‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter'' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast'': aerobic-‘famine'' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans'' and ‘Candidatus Contendobacter odensis'', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.     

PSCC: Sensitive and Reliable Population-Scale Copy Number Variation Detection Method Based on Low Coverage Sequencing

Background

Copy number variations (CNVs) represent an important type of genetic variation that deeply impact phenotypic polymorphisms and human diseases. The advent of high-throughput sequencing technologies provides an opportunity to revolutionize the discovery of CNVs and to explore their relationship with diseases. However, most of the existing methods depend on sequencing depth and show instability with low sequence coverage. In this study, using low coverage whole-genome sequencing (LCS) we have developed an effective population-scale CNV calling (PSCC) method.

Methodology/Principal Findings

In our novel method, two-step correction was used to remove biases caused by local GC content and complex genomic characteristics. We chose a binary segmentation method to locate CNV segments and designed combined statistics tests to ensure the stable performance of the false positive control. The simulation data showed that our PSCC method could achieve 99.7%/100% and 98.6%/100% sensitivity and specificity for over 300 kb CNV calling in the condition of LCS (∼2×) and ultra LCS (∼0.2×), respectively. Finally, we applied this novel method to analyze 34 clinical samples with an average of 2× LCS. In the final results, all the 31 pathogenic CNVs identified by aCGH were successfully detected. In addition, the performance comparison revealed that our method had significant advantages over existing methods using ultra LCS.

Conclusions/Significance

Our study showed that PSCC can sensitively and reliably detect CNVs using low coverage or even ultra-low coverage data through population-scale sequencing.     

A cell model to study different degrees of Hsp60 deficiency in HEK293 cells

Mitochondrial dysfunction is associated with neurodegenerative diseases and mutations in the HSPD1 gene, encoding the mitochondrial Hsp60 chaperone, are the causative factors of two neurodegenerative diseases, hereditary spastic paraplegia and MitChap60 disease. In cooperation with Hsp10, Hsp60 forms a barrel-shaped complex, which encloses unfolded polypeptides and provides an environment facilitating folding. We have generated an Hsp60 variant with a mutation (Asp423Ala) in the ATPase domain and established a stable human embryonic kidney (HEK293) cell line allowing tetracycline-controlled expression of this mutant variant. We monitored expression of the Hsp60–Asp423Ala variant protein following induction and examined its effects on cellular properties. We showed that the folding of mitochondrial-targeted green fluorescent protein, a well-known substrate protein of Hsp60, was consistently impaired in cells expressing Hsp60–Asp423Ala. The level of the Hsp60–Asp423Ala variant protein increased over time upon induction, cell proliferation stopped after 48-h induction and mitochondrial membrane potential decreased in a time-dependent manner. In summary, we have established a stable cell line with controllable expression of an Hsp60 variant, which allows detailed studies of different degrees of Hsp60 deficiency.     

Fat maintenance is a predictor of the murine lifespan response to dietary restriction

Dietary restriction (DR), one of the most robust life-extending manipulations, is usually associated with reduced adiposity. This reduction is hypothesized to be important in the life-extending effect of DR, because excess adiposity is associated with metabolic and age-related disease. Previously, we described remarkable variation in the lifespan response of 41 recombinant inbred strains of mice to DR, ranging from life extension to life shortening. Here, we used this variation to determine the relationship of lifespan modulation under DR to fat loss. Across strains, DR life extension correlated inversely with fat reduction, measured at midlife (males, r= -0.41, P<0.05, n=38 strains; females, r= -0.63, P<0.001, n=33 strains) and later ages. Thus, strains with the least reduction in fat were more likely to show life extension, and those with the greatest reduction were more likely to have shortened lifespan. We identified two significant quantitative trait loci (QTLs) affecting fat mass under DR in males but none for lifespan, precluding the confirmation of these loci as coordinate modulators of adiposity and longevity. Our data also provide evidence for a QTL previously shown to affect fuel efficiency under DR. In summary, the data do not support an important role for fat reduction in life extension by DR. They suggest instead that factors associated with maintaining adiposity are important for survival and life extension under DR.     

pH landscapes in a novel five-species model of early dental biofilm

Background

Despite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate.

Methodology/Principal Findings

Strains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours.

Conclusions/Significance

The designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth, composition and extracellular pH. Ratiometric pH analysis using C-SNARF-4 gives detailed insight into the pH landscape of living biofilms and contributes to our general understanding of metabolic processes in in-vivo-grown bacterial biofilms.     

