This chapter deals with the most central and contentious security issue in the international relations of the modern Middle East: the conflict between Arab states and Israel. It traces the characteristics of the Arab–Israel conflict and how these have changed over time. It also demonstrates how both realism and identity politics have informed the position of different parties to the conflict. The chapter explains how the 1967 war or the Arab–Israel conflict was as much about Arab identity and leadership as it was about the struggle with Israel, even at its high point. It shows how from 1948 to the present, the unresolved Palestinian question has remained at the heart of debates about regional relations, even as more Arab states have signed accords with Israel.

Exposure to ionizing radiation (IR) is a lingering threat from accidental or terroristic nuclear events, but is also widely used in cancer therapy. In both cases, host inflammatory responses to IR damage normal tissue causing morbidity and possibly mortality to the victim/patient. Opaganib, a first-in-class inhibitor of sphingolipid metabolism, has broad anti-inflammatory and anticancer activity. Opaganib elevates ceramide and reduces sphingosine 1-phosphate (S1P) in cells, conditions that increase the antitumor efficacy of radiation while concomitantly suppressing inflammatory damage to normal tissue. Therefore, opaganib may suppress toxicity from unintended IR exposure and improve patient response to chemoradiation. To test these hypotheses, we first examined the effects of opaganib on the toxicity and antitumor activity of radiation in mice exposed to total body irradiation (TBI) or IR with partial bone marrow shielding. Oral treatment with opaganib 2 h before TBI shifted the LD75 from 9.5 Gy to 11.5 Gy, and provided substantial protection against gastrointestinal damage associated with suppression of radiation-induced elevations of S1P and TNFα in the small intestines. In the partially shielded model, opaganib provided dose-dependent survival advantages when administered 4 h before or 24 h after radiation exposure, and was particularly effective when given both prior to and following radiation. Relevant to cancer radiotherapy, opaganib decreased the sensitivity of IEC6 (non-transformed mouse intestinal epithelial) cells to radiation, while sensitizing PAN02 cells to in vitro radiation. Next, the in vivo effects of opaganib in combination with radiation were examined in a syngeneic tumor model consisting of C57BL/6 mice bearing xenografts of PAN02 pancreatic cancer cells and a cross-species xenograft model consisting of nude mice bearing xenografts of human FaDu cells. Mice were treated with opaganib and/or IR (plus cisplatin in the case of FaDu tumors). In both tumor models, the optimal suppression of tumor growth was attained by the combination of opaganib with IR (± cisplatin). Overall, opaganib substantially protects normal tissue from radiation damage that may occur through unintended exposure or cancer radiotherapy.

Infants born prematurely, often associated with maternal infection, frequently exhibit breathing instabilities that require resuscitation. We hypothesized that breathing patterns during the first hour of life would be predictive of survival in an animal model of prematurity. Using plethysmography, we measured breathing patterns during the first hour after birth in mice born at term (Term 19.5), delivered prematurely on gestational day 18.5 following administration of low-dose lipopolysaccharide (LPS; 0.14 mg/kg) to pregnant dams (LPS 18.5), or delivered on gestational day 18.7 or 17.5 by caesarian section (C-S 18.5 and C-S 17.5, respectively). Our experimental approach allowed us to dissociate effects caused by inflammation, from effects due to premature birth in the absence of an inflammatory response. C-S 17.5 mice did not survive, whereas mortality was not increased in C-S 18.5 mice. However, in premature pups born at the same gestational age (day 18.5) in response to maternal LPS injection, mortality was significantly increased. Overall, mice that survived had higher birth weights and showed eupneic or gasping activity that was able to transition to normal breathing. Some mice also exhibited a "saw tooth" breathing pattern that was able to transition into eupnea during the first hour of life. In contrast, mice that did not survive showed distinct, large amplitude, long-lasting breaths that occurred at low frequency and did not transition into eupnea. This breathing pattern was only observed during the first hour of life and was more prevalent in LPS 18.5 and C-S 18.5 mice. Indeed, breath tidal volumes were higher in inflammation-induced premature pups than in pups delivered via C-section at equivalent gestational ages, whereas breathing frequencies were low in both LPS-induced and C-section-induced premature pups. We conclude that a breathing pattern characterized by low frequency and large tidal volume is a predictor for the failure to survive, and that these characteristics are more often seen when prematurity occurs in the context of maternal inflammation. Further insights into the mechanisms that generate these breathing patterns and how they transition to normal breathing may facilitate development of novel strategies to manage premature birth in humans.

