PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3-V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

rRNA and poly-beta-hydroxybutyrate dynamics in bioreactors subjected to feast and famine cycles

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-beta-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.     

Mycobacterium avium Infections of Acanthamoeba Strains: Host Strain Variability,Grazing-Acquired Infections,and Altered Dynamics of Inactivation with Monochloramine

Stable Mycobacterium avium infections of several Acanthamoeba strains were characterized by increased infection resistance of recent environmental isolates and reduced infectivity in the presence of other bacteria. Exposure of M. avium in coculture with Acanthamoeba castellanii to monochloramine yielded inactivation kinetics markedly similar to those observed for A. castellanii alone.Acanthamoebae are widely distributed in the environment (20) and generally function ecologically as predators of bacteria (23), although numerous types of bacteria resist predation (22). Acanthamoebae are very resistant to a range of disinfectants (5, 6, 8, 28), and bacteria within acanthamoebae are generally afforded extra protection (16). A notable example is the opportunistic pathogen Mycobacterium avium (10), which can survive within Acanthamoeba species trophozoites and cysts (4, 26), resulting in increased resistance to several antimicrobials (22). It has been demonstrated that many Mycobacterium spp. are able to infect the laboratory strain Acanthamoeba polyphaga (1). Acanthamoeba cultures undergo many physiological changes after several passages in the laboratory (15, 17, 21), although it is not known if prolonged cultivation of Acanthamoeba alters their capacity to be infected by M. avium. This knowledge is important for assessing the environmental relevance of associations between Acanthamoeba and M. avium. Therefore, we studied the infectivity and infection stability of M. avium with several laboratory and environmental Acanthamoeba strains for 28 days under high-nutrient (peptone-yeast extract-glucose [PYG] medium) and low-nutrient (Page''s amoeba saline [PAS]) conditions.     

Quantitative Proteomics and Metabolomics Analysis of Normal Human Cerebrospinal Fluid Samples*

