Real-Time Particle Size Analysis Using Focused Beam Reflectance Measurement as a Process Analytical Technology Tool for a Continuous Granulation–Drying–Milling Process

Focused beam reflectance measurement (FBRM) was used as a process analytical technology tool to perform inline real-time particle size analysis of a proprietary granulation manufactured using a continuous twin-screw granulation–drying–milling process. A significant relationship between D20, D50, and D80 length-weighted chord length and sieve particle size was observed with a p value of <0.0001 and R² of 0.886. A central composite response surface statistical design was used to evaluate the effect of granulator screw speed and Comil® impeller speed on the length-weighted chord length distribution (CLD) and particle size distribution (PSD) determined by FBRM and nested sieve analysis, respectively. The effect of granulator speed and mill speed on bulk density, tapped density, Compressibility Index, and Flowability Index were also investigated. An inline FBRM probe placed below the Comil-generated chord lengths and CLD data at designated times. The collection of the milled samples for sieve analysis and PSD evaluation were coordinated with the timing of the FBRM determinations. Both FBRM and sieve analysis resulted in similar bimodal distributions for all ten manufactured batches studied. Within the experimental space studied, the granulator screw speed (650–850 rpm) and Comil® impeller speed (1,000–2,000 rpm) did not have a significant effect on CLD, PSD, bulk density, tapped density, Compressibility Index, and Flowability Index (p value > 0.05).Key words: continuous granulation-drying-milling, focused beam reflectance measurement (FBRM), process analytical technology (PAT), real-time particle size measurement     

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting

Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrP^Sc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrP^Sc or consolidation of the bonding between PrP^Sc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions.     

Characterizing the Freeze–Drying Behavior of Model Protein Formulations

The freeze–drying behavior of three model proteins, namely, lysozyme, BSA, and IgG, has been studied using a variety of techniques under two different primary drying conditions (shelf temperatures of −25°C and +25°C, respectively) in an amorphous formulation. Manometric temperature measurements were used to characterize product temperature (T _pr), sublimation rates, and product resistance (R _p) during primary drying. Biophysical techniques such as circular dichroism, fluorescence, and Fourier transform infrared spectroscopy were used to study protein conformation. Size exclusion chromatography was used to monitor the formation of high-molecular-weight species (HMWS) over time on storage, and cake morphology was studied using scanning electron microscopy. The differences in the freeze–drying behavior of the three proteins were more evident at higher protein concentrations, where the protein significantly influences the behavior of the formulation matrix. However, these differences were minimized in the aggressive mode and were insignificant at lower protein concentrations where excipients dominated the freeze–drying behavior. Differences in cake morphology were observed between the two drying conditions employed as well as between the three proteins studied. The stability and the protein structure, however, were equivalent for the protein cakes generated using the two different primary drying conditions.     

Luminescence Spectroscopy – a Useful Tool in Real-Time Monitoring of Viscosity during In-Vitro Digestion

Luminescence spectroscopy coupled with molecular rotors was used in the TNO Intestinal Model-1 (TIM-1) to monitor in situ changes to luminal viscosity of three maize starch samples varying in the amylose-to-amylopectin ratio (AM: AP): normal, high amylose (AM) and high amylopectin (AP). The fluorescence intensity (FI) of Fast Green (FG), a proven micro (and bulk) viscosity probe, was monitored throughout digestion to track changes in the gastric viscosity. The FI of FG and the viscosity imparted by the starch followed a power-law relationship. The emission of the MR was unaffected by the composition of TIM-1 secretion fluids nor pH. Hence, direct measurements of digesta FI are sensitive to changing viscosity during the simulated digestion. The viscosity was highest for AP, followed by normal starch, and high AM had the lowest viscosity. In the TIM-1 gastric compartment, from highest to lowest FI, and thus viscosity was high AM > high AP > normal maize starches. We conclude the validity of the proposed method to facilitate the measurement of luminal viscosity, in vitro, when the microviscosity represents bulk viscosity (i.e., when the increase in bulk viscosity is a result of molecular crowding and the surrounding environment around the rotor is homogeneous). Careful consideration is required when foods are heterogeneous as molecular rotors report only on their local non-uniform environment.

