The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.
C9orf72 promoter hypermethylation inhibits the accumulation of pathologies which have been postulated to be neurotoxic. We tested here whether C9orf72 hypermethylation is associated with prolonged disease in C9orf72 mutation carriers. C9orf72 methylation was quantified from brain or blood using methylation-sensitive restriction enzyme digest-qPCR in a cross-sectional cohort of 118 C9orf72 repeat expansion carriers and 19 non-carrier family members. Multivariate regression models were used to determine whether C9orf72 hypermethylation was associated with age at onset, disease duration, age at death, or hexanucleotide repeat expansion size. Permutation analysis was performed to determine whether C9orf72 methylation is heritable. We observed a high correlation between C9orf72 methylation across tissues including cerebellum, frontal cortex, spinal cord and peripheral blood. While C9orf72 methylation was not significantly different between ALS and FTD and did not predict age at onset, brain and blood C9orf72 hypermethylation was associated with later age at death in FTD (brain: ? = 0.18, p = 0.006; blood: ? = 0.15, p < 0.001), and blood C9orf72 hypermethylation was associated with longer disease duration in FTD (? = 0.03, p = 0.007). Furthermore, C9orf72 hypermethylation was associated with smaller hexanucleotide repeat length (? = -16.69, p = 0.033). Finally, analysis of pedigrees with multiple mutation carriers demonstrated a significant association between C9orf72 methylation and family relatedness (p < 0.0001). C9orf72 hypermethylation is associated with prolonged disease in C9orf72 repeat expansion carriers with FTD. The attenuated clinical phenotype associated with C9orf72 hypermethylation suggests that slower clinical progression in FTD is associated with reduced expression of mutant C9orf72. These results support the hypothesis that expression of the hexanucleotide repeat expansion is associated with a toxic gain of function.
This paper tests whether in utero conditions affect long-run developmental outcomes using the 1918 influenza pandemic in Taiwan as a natural experiment. Combining several historical and current datasets, we find that cohorts in utero during the pandemic are shorter as children/adolescents and less educated compared to other birth cohorts. We also find that they are more likely to have serious health problems including kidney disease, circulatory and respiratory problems, and diabetes in old age. Despite possible positive selection on health outcomes due to high infant mortality rates during this period (18%), our paper finds a strong negative impact of in utero exposure to influenza.
Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-? (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1? was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2? remained unchanged at all Hb levels. Both kidney HIF-1? and HIF-2? increased earlier (Hb ?70-90 g/l) in response to anemia. Liver also exhibited an early HIF-? response. Carotid blood flow was increased early (Hb ?70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIF? were nNOS-dependent, our current data demonstrate that increased renal HIF? was nNOS independent. HIF-dependent RNA levels increased linearly (?10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (?100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.
Hexanucleotide repeat expansions of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal degeneration. The mutation is associated with reduced C9orf72 expression and the accumulation of potentially toxic RNA and protein aggregates. CpG methylation is known to protect the genome against unstable DNA elements and to stably silence inappropriate gene expression. Using bisulfite cloning and restriction enzyme-based methylation assays on DNA from human brain and peripheral blood, we observed CpG hypermethylation involving the C9orf72 promoter in cis to the repeat expansion mutation in approximately one-third of C9orf72 repeat expansion mutation carriers. Promoter hypermethylation of mutant C9orf72 was associated with transcriptional silencing of C9orf72 in patient-derived lymphoblast cell lines, resulting in reduced accumulation of intronic C9orf72 RNA and reduced numbers of RNA foci. Furthermore, demethylation of mutant C9orf72 with 5-aza-deoxycytidine resulted in increased vulnerability of mutant cells to oxidative and autophagic stress. Promoter hypermethylation of repeat expansion carriers was also associated with reduced accumulation of RNA foci and dipeptide repeat protein aggregates in human brains. These results indicate that C9orf72 promoter hypermethylation prevents downstream molecular aberrations associated with the hexanucleotide repeat expansion, suggesting that epigenetic silencing of the mutant C9orf72 allele may represent a protective counter-regulatory response to hexanucleotide repeat expansion.
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment.
White matter sparing after traumatic brain injury (TBI) is an important predictor of survival and outcome. Blood vessels and axons are intimately associated anatomically and developmentally. Neural input is required for appropriate vascular patterning, and vascular signaling is important for neuron development and axon growth. Owing to this codependence between endothelial cells and axons during development and the contribution of endothelial progenitor cells (EPCs) in ischemic injury, we hypothesized that EPCs are important in axonal survival after TBI. We examined the effects of allogenic-cultured EPCs on white matter protection and microvascular maintenance after midline fluid percussion injury in adult Sprague-Dawley rats. We used two in vitro models of injury, mechanical stretch and oxygen-glucose deprivation (OGD), to examine the effects of EPCs on the mechanical and ischemic components of brain trauma, respectively. Our results indicate that EPCs improve the white matter integrity and decrease capillary breakdown after injury. Cultured cortical neurons exposed to OGD had less axon degeneration when treated with EPC-conditioned media, whereas no effect was seen in axons injured by mechanical stretch. The results indicate that EPCs are important for the protection of the white matter after trauma and represent a potential avenue for therapy.Journal of Cerebral Blood Flow & Metabolism advance online publication, 4 December 2013; (2013) 0, 000-000. doi:10.1038/jcbfm.2013.216.
Acute ?-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and ?-blockade, animal studies have demonstrated that acute ?-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute ?-blockade with a highly ?(1)-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution.
