Dysfunction of the Salivary and Lacrimal Glands After Radioiodine Therapy for Thyroid Cancer: Results of the START Study After 6-Months of Follow-Up.

Background: Understanding of changes in salivary and lacrimal gland functions after radioactive iodine therapy (131I-therapy) remains limited, and, to date, no studies have evaluated dose-response relationships between absorbed dose from 131I-therapy and dysfunctions of these glands. This study investigates salivary/lacrimal dysfunctions in differentiated thyroid cancer (DTC) patients six months after 131I-therapy, identifies 131I-therapy-related risk factors for salivary/lacrimal dysfunctions, and assesses the relationships between 131I-therapy radiation dose and these dysfunctions. Methods: A cohort study was conducted involving 136 DTC patients treated by 131I-therapy of whom 44 and 92 patients received 1.1 and 3.7 GBq, respectively. Absorbed dose to the salivary glands was estimated using a dosimetric reconstruction method based on thermoluminescent dosimeter measurements. Salivary and lacrimal functions were assessed at baseline (T0, i.e., immediately before 131I-therapy) and six months later (T6) using validated questionnaires and salivary samplings, with and without stimulation of the salivary glands. Statistical analyses included descriptive analyses and random-effects multivariate logistic and linear regressions. Results: There was no difference between T0 and T6 in the level of parotid gland pain, nor was there difference in the number of patients with hyposalivation, but there were significantly more patients with dry mouth sensation and dry eyes after therapy compared with baseline. Age, menopause, depression and anxiety symptoms, history of systemic disease, and not taking painkillers in the past three months were found to be significantly associated with salivary or lacrimal disorders. Significant associations were found between 131I-exposure and salivary disorders adjusted on the previous variables: for example, per 1-Gy increase in mean dose to the salivary glands, odds ratio = 1.43 [CI 1.02 to 2.04] for dry mouth sensation, ß = -0.08 [CI -0.12 to -0.02] mL/min for stimulated saliva flow, and ß = 1.07 [CI 0.42 to 1.71] mmol/L for salivary potassium concentration. Conclusions: This study brings new knowledge on the relationship between the absorbed dose to the salivary glands from 131I-therapy and salivary/lacrimal dysfunctions in DTC patients six months after 131I-therapy. Despite the findings of some dysfunctions, the results do not show any obvious clinical disorders after the 131I-therapy. Nevertheless, this study raises awareness of the risk factors for salivary disorders, and calls for longer follow-up. Clinical Trials Registration: Number NCT04876287 on the public website (ClinicalTrials.gov).

Dietary Protein in Older Adults: Adequate Daily Intake but Potential for Improved Distribution.

Daily distribution of dietary protein may be important in protecting against sarcopenia, specifically in terms of per meal amounts relative to a proposed threshold for maximal response. The aims of this study were to determine total and per meal protein intake in older adults, as well as identifying associations with physical activity and sedentary behavior. Three-day food diaries recorded protein intake in 38 participants. Protein distribution, coefficient of variation (CV), and per meal amounts were calculated. Accelerometry was used to collect physical activity data as well as volume and patterns of sedentary time. Average intake was 1.14 g·kg-1·day-1. Distribution was uneven (CV = 0.67), and 79% of participants reported <0.4 g·kg-1 protein content in at least 2/3 daily meals. Protein intake was significantly correlated with step count (r = 0.439, p = 0.007) and negatively correlated with sedentary time (r = -0.456, p = 0.005) and Gini index G, which describes the pattern of accumulation of sedentary time (r = -0.421, p = 0.011). Total daily protein intake was sufficient; however, distribution did not align with the current literature; increasing protein intake may help to facilitate optimization of distribution. Associations between protein and other risk factors for sarcopenia may also inform protective strategies.

Time-resolved EPR immersion depth studies of a transmembrane peptide incorporated into bicelles.

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n=5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n=5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2δ AChR peptide, the depth of immersion was calculated to be 5.8Å and 3Å for Glu-1, and 21.7Å and 19Å for Ala-12 in the gel-phase (298K) and L(α)-phases (318K), respectively. The immersion depth values are consistent with the pitch of an α-helix and the structural model of M2δ AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2δ AChR peptide.

