Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument.
In cystic fibrosis (CF), the paranasal sinuses are sites of first and persistent colonization by pathogens such as Pseudomonas aeruginosa. Pathogens subsequently descend to the lower airways, with P. aeruginosa remaining the primary cause of premature death in patients with the inherited disease. Unlike conventional aerosols, vibrating aerosols applied with the PARI Sinus™ nebulizer deposit drugs into the paranasal sinuses. This trial assessed the effects of vibrating sinonasal inhalation of the antibiotic tobramycin in CF patients positive for P. aeruginosa in nasal lavage.
To evaluate the effects of sinonasal inhalation of tobramycin on P. aeruginosa quantification in nasal lavage; and on patient quality of life, measured with the Sino-Nasal Outcome Test (SNOT-20), and otologic and renal safety and tolerability.
Patients were randomized to inhalation of tobramycin (80 mg/2 mL) or placebo (2 mL isotonic saline) once daily (4 minutes/nostril) with the PARI Sinus™ nebulizer over 28 days, with all patients eligible for a subsequent course of open-label inhalation of tobramycin for 28 days. Nasal lavage was obtained before starting and 2 days after the end of each treatment period by rinsing each nostril with 10 mL of isotonic saline.
Nine patients participated, six initially receiving tobramycin and three placebo. Sinonasal inhalation was well tolerated, with serum tobramycin <0.5 mg/L and stable creatinine. P. aeruginosa quantity decreased in four of six (67%) patients given tobramycin, compared with zero of three given placebo (non-significant). SNOT-20 scores were significantly lower in the tobramycin than in the placebo group (P=0.033).
Sinonasal inhalation of vibrating antibiotic aerosols appears promising for reducing pathogen colonization of paranasal sinuses and for control of symptoms in patients with CF.
PARI Sinus; nasal lavage; SNOT-20; cystic fibrosis; Pseudomonas aeruginosa; sinonasal; upper airways
Long and short term side effects of antiretroviral drugs are not fully understood yet. Here a case of reversible blood count changes following post exposure prophylaxis with tenofovir/emtricitabin and lopinavir/ritonavir is reported. We propose that antiretroviral drugs used in post exposure prophylaxis may have a significant impact on hematopoiesis.
HIV; Post exposure prophylaxis; Leukopenia; Thrombocytopenia; Tenofovir; Emtricitabin; Lopinavir; Ritonavir
In our daily lives, auditory stream segregation allows us to differentiate concurrent sound sources and to make sense of the scene we are experiencing. However, a combination of segregation and the concurrent integration of auditory streams is necessary in order to analyze the relationship between streams and thus perceive a coherent auditory scene. The present functional magnetic resonance imaging study investigates the relative role and neural underpinnings of these listening strategies in multi-part musical stimuli. We compare a real human performance of a piano duet and a synthetic stimulus of the same duet in a prioritized integrative attention paradigm that required the simultaneous segregation and integration of auditory streams. In so doing, we manipulate the degree to which the attended part of the duet led either structurally (attend melody vs. attend accompaniment) or temporally (asynchronies vs. no asynchronies between parts), and thus the relative contributions of integration and segregation used to make an assessment of the leader-follower relationship. We show that perceptually the relationship between parts is biased towards the conventional structural hierarchy in western music in which the melody generally dominates (leads) the accompaniment. Moreover, the assessment varies as a function of both cognitive load, as shown through difficulty ratings and the interaction of the temporal and the structural relationship factors. Neurally, we see that the temporal relationship between parts, as one important cue for stream segregation, revealed distinct neural activity in the planum temporale. By contrast, integration used when listening to both the temporally separated performance stimulus and the temporally fused synthetic stimulus resulted in activation of the intraparietal sulcus. These results support the hypothesis that the planum temporale and IPS are key structures underlying the mechanisms of segregation and integration of auditory streams, respectively.
