Ontogeny and structure of the acervulate partial inflorescence in Hyophorbe lagenicaulis (Arecaceae; Arecoideae).

BACKGROUND AND AIMS: The palm tribe Chamaedoreeae displays flowers arranged in a complex partial inflorescence called an acervulus. This type of partial inflorescence has so far not been reported elsewhere in the largest palm subfamily Arecoideae, which is traditionally characterized by flowers predominantly arranged in triads of one central female and two lateral male flowers. The ontogenetic basis of the acervulus is as yet unknown and its structural diversity throughout the genera of the Chamaedoreeae poorly recorded. This study aims to provide critical information on these aspects. METHODS: Developmental series and mature inflorescences were sampled from plants cultivated in international botanical gardens and wild populations. The main techniques employed included scanning electronic microscopy and serial anatomical sectioning of resin-embedded fragments of rachillae. KEY RESULTS: Inflorescence ontogeny in Hyophorbe lagenicaulis demonstrates that the acervulus and the inflorescence rachilla form a condensed and cymose branching system resembling a coenosome. Syndesmy results from a combined process of rapid development and adnation, without or with reduced axis elongation. Acervulus diversity in the ten taxa of the Chamaedoreeae studied is displayed at the level of their positioning within the inflorescence, their arrangement, the number of floral buds and their sexual expression. CONCLUSIONS: The results show that a more general definition of the type of partial inflorescence observed within the large subfamily Arecoideae would correspond to a cyme rather than to a floral triad. In spite of their common cymose architecture, the floral triad and the acervulus present differences with respect to the number and arrangement of floral buds, the superficial pattern of development and sexual expression.

Evaluation of torque as an in-process control for granule size during twin-screw wet granulation.

The potential of torque as in-process control (IPC) to monitor granule size in twin-screw wet granulation (TSG) was investigated. An experimental set-up allowing the collection of granules at four different locations (i.e., in the wetting zone, after the first and second kneading zone and at the end of the granulator) of the granulator screws was used to determine the change in granule size, granule temperature and the contribution of each compartment to the overall torque for varying screw speed, mass feed rate and liquid-to-solid ratio. The only observed correlation was between the granule size and torque increase after the first kneading zone because the torque increase was an indication of the degree in granule growth which was consistently observed with all applied granulation process parameters. No correlation was observed in the other locations as changes of torque were accompanied to either granule breakage and/or growth. Moreover, torque increase was correlated to higher granule temperature, suggesting that energy put into the granulator was partly used to heat up the material being processed and explains additionally the lack of correlation between granule size and torque. Therefore, this study showed that torque could not be used as IPC to monitor granule size during TSG.

In-line temperature measurement to improve the understanding of the wetting phase in twin-screw wet granulation and its use in process development.

Wetting is the initial stage of wet granulation processes during which the first contact between the powder and the liquid occurs. Wetting is a critical step to allow granule growth and consolidation, but also to ensure uniform active pharmaceutical ingredient (API) distribution over all granule size fractions. A physical understanding of the wetting stage is therefore crucial to design a robust granulation process. In twin-screw granulation, wetting is physically separated from granule consolidation, growth, breakage and attrition. The present study used this particularity to investigate the wetting step in such a way that the fundamental mechanisms governing the wetting can be linked and understood. A modified granulator barrel was used allowing the collection of granules immediately after the wetting. A low drug-loaded pharmaceutical formulation containing a poorly soluble and poorly wettable API was used for this investigation. Granules obtained after the wetting zone were analysed for granule size distribution, API distribution over the different size fractions and granule temperature. It was found that "wetting efficiency" (i.e., fraction of powder being nucleated during the wetting stage) could be predicted using an energy balance based on in-line measurement of the granule temperature. Wetting efficiency could moreover be linked to final granule quality attributes (i.e., granule size distribution) at the outlet of the granulator. It was further demonstrated that granule growth and consolidation could only be achieved when complete wetting was achieved in the wetting zone of the granulator. This study suggested a methodology based on in-line temperature measurements to quickly determine wetting efficiency. The described methodology could therefore be used as a tool to gain more fundamental understanding of the wetting stage during twin-screw granulation as well as to define suitable formulation and process ranges for further granulation process development.

Managing API raw material variability in a continuous manufacturing line - Prediction of process robustness.

