Daidzein and genistein are converted to equol and 5-hydroxy-equol by human intestinal Slackia isoflavoniconvertens in gnotobiotic rats

Intestinal conversion of the isoflavone daidzein to the bioactive equol is exclusively catalyzed by gut bacteria, but a direct role in equol formation under in vivo conditions has not yet been demonstrated. Slackia isoflavoniconvertens is one of the few equol-forming gut bacteria isolated from humans and, moreover, it also converts genistein to 5-hydroxy-equol. To demonstrate the isoflavone-converting ability of S. isoflavoniconvertens in vivo, the metabolization of dietary daidzein and genistein was investigated in male and female rats harboring a simplified human microbiota without (control) or with S. isoflavoniconvertens (SIA). Feces, urine, intestinal contents, and plasma of the rats were analyzed for daidzein, genistein, and their metabolites. Equol and 5-hydroxy-equol were found in intestinal contents, feces, and urine of SIA rats but not in the corresponding samples of the control rats. 5-Hydroxy-equol was present at much lower concentrations than equol and the main metabolite produced from genistein was the intermediate dihydrogenistein. The plasma of SIA rats contained equol but no 5-hydroxy-equol. Equol formation had no effect on plasma concentrations of the insulin-like growth factor I. The concentrations of daidzein and genistein were considerably lower in all samples of the SIA rats than in those of the control rats. Male SIA rats had higher intestinal and fecal concentrations of the isoflavones and their metabolites than female SIA rats. The observed activity in the rat model indicates that S. isoflavoniconvertens is capable of contributing in vivo to the bioactivation of daidzein and genistein by formation of equol and 5-hydroxy-equol.     

mTOR-dependent translation amplifies microglia priming in aging mice

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB–dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.     

Heat shock protein 70 and tumor‐infiltrating NK cells as prognostic indicators for patients with squamous cell carcinoma of the head and neck after radiochemotherapy: A multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group (DKTK‐ROG)

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre‐activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor‐infiltrating CD56⁺ NK cells in formalin‐fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin‐based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3–4) showed significantly decreased overall survival (OS; p = 0.008), local progression‐free survival (LPFS; p = 0.034) and distant metastases‐free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0–2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16‐ (p = 0.001), p53‐ (p = 0.0003) and HPV16 DNA‐negative (p = 0.001) tumors. The absence or low numbers of tumor‐infiltrating CD56⁺ NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor‐infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor‐infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers.     

Immunglobulin repertoire analysis provides new insights into the immunopathogenesis of Sjögren's syndrome

This review focuses on the use of immunglobulin (Ig) variable region genes by B cells from patients with primary Sj?gren's syndrome (pSS) and the biologic insights that this provides. Comparison of the Ig repertoire from the blood and parotid gland of pSS patients with that of normal donors suggests that there are typical disturbances of B cell homeostasis with depletion of memory B cells from the peripheral blood and accumulation/retention of these antigen-experienced B cells in the inflamed tissue. Although there are clonally expanded B cells in the parotid gland, generalized abnormalities in the B cell repertoire are also found in pSS patients. The vast majority of the current data indicate that there is no major molecular abnormality in generating the IgV chain repertoire in patients with pSS. In contrast, disordered selection leads to considerable differences in the V(L) gene usage and V(H) CDR3 length of the B cell Ig repertoire in pSS patients. The nature of the influences that lead to disordered selection in pSS remains to be determined, but should provide important clues to the etiology of this autoimmune inflammatory disorder.     

Challenges and strategies for coping with scleroderma: implications for a scleroderma-specific self-management program

Abstract

Purpose: The purpose of this study was to explore challenges faced by patients with systemic sclerosis, also called scleroderma, in coping with their disease and the strategies they used to face those challenges.

Method: Five focus groups were held with scleroderma patients (4 groups, n?=?34) and health care professionals who have experience treating scleroderma (1 group, n?=?8). Participants’ discussions were recorded, transcribed and analyzed using thematic analysis.

Results: Participants reported challenges accessing information (e.g., knowledgeable specialists), dealing with negative emotions (e.g., stress due to misunderstandings with loved ones), and accessing resources (e.g., helpful products or devices). Strategies for overcoming challenges were also discussed (e.g., advocating for own needs).

Conclusion: When faced with significant challenges while coping with scleroderma, patients develop strategies to manage better and improve their quality of life. To help them cope, patients would benefit from easier access to supportive interventions, including tailored scleroderma self-management programs. Although the challenges experienced by patients with scleroderma are unique, findings from this study might help better understand patients’ perspectives regarding coping and disease management for other chronic diseases as well.

• Implications for Rehabilitation

• People living with rare diseases, including the rare autoimmune disease scleroderma, face unique challenges and often do not have access to disease-specific educational or other support resources.

• People with scleroderma report that they face challenges in accessing information, including knowledgeable healthcare providers; managing difficult social interactions and negative emotions; and accessing resources.

