is resistant to chemical reagents; thus, most extraction of DNA uses a commercial DNA extraction kit for obtaining DNA for further use in polymerase chain reaction (PCR). Therefore in the present study, we improved the ability to diagnose Acanthamoeba using a simplified PCR technique. Interestingly, heating at 94°C for 10 min could release DNA which is amplified with specific primers designed from 16S rRNA. The PCR product is about 180 bp. This technique is a simple and efficient method for detection of Acanthamoeba—even a single cyst—and does not require high-cost reagents or complicated procedures to extract DNA.     

Simultaneous DNA'fingerprinting', diagnosis of sex and single-gene defect status from single cells

Sex and cystic fibrosis status have been previously diagnosedseparately at the single cell level. We have developed a sensitive,reliable, accurate and rapid (within 5–6 h) system forthe simultaneous diagnosis of sex, cystic fibrosis and a DNA‘fingerprint’ within a single reaction from a varietyof single cells. As contamination cannot be totally excluded,particularly at the single cell level, DNA ‘fingerprinting’can be used to assess the risk of contamination. High sensitivitywith single cells is combined with very high specificity (estimatedmatching probability of 10^–7–10^–8), allowingthe source of the amplified cell to be identified with a veryhigh degree of probability. Fluorescent primers were multiplexedfor six tetranucleotide microsatellite sequences to determinethe DNA fingerprint; the amelogenin gene was used to diagnosesex, and primers for the CFTR region were used to determinecystic fibrosis (CF) status. Analysis of the fluorescent productwas undertaken using an automated DNA sequencer with Genescansoftware. This technique has many applications such as prenataland preimplantation diagnosis, forensic identification of smallor degraded samples, and detection of contamination sources.DNA fingerprints of single haploid spermatozoa and other cellscan be assessed, so ensuring the detection of both diploid andhaploid contamination during preimplantation diagnosis. cystic fibrosis/DNA fingerprinting/fluorescent PCR/preimplantation diagnosis/sexing     

Low-frequency physiological activation of the vestibular utricle causes biphasic modulation of skin sympathetic nerve activity in humans

We have previously shown that sinusoidal galvanic vestibular stimulation, a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, it influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. Here, we tested the hypothesis that selective stimulation of one set of otolithic organs—those located in the utricle, which are sensitive to displacement in the horizontal axis—could entrain sympathetic nerve activity. Skin sympathetic nerve activity (SSNA) was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 10 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations–decelerations (~4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz; composite movements in both directions were also applied. Subjects either reported feeling a vague sense of movement (with no sense of direction) or no movement at all. Nevertheless, cross-correlation analysis revealed a marked entrainment of SSNA for all types of movements: vestibular modulation was 97 ± 3 % for movements in the X axis and 91 ± 5 % for displacements in the Y axis. For each sinusoidal cycle, there were two major peaks of modulation—one associated with acceleration as the platform moved forward or to the side, and one associated with acceleration in the opposite direction. We interpret these observations as reflecting inertial displacement of the stereocilia within the utricle during acceleration, which causes a robust vestibulosympathetic reflex.     

Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.     

An efficient method for the assessment of DNA quality of archival microdissected specimens.

There will be an increasing need of methods for assessing the suitability of specimens for genetic-based assays as DNA markers become an integral part of molecular diagnosis. The targeting of specimens for specific analyses will require the ability to rapidly screen for DNA quality. Conventional methods such as Southern analysis and gene specific-polymerase chain reaction (PCR) often require quantities of material that represent a significant portion of the specimen, especially in microdissected samples. Here we describe a novel application of a commonly used PCR-based DNA-fingerprinting technology that requires minimal quantities of DNA to simultaneously assess multiple regions throughout the genome for DNA quality. Randomly amplified polymorphic DNA (RAPD) PCR generates DNA fragments of a broad size range with the product size reflecting the degree of sample fragmentation. Fourteen DNA samples extracted from cells microdissected from seven formalin-fixed, paraffin-embedded oral cancer biopsies were assessed for DNA quality using gene-specific PCR and RAPD-PCR. Although the more conventional assay required 2-ng DNA (or 300-cell equivalents) to examine DNA quality at a single locus, RAPD-PCR provided a more informative profile of DNA quality from the same microdissected archival specimens.     

