Genotype-phenotype associations in CRB1 bi-allelic patients: a novel mutation, a systematic review and meta-analysis.

PURPOSE: The goal of the study was to search for novel bi-allelic CRB1 mutations, and then to analyze the CRB1 literature at the genotypic and phenotypic levels. APPROACH: We screened various variables such as the CRB1 mutation types, domains, exons, and genotypes and their relation with specific ocular phenotypes. An emphasis was given to the bi-allelic missense and nonsense mutations because of their high prevalence compared to other mutation types. Finally, we quantified the effect of various non-modifiable factors over the best-corrected visual acuity oculus uterque (BCVA OU) using multivariate linear regression models and identified genetic interactions. RESULTS: A novel bi-allelic missense in the exon 9 of CRB1; c.2936G > A; p.(Gly979Asp) was found to be associated with rod-cone dystrophy (RCD). CRB1 mutation type, exons, domains, and genotype distribution varied significantly according to fundus characteristics, such as peripheral pigmentation and condition, optic disc, vessels, macular condition, and pigmentation (P < 0.05). Of the 154 articles retrieved from PubMed, 96 studies with 439 bi-allelic CRB1 patients were included. Missense mutations were significantly associated with an absence of macular pigments, pale optic disc, and periphery pigmentation, resulting in a higher risk of RCD (P < 0.05). In contrast, homozygous nonsense mutations were associated with macular pigments, periphery pigments, and a high risk of LCA (P < 0.05) and increased BCVA OU levels. We found that age, mutation types, and inherited retinal diseases were critical determinants of BCVA OU as they significantly increased it by 33% 26%, and 38%, respectively (P < 0.05). Loss of function alleles additively increased the risk of LCA, with nonsense having a more profound effect than indels. Finally, our analysis showed that p.(Cys948Tyr) and p.(Lys801Ter) and p.(Lys801Ter); p.(Cys896Ter) might interact to modify BCVA OU levels. CONCLUSION: This meta-analysis updated the literature and identified genotype-phenotype associations in bi-allelic CRB1 patients.

Systemic Treatment for Brain Metastasis and Leptomeningeal Disease in Breast Cancer Patients.

PURPOSE OF REVIEW: Breast cancer with brain metastasis (BCBM) and leptomeningeal disease (LMD) are important clinical problems. Traditionally, patients with metastases to the brain and meninges were excluded from clinical trials; hence, robust, evidence-based treatment recommendations are lacking. In this review, we outline the systemic treatment options and ongoing clinical trials. RECENT FINDINGS: Several recent studies have added to the systemic treatment options available. Antibody-drug conjugates have changed the therapeutic landscape. Combination treatment modalities that target multiple mechanisms including disruption of the blood brain barrier are increasingly being studied. Breast cancer with brain metastases and LMD is a heterogenous disease. While the prognosis remains grim, with more systemic treatment options, patients with BCBM are now living longer. Many ongoing clinical trials hold promise to further improve outcomes.

Tetrazo[1,2-b]indazoles: Straightforward Access to Nitrogen-Rich Polyaromatics from s-Tetrazines.

The straightforward access to a new class of aza-polyaromatics is reported. Starting from readily available fluorinated s-tetrazine, a cyclization process with azide leads to the formation of an unprecedented tetrazo[1,2-b]indazole or a bis-tetrazo[1,2-b]indazole (cis and trans conformers). Based on the new nitrogen core, further N-directed palladium-catalyzed ortho-C-H bond functionalization allows the introduction of halides or acetates. The physicochemical properties of these compounds were studied by a joint experimental/theoretical approach. The tetrazo[1,2-b]indazoles display solid-state π-stacking, low reduction potential, absorption in the visible range up to the near-infrared, and intense fluorescence, depending on the molecular structure.

Antitumor Activity of Ethanolic Extract from Thymbra Spicata L. aerial Parts: Effects on Cell Viability and Proliferation, Apoptosis Induction, STAT3, and NF-kB Signaling.

