Spectroscopic identification of water emission from a main-belt comet.

Main-belt comets are small Solar System bodies located in the asteroid belt that repeatedly exhibit comet-like activity (that is, dust comae or tails) during their perihelion passages, strongly indicating ice sublimation1,2. Although the existence of main-belt comets implies the presence of extant water ice in the asteroid belt, no gas has been detected around these objects despite intense scrutiny with the world's largest telescopes3. Here we present James Webb Space Telescope observations that clearly show that main-belt comet 238P/Read has a coma of water vapour, but lacks a significant CO2 gas coma. Our findings demonstrate that the activity of comet Read is driven by water-ice sublimation, and implies that main-belt comets are fundamentally different from the general cometary population. Whether or not comet Read experienced different formation circumstances or evolutionary history, it is unlikely to be a recent asteroid belt interloper from the outer Solar System. On the basis of these results, main-belt comets appear to represent a sample of volatile material that is currently unrepresented in observations of classical comets and the meteoritic record, making them important for understanding the early Solar System's volatile inventory and its subsequent evolution.

The role of routine chest radiography after implantable venous access port catheter insertion under the guide of ultrasonography and fluoroscopy.

BACKGROUND: The objective of this study is the evaluation of routine chest radiography following the placement of Implantable venous access port catheter (IVAPC) central lines using combined ultrasound and fluoroscopic guidance by a vascular surgeon in the operating room. MATERIAL AND METHOD: A prospective study of 189 consecutive patients who underwent IVAPC central line insertion in the vascular surgery operating room from 2016 to 2019. Venipuncture was performed with an 18-gauge needle under the guidance of sonography in each case, and the access site was noted. The line position was confirmed by fluoroscopy following the procedure. Multiple tries for puncture and patients under 18 were excluded from our study. Routine radiography of chest was performed for all patients and pneumothorax, hemothorax, and catheter malposition were evaluated in each case. RESULTS: There were 2 cases of asymptomatic pneumothorax, no cases of hemothorax, and all catheter tip positions were optimal or acceptable. The annual cost of chest radiography was 33,000,000IRR, 220 h of hospital and staff time, and 1.1 mSv radiation. CONCLUSION: In conclusion, when imaging guidance is used for IVAPC insertion by an experienced surgeon in a high-volume center, performing post-procedure routine chest radiography shows little benefit.

Patient posture correction and alignment using mixed reality visualization and the HoloLens 2.

PURPOSE: The purpose of this study was to develop and preliminarily test a radiotherapy system for patient posture correction and alignment using mixed reality (MixR) visualization. The write-up of this work also provides an opportunity to introduce the concepts and technology of MixR for a medical physics audience who may be unfamiliar with the topic. METHODS: A MixR application was developed for on optical-see-through head-mounted display (HoloLens 2) allowing a user to simultaneously and directly view a patient and a reference hologram derived from their simulation CT scan. The hologram provides a visual reference for the exact posture needed during treatment and is initialized in relation to the origin of a radiotherapy device using marker-based tracking. The system further provides marker-less tracking that allows the user tofreely navigate the room as they view and align the patient from various angles. The system was preliminarily tested using both a rigid (pelvis) and nonrigid (female mannequin) anthropomorphic phantom. Each phantom was aligned via hologram and accuracy quantified using CBCT and CT. RESULTS: A fully realized system was developed. Rigid registration accuracy was on the order of 3.0 ± 1.5 mm based on the performance of three users repeating alignment five times each. The lateral direction showed the most variability among users and was associated with the largest off-sets (approximately 2.0 mm). For nonrigid alignment, the MixR setup outperformed a setup based on three-point alignment and setup photos, the latter of which showed a difference in arm position of 2 cm and a torso roll of 6-7°. CONCLUSIONS: MixR visualization is a rapidly emerging domain that has the potential to significantly impact the field of medicine. The current application is an illustration of this and highlights the advantages of MixR for patient setup in radiation oncology. The key feature of the system is the way in which it transforms nonrigid registration into rigid registration by providing an efficient, portable, and cost-effective mechanism for reproducing patient posture without the use of ionizing radiation. Preliminary estimates of registration accuracy indicate clinical viability and form the foundation for further development and clinical testing.

Dopamine Receptor Antagonists, Radiation, and Cholesterol Biosynthesis in Mouse Models of Glioblastoma.