Interleukin 12B (IL12B) genetic variation and pulmonary tuberculosis: a study of cohorts from The Gambia, Guinea-Bissau, United States and Argentina

We examined whether polymorphisms in interleukin-12B (IL12B) associate with susceptibility to pulmonary tuberculosis (PTB) in two West African populations (from The Gambia and Guinea-Bissau) and in two independent populations from North and South America. Nine polymorphisms (seven SNPs, one insertion/deletion, one microsatellite) were analyzed in 321 PTB cases and 346 controls from Guinea-Bissau and 280 PTB cases and 286 controls from The Gambia. For replication we studied 281 case and 179 control African-American samples and 221 cases and 144 controls of European ancestry from the US and Argentina. First-stage single locus analyses revealed signals of association at IL12B 3′ UTR SNP rs3212227 (unadjusted allelic p = 0.04; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61–0.99]) in Guinea-Bissau and rs11574790 (unadjusted allelic p = 0.05; additive genotypic p = 0.05, OR = 0.76, 95% CI [0.58–1.00]) in The Gambia. Association of rs3212227 was then replicated in African-Americans (rs3212227 allelic p = 0.002; additive genotypic p = 0.05, OR = 0.78, 95% CI [0.61–1.00]); most importantly, in the African-American cohort, multiple significant signals of association (seven of the nine polymorphisms tested) were detected throughout the gene. These data suggest that genetic variation in IL12B, a highly relevant candidate gene, is a risk factor for PTB in populations of African ancestry, although further studies will be required to confirm this association and identify the precise mechanism underlying it.     

Glucose Metabolism and Regulation in Lactating Mink (Mustela Vison) - Effects of Low Dietary Protein Supply

Eighteen lactating mink raising litters of 6 to 7 kits were fed ad libitum from parturition on diets with 32% of ME derived from protein and decreasing fat:carbohydrate ratios [high fat:low carbohydrate (HFLC): 67:1, medium fat:medium carbohydrate (MFMC): 52:16, low fat:high carbohydrate (LFHC): 37:31]. Four weeks post partum the dams were fitted with a jugular vein catheter, and the experiment started with a 3 hours fasting period, after which the dams were fed 210kJ ME of the experimental diets. Blood samples were collected 10 and 5min before feeding and 30, 60, 90, 120, 150 and 180min postprandially. Two hours postprandially a single dose of 50µCi U-¹⁴C-labelled glucose was administered to each dam and blood samples were collected 5, 10, 20, 30, 45 and 60min after the tracer administration. Plasma concentrations of glucose and insulin 30 to 120min postprandially were higher in dams fed the LFHC diet, than in dams fed the HFLC diet, values for dams fed the MFMC diet being intermediate. Plasma glucagon concentrations were not significantly affected by dietary treatment. The glucagon:insulin ratios decreased postprandially in all dams, the response being significant in dams fed the LFHC diet. Plasma concentrations of urea were not significantly affected by dietary treatment. Plasma FFA concentrations tended to increase postprandially in dams fed the HFLC diet. Glucose turnover rates were approximately 4.0% permin in all dams, irrespective of dietary treatment. However, the daily glucose flux was lower in dams fed the HFLC diet than in dams fed the LFHC diet, and tended to be lower than in dams fed the MFMC diet. In conclusion, a dietary protein supply of 32% of ME simultaneously with a carbohydrate supply of 16% or 31% of ME had no adverse effects on glucose homeostasis or glucose metabolism in lactating mink.     

Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper,Th17- and Activated B-Cells and Correlates with Progression

Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (T_FH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4⁺ and CD8⁺T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS⁺T_FH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 T_FH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 T_FH-cells. The Th17-subset, interleukin-23-receptor⁺CD4⁺T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN⁺ and CD83⁺B-cells in SPMS. ICOS⁺T_FH-cells and DC-SIGN⁺B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4⁺T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of T_FH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated T_FH-cells in MS. The increased frequencies of Th17-cells, activated T_FH- and B-cells parallel findings from pathology studies which, along with the correlation between activated T_FH- and B-cells and disease progression, suggest a pathogenic role of systemic inflammation in progressive MS. These observations may have implications for the treatment of progressive MS.     

ADP Protects Cardiac Mitochondria under Severe Oxidative Stress

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca²⁺ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F₀F₁ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca²⁺-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.