Single Cell Deposition and Patterning with a Robotic System

Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.FiocruzBritish Council Progra

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients

<p>Abstract</p> <p>Background</p> <p>Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). <it>CFTR </it>genotype effects acquisition of <it>Pseudomonas aeruginosa (Pa)</it>, however the effect on acquisition of other infectious organisms that frequently precede <it>Pa </it>is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF.</p> <p>Methods</p> <p>Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function <it>CFTR </it>mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess <it>CFTR </it>effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with <it>Pa</it>, mucoid <it>Pa </it>or <it>Aspergillus (Asp) </it>using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories.</p> <p>Results</p> <p>Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with <it>Pa</it>, mucoid <it>Pa </it>or <it>Asp </it>were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile).</p> <p>Conclusions</p> <p>Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.</p

Pulmonary Hypertension in Elderly Patients with Diastolic Dysfunction and Preserved Ejection Fraction

Abstract: Purpose: Patients with diastolic dysfunction may have a disproportionate degree of elevation in pulmonary pressure, particularly in the elderly. Higher pulmonary vascular resistance in the elderly patients with heart failure but preserved ejection fraction suggests that beyond the post-capillary contribution of pulmonary venous congestion, a pre-capillary component of pulmonary arterial hypertension occurs. We aim to identify if pulmonary vascular resistance in elderly patients with diastolic dysfunction is disproportionately higher than patients with systolic dysfunction independent of filling pressures. Methods: 389 patients identified retrospectively between 2003- 2010; elderly with preserved ejection fraction, elderly with depressed ejection fraction, and primary arterial hypertension who underwent right-heart catheterization at Rush University. Results: No significant difference in pulmonary vascular resistance between systolic and diastolic dysfunction. The mean difference in pulmonary vascular resistance was not statistically significant at 0.40 mmHg·min/l (95 % CI-3.03 to 3.83) with similar left ventricular filling pressures with mean difference of 3.38 mmHg (95 % CI,-1.27 to 8.02). When adjusted for filling pressures, there remained no difference in pulmonary vascular resistance for systolic and diastolic dysfunction. The mean pulmonary vascular resistance is more elevated in systolic heart failure compared to diastolic heart failure with means 3.13 mmHg·min/l and 3.52 mmHg·min/l, respectively

Reductions in the Prevalence and Incidence of Geohelminth Infections following a City-wide Sanitation Program in a Brazilian Urban Centre

In the city of Salvador, a large urban centre in Northeast Brazil, a city-wide sanitation intervention started in 1997, aiming to improve the sewerage coverage of households from 26% to 80%. Our aim was to study the impact of the intervention on the prevalence and incidence of geohelminths in the school-aged population. The longitudinal study comprised two cohorts: from the beginning of 1997 to 1998, where data was collected before the intervention, and at the end of 2003 to 2004, after the intervention. Copro-parasitological examinations were carried out on every individual from both cohorts, at the start and nine months later. Demographic, socio-economic, and environmental data were collected using semi-structured questionnaires. The variables utilized to demonstrate the effects of intervention, when utilized together in a multivariate model, accounted for a 100% observed reduction in the prevalence ratio (PR) and incidence ratio (IR). As well as proving that the variables associated with the effect of the program intervention were mediators in this reduction, the reduction in the PR and IR between these periods demonstrated that modifications to the urban environment, particularly those associated with sanitary sewage systems, affected the health of the population, significantly reducing the prevalence of geohelminths