Acute ?-blockade with metoprolol has been associated with increased mortality by undefined mechanisms. Since metoprolol is a relatively high affinity blocker of ?(2)-adrenoreceptors, we hypothesized that some of the increased mortality associated with its use may be due to its abrogation of ?(2)-adrenoreceptor-mediated vasodilation of microvessels in different vascular beds. Cardiac output (CO; pressure volume loops), mean arterial pressure (MAP), relative cerebral blood flow (rCBF; laser Doppler), and microvascular brain tissue Po(2) (G2 oxyphor) were measured in anesthetized mice before and after acute treatment with metoprolol (3 mg/kg iv). The vasodilatory dose responses to ?-adrenergic agonists (isoproterenol and clenbuterol), and the myogenic response, were assessed in isolated mesenteric resistance arteries (MRAs; ?200-?m diameter) and posterior cerebral arteries (PCAs ?150-?m diameter). Data are presented as means ± SE with statistical significance applied at P < 0.05. Metoprolol treatment did not effect MAP but reduced heart rate and stroke volume, CO, rCBF, and brain microvascular Po(2), while concurrently increasing systemic vascular resistance (P < 0.05 for all). In isolated MRAs, metoprolol did not affect basal artery tone or the myogenic response, but it did cause a dose-dependent impairment of isoproterenol- and clenbuterol-induced vasodilation. In isolated PCAs, metoprolol (50 ?M) impaired maximal vasodilation in response to isoproterenol. These data support the hypothesis that acute administration of metoprolol can reduce tissue oxygen delivery by impairing the vasodilatory response to ?(2)-adrenergic agonists. This mechanism may contribute to the observed increase in mortality associated with acute administration of metoprolol in perioperative patients.
Although depression may be commonly experienced by persons living with AIDS, it may be challenging for health care providers to identify persons who are suffering from depression symptoms, particularly if they are living in the more isolated rural areas of India. The purpose of this study is to assess correlates of depression among women living with AIDS in rural Andhra Pradesh, India. A total of 68 rural women living with AIDS (WLA) completed baseline data and were assessed by means of structured instruments. Regression modeling revealed that disclosure avoidance and making at least six health care visits in the last six months were all associated with depression. Further, living with a spouse was associated with lower depressive symptom scores. Stigma was not found to be associated with depression. Understanding correlates of depression can lead the way toward designing culturally-tailored interventions that can mitigate disclosure avoidance and improve the health of women. A more comprehensive health focus may be needed to empower the women to seek quality care for both physical health, as well as mental health, symptomatology.
Perioperative beta-blockade and anemia are independent predictors of increased stroke and mortality by undefined mechanisms. This study investigated the effect of beta-blockade on cerebral tissue oxygen delivery in an experimental model of blood loss and fluid resuscitation (hemodilution).
Leptin is often regarded as a mediator of fever, even though an in-depth analysis of the dose-dependent effects of leptin on body temperature (T(b)), pro-inflammatory cytokines, and circulating leptin has never been performed. In the present study, such an analysis was performed in rats that were food deprived (lower baseline levels of leptin) or free feeding (higher baseline levels of leptin). In a relatively cool environment (22 degrees C), rats deprived of food for 24 h exhibited mild (approximately 0.5 degrees C) hypothermia. Leptin infusion (250 microg/kg iv) elevated the T(b) of the food-deprived rats to a normothermic level, an effect that peaked (120 min post-infusion) when plasma leptin was at a level (approximately 8 ng/mL) often found in leptin-responsive subjects. Increasing the leptin dose to 1000 microg/kg did not produce any further (febrile) elevation in the T(b) of food-deprived rats. The anti-hypothermic effect of leptin in food-deprived rats was not associated with any rise in the plasma levels of the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In free-feeding rats kept in a cooler (22 degrees C) or warmer (28 degrees C) environment, leptin infusion failed to alter T(b) or to produce any surge in plasma TNF-alpha or IL-6, even when the dose infused (3500 microg/kg iv) resulted in excessive, non-physiological rises in plasma leptin (approximately 542 ng/mL at 30 min; approximately 75 ng/mL at 120 min post-infusion). In contrast, free-feeding rats in the same experimental set-up were able to respond to a low dose (2 microg/kg iv) of IL-1beta with a typical biphasic fever, which was associated with surges in plasma TNF-alpha and IL-6. Collectively, our data show that an acute rise in plasma leptin to a level within or fairly above the physiological range does not induce fever. These results challenge the idea that leptin may be a mediator of fever.
Little is known about the neuroimmune mechanisms responsible for the switch from fever to hypothermia observed in severe forms of systemic inflammation. We evaluated whether bacterial lipopolysaccharide (LPS) acting directly on the brain could promote a fever-hypothermia switch as well as the hypotension that is often associated with hypothermia in models of systemic inflammation. At an ambient temperature of 22°C, freely moving rats received intracerebroventricular (i.c.v.) injections of LPS at doses ranging from 0.5 to 25?g. Despite the use of such high doses, the prevailing thermal response was fever. To investigate if a hypothermic response could be hidden within the prevailing febrile response, rats were pretreated with a cyclooxygenase-2 inhibitor (SC-236, 3.5mg/kg i.v.) known to block fever, but this strategy also failed to reveal any consistent hypothermic response following i.c.v. LPS. At the doses tested, i.c.v. LPS was similarly ineffective at inducing hypotension. Additional doses of LPS did not need to be tested because the 25-?g dose was already sufficient to induce both hypothermia and hypotension when administered peripherally (intra-arterially). An empirical 3D model of the interplay among body temperature, arterial pressure and heart rate following intra-arterial LPS reinforced the strong association of hypothermia with hypotension and, at the same time, exposed a bell-shaped relationship between heart rate and body temperature. In summary, the present study demonstrates that hypothermia and hypotension are triggered exclusively by LPS acting outside the brain and provides an integrated model of the thermal and cardiovascular responses to peripheral LPS.
Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5?atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72?h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery.
The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.
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