Many studies on continuous twin-screw granulation only focus on the granulator without linking this process step to the upstream and downstream unit operations. Product critical quality attributes (CQAs) are however not only determined by the granulation step. In this study, the possibility to manage the batch-to-batch variability of an active pharmaceutical ingredient (API) in a high drug loaded formulation on a continuous line was investigated to obtain consistent tablet CQAs. As the ultimate goal of continuous manufacturing is to produce 24/7, current study also aimed at guaranteeing long term stability of the process. To do so, previously identified API critical material attributes (CMAs) were varied together with granulation, drying and milling critical process parameters (CPPs) in a screening design of experiments to understand the influence of these factors upon product CQAs and process stability. To evaluate the factors affecting the process stability with a reduced amount of materials, process deviations recorded by process sensors were used. While product CQAs only depended on process CPPs, process stability was strongly affected by API CMAs. The effect of API batch-to-batch variability on process stability could nonetheless be managed by applying suitable granulation conditions. Therefore, appropriate ranges of CPPs were defined to ensure both product CQAs and process stability. By studying the fully integrated continuous manufacturing line, it was possible to highlight the interactions between the different unit operations and the API CMAs and to design a robust process.

Managing API raw material variability during continuous twin-screw wet granulation.

Very few studies have investigated the impact of raw material variability upon the granule critical quality attributes (CQAs) produced via twin-screw wet granulation (i.e., granule size distribution, density, flowability). In this study, the impact of the raw material variability of an active pharmaceutical ingredient (API) in a high dose formulation on the twin-screw wet granulation process and on the resulting granule quality attributes was investigated. In a previous study (Stauffer et al., 2018), eight API batches were characterized to determine the API batch-to-batch variability. Principal component analysis (PCA) was then used to analyse the raw material property differences between the API batches and to determine the causes of the batch-to-batch variability. In current study, the three principal components from that PCA model were used as factors together with twin-screw granulation process parameters (i.e., screw speed and liquid-to-solid ratio) in a D-optimal screening design of experiments to understand the influence of these factors upon the granule CQAs. It was found that the API particle size distribution and related properties (e.g., density, agglomeration profile) were critical for the granule CQAs. In a next step, the significant factors from the screening design results were used to determine the design space of the twin-screw granulation process for the studied formulation via a D-optimal optimisation design, herewith controlling the risk of failure for the potential API raw material variability. The possibility to obtain suitable granule CQAs with a risk of failure of 1% for all API batches was demonstrated. It was thus possible to identify a combination of process parameters that can manage the API batch-to-batch variability leading to granules with pre-defined suitable CQAs.

Managing active pharmaceutical ingredient raw material variability during twin-screw blend feeding.

Continuous powder feeding is a critical step in continuous manufacturing of solid dosage forms, as this unit operation should ensure the mass flow consistency at the desired powder feed rate to guarantee the process throughput and final product consistency. In this study, twin-screw feeding of a pharmaceutical formulation (i.e., blend) existing of a highly dosed very poorly flowing active pharmaceutical ingredient (API) leading to insufficient feeding capacity was investigated. Furthermore, the API showed very high batch-to-batch variability in raw material properties dominating the formulation blend properties. Formulation changes were evaluated to improve the flowability of the blends and to mitigate the impact of API batch-to-batch variability on the twin-screw feeding. Herewith, feeding evaluation tests and an extensive material characterization of the reformulated blends were performed to assess the impact of the formulation changes upon continuous twin-screw feeding. The transfer of the glidant from extra-granular to intra-granular phase allowed to improve the flowability of the blends. A sufficient feeding capacity for the downstream process and a mitigation of the impact of batch-to-batch variability of the API upon twin-screw feeding of the blends could be achieved. No effect of the formulation or of the API properties on the feeding stability was observed. The material characterization of the blends allowed identifying the material attributes which were critical for continuous twin-screw feeding (i.e., bulk density, mass charge and powder cohesiveness).

Raw material variability of an active pharmaceutical ingredient and its relevance for processability in secondary continuous pharmaceutical manufacturing.