Cerebral microbleeds (CMBs) are considered to be an imaging marker for small vessel ischemic disease associated with cerebral amyloid angiopathy. Recent evidence also suggests that white matter hyperintensities (WMHs) located in posterior brain regions are associated with Alzheimer's disease (AD). The current study was designed to explore if there is a similar trend of associations between CMBs, WMHs, and CSF levels of Aβ1-42. Imaging measures of CMBs and WMHs were collected from 62 participants who also had concomitant CSF sampling. CSF levels of Aβ1-42, indicating the presence or absence of AD-type amyloidosis pathology, were measured and fluid-attenuated inversion recovery (FLAIR) imaging and gradient recalled echo (GRE) were obtained to assess WMHs and CMBs. CMBs were visually rated using the well validated microbleed anatomical rating scale (MARS). Partial correlation and linear regression analyses were conducted to examine the association of lobar CMBs with CSF Aβ1-42. The mean age was 74.9 ± 7.4 years, 45% were male and the mean years of educational attainment were 16.9 ± 3.1 years. Partial correlation analyses showed an association between parietal lobe CMBs and lower CSF levels of Aβ1-42 (rho = - 0.27, p = 0.04). CMBs in the frontal, temporal and occipital lobes were not correlated with the CSF levels of Aβ1-42. Linear regression analysis demonstrated that parietal lobe CMBs were strongly associated with lower CSF levels of Aβ1-42 (p = 0.04, beta = - 0.29). Parietal lobe CMBs showed a strong association with lower CSF Aβ1-42, providing evidence that CMBs in the parietal lobe represent a potential surrogate imaging biomarker for sporadic AD in subjects who have not undergone direct amyloid assessments. Additional studies examining the association of lobar CMBs with regional WMHs and specific patterns of cognitive decline are under investigation currently.

This retrospective study of presurgical fMRI for brain tumors compares nonregistered images and 5 registration cost functions: Hellinger, mutual information, normalized mutual information, correlation ratio, and local Pearson correlation. To adjudicate the accuracy of coregistration, the authors edge-enhanced echo-planar maps and rated them for alignment with structural anatomy. The local Pearson correlation is a special-purpose cost function specifically designed for T2*-T1 coregistration and should be more widely incorporated into software tools as a better method for coregistration in clinical fMRI. BACKGROUND AND PURPOSE: Interpretation of fMRI depends on accurate functional-to-structural alignment. This study explores registration methods used by FDA-approved software for clinical fMRI and aims to answer the following question: What is the degree of misalignment when registration is not performed, and how well do current registration methods perform? MATERIALS AND METHODS: This retrospective study of presurgical fMRI for brain tumors compares nonregistered images and 5 registration cost functions: Hellinger, mutual information, normalized mutual information, correlation ratio, and local Pearson correlation. To adjudicate the accuracy of coregistration, we edge-enhanced echo-planar maps and rated them for alignment with structural anatomy. Lesion-to-activation distances were measured to evaluate the effects of different cost functions. RESULTS: Transformation parameters were congruent among Hellinger, mutual information, normalized mutual information, and the correlation ratio but divergent from the local Pearson correlation. Edge-enhanced images validated the local Pearson correlation as the most accurate. Hellinger worsened misalignment in 59% of cases, primarily exaggerating the inferior translation; no cases were worsened by the local Pearson correlation. Three hundred twenty lesion-to-activation distances from 25 patients were analyzed among nonregistered images, Hellinger, and the local Pearson correlation. ANOVA analysis revealed significant differences in the coronal (P < .001) and sagittal (P = .04) planes. If registration is not performed, 8% of cases may have a >3-mm discrepancy and up to a 5.6-mm lesion-to-activation distance difference. If a poor registration method is used, 23% of cases may have a >3-mm discrepancy and up to a 6.9-mm difference. CONCLUSIONS: The local Pearson correlation is a special-purpose cost function specifically designed for T2*-T1 coregistration and should be more widely incorporated into software tools as a better method for coregistration in clinical fMRI.

