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Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions. Graphic abstract: Schematic representation of the ODELAM platform.

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions. Graphic abstract: [Image: see text] Schematic representation of the ODELAM platform

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths each year. Current assays of antimicrobial resistance need knowledge of mutations that confer drug resistance, or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. ODELAM enables rapid quantitative measures of growth kinetics in as little as 30 hrs under a wide variety of environmental conditions. We demonstrate ODELAM’s utility by identifying ofloxacin resistance in cultured clinical isolates of Mtb and benchmark its performance with standard minimum inhibitory concentration (MIC) assays. ODELAM identified ofloxacin heteroresistance and the presence of drug resistant colony forming units (CFUs) at 1 per 1000 CFUs in as little as 48 hrs. ODELAM is a powerful new tool that can rapidly evaluate Mtb drug resistance in a laboratory setting.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths each year. Current assays of antimicrobial resistance need knowledge of mutations that confer drug resistance, or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. ODELAM enables rapid quantitative measures of growth kinetics in as little as 30 hours under a wide variety of environmental conditions. We show the utility of ODELAM by identifying ofloxacin resistance in clinical isolates of Mtb and benchmark its performance with standard MIC assays. In one clinical isolate, ODELAM identified ofloxacin heteroresistance and identifies the presence of drug resistant colony forming units (CFU) at 1 per 1000 CFUs in as little as 48 hours. ODELAM is a powerful new tool that can rapidly evaluate Mtb drug resistance in a laboratory setting.

Membranes and membrane proteins destined for peroxisomes are trafficked from the endoplasmic reticulum (ER) in preperoxisomal vesicles. Mast et al. define a role for ESCRT-III in releasing these vesicles from the ER., Dynamic control of peroxisome proliferation is integral to the peroxisome’s many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

Dynamic control of peroxisome proliferation is integral to the peroxisome’s many functions. A breakdown in the ability of cells to form peroxisomes is linked to many human health issues, including defense against infectious agents, cancer, aging, heart disease, obesity and diabetes, and forms the basis of a spectrum of peroxisomal genetic disorders that cause severe neuropathologies. The ER serves as a source for preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and to support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. Here we show that the evolutionarily ancient endosomal sorting complexes required for transport (ESCRT)-III are peroxisome biogenesis factors that function to cleave PPVs budding from the ER into the cytosol. Using comprehensive morphological and genetic assays of peroxisome formation and function we find that absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay we show that ESCRT-III proteins Vps20 and Snf7 are required to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and towards the cytosol, a finding that has important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM’s enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells., Author summary The integration of gene regulatory and metabolic network models is an important goal in computational biology, in order to develop methods that can identify the underlying mechanistic links in biological networks and advance metabolic engineering techniques. In this paper, we develop a framework called Integrated Deduced REgulation And Metabolism (IDREAM) that can improve our ability to predict phenotypes of microorganisms, and particularly it can address the challenges in evaluating phenotypic consequence of perturbing transcriptional regulation of metabolism in a eukaryotic cell. We compare the predictive performance of an IDREAM S. cerevisiae model with a PROM model using a TRN available from the YEASTRACT database. IDREAM outperforms PROM using any of three popular yeast metabolic models and across three experimental growth conditions, making it possible to identify subtle synthetic growth defects, and a new role for Oaf1 in the regulation of acetyl-CoA biosynthesis.

The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence.

Splenic filtration of Plasmodium falciparum-infected red blood cells has been hypothesized to influence malaria pathogenesis. We have developed a minimum cylindrical diameter (MCD) filtration model which estimates physical splenic filtration during malaria infection. The key parameter in the model is the MCD, the smallest tube or cylinder that a red blood cell (RBC) can traverse without lysing. The MCD is defined by a relationship between the RBC surface area and volume. In the MCD filtration model, the MCD filtration function represents the probability of a cell becoming physically removed from circulation. This modelling approach was implemented at a field site in Blantyre, Malawi. We analysed peripheral blood samples from 120 study participants in four clinically defined groups (30 subjects each): cerebral malaria, uncomplicated malaria, aparasitaemic coma and healthy controls. We found statistically significant differences in the surface area and volumes of uninfected RBCs when healthy controls were compared with malaria patients. The estimated filtration rates generated by the MCD model corresponded to previous observations in ex vivo spleen experiments and models of red blood cell loss during acute malaria anaemia.There were no differences in the estimated splenic filtration rates between cerebral malaria and uncomplicated malaria patients. The MCD filtration model estimates that at time of admission, one ring-stage infected RBC is physically filtered by the spleen for each parasite that remains in peripheral circulation. This field study is the first to use microfluidic devices to identify rheological diversity in RBC populations associated with malaria infection and illness in well-characterized groups of children living in a malaria endemic area.