Implicit learning (IL) occurs unconsciously and without intention. Perceptual fluency is the ease of processing elicited by previous exposure to a stimulus. It has been assumed that perceptual fluency is associated with IL. However, the role of perceptual fluency following IL has not been investigated in temporal pattern learning. Two experiments by Schultz, Stevens, Keller, and Tillmann demonstrated the IL of auditory temporal patterns using a serial reaction-time task and a generation task based on the process dissociation procedure. The generation task demonstrated that learning was implicit in both experiments via motor fluency, that is, the inability to suppress learned information. With the aim to disentangle conscious and unconscious processes, we analyze unreported recognition data associated with the Schultz et al. experiments using the sequence identification measurement model. The model assumes that perceptual fluency reflects unconscious processes and IL. For Experiment 1, the model indicated that conscious and unconscious processes contributed to recognition of temporal patterns, but that unconscious processes had a greater influence on recognition than conscious processes. In the model implementation of Experiment 2, there was equal contribution of conscious and unconscious processes in the recognition of temporal patterns. As Schultz et al. demonstrated IL in both experiments using a generation task, and the conditions reported here in Experiments 1 and 2 were identical, two explanations are offered for the discrepancy in model and behavioral results based on the two tasks: 1) perceptual fluency may not be necessary to infer IL, or 2) conscious control over implicitly learned information may vary as a function of perceptual fluency and motor fluency.
While oxidative stress is implicated in aging, the impact of oxidative stress on aging in the peripheral nervous system is not well understood. To determine a potential mechanism for age-related deficits in the peripheral nervous system, we examined both functional and morphological changes and utilized microarray technology to compare normal aging in wild-type mice to effects in copper/zinc superoxide dismutase-deficient (Sod1−/−) mice, a mouse model of increased oxidative stress. Sod1−/− mice exhibit a peripheral neuropathy phenotype with normal sensory nerve function and deficits in motor nerve function. Our data indicate that a decrease in the synthesis of cholesterol, which is vital to myelin formation, correlates with the structural deficits in axons, myelin, and the cell body of motor neurons in the Sod1+/+ mice at 30 months and the Sod1−/− mice at 20 months compared with mice at 2 months. Collectively, we have demonstrated that the functional and morphological changes within the peripheral nervous system in our model of increased oxidative stress are manifested earlier and resemble the deficits observed during normal aging.
Neoehrlichiosis caused by “Candidatus Neoehrlichia mikurensis” is an emerging zoonotic disease. In total, six patients have been described in Europe, with the first case detected in 2007. In addition, seven patients from China were described in a report published in October 2012. In 2009, we diagnosed the first human case of “Ca. Neoehrlichia mikurensis” infection in the Zurich area (Switzerland). Here, we report two additional human cases from the same region, which were identified by broad-range 16S rRNA gene PCR. Both patients were immunocompromised and presented with similar clinical syndromes, including fever, malaise, and weight loss. A diagnostic multiplex real-time PCR was developed for specific detection of “Ca. Neoehrlichia mikurensis” infections. The assay is based on the signature sequence of a 280-bp fragment of the “Ca. Neoehrlichia mikurensis” 16S rRNA gene and incorporates a “Ca. Neoehrlichia mikurensis” species, a “Ca. Neoehrlichia” genus, and an Anaplasmataceae family probe for simultaneous screening. The analytical sensitivity was determined to be below five copies of the “Ca. Neoehrlichia mikurensis” 16S rRNA gene. Our results show that the assay is suitable for the direct detection of “Ca. Neoehrlichia mikurensis” DNA in clinical samples from, for example, blood and bone marrow. In addition, it allows for monitoring treatment response during antibiotic therapy. Using the same assay, DNA extracts from 1,916 ticks collected in four forests in close proximity to the patients' residences (<3 km) were screened. At all sampling sites, the minimal prevalence of “Ca. Neoehrlichia mikurensis” was between 3.5 to 8% in pools of either nymphs, males, or females, showing a strong geographic association between the three patients and the assumed vector.
