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STUDY OBJECTIVES: To determine whether adherence to continuous positive airway pressure (CPAP) in adults with uncomplicated obstructive sleep apnea differs by rural vs urban residential address. METHODS: In this prospective cohort study, we recruited adults who initiated CPAP for uncomplicated obstructive sleep apnea that was diagnosed by a physician using sleep specialist-interpreted diagnostic testing. Participants were classified as urban (community size > 100,000) or rural (community size < 100,000) by translating residential postal code into geographic census area. The primary outcome was mean daily hours of CPAP use compared between rural and urban patients. Secondary outcomes included the proportion of patients who were adherent to CPAP, change in Epworth Sleepiness Scale score, change in EuroQOL-5D visual analog score, and Visit-Specific Satisfaction Instrument score. All outcomes were measured 3 months after CPAP initiation. RESULTS: We enrolled 242 patients (100 rural) with a mean (standard deviation) age of 51 (13) years and a respiratory event index of 24 (18) events/h. The mean (95% confidence interval) CPAP use was 3.19 (2.8–3.58) hours/night and 35% were CPAP-adherent, with no difference between urban and rural patients. Among the 65% of patients who were using CPAP at 3 months, the mean CPAP use was 4.89 (4.51–5.28) hours/night and was not different between rural and urban patients. Improvement in the Epworth Sleepiness Scale score and patient satisfaction was similar between groups, but the EuroQOL-5D score improved to a greater extent in rural patients. Urban or rural residence was not associated with CPAP adherence according to multivariable regression analysis. CONCLUSIONS: Rural vs urban residence was not associated with differences in CPAP adherence among patients with uncomplicated OSA diagnosed by a physician using specialist-interpreted sleep diagnostic testing. CITATION: Corrigan J, Tsai WH, Ip-Buting A, et al. Treatment outcomes among rural and urban patients with obstructive sleep apnea: a prospective cohort study. J Clin Sleep Med. 2022;18(4):1013–1020.

Supplementary Figure Legends

Supplementary Figure 7. INPP4B loss does not produce any morphological or cytoskeletal differences.

PDF - 14407K, Supporting histology for Olaparib treatment in vivo.

Supplementary Figure 3. INPP4B's promoter is not directly methylated.

Supplementary Figure S3. ERG binding to CHK1 promoter.

PDF - 632K, Preclinical assessment of Olaparib and BKM120 treatment in vivo.

Supplementary Figure Legends

PDF - 546K, Olaparib induces a differing response in vitro depending on the genetic background.

PDF - 2951K, Olaparib induces a differing response in vivo depending on the genetic background.

Supplementary Figure 2. Validation of INPP4B antibody.

Supplementary Figure 1. Generation of Pten+/-Inpp4b+/- and Pten+/-Inpp4b-/- mice.

PDF - 114k, Supporting western blot analysis and drug treatment in prostate cancer cell lines.

PDF - 116K, Supplementary Materials and Methods and Supplementary Figure Legends.

Supplementary Figure 4. Loss of Inpp4b in MEFs lead to increased Akt activation.

Supplementary Figure 5. Loss of INPP4B results in increased AKT2 activation.

PDF - 138K, Survival curves of the in vivo treatment of Olaparib and BKM120.

Depletion of FZR1 leads to BRAF stabilization and subsequent ERK activation. Supplementary Figure S2. Depletion of FZR1 triggers the onset of premature senescence in primary melanocytes. Supplementary Figure S3. APCFZR1 promotes BRAF ubiquitination and subsequent degradation in a D-box dependent manner. Supplementary Figure S4. FZR1 disrupts the BRAF dimerization process. Supplementary Figure S5. Phosphorylation of FZR1 N-terminus by ERK and CYCLIN D1/CDK4 inhibits the APCFZR1 E3 ubiquitin ligase activity. Supplementary Figure S6. Pharmacologically inhibiting BRAF/ERK and CDK4 restores the APCFZR1 E3 ligase activity. Supplementary Figure S7. Depletion of FZR1 co-operates with PTEN deficiency to promote co-activation of BRAF/ERK and AKT oncogenic signaling both in vitro and in vivo. Supplementary Table S1. Mutation and deletion of FZR1 and 14 APC core complex subunits identified in melanoma patients from the TCGA melanoma dataset (cbioportal.org).

