Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

Binding kinetics,potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir

Background

GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.

Methods

Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.

Results

GS-9256 and vedroprevir had measured K_i values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; K_i values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 10⁵ M^− 1 s^− 1 and 1 × 10⁶ M^− 1 s^− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10^− 5 s^− 1 and 2.6 × 10^− 4 s^− 1 were determined.

Conclusions

GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.

General Significance

The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.     

The AMPK-SIRT signaling network regulates glucose tolerance under calorie restriction conditions

Aims

SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle—the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.

Main methods

To investigate this, we used skeletal muscle-specific AMPKα1/2 knockout mice (AMPKα1/2^−/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).

Key findings

CR-AMPKα1/2^−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ^±±p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).

Significance

This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance and that these effects may be dependent on reduced SIRT1 levels.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Systemic lupus erythematosus: Molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties

Background

DNase antibodies can play an important role in the pathogenesis of different autoimmune pathologies.

Methods

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) was used. The small pools of phage particles displaying DNA binding light chains with different for DNA were isolated by affinity chromatography on DNA-cellulose and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Forty-five of 451 individual colonies were randomly chosen for a study of MLChs with DNase activity. The clones were expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography followed by gel filtration, and studied in detail.

Results

Fifteen of 45 MLChs efficiently hydrolyzed DNA, and fourteen of them demonstrated various optimal concentrations of KCl or NaCl in a 1–100 mM range and showed one or two pH optima in a 4.8–9.1 range. All MLChs were dependent on divalent metal cations: the ratio of relative DNase activity in the presence of Mn^2 +, Ca^2 +, Mg^2 +, Ni^2 +, Zn^2 +, Cu^2 +, and Co^2 + was individual for each MLCh preparation. Fourteen MLChs demonstrated a comparable affinity for DNA (260–320 nM), but different k_cat values (0.02–0.7 min^− 1).

Conclusions

These observations suggest an extreme diversity of DNase abzymes from SLE patients.

General significance

SLE light chain repertoire can serve as a source of new types of DNases.     

Contribution of PTHrP to mechanical strain-induced fibrochondrogenic differentiation in entheses of Achilles tendon of miniature pigs

Background

Fibrochondrocytes are involved in entheses repair, but their response to mechanical strain (MS) is ill known.

Objective

To determine if parathyroid hormone-related protein (PTHrP) expression in fibrochondrocytes from fibrocartilaginous entheses is modulated by MS, and to further observe the regulatory effects of human (h) PTHrP on fibrochondrocyte differentiation.

Methods

Fibrochondrocytes from fibrocartilaginous entheses of Guizhou miniature pig?s Achilles tendon were submitted or not to MS (4%, 8% or 12% cyclic tensile strain; 1 Hz). Fibrochondrocytes were also exposed to: cyclopamine (Indian hedgehog (Ihh) inhibitor) (10 μM), hPTHrP (10 nM) or cyclopamine/hPTHrP (cyclopamine 10uM+hPTHrP 10 nM). Types I, II and X collagen and PTHrP expressions were measured by real-time RT-PCR and Western blot.

Result

Under 4% strain load for 12 h, types I and II collagen mRNA expressions were increased (+324% and +659%, P<0.001), while type X collagen was decreased (−89%, P<0.001). At 12%, types I and II collagen mRNA expressions were decreased (−62% and −62%, P<0.001), while type X collagen was increased (+375%, P<0.05). Under 4% strain load, PTHrP mRNA expression was increased in relation with strain duration (from 3 to 12 h: +168%, P<0.001), while at 12%, PTHrP expression decreased with time (from 3 to 12 h: −81%, P<0.001). Using cyclopamine for 24 h, PTHrP mRNA expression was significantly decreased (−88%, P<0.05), types I and II collagen were decreased (−90% and −82%, P<0.001), and type X collagen was increased (+261%, P<0.001).

