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.     

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome–nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP–RNC complex results in GTP-independent binding of the SRP–FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP–FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP–FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.The Escherichia coli signal recognition particle (SRP) is a complex consisting of the universally conserved protein Ffh and 4.5S RNA, which adopts a hairpin structure (1). Ffh is composed of the N-terminal domain, the G domain that harbors GTPase activity, and the C-terminal methionine-rich M domain that interacts with 4.5S RNA (2, 3) and with the signal sequence (4, 5). The N and G domains form a compact structural and functional unit termed “the NG domain.” Targeting of ribosome-nascent chain complexes (RNC) containing a signal sequence depends on the interaction of the RNC–SRP complex with the SRP receptor FtsY, which is membrane associated (6–9). FtsY and Ffh interact via their homologous NG domains and form a composite GTPase active site (10, 11). Crystal structures of the M domain reveal a hydrophobic groove used to capture signal sequences (4, 5, 12).Protein targeting is driven by highly regulated conformational rearrangements of SRP and FtsY as well as GTP hydrolysis. SRP recognizes and tightly binds to RNCs displaying a signal sequence (cargo). Next, RNC-bound SRP efficiently recruits FtsY to form a nucleotide-independent, transient early state that rearranges to a GTP-stabilized closed state (13). Ultimately, in the activated state, handover of the RNC to the Sec translocon takes place, followed by GTP hydrolysis and disassembly of the SRP–FtsY complex (14–16). These distinct conformational transitions are regulated by the ribosome and translocon in the membrane, leading to a switch from cargo recognition by SRP to cargo release (17, 18).Cryo-EM structures of bacterial SRP-bound RNCs revealed a tight cargo-recognition complex (19, 20). In the SRP–FtsY early complex an overall detachment of SRP from the ribosome was observed (21). In this state, the G domain of FtsY contacts the conserved SRP RNA tetraloop, and Ffh and FtsY interact via their N domains (21) forming a pseudosymmetric V-shaped complex positioned above the ribosomal tunnel exit. The active sites of the GTPase domains are apart from each other, explaining why the early state is inactive in GTP hydrolysis (13, 21, 22).GTP is required for SRP and FtsY to rearrange into the closed state. FRET experiments indicate that, in this state, the Ffh–FtsY NG domains adopt a conformation that resembles the intimate heterodimeric architecture observed in crystal structures (10, 11, 13). The complete SRP was crystallized in complex with the FtsY NG domain in the closed/activated state showing the NG domains docked at the distal end of the RNA hairpin (23, 24). Single-molecule total internal reflection fluorescence microscopy directly demonstrated that the Ffh–FtsY NG domains need to relocate from the tetraloop to the RNA distal end to become activated for GTP hydrolysis and to progress further in the targeting reaction (24).Although the early, closed, and activated SRP–FtsY targeting complexes have been well-characterized biochemically, the generation of distinct, conformationally homogenous closed and activated ribosome–SRP–FtsY complexes for structural studies proved to be exceedingly difficult, because the ribosome stabilizes the early state (13). We overcame this challenge by developing a robust complex preparation strategy, and describe here the cryo-EM structure of the closed state of the RNC–SRP–FtsY complex at a resolution of 5.7 Å.     

Obesity and Diabetes in Mothers and Their Children: Can We Stop the Intergenerational Cycle?

Obesity prevalence in the United States has reached an alarming level. Consequently, more young women are entering pregnancy with body mass indices of at least 30 kg/m². While higher maternal weight entering pregnancy is related to several adverse pregnancy outcomes, some of the strongest and most compelling data to date have linked prepregnancy obesity to gestational diabetes mellitus (GDM). The mechanisms by which excess maternal weight influences metabolic dysfunction in pregnancy are similar to those in obese nonpregnant women; adipocytes are metabolically active and release a number of hormones implicated in insulin resistance. Heavier mothers are also more likely to have higher glucose levels that do not exceed the cutoff for GDM, but nevertheless predict poor perinatal outcomes. Longer-term complications of GDM include increased risk of maternal type 2 diabetes and offspring obesity. Promising intervention studies to decrease the intergenerational cycle of obesity and diabetes are currently underway.     