Krüppel-like Factor 5 (KLF5) is a zinc-finger transcription factor associated with cell cycle progression and cell survival. KLF5 plays a key role in mammalian intestinal epithelium development and maintenance, expressed at high levels in basal proliferating cells and low levels in terminally differentiated cells. Considering Barrett's esophagus (BE) and esophageal adenocarcinoma's (EAC) histopathological similarities to intestinal epithelium, we sought to determine KLF5’s role in BE and EAC, as well as KLF5’s possible connection to the sonic hedgehog (SHH) pathway which is highly active in BE and EAC development. Low levels of KLF5 mRNA were found in BE cell lines and tissue– similar to what has been reported in differentiated intestinal epithelium. In contrast, higher KLF5 levels were observed in EAC cells and tissues. KLF5 knockdown in EAC cells caused significant decreases in cell migration, proliferation, and EAC-associated gene expression. Moreover, KLF5 knockdown led to decreased SHH signaling. These results suggest that KLF5 is connected to the SHH pathway in BE and EAC and may represent a potential drug target in EAC; further studies are now indicated to verify these findings and elucidate underlying mechanisms involved.

Objective The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. Background The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. Methods The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. Results BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. Conclusion Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

There have been no reports describing the effects of cancer cell–derived extracellular vesicles (EVs) on three-dimensional organoids. In this study, we delineated the proneoplastic effects of esophageal adenocarcinoma (EAC)–derived EVs on gastric organoids (gastroids) and elucidated molecular mechanisms underlying these effects. EVs were identified using PKH-67 staining. Morphologic changes, Ki-67 immunochemistry, cell viability, growth rates, and expression levels of miR-25 and miR-210, as well as of their target mRNAs, were determined in gastroids co-cultured with EAC-derived extracellular vesicles (c-EVs). C-EVs were efficiently taken up by gastroids. Notably, c-EV–treated gastroids were more crowded, compact, and multilayered and contained smaller lumens than did those cultured in organoid medium alone or in EAC-conditioned medium that had been depleted of EVs. Moreover, c-EV–treated gastroids manifested increased proliferation and cellular viability relative to medium-only or EV-depleted controls. Expression levels of miR-25 and miR-210 were significantly higher, and those of PTEN and AIFM3 significantly lower, in c-EV–treated versus medium-only or EV-depleted control groups. Inhibitors of miR-25 and miR-210 reversed the increased cell proliferation induced by c-exosomes in co-cultured gastroids by lowering miR-25 and miR-210 levels. In conclusion, we have constructed a novel model system featuring the co-culture of c-EVs with three-dimensional gastroids. Using this model, we discovered that cancer-derived EVs induce a neoplastic phenotype in gastroids. These changes are due, at least in part, to EV transfer of miR-25 and miR-210.

During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.

Most deep neural networks deployed today are trained using GPUs via high-level frameworks such as TensorFlow and PyTorch. This paper describes changes we made to the GPGPU-Sim simulator to enable it to run PyTorch by running PTX kernels included in NVIDIA's cuDNN library. We use the resulting modified simulator, which has been made available publicly with this paper, to study some simple deep learning workloads. With our changes to GPGPU-Sim's functional simulation model, we find GPGPU-Sim performance model running a cuDNN enabled implementation of LeNet for MNIST reports results within 30% of real hardware. Using GPGPU-Sim's AerialVision performance analysis tool we observe that cuDNN API calls contain many varying phases and appear to include potentially inefficient microarchitecture behaviour such as DRAM partition bank camping, at least when executed on GPGPU-Sim's current performance model., Source code available at: https://github.com/gpgpu-sim/gpgpu-sim_distribution/tree/dev

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable.