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and from Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institute of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations, and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India, including a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012-2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Splenic filtration of infected red blood cells (RBCs) may contribute to innate immunity and variable outcomes of malaria infections. We show that filterability of individual RBCs is well predicted by the minimum cylindrical diameter (MCD) which is calculated from a RBC's surface area and volume. The MCD describes the smallest diameter tube or smallest pore that a cell may fit through without increasing its surface area. A microfluidic device was developed to measure the MCD from thousands of individual infected RBCs (IRBCs) and uninfected RBCs (URBCs). Average MCD changes during the blood-stage cycle of Plasmodium falciparum were tracked for the cytoadherent strain ITG and the knobless strain Dd2. The MCD values for IRBCs and URBCs raise several new intriguing insights into how the spleen may remove IRBCs: some early-stage ring-IRBCs, and not just late-stage schizont-IRBCs, may be highly susceptible to filtration. In addition, knobby parasites may limit surface area expansions and thus confer high MCDs on IRBCs. Finally, URBCs, in culture with IRBCs, show higher surface area loss which makes them more susceptible to filtration than naive URBCs. These findings raise important basic questions about the variable pathology of malaria infections and metabolic process that affect volume and surface area of IRBCs.

Silica colloids embedded with superparamagnetic iron oxide nanoparticles were synthesized by combining commercial ferrofluids with the well-known Stober process. In a typical procedure, organophilic iron oxide nanoparticles were extracted from a ferrofluid and redispersed in toluene. The suspension was then added to an alcoholic medium to produce emulsion drops consisting of aggregates of iron oxide nanoparticles and toluene. When tetraethylorthosilicate was introduced into the system, it hydrolyzed and formed silica coating around each emulsion drop. The final size of silica colloids depended on the concentration of iron oxide nanoparticles and the type of solvent used for the Stober synthesis. Larger colloids were obtained at lower concentrations of iron oxide nanoparticles and in alcohols with higher molecular weights.

Morphological control over platinum nanoparticles was realized by varying the amount of NaNO3 added to a polyol process, where H2PtCl6 was reduced by ethylene glycol to form PtCl42- and Pt0 at 160 °C. As the molar ratio between NaNO3 and H2PtCl6 was increased from 0 to 11, the morphology of Pt nanoparticles evolved from irregular spheroids with rounded profiles to tetrahedra and octahedra with well-defined facets. Absorption spectroscopy studies suggest that nitrate was reduced to nitrite by PtCl42- in the early stage of the synthesis, and the nitrite could then form stable complexes with both Pt(II) and Pt(IV) species. As a result, the reduction of Pt precursors by ethylene glycol was greatly slowed. This change in reaction kinetics altered the growth rates associated with different crystallographic directions of the Pt nanocrystals and ultimately led to the formation of different morphologies.

Single-crystal cubes and tetrahedrons of silver with truncated corners/edges have been prepared for the first time in high yields by reducing silver nitrate with ethylene glycol heated to 148 °C in the presence of poly(vinyl pyrrolidone) and a trace amount of sodium chloride. These nanoparticles were relatively monodisperse in size and shape, and their dimensions could be readily controlled in the range of 20 to 80 nm by varying the reaction time and other experimental parameters. We propose that the defects inherent in twinned nuclei of silver led to their selective etching and dissolution by chloride and oxygen (from air), leaving only the single crystalline ones to grow into nanoscale cubes and tetrahedrons.