Electron spin resonance studies of the effects of sterilization on poly(ethylene glycol) hydrogels.

The effects of several sterilization procedures on a poly(ethylene glycol) (PEG) hydrogel have been examined by electron spin resonance (ESR) spectroscopy. The crosslinked polyurethanes were synthesized by reacting PEG with a tri-functional isocyanate. The free radical concentration of unsterilized, ethylene oxide (EtO), hydrogen peroxide (H(2)O(2)), and gamma sterilized hydrogels were monitored over time. Free radical presence was observed for all the treatments, unsterilized and sterilized PEG hydrogels. The unsterilized and the EtO sterilized samples elicited similar levels of free radical intensity whereas, the H(2)O(2) and gamma sterilized samples had a significantly higher free radical concentration. The spectra reveal overlapping resonances of a peroxy and a triphenylmethyl radical. The concentration of the free radicals increase for all the treatments over time except for the gamma sterilized sample. The increase is significantly higher in the H(2)O(2) sterilized sample. A tentative model is proposed to explain the reaction pathway leading to the production of the free radicals. The observed increases in the free radical concentrations of the EtO and hydrogen peroxide sterilized hydrogels over a five-month-period make it difficult to predict properties that are affected by free radical concentrations. In that light, gamma sterilization, that does not induce a change in free radical concentrations over a five month period, could be the sterilization method of choice for PEG hydrogels that could potentially be stored for undetermined periods of time prior to application.

Determining the topology of integral membrane peptides using EPR spectroscopy.

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.

The assembly of organic radical anions between metal-borate scaffolds.

Metal-organic frameworks based on the Pb[B(Im)(4)](+) unit form layered structures analogous to those observed in clays and double layered hydroxide minerals. These layers can act as scaffolds for the organization of anionic organic guests. In this report, we use this scaffold to assemble TEMPO and PROXYL carboxylates in the interlayer spacings of Pb[B(Im)(4)](4-carboxy-TEMPO) 1 and Pb[B(Im)(4)](3-carboxy-PROXYL)(H(2)O)2, respectively. The resultant materials are paramagnetic, and the organization of the radical units differs between the two compounds. This results in changes in electronic structure of the radical unit, as observed by EPR spectroscopy.

Magnetically aligned phospholipid bilayers with large q ratios stabilize magnetic alignment with high order in the gel and L(alpha) phases.

The magnetic alignment behavior ofbicelles (magnetically alignable phospholipid bilayered membranes) as a function of the q ratio (1,2-dihexanoyl-sn-glycerol phosphatidylcholine/1,2-dimyristoyl-sn-glycerol phosphatidylcholine mole ratio) and temperature was studied by spin-labeled X-band electron paramagnetic resonance (EPR) spectroscopy and solid-state 2H and 31P NMR spectroscopy. Well-aligned bicelle samples were obtained at 45 degrees C for q ratios between 2.5 and 9.5 in both the EPR and NMR spectroscopic studies. The molecular order of the system, S(mol), increased as the q ratio increased and as the temperature decreased. For higher q ratios (> or = 5.5), bicelles maintained magnetic alignment when cooled below the main phase transition temperature (approximately 30 degrees C when in the presence of lanthanide cations), which is the first time, to our knowledge, that bicelles were magnetically aligned in the gel phase. For the 9.5 q ratio sample at 25 degrees C, S(mol) was calculated to be 0.83 (from 2H NMR spectra, utilizing the isotopic label perdeuterated 1,2-dimyristoyl-sn-glycerol phosphatidylcholine) and 0.911 (from EPR spectra utilizing the spin probe 3beta-doxyl-5alpha-cholestane). The molecular ordering of the high q ratio bicelles is comparable to literature values of S(mol) for both multilamellar vesicles and macroscopically aligned phospholipid bilayers on glass plates. The order parameter S(bicelle) revealed that the greatest degree of bicelle alignment was found at higher temperatures and larger q ratios (S(bicelle) = -0.92 for q ratio 8.5 at 50 degrees C).

Investigating magnetically aligned phospholipid bilayers with various lanthanide ions for X-band spin-label EPR studies.

