The TNM 8 M1b and M1c classification for non-small cell lung cancer in a cohort of patients with brain metastases

Background and purpose. According to the recent TNM 8 classification, patients with metastatic non-small cell lung cancer (NSCLC) and single extrathoracic metastasis should be classified as stage M1b, while those with 2 or more metastases comprise stage M1c. The purpose of this study was to analyze the impact of this classification in patients with brain metastases. Materials and methods. This retrospective study included 172 patients treated with individualized approaches. Actuarial survival was calculated. Uni- and multivariate analyses were performed. Results. Thirty patients (17%) were staged as M1b. Those with squamous cell cancer were more likely to harbor M1b disease (29%, adenocarcinoma 14%, other histology 17%, p = 0.16). Median survival was 5.4 months (8.0 months in case of M1b disease and 4.5 months in case of M1c disease, p = 0.001). Multivariate analysis confirmed the role of M1b stage. M1b patients managed with upfront surgery or radiosurgery had significantly longer median survival than those who received whole-brain irradiation (21.0 vs. 3.5 months, p = 0.0001) and the potential to survive beyond 5 years. Conclusions. We found the M1b classification to provide clinically relevant information. The multivariate analysis suggested that patients with M1b disease, better performance status and younger age have better survival (AU)

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The TNM 8 M1b and M1c classification for non-small cell lung cancer in a cohort of patients with brain metastases.

BACKGROUND AND PURPOSE: According to the recent TNM 8 classification, patients with metastatic non-small cell lung cancer (NSCLC) and single extrathoracic metastasis should be classified as stage M1b, while those with 2 or more metastases comprise stage M1c. The purpose of this study was to analyze the impact of this classification in patients with brain metastases. MATERIALS AND METHODS: This retrospective study included 172 patients treated with individualized approaches. Actuarial survival was calculated. Uni- and multivariate analyses were performed. RESULTS: Thirty patients (17%) were staged as M1b. Those with squamous cell cancer were more likely to harbor M1b disease (29%, adenocarcinoma 14%, other histology 17%, p = 0.16). Median survival was 5.4 months (8.0 months in case of M1b disease and 4.5 months in case of M1c disease, p = 0.001). Multivariate analysis confirmed the role of M1b stage. M1b patients managed with upfront surgery or radiosurgery had significantly longer median survival than those who received whole-brain irradiation (21.0 vs. 3.5 months, p = 0.0001) and the potential to survive beyond 5 years. CONCLUSIONS: We found the M1b classification to provide clinically relevant information. The multivariate analysis suggested that patients with M1b disease, better performance status and younger age have better survival.

[Perampanel as a therapy option in patients with epilepsy]. / Perampanel als Therapieoption bei Epilepsiepatienten.

BACKGROUND: This article describes a monocentric retrospective analysis of clinical experience with the latest antiepileptic drug perampanel with non-competitive modulation of postsynaptic AMPA receptors. MATERIAL AND METHODS: Evaluation of electronic medical charts of patients newly treated with perampanel between 2012 and 2014 at the epilepsy center of the University Hospital Freiburg regarding effectiveness and tolerability. RESULTS: A total of 85 patients (45 male, mean age 37.4 years, range 14-80 years) with therapy resistance to an average of 6 antiepileptic medications were newly treated with add-on perampanel. Of the patients 35 % experienced a relevant reduction in seizures. The most commonly reported side effects were tiredness (32.5 %), dizziness (24.5 %) and irritability (10.5 %). The dosages resulting in a significant reduction in seizures which varied between patients from 4 to 12 mg/day. Even multidrug-resistant patients who had not benefited from vagus nerve and deep brain stimulation, profited from add-on treatment with perampanel. CONCLUSION: In this cohort, even epilepsy patients who did not respond to multiple previous antiepileptic treatment profited from add-on therapy with the new mode of action of perampanel.

