Challenges and promise at the interface of metaproteomics and genomics: an overview of recent progress in metaproteogenomic data analysis

Introduction: The study of microbial communities based on the combined analysis of genomic and proteomic data – called metaproteogenomics – has gained increased research attention in recent years. This relatively young field aims to elucidate the functional and taxonomic interplay of proteins in microbiomes and its implications on human health and the environment.

Areas covered: This article reviews bioinformatics methods and software tools dedicated to the analysis of data from metaproteomics and metaproteogenomics experiments. In particular, it focuses on the creation of tailored protein sequence databases, on the optimal use of database search algorithms including methods of error rate estimation, and finally on taxonomic and functional annotation of peptide and protein identifications.

Expert opinion: Recently, various promising strategies and software tools have been proposed for handling typical data analysis issues in metaproteomics. However, severe challenges remain that are highlighted and discussed in this article; these include: (i) robust false-positive assessment of peptide and protein identifications, (ii) complex protein inference against a background of highly redundant data, (iii) taxonomic and functional post-processing of identification data, and finally, (iv) the assessment and provision of metrics and tools for quantitative analysis.     

Targeted disruption of the mouse gene encoding the V-ATPase accessory subunit Ac45

Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptor-mediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H(+)-ATPase (V-ATPase). How the activity of this multisubunit enzyme is regulated remains to be established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function of Ac45, we disrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (-IY) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affect the normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.     

Oxygen concentration profiles in sediments of two ancient lakes: Lake Baikal (Siberia,Russia) and Lake Malawi (East Africa)

Oxygen concentration profiles have been measured, by means of with microelectrodes in sediments of Lake Baikal and Lake Malawi, along transects allowing to give a survey of two major ancient Rift lakes: Lake Baikal (Eastern Siberia) and Lake Malawi (East Africa), along depth transects in the constitutive basins of the lakes and/or of relevant depths with regard to oxygen (including including the deepest point, 1680 m, in Lake Baikal). Sediment oxygen penetration depths (SOPs) display very different patterns, depending on the lake in the two lakes. In Lake Baikal, SOPs are variable, show no significant relationship with bathymetric depth and are surprisingly deep on Akademichesky ridge (> 50.0 mm), emphasizing the distinctive feature of this region in the lake. While the Selenga river is an important source of eutrophication, the similarity of SOP-values in the Selenga shallow with those of most other sites suggests either a dilution of organic material by allochthonous matter, or a strong south-to-north transport of particles. In Lake Malawi, available oxygen is restricted to a maximum of three millimetres of the sediment, and there is a negative relationship with bathymetric depth, as a result of a steady decline of oxygen concentration with depth through the water column. Amongst the few parameters known to affect SOPs, the oxygen consumption by the sediment seems the most significant in both lakes. SOP-values furthermore confirm differences in the trophic status of Baikal and Malawi, respectively. The importance of oxygen as a factor likely to create ecological segregation for benthic organisms is discussed. Lake Malawi offers possibilities of bathymetric segregation but no vertical segregation in the sediment. In contrast, no bathymetric segregation related to oxygen is possible in Lake Baikal, but vertical segregation in the sediment is very likely. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO

The effect of three different nutrient depletions (nitrogen, sulphur and magnesium) on the growth and pigment accumulation of the haptophyte Isochrysis aff. galbana (clone T-ISO) has been studied. Pigments were quantified based on RP-UHPLC-PDA-MSⁿ analysis. All nutrient depletions led to reduced maximal biomass concentrations. Besides, all nutrient-depleted cultures accumulated 3-hydroxyechinenone. To our knowledge, this is the first time that 3-hydroxyechinenone has been found in I. aff. galbana T-ISO. Most 3-hydroxyechinenone, as well as the most echinenone and diatoxanthin, were found in the nitrogen-limited culture in which a more severe limitation resulted in higher cellular contents. Similar to accumulation of diatoxanthin, accumulation of 3-hydroxyechinenone and echinenone may be part of a global (stress) response mechanism to oversaturating light conditions.     

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.     

Effects of dietary fibre and protein on urea transport across the cecal mucosa of piglets

In ruminants, gastrointestinal recycling of urea is acutely enhanced by fibre-rich diets that lead to high ruminal concentration of short chain fatty acids (SCFA), while high ammonia has inhibitory effects. This study attempted to clarify if urea flux to the porcine cecum is similarly regulated. Thirty-two weaned piglets were fed diets containing protein (P) of poor prececal digestibility and fibre (F) at high (H) or low levels (L) in a 2 × 2 factorial design. After slaughter, cecal content was analyzed and the cecal mucosa incubated in Ussing chambers to measure the effect of pH, SCFA and NH₄ ⁺ on the flux rates of urea, short-circuit current (I _sc) and tissue conductance (G _t). NH₄ ⁺ significantly enhanced I _sc (from 0.5 ± 0.2 to 1.2 ± 0.1 μEq cm^?2 h^?1). No acute effects of SCFA or ammonia on urea flux were observed. Tissue conductance was significantly lower in the high dietary fibre groups irrespective of the protein content. Only the HP-LF group emerged as different from all others in terms of urea flux (74 ± 6 versus 53 ± 3 nmol cm^?2 h^?1), associated with higher cecal ammonia concentration and reduced fecal consistency. The data suggest that as in the rumen, uptake of ammonia by the cecum may involve electrogenic transport of the ionic form (NH₄ ⁺). In contrast to findings in the rumen, neither a high fibre diet nor acute addition of SCFA enhanced urea transport across the pig cecum. Instead, a HP-LF diet had stimulatory effects. A potential role for urea recycling in stabilizing luminal pH is discussed.     

