Cláudia L. Silva

Solubilization of Poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)]fluorene-2,7-diyl} in Water by Nonionic Amphiphiles

In the presence of the nonionic alkyloxyethylene surfactant n-dodecylpentaoxyethylene glycol ether (C12E5), the anionic conjugated polyelectrolyte (CPE) poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)]fluorene-2,7-diyl} (PBS-PFP) dissolves in water, leading to a blue shift in fluorescence and dramatic increases in fluorescence quantum yields above the surfactant critical micelle concentration (cmc). No significant changes were seen with a poly(ethylene oxide) of similar size to the surfactant headgroup, confirming that specific surfactant-polyelectrolyte interactions are important. From UV-visible and fluorescence spectroscopy, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and electrical conductivity, together with our published NMR and small-angle neutron scattering (SANS) results, we provide a coherent model for this behavior in terms of breakup of PBS-PFP clusters through polymer-surfactant association leading to cylindrical aggregates containing isolated polymer chains. This is supported by molecular dynamics simulations, which indicate stable polymer-surfactant structures and also provide indications of the tendency of C12E5 to break up polymer clusters to form these mixed polymer-surfactant aggregates. Radial electron density profiles of the cylindrical cross section obtained from SAXS results reveal the internal structure of such inhomogeneous species. DLS and cryo-TEM results show that at higher surfactant concentrations the micelles start to grow, possibly partially due to formation of long, threadlike species. Other alkyloxyethylene surfactants, together with poly(propylene glycol) and hydrophobically modified poly(ethylene glycol), also solubilize this polymer in water, and it is suggested that this results from a balance between electrostatic (or ion-dipole), hydrophilic, and hydrophobic interactions. There is a small, but significant, dependence of the emission maximum on the local environment.

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose : A simple analysis by 13C NMR of urinary menthol glucuronide

Menthol glucuronide was isolated from the urine of a healthy 70‐kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U‐13C]glucose. Glucuronide 13C‐excess enrichment levels were 4–6% and thus provided high signal‐to‐noise ratios (SNRs) for confident assignment of 13C‐13C spin‐coupled multiplet components within each 13C resonance by 13C NMR. The [U‐13C]glucuronide isotopomer derived via direct pathway conversion of [U‐13C]glucose to [U‐13C]UDP‐glucose was resolved from [1,2,3‐13C3]‐ and [1,2‐13C2]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U‐13C]glucose. In a second study, a group of four overnight‐fasted patients (63 ± 10 kg) with severe heart failure were given peppermint oil and infused with [U‐13C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady‐state plasma [U‐13C]glucose enrichment of 4.6% ± 0.6%. Menthol glucuronide was harvested and glucuronide 13C‐isotopomers were analyzed by 13C NMR. [U‐13C]glucuronide enrichment was 0.6% ± 0.1%, and the sum of [1,2,3‐13C3] and [1,2‐13C2]glucuronide enrichments was 0.9% ± 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% ± 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% ± 10% of GP. Magn Reson Med, 2006.

Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects

During feeding, dietary galactose is a potential source of hepatic glycogen synthesis; but its contribution has not been measured to date. In the presence of deuterated water ((2)H(2)O), uridine diphosphate (UDP)-glucose derived from galactose is not enriched, whereas the remainder derived from glucose-6-phosphate (G6P) is enriched in position 2 to the same level as body water, assuming complete G6P-fructose-6-phosphate (F6P) exchange. Hence, the difference between UDP-glucose position 2 and body water enrichments reflects the contribution of galactose to glycogen synthesis relative to all other sources. In study 1, G6P-F6P exchange in 6 healthy subjects was quantified by supplementing a milk-containing breakfast meal with 10 g of [U-(2)H(7)]glucose and quantifying the depletion of position 2 enrichment in urinary menthol glucuronide. In study 2, another 6 subjects ingested (2)H(2)O and acetaminophen followed by an identical breakfast meal with 10 g of [1-(13)C]glucose to resolve direct/indirect pathways and galactose contributions to glycogen synthesis. Metabolite enrichments were determined by (2)H and (13)C nuclear magnetic resonance. In study 1, G6P-F6P exchange approached completion; therefore, the difference between position 2 and body water enrichments in study 2 (0.20% ± 0.03% vs 0.27% ± 0.03%, P < .005) was attributed to galactose glycogenesis. Dietary galactose contributed 19% ± 3% to glycogen synthesis. Of the remainder, 58% ± 5% was derived from the direct pathway and 22% ± 4% via the indirect pathway. The contribution of galactose to hepatic glycogen synthesis was resolved from that of direct and indirect pathways using a combination of (2)H(2)O and [1-(13)C]glucose tracers.

