The Strength of Selection against Neanderthal Introgression

Hybridization between humans and Neanderthals has resulted in a low level of Neanderthal ancestry scattered across the genomes of many modern-day humans. After hybridization, on average, selection appears to have removed Neanderthal alleles from the human population. Quantifying the strength and causes of this selection against Neanderthal ancestry is key to understanding our relationship to Neanderthals and, more broadly, how populations remain distinct after secondary contact. Here, we develop a novel method for estimating the genome-wide average strength of selection and the density of selected sites using estimates of Neanderthal allele frequency along the genomes of modern-day humans. We confirm that East Asians had somewhat higher initial levels of Neanderthal ancestry than Europeans even after accounting for selection. We find that the bulk of purifying selection against Neanderthal ancestry is best understood as acting on many weakly deleterious alleles. We propose that the majority of these alleles were effectively neutral—and segregating at high frequency—in Neanderthals, but became selected against after entering human populations of much larger effective size. While individually of small effect, these alleles potentially imposed a heavy genetic load on the early-generation human–Neanderthal hybrids. This work suggests that differences in effective population size may play a far more important role in shaping levels of introgression than previously thought.     

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.     

The fatty acid profile of vegetative Azotobacter vinelandii ATCC 12837: growth phase-dependence

Fatty acids of Azotobacter vinelandii ATCC 12837 were determined at various times during aerobic vegetative growth at 30°C to provide baseline data for studying the effects of chemical agents on the organism’s survival and fatty acid biosynthesis. Palmitate (16:0) was the highest at 36.7±4.3 mol% (mean±SD) after the first 5 h in fresh culture, decreasing slightly to 33.4±2.6 mol% at 49 h. The other fatty acids were therefore each normalized as a ratio of 16:0. At 5 h, as a ratio of 16:0, myristate (14:0) was 0.14±0.06, palmitoleate (16:1cΔ^9–10) 0.13±0.06, oleate (18:1cΔ^9–10) 0.21±0.12, cis-vaccenate (18:1cΔ^11–12) 0.30±0.17 and stearate (18:0) 0.68±0.02. As the growth phase advanced to 49 h, 14:0 and 16:1cΔ^9–10 increased, 18:1cΔ^9–10 decreased and cis-vaccenate reciprocally increased, whereas 18:0 decreased. These suggest that the saturated fatty acid biosynthesis pathway yielded 16:0 and 18:0 in the 5-h lag period. By desaturation, 18:0 formed the unsaturated fatty acid (UFA) 18:1cΔ^9–10. As the culture aged, the anaerobic UFA biosynthesis pathway formed 16:1cΔ^9–10, which was elongated to 18:1cΔ^11–12. These fatty acid alterations represent a homeoviscous adaptation, modulating the microbe’s membrane lipid viscosity for optimal cellular function.     

Alterations in cardiac contractile performance and sarcoplasmic reticulum function in sucrose-fed rats is associated with insulin resistance

Diabetes mellitus (DM) causes the development of a specific cardiomyopathy that results from the metabolic derangements present in DM and manifests as cardiac contractile dysfunction. Although myocardial dysfunction in Type 1 DM has been associated with defects in the function and regulation of the sarcoplasmic reticulum (SR), very little is known about SR function in Type 2 DM. Accordingly, this study examined whether abnormalities in cardiac contractile performance and SR function occur in the prestage of Type 2 DM (i.e., during insulin resistance). Sucrose feeding was used to induce whole body insulin resistance, whereas cardiac contractile performance was assessed by echocardiography and SR function was measured by SR calcium (Ca²⁺) uptake. Sucrose-fed rats exhibited hyperinsulinemia, hyperglycemia, and hyperlipidemia relative to control rats. Serial echocardiographic assessments in the sucrose-fed rats revealed early abnormalities in diastolic function followed by late systolic dysfunction and concurrent alterations in myocardial structure. The hearts of the 10-wk sucrose-fed rats showed depressed SR function demonstrated by a significant reduction in SR Ca²⁺ uptake. The decline in SR Ca²⁺ uptake was associated with a significant decrease in the cAMP-dependent protein kinase and Ca²⁺/calmodulin-dependent protein kinase II-mediated phosphorylation of phospholamban. The results show that abnormalities in cardiac contractile performance and SR function occur at an insulin-resistant stage before the manifestation of overt Type 2 DM. cardiomyopathy; diabetes mellitus; echocardiography     

Thyroxine-thyroid hormone receptor interactions

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.     