Diversity and Structure of Intergenerational Relationships: Elderly Parent–Adult Child Relations in Korea

Korean society has undergone a rapid demographic transition that has challenged traditional patterns of family exchanges. The structure and directions of support flows have become more complex as multiple generations coexist. This article examines the complexity of contemporary Korean intergenerational relationships. The study analyzed two different samples to address anticipated differences in perceptions of and attitudes toward relationships between adult children and elderly parents. The researchers used maximum likelihood latent structure analysis to discover the latent patterns of the association among three main subdimensions of intergenerational relationships: geographic proximity, exchange of support, and cultural norms of family support. Results show that the perspectives on intergenerational relationships differ significantly between middle-aged children and elderly parents. Intergenerational relationships among middle-aged adults comprise five distinct patterns: strong reciprocal, strong traditional, intermediate normative, intermediate circumstantial, and weak. The interpretation of intergenerational relationships from the elders’ perspectives is more straightforward, with only three patterns: traditional, reciprocal, and weak. Along with significant socioeconomic differences in the prevalent patterns of intergenerational relationships, these results emphasize the complex interplay of contingency and path dependency in diversifying the value and support exchanges of intergenerational relationships.     

L596–W733 bond between the start of the S4–S5 linker and the TRP box stabilizes the closed state of TRPV4 channel

Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4–S5 linker helix. By homology modeling, we identified the corresponding L596–W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4–S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar–nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.Transient receptor potential (TRP) channels are Ca²⁺-permeable nonselective cation channels found in nearly all eukaryotes, including yeast, worm, and fly (1). Mammalian TRPs comprise seven subfamilies (2, 3), of which the vanilloid subfamily (TRPV) is perhaps best known with its founding member TRPV1 being a painful heat sensor (4). Each TRP is polymodal, receiving and integrating a variety of physical or chemical stimuli. For example, TRPV4, a TRPV1 homolog, can be activated by mild heat, cell swelling, endogenous chemicals (e.g., anandamide, arachidonic acid), and synthetic agonists (e.g., 4-αPDD, GSK1016790A) (5). How TRPs detect and integrate multiple sensory inputs is unclear. Mutations in several TRP channels are known to cause heritable diseases in human (6–8).Sequence-based algorithms indicate that TRP channels have architecture similar to that of voltage-gated cation channels such as the K⁺ channel (Kv) (9, 10). The TRPV1 structure has been determined recently by single-particle cryo-electron microscopy (cryo-EM) at 3.4-Å resolution (11, 12), providing an invaluable basis for structure–function analyses of TRP channels in general. TRPV1 is a tetramer, and the transmembrane region of each TRPV1 subunit has an arrangement of a peripheral (S1–S4) and a pore (S5–S6) domain similar to that of Kv (Fig. S1A). In Kv, the electromechanical coupling between the voltage sensor in S4 and the inner S6 gate occurs through the movement of the long α-helical S4–S5 linker that connects the peripheral and the pore domain (10). In TRPV1, this linker is a four-turn amphipathic helix beginning with F559, from which the linker juts away sharply from S4, forming an elbow that suggests a pivot.By sequence similarity, a block of ∼25 residues following S6 has been identified as the TRP domain, which is found in all mammalian TRPs except TRPAs and TRPPs (2, 3, 13). The main function of the TRP domain is unclear. It includes a highly conserved TRP box of five or six residues including an invariant tryptophan. In the cryo-EM structures of TRPV1 (11), the TRP domain forms a helix not seen in Kvs. This five-turn helix immediately follows S6 after a sharp bend and lies parallel to the inner leaflet of the bilayer membrane. It is amphipathic in nature, with its hydrophilic side facing toward the cytoplasm and with the invariant tryptophan (W697) located near the center of this TRP helix facing away from the cytoplasm. Although the TRP helix bonds with the pre-S1 and the peripheral domain of the same subunit and potentially with the S5 of a neighboring subunit, we are most intrigued by the intrasubunit hydrogen bond between the TRP box W697 and F559 at the elbow that begins the S4–S5 linker. Note that the indole ring of W697 bonds not with the side chain of F559 but with its backbone carbonyl oxygen (Fig. S1B).TRPV4 is the closet homolog of TRPV1, with 41% of sequence identity. Aside from its interesting responses to a variety of stimuli, including mechanical force, it draws attention because more than 50 TRPV4 mutations now are known to cause peripheral neuropathy and/or skeletal dysplasia (6). The interference with bone development can range from dwarfism to neonatal or even prenatal death. Our previous research suggested that these are likely to be gain-of-function (GOF) phenotypes, with disease severity corresponding to the extent of leakage caused by the increased constitutive open probability (Po) of the mutant channels (14). By homology modeling, the TRPV4 bond that corresponds to TRPV1’s W697–F559 bond is between the TRP box W733 and the carbonyl oxygen of L596 that starts the S4–S5 linker (Fig. 1 A and B and Figs. S2 and S3). Among the many human skeletal-dysplasia alleles is L596P (15), which causes Kozlowski-type spondylometaphyseal dysplasia, a clinically distinguishable form of progressive bone abnormality.Open in a separate windowFig. 1.Locations of L596 and W733 and the activity of their mutant protein channels. (A) Locations of L596 and W733 mapped onto the predicted membrane topology of a TRPV4 subunit. L596 is located at the elbow between the S4 and S4–S5 linker helix. W733 is located in the middle of TRP helix. (B) The main chain carbonyl oxygen of L596 forms a hydrogen bond with the indol side chain of W733 in the closed state of a TRPV4 homology model based on TRPV1. (C) Representative current traces from the oocytes injected with 5 ng of WT cRNA (Upper Left), 1 ng of L596P (Lower Left), or 0.1 ng of W733R cRNA (Lower Right) and an uninjected oocyte as negative control (NC, Upper Right). Oocytes were recorded 3 or 4 d after cRNA injection. Currents were evoked by voltage steps from a holding potential of −60 mV to a test potential between −100 and +60 mV in 20 mV increments (diagramed as in the Inset). (D) The mean peak currents of the negative control (gradient bar), WT (white bars), L596P (gray bar), and W733R (black bar) with different amounts of injected cRNAs. Peak currents were recorded during 400-ms depolarization to 60 mV from a holding potential of −60 mV. The numbers of oocytes tested are indicated. Data are represented as means ± SEM.We also have expanded the experimental arena to study TRP channels by functionally expressing rat TRPV4 in the budding yeast Saccharomyces cerevisiae (16). Therein strong GOF mutations can be selected that stop yeast growth, presumably by ion leakage such as cytoplasmic Ca²⁺ poisoning (17). The mutations examined have nearly saturated constitutive opening. Note that these mutations were selected, after random mutageneses, by a biological phenotype (toxicity to yeast) without a preconceived bias and were selected before detailed structural knowledge (11) that allows intelligent mutant engineering. Revealed by our homology model of TRPV4 (Figs. S2 and S3), these selected mutations occurred at key structural elements. Most interestingly, in two independent mutageneses, W773R was selected among the harvests. This finding, together with the L596P human mutation, strongly indicates that the W733–L596 bond (Fig. 1 A and B, Figs. S2 and S3, and Movie S1) is crucial in TRPV4 gating. Our examination of these and related mutations discussed below indicate that this bond stabilizes the closed and the inactivated state of TRPV4. Breaking or distorting this bond increases basal channel opening, explaining the pathologies in yeast and in human.     

