A high yield multi-method extraction protocol for protein quantification in activated sludge

A multi-method extraction protocol based on mechanical, ionic and hydrophobic methods was investigated on two types of activated sludge samples. Extraction methods were chosen with regards to optimal protein yield without cell disruption. Sonication, EDTA and Tween extraction methods were selected and combined. The total amount of protein released by the multi-method protocol sums up to 191 and 264 mg equiv. BSA/g VSS for the two different sludge samples. Protocol repetition on the same sample showed that protein yield after each successive protocol fitted an exponential curve model. The total amount of extractable proteins was evaluated by model predictions, 423 and 516 mg equiv. BSA/g VSS for the two sludge samples. The multi-method extraction protocol appears relevant for harvesting a representative quantity of proteins from the original sample (45-49%), moreover the multi-method criterion of the protocol also offers a heterogeneous pool of proteins. Thus, further qualitative studies may not be biased by the extraction protocol.     

Conserved glutamate residues are critically involved in Na+/nucleoside cotransport by human concentrative nucleoside transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.     

Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.     

Rapid growth rates of aerobic anoxygenic phototrophs in the ocean

We analysed bacteriochlorophyll diel changes to assess growth rates of aerobic anoxygenic phototrophs in the euphotic zone across the Atlantic Ocean. The survey performed during Atlantic Meridional Transect cruise 16 has shown that bacteriochlorophyll in the North Atlantic Gyre cycles at rates of 0.91-1.08 day(-1) and in the South Atlantic at rates of 0.72-0.89 day(-1). In contrast, in the more productive equatorial region and North Atlantic it cycled at rates of up to 2.13 day(-1). These results suggest that bacteriochlorophyll-containing bacteria in the euphotic zone of the oligotrophic gyres grow at rates of about one division per day and in the more productive regions up to three divisions per day. This is in striking contrast with the relatively slow growth rates of the total bacterial community. Thus, aerobic anoxygenic phototrophs appear to be a very dynamic part of the marine microbial community and due to their rapid growth, they are likely to be larger sinks for dissolved organic matter than their abundance alone would predict.     

A baboon model for in vivo assessment of mucociliary lung clearance

A suitable baboon model (Papio ursinus) for assessing inhibitory effects on mucociliary lung clearance was required. Clearance of various dimensions of nebulized particles (99mTc-labelled) was monitored with the animals (n = 6) under either ketamine or pentobarbitone anaesthesia. The best prospect of substantial and reproducible clearance in spite of the inhibition by the anaesthesia were obtained with pentobarbitone, and using nebulized radiolabelled particles of diameter range between 10 and 45 microns, thus avoiding trapping in the non-ciliary alveoli.     

13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-¹³C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a ¹³C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of ¹³C-labeled primary metabolites are reported for P. chrysogenum and used for a ¹³C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h⁻¹ yielded comparable values for the gluconate tracer method and the ¹³C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the ¹³C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the ¹³C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Using a multiple-delivery-mode training approach to develop local capacity and infrastructure for advanced bioinformatics in Africa

With more microbiome studies being conducted by African-based research groups, there is an increasing demand for knowledge and skills in the design and analysis of microbiome studies and data. However, high-quality bioinformatics courses are often impeded by differences in computational environments, complicated software stacks, numerous dependencies, and versions of bioinformatics tools along with a lack of local computational infrastructure and expertise. To address this, H3ABioNet developed a 16S rRNA Microbiome Intermediate Bioinformatics Training course, extending its remote classroom model. The course was developed alongside experienced microbiome researchers, bioinformaticians, and systems administrators, who identified key topics to address. Development of containerised workflows has previously been undertaken by H3ABioNet, and Singularity containers were used here to enable the deployment of a standard replicable software stack across different hosting sites. The pilot ran successfully in 2019 across 23 sites registered in 11 African countries, with more than 200 participants formally enrolled and 106 volunteer staff for onsite support. The pulling, running, and testing of the containers, software, and analyses on various clusters were performed prior to the start of the course by hosting classrooms. The containers allowed the replication of analyses and results across all participating classrooms running a cluster and remained available posttraining ensuring analyses could be repeated on real data. Participants thus received the opportunity to analyse their own data, while local staff were trained and supported by experienced experts, increasing local capacity for ongoing research support. This provides a model for delivering topic-specific bioinformatics courses across Africa and other remote/low-resourced regions which overcomes barriers such as inadequate infrastructures, geographical distance, and access to expertise and educational materials.