Expert ensemble musicians produce exquisitely coordinated sounds, but rehearsal is typically required to do so. Ensemble coordination may thus be influenced by the degree to which individuals are familiar with each other's parts. Such familiarity may affect the ability to predict and synchronize with co-performers' actions. Internal models related to action simulation and anticipatory musical imagery may be affected by knowledge of (1) the musical structure of a co-performer's part (e.g., in terms of its rhythm and phrase structure) and/or (2) the co-performer's idiosyncratic playing style (e.g., expressive micro-timing variations). The current study investigated the effects of familiarity on interpersonal coordination in piano duos. Skilled pianists were required to play several duets with different partners. One condition included duets for which co-performers had previously practiced both parts, while another condition included duets for which each performer had practiced only their own part. Each piece was recorded six times without joint rehearsal or visual contact to examine the effects of increasing familiarity. Interpersonal coordination was quantified by measuring asynchronies between pianists' keystroke timing and the correlation of their body (head and torso) movements, which were recorded with a motion capture system. The results suggest that familiarity with a co-performer's part, in the absence of familiarity with their playing style, engenders predictions about micro-timing variations that are based instead upon one's own playing style, leading to a mismatch between predictions and actual events at short timescales. Predictions at longer timescales—that is, those related to musical measures and phrases, and reflected in head movements and body sway—are, however, facilitated by familiarity with the structure of a co-performer's part. These findings point to a dissociation between interpersonal coordination at the level of keystrokes and body movements.
interpersonal coordination; body movement; music; ensembles; sensorimotor synchronization
We recently described the novel species Streptococcus tigurinus sp. nov. belonging to the Streptococcus mitis group. The type strain AZ_3aT of S. tigurinus was originally isolated from a patient with infective endocarditis. According to its phenotypic and molecular characteristics, S. tigurinus is most closely related to Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus oralis, and Streptococcus infantis. Accurate identification of S. tigurinus is facilitated by 16S rRNA gene analysis. We retrospectively analyzed our 16S rRNA gene molecular database, which contains sequences of all clinical samples obtained in our institute since 2003. We detected 17 16S rRNA gene sequences which were assigned to S. tigurinus, including sequences from the 3 S. tigurinus strains described previously. S. tigurinus originated from normally sterile body sites, such as blood, cerebrospinal fluid, or heart valves, of 14 patients and was initially detected by culture or broad-range 16S rRNA gene PCR, followed by sequencing. The 14 patients had serious invasive infections, i.e., infective endocarditis (n = 6), spondylodiscitis (n = 3), bacteremia (n = 2), meningitis (n = 1), prosthetic joint infection (n = 1), and thoracic empyema (n = 1). To evaluate the presence of Streptococcus tigurinus in the endogenous oral microbial flora, we screened saliva specimens of 31 volunteers. After selective growth, alpha-hemolytic growing colonies were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and subsequent molecular methods. S. tigurinus was not identified among 608 strains analyzed. These data indicate that S. tigurinus is not widely distributed in the oral cavity. In conclusion, S. tigurinus is a novel agent of invasive infections, particularly infective endocarditis.
A constantly changing environment requires precise yet flexible timing of movements. Sensorimotor synchronization (SMS)—the temporal coordination of an action with events in a predictable external rhythm—is a fundamental human skill that contributes to optimal sensory-motor control in daily life. A large body of research related to SMS has focused on adaptive error correction mechanisms that support the synchronization of periodic movements (e.g., finger taps) with events in regular pacing sequences. The results of recent studies additionally highlight the importance of anticipatory mechanisms that support temporal prediction in the context of SMS with sequences that contain tempo changes. To investigate the role of adaptation and anticipatory mechanisms in SMS we introduce ADAM: an ADaptation and Anticipation Model. ADAM combines reactive error correction processes (adaptation) with predictive temporal extrapolation processes (anticipation) inspired by the computational neuroscience concept of internal models. The combination of simulations and experimental manipulations based on ADAM creates a novel and promising approach for exploring adaptation and anticipation in SMS. The current paper describes the conceptual basis and architecture of ADAM.