A novel model used to compare water–perfused and solid–state anorectal manometry

Background Anal pressures are commonly measured using water–perfused and solid–state manometers. We constructed a dynamic model of the anus to compare the agreement and reproducibility of the two types of manometers. Methods The model system was constructed using a pig anorectum together with an inflatable bowel sphincter. The pig anorectum was mounted on a jig and the sphincter was inserted external to the internal sphincter. The sphincter pressure was adjusted over the range 20 to 185 mmHg. At each of 24 constant sphincter pressures, triplicate readings were carried out with both manometers. The first measurement by each method was used for the comparison. The replicate measurements were used to calculate measures of repeatability for each method. Results Measurements by the two manometers were highly correlated (r=0.97). Measurements by the solid state manometer were higher than the water–perfused manometer by 8.1±12.2 mmHg (mean±SD). Precision (coefficient of variation) for the solid–state manometer (2.8%) was better than for the water–perfused manometer (8.3%). Conclusions The new model of the anal canal shows promise as a tool for assessing physiological interventions. The solid–state manometer has many advantages over the water–perfused manometer, providing more consistent measurements at clinically relevant pressures.     

Iron and pregnancy—a delicate balance

The review focuses on iron balance during pregnancy and postpartum in the Western affluent societies. Iron status and body iron can be monitored using serum ferritin, haemoglobin, serum soluble transferrin receptors (sTfR) and the sTfR/ferritin ratio. Requirements for absorbed iron increase during pregnancy from 0.8 mg/day in the first trimester to 7.5 mg/day in the third trimester. Average requirement during the entire gestation is ~4.4 mg/day. Intestinal iron absorption increases during pregnancy, but women with ample body iron reserves have lower absorption than those with depleted reserves, so increased absorption is, in part, due to progressive iron depletion. Apparently, women do not change dietary habits when they become pregnant. Non-pregnant Scandinavian women have a median dietary iron intake of ~9 mg/day, i.e. more than 90% of the women have an intake below the recommended ~18 mg/day. Non-pregnant women have a low iron status, 42% have serum ferritin levels ≤30 μg/l, i.e. small or depleted iron reserves and 2–4% have iron deficiency anaemia; only 14–20% have ferritin levels >70 μg/l corresponding to body iron of ≥500 mg. The association between high haemoglobin during gestation and a low birth weight of the newborns is caused by inappropriate haemodilution. In placebo-controlled studies on healthy pregnant women, there is no relationship between the women’s haemoglobin and birth weight of the newborns and no increased frequency of preeclampsia in women taking iron supplements.     

PKC-delta and PKC-epsilon: foes of the same family or strangers?

Protein kinase C (PKC) is a family of 10 serine/threonine kinases divided into 3 subfamilies, classical, novel and atypical classes. Two PKC isozymes of the novel group, PKCε and PKCδ, have different and sometimes opposite effects. PKCε stimulates cell growth and differentiation while PKCδ is apoptotic. In the heart, they are among the most expressed PKC isozymes and they are opposed in the preconditioning process with a positive role of PKCε and an inhibiting role of PKCδ. The goal of this review is to analyze the structural differences of these 2 enzymes that may explain their different behaviors and properties.     