Sarcoid-like lesions associated with the immune restoration inflammatory syndrome in AIDS: absence of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection

Highly active antiretroviral therapy (HAART)-treated human immunodeficiency virus (HIV)-positive patients can develop granulomatous lesions within the first few weeks of initiating therapy. This immune syndrome, called immune restoration inflammatory syndrome (IRIS), can induce sarcoid-like lesions in tissues. The pathogenesis of these granulomas is currently unknown because no pathogen has been identified to date in the lesions using morphological and/or microbiological approaches. However, the role of certain microbes, such as Mycobacterium tuberculosis, is still debated. The aim of this study was to look for the presence of M. tuberculosis in sarcoid-like lesions occurring in 14 AIDS patients treated with HAART. We used the PCR DNA amplification method in granulomas microdissected from sections stained by hematoxylin–eosin from formalin-fixed, paraffin-embedded specimens. Results were compared to those obtained from microdissected tuberculosis (TB) granulomas (15 patients) and from microdissected sarcoidosis granulomas (12 patients). M. tuberculosis DNA was undetectable from the microdissected sarcoid-like granulomas, whereas DNA from M. tuberculosis was isolated in all the microdissected TB granulomas and was absent in the microdissected sarcoidosis granulomas. Taken together, these data showed that M. tuberculosis DNA is not associated with the presence of sarcoid-like lesions occurring in HIV-positive patients treated with HAART.     

Characteristics of a micro-biolistic system for murine immunological studies

With an advanced computational fluid dynamics (CFD) technique, we have numerically developed and examined a micro-biolistic system for delivering particles to murine target sites. The micro-particles are accelerated by a high speed flow initiated by a traveling shock wave, so that they can attain a sufficient momentum to penetrate in to the cells of interest within murine skin (or mucosa). In immunization application, powdered vaccines are directly delivered into the antigen presenting cells (APCs) within the epidermis/dermis of the murine skin with a narrow and highly controllable velocity range (e.g., 699±5.6 m/s for 1.8 μm modeled gold particles) and a uniform spatial distribution over a diameter of ~ 4 mm target area. Key features of gas dynamics and gas-particle interaction are presented. Importantly, the particle impact velocity conditions are quantified as a function of: stand-off distance (2–15 mm), driver gas species (air/helium mixtures), particle density (1,050 kg/m³ and 19,320 kg/m³) and particle size (1–5 μm for gold particles and 10–50 μm for less dense particles, respectively). The influential parameters—representative of immunotherapeutic (e.g., DNA vaccination) and protein (e.g., lidocaine) biolistic applications—are studied in detail.     

Analysis of two strains of <Emphasis Type="Italic">Peanut stunt virus</Emphasis>: satRNA-associated and satRNA free

Peanut stunt virus (PSV) is a pathogen of legumes, vegetables, trees, and weeds occurring worldwide. The species is characterized by significant genetic variability. PSV strains are classified into four subgroups on the basis of their nucleotide sequence homology. Here, we are presenting two further, fully sequenced PSV strains—PSV-Ag and PSV-G, that could be considered as I subgroup representatives. However, their sequence homology with other typical I subgroups members, similarly as another strain—PSV-P, characterized by our group previously, is lower than 90%. This lead us to propose further subdivision of the I subgroup into IA, IB, and IC units, and to classify PSV-Ag and PSV-G strains to the last one. In this article, we are showing that identity level of PSV-Ag and PSV-G is very high and apart from the presence of satRNA in the first one, they differ only by a few nucleotides in their genomic RNAs. Nevertheless, symptoms they cause on host plants might differ significantly, just as the levels in infected plants. Effect of single amino acid changes between strains on the three-dimensional structure of viral proteins was analyzed. Differences occur mainly on the protein surfaces which can possibly affect protein–protein interaction in infected cells, which is discussed.     