• Strategies employed by scleroderma patients to overcome these challenges include seeking connections to other people with scleroderma or scleroderma patient organizations, actively seeking out local resources, and learning to communicate and advocate more effectively.

• Rehabilitation professionals can support people with scleroderma by providing them with information on connecting with scleroderma patient organizations or by facilitating local patient support networks.

     

Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet–triplet energy transfer at high pressure to determine volume changes associated with the helix–coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (−0.23 cm³ per mol = −0.38 ų per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm³/mol (3.7 ų per molecule) for adding and 2.4 cm³/mol (4.0 ų per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.Understanding the effect of pressure on protein stability and dynamics provides insight into fundamental principles and mechanisms of protein folding (1). For most proteins, increasing pressure shifts the folding equilibrium toward the unfolded state, which, according to Le Chatelier’s principle, shows that the native state has a larger volume than the unfolded state (2–4). The origin of the volume increase upon folding has been discussed controversially for a long time. The major obstacle in the interpretation of volume changes is opposing contributions from different effects. Analysis of high-resolution X-ray structures suggested that formation of intramolecular hydrogen bonds and van der Waals interactions in the native state lead to a decrease in atomic volumes and thus to a decrease in protein volume upon folding (5). Further, water around hydrophobic groups has a larger volume than bulk water, which also leads to a decrease in volume upon burial of hydrophobic groups in the native protein (6). On the other hand, formation of ordered water structures around charged groups (7) and solvation of the peptide backbone decrease the water volume (6), which leads to a volume increase upon folding. Recent experimental results suggested that volume changes associated with transfer of groups from solvent to the protein interior upon folding are small and that the formation of void volumes in native proteins is the major origin of the volume increase upon folding (8).Volume changes associated with the formation of protein folding transition states are only poorly characterized. High-pressure stopped-flow experiments on tendamistat (9) and cold shock protein (10), as well as pressure-jump experiments on cold-shock protein (10) and an ankyrin repeat domain (11), revealed that volumes of protein folding transition states are close to the volume of the native state and may even exceed the native state volume under some conditions (9–11). Similar results were found for the folding and unfolding reactions of ribonuclease A (12), which are both complex and dominated by kinetic coupling between slow prolyl isomerization and protein folding reactions (13–16). All folding and unfolding reactions of RNase A reveal positive activation volumes, indicating that the transition state volumes are larger than those of the native and unfolded state (12).To understand the origin of volume changes associated with protein folding it is important to dissect contributions of intramolecular interactions, such as secondary structure formation, from contributions of packing deficiencies in the native state. Volume changes associated with the formation of protein α-helices can be obtained by studying the effect of pressure on stability and folding–unfolding dynamics of α-helical peptides. Alanine-based peptides form helical structures with intramolecular hydrogen bonds and solvent-accessible side chains and are thus well-suited to study the effect of pressure on secondary structure formation in the absence of void volumes formed in the protein core of native proteins. The effect of pressure on the stability of alanine-based helical peptides has been addressed both experimentally and in simulations albeit with different conclusions. FTIR experiments revealed an increase in helix stability with increasing pressure, indicating a smaller volume of the helical state compared with the unfolded state (17). Molecular dynamics simulations, in contrast, suggested that the unfolded state of Ala-based peptides has a slightly smaller volume than the helical state (18). The simulations further revealed a change in geometry and length of hydrogen bonds with increasing pressure, which should have an effect on FTIR bands and NMR chemical shifts. Changes in hydrogen bond length in protein secondary structures with increasing pressure were experimentally confirmed in high-pressure NMR studies on ubiquitin (19).To determine the effect of pressure on stability and dynamics of α-helices in the absence of tertiary structure and without contributions from changes in spectroscopic properties, we measured triplet–triplet energy transfer (TTET) at various pressures in an Ala-based 21-amino-acid helical peptide. TTET coupled to a folding–unfolding equilibrium yields information on local conformational stability and dynamics on the nanoseconds to microseconds time scale (20–22). Intrachain TTET in polypeptide chains occurs through loop formation and is based on van der Waals contact between a triplet donor and a triplet acceptor group (23, 24). If the triplet labels are introduced in proteins and peptides at sites that are not in contact in the folded structure, unfolding in the region between the labels has to occur before TTET. The resulting TTET kinetics yield information on the local folding–unfolding dynamics and on the local stability in the region between the TTET labels (20–22). In our previous work we introduced the triplet donor xanthone (Xan) and the triplet acceptor naphthylalanine (Nal) into α-helical peptides with i,i+6 spacing, which places them at opposite sides of the helix and prevents TTET in the helical state (Fig. 1). Thus, at least partial unfolding of the helix in the region between the labels is required before TTET can occur (20, 22). The overall reaction can be described by the three-state model shown in Fig. 1. Because the folding–unfolding dynamics and loop formation occur on a similar time scale (20, 22) and both the folded and the unfolded state are populated to significant amounts in equilibrium, the two observable rate constants λ₁ and λ₂ for TTET and their corresponding amplitudes A₁ and A₂ yield the rate constants for local helix formation and unfolding between the labels k_f and k_u, as well as the rate constant for loop formation (k_c) (SI Text). In addition, the local equilibrium constant (K_eq) for helix stability in the region between the labels can be obtained from K_eq = k_f/k_u. TTET experiments do not require perturbation of the helix–coil equilibrium and thus yield information on equilibrium fluctuations of the helix. We have previously investigated local stability and dynamics in various regions of the 21-amino-acid Ala-based helical peptide, which showed a position-independent helix formation rate constant (k_f) and a position-dependent helix unfolding rate constant (k_u) with faster unfolding at the termini compared with the center (20). This results in higher local helix stability in the center compared with the ends, which is expected from helix–coil theory based on a linear Ising model and in agreement with previous results from hydrogen–deuterium exchange experiments (25).Open in a separate windowFig. 1.Schematic representation of TTET coupled to a helix–coil equilibrium. The equilibrium between a folded (F) and an unfolded or partially unfolded (U) conformation between the labels is monitored by fast and irreversible TTET through loop formation in the ensemble of unfolded conformations (U*). The triplet labels xanthonic acid (Xan, blue) and 1-naphthylalanine (Nal, red) are placed on opposite sides in the central region of the helix with i,i+6 spacing.Because TTET coupled to the helix–coil transition gives information on local equilibrium constants, independent of changes in spectroscopic properties of the helix, this method is perfectly suited to study the effect of pressure on local helix stability. In addition, pressure-dependent TTET experiments yield the activation volumes for helix folding and unfolding. Here, we investigate the effect of pressures on stability and dynamics of the α-helix–coil transition in the center of the 21-amino-acid Ala-based peptide using a high-pressure laserflash setup. The results reveal that the helical structure becomes stabilized with increasing pressure indicating a volume decrease upon helix formation. The dynamics of helix formation and unfolding both slow down with increasing pressure, which shows that the transition state for growth and shrinking of the helical structure has a larger volume than both helical and unfolded state and points at a non-hydrogen-bonded transition state structure.     