BACKGROUND: Glioblastoma is the deadliest brain tumor in adults, and the standard of care consists of surgery followed by radiation and treatment with temozolomide. Overall survival times for patients suffering from glioblastoma are unacceptably low indicating an unmet need for novel treatment options. METHODS: Using patient-derived HK-157, HK-308, HK-374, and HK-382 glioblastoma lines, the GL261 orthotopic mouse models of glioblastoma, and HK-374 patient-derived orthotopic xenografts, we tested the effect of radiation and the dopamine receptor antagonist quetiapine on glioblastoma self-renewal in vitro and survival in vivo. A possible resistance mechanism was investigated using RNA-sequencing. The blood-brain-barrier-penetrating statin atorvastatin was used to overcome this resistance mechanism. All statistical tests were 2-sided. RESULTS: Treatment of glioma cells with the dopamine receptor antagonist quetiapine reduced glioma cell self-renewal in vitro, and combined treatment of mice with quetiapine and radiation prolonged the survival of glioma-bearing mice. The combined treatment induced the expression of genes involved in cholesterol biosynthesis. This rendered GL261 and HK-374 orthotopic tumors vulnerable to simultaneous treatment with atorvastatin and further statistically significantly prolonged the survival of C57BL/6 (n = 10 to 16 mice per group; median survival not reached; log-rank test, P < .001) and NOD Scid gamma mice (n = 8 to 21 mice per group; hazard ratio = 3.96, 95% confidence interval = 0.29 to 12.40; log-rank test, P < .001), respectively. CONCLUSIONS: Our results indicate promising therapeutic efficacy with the triple combination of quetiapine, atorvastatin, and radiation treatment against glioblastoma without increasing the toxicity of radiation. With both drugs readily available for clinical use, our study could be rapidly translated into a clinical trial.

The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma.

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine treatment after brain irradiation preserves cognitive function in mice.

BACKGROUND: Normal tissue toxicity is an inevitable consequence of primary or secondary brain tumor radiotherapy. Cranial irradiation commonly leads to neurocognitive deficits that manifest months or years after treatment. Mechanistically, radiation-induced loss of neural stem/progenitor cells, neuroinflammation, and demyelination are contributing factors that lead to progressive cognitive decline. METHODS: The effects of 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP) on irradiated murine neurospheres, microglia cells, and patient-derived gliomaspheres were assessed by sphere-formation assays, flow cytometry, and interleukin (IL)-6 enzyme-linked immunosorbent assay. Activation of the hedgehog pathway was studied by quantitative reverse transcription PCR. The in vivo effects of NSPP were analyzed using flow cytometry, sphere-formation assays, immunohistochemistry, behavioral testing, and an intracranial mouse model of glioblastoma. RESULTS: We report that NSPP mitigates radiation-induced normal tissue toxicity in the brains of mice. NSPP treatment significantly increased the number of neural stem/progenitor cells after brain irradiation in female animals, and inhibited radiation-induced microglia activation and expression of the pro-inflammatory cytokine IL-6. Behavioral testing revealed that treatment with NSPP after radiotherapy was able to successfully mitigate radiation-induced decline in memory function of the brain. In mouse models of glioblastoma, NSPP showed no toxicity and did not interfere with the growth-delaying effects of radiation. CONCLUSIONS: We conclude that NSPP has the potential to mitigate cognitive decline in patients undergoing partial or whole brain irradiation without promoting tumor growth and that the use of this compound as a radiation mitigator of radiation late effects on the central nervous system warrants further investigation.

Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine.

The objective of the study was to identify the mechanism of action for a radiation mitigator of the gastrointestinal (GI) acute radiation syndrome (ARS), identified in an unbiased high-throughput screen. We used mice irradiated with a lethal dose of radiation and treated with daily injections of the radiation mitigator 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine to study its effects on key pathways involved in intestinal stem cell (ISC) maintenance. RNASeq, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry were performed to identify pathways engaged after drug treatment. Target validation was performed with competition assays, reporter cells, and in silico docking. 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine activates Hedgehog signaling by binding to the transmembrane domain of Smoothened, thereby expanding the ISC pool, increasing the number of regenerating crypts and preventing the GI-ARS. We conclude that Smoothened is a target for radiation mitigation in the small intestine that could be explored for use in radiation accidents as well as to mitigate normal tissue toxicity during and after radiotherapy of the abdomen.

Effects of Brain Irradiation in Immune-Competent and Immune-Compromised Mouse Models.

