Resuspension studies in cylindrical microcosms: Effects of stirring velocity on the dynamics of redox sensitive elements in a coastal sediment

Effects of resuspension on the release of dissolved, redox sensitive elements (Fe, Mn) was studied in cylindrical microcosms. Effects from changing water stirring velocity in sediment pools were evaluated through measurements of pore water profiles of dissolved Mn, Fe and redox potential. Mn was a good natural marker to follow such effects. At current velocities below the threshold velocity for resuspension (37 cm s-1), Mn release rates to overlying water were 100 times higher compared to steady-state values. Pulse increases in Mn concentration were the result of convective currents inside flow chambers. These results were strongly supported by measurements of Eh profiles in the sediment pore water. Furthermore, impacts from increasing stirring velocity were found down to 1.9 cm depth below the resuspended layer of sediment.     

Role of Mannose-Binding Lectin Deficiency in HIV-1 and Schistosoma Infections in a Rural Adult Population in Zimbabwe

Background

Polymorphism in the MBL2 gene lead to MBL deficiency, which has been shown to increase susceptibility to various bacterial, viral and parasitic infections. We assessed role of MBL deficiency in HIV-1 and schistosoma infections in Zimbabwean adults enrolled in the Mupfure Schistosomiasis and HIV Cohort (MUSH Cohort).

Methods

HIV-1, S. haematobium and S. mansoni infections were determined at baseline. Plasma MBL concentration was measured by ELISA and MBL2 genotypes determined by PCR. We calculated and compared the proportions of plasma MBL deficiency, MBL2 structural variant alleles B (codon 54A>G), C (codon 57A>G), and D (codon 52T>C) as well as MBL2 promoter variants -550(H/L), -221(X/Y) and +4(P/Q) between HIV-1 and schistosoma co-infection and control groups using Chi Square test.

Results

We assessed 379 adults, 80% females, median age (IQR) 30 (17–41) years. HIV-1, S. haematobium and S. mansoni prevalence were 26%, 43% and 18% respectively in the MUSH baseline survey. Median (IQR) plasma MBL concentration was 800μg/L (192-1936μg/L). Prevalence of plasma MBL deficiency was 18% with high frequency of the C (codon 57G>A) mutant allele (20%). There was no significant difference in median plasma MBL levels between HIV negative (912μg/L) and HIV positive (688μg/L), p = 0.066. However plasma MBL levels at the assay detection limit of 20μg/L were more frequent among the HIV-1 infected (p = 0.007). S. haematobium and S. mansoni infected participants had significantly higher MBL levels than uninfected. All MBL2 variants were not associated with HIV-1 infection but promoter variants LY and LL were significantly associated with S. haematobium infection.

Conclusion

Our data indicate high prevalence of MBL deficiency, no evidence of association between MBL deficiency and HIV-1 infection. However, lower plasma MBL levels were protective against both S. haematobium and S. mansoni infections and MBL2 promoter and variants LY and LL increased susceptibility to S. haematobium infection.     

Behavioural Effects of Tourism on Oceanic Common Dolphins,Delphinus sp., in New Zealand: The Effects of Markov Analysis Variations and Current Tour Operator Compliance with Regulations

Common dolphins, Delphinus sp., are one of the marine mammal species tourism operations in New Zealand focus on. While effects of cetacean-watching activities have previously been examined in coastal regions in New Zealand, this study is the first to investigate effects of commercial tourism and recreational vessels on common dolphins in an open oceanic habitat. Observations from both an independent research vessel and aboard commercial tour vessels operating off the central and east coast Bay of Plenty, North Island, New Zealand were used to assess dolphin behaviour and record the level of compliance by permitted commercial tour operators and private recreational vessels with New Zealand regulations. Dolphin behaviour was assessed using two different approaches to Markov chain analysis in order to examine variation of responses of dolphins to vessels. Results showed that, regardless of the variance in Markov methods, dolphin foraging behaviour was significantly altered by boat interactions. Dolphins spent less time foraging during interactions and took significantly longer to return to foraging once disrupted by vessel presence. This research raises concerns about the potential disruption to feeding, a biologically critical behaviour. This may be particularly important in an open oceanic habitat, where prey resources are typically widely dispersed and unpredictable in abundance. Furthermore, because tourism in this region focuses on common dolphins transiting between adjacent coastal locations, the potential for cumulative effects could exacerbate the local effects demonstrated in this study. While the overall level of compliance by commercial operators was relatively high, non-compliance to the regulations was observed with time restriction, number or speed of vessels interacting with dolphins not being respected. Additionally, prohibited swimming with calves did occur. The effects shown in this study should be carefully considered within conservation management plans, in order to reduce the risk of detrimental effects on common dolphins within the region.     