A solution-phase, precursor method has been demonstrated for the large-scale synthesis of single crystalline nanowires of lead with uniform diameters in the range 50−90 nm and lengths up to several...

This paper describes a solution-phase route to the facile synthesis of lead nanowires with lateral dimensions as thin as 35 nm and lengths up to 250 μm. These lead nanowires were single crystalline in structure, and electron transport measurements on individual nanowires (using the four-probe method) indicate a phase transition to the superconducting state around 7.13 K, a temperature similar to the value (7.20 K) reported for bulk lead. Different from polycrystalline samples, no significant variation in the transition temperature was observed for nanowires with diameters down to 50 nm.

We have recently demonstrated an approach based on the polyol process for the large-scale synthesis of silver nanowires with uniform diameters (see Sun, Y.; Gates, B.; Mayers, B.; Xia, Y. Nano Lett. 2002, 2, 165. Sun, Y.; Xia, Y. Adv. Mater. 2002, 14, 833. Sun, Y.; Yin, Y.; Mayers, B. T.; Herricks, T.; Xia, Y. Chem. Mater. 2002, 14, 4736). Although the capability and feasibility of this method have been successfully illustrated with the production of silver nanowires 30−60 nm in diameter and 1−50 μm in length, the growth mechanism of this process is yet to be elucidated. Here we report some progress on this matter: First, electron microscopy studies on microtomed samples indicated that the cross sections of such silver nanowires had a pentagonal shape, together with a 5-fold twinned crystal structure. Second, the side surfaces (bounded by {100} facets) and the ends (bounded by {111} facets) of each nanowire were shown to have significant difference in reactivity toward dithoil molecules, with the side su...

This paper describes a vapor-phase approach to the facial synthesis of cupric oxide (CuO) nanowires supported on the surfaces of various copper substrates that include grids, foils, and wires. A typical procedure simply involved the thermal oxidation of these substrates in air and within the temperature range from 400 to 700 °C. Electron microscopic studies indicated that these nanowires had a controllable diameter in the range of 30−100 nm with lengths of up to 15 μm by varying the temperature and growth time. Electron diffraction and high-resolution TEM studies implied that each CuO nanowire was a bicrystal divided by a (111) twin plane in its middle along the longitudinal axis. A possible mechanism was also proposed to account for the growth of these CuO nanowires.

A solution-phase approach has been demonstrated for the large-scale synthesis of silver nanowires with diameters in the range of 30−40 nm, and lengths up to ∼50 μm. The first step of this process involved the formation of Pt (or Ag) nanoparticles by reducing PtCl2 (or AgNO3) with ethylene glycol (EG) heated to ∼160 °C. These Pt (or Ag) nanoparticles could serve as seeds for the heterogeneous nucleation and growth of silver (formed by reducing AgNO3 with EG) because of their close match in crystal structure and lattice constants. In the presence of poly(vinyl pyrrolidone) (PVP), the growth of silver could be directed into a highly anisotropic mode to form uniform nanowires with aspect ratios as high as ∼1000. UV−visible spectroscopy, SEM, TEM, XRD, and electron diffraction were used to characterize these silver nanowires, indicating the formation of a highly pure phase, as well as a uniform diameter and bicrystalline structure. Both morphology and aspect ratios of these silver nanostructures could be varie...

Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ∼5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding.

This letter describes a simple procedure for fabricating randomly and uniaxially aligned nanofibers of nickel ferrite by electrospinning. Polycrystalline nanofibers of NiFe2O4 with an average diameter of 46 nm have been prepared by electrospinning a solution that contained poly(vinyl pyrrolidone) and alkoxide precursors to nickel and iron oxides, followed by hydrolysis and calcination at 550 °C in air. Magnetic hysteresis scans were performed on these nanofibers, in comparison with a powder sample prepared using the conventional sol–gel process. Significant differences in magnetic properties were noted between these two samples, and these differences seemed to be associated with the size and morphological differences between the nanofibers and the powders.