Active Pharmaceutical Ingredients (API) raw material variability is not always thoroughly considered during pharmaceutical process development, mainly due to low quantities of drug substance available. However, synthesis, crystallization routes and production sites evolve during product development and product life cycle leading to changes in physical material attributes which can potentially affect their processability. Recent literature highlights the need for a global approach to understand the link between material synthesis, material variability, process and product quality. The study described in this article aims at explaining the raw material variability of an API using extensive material characterization on a restricted number of representative batches using multivariate data analysis. It is part of a larger investigation trying to link the API drug substance manufacturing process, the resulting physical API raw material attributes and the drug product continuous manufacturing process. Eight API batches produced using different synthetic routes, crystallization, drying, delumping processes and processing equipment were characterized, extensively. Seventeen properties from seven characterization techniques were retained for further analysis using Principal Component Analysis (PCA). Three principal components (PCs) were sufficient to explain 92.9% of the API raw material variability. The first PC was related to crystal length, agglomerate size and fraction, flowability and electrostatic charging. The second PC was driven by the span of the particle size distribution and the agglomerates strength. The third PC was related to surface energy. Additionally, the PCA allowed to summarize the API batch-to-batch variability in only three PCs which can be used in future drug product development studies to quantitatively evaluate the impact of the API raw material variability upon the drug product process. The approach described in this article could be applied to any other compound which is prone to batch-to-batch variability.

The use of partially hydrolysed polyvinyl alcohol for the production of high drug-loaded sustained release pellets via extrusion-spheronisation and coating: In vitro and in vivo evaluation.

Partially hydrolysed polyvinyl alcohol (PVA) was evaluated as a pelletisation aid for the production of pellets with a high acetaminophen and metformin hydrochloride concentration (>70%, w/w). Mixtures with varying drug concentration and PVA/microcrystalline cellulose (MCC) ratios were processed via extrusion-spheronisation, either after addition of PVA as a dry powder or as an aqueous solution. Finally, high drug- loaded metformin pellets were coated with a methacrylic acid copolymer (Eudragit™ NM 30D) and evaluated for their sustained release potency in vitro and in vivo. The plasticity index of the wet mass increased by the addition of PVA to the formulation, which resulted in enhanced extrusion-spheronisation properties, even at a high drug load. Although the MCC concentration was successfully lowered by adding PVA, the inclusion of MCC in the formulation was essential to overcome problems related to the tackiness effect of PVA during extrusion. Overall, wet addition of PVA was superior to dry addition, as pellets with a higher mechanical strength and narrower particle size distribution were obtained. Pellets containing 87% (w/w) metformin hydrochloride were successfully layered with 20% (w/w) coating material, yielding sustained release pellets with a final drug load of 70% (w/w). In addition, the sustained release characteristics of the PVA-based pellets with a high drug content were confirmed in vivo as no difference with the Glucophage™ SR reference formulation was observed.

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors.

The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.

The dermatoscope: a potential source of nosocomial infection?

Dermatoscopes are directly placed on the skin of patients and therefore may harbour potentially pathogenic bacteria. In this study we assessed bacterial contamination of dermatoscopes during routine use and examined the effectiveness of isopropyl alcohol and disposable lens covers. Aerobic bacterial cultures were performed on randomly selected dermatoscopes after examination of 39 patients. In addition, bacterial contamination of dermatoscopes after cleaning with 70% isopropyl alcohol and after using a disposable transparent lens cover was tested. Bacteria such as Staphylococcus epidermidis, Micrococcus species and Corynebacterium species, which are usually present on the skin, were repeatedly identified. In addition, rare bacteria such as Moraxella species were found. Known nosocomial micro-organisms such as Staphylococcus aureus were recovered from hospitalized patients. Disinfection with 70% isopropyl alcohol and the use of a disposable lens cover significantly reduced the number of bacteria. Image quality was identical with and without the use of a transparent lens cover. Dermatoscopes harbour potential pathogens and may be a source of bacterial transmission. Isopropyl alcohol is an effective cleaning agent for dermatoscopes. Disposable lens covers completely prevented bacterial transmission and are therefore recommended for routine dermatoscopy, especially in hospitalized patients.

Microbiological irrigation water quality of the Marchfeld canal system.