Telomerase activation protects cells from telomere damage by delaying senescence and inducing cell immortalization, whereas telomerase inhibition mediates rapid senescence or apoptosis. However, the cellular mechanisms that determine telomere damage-dependent senescence versus apoptosis induction are largely unknown. Here, we demonstrate that telomerase instability mediated by silencing of sphingosine kinase 2 (SPHK2) and sphingosine 1-phosphate (S1P), which binds and stabilizes telomerase, induces telomere damage-dependent caspase-3 activation and apoptosis, but not senescence, in p16-deficient lung cancer cells or tumors. These outcomes were prevented by knockdown of a tumor-suppressor protein, transcription factor 21 (TCF21), or by ectopic expression of WT human telomerase reverse transcriptase (hTERT) but not mutant hTERT with altered S1P binding. Interestingly, SphK2-deficient mice exhibited accelerated aging and telomerase instability that increased telomere damage and senescence via p16 activation especially in testes tissues, but not in apoptosis. Moreover, p16 silencing in SphK2(-/-) mouse embryonic fibroblasts activated caspase-3 and apoptosis without inducing senescence. Furthermore, ectopic WT p16 expression in p16-deficient A549 lung cancer cells prevented TCF21 and caspase-3 activation and resulted in senescence in response to SphK2/S1P inhibition and telomere damage. Mechanistically, a p16 mutant with impaired caspase-3 association did not prevent telomere damage-induced apoptosis, indicating that an association between p16 and caspase-3 proteins forces senescence induction by inhibiting caspase-3 activation and apoptosis. These results suggest that p16 plays a direct role in telomere damage-dependent senescence by limiting apoptosis via binding to caspase-3, revealing a direct link between telomere damage-dependent senescence and apoptosis with regards to aging and cancer.

Tolyporphins are glycosylated macrocycles isolated from lipophilic soil extracts of the cyanobacterium, Tolypothrix nodosa, and found to potentiate the cytotoxicity of antitumor drugs like vinblastine and adriamycin. Here we find that, unlike porphyrins, tolyporphins are not able to form complexes with most metal ions. However, they do react strongly with copper(II) and silver(II), forming square-planar metal complexes with an unpaired electron in a dx2-y2 orbital of the metal delocalized onto the ligating tolyporphin nitrogen atoms. Complexes were characterized by visible absorption spectra, mass spectrometry (EI, FAB, ESI, LDI-TOF, and MALDI-TOF) and multifrequency continuous-wave electron paramagnetic resonance spectra. Copper(II) and silver(II) complexes of tolyporphins A and E were found to have the interesting property of reversing multidrug resistance (MDR), with the copper complexes being less toxic than free tolyporphins. Reactive oxygen-free radicals were implicated in both the cytotoxic and MDR-reversing effects of free and metalated tolyporphins.