Splenic filtration of Plasmodium falciparum infected red blood cells has been hypothesized to influence malaria pathogenesis. We have developed a minimum cylindrical diameter (MCD) filtration model which estimates physical splenic filtration during malaria infection. The key parameter in the model is the minimum cylindrical diameter (MCD), the smallest tube or cylinder that a red blood cell (RBC) can traverse without lysing. The MCD is defined by a relationship between the RBC surface area and volume. In the MCD filtration model, the MCD filtration function represents the probability of a cell becoming physically removed from circulation. This modeling approach was implemented at a field site in Blantyre, Malawi. We analyzed peripheral blood samples from 120 study participants in 4 clinically defined groups (30 subjects each): cerebral malaria, uncomplicated malaria, aparasitemic coma, and healthy controls. We found statistically significant differences in the surface area and volumes of uninfected RBCs when healthy controls were compared to malaria patients. The estimated filtration rates generated by the MCD model corresponded to previous observations in ex vivo spleen experiments and models of red blood cell loss during acute malaria anemia. There were no differences in the estimated splenic filtration rates between cerebral malaria and uncomplicated malaria patients. The MCD filtration model estimates that at time of admission, 1 ring-stage infected RBC is physically filtered by the spleen for each parasite that remains in peripheral circulation. This field study is the first to use microfluidic devices to identify rheological diversity in RBC populations associated with malaria infection and illness in well characterized groups of children living in a malaria endemic area.

Malaria is a major poverty-related human infectious disease of the world. Over a billion individuals are under threat and several million die from malaria every year. The nature of disease, especially fatal disease, has been the subject of many studies. The consensus is that parasite-induced cytoadherance of red blood cells precipitates capillary blockage and inflammatory responses in affected organs. Reduced deformability of infected erythrocytes may also contribute to disease. What is not very clear is why people with significant parasite burdens display large variations in disease outcomes. Technologies which allow a detailed description of the cytoadherance properties of infected erythrocytes in individual patients, and which allow a complete description of the flow capabilities of red blood cell populations in that patient, would be very useful. Here we review the recent introduction of microfluidic technology to study malaria pathogenesis, including the fabrication processes. The devices are cheap, versatile, portable and require very small patient samples. With greater use in research laboratories and field sites, we eventually expect microfluidic methods to play important roles in malaria diagnosis, as well as prognosis.

We have successfully incorporated iron oxide nanoparticles into monodispersed amorphous selenium (a-Se) colloids by regulating the reaction temperature during the synthesis of a-Se. The surfaces of these a-Se colloids could be coated with conformal and smooth shells made of Pt and SiO2. The Se cores could then be removed by etching with hydrazine. The spherical morphology and superparamagnetism were maintained in all these synthetic steps. The presence of Pt and SiO2 on the outer surfaces of these colloidal particles allows one to control their surface functionalities through the formation of alkanethiolate and siloxane monolayers, respectively.

Platinum nanowires of approximately 100 nm in length and approximately 5 nm in diameter have been synthesized by reducing H(2)PtCl(6) with ethylene glycol in the presence of poly(vinyl pyrrolidone) (PVP) and a trace amount of Fe(3+) or Fe(2+). The wires were generated at the final stage of the synthesis, which involved the formation of several intermediate species. The Fe(3+) or Fe(2+) ions had dual functions in the synthesis: they induced aggregation of Pt nanoparticles into larger structures that served as the nucleation sites, and they greatly reduced the reaction rate and supersaturation level to induce anisotropic growth. The reaction mechanism was studied by X-ray photoelectron spectroscopy (XPS) and UV-vis spectral analysis. The Pt nanowires could be readily separated from the surfaces of the agglomerates by sonication and obtained as pure samples by centrifugation.

The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria.

Nanofibers consisting of enzyme–polymer composites have been prepared by directly electrospinning a solution of surfactant-stabilized enzyme and polymer in toluene. Additional treatment with glutaraldehyde could greatly stabilize the enzyme activity of the fibers, which could be maintained in a buffer under shaking conditions for more than two weeks. The nanofibers also showed great improvement in the enzyme activity over bulk films as a result of increased mass-transfer for substrate molecules to and from the enzyme reactive sites. These stable and catalytically active nanofiber-based mats were highly durable and could be easily recovered from solution, making them ideal candidates for large scale applications.