Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.     

Increased Lymph Node Yield in Colorectal Cancer Is Not Necessarily Associated with a Greater Number of Lymph Node Positive Cancers

The presence of lymph node metastasis is a key prognostic factor in colorectal cancer and lymph node yield is an important parameter in assessing the quality of histopathology reporting of colorectal cancer excision specimens. This study assesses the trend in lymph node evaluation over time in a single institution and the relationship with the identification of lymph node positive tumours. It compares the lymph node yield of a contemporary dataset compiled from the histopathology reports of 2178 patients who underwent surgery for primary colorectal cancer between 2005 and 2012 with that of a historic dataset compiled from the histopathology reports of 1038 patients who underwent surgery for colorectal cancer at 5 yearly intervals from 1975 to 2000. The mean lymph node yield was 14.91 in 2005 rising to 21.38 in 2012. In 2012 92.9% of all cases had at least 12 lymph nodes examined. Comparison of the mean lymph node yield and proportion of Dukes C cases shows a significant increase (Pearson correlation = 0.927, p = 0.001) in lymph node yield while there is no corresponding significant trend in the proportion of Dukes C cases (Pearson correlation = −0.138, p = 0.745). This study shows that there is increasing yield of lymph nodes from colorectal cancer excision specimens. However, this is not necessarily associated with an increase number of lymph node positive cancers. Further risk stratifying of colorectal cancer requires consideration of other pathological parameters especially the presence of extramural venous invasion and relevant biomarkers.     

Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines.     

Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif ^P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif ^P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.     

Succination of Thiol Groups in Adipose Tissue Proteins in Diabetes: SUCCINATION INHIBITS POLYMERIZATION AND SECRETION OF ADIPONECTIN*

