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.     

Gene dosage effect on gamma-secretase component Aph-1b in a rat model for neurodevelopmental disorders

A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Here, we report microarray analyses revealing in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple (neuro)developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregated with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype.     

Insulin Resistance in Relation to Lipids and Inflammation in Type-2 Diabetic Patients and Non-Diabetic People

BackgroundWe demonstrated in experimental studies that hypercholesterolaemia enhances the proliferation of haematopoietic stem cells and the subsequent differentiation to neutrophils, whereas HDL-cholesterol inhibits these processes. To translate our experimental findings to clinical practice, we investigated in Chinese type-2 diabetic patients and in Flemish non-diabetic people the independent and joint associations of insulin resistance with markers of dyslipidaemia and inflammation, while looking for consistency between ethnicities and across the spectrum of insulin resistance.MethodsWe studied 798 Chinese patients with type-2 diabetes (53.6% women; mean age, 60.6 years) admitted to a tertiary referral centre and 1060 white Flemish (50.5%; 51.1 years) randomly recruited in Northern Belgium. Fasting insulin resistance (HOMA-IR) was derived from C-peptide in Chinese and from insulin in Flemish using the Homeostasis Model of Assessment algorithm. In multivariable-adjusted analyses, HOMA-IR was regressed on triglycerides, HDL-cholesterol and neutrophil count.ResultsIn Chinese patients, the percentage changes in HOMA-IR associated with triglycerides, HDL-cholesterol and neutrophils (per 1-SD increment) amounted to 8.1 (95% confidence interval, 3.0 to 13.4; p = 0.0015), -8.7 (-13.0 to -4.2; p = 0.0002) and 5.6 (1.0 to 10.4; p = 0.017). In non-diabetic Flemish, the corresponding estimates were 11.7 (8.3 to 15.1; p<0.0001), -1.7 (-4.6 to 1.4; p = 0.28) and 3.3% (0.5 to 6.3; p = 0.022), respectively. None of the interaction terms between the three explanatory variables reached significance in Chinese or Flemish (p≥0.10).ConclusionsInsulin resistance increases with the serum level of triglycerides and the blood neutrophil count, but decreases with serum HDL-cholesterol concentration. These associations were consistent in Chinese type-2 diabetic patients and non-diabetic Flemish people and were independent from one another. The clinical implications are that future studies should focus on intervening with serum triglyceride and HDL-cholesterol levels or controlling inflammation as a way to prevent or treat insulin resistance.