Experimental study. The objective of this study was to establish a non-invasive model to produce pressure ulcers of varying severity in animals with spinal cord injury (SCI). The study was conducted at the Johns Hopkins Hospital in Baltimore, Maryland, USA. A mid-thoracic (T7–T9) left hemisection was performed on Sprague-Dawley rats. At 7 days post SCI, rats received varying degrees of pressure on the left posterior thigh region. Laser Doppler Flowmetry was used to record blood flow. Animals were killed 12 days after SCI. A cardiac puncture was performed for blood chemistry, and full-thickness tissue was harvested for histology. Doppler blood flow after SCI prior to pressure application was 237.808±16.175 PFUs at day 7. Following pressure application, there was a statistically significant decrease in blood flow in all pressure-applied groups in comparison with controls with a mean perfusion of 118.361±18.223 (P

As operational and information technologies converge to allow for remote and real-time access to plant operating data and control functions, the process industry could become increasingly susceptible to cyber-attacks. Traditional hazard and risk analysis methods appear inadequate to identify, prevent, and mitigate such attacks. This paper discusses the significance of incorporating cybersecurity vulnerability analysis not just as part of process hazard analysis (PHA), but also in terms of protecting the process control network and implementing adequate safeguards in general against cyber threats. A layer of protection analysis (LOPA) is adapted to evaluate potential weaknesses and ensure safeguards for critical applications would be resistant to cyber-attacks. Integrating cybersecurity into hazard and risk analyses as well as other elements of process safety management (PSM) is demonstrated with examples, making the plant more resilient against both traditional and cyber threats.

Acute stroke care systems in Southeast Asian countries are at various stages of development, with disparate treatment availability and practice in terms of intravenous thrombolysis and endovascular therapy. With the advent of successful endovascular therapy stroke trials over the past decade, the pressure to revise and advance acute stroke management has greatly intensified. Southeast Asian patients exhibit unique stroke features, such as increased susceptibility to intracranial atherosclerosis and higher prevalence of intracranial haemorrhage, likely secondary to modified vascular risk factors from differing dietary and lifestyle habits. Accordingly, the practice of acute endovascular stroke interventions needs to take into account these considerations. Acute stroke care systems in Southeast Asia also face a unique challenge of huge stroke burden against a background of ageing population, differing political landscape and healthcare systems in these countries. Building on existing published data, further complemented by multi-national interaction and collaboration over the past few years, the current state of acute stroke care systems with existing endovascular therapy services in Southeast Asian countries are consolidated and analysed in this review. The challenges facing acute stroke care strategies in this region are discussed.

See related articles at [[www.cmaj.ca/lookup/doi/10.1503/cmaj.171076][2]][2] and [[www.cmaj.ca/lookup/doi/10.1503/cmaj.170371][3]][3] KEY POINTS Against a background of growing concern that rates of cesarean delivery in Canada and globally are rising, trial of labour after previous cesarean

During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.

The harvesting of game birds, such as grouse, with small arms using lead ammunition continues to be common practice in Alberta, Canada. Grouse are routinely consumed as a subsistence traditional food by First Nations in Alberta and the use of lead ammunition increases the potential lead exposure through the consumption of residual bullet or shot fragments in the edible portion of the meat. After removal of visible shot and ammunition fragments, similar to how the samples would be prepared for consumption, impacted and non-impacted grouse breast meat samples were analyzed to characterize the lead content. Using publicly available consumption rates, the potential lead exposures from consumption of both impacted and non-impacted grouse breast meat were estimated and the corresponding health risks were predicted. Comparison of the predicted health risks revealed a significantly higher risk (p

Aggressive T-cell large granular lymphocytic leukemia (T-LGL) is a rare entity with a poor prognosis, the main clinical findings being splenomegaly, lymphadenopathy, thrombocytopenia and a CD3 + /CD56 + phenotype. A 21-year-old lady presented with hepatosplenomegaly, lymphadenopathy, “B” symptoms and thrombocytopenia. She subsequently developed jaundice, ascites and deranged liver function. LGL was detected in the peripheral blood and marrow. They had an aberrant CD3 + /CD4 - /CD8dim/CD56 + phenotype. Karyotyping showed trisomy 8 which has not previously been reported in aggressive T-LGL. A liver biopsy showed hepatic fibrosis with a sinusoidal LGL infiltrate. Her ascitic fluid also showed involvement by T-LGL. She had a poor response to cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) chemotherapy and opted for palliation. We performed targeted sequencing to detect mutations in the STAT3 , STAT5b and PTPRT genes which were absent, suggesting the presence of an alternative genetic driver in our patient. We report an unusual case of aggressive T-LGL with an aberrant immunophenotype, trisomy 8 and hepatic fibrosis with absence of mutations in the STAT pathway. Our case describes novel features of aggressive T-LGL and the need for further studies to elucidate the genetic basis of this rare disease. J Hematol. 2015;4(3):214-218 doi: http://dx.doi.org/10.14740/jh223w