Conclusions

Dynamic MS modulate PTHrP expressions. Thus, PTHrP might play an important role in fibrochondrocyte differentiation, indirectly revealing a role in entheses? formation and repair.     

Transferrin receptor-1 iron-acquisition pathway — Synthesis,kinetics, thermodynamics and rapid cellular internalization of a holotransferrin–maghemite nanoparticle construct

Background

Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy?

Methods

Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe₂) by amide bonds (TFe₂–NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe₂–NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy.

Results

In-vitro, R1 interacts with TFe₂–NP with an overall dissociation constant K_D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe₂–NP interacts with R1 in 50 μs: second-order rate constant, k₁ = 6 × 10¹⁰ M^− 1 s^− 1; first-order rate constant, k_− 1 = 9 × 10⁴ s^− 1; dissociation constant, K_1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe₂. In HeLa cells, TFe₂–NP is internalized in the cytosol in less than 15 min.

Conclusion

This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin.

General significance

TFe₂–NP behaves as free TFe₂ and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM,FTIR and electrocatalysis

Background

Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes.

Methods

In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry.

Results

Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K_M values of 52 μM and 27 μM, with V_max of 8 nmol min^− 1 mg^− 1 and 4 nmol min^− 1 mg^− 1, respectively, which are in agreement with data obtained with the microsomal enzyme.

Conclusions

The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme.

General significance

This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase

Background

The p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α.

Methods

A tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry.

Results

The percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the K_D and IC₅₀ of the selective peptide as 7.22 × 10^− 9 M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC₅₀ value of 10 μM and induced apoptosis by activating Caspase 3 and 7.

Conclusions

VWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC.

General significance

VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.     

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

Background

Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.

Methods

We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10^− 8 M and 10^− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.

Results

Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10^− 8 M and 10^− 5 M BPA, respectively, for 24 h. Exposure to 10^− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10^− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.

Conclusions

Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.

General significance

Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Population-based and family-based studies on the protein tyrosine phosphatase non-receptor 22 gene polymorphism and type 1 diabetes: A meta-analysis

Purpose

Studies investigating the association between PTPN22 gene C1858T polymorphism and type 1 diabetes (T1D) susceptibility among Caucasian population have reported conflicting results. To investigate this inconsistency, we performed a meta-analysis of all available studies dealing with the relationship between the PTPN22 C1858T polymorphism and T1D.

Methods

Databases including PubMed, Web of Science, and EMBASE were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

In total, 33 population-based studies with 22, 485 cases and 35, 292 controls, 9 family-based studies involving 7276 families were included. Under the random-effects model, the per-allele overall OR of the C1858T polymorphism for T1D was 1.89 (95% CI: 1.76–2.02, P < 10^− 5) by pooling all available case–control studies. In addition, we found significant evidence for overtransmission of the risk T allele in family-based studies (overall OR _TDT = 1.58, 95% CI: 1.43–1.74; P < 10^− 5). The summary OR from case–control and family-based association studies was 1.81 (95% CI: 1.70–1.93, P < 10^− 5).

Conclusions

In conclusion, this meta-analysis suggests that C1858T polymorphism in PTPN22 is associated with elevated T1D risk among Caucasian population.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

Genetic association between IL-17F gene polymorphisms and the pathogenesis of Graves' Disease in the Han Chinese population

Background

Graves' Disease (GD) is a common and complex disorder, with a strong hereditary component. IL-17F is a potent cytokine and a potential contributor to the etiology of various human autoimmune diseases. In the present study, we focused on the relationship between polymorphisms in the IL-17F gene and GD susceptibility through a case–control association study in two independent Chinese cohorts.

Methods

Our pilot study was performed on a cohort from Shanghai, which included 757 GD patients and 741 healthy controls. Our replication cohort was from Xiamen, consisting of 434 GD patients and 420 healthy controls. We selected four tag SNPs (rs763780, rs2397084, rs9463772 and rs761167) within the IL-17F gene to conduct a genotyping analysis.