Patient-derived orthotopic xenografts (PDOXs) closely recapitulate primary human glioblastoma (GBM) tumors in terms of histology and genotype. Compared to other mouse strains, NOD-scid IL2Rgammanull (NSG) mice show excellent tumor take rates, which makes them an ideal host for PDOXs. However, NSG mice harbor a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), which renders them relatively radiosensitive. This has been a frequently voiced concern in studies involving ionizing radiation. In this study, we assessed brain toxicity in NSG mice compared to three other different mouse strains frequently used in radiation studies at radiation doses commonly used in experimental combination therapy studies. C3H/Sed/Kam, C57Bl/6, nude and NOD-scid IL2Rgammanull mice received a single dose of 4 Gy to the right brain hemispheres using an image-guided small animal irradiator. Brains were stained using H&E, luxol fast blue, and antibodies against IBA1 and GFAP one, two, four or six months postirradiation. Additional animals of all four strains were exposed to five daily fractions of 2 Gy (5 × 2 Gy), and tissue sections were stained 72 h later against gH2AX, NeuN, GFAP and IBA1. None of the mouse strains displayed radiation-induced toxicity at any of the time points tested. Radiation doses relevant for testing combination therapies can be safely applied to the brains of NSG mice without the occurrence of radiation-induced normal tissue toxicity.

1-(4-nitrobenzenesulfonyl)-4-penylpiperazine increases the number of Peyer's patch-associated regenerating crypts in the small intestines after radiation injury.

OBJECTIVE: Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS: C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS: Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly, 96 h after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION: In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.

An advanced uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) technique used in the sensitive and specific detection of Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium meleagridis in AIDS patients.

The rapid and accurate detection of Cryptosporidium spp. is critically important for the prevention and timely treatment of cryptosporidiosis in AIDS patients (APs). This study was conducted to examine a UDG-LAMP technique for the first time to diagnose cryptosporidiosis in APs. After collecting demographic and clinical data, three stool samples were collected from the participants (120 volunteering APs). The microscopic examination of stained smears using the acid-fast method and the UDG-LAMP assay were performed for each sample. 10% of APs were infected with Cryptosporidium spp. The number of detected cryptosporidiosis cases using the acid-fast staining and UDG-LAMP methods were significantly different (P < 0.001). Diarrhea and weight loss were found to be significantly associated with cryptosporidiosis in patients (P < 0.05). The pretreatment of LAMP reagents with UDG successfully eliminated the likelihood of product re-amplification remaining from previous reactions. The UDG-LAMP technique could detect cryptosporidiosis in APs with high sensitivity and rapidity without carryover contamination.

EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance through Impaired Replication Fork-Associated DNA Damage Repair.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition.Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503-16. ©2017 AACR.

Prevalence of HIV, HBV, and HCV and Related Risk Factors amongst Male Homeless People in Lorestan Province, the West of Iran.

BACKGROUND: Homeless people are at a higher risk of blood-born infectious diseases. The aim of this study was to estimate the prevalence of HIV, HBV, HCV and related risk factors among male homeless people. STUDY DESIGN: A cross-sectional study. METHODS: This study was conducted in Khorramabad City, western Iran from January to June 2015. A pre-designed validated questionnaire was used to collect the data on behavioral and other potential risk factors. Blood samples were taken in order to diagnose HIV, hepatitis B and C infections. The prevalence of HIV, hepatitis B, C and related risk factors was reported with a 95% confidence interval (CI). RESULTS: The participants were 307 male homeless people with a mean (±SD) age of 35.86 (±9.62) yr. The prevalence of HIV, HBs Ag, and HCV Ab positive cases was 6.51% (95% CI: 4.23, 9.90), 0.98% (95% CI: 0.31, 3.00), and 31.27% (26.31, 36.71), respectively. The prevalence of co-infections of HIV and HCV Ab+ was 5.76% (95% CI: 1.34, 8.51). The most common recently used drugs were heroin, methamphetamine, and opium with a prevalence of 44.30%, 41.04%, and 24.76%, correspondingly. CONCLUSIONS: According to this study, prevalence of HIV and hepatitis C among homeless people was considerable. Abusing heroin, methamphetamine, and industrial drugs was also significant. Considering the association between drug abuse, HIV, and hepatitis C infections, planning for effective control and preventive interventions is important in homeless people.

DNA damage related crosstalk between the nucleus and mitochondria.

The electron transport chain is the primary pathway by which a cell generates energy in the form of ATP. Byproducts of this process produce reactive oxygen species that can cause damage to mitochondrial DNA. If not properly repaired, the accumulation of DNA damage can lead to mitochondrial dysfunction linked to several human disorders including neurodegenerative diseases and cancer. Mitochondria are able to combat oxidative DNA damage via repair mechanisms that are analogous to those found in the nucleus. Of the repair pathways currently reported in the mitochondria, the base excision repair pathway is the most comprehensively described. Proteins that are involved with the maintenance of mtDNA are encoded by nuclear genes and translocate to the mitochondria making signaling between the nucleus and mitochondria imperative. In this review, we discuss the current understanding of mitochondrial DNA repair mechanisms and also highlight the sensors and signaling pathways that mediate crosstalk between the nucleus and mitochondria in the event of mitochondrial stress.

Epidemiological Study of Animal Bites and Rabies in Lorestan Province in West of Iran During 2004-2014 for Preventive Purposes.