Thyme-like plants including Thymbra spicata L. are widely used as food and folk medicinal remedies in the Mediterranean area. This study aimed to explore the in vitro antitumor potential of polyphenol-enriched extracts from aerial parts of T. spicata. The ethanolic extract significantly inhibited proliferation of different human tumor cell lines, without significant effects on non-neoplastic cells. A deeper investigation of the molecular mechanism sustaining the in vitro antitumor activity of the extract was carried on the human breast cancer cells MCF-7 in comparison with the normal breast cells MCF-10A. The effects on MCF-7 cells were associated with the following: (i) production of reactive oxygen species (ROS) and release of nitric oxide; (ii) apoptosis induction; and (iii) reduction in STAT3 and NF-kB phosphorylation. The ethanolic extract from T. spicata leaves might represent a novel therapeutic tool in combination with conventional chemotherapy to reduce the adverse side effects and drug resistance.

Bridge-Clamp Bis(tetrazine)s with [N]8 π-Stacking Interactions and Azido-s-Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines.

Click chemistry at a tetrazine core is useful for bioorthogonal labeling and crosslinking. Introduced here are two new classes of doubly clickable s-aryl tetrazines synthesized by Cu-catalyzed cross-coupling. Homocoupling of o-brominated s-aryl tetrazines leads to bis(tetrazine)s structurally characterized by tetrazine cores arranged face-to-face. [N]8 π-stacking interactions are essential to the conformation. Upon inverse electron demand Diels-Alder (iEDDA) cycloaddition, the bis(tetrazine)s produce a unique staple structure. The o-azidation of s-aryl tetrazines introduces a second proximal intermolecular clickable function that leads to double click chemistry opportunities. The stepwise introduction of fluorophores and then iEDDA cycloaddition, including bioconjugation to antibodies, was achieved on this class of tetrazines. This method extends to (thio)etherification, phosphination, trifluoromethylation and the introduction of various bioactive nitrogen-based heterocycles.

Molecular engineering of 3-arylated tetrazo[1,2-b]indazoles: divergent synthesis and structure-property relationships.

The synthetic scope of 3-arylated tetrazo[1,2-b]indazoles is reported based on a Pd-catalyzed Liebeskind-Srogl cross-coupling reaction followed by an N-cyclisation process. The reactivity of the nitrogen atoms was used to further diversify these N-rich polyaromatic tetrazo[1,2-b]indazoles in a panel of reactions (protonation, selective oxidation, metallations). Selective ortho-C-H activation/functionalization on the heterocycle was also demonstrated with three transition metals (TM = Pd, Ir and Rh). The effects of all these molecular engineering strategies, particularly the N-modifications, on the optical and redox properties of the 3-arylated tetrazoindazoles were studied experimentally and theoretically. This study highlights the diversity of molecular structures and electronic properties offered by the tetrazo[1,2-b]indazole platform.

Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia.

BACKGROUND: MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet. METHODS: TaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). RESULTS: The plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119- 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796-0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819-0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls. CONCLUSIONS: Our findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.

Chemical composition, in vitro cytotoxicity and anti-free radical properties of six extracts from Lebanese Trigonella berythea Boiss.

This study represents an original work aimed to recognize the main constitution of Trigonella berythea. The total phenolic content and total flavonoid content of leaves and stems of T. berythea have been estimated. An extraction and purification of phenolic mixture compounds from the leaves and stems of this plant have been performed and their antioxidant potential using the DPPH, H2O2 and chelating of ferrous ions tests has been evaluated. Then, their cytotoxicity on the MCF7 breast cancer cell line by the XTT Cell Viability technique has been studied. Our results demonstrated that leaves of T. berythea had higher total phenolic content and total flavonoid content than stems. On the other hand, the six extract from leaves and stems of this plant demonstrated a high antioxidant potential reaches more than 80%. Also, extracts from leaves of T. berythea had the highest inhibitory effect on MCF7 and U937 cell growth than that of stems. This inhibition was more than 60% for all extracts. These results provide new insight into the health functions of leaves and stems and demonstrate that T. berythea extracts can potentially have health benefits.

Impact of GSM-EMW exposure on the markers of oxidative stress in fetal rat liver.