Results

In the Shanghai cohort, the rs9463772 polymorphism showed a significant association with GD and Graves' Disease-associated Ophthalmopathy (GO) patients (P_allele = 7 × 10^− 5 and 7.4 × 10^− 3 for GD and GO patients, respectively). The rs763780 polymorphism was found to have only a difference in genotype distribution between GD individuals and healthy controls (P = 0.017). In the replication study, we confirmed the association between the rs9463772 polymorphism and GD susceptibility. Haplotype analysis showed that the haplotype of the four SNPs (GCTT) was associated with a significant risk of GD in the Shanghai cohort (P = 7.9 × 10^− 3).

Conclusion

Our results suggest that polymorphisms in the IL-17F gene increase the risk of Graves' Disease and that IL-17F is therefore a good candidate gene for Graves' Disease prediction in the Han Chinese population.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.     

Association of functional FEN1 genetic variants and haplotypes and breast cancer risk

Aim

As a tumor suppressor, FEN1 plays an essential role in preventing tumorigenesis. Two functional germline variants (-69G > A and 4150G > T) in the FEN1 gene have been associated with DNA damage levels in coke-oven workers and multiple cancer risk in general populations. However, it is still unknown how these genetic variants are involved in breast cancer susceptibility.

Methods

We investigated the association between these polymorphisms and breast cancer risk in two independent case–control sets consisted of a total of 1100 breast cancer cases and 1400 controls. The influence of these variations on FEN1 expression was also examined using breast normal tissues.

Results

It was found that the FEN1-69GG genotypes were significantly correlated to increased risk for developing breast cancer compared with the -69AA genotype in both sets [Jinan set: odds ratios (OR) = 1.41, 95% confidence interval (CI) = 1.20–1.65, P = 1.9×10^− 5; Huaian set: OR = 1.51, 95% CI = 1.22–1.86, P = 1.7×10^− 4]. Similar results were observed for 4150G > T polymorphism. The genotype–phenotype correlation analyses demonstrated that the -69G or 4150G allele carriers had more than 2-fold decreased FEN1 expression in breast tissues compared with -69A or 4150T carriers, suggesting that lower FEN1 expression may lead to higher risk for malignant transformation of breast cells.

Conclusion

Our findings highlight FEN1 as an important gene in human breast carcinogenesis and genetic variants in FEN1 confer susceptibility to breast cancer.     

Inhibition of TNF-α, and NF-κB and JNK pathways accounts for the prophylactic action of the natural phenolic,allylpyrocatechol against indomethacin gastropathy

Background

The gastro-intestinal disorders, induced by the NSAIDs including indomethacin (IND) remain unresolved medical problems. Herein, we disclose allylpyrocatechol (APC) as a potential agent against IND-gastropathy and rationalize its action mechanistically.

Methods

Mice were pre-treated with APC for 1 h followed by IND (18 mg kg^− 1) administration, and the ulcer-prevention capacity of APC was evaluated on the 3rd day by histology. Its effect on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COX, prostaglandins, growth factors and their receptors) and signaling parameters (NF-κB and MAPKs) were assessed by immunoblots/mRNA, and ELISA at the time points of their maximal changes due to IND administration.

Results

IND induced oxidative stress, triggering mucosal TNF-α that activated NF-κB and JNK MAPK signaling in mice. These increased the pro-inflammatory biochemical parameters, but reduced the healing factors. APC reversed all the adverse effects to prevent gastric ulceration. APC (5 mg kg^− 1), trolox (50 mg kg^− 1) and NAC (250 mg kg^− 1) showed similar protection that was better than that by misoprostol (5 μg kg^− 1) and omeprazole (3 mg kg^− 1).

Conclusions

The anti-ulcer effect of APC can be primarily attributed to its antioxidant action that helped in controlling various inflammatory parameters and augmenting angiogenesis.

General significance

Given that APC is an effective, non-toxic antioxidant with appreciable natural abundance, further evaluation of its pharmacokinetics and dynamics would help in promoting it as a new anti-inflammatory agent.