Gait adaptability training is affected by visual dependency

As part of a larger gait adaptability training study, we designed a program that presented combinations of visual flow and support-surface manipulations to investigate the response of healthy adults to walking on a treadmill in novel discordant sensorimotor conditions. A visual dependence score was determined for each subject, and this score was used to explore how visual dependency was linked to locomotor performance (1) during three training sessions and (2) in a new discordant environment presented at the conclusion of training. Performance measures included reaction time (RT), stride frequency (SF), and heart rate (HR), which respectively served as indicators of cognitive load, postural stability, and anxiety. We hypothesized that training would affect performance measures differently for highly visually dependent individuals than for their less visually dependent counterparts. A seemingly unrelated estimation analysis of RT, SF, and HR revealed a significant omnibus interaction of visual dependency by session (p < 0.001), suggesting that the magnitude of differences in these measures across training day 1 (TD1), training day 3 (TD3), and exposure to a novel test is dependent on subjects’ levels of visual dependency. The RT result, in particular, suggested that highly visually dependent subjects successfully trained to one set of sensory discordant conditions but were unable to apply their adapted skills when introduced to a new sensory discordant environment. This finding augments rationale for developing customized gait training programs that are tailored to an individual. It highlights one factor—personal level of visual dependency—to consider when designing training conditions for a subject or patient. Finally, the link between visual dependency and locomotor performance may offer predictive insight regarding which subjects in a normal population will require more training when preparing for specific novel locomotor conditions.     

Modulation of muscle sympathetic bursts by sinusoidal galvanic vestibular stimulation in human subjects

There is controversy as to whether the vestibulosympathetic reflexes demonstrated in experimental animals actually exist in human subjects. While head-down neck flexion and off-vertical axis rotation can increase muscle sympathetic nerve activity (MSNA) in awake subjects, we recently showed that bipolar galvanic vestibular stimulation (GVS) does not. However, it is possible that our stimuli (2 mA, 1 s)—although capable of causing strong postural and occulomotor responses—were too brief. To address this issue we activated vestibular afferents using continuous sinusoidal (0.5–0.8 Hz, 60–100 cycles, ±2 mA) bipolar binaural GVS in 11 seated subjects. Sinusoidal GVS evoked robust vestibular illusions of “rocking in a boat” or “swinging from side to side.” Cross-correlation analysis revealed a cyclic modulation of MSNA ranging from 31 to 86% across subjects (mean ± SE 58 ± 5%), with total MSNA increasing by 156 ± 19% (P = 0.001). Furthermore, we documented de novo synthesis of sympathetic bursts that were coupled to the sinusoidal input, such that two bursts—rather than the obligatory single burst—could be generated within a cardiac interval. This demonstrates that the human vestibular apparatus exerts a potent facilitatory influence on MSNA that potentially operates independently of the baroreceptor system.     

Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films

An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and—in general—in the field of bio-hybrid devices.     

A Numerical Model of Skin Electropermeabilization Based on In Vivo Experiments

As an alternative to viral methods that are controversial because of their safety issues, chemical and physical methods have been developed to enhance gene expression in tissues. Reversible increase of the cell membrane permeability caused by the electric field—electroporation—is currently one of the most efficient and simple non-viral methods of gene transfer. We performed a series of in vivo experiments, delivering plasmids to rat skin using external plate electrodes. The experiments showed that skin layers below stratum corneum can be permeabilized in this way. In order to study the course of skin tissue permeabilization by means of electric pulses, a numerical model using the finite element method was made. The model is based on the tissue-electrode geometry and electric pulses used in our in vivo experiments. We took into account the layered structure of skin and changes of its bulk electrical properties during electroporation, as observed in the in vivo experiments. We were using tissue conductivity values found in literature and experimentally determined electric field threshold values needed for tissue permeabilization. The results obtained with the model are in good agreement with the in vivo results of gene transfection in rat skin. With the model presented we used the available data to explain the mechanism of the tissue electropermeabilization propagation beyond the initial conditions dictated by the tissue initial conductivities, thus contributing to a more in-depth understanding of this process. Such a model can be used to optimize and develop electrodes and pulse parameters.     