The current study investigated the effects of 24 h/day prenatal exposure to global system for mobile communication electromagnetic fields (GSM-EMFs), 900 MHZ-induced electromagnetic radiation (EMR), on oxidative stress (OS) status, apoptotic, and inflammatory changes in liver of rats during their fetal development period. Fifty-two Sprague-Dawley pregnant rats were equally divided into control and exposed groups. Whole embryos were removed at 7.5 dpc (days post coitus), while liver tissues were extracted from embryos at 11.5, 15.5, and 19.5 dpc. For exposed animals, results showed an increased OS reflected by high levels of malondialdehyde (MDA), a decrease in cytosolic superoxide dismutase (cytoSOD) activity, in mitochondrial superoxide dismutase (mitoSOD) levels and catalase (CAT) mRNA expression but also in hepatic nuclear factor erythroïd 2-related Factor 2 (Nrf-2), protein kinase B (Akt1), and intercellular adhesion molecule-1 (ICAM-1) mRNA expression at 15.5 dpc. Moreover, GSM-EMR exposure was shown to significantly decrease mitoSOD and CAT activities at almost all studied ages. Thus, rat embryos may be protected by their mothers from OS, apoptotic, and pro-inflammatory responses till a sensitive developmental stage, during a continuous prenatal EMR exposure. This protection could be then created from the embryos themselves.

Alkali halides as nucleophilic reagent sources for N-directed palladium-catalysed ortho-C-H halogenation of s-tetrazines and other heteroaromatics.

A general palladium-catalysed selective C-H halogenation reaction is reported, which was successfully achieved for a large variety of functionalized aromatic rings incorporating diverse N-directing groups. By using simple alkali halides of MX type as the nucleophilic reagent source (M = Li, Na, K, Cs and X = I, Br and Cl), and phenyliodanediacetate oxidant, clean C-H-iodination, bromination and chlorination reactions were performed. This general protocol of selective ortho-monohalogenation, which complements but contrasts with the classical methods using electrophilic reagents, is achievable in a short time (30 min) with microwave irradiation assistance. The reaction was extended to substrates bearing N-directing pyridine, pyrimidine, pyrazole, oxazoline, naphtho[1,2-d]thiazole, and azobenzene groups. Notably, the topical and selectivity-challenging s-tetrazine, as a nitrogen-rich heteroaromatic, was successfully halogenated by this protocol.

Effects of continuous prenatal and postnatal global system for mobile communications electromagnetic waves (GSM-EMW) exposure on the oxidative stress biomarkers in female rat liver.

In light of the increased use of communication technologies, the harm caused by continuous exposure to emitted radiation on pregnancy and developing newborns is among the public concerns. Using Sprague-Dawley rats, our study investigates the effects of 24 h/day prenatal and postnatal 900 MHz radiofrequency electromagnetic radiation (RF-EMR) exposure of female rats on liver oxidative stress (OS) and other hepatic parameters at postnatal days (PND) 1, 9, and 21. Our results showed that RF-EMR exposure led to an increase in oxidative stress status as indicated by a significant elevation in MDA level at PND9 and PND21, a decrease in catalase (CAT) activity at all ages, a reduction (PND1 and PND9) in catalase amounts and mRNA expression, in addition to a decrease in GPx activity at PND21 in the exposed group. Current findings also showed a significant increase in cytoSOD at PND9 and 21 and a reduction in mitoSOD at PND21 in the exposed groups compared to the control groups. However, significant increases in glutathione peroxidase (GPx) level and mitoSOD activity were observed at all studied ages. Furthermore, cytoSOD activity showed a significant reduction in PND1, whereas in PND9 the value of this parameter increased compared to the non-exposed group. Moreover, while SOD1 mRNA expression increased at PND1, it decreased at PND9 and 21. However, GPx1 expression was shown to be always decreased in the exposed group. In addition, at PND1 and 9, exposed rats showed a similar response on Akt1, nuclear factor erythroïd 2-related factor 2 (Nrf-2), and intercellular adhesion molecule-1 (ICAM-1) expression. Therefore, an increased oxidative stress status produced from a continuous (24 h/day) GSM-modulated 900 MHz radiofrequency electromagnetic radiation (RF-EMR) exposure during the prenatal and postnatal periods may result in adverse health effects during future life stages.