BACKGROUND: Despite the progress made, animal bites and rabies are one of the important health problems in the country. The purpose of this study was to investigate the epidemiology of animal bites and rabies during 2004-2014 in Lorestan Province to prevent them in population of the province for the future prospective aspects. MATERIALS AND METHODS: In a descriptive cross-sectional study, all those cases bitten in the province, during 2004 and 2014, were studied. The required information about the age, sex, the bitten organ, type of the invasive animal time, and location of the event were collected in questionnaires and then analyzed. RESULTS: The total number of cases of animal rabies during the period of study was 43,892, shown at the rate of 223.23 in 100,000 people. Seventy-eight percent of animal bites in rural areas, 41.42% in the ages 10-29-year-old, 26.8% of cases were students, 56.77% leg bites, and 82.5% of dog bites. Four cases of human rabies were observed during this period. CONCLUSIONS: Rate of animal bites and rabies is high in Lorestan Province. Controlling animals such as dogs and cats in the province through training people at risk, especially among the students, rural areas and inter-sectorial coordination to eliminate stray animals should be considered over and over. Preventive actions to avoid bites are a priority.

Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.

Comparison of fumaric acid 5% cream versus triamcinolone 0.1% cream in the treatment of hand eczema.

Hand eczema is a common distressing skin problem. It is an immune reaction to haptens. Thus, substances that inhibit Immune system can be effective in the treatment of hand eczema. In this study, topical fumaric acid 5% cream is compared with topical steroid in the treatment of hand eczema. Patients with hand eczema were randomly divided into two groups. One group received fumaric acid 5% in a cream base, and the other received triamcinolone 0.1% in the same cream base. Both groups used creams twice daily for one month. Patients were checked for erythema, excoriation, population and lichenification, EASI score, and pruritus before and after treatment. In both groups, the mean of all signs of the disease and EASI score decreased after one month of treatment. There was no significant difference between the two treatments in decreasing erythema, but excoriation, population, lichenification, EASI score and itching were all decreased more in triamcinolone 0.1 % group. Although fumaric acid can inhibit the immune system; it was less effective for the treatment of all signs of hand eczema except erythema in comparison to triamcinolone. These results may be justified for two reasons: low penetration of topical fumaric acid through the skin or a low concentration used in this study.

Cancer stem cell characteristics of circulating tumor cells.

PURPOSE: To review the results from previous studies aiming at the molecular characterization of circulating tumor cells (CTC), to specifically address the role of epithelial-mesenchymal-transition (EMT) and stemness features of CTC in their contribution to tumor progression and to summarize potential interference of CTC with the efficacy of radiotherapy. CONCLUSIONS: Detection of CTC has been reported for most epithelial tumors and has been associated with an increased risk of local and regional recurrence as well as the development of distant metastases. Given a causal relationship between the presence of CTC and tumor progression at the primary or distant sites, several distinct features have to be postulated for these cells: First, a change from an epithelial to a mesenchymal cell-like phenotype which should alleviate the disconnection of individual tumor cells from tight cell-to-cell junctions within the epithelial cell layer and endow single tumor cells with the capacity to migrate into blood vessels; secondly, the presence of stem-cell properties which contribute to the re-establishment of bulk tumor tissue at the primary or metastatic site upon tumor recurrence or distant progression, respectively. Indeed, EMT and stem-cell features were frequently observed in CTC and the phenotype of CTC was established as a stronger predictor of outcome than sole enumeration of CTC in a defined volume of blood. The exploitation of CTC above their use as prognostic marker is still a subject of many ongoing investigations as are the identification of suitable therapeutic targets for this small cell subpopulation.

ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition.

K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors.

Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors.

BACKGROUND/AIMS: Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NFκB-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NFκB and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age. METHODS: Human erythrocytes were separated into five fractions, based on age-associated characteristics cell density and volume. NFκB compared to ß-actin protein abundance was estimated by Western blotting and cell volume from forward scatter. Phosphatidylserine exposure was identified using annexin-V binding. RESULTS: NFκB was most abundant in young erythrocytes but virtually absent in aged erythrocytes. A 24h or 48h exposure to Ringer resulted in spontaneous decrease of forward scatter and increase of annexin V binding, effects more pronounced in aged than in young erythrocytes. Both, Bay 11-7082 (20 µM) and parthenolide (100 µM) triggered eryptosis, effects again most pronounced in aged erythrocytes. CONCLUSION: NFκB protein abundance is lowest and spontaneous eryptosis as well as susceptibility to Bay 11-7082 and parthenolide highest in aged erythrocytes. Thus, inhibition of NFκB signalling alone is not responsible for the stimulation of eryptosis by parthenolide or Bay 11-7082.