sensorimotor synchronization; computational model; temporal adaptation; error correction; temporal anticipation; predictive internal models
Musical ensemble performance requires temporally precise interpersonal action coordination. To play in synchrony, ensemble musicians presumably rely on anticipatory mechanisms that enable them to predict the timing of sounds produced by co-performers. Previous studies have shown that individuals differ in their ability to predict upcoming tempo changes in paced finger-tapping tasks (indexed by cross-correlations between tap timing and pacing events) and that the degree of such prediction influences the accuracy of sensorimotor synchronization (SMS) and interpersonal coordination in dyadic tapping tasks. The current functional magnetic resonance imaging study investigated the neural correlates of auditory temporal predictions during SMS in a within-subject design. Hemodynamic responses were recorded from 18 musicians while they tapped in synchrony with auditory sequences containing gradual tempo changes under conditions of varying cognitive load (achieved by a simultaneous visual n-back working-memory task comprising three levels of difficulty: observation only, 1-back, and 2-back object comparisons). Prediction ability during SMS decreased with increasing cognitive load. Results of a parametric analysis revealed that the generation of auditory temporal predictions during SMS recruits (1) a distributed network of cortico-cerebellar motor-related brain areas (left dorsal premotor and motor cortex, right lateral cerebellum, SMA proper and bilateral inferior parietal cortex) and (2) medial cortical areas (medial prefrontal cortex, posterior cingulate cortex). While the first network is presumably involved in basic sensory prediction, sensorimotor integration, motor timing, and temporal adaptation, activation in the second set of areas may be related to higher-level social-cognitive processes elicited during action coordination with auditory signals that resemble music performed by human agents.
temporal prediction; sensorimotor synchronization; medial prefrontal cortex; motor timing; dual-task interference
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
When people play music and dance together, they engage in forms of musical joint action that are often characterized by a shared sense of rhythmic timing and affective state (i.e., temporal and affective entrainment). In order to understand the origins of musical joint action, we propose a model in which entrainment is linked to dual mechanisms (motor resonance and action simulation), which in turn support musical behavior (imitation and complementary joint action). To illustrate this model, we consider two generic forms of joint musical behavior: chorusing and turn-taking. We explore how these common behaviors can be founded on entrainment capacities established early in human development, specifically during musical interactions between infants and their caregivers. If the roots of entrainment are found in early musical interactions which are practiced from childhood into adulthood, then we propose that the rehearsal of advanced musical ensemble skills can be considered to be a refined, mimetic form of temporal and affective entrainment whose evolution begins in infancy.
music; joint action; entrainment; ensemble skills; development; dance
Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-γ)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.
Recruitment of platelets (PLT) during donor PLT apheresis may facilitate the harvest of multiple units within a single donation.
We compared two PLT apheresis procedures (Amicus and Trima Accel) in a prospective, randomized, paired cross-over study in 60 donors. The 120 donations were compared for depletion of circulating PLT in the donors, PLT yields and PLT recruitment. A recruitment was defined as ratio of total PLT yield and donor PLT depletion > 1.
Despite comparable differences of pre- and post-apheresis PLT counts (87 × 109/l in Trima Accel vs. 92 × 109/l in Amicus, p = 0.383), PLT yields were higher with Trima Accel (7.48 × 1011 vs. 6.06 × 1011, p < 0.001), corresponding to a higher PLT recruitment (1.90 vs. 1.42, p < 0.001). We observed a different increase of WBC counts after aphereses, which was more pronounced with Trima Accel than with Amicus (1.30 × 109/l vs. 0.46 × 109/l, p < 0.001).
Both procedures induced PLT recruitment. This was higher in Trima Accel, contributing to a higher yield in spite of a comparable depletion of circulating PLT in the donors. This recruitment facilitates the harvest of multiple units within a single donation and seems to be influenced by the procedure utilized. The different increases of circulating donor white blood cells after donation need further investigation.