Repressive effect of Rhus coriaria L. fruit extracts on microglial cells-mediated inflammatory and oxidative stress responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhus coriaria L. represents a herbal shrub that is used widely in traditional medicine in the Middle East region to treat different diseases including inflammation-related disorders. R. coriaria extracts have been well characterized in terms of their biological activities, pharmacological potential and phytochemical components. However, the effect of R. coriaria on neuro-inflammation has not been studied previously in detail. AIM OF THE STUDY: In the present study, we performed a qualitative phytochemical analysis and investigated the antioxidant and anti-neuro-inflammatory potential of R. coriaria extracts on BV-2 microglial cells. MATERIALS AND METHODS: R. coriaria extracts were prepared using two different solvents: distilled water and ethanol. Phytochemical screening was performed to determine the principal bioactive components. The radical scavenging activity was assessed by DPPH method (2,2-diphenyl-1-picrylhydrazyl). The effect of R. coriaria on neuro-inflammation was studied upon measuring the production of oxidative stress and inflammatory factors using DCF (2',7'-dichlorofluorescein) and Nitric oxide (NO) assays respectively, and by analyzing the mRNA (TNFα, IL-10, iNOS and COX-2) and protein (NFκß) levels of genes involved BV-2 microglia cells-mediated inflammation using quantitative Real Time PCR and Western blot, respectively. RESULTS: We found that R. coriaria extracts contain high phenolic and flavonoid contents. Interestingly, the ethanolic extract exerted a potent anti-inflammatory potential on insulted BV-2 cells manifested by: i) inhibition of Reactive Oxygen species (ROS) production and nitric oxide (NO) release; ii) suppressing TNFα, iNOS and COX-2 mRNA levels; iii) reducing NFκß activation; and iiii) enhancing IL-10 transcription levels. CONCLUSION: Our results indicate that the neuro-inflammation inhibitory activity of R. coriaria extracts involves the inhibition of NF-κB signaling pathway. These findings suggest that R. coriaria might carry therapeutic potential against neurodegenerative diseases.

Protective effects of extracts from Ephedra foeminea Forssk fruits against oxidative injury in human endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedra foeminea is a member of the Ephedraceae family which is widespread in the eastern Mediterranean area. In Lebanon, Ephedra is a popular remedy in traditional medicine to prevent and/or counteract many stress oxidative-related diseases like inflammation and bacterial infections. AIM OF THE STUDY: Oxidative stress leads to endothelial cell dysfunction, and is a major factor contributing to etiology of atherosclerosis and related diseases. This study aims to investigate the antioxidant and cytoprotective potential of extracts from E. foeminea fruits on human endothelial cells exposed to hydrogen peroxide (H2O2) to mimic in vitro vascular endothelium dysfunction. MATERIALS AND METHODS: Different extracts of E. foeminea fruits were prepared using pure ethanol (EE), methanol/water (EMW), pure hexane (Ehex) or ethyl acetate/water (Epoly) as extraction solvents. The phenolome profile of each extract was characterized using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Total phenolic and flavonoid content, and radical scavenging properties of the extracts were assessed spectrophotometrically. Then, the effects on human endothelial cells HECV were evaluated. RESULTS: Epoly extract showed the highest phenol and flavonoid content, and the highest radical scavenging capacity. On H2O2-insulted HECV cells Epoly was able: (i) to counteract the ROS/RNS production and lipid peroxidation; (ii) to rescue the ROS-dependent decrease in the mitochondrial membrane potential; (iii) to counteract the apoptosis induction; (iv) to restore endothelial cell viability and migration. CONCLUSIONS: The findings indicated that the polyphenol-enriched extract Epoly from E. foeminea fruits is endowed with in vitro anti-oxidant and anti-apoptotic effects and might be used as nutraceutical for treating ROS-related endothelium dysfunction and inflammation.

UVB upregulates the bax promoter in immortalized human keratinocytes via ROS induction of Id3.

Id3 belongs to the inhibitor of differentiation family of helix-loop-helix transcription factors, important in proliferation, differentiation and apoptosis. We showed that Id3, but not Id2 or Id1, mediates the UVB-sensitization of immortalized keratinocytes by inducing caspase 9-dependent apoptosis. In this study, quantitative PCR analysis revealed a time-dependent increase in Id3 mRNA induced by UVB, dependent on reactive oxygen species. UVB upregulated promoter activity of Id3, but not Id2, at early time points, as shown by reporter assays and also stabilized Id3 mRNA, increasing its half-life from 10 to approximately 60 min. We next examined downstream events related to UVB-induced Id3 upregulation and investigated the effects of UVB or ectopic expression of Id3 on bax promoter activity. Regulatory elements in the bax promoter that mediate transcriptional activation by UVB and Id3, in the absence of p53, were identified. Bax promoter deletion analysis revealed that transcriptional activation by UVB involves a 738-bp region upstream from the transcription start site of bax. Mimicking the effects of UVB, ectopic expression of Id3 also upregulated bax mRNA and activated this 738-bp fragment. Mutational analysis of the transcription binding sites further showed that point mutations of the E-box region found in the 738-bp fragment, but not in a 174-bp fragment, completely abolished Id3- and UVB-inducible bax promoter activity, thus confirming the importance of Id3 and UVB-mediated Id3 upregulation in activating the bax promoter. These results suggest a mechanism whereby reactive oxygen species upregulation of Id3 relieves repression of bax via E-box-binding factors.

