Crude Suspensions Research Articles

Optimization and Appraisal of Nintedanib-Loaded Mixed Polymeric Micelles as a Potential Nanovector for Non-Invasive Pulmonary Fibrosis Mitigation.

Nintedanib (NTD), a triple tyrosine kinase receptor inhibitor, is the recommended first-line tackling option for idiopathic pulmonary fibrosis (IPF). Nevertheless, the adequacy of NTD is curtailed by issues associated with its low solubility, first-pass effect, poor bioavailability, and liver toxicity. The objective of our work was to develop a non-invasive intratracheal (i.t.) nanoparadigm based on NTD-loaded polymeric mixed micelles (NTD-PMMs) that can effectively treat IPF by sustaining the release of NTD, and snowballing its bioavailability, solubility, and efficacy. Design-Expert® software was used to optimize various NTD-PMMs formulations via Box-Behnken design adopting the thin-film hydration technique. The optimum formulation was chosen and in vivo tested in a rat model to explore its comparative bioavailability and toxicity. The formulation composition with 309.217 mg of Soluplus, 150 mg of Tween 80, and 40 mg of sodium deoxycholate was found to fulfill the requisites of an optimum NTD-PMMs formulation. The optimum NTD-PMMs formulation divulged 90.26% entrapment efficiency with a surface charge of -14.72 mV and a nanoscale diameter of 61.36 nm. Also, it substantially sustained the release of NTD by 66.84% after 24 h and manifested a pronounced stability. In vivo histopathology investigations verified the safety of NTD-PMMs delivered intratracheally. Moreover, pharmacokinetic analyses disclosed accentuated relative bioavailability of the optimized NTD-PMMs by 2.4- and 3.82-fold as compared with both the i.t. and oral crude NTD suspensions, respectively. Overall, the current results elicited the potential of PMMs to serve as a promising pulmonary nanovector for the targeted delivery of NTD.

Obtaining absorbance spectra from turbid retinal cell and tissue suspensions - Beating the light-scatter problem

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a “Dark-Interval” between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec−1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.

From Impure to Purified Silver Nanoparticles: Advances and Timeline in Separation Methods.

AgNPs have exceptional characteristics that depend on their size and shape. Over the past years, there has been an exponential increase in applications of nanoparticles (NPs), especially the silver ones (AgNPs), in several areas, such as, for example, electronics; environmental, pharmaceutical, and toxicological applications; theragnostics; and medical treatments, among others. This growing use has led to a greater exposure of humans to AgNPs and a higher risk to human health and the environment. This risk becomes more aggravated when the AgNPs are used without purification or separation from the synthesis medium, in which the hazardous synthesis precursors remain unseparated from the NPs and constitute a severe risk for unnecessary environmental contamination. This review examines the situation of the available separation methods of AgNPs from crude suspensions or real samples. Different separation techniques are reviewed, and relevant data are discussed, with a focus on the sustainability and efficiency of AgNPs separation methods.

Effect of carbon-based nanoparticles on the ignition, combustion and flame characteristics of crude oil droplets

The use of in-situ burning (ISB) as a clean-up response in the event of an oil spill has generated controversy because of unburned hydrocarbons and products of incomplete combustion left behind on an ISB site. These substances threaten marine life, both in the ocean and on the ocean floor. Treating crude oil as a multicomponent liquid fuel, this manuscript investigates the effect of carbon-based nanomaterials, acetylene black (AB) and multi-walled carbon nanotube (MWNT), on the combustion and flame characteristics of crude sourced from the Bakken formation (ND, USA). Sub-millimeter droplets of colloidal suspensions of Bakken crude and nanomaterials at various particle loadings were burned, and the process was captured with CMOS and CCD cameras. The resulting images were post-processed to generate burning rate, ignition delay, total combustion time, and flame stand-off (FSR) ratio data for the various crude suspensions. A maximum combustion rate enhancement of 39.5% and 31.1% was observed at a particle loading of 0.5% w/w acetylene black nanoparticles and 0.5% w/w multi-walled carbon nanotubes, respectively. Generally, FSR for pure Bakken was noted as larger than for Bakken with nanoparticle additives. These results are expected to spur further investigations into the use of nanomaterials for ISB crude oil clean-ups.