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Downscaling Drug Nanosuspension Production: Processing Aspects and Physicochemical Characterization

In this study, scaling down nanosuspension production to 10 mg of drug compound and evaluation of the nanosuspensions to 1 mg of drug compound per test were investigated. Media milling of seven model drug compounds (cinnarizine–indomethacin–itraconazole–loviride–mebendazole–naproxen–phenytoin) was evaluated in a 96-well plate setup (10, 20, and 30 mg) and a glass-vial-based system in a planetary mill (10, 100, and 1,000 mg). Physicochemical properties evaluated on 1 mg of drug compound were drug content (high-performance liquid chromatography), size [dynamic light scattering (DLS)], morphology (scanning electron microscopy), thermal characteristics (differential scanning calorimetry), and X-ray powder diffraction (XRPD). Scaling down nanosuspension production to 10 mg of drug compound was feasible for the seven model compounds using both designs, the planetary mill design being more robust. Similar results were obtained for both designs upon milling 10 mg of drug compound. Drug content determination was precise and accurate. DLS was the method of choice for size measurements. Morphology evaluation and thermal analysis were feasible, although sample preparation had a big influence on the results. XRPD in capillary mode was successfully performed, both in the suspended state and after freeze-drying in the capillary. Results obtained for the latter were superior. Both the production and the physicochemical evaluation of nanosuspensions can be successfully downscaled, enabling nanosuspension screening applications in preclinical development settings.     

Antibodies raised against tobacco aquaporins of the PIP2 class label viscin tissue of the explosive dwarf mistletoe fruit

Dwarf mistletoes, genus Arceuthobium , are parasitic flowering plants and forest pests. In western North America, Arceuthobium americanum (lodgepole pine dwarf mistletoe) is principally found on Pinus contorta var. latifolia (lodgepole pine). Dwarf mistletoes disperse their seeds by an explosive process that involves the buildup of hydrostatic pressure within a mucilaginous fruit tissue called the 'viscin'. Living viscin tissue envelops the discharged seeds. This study examined the possibility that aquaporins, critical in plant water relations, might be found in the dwarf mistletoe fruit, specifically the viscin cells. An antibody raised against a tobacco plasma membrane intrinsic 2 (PIP2) aquaporin was used with a gold-labeled secondary antibody to probe dwarf mistletoe fruit at various developmental stages. Viscin cell plasma membranes were successfully labeled with the anti-tobacco probe, and the validity of the immunolabeling was supported by Western blot analysis, showing a strong signal at about 30 kDa, which is at the expected size of a PIP2. A definitive immunolabeling pattern, supported by quantification of gold signal per membrane length, was observed: viscin cells sampled early in development had abundant gold label at their plasma membranes (1.93 ± 0.13 to 2.13 ± 0.33 gold particles per μm membrane), while other areas of the cells had no discernible label. Viscin cells sampled near the time of explosive discharge had significantly less label at the plasma membrane (0.21 gold particles ± 0.11 per μm membrane, P   <   0.05), and label was seen at vesicular membranes. Aquaporins likely have a role in directing water to the viscin mucilage early in development, but are retrieved via endocytosis to prevent excess water loss from viscin cells when discharge is imminent.     

Tryptophan-dependent biosynthesis of auxins in soil

The presence of auxins in soil may have an ecological impact affecting plant growth and development. A rapid and simple colorimetric method was used to assess California soils for their potential to produce auxins upon the addition of L-tryptophan (L-TRP). The auxin content measured by colorimetry was expressed as indole-3-acetic acid (IAA)-equivalents. A substrate (L-TRP) concentration of 5.3 g kg^-1, glucose concentration of 6.7 g kg^-1, no nitrogen, pH 7.0, 40°C, shaking (aeration) and 48 h incubation time were selected as standardized conditions to assay for auxin biosynthesis in soil. IAA was confirmed as a major microbial metabolite derived from L-TRP in soil by use of high performance liquid chromatography (HPLC). Under standardized conditions, L-TRP-derived auxins in 19 soils varied greatly ranging from 18.2 to 303.2 mg IAA equivalents (auxins) kg^-1 soil. This study suggests that the phenotypic character of the soil microbiota has more of an influence on auxin production than the soil physicochemical properties (e.g., pH, organic C content, CEC, etc.).