Canonical Family Research Articles

Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells.

BackgroundMycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.ResultsParallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.ConclusionWe conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1569-2) contains supplementary material, which is available to authorized users.

Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose.

Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.

Invariant Solutions to the Strominger System on Complex Lie Groups and Their Quotients

Using canonical 1-parameter family of Hermitian connections on the tangent bundle, we provide invariant solutions to the Strominger system on complex Lie groups. Both flat and non-flat cases are discussed in detail.

Traces of singular values of Hauptmoduln

In an important paper, Zagier proved that certain half-integral weight modular forms are generating functions for traces of polynomials in the j-function. It turns out that Zagier's work makes it possible to algorithmically compute Hilbert class polynomials using a canonical family of modular forms of weight [Formula: see text]. We generalize these results and consider Hauptmoduln for levels 1, 2, 3, 5, 7, and 13. We show that traces of singular values of polynomials in Hauptmoduln are again described by coefficients of half-integral weight modular forms. This realization makes it possible to algorithmically compute class polynomials.

Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle.

Alopecia areata (AA) is an autoimmune hair loss disease caused by a cell-mediated immune attack of the lower portion of the cycling hair follicle. Feeding mice 3-7 times the recommended level of dietary vitamin A accelerated the progression of AA in the graft-induced C3H/HeJ mouse model of AA. In this study, we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells (SCs) to induce the development and growth phase of the hair cycle (anagen), which may have made the hair follicle more susceptible to autoimmune attack. Our purpose here is to determine the mechanism by which dietary vitamin A regulates the hair cycle. We found that vitamin A in a dose-dependent manner increased nuclear localized beta-catenin (CTNNB1; a marker of canonical wingless-type Mouse Mammary Tumor Virus integration site family (WNT) signaling) and levels of WNT7A within the hair follicle bulge in these C3H/HeJ mice. These findings suggest that feeding mice high levels of dietary vitamin A increases WNT signaling to activate hair follicle SCs.

IKKβ regulates endothelial thrombomodulin in a Klf2‐dependent manner

Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression. TNFα has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-κB). However, because the TM promoter lacks an NF-κB binding site, the direct involvement of NF-κB has been controversial. We investigated the role of the upstream regulatory serine kinase, inhibitory kappa-B kinase-β (IKKβ), in TM expression and function with or without TNFα treatment. Inhibition of IKKβ was achieved by specific chemical inhibitors, siRNA or shRNA. TM expression was assessed by qRT-PCR, Western blot, flow cytometry, luciferase reporter assay and chromatin immune-precipitation (ChIP) assay. TM function was estimated by generation of activated protein C (APC). NF-κB activation was determined by immunocytochemistry. IKKβ inhibition increased TM expression and function, and attenuated TNFα-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-κB protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKKβ inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-κB pathway, respectively, resulted in TM over-expression. IKKβ inhibition caused over-expression, increased promoter activity and enhanced binding of Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKβ inhibition-mediated TM up-regulation. We conclude that IKKβ regulates TM in a Klf2-dependent manner.

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology.

BackgroundThe search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.ResultsXylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.ConclusionsMpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

Abstract 77: Distinct Profiles of Transient Receptor Potential Canonical (TRPC) Channel Expression in Biventricular Failure

Heart failure is a major cause of morbidity and mortality. The transient receptor potential canonical (TRPC) family of channels mediate pathologic cardiac remodeling. In particular, TRPC6 participates in a self-propagating circuit that amplifies cardiac hypertrophy and fibrosis. The objective of this study was to explore biventricular expression of TRPCs in advanced heart failure. Methods: Viable left (LV) and right (RV) ventricular free wall tissue was obtained from human subjects with end-stage heart failure (n=12) referred for transplantation or biventricular assist devices. Control LV and RV tissue was obtained from the National Disease Research Interchange (n=3/group). To explore TRPC expression in a murine model, adult male C57BL/6 mice underwent thoracic aortic constriction (TAC) for 10 weeks (n=6/group). Biventricular tissue was analyzed by real-time polymerase chain reaction. Results: Compared to normal LV and RV, levels of TRPC 1, 3, 4 and 6 were increased in failing LV and RV samples, respectively. Levels of TRPC1 and TRPC6 were greater in failing RV than failing LV samples. TRPC 5 and 7 expression were not consistently detected in normal or failing tissue samples. Compared to sham LV, levels of TPRC 1, 4 and 6 increased in the LV after TAC. Compared to sham RV, levels of TRPC 3, 4, and 6 increased in the RV after TAC. Levels of TRPC3 were greater in the RV than LV after TAC. Conclusions: Our results identify distinct profiles of TRPC expression in the RV versus LV in both human tissue and in a murine model of advanced biventricular failure. Levels of select TRPCs are higher in the failing RV compared to LV, suggesting a potentially important role for TRPCs in RV remodeling.