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of >40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

Effects of temperature on germination of sporangia, infection and protein secretion by Phytophthora kernoviae

Phytophthora kernoviae is a pathogen on a wide range of plants, but little is known of optimal infection conditions. Rhododendron ponticum leaves were inoculated with six different isolates of P. kernoviae sporangia and incubated at different temperatures from 10 to 28 °C. After 1 week, lesion development and pathogen recovery were only observed from all isolates at 15 and 20 °C and a few isolates at 10 °C. In an experiment with temperatures ranging from 20 to 25 °C, lesion development and pathogen recovery on R. ponticum, Magnolia stellata and Viburnum tinus occurred consistently at 20 and 21 °C, was limited at 22 °C, and did not occur at 23 °C and above. There was no difference in sporangia and zoospore germination at 20–25 °C. In a temperature fluctuation experiment, the necrotic area of inoculated R. ponticum leaves increased with longer incubation at 20 °C and decreased with longer incubation at 24 °C. Crude extracts of secreted proteins from P. kernoviae cultures grown at 20 and 24 °C were compared to determine any effects of temperature on pathogenicity. When spot tested on R. ponticum leaves, crude protein suspensions from cultures grown at 20 °C induced necrosis, while proteins from cultures grown at 24 °C did not. Proteomic analysis confirmed that a 10 kDa protein secreted at both 20 and 24 °C shared sequence homology to the conserved domains of known elicitins of other Phytophthora spp. The protein secreted at 20 °C that was responsible for necrosis has not been identified.

Formulation and characterization of biocompatible and stable I.V. itraconazole nanosuspensions stabilized by a new stabilizer polyethylene glycol-poly(β-Benzyl-l-aspartate) (PEG-PBLA)

Amphiphilic block copolymers, PEG-PBLA with different molecular weights, were synthesized and used as new stabilizers for Itraconazole nannosuspensions (ITZ-PBLA-Nanos). ITZ-PBLA-Nanos were prepared by the microprecipitation-high pressure homogenization method, and the particle size and zeta potential were measured using a ZetaSizer Nano-ZS90. Morphology and crystallinity were studied using TEM, DSC and powder X-ray. The effect of the PEG-to-PBLA ratio, and the drug-to-stabilizer ratio were investigated to obtain the optimal formulation. It was found that the optimal length of hydrophobic block was 25 BLA-NCA molecules and the optimal ratio of drug/stabilizer was 1:1, where the resulted average particle size of ITZ-PBLA-Nanos was 262.1±7.13nm with a PDI value of 0.163±0.011. The images of TEM suggest that ITZ-PBLA-Nanos were rectangular in shape. ITZ existed as crystals in the nanoparticles as suggested by the DSC and XRD results. Compared with the crude drug suspensions, the dissolution rate of ITZ nanocrystals, was significantly increased and was similar to Sporanox® injection. The ITZ-PBLA-Nanos also demonstrated better dilution stability and storage stability compared with ITZ-F68-Nanos. The particle size of ITZ-PBLA-Nanos did not change significantly after incubated in rat plasma for 24h which is a good attribute for I.V. administration. Acute toxicity tests showed that ITZ-PBLA-Nanos has the highest LD50 compared with ITZ-F68-Nanos and Sporanox® injection. ITZ-PBLA-Nanos also showed stronger inhibiting effect on the growth of Candida albicans compared with Sporanox® injection. Therefore, PEG-PBLA has a promising potential as a biocompatible stabilizer for ITZ nanosuspensions and potentially for other nanosuspensions as well.