     

Continuous production of ethanol in a two-stage fermentation process using a protein — Phospholipid complex as a protective agent

A two-stage continuous fermentation process, using a continuous stirred tank fermenter (CSTF) and a tower fermenter (TF) connected in series, has been studied for ethanol production from d-glucose. The addition of a protein-phospholipid complex as a protective agent (PA) in the TF led to a three-fold increase in ethanol productivity and a 23.63% increase in final ethanol concentration in the tower effluent. The results are consistent with our previous findings on a cascade operation of CSTFs, namely, that the addition of PA to the tower increases cell tolerance towards ethanol at ethanol concentrations up to 70 gl^?1. Both studies indicate that beyond this experimentally determined critical ethanol concentration, ethanol production is significantly inhibited. Mathematical modelling of the behaviour of a single flocculated yeast floc suggested that, for yeast flocs up to 1 mm diameter, neither internal nor external diffusion of substrate is limiting. Therefore, a simplified mathematical method was developed for the analysis of the TF system. By plotting the calculated values from the derived performance equation and the experimental values of substrate conversion vs. residence time, good agreement was obtained between these two for the addition of PA. However, a small deviation was observed for the PA-free system.     

Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF- were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF- exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF- exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.     

Effect of Drying Methods on Swelling,Erosion and Drug Release from Chitosan–Naproxen Sodium Complexes

The purpose of this research was to explore theapplication of ionic interactions between naproxen sodium (NS) and chitosan (CH) in complexes (NSC) prepared by tray drying (TD) and spray drying (SD) methods. Drug–polymer ratio (1:1) in the NSC was optimized on the basis of dialysis studies. The particulate systems of NSC were prepared by tray drying (TD) and spray drying (SD) methods. Release retarding polymers were added to the NSC and to the physical mixtures containing NS–CH and their effects on water uptake, matrix erosion and drug release at different pH were compared. Spray dried complexes (SDC) were spherical, free flowing, light and fine amorphous particles in contrast to the crystalline, hard, tenacious, irregularly shaped, denser tray dried complexes (TDC) with poor flowability. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared (FTIR) patterns confirm the conversion of crystalline to high energy amorphous phase suitable for ionic interactions in NSC. Presence of release retarding polymers, kappa carrageenan and hydroxypropylmethylcellulose (HPMC) in the NSC compacts retarded the drug release and improved the matrix integrity. Carrageenan matrices exhibited more retardation than HPMC tablets. FTIR patterns, erosion, swelling and drug release from matrices support ionic interactions between NS and CH in NSC. The reasons for retarded drug release from the chitosan matrices at acidic pH include poor solubility of drug at acidic pH, formation of a rate limiting polymer gel barrier along the periphery of matrices and the ionic interactions between oppositely charged moieties.     

Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Transforming growth factor-β-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain αG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic αG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of αG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.     

Study of Oxidative Damage in Growing–Finishing Pigs with Continuous Excess Dietary Chromium Picolinate Intake

Chromium picolinate (CrPic) is a popular nutritional supplement; however, its safety has been questioned as it may be a source of oxidative stress that induces genotoxicity. The current work investigated the effect of excessive CrPic intake on oxidative damage in growing-finishing pigs. Thirty castrated male pigs, weighing approximately 30 kg each, were randomly divided into five groups and fed a diet with 0, 200, 800, 1,600, 3,200 microg of Cr/kg feed as CrPic for 80 days (approximately the entire growing-finishing period). High CrPic dose significantly decreased superoxide dismutase activity in serum at 80 days as well as the catalase activity in kidney (p < 0.05); however, compared to controls, malondialdehyde in tissue and serum, urinary 8-hydroxydeoxyguanosine level, and DNA strand breaks in liver and kidney had no notable differences (p > 0.05). These results suggested that long-term exposure to different doses of CrPic in feed did not increase the formation of biomarkers of oxidative damage in growing-finishing pigs. However, taking into account the changes of antioxidant enzymes activity, excessive dietary CrPic intake was not recommended in this study.     

