Time and Force Dependence of the Rupture of Glycoprotein IIb-IIIa-Fibrinogen Bonds between Latex Spheres

AbstractWe studied the shear-induced breakup of doublets of aldehyde/sulfate (A/S) latex spheres covalently linked with purified platelet GPIIb-IIIa receptor, and cross-linked by fibrinogen. Flow cytometry with fluorescein isothiocyanate-fibrinogen showed than an average of 22,500 molecules of active GPIIb-IIIa were captured per sphere, with a mean Kd=56nM for fibrinogen binding. The spheres, suspended in buffered 19% Ficoll 400 containing 120 or 240pM fibrinogen, were subjected to Couette flow in a counter-rotating cone-plate rheoscope. Doublets, formed by two-body collisions at low shear rate (G=8s−1) for ≤15min, were subjected to shear stress from 0.6 to 2.9 Nm−2, their rotations recorded until they broke up or were lost to view. Although breakup was time dependent, occurring mostly in the first 2 rotations after the onset of shear, the percentage of doublets broken up after 10 rotations were almost independent of normal hydrodynamic force, Fn: at 240 pN, 15.6, 16.0, and 17.0% broke up in the force range 70–150 pN, 150–230 pN, and 230–310 pN. Unexpectedly, at both [fibrinogen], the initial rate of breakup was highest in the lowest force range, and computer simulation using a stochastic model of breakup was unable to simulate the time course of breakup. When pre-sheared at low G for >15min, no doublets broke up within 10 rotations at 70<Fn<310 pN; it required >3min shear (>1110 rotations) at Fn=210 pN for significant breakup to occur. Other published work has shown that binding of fibrinogen to GPIIb-IIIa immobilized on plane surfaces exhibits an initial fast reversible process with relative low affinity succeeded by transformation of GPIIb-IIIa to a stable high-affinity complex. We postulate that most doublet breakups observed within 10 rotations were from a population of young doublets having low numbers of bonds, by dissociation of the initial receptor complex relatively unresponsive to force. The remaining, older doublets with GPIIb-IIIa in the high-affinity complex were not broken up in the time or range of forces studied

Tuberculosis and homelessness in Montreal: a retrospective cohort study

BACKGROUND: Montreal is Canada's second-largest city, where mean annual tuberculosis (TB) incidence from 1996 to 2007 was 8.9/100,000. The objectives of this study were to describe the epidemiology of TB among homeless persons in Montreal and assess patterns of transmission and sharing of key locations. METHODS: We reviewed demographic, clinical, and microbiologic data for all active TB cases reported in Montreal from 1996 to 2007 and identified persons who were homeless in the year prior to TB diagnosis. We genotyped all available Mycobacterium tuberculosis isolates by IS6110 restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping, and used a geographic information system to identify potential locations for transmission between persons with matching isolates. RESULTS: There were 20 cases of TB in homeless persons, out of 1823 total reported from 1996-2007. 17/20 were Canadian-born, including 5 Aboriginals. Homeless persons were more likely than non-homeless persons to have pulmonary TB (20/20), smear-positive disease (17/20, odds ratio (OR) = 5.7, 95% confidence interval (CI): 1.7-20), HIV co-infection (12/20, OR = 14, 95%CI: 4.8-40), and a history of substance use. The median duration from symptom onset to diagnosis was 61 days for homeless persons vs. 28 days for non-homeless persons (P = 0.022). Eleven homeless persons with TB belonged to genotype-defined clusters (OR = 5.4, 95%CI: 2.2-13), and ten potential locations for transmission were identified, including health care facilities, homeless shelters/drop-in centres, and an Aboriginal community centre. CONCLUSIONS: TB cases among homeless persons in Montreal raise concerns about delayed diagnosis and ongoing local transmission

Phylogenetic Relationships of Southern African West Nile Virus Isolates

Phylogenetic relationships were examined for 29 southern African West Nile virus (formal name West Nile virus [WNV]) isolates from various sources in four countries from 1958 to 2001. In addition sequence data were retrieved from GenBank for another 23 WNV isolates and Kunjin and Japanese encephalitis viruses. All isolates belonged to two lineages. Lineage 1 isolates were from central and North Africa, Europe, Israel, and North America; lineage 2 isolates were from central and southern Africa and Madagascar. No strict correlation existed between grouping and source of virus isolate, pathogenicity, geographic distribution, or year of isolation. Some southern African isolates have been associated with encephalitis in a human, a horse, and a dog and with fatal hepatitis in a human and death of an ostrich chick

