A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental <Emphasis Type="Italic">Aeromonas</Emphasis> Isolates

The colonization rates of 10 different environmental Aeromonas isolates were determined using a novel mouse–streptomycin pretreatment method. As demonstrated, alterations to the colon flora of mice pretreated with streptomycin allowed transient colonization by bacterial species normally excluded by host competition. A novel procedure is described for determining the colonization abilities of Aeromonas isolates under these conditions. The colonization rates of A. salmonicida, A. encheleia, and A. allosaccharophila were either negative or occurred randomly at low levels with respect to concentrations of the dosage consumed by the animals. In contrast, A. hydrophila, A. veronii biovar sobria, and A. caviae exhibited relatively high rates of mouse colon tissue colonization.     

Leaf anatomical characteristics of Ugandan species of Festuca L. (Poaceae)

The importance of leaf anatomical characters in the taxonomy of Ugandan Festuca species was investigated. In particular, leaf cross sections were studied for variation in disposition of the sclerenchyma tissue to determine whether this is as taxonomically useful as is implied in much of the literature. Also the leaf epidermis was investigated for the presence of diagnostic features. A lot of variation in disposition of sclerenchyma tissue was found in F. abyssinica, which did not support the recognition of F. elgonensis and F. richardii as good species, yet they were described mainly based on leaf anatomy. The ring of sclerenchyma in F. pilgeri and the presence of papillae on the epidermis of F. claytonii supported them to be good species. The presence of silica bodies on the epidermis of Ugandan species of Festuca was investigated for the first time and is confirmed using energy dispersive X-ray analysis. In conclusion, leaf anatomy should be used together with other non-anatomical characters before recognizing species.     

Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination

A 1.5 μL ion exchange chromatography column to accommodate resins used for biopharmaceutical processing has been designed to produce breakthrough curves and to quantify dynamic and maximum protein binding capacities. Channels within a glass chip were fabricated using photolithography and isotropic etching. The design includes a 1 cm long microfluidic column in which compressible, polydispersed porous agarose beads (70 μm mean diameter) were packed using a keystone method where particles aggregate in a narrow channel. The depth of the column is such that two bead layers exist. The fabrication technique used forms Cartesian geometries as opposed to circular cross sections found in standard columns. The voidage was therefore higher than standard values when measured by 3D confocal microscopy. In conjunction with microscopic techniques, the column allows visualization of events within the bed such as adsorption profiles that would otherwise be difficult to observe. In this work, the binding of fluorescently labeled protein during isocratic loading was used to generate breakthrough from the microcolumn. Useful breakthrough curves were achieved using mobile phase velocities from 60 to 270 cm h^?1. Calculated dynamic binding capacities were compared well with previously published data on conventional scale columns. The microfluidic chromatography column described here thus allows study of process scale chromatography behavior at scales 20,000 times smaller than in current practice. The work described in this article is representative of the proof of principle of a potentially powerful tool for the generation of microfluidic process bed data for the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Microscale bioprocess optimisation

Microscale processing techniques offer the potential to speed up the delivery of new drugs to the market, reducing development costs and increasing patient benefit. These techniques have application across both the chemical and biopharmaceutical sectors. The approach involves the study of individual bioprocess operations at the microlitre scale using either microwell or microfluidic formats. In both cases the aim is to generate quantitative bioprocess information early on, so as to inform bioprocess design and speed translation to the manufacturing scale. Automation can enhance experimental throughput and will facilitate the parallel evaluation of competing biocatalyst and process options.     

