Molecular indicators of microbial diversity in oolitic sands of Highborne Cay,Bahamas

Microbialites (stromatolites and thrombolites) are mineralized mat structures formed via the complex interactions of diverse microbial‐mat communities. At Highborne Cay, in the Bahamas, the carbonate component of these features is mostly comprised of ooids. These are small, spherical to ellipsoidal grains characterized by concentric layers of calcium carbonate and organic matter and these sand‐sized particles are incorporated with the aid of extra‐cellular polymeric substances (EPS), into the matrix of laminated stromatolites and clotted thrombolite mats. Here, we present a comparison of the bacterial diversity within oolitic sand samples and bacterial diversity previously reported in thrombolitic and stromatolitic mats of Highborne Cay based on analysis of clone libraries of small subunit ribosomal RNA gene fragments and lipid biomarkers. The 16S‐rRNA data indicate that the overall bacterial diversity within ooids is comparable to that found within thrombolites and stromatolites of Highborne Cay, and this significant overlap in taxonomic groups suggests that ooid sands may be a source for much of the bacterial diversity found in the local microbialites. Cyanobacteria were the most diverse taxonomic group detected, followed by Alphaproteobacteria, Gammaproteobacteria, Planctomyces, Deltaproteobacteria, and several other groups also found in mat structures. The distributions of intact polar lipids, the fatty acids derived from them, and bacteriohopanepolyols provide broad general support for the bacterial diversity identified through analysis of nucleic acid clone libraries.     

Macrophage inhibitory cytokine‐1 is associated with cognitive impairment and predicts cognitive decline – the Sydney Memory and Aging Study

Higher levels of macrophage inhibitory cytokine‐1, also known as growth differentiation factor 15 (MIC‐1/GDF15), are associated with adverse health outcomes and all‐cause mortality. The aim of this study was to examine the relationships between MIC‐1/GDF15 serum levels and global cognition, five cognitive domains, and mild cognitive impairment (MCI), at baseline (Wave 1) and prospectively at 2 years (Wave 2), in nondemented participants aged 70–90 years. Analyses were controlled for age, sex, education, Framingham risk score, history of cerebrovascular accident, acute myocardial infarction, angina, cancer, depression, C‐reactive protein, tumor necrosis factor‐α, interleukins 6 and 12, and apolipoprotein ε4 genotype. Higher MIC‐1/GDF15 levels were significantly associated with lower global cognition at both waves. Cross‐sectional associations were found between MIC‐1/GDF15 and all cognitive domains in Wave 1 (all P < 0.001) and between processing speed, memory, and executive function in Wave 2 (all P < 0.001). Only a trend was found for the prospective analyses, individuals with high MIC‐1/GDF15 at baseline declined in global cognition, executive function, memory, and processing speed. However, when categorizing MIC‐1/GDF15 by tertiles, prospective analyses revealed statistically significant lower memory and executive function in Wave 2 in those in the upper tertile compared with the lower tertile. Receiver operating characteristics (ROC) analysis was used to determine MIC‐1/GDF15 cutoff values associated with cognitive decline and showed that a MIC‐1/GDF15 level exceeding 2764 pg/ml was associated with a 20% chance of decline from normal to MCI or dementia. In summary, MIC‐1/GDF15 levels are associated with cognitive performance and cognitive decline. Further research is required to determine the pathophysiology of this relationship.     

