Genome-wide association identifies a common variant in the reelin gene that increases the risk of schizophrenia only in women

Sex differences in schizophrenia are well known, but their genetic basis has not been identified. We performed a genome-wide association scan for schizophrenia in an Ashkenazi Jewish population using DNA pooling. We found a female-specific association with rs7341475, a SNP in the fourth intron of the reelin (RELN) gene (p = 2.9 × 10⁻⁵ in women), with a significant gene-sex effect (p = 1.8 × 10⁻⁴). We studied rs7341475 in four additional populations, totaling 2,274 cases and 4,401 controls. A significant effect was observed only in women, replicating the initial result (p = 2.1 × 10⁻³ in women; p = 4.2 × 10⁻³ for gene-sex interaction). Based on all populations the estimated relative risk of women carrying the common genotype is 1.58 (p = 8.8 × 10⁻⁷; p = 1.6 × 10⁻⁵ for gene-sex interaction). The female-specific association between RELN and schizophrenia is one of the few examples of a replicated sex-specific genetic association in any disease.     

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL^−/−) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3^−/− and MLKL^−/− mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3^−/−, but not MLKL^−/− mice, were protected against postinjury motor and cognitive deficits at 1–4 weeks (RIPK3^−/− vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3^−/− mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24–48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.Subject terms: Molecular neuroscience, Brain injuries     

Spontaneous K-Complex Density in Slow-Wave Sleep

Purpose

To study spontaneous K-complex (KC) densities during slow-wave sleep. The secondary objective was to estimate intra-non-rapid eye movement (NREM) sleep differences in KC density.

Materials and Methods

It is a retrospective study using EEG data included in polysomnographic records from the archive at the sleep research laboratory of the Centre for Physiotherapy and Rehabilitation Sciences, Jamia Millia Islamia, India. The EEG records of 4459 minutes were used. The study presents a manual identification investigation of KCs in 17 healthy young adult male volunteers (age = 23.82±3.40 years and BMI = 23.42±4.18 kg/m²).

Results

N3 had a higher KC density than N2 (Z = -2.485, p = 0.013) for all of the probes taken together. Four EEG probes had a higher probe-specific KC density during N3. The inter-probe KC density differed significantly during N2 (χ² = 67.91, p < .001), N3 (χ² = 70.62, p < .001) and NREM (χ² = 68.50, p < .001). The percent distribution of KC decreased uniformly with sleep cycles.

Conclusion

The inter-probe differences during N3 establish the fronto-central dominance of the KC density regardless of sleep stage. This finding supports one local theory of KC generation. The significantly higher KC density during N3 may imply that the neuro-anatomical origin of slow-wave activity and KC is the same. This temporal alignment with slow-wave activity supports the sleep-promoting function of the KC.     

APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans

Myopia is the most common vision disorder and the leading cause of visual impairment worldwide. However, gene variants identified to date explain less than 10% of the variance in refractive error, leaving the majority of heritability unexplained (“missing heritability”). Previously, we reported that expression of APLP2 was strongly associated with myopia in a primate model. Here, we found that low-frequency variants near the 5’-end of APLP2 were associated with refractive error in a prospective UK birth cohort (n = 3,819 children; top SNP rs188663068, p = 5.0 × 10⁻⁴) and a CREAM consortium panel (n = 45,756 adults; top SNP rs7127037, p = 6.6 × 10⁻³). These variants showed evidence of differential effect on childhood longitudinal refractive error trajectories depending on time spent reading (gene x time spent reading x age interaction, p = 4.0 × 10⁻³). Furthermore, Aplp2 knockout mice developed high degrees of hyperopia (+11.5 ± 2.2 D, p < 1.0 × 10⁻⁴) compared to both heterozygous (-0.8 ± 2.0 D, p < 1.0 × 10⁻⁴) and wild-type (+0.3 ± 2.2 D, p < 1.0 × 10⁻⁴) littermates and exhibited a dose-dependent reduction in susceptibility to environmentally induced myopia (F(2, 33) = 191.0, p < 1.0 × 10⁻⁴). This phenotype was associated with reduced contrast sensitivity (F(12, 120) = 3.6, p = 1.5 × 10⁻⁴) and changes in the electrophysiological properties of retinal amacrine cells, which expressed Aplp2. This work identifies APLP2 as one of the “missing” myopia genes, demonstrating the importance of a low-frequency gene variant in the development of human myopia. It also demonstrates an important role for APLP2 in refractive development in mice and humans, suggesting a high level of evolutionary conservation of the signaling pathways underlying refractive eye development.     

