Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.     

Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors

B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi.     

Ocean acidification leads to counterproductive intestinal base loss in the gulf toadfish (opsanus Beta)

Abstract Oceanic CO(2) has increased from 280 to 380 μatm since preindustrial times and is expected to reach 1,900 μatm by 2300. In addition, regional upwelling zones exhibit levels up to 2,300 μatm, making exploration at future global projected CO(2) levels ecologically relevant today. Recent work has demonstrated that CO(2) exposure as low as 1,000 μatm induces acidosis in toadfish (Opansus beta), leading to metabolic compensation by retention of blood [Formula: see text] in an effort to defend pH. Since increased serosal [Formula: see text] translates to increased [Formula: see text] secretion rates in isolated intestinal tissue, we predicted that blood elevation of [Formula: see text] and Pco(2) during exposure to 1,900 μatm CO(2) would increase in vivo base secretion rates. Rectal fluid and CaCO(3) excretions were collected from toadfish exposed to 380 (control) and 1,900 μatm CO(2) for 72 h. Fluids were analyzed for pH, osmolality, ionic composition, and total CO(2). Precipitated CaCO(3) was analyzed for titratable alkalinity, Mg(2+), and Ca(2+) content. Fish exposed to 1,900 μatm CO(2) exhibited higher rectal base excretion rates, higher rectal fluid [Formula: see text] (mmol L(-1)), and lower fluid Cl(-) (mmol L(-1)) than controls, suggesting increased intestinal anion exchange as a result of the compensated respiratory acidosis. This study verifies that imminent projected CO(2) levels expected by the year 2300 lead to greater intestinal [Formula: see text] loss, a process that acts against compensation for a CO(2)-induced acidosis.     

The relationship between isometric force-time curve characteristics and club head speed in recreational golfers

ABSTRACT: Leary, BK, Statler, J, Hopkins, B, Fitzwater, R, Kesling, T, Lyon, J, Phillips, B, Bryner, RW, Cormie, P, and Haff, GG. The relationship between isometric force-time curve characteristics and club head speed in recreational golfers. J Strength Cond Res 26(10): 2685-2697, 2012-The primary purpose of the present investigation was to examine the relationships between club head speed, isometric midthigh pull performance, and vertical jump performance in a cohort of recreational golfers. Twelve recreational golfers (age, 20.4 ± 1.0 years; weight, 77.0 ± 9.8 kg; height, 177.8 ± 6.3 cm; body fat, 17.1 ± 7.6%; handicap, 14.5 ± 7.3; experience, 8.9 ± 3.6 years) completed 3 testing sessions: (a) familiarization session and body composition measurements; (b) measurement of force-time curves in the isometric midthigh pull, countermovement, and static vertical jump (SJ); and (c) measurement of club head speed. During sessions 1 and 2, subjects performed 5 countermovement jumps, 5 SJ, and 2 isometric midthigh pulls. Isometric peak force was measured at 30, 50, 90, 100, 200, and 250 milliseconds. Rate of force development was measured among 0-30, 0-50, 0-90, 0-100, 0-200, and 0-250 milliseconds. Peak rate of force development was determined as the highest value in a 10-millisecond sampling windows. During session 3, subjects performed 10 maximal golf swings with a driver to measure club head speed; peak and average club head speed were analyzed across the 10 swings. Golf handicap was moderately correlated with average (r = -0.52, p = 0.04) and maximal club head speed (r = -0.45, p = 0.07). Force at 150 milliseconds during the isomeric midthigh pull test was moderately correlated with average (r = 0.46, p = 0.07) and maximal club head speed (r = 0.47, p = 0.06). Moderate correlations were also found between the rate of force development from 0 to 150 milliseconds and average (r = 0.38, p = 0.11) and maximal club head speed (r = 0.36, p = 0.12). The present findings suggest that the ability to exhibit high ground reaction forces in time frames <200 milliseconds are related to high club head speeds.     

The Effects of Juvenile Stress on Anxiety,Cognitive Bias and Decision Making in Adulthood: A Rat Model

Stress experienced in childhood is associated with an increased risk of developing psychiatric disorders in adulthood. These disorders are particularly characterized by disturbances to emotional and cognitive processes, which are not currently fully modeled in animals. Assays of cognitive bias have recently been used with animals to give an indication of their emotional/cognitive state. We used a cognitive bias test, alongside a traditional measure of anxiety (elevated plus maze), to investigate the effects of juvenile stress (JS) on adulthood behaviour using a rodent model. During the cognitive bias test, animals were trained to discriminate between two reward bowls based on a stimulus (rough/smooth sandpaper) encountered before they reached the bowls. One stimulus (e.g. rough) was associated with a lower value reward than the other (e.g. smooth). Once rats were trained, their cognitive bias was explored through the presentation of an ambiguous stimulus (intermediate grade sandpaper): a rat was classed as optimistic if it chose the bowl ordinarily associated with the high value reward. JS animals were lighter than controls, exhibited increased anxiety-like behaviour in the elevated plus maze and were more optimistic in the cognitive bias test. This increased optimism may represent an optimal foraging strategy for these underweight animals. JS animals were also faster than controls to make a decision when presented with an ambiguous stimulus, suggesting altered decision making. These results demonstrate that stress in the juvenile phase can increase anxiety-like behaviour and alter cognitive bias and decision making in adulthood in a rat model.     

Tamoxifen-induced epigenetic silencing of oestrogen-regulated genes in anti-hormone resistant breast cancer

In the present study, we have taken the novel approach of using an in vitro model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.     

High avidity cytotoxic T lymphocytes can be selected into the memory pool but they are exquisitely sensitive to functional impairment

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 μg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.