Distribution of Tardigrades within a Moss Cushion: Do Tardigrades Migrate in Response to Changing Moisture Conditions?

The distribution of tardigrades within the layers of the cushion moss Grimmia alpicola Hedwig, 1801 was investigated. The aim of this study was to determine the tardigrade species present within the moss layers during both wet and dry periods and to determine if migration occurred in response to changing moisture conditions. Samples of the moss were removed from concrete caps on brick fence posts before and after rainfall and separated into two sections (top and bottom). The tardigrades from each layer and moisture condition were identified to species. Data for each species were statistically analyzed with a two-way analysis of variance (ANOVA) to compare the numbers of individuals present in the top and bottom layers of the moss under both wet and dry conditions.

Five tardigrade species were identified, including two species new to science: Macrobiotus sp. n.; Milnesium tardigradum Doyère, 1840; Echiniscus viridissimus Peterfi, 1956; Echiniscus perviridis Ramazzotti, 1959; and Echiniscus sp. n. The new species will be described in a forthcoming paper. No significant differences were found in the numbers of individuals of four of the five species in each layer within the moss or for each moisture condition. Only one species, E. viridissimus, was significantly more frequent in the top layer of the moss, regardless of moisture condition.

Migration within the moss cushion was not detected in any of these five species as a result of changes in moisture conditions. In xeric moss species, it may not be beneficial for tardigrades to migrate to avoid desiccation. Instead, they apparently undergo anhydrobiosis in both the top and bottom layers of the moss cushion.     

Coevolution of dispersal in a parasitoid–host system

Interspecific interactions and the evolution of dispersal are both of interest when considering the potential impact of habitat fragmentation on community ecology, but the interaction between these processes is not well studied. We address this by considering the coevolution of dispersal strategies in a host–parasitoid system. An individual-based host–parasitoid metapopulation model was constructed for a patchy environment, allowing for evolution in dispersal rates of both species. Highly rarefied environments with few suitable patches selected against dispersal in both species, as did relatively static environments. Provided that parasitoids persist, all the variables studied led to stable equilibria in dispersal rates for both species. There was a tendency toward higher dispersal rates in parasitoids because of the asymmetric relationships of the two species to the patches: vacant patches are most valuable for hosts, but unsuitable for parasitoids, which require an established host population to reproduce. High host dispersal rate was favoured by high host population growth rate, and in the parasitoid by high growth rates in both species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biodegradation of ketoprofen using a microalgal–bacterial consortium

Objective

To test the toxicity of ketoprofen (a commonly-used NSAIDs) using two microalgal strains and Artemia sp. following the isolation of bacterial and microalgal strains and testing their ability to biodegrade and tolerate ketoprofen.

Results

Chlorella sp. was the most resistant to ketoprofen. A defined bacterial consortium (K₂) degraded 5 mM ketoprofen as a sole carbon source both in the dark or continuous illumination. Ketoprofen did not undergo photodegradation. In the dark, biodegradation was faster with a lag phase of 10 h, 41% COD removal and 82 % reduction in toxicity. The consortium degraded up to 16 mM ketoprofen. The consortium was composed of four bacterial isolates that were identified. MS/MS analysis suggested a ketoprofen biodegradation pathway that has not been previously reported. Combining Chlorella sp. and the K₂ consortium, ketoprofen was degraded within 7 days under a diurnal cycle of 12 h light/12 h dark.

Conclusion

The feasibility of using a microalgal–bacterial system to treat pharmaceutical wastewater is promising for the reduction of the process cost and providing a safer technology for pharmaceutical wastewater treatment.