Short-term adaptation during propagation improves the performance of xylose-fermenting <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in simultaneous saccharification and co-fermentation

Background

Inhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation.

Results

The presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield.

Conclusions

Short-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

     

Pharmacokinetics of Isoniazid,Pyrazinamide, and Ethambutol in Newly Diagnosed Pulmonary TB Patients in Tanzania

Exposure to lower-than-therapeutic levels of anti-tuberculosis drugs is likely to cause selection of resistant strains of Mycobacterium tuberculosis and treatment failure. The first-line anti-tuberculosis (TB) regimen consists of rifampicin, isoniazid, pyrazinamide, and ethambutol, and correct management reduces risk of TB relapse and development of drug resistance. In this study we aimed to investigate the effect of standard of care plus nutritional supplementation versus standard care on the pharmacokinetics of isoniazid, pyrazinamide and ethambutol among sputum smear positive TB patients with and without HIV. In a clinical trial in 100 Tanzanian TB patients, with or without HIV infection, drug concentrations were determined at 1 week and 2 months post initiation of anti-TB medication. Data was analysed using population pharmacokinetic modelling. The effect of body size was described using allometric scaling, and the effects of nutritional supplementation, HIV, age, sex, CD4+ count, weight-adjusted dose, NAT2 genotype, and time on TB treatment were investigated. The kinetics of all drugs was well characterised using first-order elimination and transit compartment absorption, with isoniazid and ethambutol described by two-compartment disposition models, and pyrazinamide by a one-compartment model. Patients with a slow NAT2 genotype had higher isoniazid exposure and a lower estimate of oral clearance (15.5 L/h) than rapid/intermediate NAT2 genotype (26.1 L/h). Pyrazinamide clearance had an estimated typical value of 3.32 L/h, and it was found to increase with time on treatment, with a 16.3% increase after the first 2 months of anti-TB treatment. The typical clearance of ethambutol was estimated to be 40.7 L/h, and was found to decrease with age, at a rate of 1.41% per year. Neither HIV status nor nutritional supplementations were found to affect the pharmacokinetics of these drugs in our cohort of patients.     

Accurate Protein Complex Retrieval by Affinity Enrichment Mass Spectrometry (AE-MS) Rather than Affinity Purification Mass Spectrometry (AP-MS)