This paper reports the EPR spectroscopic characterization of a model membrane system that magnetically aligns with a variety of different lanthanide ions in the applied magnetic field (<1 T) of an X-band EPR spectrometer. The ability to align phospholipid bilayer systems is valuable because the anisotropic spectra provide a more detailed and complete description of the structural and motional properties of the membrane-associated spin label when compared to randomly dispersed EPR spectra. The nitroxide spin probe 3beta-doxyl-5alpha-cholestane (cholestane or CLS) was inserted into the bilayer discs to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine splittings. The effects of different lanthanide ions with varying degrees of magnetic susceptibility anisotropy and relaxation properties were examined. For X-band EPR studies, the minimal amounts of the Tm(3+), Yb(3+), and Dy(3+) lanthanide ions needed to align the phospholipid bilayers were determined. Power saturation EPR experiments indicate that for the sample compositions described here, the spin-lattice relaxation rate of the CLS spin label was increased by varying amounts in the presence of different lanthanide (Gd(3+), Dy(3+), Er(3+), Yb(3+), and Tm(3+)) ions, and in the presence of molecular oxygen. The addition of Gd(3+) caused a significant increase in the spin-lattice relaxation rate of CLS when compared to the other lanthanide ions tested.

Magnetically aligned phospholipid bilayers in weak magnetic fields: optimization, mechanism, and advantages for X-band EPR studies.

Our lab is developing a spin-labeled EPR spectroscopic technique complementary to solid-state NMR studies to study the structure, orientation, and dynamics of uniaxially aligned integral membrane proteins inserted into magnetically aligned discotic phospholipid bilayers, or bicelles. The focus of this study is to optimize and understand the mechanisms involved in the magnetic alignment process of bicelle disks in weak magnetic fields. Developing experimental conditions for optimized magnetic alignment of bicelles in low magnetic fields may prove useful to study the dynamics of membrane proteins and its interactions with lipids, drugs, steroids, signaling events, other proteins, etc. In weak magnetic fields, the magnetic alignment of Tm(3+)-doped bicelle disks was thermodynamically and kinetically very sensitive to experimental conditions. Tm(3+)-doped bicelles were magnetically aligned using the following optimized procedure: the temperature was slowly raised at a rate of 1.9K/min from an initial temperature being between 298 and 307K to a final temperature of 318K in the presence of a static magnetic field of 6300G. The spin probe 3beta-doxyl-5alpha-cholestane (cholestane) was inserted into the bicelle disks and utilized to monitor bicelle alignment by analyzing the anisotropic hyperfine splitting for the corresponding EPR spectra. The phases of the bicelles were determined using solid-state 2H NMR spectroscopy and compared with the corresponding EPR spectra. Macroscopic alignment commenced in the liquid crystalline nematic phase (307K), continued to increase upon slowly raising the temperature, and was well-aligned in the liquid crystalline lamellar smectic phase (318K).

Synthesis and conformational studies of a transmembrane domain from a diverged microsomal Delta(12)-desaturase.

Transmembrane domains of the acyl-coenzyme A and acyl phosphatidylcholine-utilizing desaturases may control interactions with electron transport domains, be involved in substrate specificity and/or serve as a structural foundation for the enzyme. To experimentally define these domains and as a prelude to detailed NMR studies, a segment of the microsomal Delta(12)-desaturase/acetylenase CREP-1 predicted to contain the amino-proximate transmembrane domain TM-A was chemically synthesized. A modified 9-fluorenylmethoxycarbonyl procedure was used that ensured complete deprotections at each homologation and the peptide was purified in good yield by reverse-phase high-performance liquid chromatography. Conformational studies of the hydrophobic peptide TM-A demonstrated its strong propensity for folding into an alpha-helical secondary structure. The helical content was 58-65% in aqueous solutions containing 40-80% 2,2,2-trifluoroethanol, a lipomimetic solvent, and was maximal at low temperatures. The peptide assumed a largely helical character when incorporated into phospholipid bilayers and detergent micelles. Experimental evidence is in agreement with neural network predictions that a transmembrane domain exists between residues R-44 and I-67 in this diverged Delta(12)-desaturase.