     

Dermatophyte–host relationship of a murine model of experimental invasive dermatophytosis

Recognizing the invasive potential of the dermatophytes and understanding the mechanisms involved in this process will help with disease diagnosis and with developing an appropriate treatment plan. In this report, we present the histopathological, microbiological and immunological features of a model of invasive dermatophytosis that is induced by subcutaneous infection of Trichophyton mentagrophytes in healthy adult Swiss mice. Using this model, we observed that the fungus rapidly spreads to the popliteal lymph nodes, spleen, liver and kidneys. Similar to the human disease, the lymph nodes were the most severely affected sites. The fungal infection evoked acute inflammation followed by a granulomatous reaction in the mice, which is similar to what is observed in patients. The mice were able to mount a Th1-polarized immune response and displayed IL-10-mediated immune regulation. We believe that the model described here will provide valuable information regarding the dermatophyte–host relationship and will yield new perspective for a better understanding of the immunological and pathological aspects of invasive dermatophytosis.     

Determinants of annual–perennial plant zonation across a salt–fresh marsh interface: a multistage assessment

Vegetation zonation patterns in coastal marshes are hypothesized to be the result of both physical stress and competitive interactions. How these patterns may be driven by these factors at different life history stages remains poorly understood. We investigated the relative importance of species tolerance (response to physical stress) and competitive ability in determining the distributions of two dominant marsh species across a salt–fresh marsh interface in the Yellow River Estuary, China. There is a steep gradient in salinity across this interface and Suaeda salsa, an annual, dominates the saline side of the interface, while Phragmites australis, a perennial species, dominates the freshwater side. Using a series of field transplants, we examined the roles of physical stress and competition in mediating this zonation at different life history stages. Suaeda salsa performed well in its home zone, but seedling emergence, seedling survival, adult survival and adult growth were significantly suppressed by competition in the freshwater P. australis zone. Emergence, survival and growth of P. australis were inhibited in the saline S. salsa zone, regardless of neighbor treatments, but it performed well in its home zone. The magnitude of the competitive effect on the performance of S. salsa differed among the life history stages. Competition from P. australis had a much stronger effect on S. salsa seedling emergence and adult growth than on seedling survival and adult survival. Our results reveal that both physical stress and competition contributed to the observed zonation patterns in this marsh system. However, for S. salsa, the effect of competition varied with life-history stage. Insight into these ecological processes is critical to understanding how the zonation pattern in the marsh system is formed and maintained.     

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using ¹³C-detection experiments.     

First Report of Fungal Strains from Afşin–Elbistan Mine for Microbial Lignite Process

Abstract

Today, the need for energy is increasing and fossil fuels are constituting one of the most important energy sources in the world. Therefore, the efficient use of fossil fuels has become inevitable. In order to better use the opportunities offered by nature, we have turned to nature’s own resources. In this study, fungal strains of potential use in coal processing technology, isolated from Turkey’s largest lignite mine and identified. Our goal was to obtain active isolates for the biotechnological applications using the mine’s own source. For this aim, lignite samples were collected from different regions of the Af?in–Elbistan lignite mine, isolation studies were done and eventually, distinguished fungal isolates were identified by using conventional and molecular techniques. Hereby, fungal biodiversity of Turkey’s largest lignite mine has been investigated for the first time. Furthermore, 20 fungal isolates belong to Alternaria, Aspergillus, and Penicillium genera were cultured to provide resources for the development of future biotechnological coal processing techniques.     

Determination and pharmacokinetics of a furosemide–amiloride drug combination

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30–3000 ng/ml and 0.5–30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.     

Enhanced cytotoxicity of a polymer–drug conjugate with triple payload of paclitaxel

The development of targeting approaches to selectively release chemotherapeutic drugs into malignant tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer–drug conjugate with an AB₃ self-immolative dendritic linker. HPMA copolymers are known to accumulate selectively in tumors. The water-soluble polymer–drug conjugate was designed to release a triple payload of the hydrophobic drug paclitaxel as a result of cleavage by the endogenous enzyme cathepsin B. The polymer–drug conjugate exhibited enhanced cytotoxicity on murine prostate adenocarcinoma (TRAMP C2) cells in comparison to a classic monomeric drug–polymer conjugate.