The Marchfeld basin with a size of approximately 1000 km2 represents the Austrian "granary". To prevent shortage of water as a result of increased ground water removal for irrigation, industrial purposes and drinking water supply, a canal being 18 km in length was constructed from the Danube to the center of the Marchfeld. From there, water is further distributed via two creeks (Russbach and Stempfelbach). This study was intended to evaluate whether the surface water of the Marchfeld canal system can be classified into hygienic-microbiological categories as proposed by DIN (Deutsche Industrienorm) standards for irrigation water. For this purpose, water sampled monthly from three different sampling sites from 1996 to 1999 was examined for E. coli and enterococci. In addition, water samples were examined for salmonella twice a year from 1996 to 1998 and for cryptosporidia six times during the year 1999. Though the water showed varying degrees of fecal load, the results of the examinations revealed that only one of the three sampling sites showed constant quality levels according to the DIN classification system over prolonged periods of time. However, exceeding of the limit values was occasionally observed indicating the need for regular bacteriological examinations. The high variation of the results from the other sampling sites hardly permits a definite classification in one of the quality classes.

Mycobacterium xenopi infection of a 50-year-old oil plombage complicated by bronchopleural and pleurocutaneous fistulas.

We report the rare combination of simultaneous bronchopleural and pleurocutaneous fistulas 50 years after oil plombage, together with infection of both the plombage and the contralateral lung with Mycobacterium xenopi. Our case documents imaging patterns of complex fistula formation and subsequent infection resulting from oil plombage. Our case also emphasizes the infectious potential of Mycobacterium xenopi.

Radiographic findings in patients with acquired immunodeficiency syndrome, pulmonary infection, and microbiologic evidence of Mycobacterium xenopi.

The authors studied radiographs and clinical histories of 29 patients with acquired immunodeficiency syndrome, symptoms of pulmonary infection, and simultaneous microbiologic evidence of Mycobacterium xenopi in the respiratory tract. The presence, nature, and distribution of radiographic abnormalities were determined and analyzed in accord with clinical information. In 26 (90%) patients, M. xenopi was the only microorganism that could be isolated. Chest radiographs were normal in 13 patients (45%) and abnormal in 16 patients (55%). Radiographic abnormalities were bilateral in 94% of cases and predominantly involved the lower lobes. Patchy peribronchial opacities (44%) and miliary nodules (24%) were the most common abnormalities. Reticular opacities and parenchymal consolidation were seen in 12% of patients. Pleural effusion was seen in 18% of patients. No patients had cavitations or adenopathy. There was no statistically significant difference regarding the mean age (38.7+/-7.3 years vs. 40.2+/-11.0 years), the duration of clinically evident human immunodeficiency virus infection (2.7+/-1.2 years vs. 2.8+/-1.4 years), and the mean of CD4 cell counts (50.6+/-15.3 cells/ml vs. 47.4+/-15.9 cells/ml) between the patients with and without abnormalities on chest radiographs. In patients with acquired immunodeficiency syndrome, pulmonary infection, and simultaneous microbiologic evidence of M. xenopi, chest radiographs can be normal in a substantial number of cases. When radiographic abnormalities are present, they differ from those seen in patient not infected with the human immunodeficiency virus who had pulmonary infection caused by M. xenopi and from patients with acquired immunodeficiency syndrome and pulmonary infection with nontuberculous mycobacteria other than M. xenopi. Although these findings are not specific, they may be of importance in the imaging of patients with acquired immunodeficiency syndrome, notably in areas where M. xenopi is endemic.

[The resistance status of Mycobacterium tuberculosis in Austria 1992 and 1993]. / Resistenzsituation von Mycobacterium tuberculosis in Ostösterreich in den Jahren 1992 und 1993.

Tuberculosis caused by resistant Mycobacterium tuberculosis strains seems to be of increasing importance in some parts of the world. Aim of this study was to investigate the frequency and nature of the resistance occurring among the strains isolated in Eastern Austria in 1992 and 1993. 7.4% of all strains isolated from 608 patients in 1992 and 6.2% of those isolated from 497 patients in 1993 were resistant to one or more tuberculostatic drugs, 2.3% in 1992 and 3.0% in 1993 of the strains were resistant to isoniazid, 0.5 and < 0.1 respectively, to rifampicin. Multidrug-resistant strains were found in 2.8% of the isolates in 1992 and 2.2% in 1993.

Implementation of a molecular typing system to support epidemiological investigations in the tuberculosis health care system in Vienna.