Antisteatotic and antioxidant activities of Thymbra spicata L. extracts in hepatic and endothelial cells as in vitro models of non-alcoholic fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymbra spicata, a member of the Lamiaceae family, is native to eastern Mediterranean area. Leaves of this plant are rich in phenolic compounds and are a popular remedy of traditional medicine in Lebanon to prevent and/or counteract hyperlipidemia and hyperglycemia. AIM OF THE STUDY: To evaluate the antisteatotic and antioxidant activities of extracts from leaves of Thymbra spicata L. using in vitro models of non-alcoholic fatty liver disease (NAFLD), a leading cause of liver-related morbidity and mortality worldwide, for whom no effective treatments are still available. MATERIALS AND METHODS: Two different extracts from Thymbra spicata L. aerial parts were prepared using water (TW) or ethanol (TE) as solvent. Their chemical composition was characterized by gas and liquid chromatography coupled with mass spectrometry. Both extracts were tested on cultured hepatic and endothelial cells treated to mimic in vitro a multisistemic pathology such as NAFLD. We assayed the effects on lipid accumulation, free radical production, lipid peroxidation, cell migration. RESULTS: Both the total phenolic and the total flavonoid contents were higher in the ethanolic extract. Rosmarinic acid was the most abundant polyphenol in TW, while TE was richer in carvacrol. Our findings demonstrated that both extracts ameliorated lipid accumulation, oxidative stress and inflammation in the NAFLD cellular models. However, the aqueous extract was more effective to reduce hepatic steatosis, and the ethanolic extract had higheranti-oxidant potential and wound healing activity. CONCLUSIONS: T. spicata extracts could be promising bioactive products to develop natural therapeutic agents or dietary supplements to treat NAFLD and obesity-related metabolic disease. Our findings suggest that while the ethanolic extract might be used in preventing endothelium dysfunction, the aqueous extract would act better as lipid-lowering agent.

Sphingomyelin Synthase 1 (SMS1) Downregulation Is Associated With Sphingolipid Reprogramming and a Worse Prognosis in Melanoma.

Sphingolipid (SL) metabolism alterations have been frequently reported in cancer including in melanoma, a bad-prognosis skin cancer. In normal cells, de novo synthesized ceramide is mainly converted to sphingomyelin (SM), the most abundant SL, by sphingomyelin synthase 1 (SMS1) and, albeit to a lesser extent, SMS2, encoded by the SGMS1 and SGMS2 genes, respectively. Alternatively, ceramide can be converted to glucosylceramide (GlcCer) by the GlcCer synthase (GCS), encoded by the UGCG gene. Herein, we provide evidence for the first time that SMS1 is frequently downregulated in various solid cancers, more particularly in melanoma. Accordingly, various human melanoma cells displayed a SL metabolism signature associated with (i) a robust and a low expression of UGCG and SGMS1/2, respectively, (ii) higher in situ enzyme activity of GCS than SMS, and (iii) higher intracellular levels of GlcCer than SM. SMS1 was expressed at low levels in most of the human melanoma biopsies. In addition, several mutations and increased CpG island methylation in the SGMS1 gene were identified that likely affect SMS1 expression. Finally, low SMS1 expression was associated with a worse prognosis in metastatic melanoma patients. Collectively, our study indicates that SMS1 downregulation in melanoma enhances GlcCer synthesis, triggering an imbalance in the SM/GlcCer homeostasis, which likely contributes to melanoma progression. Evaluating SMS1 expression level in tumor samples might serve as a biomarker to predict clinical outcome in advanced melanoma patients.

Rapid identification and validation of novel targeted approaches for Glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.