Factors Influencing Parent Reports on Quality of Life for Children with Asthma

Children and parents often differ in their perceptions of a complex disease such as asthma. This ancillary study of children with mild to moderate asthma that was conducted at four of the eight clinics in the Childhood Asthma Management Program had two aims: (1) to relate quality of life to asthma symptoms, sociodemographic characteristics, child psychosocial adjustment and family social support and (2) to relate agreement between child- and parent-reported quality of life to child age. For this study participants completed the Caregiver's Pediatric Asthma Quality of Life Questionnaire (C-PAQLQ), Pediatric Asthma Quality of Life Questionnaire, and a battery of psychosocial health outcomes questionnaires at the 12-month follow-up. The average asthma symptom score from diary cards for the 2 weeks before the visit was also collected. Parent-reported family burden was the strongest correlate of C-PAQLQ scores. Although the average asthma symptom score was not associated with parent-reported quality of life, the study had low power to detect a relationship because of the few episodes of asthma reported during the assessment period. The correlation between child and parent reported quality of life improved with increasing age of the child, suggesting increased child-parent agreement about quality of life outcomes was associated with increasing age of the child. Family characteristics, such as degree of cohesion among family members, are not associated with quality of life ratings.     

Balancing diversity and tolerance: lessons from patients with systemic lupus erythematosus

The autoimmune disease systemic lupus erythematosus (SLE) is caused by a failure of B cell tolerance. Recent studies in mouse models of SLE have identified several distinct tolerance checkpoints that must each function appropriately to protect against disease. However, studies of B cell repertoire selection in humans are essential to understand which checkpoints are defective in human autoimmune diseases.     

Framing health literacy: A comparative analysis of national action plans

Population and individual deficits in health literacy, and their associated negative health outcomes, have received growing attention in the political arena in recent years. In order to respond to the problem, several governments have adopted national action plans, which outline strategies to improve health literacy. Drawing on the action plans of the USA, Australia, Scotland, and Wales and applying Entman’s concept of framing, this paper analyses how health literacy debates are framed within the political arena as well as the factors that influence framing. Analysing data from policy documents and in-depth expert interviews, this paper identifies relevant frames developed to (i) define the problem of limited health literacy, (ii) provide causal explanations, (iii) rationalise why health literacy requires political action, and (iv) present solutions. The findings indicate that the malleability of the concept allows that a diversity of frames and solutions are promoted, yet risks that debates remain vague. Health literacy seems to have been successfully used to instigate political debates about health system reforms, patient empowerment, and shared decision making. The analysis suggests that health literacy might, if applied strategically, help to focus policy debates on key public health problems and the development of systemic solutions.