Tuberculosis continues to be one of the predominant infectious diseases. Effective control of its spread requires that sources of infection and routes of transmission be disclosed as quickly as possible. At present such investigations are still performed by conventional epidemiological methods. In the recent past, however, molecular typing systems were added to the spectrum of epidemiological tools. Unfortunately, they were applied to retrospective investigations rather than used as an aid in the health care system. In this study, 515 Mycobacterium tuberculosis strains isolated during 1997 and 1998 in Vienna were analysed by spoligotyping, a molecular technique requiring no further cultivation of mycobacteria. The study was aimed to assess the suitability of the method as a quick means of disclosing new cases. Thus, clusters obtained by spoligotyping were analysed along with demographic and epidemiological data and compared with clusters obtained by conventional epidemiological techniques alone. In addition, spoligotype-forming clusters were matched with an international database containing spoligotypes from four different studies. Of 515 isolates, 107 showed an unique pattern. The remaining 408 isolates were distributed into two large clusters of 82 and 73 isolates and into 49 smaller ones consisting of 2 to 33 isolates each. The two spoligotypes forming the large clusters were identical with the most prevalent spoligotypes in the world. Therefore, for the tuberculosis authorities, information was only gained by excluding rather than tracing possible ways of transmission. Twenty-two of the 49 spoligotypes forming smaller clusters were identical with strains found in other parts of the world. Seventeen of 22 infection chains assumed by conventional investigations were confirmed by spoligotyping. In small clusters, an additional 24 infections were assumed due to similarities such as living conditions or socioeconomic status. In 27 clusters, all patients sharing the same strain belong to the same country or geographical area. In conclusion, spoligotyping proved suitable as an early guide in conventional investigations to trace routes of M. tuberculosis transmission in a community. However, when a strain isolated from a patient belongs to a spoligotype shared by many isolates, a second molecular typing method is required.

Mycobacteria other than tuberculosis with an emphasis on Mycobacterium xenopi in clinical specimens from AIDS patients at the University Hospital of Vienna from 1989 to 1996.

This study was carried out to provide an overview of the frequency of various mycobacterial species isolated from AIDS patients at the University Hospital of Vienna from 1989 to 1996. Mycobacterium xenopi was found to be the second most common nontuberculous mycobacterial species (92 specimens from 30 patients) and was cultured predominantly from respiratory tract specimens. In 55% of patients, chest X-rays taken at the time of isolation demonstrated pathologic changes which could not be attributed to another cause. Therefore, according to our results, Mycobacterium xenopi should be viewed as an infectious agent rather than a contaminant in AIDS patients.

The role of prior model calibration on predictions with ensemble Kalman filter.

This paper, based on a real world case study (Limmat aquifer, Switzerland), compares inverse groundwater flow models calibrated with specified numbers of monitoring head locations. These models are updated in real time with the ensemble Kalman filter (EnKF) and the prediction improvement is assessed in relation to the amount of monitoring locations used for calibration and updating. The prediction errors of the models calibrated in transient state are smaller if the amount of monitoring locations used for the calibration is larger. For highly dynamic groundwater flow systems a transient calibration is recommended as a model calibrated in steady state can lead to worse results than a noncalibrated model with a well-chosen uniform conductivity. The model predictions can be improved further with the assimilation of new measurement data from on-line sensors with the EnKF. Within all the studied models the reduction of 1-day hydraulic head prediction error (in terms of mean absolute error [MAE]) with EnKF lies between 31% (assimilation of head data from 5 locations) and 72% (assimilation of head data from 85 locations). The largest prediction improvements are expected for models that were calibrated with only a limited amount of historical information. It is worthwhile to update the model even with few monitoring locations as it seems that the error reduction with EnKF decreases exponentially with the amount of monitoring locations used. These results prove the feasibility of data assimilation with EnKF also for a real world case and show that improved predictions of groundwater levels can be obtained.

Viral inactivation based on inhibition of membrane fusion: understanding the role of histidine protonation to develop new viral vaccines.

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells, what makes it a potentially attractive target for viral inactivation approaches. Fusion is mediated by viral surface glycoproteins that undergo conformational changes triggered by interaction with specific cellular receptors or by the exposition to low pH of endossomal medium. Here we review how several studies on the structural rearrangements of vesicular stomatitis virus (VSV) glycoprotein G during cellular recognition and fusion led us to propose a crucial role of the protonation of His residues for G protein activity. Moreover, we demonstrated that using diethylpyrocarbonate (DEPC), a histidine-modifying compound, it was possible to abolish viral infectivity and pathogenicity in mice, and to elicit neutralizing antibodies that confer protection in these animals against challenge using lethal doses of the virus. The presence of conserved His residues in a wide range of viral fusion proteins and the use of DEPC as a more general means for vaccine development will be also discussed.