Anticipatory postural adjustments in arm muscles associated with movements of the contralateral limb and their possible role in interlimb coordination

While sitting on a turnable stool, with both shoulders flexed at 90° or, alternatively, with arms parallel to the trunk and the elbows flexed at 90°—the hands being semisupine—subjects performed unidirectional and cyclic movements on the horizontal plane of the right arm (adduction–abduction) or hand (flexion–extension). The left arm was still, in a position symmetrical to that of the right limb and with the hand contacting a fixed support by the palmar or dorsal surface. During both unidirectional and cyclic arm or hand movements, activation of the prime mover muscles (right Pectoralis Major for arm adduction and Infraspinatus for abduction; right Flexor Carpi Radialis and Extensor Carpi Radialis for the hand movements) was accompanied by activation of the homologous muscles of the contralateral arm and inhibition of antagonists. The contralateral activities (1) regularly preceded the burst in the movement prime movers and (2) were organised in fixation chains that, exerting forces on the hand fixed support, will counterbalance the rotatory action exerted on the trunk by the primary movement. Based on these features, these activities may be classified as anticipatory postural adjustments (APAs). The observed APAs distribution is such as to favour the preferential (mirror symmetrical) coupling of upper limb movements on the horizontal plane. The possible role of these APAs in determining the different constraints experienced when performing mirror symmetrical versus isodirectional coupling is discussed.     

Asymmetric interlimb transfer of concurrent adaptation to opposing dynamic forces

Interlimb transfer of a novel dynamic force has been well documented. It has also been shown that unimanual adaptation to opposing novel environments is possible if they are associated with different workspaces. The main aim of this study was to test if adaptation to opposing velocity dependent viscous forces with one arm could improve the initial performance of the other arm. The study also examined whether this interlimb transfer occurred across an extrinsic, spatial, coordinative system or an intrinsic, joint based, coordinative system. Subjects initially adapted to opposing viscous forces separated by target location. Our measure of performance was the correlation between the speed profiles of each movement within a force condition and an ‘average’ trajectory within null force conditions. Adaptation to the opposing forces was seen during initial acquisition with a significantly improved coefficient in epoch eight compared to epoch one. We then tested interlimb transfer from the dominant to non-dominant arm (D → ND) and vice-versa (ND → D) across either an extrinsic or intrinsic coordinative system. Interlimb transfer was only seen from the dominant to the non-dominant limb across an intrinsic coordinative system. These results support previous studies involving adaptation to a single dynamic force but also indicate that interlimb transfer of multiple opposing states is possible. This suggests that the information available at the level of representation allowing interlimb transfer can be more intricate than a general movement goal or a single perceived directional error.     

Anatomical landmark localization in breast dynamic contrast-enhanced MR imaging

In this article, we present a novel approach to localize anatomical features—breast costal cartilage—in dynamic contrast-enhanced MRI using level sets. Current breast MRI diagnosis involves magnetic-resonance compatible needles for localization [12]. However, if the breast costal cartilage structure can be used as an alternative to the MR needle, this will not only assist in avoiding invasive procedures, but will also facilitate monitoring of the movement of breasts caused by cardiac and respiratory motion. This article represents a novel algorithm for achieving reliable detection and extraction of costal cartilage structures, which can be used for the analysis of motion artifacts, with possible shape variations of the structure caused by uptake of contrast agent, as well as a potential for the registration of breast. The algorithm represented in this article is to extract volume features from post-contrast MR images at three different time slices for the analysis of motion artifacts, and we validate the current algorithm according to the anatomic structure. This utilizes the level-set method [18] for the size selection of the region of interest. The variable shape of contours acquired from a level-set-based segment image actually determines the feature region of interest, which is used as a guide to achieve initial masks for feature extraction. Following this, the algorithm uses a K-means method for classification of the feature regions from other types of tissue and morphological operations with a choice of an appropriate structuring element to achieve reliable masks and extraction of features. The segments of features can be therefore obtained with the application of extracted masks for subsequent motion analysis of breast and for potential registration purposes.     