Tumor heterogeneity is a major factor in glioblastoma's poor response to therapy and seemingly inevitable recurrence. Only two glioblastoma drugs have received Food and Drug Administration approval since 1998, highlighting the urgent need for new therapies. Profiling "omics" analyses have helped characterize glioblastoma molecularly and have thus identified multiple molecular targets for precision medicine. These molecular targets have influenced clinical trial design; many "actionable" mutation-focused trials are underway, but because they have not yet led to therapeutic breakthroughs, new strategies for treating glioblastoma, especially those with a pharmacological functional component, remain in high demand. In that regard, high-throughput screening that allows for expedited preclinical drug testing and the use of GBM models that represent tumor heterogeneity more accurately than traditional cancer cell lines is necessary to maximize the successful translation of agents into the clinic. High-throughput screening has been successfully used in the testing, discovery, and validation of potential therapeutics in various cancer models, but it has not been extensively utilized in glioblastoma models. In this report, we describe the basic aspects of high-throughput screening and propose a modified high-throughput screening model in which ex vivo and in vivo drug testing is complemented by post-screening pharmacological, pan-omic analysis to expedite anti-glioma drugs' preclinical testing and develop predictive biomarker datasets that can aid in personalizing glioblastoma therapy and inform clinical trial design.

Patient With 2 Hematologic Malignancies Presenting as Neurolymphomatosis.

Peripheral nervous system damage from hematologic malignancies is related to neoplastic cells infiltration of peripheral nerves or to monoclonal antibody production cross-reacting with peripheral nerves' antigens. Neurolymphomatosis (NL), a rare manifestation of hematologic malignancies, occurs when malignant cells invade the peripheral nerves leading to various manifestations. Here, we report a case of NL with 2 hematologic malignancies in a 79-year-old woman presenting with lower extremity pain/weakness. Investigation revealed anemia, IgM kappa monoclonal gammopathy, and elevated anti-MAG titer. Electrodiagnostic studies were consistent with mononeuropathy multiplex while imaging suggested malignancy in her ovaries and right S1 nerve root. Bone marrow and ovarian biopsies revealed chronic myelomonocytic leukemia, Waldenstrom macroglobulinemia, and diffuse large B-cell lymphoma. She received standard chemotherapy resulting in radiographic resolution of disease and symptomatic relief. NL can precede the diagnosis of hematologic malignancy but its symptoms are not easily identifiable, whereas management depends on the treatment of the underlying tumor.

Gefitinib inhibits the growth and invasion of urothelial carcinoma cell lines in which Akt and MAPK activation is dependent on constitutive epidermal growth factor receptor activation.

PURPOSE: Abnormally high levels of epidermal growth factor receptor (EGFR) protein are associated with advanced tumor stage/grade. The objective of this study was to evaluate the effects of the specific EGFR tyrosine kinase inhibitor gefitinib on activation of the Akt and mitogen-activated protein kinase (MAPK) pathways in human urothelial cell carcinoma (UCC) cell lines and to identify potential markers of gefitinib responsiveness in biopsy samples of UCC. EXPERIMENTAL DESIGN: Changes in markers of UCC growth and invasion after exposure to gefitinib were studied in six human UCC cell lines expressing various levels of EGFR. The findings were related to activation of Akt and MAPK. We studied the influence of gefitinib on intraepithelial expansion of the responsive 1207 cell line. EGFR, Akt, and MAPK activation was studied by Western blot analysis of a panel of 57 human UCC. RESULTS: Gefitinib had a growth-inhibitory and anti-invasive effect in two of six UCC cell lines (i.e., 647V and 1207). Gefitinib was also able to block the expansion of 1207 at the expense of normal urothelial cells. These effects did not depend on the level of expression of EGFR but they were associated with the down-regulation of MAPK and Akt activity; in 1207 cells, gefitinib activity was associated with p27 up-regulation and p21 and matrix metalloproteinase-9 down-regulation. Similarly, the Akt and MAPK pathways were found to be strongly phosphorylated in association with EGFR activation in a subset of human UCC specimens. CONCLUSIONS: Activation of EGFR, Akt, and MAPK defines a subset of UCC which might provide information for the identification of gefitinib responders.

Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L.

Sphingomyelin synthases 1 and 2 convert the anti-oncometabolite ceramide to sphingomyelin, the most abundant sphingolipid in plasma membrane. CD95L-induced ceramide increase is associated with the caspase-dependent inhibition of sphingomyelin synthesis, which enhances the mitochondrial route to apoptosis. Knocking down sphingomyelin synthase 1 or inhibiting sphingomyelin synthesis facilitates ceramide accumulation, cytochrome c release from mitochondria, and caspase-9 activation in cancer cell upon CD95L treatment. Here, we describe a method to monitor in situ sphingomyelin synthase activity changes triggered by CD95L.