Unraveling the novel function of the DNA repair enzyme 8-oxoguanine-DNA glycosylase in activating key signaling pathways

Unraveling the novel function of the DNA repair enzyme 8-oxoguanine-DNA glycosylase in activating key signaling pathways

Increasing Supply Chain Robustness Through Process Flexibility and Inventory

We study a hybrid strategy that uses both process flexibility and inventory for supply chain risk mitigation. The interplay between process flexibility and inventory is modeled as a two-stage robust optimization problem; in the first stage, the firm allocates inventory, and in the second stage, after uncertainties are realized, the firm schedules its flexible production to minimizes its cost. By taking advantage of the structure of the second stage recourse problem, we develop an effective constraint generation algorithm for solving the two-stage robust optimization problem. To further improve the algorithm's performance, we develop a method for generating Pareto cuts. We apply our algorithm numerically to study risk mitigation problems in automotive and clothing industries. Our numerical analysis provides insights regarding the impact of process flexibility on total inventory level and inventory allocation pattern. Finally, we verify our insights for a general class of uncertainty sets under a canonical family of flexibility designs known as the K-chains.

Quasi-isometry and deformations of Calabi–Yau manifolds

We prove several formulas related to Hodge theory and the Kodaira–Spencer–Kuranishi deformation theory of Kahler manifolds. As applications, we present a construction of globally convergent power series of integrable Beltrami differentials on Calabi–Yau manifolds and also a construction of global canonical family of holomorphic $$(n,0)$$ -forms on the deformation spaces of Calabi–Yau manifolds. Similar constructions are also applied to the deformation spaces of compact Kahler manifolds.

Coincident intracellular calcium rise is essential for Gi/o‐mediated activation of TRPC4 (843.3)

TRPC4 is a member of the Canonical Transient Receptor Potential family of non‐selective cation channels. Na+ and Ca2+ influx through the active TRPC4 channel elicits membrane depolarization and intracellular Ca2+ signaling in various cell types including neurons, vascular endothelium and smooth muscle cells. It has been well‐established that homomeric TRPC4 channels are synergistically activated by co‐stimulation of two separate G protein pathways, Gq/11 and Gi/o. However, it remains unclear which step(s) or constituent(s) of the Gq/11 pathway is(are) essential for synergizing with Gi/o proteins to maximally activate TRPC4 channels. Using patch‐clamp recordings and fluorescence membrane potential measurements in HEK293 cells heterologously coexpressing mouse TRPC4β and Gi/o‐coupled µ opioid receptor, we have identified intracellular Ca2+ rise as the primary event associated with Gq/11 activation that synergizes with Gi/o stimulation to elicit maximal TRPC4 current. We also show that a signification portion of this effect is independent of calmodulin although Ca2+‐calmodulin can enhance channel activation. Our experiments demonstrate the contribution of phosphoinositide hydrolysis and IP3 receptors but rule them out as essential players. These findings argue for a critical role of intracellular Ca2+ rise in TRPC4 activation by Gi/o‐coupled receptors and illustrate the key requirements that support TRPC4 as a coincidence sensor of multiple signaling pathways.Grant Funding Source: American Heart Association

Ca2+ Facilitates TRPC4 Activation by Gi/O Signaling in Both Calmodulin Dependent and Independent Manner