Dosage compensation and gene expression on the mammalian X chromosome: one plus one does not always equal two

Counting chromosomes is not just simple math. Although normal males and females differ in sex chromosome content (XY vs. XX), X chromosome imbalance is tolerated because dosage compensation mechanisms have evolved to ensure functional equivalence. In mammals this is accomplished by two processes—X chromosome inactivation that silences most genes on one X chromosome in females, leading to functional X monosomy for most genes in both sexes, and X chromosome upregulation that results in increased gene expression on the single active X in males and females, equalizing dosage relative to autosomes. This review focuses on genes on the X chromosome, and how gene content, organization and expression levels can be influenced by these two processes. Special attention is given to genes that are not X inactivated, and are not necessarily fully dosage compensated. These genes that “escape” X inactivation are of medical importance as they explain phenotypes in individuals with sex chromosome aneuploidies and may impact normal traits and disorders that differ between men and women. Moreover, escape genes give insight into how X chromosome inactivation is spread and maintained on the X.     

Efficacy of toltrazuril (Baycox? 5% suspension) in natural infections with pathogenic Eimeria spp. in housed lambs

A field study was conducted to evaluate the effect of a single oral treatment with 20 mg/kg body weight (BW) of toltrazuril (Baycox? 5% suspension)—TOL—in comparison to a single oral treatment with 1 mg/kg BW of diclazuril (Vecoxan? suspension orale, 2.5 mg/ml)—DIC—and an untreated control group (CTRL) on naturally acquired Eimeria infections in lambs. On a French sheep farm with a known history of coccidiosis, 75 housed lambs aged 10–14 days were randomised and allocated to one of three groups. During an observation period of 60 days after treatment, clinical (faecal consistency, BW) and parasitological parameters (oocyst excretion) were evaluated. Excretion in the negative control group started 3 days after treatment and peaked on the 31st day with a prevalence of 80%. Animals were predominantly infected with Eimeria ovinoidalis. Treatment with toltrazuril, but not with diclazuril, resulted in significantly reduced numbers of excreting animals. The number of excretion days and the average oocyst excretion decreased significantly in both the TOL and the DIC groups compared to the CTRL, with the TOL group showing significantly fewer excretion days and excretion intensities than the DIC group. Changes in the faecal consistency were moderate throughout the study and not significantly different between the groups. Daily weight gains were higher in the TOL group compared to the DIC and CTRL groups which did not differ. This study demonstrates the good efficacy of toltrazuril administered orally to lambs in the prepatent period in subclinical natural Eimeria infections in housed lambs.     

Ubiquitination of plasma membrane ectophosphatase in bloodstream forms of Trypanosoma brucei

Bloodstream forms of Trypanosoma brucei contain plasma-membrane-integral acidic ectophosphatase. Here, it is shown by N-terminal sequencing that the ectophosphatase found in ricin-binding material was modified by ubiquitin. Three different ubiquitinated species corresponding to single, double and triple ubiquitinated forms of the enzyme were identified. Immunofluorescence studies with live bloodstream-form parasites showed that the ectophosphatase was localized in the flagellar pocket—the sole site for endocytosis in trypanosomes. As ubiquitin modification of plasma membrane proteins serves as an internalization signal, it is suggested that ubiquitinated ectophosphatase is labelled for endocytosis. This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technik, Schwerpunkt für tropenmedizinische Forschung in Heidelberg (01 KA 9301/3).     

Differential human brain activation by vertical and horizontal global visual textures

Mid-level visual processes which integrate local orientation information for the detection of global structure can be investigated using global form stimuli of varying complexity. Several lines of evidence suggest that the identification of concentric and parallel organisations relies on different underlying neural substrates. The current study measured brain activation by concentric, horizontal parallel, and vertical parallel arrays of short line segments, compared to arrays of randomly oriented segments. Six subjects were scanned in a blocked design functional magnetic resonance imaging experiment. We compared percentage BOLD signal change during the concentric, horizontal and vertical blocks within early retinotopic areas, the fusiform face area and the lateral occipital complex. Unexpectedly, we found that vertical and horizontal parallel forms differentially activated visual cortical areas beyond V1, but in general, activations to concentric and parallel forms did not differ. Vertical patterns produced the highest percentage signal change overall and only area V3A showed a significant difference between concentric and parallel (horizontal) stimuli, with the former better activating this area. These data suggest that the difference in brain activation to vertical and horizontal forms arises at intermediate or global levels of visual representation since the differential activity was found in mid-level retinotopic areas V2 and V3 but not in V1. This may explain why earlier studies—using methods that emphasised responses to local orientation—did not discover this vertical–horizontal anisotropy.