SummaryTumor cells metastasize to distant organs through genetic and epigenetic alterations, including changes in microRNA (miR) expression. Here we find miR-22 triggers epithelial-mesenchymal transition (EMT), enhances invasiveness and promotes metastasis in mouse xenografts. In a conditional mammary gland-specific transgenic (TG) mouse model, we show that miR-22 enhances mammary gland side-branching, expands the stem cell compartment, and promotes tumor development. Critically, miR-22 promotes aggressive metastatic disease in MMTV-miR-22 TG mice, as well as compound MMTV-neu or -PyVT-miR-22 TG mice. We demonstrate that miR-22 exerts its metastatic potential by silencing antimetastatic miR-200 through direct targeting of the TET (Ten eleven translocation) family of methylcytosine dioxygenases, thereby inhibiting demethylation of the mir-200 promoter. Finally, we show that miR-22 overexpression correlates with poor clinical outcomes and silencing of the TET-miR-200 axis in patients. Taken together, our findings implicate miR-22 as a crucial epigenetic modifier and promoter of EMT and breast cancer stemness toward metastasis.

Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC. Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance. Citation Format: Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3077.

Helicobacter pylori prevalence is higher in developing countries than in industrialized countries, and within the latter, higher among immigrants than among nativeborn residents. Using a point-prevalence survey, we sought to identify risk factors for H. pylori seropositivity in US urban East Asian-born populations. At a clinic in New York City, we consecutively enrolled 194 East Asian-born adults, who then responded to a survey and provided a blood sample. Assays were performed to detect IgG antibodies against whole cell (WC) and cytotoxin associated gene A (CagA) antigens of H. pylori. For this group (mean age 50.2+/-14.7 years), the mean period of residence in the United States was 11.9+/-7.7 years. The total H. pylori seroprevalence was 70.1%, with highest (81.4%) in Fujianese immigrants. Multiple logistic regression analysis indicated an independent association of H. pylori seropositivity with Fujianese origin [odds ratios (OR) =2.3, 95% confidence interval (95% CI) =1.05-5.0] and inverse associations with period in the United States (OR per year of residency in the United States =0.95, 95% CI =0.91-0.99) and with a history of dyspepsia (OR for a history of stomach pain =0.52, 95% CI =0.3-1.0). We conclude that H. pylori is highly prevalent among recent East Asian immigrants, especially among Fujianese. The protective effects of history of dyspepsia and duration in the United States suggest that these may be markers for antibiotic therapies.

Case of cerebrofacial arteriovenous metameric syndrome (CAMS) in a 9-year-old boy is described with arteriovenous malformation simultaneously involving the brain and face, with characteristic CAMS type 1 and 2 involvement. This patient demonstrates the wide spectrum of clinical manifestations of CAMS, and in this particular case, the patient exhibits features of hypopituitarism—an association that was not previously described in the literature to our knowledge. Awareness of the underlying embryological abnormality and recognition of resultant clinical and radiological presentations are paramount for diagnosis and treatment.

Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound loss is often observed in advanced prostate cancer. Here, we show that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the prosurvival PI3K–AKT signaling pathway limits the efficacy of a PARP single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human prostate cancer cell lines and in a Pten/Trp53–deficient mouse model of advanced prostate cancer. Our findings, therefore, identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient prostate cancer. Significance: The paucity of therapeutic options in advanced prostate cancer displays an urgent need for the preclinical assessment of novel therapeutic strategies. We identified differential therapeutic vulnerabilities that emerge upon the loss of both PTEN and p53, and observed that combined inhibition of PARP and PI3K provides increased efficacy in hormone-insensitive advanced prostate cancer. Cancer Discov; 4(8); 896–904. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 855

Summary Deregulation of mitogen-activated protein kinase (MAPK) signaling leads to development of pancreatic cancer. Although Ras -mutation-driven pancreatic tumorigenesis is well understood, the underlying mechanism of Ras-independent MAPK hyperactivation remains elusive. Here, we have identified a distinct function of PCNA-associated factor (PAF) in modulating MAPK signaling. PAF is overexpressed in pancreatic cancer and required for pancreatic cancer cell proliferation. In mouse models, PAF expression induced pancreatic intraepithelial neoplasia with expression of pancreatic cancer stem cell markers. PAF-induced ductal epithelial cell hyperproliferation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation independently of Ras or Raf mutations. Intriguingly, PAF transcriptionally activated the expression of late endosomal/lysosomal adaptor, MAPK and mTOR activator 3 ( LAMTOR3 ), which hyperphosphorylates MEK and ERK and is necessary for pancreatic cancer cell proliferation. Our results reveal an unsuspected mechanism of mitogenic signaling activation via LAMTOR3 and suggest that PAF-induced MAPK hyperactivation contributes to pancreatic tumorigenesis.

With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5'-GC and 5'-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO > or = ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene.

The efficacy and safety of a single tablet regimen (STR) of elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF) were analyzed in Phase 3 clinical trials in antiretroviral therapy (ART)-naïve and ART-experienced Asian subjects infected with human immunodeficiency virus (HIV)-1. Studies GS-US-236-102 and GS-US-236-103 were randomized, double-blind, placebo-controlled, 144-week studies conducted in ART-naïve subjects, comparing E/C/F/TDF versus efavirenz (EFV)/F/TDF or ritonavir-boosted atazanavir (ATV+RTV) plus emtricitabine/tenofovir DF (F/TDF), respectively. Studies GS-US-236-115 and GS-US-236-121 were randomized, open-label, 96-week long conducted in ART-experienced subjects, who switched to E/C/F/TDF from ritonavir-boosted protease inhibitors (PI+RTV)+F/TDF, or non-nucleoside reverse transcriptase inhibitors (NNRTI)+F/TDF regimens. The E/C/F/TDF appeared to have sustained efficacy and safety and was well tolerated in the small number of ART-naïve and ART-experienced Asian subjects.

The cytoplasmic membrane protein TonB spans the periplasm of the Gram-negative bacterial cell envelope, contacts cognate outer membrane receptors, and facilitates siderophore transport. The outer membrane receptor FhuA from Escherichia coli mediates TonB-dependent import of ferrichrome. We report the 3.3 angstrom resolution crystal structure of the TonB carboxyl-terminal domain in complex with FhuA. TonB contacts stabilize FhuA's amino-terminal residues, including those of the consensus Ton box sequence that form an interprotein β sheet with TonB through strand exchange. The highly conserved TonB residue arginine-166 is oriented to form multiple contacts with the FhuA cork, the globular domain enclosed by the β barrel.