Monocyte–macrophage polarization balance in pre-diabetic individuals

Pre-diabetes is characterized by increased cardiovascular risk and chronic inflammation. The activation of monocyte–macrophages plays major roles in vascular biology. Herein, we aimed to analyze monocyte–macrophage polarization status in subjects with IFG and/or IGT compared with normal glucose tolerant (NGT) individuals. We enrolled 87 middle-aged individuals with low prevalence of cardiovascular disease. Based on OGTT, they were divided into 49 NGT and 38 pre-diabetic (IFG and/or IGT). Using flow cytometry analysis of peripheral blood cells, we quantified traditional monocyte subsets based on CD14 and CD16 expression as well as novel monocyte–macrophage pro-inflammatory CD68⁺CCR2⁺ M1 and anti-inflammatory CX3CR1⁺CD163⁺/CD206⁺ M2 phenotypes. The M1/M2 ratio was taken to represent the polarization balance. There were no differences in traditional classical (CD14⁺⁺CD16^?), intermediate (CD14⁺⁺CD16⁺) and nonclassical (CD14⁺CD16⁺) monocytes between groups. Rather, compared to NGT, pre-diabetic subjects showed a significant increase in pro-inflammatory M1 cells and percent expression of the oxLDL scavenger receptor CD68, without changes in anti-inflammatory M2 cells. M1 levels and CD68 expression were directly correlated with HbA_1c. We show for the first time that otherwise healthy pre-diabetic subjects have excess M1 inflammatory cells in peripheral blood, which may contribute to cardiovascular risk.     

Allergen immunotherapy: how to balance the different views from pulmonologists and allergists?

Allergen immunotherapy (AIT) is the treatment characterizing the allergological approach to respiratory allergy. Unfortunately, most available data from the literature and current practice indicate that pulmonologists do no consider AIT when choosing the treatment strategy in patients with asthma. Indeed AIT, from its introduction in 1911 to nowadays, was unceasingly improved and has accumulated clear evidence on its effectiveness. Moreover, AIT has a characteristic not shared by drugs in the capacity to modify the natural history of asthma, due to its immunologic mechanisms of actions, and thus also works after the treatment withdrawal. This also makes AIT a clearly cost-effective treatment over time. It is surprising that pulmonologists, for whom asthma is a major disease to manage, do not consider AIT when choosing the optimal treatment in single patients. The insufficient information on AIT and the availability of allergen extracts with less than good quality are likely to be the most important factors influencing such an attitude. The current development of standardized, pharmaceutical-grade products for AIT seems capable of making allergen extracts comparable to drugs and to stimulate a rethinking of AIT's role in the treatment of asthma in pulmonologists. A reappraisal of the significance of the allergen-specific bronchial challenge could represent a further factor suggesting AIT as a reliable option.     

Expression and potential role of apolipoprotein D on the death–survival balance of human colorectal cancer cells under oxidative stress conditions

Purpose

Inverse correlations of apolipoprotein D (ApoD) expression with tumor growth have been shown, therefore proposing ApoD as a good prognostic marker for diverse cancer types, including colorectal cancer (CRC). Besides, ApoD expression is boosted upon oxidative stress (OS) in many pathological situations. This study aims at understanding the role of ApoD in the progression of human CRC.

Methods

Samples of CRC and distant normal tissue (n?=?51) were assayed for levels of lipid peroxidation, expression profile of OS-dependent genes, and protein expression. Three single-nucleotide polymorphisms in the ApoD gene were analyzed (n?=?139), with no significant associations found. Finally, we assayed the effect of ApoD in proliferation and apoptosis in the CRC HT-29 cell line.

Results

In CRC, lipid peroxides increase while ApoD messenger RNA and protein decrease through tumor progression, with a prominent decrease in stage I. In normal mucosa, ApoD protein is present in lamina propia and enteroendocrine cells. In CRC, ApoD expression is heterogeneous, with low expression in stromal cells commonly associated with high expression in the dysplastic epithelium. ApoD promoter is basally methylated in HT-29 cells but retains the ability to respond to OS. Exogenous addition of ApoD to HT-29 cells does not modify proliferation or apoptosis levels in control conditions, but it promotes apoptosis upon paraquat-induced OS.

Conclusion

Our results show ApoD as a gene responding to OS in the tumor microenvironment. Besides using ApoD as marker of initial stages of tumor progression, it can become a therapeutic tool promoting death of proliferating tumor cells suffering OS.     