A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion.The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. An effective method for the detection of posttranslational modifications of proteins is the metabolic incorporation of modified biomolecules such as tagged carbohydrates or lipids (1). Reversible interactions of enzyme inhibitors, natural products, or drugs can be detected when one appends photocrosslinking agents, thereby facilitating target discovery (2, 3). A particularly interesting example of protein labeling is activity-based protein profiling (ABPP)¹ (4, 5), which utilizes the intrinsic catalytic activity of a target enzyme for the covalent attachment of an affinity or visualization tag. ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A). DCG-04, for example, is based on the naturally occurring inhibitor E-64 and targets the papain family of cysteine proteases via covalent attachment of the epoxysuccinate group to the active site cysteine (Fig. 1B) (6).Open in a separate windowFig. 1.The cleavable linker strategy in ABPP. A, the elements of an ABP. B, the example ABP DCG-04, an epoxysuccinate-containing probe for clan CA cysteine proteases. DCG-04 is based on the naturally occurring protease inhibitor E-64. C, schematic strategy of cleavable linker-mediated target identification. D, the cleavage mechanism of a vicinal diol.Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding. Moreover, they can negatively influence the cell permeability of probes, which therefore limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation (7) and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) (8), allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. Consequently, the probes themselves only carry azide or alkyne groups as “mini-tags.” Tandem labeling using bioorthogonal chemistry has now become a widely used strategy to label biomolecules in lysates and in live cells (9–11).An essential step in ABPP, as well as in other chemical proteomics approaches, is the elucidation of the tagged proteins. This usually involves a biotin-mediated enrichment step followed by mass-spectrometry-based identification. Although the streptavidin-biotin interaction allows efficient enrichment as a result of the strong binding affinity (K_d ∼ 10⁻¹⁵ m), it also has limitations. The quantitative elution of biotinylated proteins requires harsh conditions (12), which lead to contamination of the sample by endogenous biotinylated and non-specifically bound proteins. These other proteins will be identified together with the real protein targets. Given that subsequent target validation with secondary assays can be a costly and time-consuming process, a reduction in false positive identifications is highly desirable. For cleaner protein identification, cleavable linker strategies (13) that allow the selective release of target proteins have been developed (Fig. 1C). The commercially available disulfide linker can be cleaved under mild conditions, but it suffers from premature cleavage in reducing media such as the intracellular environment and reducing buffers used for click chemistry and in vitro reactions of cysteine proteases. Therefore, a variety of alternative linkers for proteomics applications have been reported, including a sterically hindered disulfide (14), diazobenzenes (15–19), hydrazones (20, 21), silanes (22), light sensitive linkers (23–25), tobacco etch virus protease sensitive linkers (26, 27), and a levulinoyl-based linker (28). The synthesis of some of these linkers is lengthy or difficult to scale up, which limits their general application in chemical proteomics.Ideally, a cleavable linker is stable under a wide variety of conditions, is efficiently and selectively cleaved, and can be synthesized in a low number of easy chemical transformations. We aimed to meet these requirements by using a vicinal diol as a cleavable linker system. When vicinal diols are treated with sodium periodate (NaIO₄), the carbon–carbon bond is cleaved (Fig. 1D). Periodate treatment of proteins can result in side-reactions, such as the cleavage of linked carbohydrates or the oxidation of N-terminal serine and threonine residues. However, these N-termini rarely occur in proteins and are therefore of minor concern. In general, the mild, neutral conditions of periodate cleavage are compatible with proteins. This has been illustrated in the past, for example, by its application in the detection of protein–protein interactions (29) and the creation of unliganded MHC class I molecules (30). In this article, we report the chemical proteomics application of diol cleavable linker probes. We show that the synthesis of the linker and its probe derivatives is straightforward, that the linker is compatible with tandem click labeling, that enrichment and release of probe targets is efficient, and that the identification of targets takes place with significantly lower background than in on-bead digestion protocols.     

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background

An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods

Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results

Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion

We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.     

Mammalian Target of Rapamycin Complex 2 (mTORC2) Is a Critical Determinant of Bladder Cancer Invasion

Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.     

MRI Plaque Imaging Detects Carotid Plaques with a High Risk for Future Cerebrovascular Events in Asymptomatic Patients

Purpose

The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I–VIII). Within these lesion types, lesion types IV–V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones.

Methods

Eighty-three consecutive patients (45 male (54.2%); age 54–88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50–99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I–VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance.

Results

Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months.During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV–V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV–V and VI) (log rank test P<0.0001).

Conclusions

MRI plaque imaging has the potential to identify patients with asymptomatic carotid stenosis who are particularly at risk of developing future cerebral ischemia. MRI could improve selection criteria for invasive therapy in the future.