Sources of hepatic glycogen synthesis during an oral glucose tolerance test: Effect of transaldolase exchange on flux estimates

Sources of hepatic glycogen synthesis during an oral glucose tolerance test were evaluated in six healthy subjects by enrichment of a 75‐g glucose load with 6.67% [U‐13C]glucose and 3.33% [U‐2H7]glucose and analysis of plasma glucose and hepatic uridine diphosphate–glucose enrichments (sampled as urinary menthol glucuronide) by 2H and 13C nuclear magnetic resonance. The direct pathway contribution, as estimated from the dilution of [U‐13C]glucose between plasma glucose and glucuronide, was unexpectedly low (36 ± 5%). With [U‐2H7]glucose, direct pathway estimates based on the dilution of position 3 2H‐enrichment between plasma glucose and glucuronide were significantly higher (51 ± 6%, P = 0.05). These differences reflect the exchange of the carbon 4, 5, and 6 moiety of fructose‐6‐phosphate and glyceraldehyde‐3‐phosphate catalyzed by transaldolase. As further evidence of this exchange, 2H‐enrichments in glucuronide positions 4 and 5 were inferior to those of position 3. From the difference in glucuronide positions 5 and 3 enrichments, the fraction of direct pathway carbons that experienced transaldolase exchange was estimated at 21 ± 4%. In conclusion, the direct pathway contributes only half of hepatic glycogen synthesis during an oral glucose tolerance test. Glucose tracers labeled in positions 4, 5, or 6 will give significant underestimates of direct pathway activity because of transaldolase exchange. Magn Reson Med, 2009.

Subset-specific alterations in frequencies and functional signatures of γδ T cells in systemic sclerosis patients

Objective and designHere, we evaluated the distribution and functional profile of circulating CD27+ and CD27− γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder.Materials and methodsPeripheral blood from 39 SSc patients and 20 healthy individuals was used in this study. The TCR-γδ repertoire, cytokine production and cytotoxic signatures of circulating γδ T-cell subsets were assessed by flow cytometry. Gene expression of EOMES, NKG2D and GZMA was evaluated by quantitative RT-PCR in both purified γδ T-cell subsets.ResultsAbsolute numbers of γδ T-cell subsets were significantly decreased in SSc groups, likely reflecting their mobilization to the inflamed skin. Both γδ T-cell subsets preserved their relative proportions and Th1-type cytokine responses. However, cytotoxic properties showed significant disease-associated and subset-specific changes. SSc patients exhibited increased percentages of CD27+ γδ T cells expressing granzyme B or perforin and upregulated GZMA expression in diffuse cutaneous SSc. Conversely, EOMES and NKG2D were downregulated in both SSc γδ T-cell subsets vs. normal controls. Interestingly, patients with pulmonary fibrosis showed a biased TCR repertoire, with a selected expansion of effector Vγ9+ γδ T cells associated with increased frequency of cells expressing granzyme B, but decreased IFN-γ production.ConclusionsSignificant alterations on circulating γδ T-cell subsets suggest a deregulated (increased) cytotoxic activity and thus enhanced pathogenic potential of CD27+ γδ T cells in SSc.