Frequent adaptation and the McDonald–Kreitman test

Population genomic studies have shown that genetic draft and background selection can profoundly affect the genome-wide patterns of molecular variation. We performed forward simulations under realistic gene-structure and selection scenarios to investigate whether such linkage effects impinge on the ability of the McDonald–Kreitman (MK) test to infer the rate of positive selection (α) from polymorphism and divergence data. We find that in the presence of slightly deleterious mutations, MK estimates of α severely underestimate the true rate of adaptation even if all polymorphisms with population frequencies under 50% are excluded. Furthermore, already under intermediate rates of adaptation, genetic draft substantially distorts the site frequency spectra at neutral and functional sites from the expectations under mutation–selection–drift balance. MK-type approaches that first infer demography from synonymous sites and then use the inferred demography to correct the estimation of α obtain almost the correct α in our simulations. However, these approaches typically infer a severe past population expansion although there was no such expansion in the simulations, casting doubt on the accuracy of methods that infer demography from synonymous polymorphism data. We propose a simple asymptotic extension of the MK test that yields accurate estimates of α in our simulations and should provide a fruitful direction for future studies.The relative importance of natural selection and random genetic drift in shaping molecular evolution is a matter of a longstanding dispute. Whereas the neo-Darwinian synthesis placed natural selection as the dominant force (1), from the late 1960s on it became popular to assume that the bulk of molecular variation is selectively neutral or at most weakly selected (2). The “neutral theory” of molecular evolution enabled development of analytical approaches, based on the diffusion approximation, for calculating the expected frequency spectra and fixation probabilities of polymorphisms of varying selective effect. Most of the currently available approaches for estimating selection and demography from population genetic data rest upon these results.Recent studies have strongly challenged key assumption of the neutral theory. First, in many species the rate of adaptation appears to be very high with, for example, in Drosophila melanogaster more than 50% of the amino acid changing substitutions, and similarly large proportions of noncoding substitutions, driven to fixation by positive selection (3). Importantly, it appears that frequent adaptation strongly affects the genome-wide patterns of polymorphism (3–6). These results imply that the dynamics of a given polymorphism is not only affected by genetic drift and purifying selection acting at its particular site, but also by the so-called genetic draft (7), which describes the stochastic effects generated by recurrent selective sweeps at closely linked sites. Second, there is accumulating evidence that many polymorphisms in natural populations are slightly deleterious (8–11), and such polymorphisms are expected to generate another kind of interference among linked sites, known as background selection (12, 13). It is becoming increasingly clear that the assumption of independence between sites is violated in most cases in one way or another. What we do not yet fully understand is the extent to which these violations affect population genetic methods.Here, we focus on the investigation of one of the primary methods to test the neutral theory and to estimate the rate of adaptation at the molecular level, introduced by McDonald and Kreitman in 1991 (14). The McDonald–Kreitman (MK) test contrasts levels of polymorphism and divergence at neutral and functional sites and uses this contrast to estimate the fraction of substitutions at the functional sites that were driven to fixation by positive selection. The MK test has been applied in many organisms with estimates of the rate of adaptation varying from extremely high in Drosophila (3) and Escherichia coli (15), to virtually zero in yeast (16) and humans (8, 17). These differences might reflect true variation in the rate of adaptation in different lineages or indicate that the test is biased to different extent, and possibly in different direction, in those lineages (18).By using closely interdigitated sites, the MK test is robust to many sources of error, such as variation of mutation rate across the genome and variation in coalescent histories at different genomic locations. It can be confounded, however, by slightly deleterious mutations and demography (18). Much work has thus gone into the development of sophisticated extensions of the MK test that use the frequency distribution of polymorphisms to estimate the demographic history of the organism in question, to assess the distribution of deleterious effects at the functional sites, and to correct for both in estimating the rate of adaptation (8, 16, 19–26). However, all of these extensions are still based on the assumption that evolutionary dynamics at different sites can be modeled independently of each other. In light of the recent findings that genetic draft and background selection might often be important, it is essential to verify that these methods are robust to the linkage effects from advantageous and weakly deleterious polymorphisms and their interactions.