Heterologous Production of 1-Tuberculosinyladenosine in Mycobacterium kansasii Models Pathoevolution towards the Transcellular Lifestyle of Mycobacterium tuberculosis

ABSTRACT Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2−/− mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation. IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen

High Efficiency Planar Geometry Germanium-on-silicon Single-photon Avalanche Diode Detectors

This paper presents the performance of 26 μm and 50 μm diameter planar Ge-on-Si single-photon avalanche diode (SPAD) detectors. The addition of germanium in these detectors extends the spectral range into the short-wave infrared (SWIR) region, beyond the capability of already well-established Si SPAD devices. There are several advantages for extending the spectral range into the SWIR region including: reduced eye-safety laser threshold, greater attainable ranges, and increased depth resolution in range finding applications, in addition to the enhanced capability to image through obscurants such as fog and smoke. The time correlated single-photon counting (TCSPC) technique has been utilized to observe record low dark count rates, below 100 kHz at a temperature of 125 K for up to a 6.6 % excess bias, for the 26 μm diameter devices. Under identical experimental conditions, in terms of excess bias and temperature, the 50 μm diameter device consistently demonstrates dark count rates a factor of 4 times greater than 26 μm diameter devices, indicating that the dark count rate is proportional to the device volume. Single-photon detection efficiencies of up to ~ 29 % were measured at a wavelength of 1310 nm at 125 K. Noise equivalent powers (NEP) down to 9.8 × 10-17 WHz-1/2 and jitters &lt; 160 ps are obtainable, both significantly lower than previous 100 μm diameter planar geometry devices, demonstrating the potential of these devices for highly sensitive and high-speed imaging in the SWIR

Template-free 13-protofilament microtubule–MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs

Penetrance estimates for BRCA1 and BRCA2 based on genetic testing in a Clinical Cancer Genetics service setting: Risks of breast/ovarian cancer quoted should reflect the cancer burden in the family

<p>Abstract</p> <p>Background</p> <p>The identification of a <it>BRCA1 </it>or <it>BRCA2 </it>mutation in familial breast cancer kindreds allows genetic testing of at risk relatives. However, considerable controversy exists regarding the cancer risks in women who test positive for the family mutation.</p> <p>Methods</p> <p>We reviewed 385 unrelated families (223 with <it>BRCA1 </it>and 162 with <it>BRCA2 </it>mutations) ascertained through two regional cancer genetics services. We estimated the penetrance for both breast and ovarian cancer in female mutation carriers (904 proven mutation carriers – 1442 females in total assumed to carry the mutation) and also assessed the effect on penetrance of mutation position and birth cohort.</p> <p>Results</p> <p>Breast cancer penetrance to 70 and to 80 years was 68% (95%CI 64.7–71.3%) and 79.5% (95%CI 75.5–83.5%) respectively for <it>BRCA1 </it>and 75% (95%CI 71.7–78.3%) and 88% (95%CI 85.3–91.7%) for <it>BRCA2</it>. Ovarian cancer risk to 70 and to 80 years was 60% (95%CI 65–71%) and 65% (95%CI 75–84%) for <it>BRCA1 </it>and 30% (95%CI 25.5–34.5%) and 37% (95%CI 31.5–42.5%) for <it>BRCA2</it>. These risks were borne out by a prospective study of cancer in the families and genetic testing of unaffected relatives. We also found evidence of a strong cohort effect with women born after 1940 having a cumulative risk of 22% for breast cancer by 40 years of age compared to 8% in women born before 1930 (p = 0.0005).</p> <p>Conclusion</p> <p>In high-risk families, selected in a genetics service setting, women who test positive for the familial <it>BRCA1/BRCA2 </it>mutation are likely to have cumulative breast cancer risks in keeping with the estimates obtained originally from large families. This is particularly true for women born after 1940.</p

Reduced Transmissibility of East African Indian Strains of Mycobacterium tuberculosis