Background: Slightly less than half of the patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF) do not have specific gene mutations. Recent discovery of CALR mutation in JAK2V617F-negative ET and PMF patients have accounted for 20-25% of ET and PMF patients, and therefore redefining diagnostic approach to ET or PMF. Here we reported the incidence of CALR mutation and clinical features of clinically diagnosed Jak2V617F-negative ET and PMF in Singapore. Method: We retrospectively identified 140 cases of Jak2V617F-negative ET and PMF from two healthcare institutions in Singapore since 2006. Sanger sequencing was performed for the targeted detection of CALR exon 9 mutations. Clinicopathologic correlation was carried out by assessing relevant clinical data and the CALR mutation status, and compared with JAK2-positive cases (25 cases of ET and 9 cases of PMF). Results: The Jak2V617-negative ET/PMF patients were ethnic Chinese (76.2%), Malay (13.9%) and others (9.9%). CALR mutations were detected in 32.8% (40/122) of Jak2V617-negative ET and 55.6% (10/18) Jak2V617-negative PMF patients. Higher platelet count was observed in CALR-positive ET patients with median platelet count of 1056.5 x 109/L and 637.5 x 109/L for CALR-positive and -negative patients respectively (p1% in CALR-positive ET/PMF (12% in CALR-positive and 3.3% in CALR-negative group, p=0.045). Blast transformation occurred in 6% of patients with CALR-positive PMF while only 1.1% in CALR-negative PMF. Besides the two commonly reported mutations (CALR52del, 30.8%; CALR5ins, 33.3%) in our screening of the CALR exon 9 in Jak2V617F-negative ET and PMF patients, 7 other novel mutations (c.1098_1137delinsTTTCTT, c.1102_1104delAAG, c.1122_1155delinsGGGCCAGGCACTTGTCG, c.1129_1153delinsCTTTGCGTTTCTTTT, c.1132_1155delinsTGTCG, c.1139delA, c.1141delG) were also found individually. All mutant CALR proteins possess an altered C-terminus with a longer peptide stretch caused by a disrupted reading frame due to these frameshift mutations. Conclusion: CALR mutation is common in Jak2V617F-negative PMF and ET in this multiracial Singaporean cohort, and has similar frequency when compared with Western report. It was associated with higher platelet count in ET, higher level of LDH and possibly a higher risk of blast transformation in PMF. 7 novel CALR mutations were identified and more subtypes of CALRmutation variants are expected to be uncovered in the near future. Disclosures No relevant conflicts of interest to declare.

The discovery that bifunctional alkylating agents have antitumor activity led to the development of cancer chemotherapy over 50 years ago. While these compounds form a variety of cellular lesions, DNA interstrand cross-links are believed to be the most lethal, impeding both replication and expression of the genetic material. We are characterizing the mechanisms by which diepoxybutane (DEB) and the structurally related compound epichlorohydrin (ECH) exert their cytotoxic effects in cultured cells. Our first goal is to determine the relationship between interstrand cross-linking and cytotoxicity. We are assaying cross-linking ability in cultured cells using Hoechst 33258 to determine the amount of duplex DNA following alkaline denaturation. Only cross-linked DNA reanneals rapidly and interacts with the dye, which is highly fluorescent when bound to duplex DNA. Preliminary results in chicken 6C2 and human HL60 cells suggest a correlation between cross-linking and LD50 values for these compounds. Our second goal is to characterize the pathways by which these compounds induce apoptosis. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB and ECH under conditions determined to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, PUMA and APAF1. Finally, we are identifying the covalent structure of the ECH-cross-linked lesion via electrospray LC/MS. Characterization of the DNA modifications induced by these agents is an important step in understanding how these compounds exert their cytotoxic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2011-4904

The bifunctional alkylating agents are an important class of drugs for the treatment of cancer, psoriasis, and some anemias. These compounds are widely believed to exert their cytotoxic effects through reactions with biological nucleophiles, with DNA interstrand cross-links considered to be the most lethal lesions. Indeed, for many families of bifunctional alkylators, DNA interstrand cross-linking correlates with cytotoxicity. Moreover, apoptotic potential may play a role in antitumor chemotherapeutic utility. We have established previously that diepoxybutane (DEB) and the structurally related compounds epichlorohydrin (ECH) and 1-(chloroethenyl)oxirane (CEO) form DNA interstrand cross-links in vitro. With the goal of elucidating the molecular and cellular mechanisms by which this family of epoxides exerts its cytotoxic effects, we are determining the relationships between interstrand cross-linking, LD50 values, and apoptotic potentials for DEB, ECH, and CEO in chicken 6C2 and human HL-60 cells. Preliminary results in 6C2 cells suggest that cytotoxicity follows the order DEB > CEO > ECH. Furthermore, flow cytometry with Annexin V-FITC/PI dual staining has revealed that the apoptotic potentials follow the order DEB >> ECH > CEO. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB under conditions known to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, and APAF1. Further work is underway to characterize the pathways by which ECH and CEO exert their apoptotic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1695.