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200‐μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His₆‐tagged enzymes via Ni‐NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop‐flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK‐catalysed synthesis of L ‐erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis–Menten model. Results show that the TK kinetic parameters in the IEMR (V_max(app) = 0.1 ± 0.02 mmol min^–1, K_m(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the k_cat for the microreactor of 4.1 × 10⁵ s^?1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His₆‐immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell‐based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of Prolonged Chlorine Exposures upon PCR Detection of Helicobacter pylori DNA

The effect of low doses of free chlorine on the detection of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. Detection of sequences targeted to the ureA gene from preparations containing 107 cells/ml decreased about 2-4 logs by days 9 and 14, respectively. When duplicate suspensions of the 107 cells/ml were exposed to higher levels of chlorine, 0.2-2.2 mg/l, by day 9 and 14 there were 5 and 6 log decreases, respectively, in the detection of ureA gene. H. pylori target sequences (within suspended, intact cells at densities of 102-103 cells /ml) were rendered undetectable by qPCR analysis after 17 h of continuous exposure to low chlorine levels common to treated drinking water distribution systems. The persistence of DNA sequences within treated distribution systems detectable by qPCR may be as brief as 17 h especially for bacteria such as H. pylori which are known to occur in very low numbers within treated distribution systems. This study suggests that degradation of H. pylori DNA target sequences by chlorine levels commonly found within treated water distribution systems occurs within the average water retention times (2-3 days) commonly found in these systems.     

Predictive Tools for Severe Dengue Conforming to World Health Organization 2009 Criteria

Background

Dengue causes 50 million infections per year, posing a large disease and economic burden in tropical and subtropical regions. Only a proportion of dengue cases require hospitalization, and predictive tools to triage dengue patients at greater risk of complications may optimize usage of limited healthcare resources. For severe dengue (SD), proposed by the World Health Organization (WHO) 2009 dengue guidelines, predictive tools are lacking.

Methods

We undertook a retrospective study of adult dengue patients in Tan Tock Seng Hospital, Singapore, from 2006 to 2008. Demographic, clinical and laboratory variables at presentation from dengue polymerase chain reaction-positive and serology-positive patients were used to predict the development of SD after hospitalization using generalized linear models (GLMs).

Principal findings

Predictive tools compatible with well-resourced and resource-limited settings – not requiring laboratory measurements – performed acceptably with optimism-corrected specificities of 29% and 27% respectively for 90% sensitivity. Higher risk of severe dengue (SD) was associated with female gender, lower than normal hematocrit level, abdominal distension, vomiting and fever on admission. Lower risk of SD was associated with more years of age (in a cohort with an interquartile range of 27–47 years of age), leucopenia and fever duration on admission. Among the warning signs proposed by WHO 2009, we found support for abdominal pain or tenderness and vomiting as predictors of combined forms of SD.

Conclusions

The application of these predictive tools in the clinical setting may reduce unnecessary admissions by 19% allowing the allocation of scarce public health resources to patients according to the severity of outcomes.     

Predictive Value of Proteinuria in Adult Dengue Severity

Background

Dengue is an important viral infection with different presentations. Predicting disease severity is important in triaging patients requiring hospital care. We aim to study the value of proteinuria in predicting the development of dengue hemorrhagic fever (DHF), utility of urine dipstick test as a rapid prognostic tool.

Methodology and principal findings

Adult patients with undifferentiated fever (n = 293) were prospectively enrolled at the Infectious Disease Research Clinic at Tan Tock Seng Hospital, Singapore from January to August 2012. Dengue infection was confirmed in 168 (57%) by dengue RT-PCR or NS1 antigen detection. Dengue cases had median fever duration of 6 days at enrolment. DHF was diagnosed in 34 cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger and were mostly seen in the outpatient setting with higher platelet level. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p<0.001). We obtained a UPCR cut-off value of 29 mg/mmol based on maximum AUC in ROC curves of peak UPCR for DF versus DHF, corresponding to 76% sensitivity and 60% specificity. Multivariate analysis with other readily available clinical and laboratory variables increased the AUC to 0.91 with 92% sensitivity and 80% specificity. Neither urine dipstick at initial presentation nor peak urine dipstick value during the entire illness was able to discriminate between DF and DHF.

Conclusions

Proteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.