Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.     

Rapid and effective detection of anthrax spores in soil by PCR

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.     

Fine-scale genetic structure among genetic individuals of the clone-forming monotypic genus Echinosophora koreensis (Fabaceae)

• Background and Aims For rare endemics or endangered plantspecies that reproduce both sexually and vegetatively it iscritical to understand the extent of clonality because assessmentof clonal extent and distribution has important ecological andevolutionary consequences with conservation implications. Asurvey was undertaken to understand clonal effects on fine-scalegenetic structure (FSGS) in two populations (one from a disturbedand the other from an undisturbed locality) of Echinosophorakoreensis, an endangered small shrub belonging to a monotypicgenus in central Korea that reproduces both sexually and vegetativelyvia rhizomes. • Methods Using inter-simple sequence repeats (ISSRs) asgenetic markers, the spatial distribution of individuals wasevaluated using Ripley's L(d)-statistics and quantified thespatial scale of clonal spread and spatial distribution of ISSRgenotypes using spatial autocorrelation analysis techniques(join-count statistics and kinship coefficient, F_ij) for totalsamples and samples excluding clones. • Key Results A high degree of differentiation betweenpopulations was observed (_ST(g) = 0·184, P < 0·001).Ripley's L(d)-statistics revealed a near random distributionof individuals in a disturbed population, whereas significantaggregation of individuals was found in an undisturbed site.The join-count statistics revealed that most clones significantlyaggregate at 6-m interplant distance. The Sp statistic reflectingpatterns of correlograms revealed a strong pattern of FSGS forall four data sets (Sp = 0·072–0·154), butthese patterns were not significantly different from each other.At small interplant distances (2 m), however, jackknifed 95% CIs revealed that the total samples exhibited significantlyhigher F_ij values than the same samples excluding clones. • Conclusion The strong FSGS from genets is consistentwith two biological and ecological traits of E. koreensis: bee-pollinationand limited seed dispersal. Furthermore, potential clone matesover repeated generations would contribute to the observed highF_ij values among genets at short distance. To ensure long-termex situ genetic variability of the endangered E. koreensis,individuals located at distances of 10–12 m should becollected across entire populations of E. koreensis.     

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3-7 and 7-23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10-40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.     

High-resolution analysis of neuronal growth cone morphology by comparative atomic force and optical microscopy

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.     

Rescue of infectious particles from preassembled alphavirus nucleocapsid cores

Alphaviruses are small, spherical, enveloped, positive-sense, single-stranded, RNA viruses responsible for considerable human and animal disease. Using microinjection of preassembled cores as a tool, a system has been established to study the assembly and budding process of Sindbis virus, the type member of the alphaviruses. We demonstrate the release of infectious virus-like particles from cells expressing Sindbis virus envelope glycoproteins following microinjection of Sindbis virus nucleocapsids purified from the cytoplasm of infected cells. Furthermore, it is shown that nucleocapsids assembled in vitro mimic those isolated in the cytoplasm of infected cells with respect to their ability to be incorporated into enveloped virions following microinjection. This system allows for the study of the alphavirus budding process independent of an authentic infection and provides a platform to study viral and host requirements for budding.     

Microfluidics-assisted rapid generation of tubular cell-laden microgel inside glass capillaries

Drug screening using engineered blood vessels (EBVs) faces considerable barriers in approximating the conditions of an in vivo environment. To address this issue, we have introduced a microfluidic system for cell-laden tubular microgels. N-Carboxyethyl chitosan crosslinked with oxidized dextran was used for in situ gelable tubular scaffolds. The microfluidic system consisted of four glass capillaries that generated a coaxial flow of pre-polymer and phosphate buffered solutions. It rapidly produced cell-laden tubular microgels inside glass capillaries. The mechanical strength of the tubular microgels was suitable for their application as EBVs, with a maximum Young’s modulus of 12.2 ± 1.9 kPa. In vitro cell studies using human umbilical vein endothelial cells verified the biocompatibility and non-cytotoxicity of the gelation and fabrication process. Thus, in situ gelable cell-laden tubular microgels can be a potential platform for screening drugs to treat blood vessel diseases.     

Inverted Tandem Polymer Solar Cells with Polyethylenimine‐Modified MoOX/Al2O3:ZnO Nanolaminate as the Charge Recombination Layers

A new charge recombination layer for inverted tandem polymer solar cells is reported. A bilayer of MoO_X/Al₂O₃:ZnO nanolaminate is shown to enable efficient charge recombination in inverted tandem cells. A polymer surface modification on the MoO_X/Al₂O₃:ZnO nanolaminate bilayer increases the work function contrast between the two outward surfaces of the charge recombination layer, further improving the performance of tandem solar cells. An analysis of the electrical, optical, and surface properties of the charge recombination layer is presented. Inverted tandem polymer solar cells, with two photoactive layers comprising poly (3‐hexylthiophene) (P3HT):indene‐C₆₀ bisadduct (IC₆₀BA) for the bottom cell and poly[(4,8‐bis‐(2‐ethylhexyloxy)‐benzo[1,2‐b:4,5‐b']dithiophene)‐2,6‐diyl‐alt‐(4‐(2‐ethylhexanoyl)‐thieno[3,4‐b]thiophene))‐2,6‐diyl] (PBDTTT‐C):[6,6]‐phenyl C₆₁ butyric acid methyl ester (PC₆₀BM) for the top cell, yield an open‐circuit voltage of 1481 mV ± 15 mV, a short‐circuit current density of 7.1 mA cm^?2 ± 0.1 mA cm^?2, and a fill factor of 0.62 ± 0.01, resulting in a power conversion efficiency of 6.5% ± 0.1% under simulated AM 1.5G, 100 mW cm^?2 illumination.