Abstract Sphingosine 1-phosphate (S1P), a pro-proliferative sphingolipid generated by Sphingosine Kinases is upregulated in many cancers. Telomerase (hTERT), is a ribonucleoprotein that extends the ends of chromosomes (telomeres), to promote lung cancer growth. We discovered that SK2 generated S1P binds and stabilizes telomerase in the nuclear periphery by allosterically mimicking protein phosphorylation. Mechanistically, S1P binding protected telomerase from MKRN1 mediated degradation thereby preventing telomere dysfunction and senescence. The objective of this study is to delineate the molecular mechanism of SK2-S1P mediated telomere dysfunction in aging and lung cancer. SK2 knockout mice tissues showed increased telomeric DNA damage and senescence associated-beta galactosidase activity. Moreover, SK2 knockout mice displayed aging phenotypes by reduced subcutaneous fat, atrophy in spleens and hypoplasia in mice testes. Also, SK2 knockout fibroblasts showed increased senescence and robust p16 expression and interestingly, p16 ablation rescued SK2 knockout fibroblasts from senescence. Importantly, we found SK2 and telomerase protein levels to be upregulated in lung cancer tumor tissues (n=48). Mechanistically, pharmacological inhibition of SK2 by ABC294640 (in Phase II clinical trials) leads to telomere dysfunction in A549 cells and interestingly wild type and a phosphomimicking S921D-hTERT mutant of human telomerase overexpression prevented telomere dysfunction whereas S1P defective mutant D684A-hTERT did not. In vivo, inhibition of SK2 by ABC294640 or shRNA knockdown displayed reduced tumor volume, decreased telomerase protein levels and increased TUNEL positive cells. Interestingly, qPCR based gene array revealed robust increase in TCF21 tumor suppressor in stable shSK2 tumors. Furthermore, shRNA against TCF21 following SCID mice xenograft studies showed protection against ABC294640 induced tumor suppression. Interestingly, SK2 inhibition showed ATM/ATR kinase mediated p-CHK1 increase and subsequent activation of caspase-3 in both in vivo and in vitro experiments and stable TCF21 knockdown prevents apoptotic cell death. Importantly, p16 overexpression in p16 deficient A549 lung cancer cells prevented caspase-3 activation and leading to senescence induction following inhibition of SK2-S1P by ABC294640 or shRNA against SK2. Overall, our data suggest that SK2-S1P regulates telomere dysfunction through p16 mediated aging in normal somatic tissues and TCF21 mediated tumor suppression in lung cancer and interestingly SK2-S1P and p16 acts as a molecular switch between senescence and apoptosis in normal and cancer cells respectively. Citation Format: Shanmugam Panneer Selvam, Marion Cooley, Kristi Helke, Elizabeth Garrett-Mayer, Charles D. Smith, Besim Ogretmen. Sphingosine kinase 2/sphingosine 1-phosphate signaling regulates p16 mediated accelerated aging in normal somatic tissues and tcf21 mediated tumor suppression in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2017-5474