Liveable residential space,residential density,and hypertension in Hong Kong: A population-based cohort study

BackgroundHypertension is a leading preventable risk factor of chronic disease and all-cause mortality. Housing is a fundamental social determinant of health. Yet, little is known about the impacts of liveable residential space and density on hypertension.Methods and findingsThis retrospective observational study (median follow-up of 2.2 years) leveraged the FAMILY Cohort, a large territory-wide cohort in Hong Kong, Special Administrative Region, People’s Republic of China to quantify associations of objectively measured liveable space and residential density with blood pressure outcomes among adults aged ≥16 years. Blood pressure outcomes comprised diastolic blood pressure (DBP), systolic blood pressure (SBP), mean arterial pressure (MAP), and hypertension. Liveable space was measured as residential floor area, and density was assessed using the number of residential units per building block and neighborhood residential unit density within predefined catchments. Multivariable regression models examined associations of liveable floor area and residential density with prevalent and incident hypertension. We investigated effect modifications by age, sex, income, employment status, and housing type. Propensity score matching was further employed to match a subset of participants who moved to smaller residences at follow-up with equivalent controls who did not move, and generalized linear models examined the impact of moving to smaller residences upon blood pressure outcomes. Our fully adjusted models of prevalent hypertension outcomes comprised 30,439 participants at baseline, while 13,895 participants were available for incident models at follow-up. We found that each interquartile range (IQR) increment in liveable floor area was associated with lower DBP (beta [β] = −0.269 mm Hg, 95% confidence interval [CI]: −0.419 to −0.118, p < 0.001), SBP (β = −0.317 mm Hg, −0.551 to −0.084, p = 0.008), MAP (β = −0.285 mm Hg, −0.451 to −0.119 with p < 0.001), and prevalent hypertension (odds ratio [OR] = 0.955, 0.918 to 0.993, p = 0.022) at baseline. Each IQR increment in residential units per building block was associated with higher DBP (β = 0.477 mm Hg, 0.212 to 0.742, p = <0.001), SBP (β = 0.750 mm Hg, 0.322 to 1.177, p = <0.001), MAP (β = 0.568 mm Hg, 0.269 to 0.866, p < 0.001), and prevalent hypertension (OR = 1.091, 1.024 to 1.162, p = 0.007). Each IQR increase in neighborhood residential density within 0.5-mi street catchment was associated with lower DBP (β = −0.289 mm Hg, −0.441 to −0.137, p = <0.001), SBP (β = −0.411 mm Hg, −0.655 to −0.168, p < 0.001), MAP (β = −0.330 mm Hg, −0.501 to −0.159, p = <0.001), and lower prevalent hypertension (OR = 0.933, 0.899 to 0.969, p < 0.001). In the longitudinal analyses, each IQR increment in liveable floor area was associated with lower DBP (β = −0.237 mm Hg, −0.431 to −0.043, p = 0.016), MAP (β = −0.244 mm Hg, −0.444 to −0.043, p = 0.017), and incident hypertension (adjusted OR = 0.909, 0.836 to 0.988, p = 0.025). The inverse associations between larger liveable area and blood pressure outcomes were more pronounced among women and those residing in public housing. In the propensity-matched analysis, participants moving to residences of lower liveable floor area were associated with higher odds of incident hypertension in reference to those who did not move (OR = 1.623, 1.173 to 2.199, p = 0.002). The major limitations of the study are unmeasured residual confounding and loss to follow-up.ConclusionsWe disentangled the association of micro-, meso-, and macrolevel residential densities with hypertension and found that higher liveable floor area and neighborhood scale residential density were associated with lower odds of hypertension. These findings suggest adequate housing in the form of provisioning of sufficient liveable space and optimizing residential density at the building block, and neighborhood levels should be investigated as a potential population-wide preventive strategy for lowering hypertension and associated chronic diseases.

In a cohort study, Dr. Chinmoy Sarkar and colleagues investigate the association between liveable residential space, residential density and hypertension in Hong Kong.     

Study of EEG microstates in Parkinson’s disease: a potential biomarker?

The spontaneous activity of the brain is dynamic even at rest and the deviation from this normal pattern of dynamics can lead to different pathological states. EEG microstate analysis of resting-state neuronal activity in Parkinson’s disease (PD) could provide insight into altered brain dynamics of patients exhibiting dementia. Resting-state EEG microstate maps were derived from 128 channel EEG data in 20 PD without dementia (PDND), 18 PD with dementia (PDD) and 20 Healthy controls (CON) using Cartool and sLORETA softwares. Microstate map parameters including global explained variance, mean duration, frequency of occurrence (TF) and time coverage were compared statistically among the groups. Eight maps that explained 72% of the topographic variance were identified and only three maps differed significantly across the groups. TF of Map1 was lower in both PDND and PDD (p < 0.001) and that of Map3 (p = 0.02) in PDND compared to control. Cortical sources showed higher activation in precuneus, cuneus and superior parietal lobe (Threshold: Log-F = 1.74, p < 0.05) with maximum activity in the precuneus region (MNI co-ordinates: − 25, − 75, − 40; Log-F = 1.9) in PDND compared to control only for Map1. Lower TF of Map1 (prototypical microstate D) may potentially serve as a biomarker for PD with or without dementia whereas higher activation of precuneus, cuneus and superior parietal lobe at resting-state could favour signal processing, lack of which could be associated with dementia in Parkinson’s disorder.     

Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10⁻⁶ mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10⁻¹⁹ ≤ P ≤ 4.5 × 10⁻¹⁸) and D6D (FADS2) (6.05 × 10⁻⁸ ≤ P ≤ 4.20 × 10⁻⁷) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10⁻¹⁶). The significance of haplotype association with D5D activity (P = 2.19 × 10⁻¹⁷) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10⁻²⁸) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10⁻⁹) and decreased D6D activity (P = 9.16 × 10⁻⁶) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.     

Morphine Induces Hyperalgesia without Involvement of μ-Opioid Receptor or Morphine-3-glucuronide

Opioid-induced hyperalgesia (OIH) is a paradoxical increase in pain perception that may manifest during opioid treatment. For morphine, the metabolite morphine-3-glucuronide (M3G) is commonly believed to underlie this phenomenon. Here, in three separate studies, we empirically assess the role of M3G in morphine-induced hyperalgesia. In the first study, CD-1 mice injected with morphine (15 mg/kg subcutaneously) after pretreatment with the opioid receptor antagonist naltrexone (NTX) (15 mg/kg) showed tail withdrawal latency reductions indicative of hyperalgesia (2.5 ± 0.1 s at t = 30 min, P < 0.001 versus baseline). In these mice, the morphine/M3G concentration ratios versus effect showed a negative correlation (r_p = −0.65, P < 0.001), indicating that higher morphine relative to M3G concentrations are associated with increased OIH. In the second study, similar hyperalgesic responses were observed in mice lacking the multidrug resistance protein 3 (MRP3) transporter protein (Mrp3^−/− mice) in the liver and their wild-type controls (FVB mice; latency reductions: 3.1 ± 0.2 s at t = 30 min, P < 0.001 versus within-strain baseline). In the final study, the pharmacokinetics of morphine and M3G were measured in Mrp3^−/− and FVB mice. Mrp3^−/− mice displayed a significantly reduced capacity to export M3G into the systemic circulation, with plasma M3G concentrations just 7% of those observed in FVB controls. The data confirm previous literature that morphine causes hyperalgesia in the absence of opioid receptor activation but also indicate that this hyperalgesia may occur without a significant contribution of hepatic M3G. The relevance of these data to humans has yet to be demonstrated.     

Effects of a High-Fat Diet on Spontaneous Metastasis of Lewis Lung Carcinoma in Plasminogen Activator Inhibitor-1 Deficient and Wild-Type Mice

This study investigated the effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma (LLC) in plasminogen activator inhibitor-1 deficient (PAI-1^−/−) and wild-type mice. The high-fat diet increased the number of pulmonary metastases by 60% (p<0.01), tumor cross-sectional area by 82% (p<0.05) and tumor volume by 130% (p<0.05) compared to the AIN93G diet. Deficiency in PAI-1 reduced the number of metastases by 35% (p<0.01) compared to wild-type mice. In mice fed the high-fat diet, PAI-1 deficiency reduced tumor cross-sectional area by 52% (p<0.05) and tumor volume by 61% (p<0.05) compared to their wild-type counterparts; however, PAI-1 deficiency affected neither area nor volume in mice fed the AIN93G diet. Adipose and plasma concentrations of PAI-1 were significantly higher in high-fat fed wild-type mice than in their AIN93G-fed counterparts. Adipose and plasma PAI-1 were not detectable in PAI-1^−/− mice regardless of the diet. Mice deficient in PAI-1 showed significantly greater plasma concentrations of monocyte chemotactic protein-1, tumor necrosis factor-α, leptin, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-1 and insulin compared to wild-type mice, indicating a compensatory overproduction of inflammatory cytokines, angiogenic factors and insulin in the absence of PAI-1. We conclude that PAI-1 produced by the host, including that by adipose tissue, promotes high-fat enhanced metastasis of LLC.     

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

Background

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.

Aim

To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.

Methods

We measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1^−/−, hMSH6^−/−, and hMSH3^−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.

Results

D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1^−/− and hMSH3^−/− cells, respectively, than in hMSH6^−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.

Conclusion

EMAST is dependent upon the MMR background, with hMSH3^−/− more prone to frameshift mutations than hMSH6^−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3^−/− mimics complete MMR failure (hMLH1^−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)⁺ and CD69⁺ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL⁻ natural killer cells that were adoptively transferred into Rag-2^−/− γ chain^−/− mice could convert into TRAIL⁺ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.     