Mechanism of in vitro antagonism of phytopathogenic Scelrotium rolfsii by actinomycetes

Sclerotium rolfsii (Sr), a soil-borne fungal pathogen, causes disease in a wide range of crops. Recently, we identified five actinomycetes (Streptomyces globisporus subsp. globisporus, S. globisporus, S. flavotricini, S. pactum, and S. senoensis) showing significant inhibitory effects on plant pathogens. In this study, the effects of the five actinomycetes for the biocontrol of Sr were investigated using the plate culture method and microscopy examination. Two actinomycetes with higher inhibitory effect were subsequently examined for the inhibition of sclerotial germination of Sr in unsterile soil in vitro. The cell-free cultures of five actinomycetes mediated significant inhibition of hyphal growth and sclerotial formation and germination of Sr. All actinomycete strains exhibited the ability to produce extracellular cell wall degrading enzymes in the culture conditions. The crude enzyme suspensions of S. flavotricini and S. pactum hydrolyzed the cell wall of Sr. At a dose of 1 g per kilogram soil, the solid formulations of S. flavotricini and S. senoensis prevented germination of 24% and 68% of sclerotia, respectively. Our results provide evidence of effective strains for the biocontrol of Sr, in addition to a further understanding of the underlying mechanism.

Sexual variation of bacterial microbiota of Dendroctonus valens guts and frass in relation to verbenone production

Gut microbiota are widely involved in insect biology, and many factors can influence the microbiota in guts and frass. Dendroctonus valens is a very destructive forest pest in China, and the mass-attacking behavior is regulated by several semiochemicals, including verbenone, a multifunctional pheromone. The beetle harbors a variety of bacteria in its guts and frass and some of them are capable of verbenone production. D. valens is characterized by monogamy and female-initiated attacking behavior. Whether the bacterial communities fluctuate according to sex, and whether the variation influences the verbenone production, remains to be determined. In this study, the bacterial microbiota in D. valens guts and frass were analyzed, and verbenone production by their crude bacterial suspensions was compared in vitro. Bacterial diversity in female frass is more abundant compared to male frass, and the percentages and total amounts of main genera like Lactococcus and Pseudomonas in female frass are significantly higher than those in male frass. The verbenone produced by the female frass suspension is significantly higher than male frass. This study presents a comprehensive comparison of bacterial communities in guts and frass between both sexes of D. valens, highlighting the potential significance of female frass microbiota in verbenone production.

Fast and direct extraction of cell-associated hepatotoxins from toxic cyanobacteria.

Microcystins are an important group of toxins produced by cyanobacteria of different genera. An increasing number of water contaminations by this class of toxins have been reported that are susceptible to generate important public health problems. We designed an efficient method for extracting these toxins on-site for a rapid testing of potentially contaminated water. The extraction parameters have been optimized using Microcystis aeruginosa and the technique successfully applied to different laboratory cultures and field samples. The procedure employs a simple and stable reagent mix of propanol, Tween 20, and trifluoroacetic acid. It is directly active on crude cell suspensions without any pre-treatment. Extraction yields measured by immunological quantification were at least equal to the best values obtained with the most commonly used laboratory techniques. An additional simple concentration/extraction step is also described that allows measurements on samples too dilute for direct detection by immunochromatographic techniques.

New Technique to Create a Suspension Containing Bacteriophages and How It Can Be Used to Control Cabbage Leaf Spot Caused by <i>Xanthomonas campestris</i> PV. <i>campestris</i>

The purpose of the present study was to develop a new method for producing and identifying a bacteriophage suspension and research its potential against plant bacteria. The suspension obtained with the new technique was effective in controlling Xanthomonas campestris PV. campestris both in vitro and in vivo. A difference was observed between the suspension with pathogen (PLP) and crude suspension with lesioned plant without the pathogen originated from it (PLN). Clear zones were observed at 48 h/28°C in both (PLS) suspension added saccharose and (PLP). Although PLP and PLS treatments showed no statistical difference in CFU values (5 × 105 and 1 × 105, respectively), they differed from the crude suspension (PLN) and the control. Then, in vivo assessment of suspensions with clear zones in the dishes was performed. In vivo (PLPS) and (PLPP) treatments consisting of inoculum were added to the crude suspensions (PLS and PLP) to increase bacteriophages. A decrease in the disease severity index compared to the control was observed, showing that treatment with the phage suspension protects the plant. The lowest disease indexes were reported with (PLP) and (PLS), with values of 1.46 and 1.40, respectively. The highest averages in dry matter (5.1 inboth treatments) were obtained with (PLP) and (PLS) treatments.