Transcriptional CDK inhibitors,CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.Subject terms: Cancer models, Apoptosis, RNAi     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

Fas-Mediated Apoptosis Is Regulated by the Extracellular Matrix Protein CCN1 (CYR61) In Vitro and In Vivo

Although Fas ligand (FasL) is primarily expressed by lymphoid cells, its receptor Fas (CD95/Apo-1) is broadly expressed in numerous nonlymphoid tissues and can mediate apoptosis of parenchymal cells upon injury and infiltration of inflammatory cells. Here we show that CCN1 (CYR61) and CCN2 (CTGF), matricellular proteins upregulated at sites of inflammation and wound repair, synergize with FasL to induce apoptosis by elevating cellular levels of reactive oxygen species (ROS). CCN1 acts through engagement of integrin α₆β₁ and cell surface heparan sulfate proteoglycans, leading to ROS-dependent hyperactivation of p38 mitogen-activated protein kinase in the presence of FasL to enhance mitochondrial cytochrome c release. We show that CCN1 activates neutral sphingomyelinase, which functions as a key source of CCN1-induced ROS critical for synergism with FasL. Furthermore, Fas-dependent hepatic apoptosis induced by an agonistic monoclonal anti-Fas antibody or intragastric administration of alcohol is severely blunted in knock-in mice expressing an apoptosis-defective Ccn1 allele. These results demonstrate that CCN1 is a physiologic regulator of Fas-mediated apoptosis and that the extracellular matrix microenvironment can modulate Fas-dependent apoptosis through CCN1 expression.Cell adhesion to several abundant extracellular matrix (ECM) proteins via engagement of integrin receptors is known to induce potent prosurvival signals, whereas detachment from the ECM triggers many cell types to undergo anoikis, a form of apoptotic cell death (13). This regulation of cell survival through integrin-mediated cell adhesion plays a critical role in controlling homeostasis and the integrity of tissue architecture, whereas unligated or inappropriately ligated integrins may elicit apoptotic signals (12). However, during embryogenesis, inflammation, tissue remodeling, and wound repair, death-inducing factors can provoke programmed or apoptotic death in normal cells without requiring their detachment from the ECM (4).Fas (CD95/APO-1) is a member of the tumor necrosis factor (TNF) receptor family of cell surface death receptors that mediates apoptotic signals upon binding to its specific ligand, FasL. Ligation of Fas to FasL or its agonistic antibodies results in receptor clustering, recruitment of the adaptor protein FADD, and activation of the proteolytic caspase cascade (19, 50). Whereas FasL is primarily expressed in activated T lymphocytes, natural killer cells, and tissues of immune privilege, Fas is broadly expressed in most lymphoid and nonlymphoid tissues (50). Fas-mediated apoptosis is critical for the regulation of the immune response, including deletion of activated T and B lymphocytes, cell death-inducing activity of cytotoxic T cells, and removal of infiltrating lymphocytes in immune-privileged tissues (19, 50). Fas also plays an important role in parenchymal cell apoptosis in many organs during tissue injury and upon inflammatory infiltration of lymphocytes (7, 20, 38, 42, 46). Consistent with the notion that cell adhesion promotes cell survival, integrin-matrix interactions inhibit Fas-dependent apoptosis in a variety of cell types (22, 32). Thus, optimal apoptotic responses to Fas/FasL signaling in adherent parenchymal cells must override the cytoprotective effects of integrin-mediated cell adhesion. In these instances, dynamic changes in the ECM induced by inflammation or injury repair may establish conditions that are permissive of, or conducive to, the apoptotic responses to FasL.Recent studies have described the emergence of ECM proteins that can induce or promote apoptosis (49, 60, 65). Among them are members of the CCN family (9), which are secreted cysteine-rich proteins that serve regulatory rather than structural roles in the ECM and are therefore considered matricellular proteins (6). CCN1 (CYR61) and CCN2 (CTGF) support cell adhesion, stimulate cell migration, induce angiogenesis, and promote chondrogenic differentiation, exerting their functions primarily through direct binding to integrin receptors. CCN1 and CCN2 promote the survival of endothelial cells through integrin α_vβ₃ but induce apoptosis in p21-deficient fibroblasts through α₆β₁ via a caspase-8-independent mechanism (3, 40, 60). CCN1 and CCN2 are also critical for embryonic development, as Ccn1-null mice die during midgestation due to cardiovascular abnormalities and Ccn2-deficient mice perish perinatally as a consequence of severe skeletal malformations (29, 47, 48). In the adult, CCN proteins are highly expressed at sites of inflammation, injury repair, and tissue remodeling and are implicated in diseases where inflammation plays a role, including fibrosis, atherosclerosis, arthritis, and cancer (9). Furthermore, the presence of CCN1 in the ECM enables TNF-α to induce apoptotic death in normal cells without inhibition of NF-κB signaling or de novo protein synthesis, conditions thought to be necessary for TNF-α to be cytotoxic (10).Here we show that CCN1 and CCN2 can synergize with FasL and significantly enhance FasL-induced apoptosis in fibroblasts. Mechanistically, CCN1 engages integrin α₆β₁ and cell surface heparan sulfate proteoglycans (HSPGs), leading to the reactive oxygen species (ROS)-dependent hyperactivation of p38 mitogen-activated protein kinase (MAPK) in the presence of FasL, which greatly enhances mitochondrial cytochrome c release and apoptosis. We show that CCN1 is a novel activator of neutral sphingomyelinase (nSMase), which is an essential contributor to CCN1-induced ROS. Further, Fas-dependent hepatic cell death is greatly diminished in knock-in mice expressing an apoptosis-defective mutant of CCN1 that is unable to bind α₆β₁-HSPGs. Together, these results show that CCN1 is a physiologic regulator of Fas-mediated apoptosis and indicate that Fas-dependent cell death at sites of inflammation and injury repair may be controlled by the matrix microenvironment through CCN1 expression.     

TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)β(5), α(6)β(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.     

Prevention of concentric hypertrophy and diastolic impairment in aortic-banded rats treated with resveratrol

This study was designed to examine the effects of the antioxidant resveratrol on cardiac structure and function in pressure overload (PO)-induced cardiac hypertrophy. Male Sprague-Dawley rats were subjected to sham operation and the aortic banding procedure. A subgroup of sham control and aortic-banded rats were treated with resveratrol for 2 wk after surgery. Echocardiographic analysis of cardiac structure and function along with Western blot analysis of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and redox factor-1 (ref-1) were performed in all groups after 4 wk of surgery. Banded rats showed significantly increased left ventricle-to-body weight ratio. Echocardiographic analysis showed that the interventricular septal wall thickness and left ventricular posterior wall thickness at systole and diastole were significantly increased in banded rats. Also, a significant increase in isovolumic relaxation time was observed in banded rats. Measured eNOS, iNOS, and ref-1 protein levels were significantly reduced in banded rats. Resveratrol treatment prevented the above changes in cardiac structure, function, and protein expression in banded rats. Aortic banding after 4 wk resulted in concentric remodeling and impaired contractile function due to PO on the heart. The 2-wk treatment with resveratrol was found to abolish PO-induced cardiac hypertrophy. Resveratrol may therefore be beneficial against PO-induced cardiac hypertrophy found in clinical settings of hypertension and aortic valve stenosis.