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.The analysis of CSF¹ is indispensable in the diagnosis and understanding of various neurodegenerative CNS disorders (1–3). CSF is a fluid that has different functions, such as the protection of the brain from outside forces, transport of biological substances, and excretion of toxic and waste substances. It is in close contact with the extracellular fluid of the brain. Therefore, the composition of CSF can reflect biological processes of the brain (4). By discovering the characterization of the proteome and metabolome of CSF we may gain better insight on the pathogenesis of CNS disorders. This would be significant because, for many of these disorders, the etiology is still unclear.CSF is produced in the ventricles of the brain and in the subarachnoidal spaces. Humans normally produce around 500 mL of CSF each day, and the total volume of CSF at a given time is approximately 150 mL. CSF reflects the composition of blood plasma, although the concentrations of most proteins and metabolites in CSF are lower. However, individual proteins and metabolites can act differently. Active transport from blood and secretion from the brain contribute to the specific composition of CSF. This composition can be disturbed in neurological disorders (5–6). Since CNS-specific proteins and metabolites are typically low in abundance compared with their levels in blood, this change in composition is more likely to be found in CSF because in blood the more abundant plasma proteins can completely mask the signal of the less abundant proteins. Also, if the disease markers do not cross the blood-brain-barrier, then the CSF is the only viable biofluid source. Therefore, CSF might be an excellent source for biomarker discovery for CNS disorders if we follow the hypothesis that neurological diseases induce alterations in CSF protein and metabolite levels.Analysis of metabolites in CSF has been common practice in clinical chemistry for decades to analyze biomarkers for inborn errors of metabolism. The approaches used are either metabolite profiling of CSF using NMR (7), or targeted analysis of one or a few metabolites using specific analytical methods (8). Metabolomics includes the analysis of metabolites in biofluids by NMR or MS-based approaches, i.e. LC-MS or GC-MS. Several metabolite profiling studies were performed on CSF using NMR, some of which were published only recently (9,10). Surprisingly, very few metabolomics studies using MS-based methods have been performed on CSF to date (11,12). One of the reasons is the fact that the human CSF metabolome has not yet been characterized very well. Many CSF metabolites remain unidentified, and for those that have been identified there is not much known about normal concentration ranges. A systematic categorization of the CSF metabolome is necessary and expected to be beneficial for future biomarker discoveries. Recently, Wishart et al. made a good start in exploring the human CSF metabolome with their computer-aided literature survey that resulted in 308 detectable metabolites in human CSF (13).The CSF proteome has been characterized to a much larger extent than the CSF metabolome and is currently the topic of investigations in several research groups worldwide. Recently, studies have been published with numerous identities and quantities of CSF proteins. Pan and co-workers were able to identify 2,594 proteins in well-characterized pooled human CSF samples using strict proteomics criteria with a combination of linear trap quadrupole LTQ-FT (Thermo Fisher Scientific, Bremen, Germany) and MALDI TOF/TOF equipment (14). They were also able to quantify several proteins using a targeted LC MALDI TOF/TOF approach (15). Hu et al. have studied the intra- and inter-individual variation in human CSF and found large variations in protein concentrations in six patients by means of two dimensional–gel electrophoresis (16), focusing mainly on the variations within individuals at two different time-points. Although only a limited number of proteins was analyzed, the variation between the time-points was profound, exceeding 200% for seven proteins.Unique CSF biomarkers may contribute to a deeper understanding of the mechanisms of CNS disorders. However, for this assumption to come true, there are still challenges ahead. Although CSF is not as complex as blood (almost missing the cellular part and the clotting system present in blood), it is expected to consist of thousands of organic- and non-organic salts, sugars, lipids, and proteins. A large part of the CSF consists of a few highly abundant metabolites and proteins, which hamper, if no precautions are undertaken, the identification and quantification of metabolites and proteins that occur in lower amounts. The analysis of the CSF metabolome is complicated because of the diverse chemical nature of metabolites and the lower concentration of metabolites compared with blood. Analytical method development is still required because it is not possible to identify the entire range of CSF metabolites with one single analytical method. Although in proteome research efforts have been made to quantify proteins, metabolomics studies up to now either do not provide quantitative information or they only give information for the most abundant metabolites.Another challenge is the sample amount obtained by lumbar puncture to collect CSF. Lumbar puncture is an invasive method that is not performed as frequently as blood sampling. However, often after the analysis of various clinical parameters, only a limited amount of CSF sample is available for biomarker discovery. Metabolomics studies are hampered by limited CSF sample amount. Therefore, analytical methods are required that are suitable to handle relatively small sample volumes.The main objectives of this study were (1) to analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples by multiple analytical platforms; and (2) to integrate metabolomics and proteomics to present biological variations in metabolite and protein abundances and compare these with technical variations with the currently used analytical methods. The results will facilitate and increase the application of CSF for future biomarker discovery studies in the field of neurodegenerative diseases and neuro-oncology.     

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.     

Embryonic developmental plasticity of the chick: increased CO(2) during early stages of incubation changes the developmental trajectories during prenatal and postnatal growth

This study investigated the effect of non-ventilation of the incubator during the first 10 days of incubation on carbon dioxide (CO(2)) concentrations in the incubator and its effects on the embryonic and post-hatch development of the chicken (Gallus gallus). Two different incubation conditions were created, one incubator was kept at standard conditions, with adequate ventilation (V) and a second incubator was non-ventilated (NV) during the first ten days of incubation, allowing the CO(2) to rise. After the first 10 days, both incubations were continued under standard conditions. The experiment was repeated twice with different ages of the breeders (45 and 60 wks) which resulted in different CO(2) levels at ED10 (1.5 and 1%). The CO(2) concentration in the V incubators remained below 0.1% in these first 10 days. The eggs of the NV incubation showed higher pCO(2) levels in the air cell from ED10 until ED14 compared to the eggs of the V group. The NV embryos had significantly higher absolute and relative (to egg weight) body weights from ED10 until ED18, pointing to an accelerated embryonic growth. At internal pipping, the NV chick embryos had higher plasma corticosterone and T(3) levels and higher pCO(2) in the air cell. Chicks incubated under NV conditions hatched 10 h earlier in the first and 15 h earlier in the second experiment and the spread of hatch was narrower. During the post-hatch period, the NV chickens had a higher body weight compared to the V chickens. From these results, it is clear that higher levels of CO(2) during the first ten days of incubation have persistent (epigenetic) effects during the incubation and early post-hatch period.     

Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.