S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219–34228). We show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for ∼7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.The accumulation of sugar and lipid-derived chemical modifications on proteins is associated with the etiology of several age-related diseases, including diabetes and its complications (1, 2). The irreversible adducts formed, termed advanced glycation/lipoxidation end products (AGE/ALEs),² accumulate over time on long lived proteins, such as collagens, affecting the solubility, elasticity, and proteolytic digestibility of the protein (3). AGE/ALEs are considered important mediators of the pathogenesis of diabetic complications through engagement of scavenger receptors, such as RAGE (receptor for AGE) and activation of proinflammatory signaling pathways (4). To date, the study of AGE/ALEs has focused mainly on modification of lysine and arginine residues in proteins by reactive carbonyl intermediates formed during metabolism or autoxidation of carbohydrates and lipids (2, 5). However, free cysteine is more abundant on intracellular proteins and, because of its greater nucleophilicity, is a more likely target for chemical modification by intracellular electrophiles.We recently identified S-(2-succinyl)-cysteine (2SC), a cysteine modification formed by a Michael addition reaction between the Krebs cycle intermediate fumarate and free sulfhydryl groups on proteins (6). This reaction, in which a thioether bond is formed, is described as succination of protein in order to distinguish it from succinylation, which leads to formation of amide, ester, or thioester bonds. 2SC was detected in human serum albumin and skin collagen and was increased in skeletal muscle protein and urine of diabetic rats. We also identified glyceraldehyde-3-phosphate dehydrogenase as one protein that is significantly modified by 2SC in skeletal muscle, resulting in the decrease in specific activity of glyceraldehyde-3-phosphate dehydrogenase in muscle of diabetic rats (7). We have proposed that 2SC may accumulate as a result of mitochondrial nutrient “flooding” because of an excess of carbohydrate and lipid fuels in diabetes and may be a biomarker of mitochondrial stress in disease.To gain further insight into the role of succination in the regulation of metabolism, we studied the maturation of 3T3-L1 fibroblasts to adipocytes, an in vitro system in which fumarate and other Krebs cycle intermediates increase severalfold during adipogenesis in high (30 mm) glucose) medium (8, 9). Adipogenesis under these conditions is associated with a substantial increase in oxidative stress as a result of mitochondrial superoxide production (10). We also observed a ≥5-fold increase in fumarate and a ≥10-fold increase in intracellular 2SC accumulation during adipogenesis and identified several of the major proteins modified by 2SC (9). This set of proteins included cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein, suggesting that succination may have wide ranging effects on the structure of the cytoskeleton and the regulation of metabolism.The adipocyte is increasingly recognized not only for its role in triglyceride storage but also as an active endocrine organ, secreting hormones and cytokines that orchestrate key metabolic processes in tissues, such as heart, liver, and muscle. All of the adipokines work as part of a greater metabolic regulatory network. Adiponectin and leptin are considered positive regulators of energy intake and expenditure, whereas resistin, interleukin-6, tumor necrosis factor-α, and PAI-1 are implicated in the development of inflammation and insulin resistance. Imbalances in adipokine metabolism are central to adipocyte dysfunction and the ensuing events leading to insulin resistance and diabetes (11, 12).Adiponectin has received particular attention as the most abundant adipokine, circulating at high levels in human blood. It is an ∼30-kDa glycoprotein that associates intracellularly into trimeric, hexameric (also known as low molecular weight (LMW)), and other high molecular weight (HMW) complexes consisting of 18–36 monomers (13, 14). The various molecular weight species differentially stimulate their target tissues; trimeric adiponectin stimulates muscle fatty acid oxidation through activation of AMP-activated protein kinase, whereas HMW forms act to enhance insulin-mediated inhibition of gluconeogenesis in the liver (15, 16). Plasma adiponectin concentration is reduced in diabetes, in general, as is the ratio of HMW forms to total adiponectin (16).The N-terminal hypervariable domain of adiponectin contains a single cysteine residue followed by a collagenous region containing several conserved lysine and proline residues. Several of these lysines and prolines are subject to modification by hydroxylation and/or glycosylation (17, 18). The cysteine at position 39 in mouse adiponectin is involved in the formation of the oligomeric species of adiponectin through disulfide bonding of monomers and trimers (Fig. 1). Cys-39 is critical for the generation of all higher order complexes, since its mutation to serine inhibits the formation of both trimer and larger species. The only other cysteine present in adiponectin is located in the C-terminal globular domain, and crystallographic studies indicate that it is unlikely to be involved in disulfide bonding of oligomers (14). In this study, we show that adiponectin is a major target of succination in both 3T3-L1 adipocytes and adipose tissue of diabetic (db/db) mice, that Cys-39 is a major site of cysteine succination, and that succinated adiponectin is neither incorporated into polymeric forms in the cell nor secreted from the cell. We propose that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.Open in a separate windowFIGURE 1.Structure of adiponectin. Two cysteines are highly conserved in adiponectin monomer: one in the hypervariable region adjacent to the N terminus (Cys-39) and the other in the C-terminal globular head domain (Cys-155) (A). Adiponectin monomers associate into trimers through disulfide bonding, and trimers associate through disulfide bonds to form LMW and HMW multimers, which are secreted from the adipocyte. Succination of Cys-39 blocks incorporation of adiponectin monomer into trimer and higher molecular weight secreted forms of the protein (B).     

A systematic review of population based epidemiological studies in Myasthenia Gravis

Background  

The aim was to collate all myasthenia gravis (MG) epidemiological studies including AChR MG and MuSK MG specific studies. To synthesize data on incidence rate (IR), prevalence rate (PR) and mortality rate (MR) of the condition and investigate the influence of environmental and technical factors on any trends or variation observed.     

5th BSPR‐EBI Meeting,Proteomics: From Technology to New Biology 8–10 July 2008, Wellcome Trust Conference Centre,Hinxton, Cambridge,UK

This paper reports on the 5^th joint British Society for Proteome Research (BSPR) and European Bioinformatics Institute (EBI) meeting which took place at the Wellcome Trust Conference Centre, Cambridge, UK, from the 8^th to 10^th July, 2008. As in previous years, the meeting attracted leading experts in the field who presented the latest cutting edge in proteomics. The meeting was entitled “Proteomics: From Technology to New Biology” taking into account the major transition proteomics has undergone in the past few years. In particular, the use of multiple reaction monitoring (MRM)‐based targeted experiments for absolute quantification and validation of proteins was the hot topic of the meeting. Attended by some 250 delegates, the conference was extremely well organised and provided a great opportunity for discussion and initiation of new collaborations.