BACKGROUND: Mycobacterium tuberculosis (MTB) has been classified into 4 main lineages. Some reports have associated certain lineages with particular clinical phenotypes, but there is still insufficient information regarding the clinical and epidemiologic implications of MTB lineage variation. METHODS: Using large sequence polymorphisms we classified MTB isolates from a population-based study in Montreal, Canada into the 4 major lineages, and identified the associated clinical and epidemiologic features. In addition, IS6110-RFLP and spoligotyping were used as indicators of recent TB transmission. The study population was divided into a derivation cohort, diagnosed between 2001 and 2007, and a separate validation cohort, diagnosed between 1996 and 2000. RESULTS: In the derivation cohort, when compared to the other MTB lineages, the East African-Indian (EAI) lineage was associated with lower rates of TB transmission, as measured by: positive TST among close contacts of pulmonary TB cases (adjusted odds ratio 0.6: [95% confidence interval 0.4-0.9]), and clustered TB cases (0.3: [<0.001-0.6]). Severe forms of TB were also less likely among the EAI group (0.4: [<0.001-0.8]). There were no significant differences when comparing patients with the other MTB lineages. In the validation cohort, the EAI lineage was associated with lower rates of positive TST among contacts (0.5: [0.3-0.9]) and a trend towards less clustered TB cases (0.5: [0.1-1.8]) when compared to the other lineages. Disease severity among the different groups was not significantly different in the validation cohort. CONCLUSIONS: We conclude that in Montreal, EAI strains were associated with reduced transmission compared to other MTB lineages

Simulating rewetting events in intermittent rivers and ephemeral streams: A global analysis of leached nutrients and organic matter

Climate change and human pressures are changing the global distribution and the ex‐ tent of intermittent rivers and ephemeral streams (IRES), which comprise half of the global river network area. IRES are characterized by periods of flow cessation, during which channel substrates accumulate and undergo physico‐chemical changes (precon‐ ditioning), and periods of flow resumption, when these substrates are rewetted and release pulses of dissolved nutrients and organic matter (OM). However, there are no estimates of the amounts and quality of leached substances, nor is there information on the underlying environmental constraints operating at the global scale. We experi‐ mentally simulated, under standard laboratory conditions, rewetting of leaves, river‐ bed sediments, and epilithic biofilms collected during the dry phase across 205 IRES from five major climate zones. We determined the amounts and qualitative character‐ istics of the leached nutrients and OM, and estimated their areal fluxes from riverbeds. In addition, we evaluated the variance in leachate characteristics in relation to selected environmental variables and substrate characteristics. We found that sediments, due to their large quantities within riverbeds, contribute most to the overall flux of dis‐ solved substances during rewetting events (56%–98%), and that flux rates distinctly differ among climate zones. Dissolved organic carbon, phenolics, and nitrate contrib‐ uted most to the areal fluxes. The largest amounts of leached substances were found in the continental climate zone, coinciding with the lowest potential bioavailability of the leached OM. The opposite pattern was found in the arid zone. Environmental vari‐ ables expected to be modified under climate change (i.e. potential evapotranspiration, aridity, dry period duration, land use) were correlated with the amount of leached sub‐ stances, with the strongest relationship found for sediments. These results show that the role of IRES should be accounted for in global biogeochemical cycles, especially because prevalence of IRES will increase due to increasing severity of drying event

Visuospatial working memory in children and adolescents with 22q11.2 deletion syndrome; an fMRI study

22q11.2 deletion syndrome (22q11DS) is a genetic disorder associated with a microdeletion of chromosome 22q11. In addition to high rates of neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder, children with 22q11DS have a specific neuropsychological profile with particular deficits in visuospatial and working memory. However, the neurobiological substrate underlying these deficits is poorly understood. We investigated brain function during a visuospatial working memory (SWM) task in eight children with 22q11DS and 13 healthy controls, using fMRI. Both groups showed task-related activation in dorsolateral prefrontal cortex (DLPFC) and bilateral parietal association cortices. Controls activated parietal and occipital regions significantly more than those with 22q11DS but there was no significant between-group difference in DLPFC. In addition, while controls had a significant age-related increase in the activation of posterior brain regions and an age-related decrease in anterior regions, the 22q11DS children showed the opposite pattern. Genetically determined differences in the development of specific brain systems may underpin the cognitive deficits in 22q11DS, and may contribute to the later development of neuropsychiatric disorders