Platelet apheresis; Platelet recruitment; Cell separator
The ability to evaluate spontaneity in human behavior is called upon in the esthetic appreciation of dramatic arts and music. The current study addresses the behavioral and brain mechanisms that mediate the perception of spontaneity in music performance. In a functional magnetic resonance imaging experiment, 22 jazz musicians listened to piano melodies and judged whether they were improvised or imitated. Judgment accuracy (mean 55%; range 44–65%), which was low but above chance, was positively correlated with musical experience and empathy. Analysis of listeners’ hemodynamic responses revealed that amygdala activation was stronger for improvisations than imitations. This activation correlated with the variability of performance timing and intensity (loudness) in the melodies, suggesting that the amygdala is involved in the detection of behavioral uncertainty. An analysis based on the subjective classification of melodies according to listeners’ judgments revealed that a network including the pre-supplementary motor area, frontal operculum, and anterior insula was most strongly activated for melodies judged to be improvised. This may reflect the increased engagement of an action simulation network when melodic predictions are rendered challenging due to perceived instability in the performer's actions. Taken together, our results suggest that, while certain brain regions in skilled individuals may be generally sensitive to objective cues to spontaneity in human behavior, the ability to evaluate spontaneity accurately depends upon whether an individual's action-related experience and perspective taking skills enable faithful internal simulation of the given behavior.
music; improvisation; spontaneity; uncertainty; amygdala; action simulation; human fMRI
Typical topics and problems encountered during data processing of diffraction experiments are discussed and the tools provided in the autoPROC software are described.
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
autoPROC; data processing
Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease.
This study investigated cognitive and emotional effects of syncopation, a feature
of musical rhythm that produces expectancy violations in the listener by
emphasising weak temporal locations and de-emphasising strong locations in
metric structure. Stimuli consisting of pairs of unsyncopated and syncopated
musical phrases were rated by 35 musicians for perceived complexity, enjoyment,
happiness, arousal, and tension. Overall, syncopated patterns were more enjoyed,
and rated as happier, than unsyncopated patterns, while differences in perceived
tension were unreliable. Complexity and arousal ratings were asymmetric by
serial order, increasing when patterns moved from unsyncopated to syncopated,
but not significantly changing when order was reversed. These results suggest
that syncopation influences emotional valence (positively), and that while
syncopated rhythms are objectively more complex than unsyncopated rhythms, this
difference is more salient when complexity increases than when it decreases. It
is proposed that composers and improvisers may exploit this asymmetry in
perceived complexity by favoring formal structures that progress from
rhythmically simple to complex, as can be observed in the initial sections of
musical forms such as theme and variations.
syncopation; serial asymmetry; affective response; cognition; rhythm; emotion; musical form
We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.
We have repeatedly detected Candidatus Neoehrlichia mikurensis, a bacterium first described in Rattus norvegicus rats and Ixodes ovatus ticks in Japan in 2004 in the blood of a 61-year-old man with signs of septicemia by 16S rRNA and groEL gene PCR. After 6 weeks of therapy with doxycycline and rifampin, the patient recovered.
Candidatus Neoehrlichia mikurensis; septicemia; human infection; 16S rRNA gene PCR; therapy; tick-borne pathogen; bacteria; dispatch
The influence of integrated goal representations on multilevel coordination stability was investigated in a task that required finger tapping in antiphase with metronomic tone sequences (inter-agent coordination) while alternating between the two hands (intra-personal coordination). The maximum rate at which musicians could perform this task was measured when taps did or did not trigger feedback tones. Tones produced by the two hands (very low, low, medium, high, very high) could be the same as, or different from, one another and the (medium-pitched) metronome tones. The benefits of feedback tones were greatest when they were close in pitch to the metronome and the left hand triggered low tones while the right hand triggered high tones. Thus, multilevel coordination was facilitated by tones that were easy to integrate with, but perceptually distinct from, the metronome, and by compatibility of movement patterns and feedback pitches.
Motor Coordination; Auditory Feedback; Perceptual Motor Processes; Finger Tapping
The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy.
As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA.
According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.
A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA− cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA− blood cells. The loss of GPI-linked proteins occurred mainly in c-kit+CD34+Lin− progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA− cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
hematopoiesis; stem cell; c-fes; paroxysmal nocturnal hemoglobinuria; conditional gene inactivation