Protein–protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein–protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.Protein–protein interactions are key to protein-mediated biological processes and influence all aspects of life. Therefore, considerable efforts have been dedicated to the mapping of protein–protein interactions. A classical experimental approach consists of co-immunoprecipitation of protein complexes combined with SDS-PAGE followed by Western blotting to identify complex members. More recently, high-throughput techniques have been introduced; among these affinity purification-mass spectrometry (AP-MS)¹ (1–3) and the yeast two-hybrid (Y2H) approach (4–6) are the most prominent. AP-MS, in particular, has great potential for detecting functional interactions under near-physiological conditions, and has already been employed for interactome mapping in several organisms (7–15). Various AP-MS approaches have evolved over time, that differ in expression, tagging, and affinity purification of the bait protein; fractionation, LC-MS measurement, and quantification of the sample; and in data analysis. Recent progress in the AP-MS field has been driven by two factors: A new generation of mass spectrometers (16) providing higher sequencing speed, sensitivity, and mass accuracy, and the development of quantitative MS strategies.In the early days of AP-MS, tagged bait proteins were mostly overexpressed, enhancing their recovery in the pull-down. However, overexpression comes at the cost of obscuring the true situation in the cell, potentially leading to the detection of false interactions (17). Today, increased MS instrument power helps in the detection of bait proteins and interactors expressed at endogenous levels, augmenting the chances to detect functional interactions. In some simple organisms like yeast, genes of interest can directly be tagged in their genetic loci and expressed under their native promoter. In higher organisms, tagging proteins in their endogenous locus is more challenging, but also for mammalian cells, methods for close to endogenous expression are available. For instance, in controlled inducible expression systems, the concentration of the tagged bait protein can be titrated to close to endogenous levels (18). A very powerful approach is BAC transgenomics (19), as used in our QUBIC protocol (20), where a bacterial artificial chromosome (BAC) containing a tagged version of the gene of interest including all regulatory sequences and the natural promoter is stably transfected into a host cell line.The affinity purification step has also been subject to substantial changes over time. Previously, AP has been combined with nonquantitative MS as the readout, meaning all proteins identified by MS were considered potential interactors. Therefore, to reduce co-purifying “contaminants,” stringent two-step AP protocols using dual affinity tags like the TAP-tag (21) had to be employed. However, such stringent and multistep protocols can result in the loss of weak or transient interactors (3), whereas laborious and partially subjective filtering still has to be applied to clean up the list of identified proteins. The introduction of quantitative mass spectrometry (22–25) to the interactomics field about ten years ago was a paradigm shift, as it offered a proper way of dealing with unspecific binding and true interactors could be directly distinguished from background binders (26, 27). Importantly, quantification enables the detection of true interactors even under low-stringent conditions (28). In turn, this allowed the return to single-step AP protocols, which are milder and faster, and hence more suitable for detecting weak and transient interactors.Despite these advances, nonquantitative methods—often in combination with the TAP-tagging approach—are still popular and widely used, presumably because of reagent expenses and labeling protocols used in label-based approaches. However, there are ways to determine relative protein abundances in a label-free format. A simple, semiquantitative label-free way to estimate protein abundance is spectral counting (29). Another relative label-free quantification strategy is based on peptide intensities (30). In recent years high resolution MS has become much more widely accessible and there has been great progress in intensity-based label-free quantification (LFQ) approaches. Together with development of sophisticated LFQ algorithms, this has boosted obtainable accuracy. Intensity-based LFQ now offers a viable and cost-effective alternative to label-based methods in most applications (31). The potential of intensity-based LFQ approaches as tools for investigating protein–protein interactions has already been demonstrated by us (20, 32, 33) and others (34, 35). We have further refined intensity-based LFQ in the context of the MaxQuant framework (36) using sophisticated normalization algorithms, achieving excellent accuracy and robustness of the measured “MaxLFQ” intensities (37).Another important advance in AP-MS, again enabled by increased MS instrument power, was the development of single-shot LC-MS methods with comprehensive coverage. Instead of extensive fractionation, which was previously needed to reduce sample complexity, nowadays even entire model proteomes can be measured in single LC-MS runs (38). The protein mixture resulting from pull-downs is naturally of lower complexity compared with the entire proteome. Therefore, modern MS obviates the need for gel-based (or other) fractionation and samples can be analyzed in single runs. Apart from avoiding selection of gel bands by visual examination, this has many advantages, including decreased sample preparation and measurement time, increased sensitivity, and higher quantitative accuracy in a label-free format.In this work, we build on many of the recent advances in the field to establish a state of the art LFQ AE-MS method. Based on our previous QUBIC pipeline (20), we developed an approach for investigating protein–protein interactions, which we exemplify in Saccharomyces cerevisiae. We extended the data analysis pipeline to extract the wealth of information contained in the LFQ data, by establishing a novel concept that specifically makes use of the signature of background binders instead of eliminating them from the data set. The large amount of unspecific binders detected in our experiments rendered the use of a classic untagged control strain unnecessary and enabled comparing to a control group consisting of many unrelated pull-downs instead. Our protocol is generic, practical, and fast, uses low input amounts, and identifies interactors with high confidence. We propose that single-step pull-down experiments, especially when coupled to high-sensitivity MS, should now be regarded as affinity enrichment rather than affinity purification methods.