The Ileal Lipid Binding Protein Is Required for Efficient Absorption and Transport of Bile Acids in the Distal Portion of the Murine Small Intestine

The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6^−/− mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05) in female but not male Fabp6^−/− mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05) but not in male Fabp6^−/− mice. The amount of [³H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (P<0.025) higher for female Fabp6^−/− mice whereas it was 57.3% (P<0.01) lower for male Fabp6^−/− mice, compared to wild-type mice. The retained fraction of the [³H]TCA localized in the small and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6^−/− mice relative wild-type mice, whereas no changes were seen in female Fabp6^−/− mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6^−/− mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.     

Effects of a Brown Beans Evening Meal on Metabolic Risk Markers and Appetite Regulating Hormones at a Subsequent Standardized Breakfast: A Randomized Cross-Over Study

Background

Dietary prevention strategies are increasingly recognized as essential to combat the current epidemic of obesity and related metabolic disorders. The purpose of the present study was to evaluate the potential prebiotic effects of indigestible carbohydrates in Swedish brown beans (Phaseolus vulgaris var. nanus) in relation to cardiometabolic risk markers and appetite regulating hormones.

Methods

Brown beans, or white wheat bread (WWB, reference product) were provided as evening meals to 16 healthy young adults in a randomised crossover design. Glucose, insulin, appetite regulatory hormones, GLP-1, GLP-2, appetite sensations, and markers of inflammation were measured at a following standardised breakfast, that is at 11 to 14 h post the evening meals. Additionally, colonic fermentation activity was estimated from measurement of plasma short chain fatty acids (SCFA, including also branched chain fatty acids) and breath hydrogen (H₂) excretion.

Results

An evening meal of brown beans, in comparison with WWB, lowered blood glucose (−15%, p<0.01)- and insulin (−16%, p<0.05) responses, increased satiety hormones (PYY 51%, p<0.001), suppressed hunger hormones (ghrelin −14%, p<0.05), and hunger sensations (−15%, p = 0.05), increased GLP-2 concentrations (8.4%, p<0.05) and suppressed inflammatory markers (IL-6 −35%, and IL-18 −8.3%, p<0.05) at a subsequent standardised breakfast. Breath H₂ (141%, p<0.01), propionate (16%, p<0.05), and isobutyrate (18%, P<0.001) were significantly increased after brown beans compared to after WWB, indicating a higher colonic fermentative activity after brown beans.

Conclusions

An evening meal with brown beans beneficially affected important measures of cardiometabolic risk and appetite regulatory hormones, within a time frame of 11–14 h, in comparison to a WWB evening meal. Concentrations of plasma SCFA and H₂ were increased, indicating involvement of colonic fermentation. Indigestible colonic substrates from brown beans may provide a preventive tool in relation to obesity and the metabolic syndrome.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01706042","term_id":"NCT01706042"}}NCT01706042     

Targeting Serum Glucocorticoid-Regulated Kinase-1 in Squamous Cell Carcinoma of the Head and Neck: A Novel Modality of Local Control

Purpose

The inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC).

Experimental Design

Human head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses.

Results

At the end of the experiment mean tumor sizes were 122.33+/−105.86, 76.73+/−36.09, 94.52+/−75.92, and 25.76+/−14.89 mm² (mean +/− SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4.

Conclusions

SGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.     

Inhibition of Propofol Anesthesia on Functional Connectivity between LFPs in PFC during Rat Working Memory Task

Working memory (WM) refers to the temporary storage and manipulation of information necessary for performance of complex cognitive tasks. There is a growing interest in whether and how propofol anesthesia inhibits WM function. The aim of this study is to investigate the possible inhibition mechanism of propofol anesthesia based on the functional connections of multi-local field potentials (LFPs) and behavior during WM tasks. Adult SD rats were randomly divided into 3 groups: pro group (0.5 mg·kg⁻¹·min⁻¹,2 h), PRO group (0.9 mg·kg⁻¹·min⁻¹, 2 h) and control group. The experimental data were 16-channel LFPs obtained at prefrontal cortex with implanted microelectrode array in SD rats during WM tasks in Y-maze at 24, 48, 72, 96, 120 hours (day 1-day 5) after propofol anesthesia, and the behavior results of WM were recoded at the same time. Directed transfer function (DTF) method was applied to analyze the connections among LFPs directly. Furthermore, the causal networks were identified by DTF. The clustering coefficient (C), network density (D) and global efficiency (E_global) were selected to describe the functional connectivity quantitatively. The results show that: comparing with the control group, the LFPs functional connectivity in pro group were no significantly difference (p>0.05); the connectivity in PRO group were significantly decreased (p<0.05 at 24 hours, p<0.05 at 48 hours), while no significant difference at 72, 96 and 120 hours for rats (p>0.05), which were consistent with the behavior results. These findings could lead to improved understanding the mechanism of inhibition of anesthesia on WM functions from the view of connections among LFPs.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.