Osteoclast and Osteoblast Activities on Carbonate Apatite Plates in Cell Cultures

Previous studies have demonstrated that carbonate apatite (CA) is superior to hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP) with regard to osteoclastic resorption, but evidence on osteoclast and osteoblast response remains controversial. In the present study, the expression of bone related mRNA is examined on CA, HA, β-TCP, and titanium plates. ICR mouse osteoblast cells are cocultured with ICR mouse bone marrow cells. Crude osteoclast-like cell-rich suspensions are then seeded onto plates and cultured for 48 h. Total RNA is extracted and mRNA expression is examined by real-time RT-PCR. Amounts of vacuolar-type ATPase, cathepsin K, and TRAP mRNA are significantly greater on CA than on the other plates. The amount of osteoprotegerin mRNA is significantly greater on CA than on the other plates. RANKL mRNA expression, which is generally regarded as an osteoblast maker, varies with material, but shows no significant differences between CA and the other plates. The formation and activity of osteoclasts is greater with CA than with the other plates. Thus, CA is superior to β-TCP as a bioresorbable bone substitute for tissue engineering.

Genotoxicity of unmodified and organo-modified montmorillonite

The natural clay mineral montmorillonite (Cloisite ® Na +) and an organo-modified montmorillonite (Cloisite ® 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-μm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 μg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite ® Na + suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite ® 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples ( p < 0.01 and p < 0.001) and ( p < 0.05 and p < 0.01), respectively. The unfiltered samples were tested up to concentrations of 170 μg/ml and the filtered samples up to 216 μg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 μg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite ® 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite ® 30B.

Susceptibility of Crude and Semi-Purified Extracts of Nucleopolyhedrovirus Isolates of Helicoverpa armigera (Hubner) to Simulated Sunlight

The stability of both crude and semi-purified extracts of different HaNPV isolates under simulated sunlight was studied. It was realized that the persistence of both crude and semi-purified suspensions of Negamum isolate collected from Tamil Nadu was most tolerant to simulated sunlight among all the isolates tested in this study. The highest larval mortality (70.12%) was obtained from crude extract of the Negamum isolate as compared with that of semi-purified suspension (54.44%). The order of inhibition of crude viral extracts of HaNPV isolates under simulated sunlight irradiation at 500 W/m 2 for 90 minutes was Negamum < Ooty < Coimbatore < Mumbai < Hyderabad < Parbhani < Rahuri, which was similar with that of the semi-purified suspensions with the exception of Parbhani < Hyderabad.

A simple method to measure effective catalase activities: Optimization, validation, and application in green coffee

Oxidative metabolism in coffee cherries during maturation appears to be regulated by the timely expression of redox enzymes such as catalase (CAT), peroxidase (POD), and polyphenoloxidase (PPO). Among these enzymes, CAT is suspected to contribute significantly in setting the redox status of the healthy cherry and the processed bean. The initial redox status of the green bean might further control the nature and dynamics of reactions induced by roasting and eventually quality aspects of the end product. In this respect, Arabica ( Coffea arabica) and Robusta ( Coffea canephora) typically differ by their cup coffee flavor profiles. We developed an assay that allowed us to screen numerous green coffee samples for effective CAT activities. The proposed assay, which monitors CAT activities by online oxygen sensing in green coffee crude suspensions incubated with H 2O 2, seeks to integrate potential effects of endogenous inhibitors and activators. After optimization and validation of the assay, 23 Arabicas, 23 Robustas, and 8 Arabustas were analyzed. Nearly all Arabicas (22 of 23) harbored high CAT activity levels, whereas all Robustas harbored low ones. Arabustas performed like Arabicas of the lower CAT activity range. The traditional spectrophotometric assay did not reveal these specificities. Because of its simplicity, our assay might be valuable for assessing effective CAT activities in various plant tissues.