TRPC4 is a member of the Canonical Transient Receptor Potential family of non-selective ion channels and is regulated by a set of intracellular and extracellular factors including G protein coupled receptor activation, calcium and membrane phospholipids. Sodium (Na+) and calcium (Ca2+) influx through the active TRPC4 channel elicits membrane depolarization and intracellular Ca2+ signaling in neurons, vascular endothelium and smooth muscle cells. Uniquely, TRPC4 is maximally activated only when two separate G protein pathways, Gq/11 and Gi/o, are co-stimulated, making it a coincidence detector of Gq/11- and Gi/o-coupled receptor co-activation. This function may be important for coordinating the actions of sympathetic (e.g. adrenergic) and parasympathetic (e.g. cholinergic) innervations. While a direct interaction between Gi/o-alpha subunits and the cytoplasmic C-terminal domain of TRPC4 has been suggested to underlie the activation mechanism by Gi/o proteins, the PLC-IP3-IP3R pathway is thought to be critical for the action of Gq/11. Using electrophysiological recordings of HEK293 cells heterologously expressing mouse TRPC4-beta, we examined the interdependence of Gi/o and Gq/11 pathways on TRPC4 activation. We show that whereas Gi/o (but not Gq/11) stimulation is necessary, it does not fully activate TRPC4 unless accompanied by a simultaneous rise in intracellular free Ca2+, which may or may not require activation of the Gq/11 pathway. Paradoxically, inhibiting calmodulin also facilitated TRPC4 activation by Gi/o signaling, but not to the full extent as that facilitated by increasing intracellular Ca2+ levels, indicating an inhibitory role played by calmodulin on TRPC4 activation and a calmodulin-independent positive regulation by Ca2+. Our findings indicate that TRPC4 is a coincidence sensor of (A) Gi/o stimulation and (B) intracellular [Ca2+] rise that can be triggered by receptor-PLC pathways, lysosomal Ca2+ release, ER Ca2+ store release and plasma membrane Ca2+ influx mechanisms.

The Kontsevich integral and re-normalized link invariants arising from Lie superalgebras

We show that the coefficients of the re-normalized link invariants of the paper Multivariable link invariants arising from Lie superalgebras of type I are Vassiliev invariants which give rise to a canonical family of weight systems.

Scaffold State Switching Amplifies, Accelerates, and Insulates Protein Kinase C Signaling

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.

NEURAL NETWORK AIDED GLITCH-BURST DISCRIMINATION AND GLITCH CLASSIFICATION

We investigate the potential of neural-network based classifiers for discriminating gravitational wave bursts (GWBs) of a given canonical family (e.g. core-collapse supernova waveforms) from typical transient instrumental artifacts (glitches), in the data of a single detector. The further classification of glitches into typical sets is explored. In order to provide a proof of concept, we use the core-collapse supernova waveform catalog produced by H. Dimmelmeier and co-Workers, and the data base of glitches observed in laser interferometer gravitational wave observatory (LIGO) data maintained by P. Saulson and co-Workers to construct datasets of (windowed) transient waveforms (glitches and bursts) in additive (Gaussian and compound-Gaussian) noise with different signal-to-noise ratios (SNR). Principal component analysis (PCA) is next implemented for reducing data dimensionality, yielding results consistent with, and extending those in the literature. Then, a multilayer perceptron is trained by a backpropagation algorithm (MLP-BP) on a data subset, and used to classify the transients as glitch or burst. A Self-Organizing Map (SOM) architecture is finally used to classify the glitches. The glitch/burst discrimination and glitch classification abilities are gauged in terms of the related truth tables. Preliminary results suggest that the approach is effective and robust throughout the SNR range of practical interest. Perspective applications pertain both to distributed (network, multisensor) detection of GWBs, where some intelligence at the single node level can be introduced, and instrument diagnostics/optimization, where spurious transients can be identified, classified and hopefully traced back to their entry points.

JFEC Invited Paper: Gaussian Macro-Finance Term Structure Models with Lags

This paper develops a new family of Gaussian macro-dynamic term structure models (MTSMs) in which bond yields follow a low-dimensional factor structure and the historical distribution of bond yields and macroeconomic variables is characterized by a vector-autoregression with order p > 1. Most formulations of MTSMs with p > 1 are shown to imply a much higher dimensional factor structure for yields than what is called for by historical data. In contrast, our \asymmetric arbitrage-free MTSM gives modelers the exibility to match historical lag distributions with p > 1 while maintaining a parsimonious factor representation of yields. Using our canonical family of MTSMs we revisit: (i) the impact of no-arbitrage restrictions on the joint distribution of bond yields and macro risks, comparing models with and without the restriction that macro risks are spanned by yield curve information; and (ii) the identication of the policy parameters in Taylor-style monetary policy rules within MTSMs with macro risk factors and lags.

Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum

Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel®, which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in α-l-arabinofuranosidases (α-l-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family α-l-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to microcrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50°C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40°C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3–5°C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.

DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios

Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355–369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225–17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein–protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-013-0448-5) contains supplementary material, which is available to authorized users.

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.