Sphingosine 1‐phosphate (S1P), a pro‐proliferative sphingolipid generated by sphingosine kinases (SK1 and SK2) has been shown to be up regulated in many cancers. Telomerase, a ribonucleoprotein extends the ends of chromosomes (telomeres), which is involved in cancer pathogenesis and hTERT, the catalytic subunit of telomerase localizes to nucleus to promote lung cancer growth/metastasis. Previously, we have demonstrated that SK2 generated S1Pbinds and stabilizes telomerase in the nuclear periphery by C′3‐OH of S1P and D684 residue of hTERT. Mechanistically, nuclear S1P binding prevented E3ubiquitin ligase MKRN1‐hTERT association, and protected hTERT from degradation, preventing telomere damage, and delaying senescence in non‐cancerous lung fibroblasts (Panneer Selvam et al , Sci Signal, 2015). The objective of the following study is to understand the molecular mechanisms of lung tumor suppression mediated by SK2‐S1P pathway. First, we identified the protein levels of SK2 and hTERT to be upregulated in both non‐small cell (N=48) and small cell lung cancer (N=40) tumor tissues. Mechanistically, pharmacological inhibition of SK2 by ABC294640 drug (in Phase I clinical trials) leads to telomere dysfunction induced foci (TIF) formation in A549 cells as observed by colocalization of γ‐H2AX and TRF‐2 and interestingly wild type hTERT overexpression prevented TIF formation whereas hTERT D684A expression (lacks S1P binding) did not. In vivo , shRNA mediated SK2 knockdown tumors displayed reduced tumor volume, decreased hTERT protein levels and increased TUNEL positive cells. Moreover, pharmacological inhibition of SK2 by ABC294640 (100mg/kg/day body weight for 16days) in an orthotopic mice model for lung cancer showed decreased cancer proliferation as measured by bioluminescence. Interestingly, qPCR based gene array studies revealed ~5 fold increase in the expression levels of epigenetically silenced tumor suppressor TCF21 in stable shSK2 tumors compared to scrambled control tumors. Moreover, we observed increased protein expression of GADD45 (growth arrest and DNA damage response protein), an upstream regulator of TCF21 with concomitant increase in the mRNA levels of TCF21 following SK2 inhibition. Interestingly, Stable shRNA mediated knockdown of TCF21 in A549cells following xenograft studies in SCID mice showed significant protection against ABC294640 mediated tumor suppression. Interestingly, in in vitro experiments we found caspase‐3activation following treatment with ABC294640 and stable shRNA mediated knockdown of GADD45 and TCF21 prevented caspase‐3 activation suggesting caspase‐3activation downstream of GADD45 and TCF21. Also, ABC294640 treated cells showed decreased cell viability and shGADD45 and shTCF21 cells showed significant protection against cell death. Overall, our data suggest that SK2 generated S1Pregulates telomere dysfunction in cancer cells through hTERT thereby leading to GADD45 and TCF21 mediated tumor suppression in lung cancer. Support or Funding Information NCI 88932‐06

During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. We found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C'3-OH) in S1P and Asp(684) in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with makorin ring finger protein 1 (MKRN1), an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth.

Valosin containing protein (VCP) disease associated with inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype–phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring 10 missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and noncarriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter‐familial variation; and significant genotype–phenotype correlations were difficult to establish because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with an earlier onset of myopathy and Paget (p = 0.03). Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p = 0.03).We identified amyotrophic lateral sclerosis (ALS) was diagnosed in 13 individuals (8.9%) and Parkinson's disease in five individuals (3%); however, there was no genotypic correlation. This study represents the largest dataset of patients with VCP disease and expands our understanding of the natural history and provides genotype–phenotype correlations in this unique disease.

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50 μM of each tetracycline-derived compound 20 min prior to exposure to 500 μM iodoacetic acid plus 1 mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH 6.2 for 4 h prior to reoxygenation at pH 7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca2 + uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca2 + uptake and suppressed the Ca2 +-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca2 + uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

Background: Positive associations between pain and depression in the general population have been well characterized; however, the interplay between pain, depression, and early cognitive decline, characterized as mild cognitive impairment (MCI), is poorly understood. Methods: The current study examined the association of self-reported pain complaints (measured by the 36-item Short Form Health Survey) and self-reported depressive symptoms (measured by the 30-item Geriatric Depression Scale) in cognitively intact participants (n = 492) and participants with a clinical diagnosis of MCI (n = 83). Results: Depressive symptoms and subjective reports of pain were significantly associated in the entire sample (r = .29; P < .0001). Multiple logistic regression modeling (adjusted for age, education, and APOE4 status as covariates) demonstrated that while depressive symptoms were positively associated with the diagnosis of MCI (P < .001), subjective pain reports were negatively associated with MCI (P < .002). Conclusion: While the negative association of subjective pain complaints with MCI might arguably be explained by the development of anosognosia, self-reports of depressive symptoms were actually increased in these participants, suggesting preserved insight into cognitive decline–associated symptoms. It is possible that preferential involvement of limbic circuitry in MCI could explain these findings. Future studies are needed to elucidate the reasons for the dissociation of pain and depressive symptoms in MCI described in the present article.