Professor Walter Morgan

Professor Walter Morgan

Biological Characterization of a Less Virulent Taura Syndrome in Pacific White Shrimp Litopenaeus vannamei (Crustacea: Decapoda): Gross Signs, Histopathological Lesions, and Mortalities

Taura syndrome (TS) was first recognized in shrimp farms located near the mouth of the Taura River in the Gulf of Guayaquil, Ecuador in mid1992 (JimCnez 1992; Lightner et al. 1995), where the disease caused catastrophic losses with cumulative mortality rates from 60% to >90% of affected pond-cultured, juvenile Pacific white shrimp Litopenaeus vannamei (Perez Farfante and Kensley 1997). The geographic distribution of the disease now includes Ecuador, the adjacent region of Peru, the Pacific and Caribbean coast of Colombia, Honduras, Guatemala, El Salvador, northeast Brazil, Nicaragua, Belize, the states of Hawaii, Florida, and Texas in the USA, the Mexican states of Sonora, Sinaloa, Chiapas, and Guerrero, and recently Taiwan (Lightner 1996; Chien et al. 1999). The epizootiological and laboratory studies that followed its discovery and spread from Ecuador showed that Taura syndrome (TS) had a viral etiology in the American (L. vannamei, L. stylirostris, L. schmitti, L. setiferus, Farfuntepenaeus aztecus) and Asian (Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis) penaeid species naturally or experimentally infected by the virus. Litopenaeus vannamei was by far the most severely affected (Brock et al. 1995, 1997; Hasson et al. 1995, 1999b; Lightner 1996; Overstreet et al. 1997). Infectivity studies demonstrated that TS could be induced in specific-pathogen-free (SPF) L. vannamei. Shrimp started dying after being fed TSV-infected tissue between day 4 or 5, with mortalities of 80% (Brock et al. 1995). Test animals were also infected by intramuscular injection with crude homogenate and cell-free suspensions, from TSV-infected Ecuatorian L. vannamei, causing mortalities of 73% to 87% among treated groups. All moribund shrimp demonstrated moderate to severe pathognomonic TS lesions (Hasson et al. 1995), which consist of pyknotic and karyorrhectic nuclei and generally spherical cytoplasmic inclusions with multifocal areas of necrosis of the cuticular epithelium giving TS lesions a peppered or buckshot-riddled appearance (Brock et al. 1995; Lightner et al. 1995; Hasson et al. 1995; Lightner 1999). The hemispheric geographic distribution of TS indicates that the same virus or a closely related one is responsible for the TS epizootics that have occurred throughout America since 1992 (Hasson et al. 1999a). Ecuatorian and Hawaiian TS virus isolates were found to be identical in their biophysical, biochemical, and biological characteristics, and they were considered to be the same virus (Bonami et al. 1997). In early 1995, TSV was detected in wild adult L. vannamei collected from the offshore fishery of the southern Mexican state of Chiapas, near its border with Guatemala and near where TS had first appeared in

Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures.

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Three-Phase Affinity Partitioning of Proteins

Three-phase partitioning is an elegant way to separate proteins directly from even the large volumes of crude suspensions. It was found that interfacing it with a metal-affinity-based step makes the technique highly selective. As “proof of the concept,” soybean trypsin inhibitor was purified 13-fold with 72% recovery. As recombinant proteins (via their polyhistidine tags) and many other naturally occurring proteins are often purified by immobilized metal ion affinity chromatography, the technique described here should prove valuable in purifying biotechnologically important proteins at a large scale.

Solid-phase enolate chemistry investigated using HR-MAS NMR spectroscopy.

Supported P4-t-Bu enolate chemistry of phenylacetyloxymethyl polystyrene (PS) resin was investigated using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Direct analysis of the crude reaction suspensions through the use of a diffusion filter (DF) allowed a rapid selection of the optimal experimental conditions, but also the characterization of the enolate on the solid phase. Comparison with solution experiments and literature data allowed us to address partially the structure of the enolate. HR-MAS NMR spectra of the enolate revealed also a tight interaction of P4-t-Bu base with the polymer matrix.