Colorectal cancer metastatic to the brain: analysis of prognostic factors and impact of KRAS mutations on presentation and outcome

Background. Treatment concepts for metastatic colorectal cancer continue to evolve. While the presence of RAS mutations impacts systemic therapy, little is known about the influence of such mutations in patients with brain metastases. Patients and methods. Pooled retrospective analysis was conducted of 57 patients with brain metastases from colorectal cancer treated in two different institutions (2005-2013). Results. The only mutations analyzed in a relatively large subgroup were KRAS mutations (14 wild type, 12 mutated). Mutation status was not associated with baseline characteristics such as number or location of metastases, and did not impact prognosis. Three factors were significantly associated with survival in multivariate analysis: Karnofsky Performance Status (KPS), management strategy, and systemic treatment. Median survival was 0.6 months with best supportive care, 3.0 months with initial whole-brain radiotherapy (WBRT), and 12.7 months if initial treatment included surgery or stereotactic radiosurgery (SRS), p = 0.0001. The survival difference between the WBRT and surgery/SRS groups was largest in patients with KPS 80-100. Conclusion. Effective local treatment was a prerequisite for improved survival. The only significant prognostic baseline factor was KPS, which forms the basis of the diagnosis-specific graded prognostic assessment (DS-GPA) score. Thus, our results validate the DS-GPA in this patient population. So far, neither this nor other studies suggest a clinically important impact of KRAS mutations beyond their previously reported association with development of brain metastases. Studies focusing on patients who develop brain metastases early during the course of metastatic disease might be warranted, because the influence of different systemic therapies might be larger in this subgroup (AU)

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Colorectal cancer metastatic to the brain: analysis of prognostic factors and impact of KRAS mutations on presentation and outcome.

BACKGROUND: Treatment concepts for metastatic colorectal cancer continue to evolve. While the presence of RAS mutations impacts systemic therapy, little is known about the influence of such mutations in patients with brain metastases. PATIENTS AND METHODS: Pooled retrospective analysis was conducted of 57 patients with brain metastases from colorectal cancer treated in two different institutions (2005-2013). RESULTS: The only mutations analyzed in a relatively large subgroup were KRAS mutations (14 wild type, 12 mutated). Mutation status was not associated with baseline characteristics such as number or location of metastases, and did not impact prognosis. Three factors were significantly associated with survival in multivariate analysis: Karnofsky Performance Status (KPS), management strategy, and systemic treatment. Median survival was 0.6 months with best supportive care, 3.0 months with initial whole-brain radiotherapy (WBRT), and 12.7 months if initial treatment included surgery or stereotactic radiosurgery (SRS), p = 0.0001. The survival difference between the WBRT and surgery/SRS groups was largest in patients with KPS 80-100. CONCLUSION: Effective local treatment was a prerequisite for improved survival. The only significant prognostic baseline factor was KPS, which forms the basis of the diagnosis-specific graded prognostic assessment (DS-GPA) score. Thus, our results validate the DS-GPA in this patient population. So far, neither this nor other studies suggest a clinically important impact of KRAS mutations beyond their previously reported association with development of brain metastases. Studies focusing on patients who develop brain metastases early during the course of metastatic disease might be warranted, because the influence of different systemic therapies might be larger in this subgroup.

Apoptosis in sulfur mustard treated A549 cell cultures.

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

[Isoprenoid pigments in representatives of the family Microbacteriaceae].

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.

[Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups].

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

The mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in many eubacteria, plants, and the malaria parasite. Using genetically engineered Escherichia coli cells able to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway we demonstrate that the lytB gene is involved in the trunk line of the MEP pathway. Cells deleted for the essential lytB gene were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of lytB.

GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli.

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.

Identification of a new C-type lectin, TES-70, secreted by infective larvae of Toxocara canis, which binds to host ligands.

Infective larvae of the dog roundworm Toxocara canis survive in the tissues of their hosts for extended periods in a state of developmental arrest, successfully evading immune destruction. This survival strategy is thought to be mediated by T. canis excretory/secretory (TES) products which downregulate or divert the immune response. We purified one of the major TES products, TES-70 and gained amino acid sequence from 4 tryptic peptides. These peptides were matched to a predicted protein from a cDNA that was isolated by expression screening a T. canis cDNA library with mouse anti-TES serum. The predicted protein (Tc-CTL-4) is similar to, but larger than, Tc-CTL-1, a 32-kDa C-type lectin secreted by T. canis larvae. Tc-CTL-4 has a signal peptide, 2 Cys-rich domains and a C-terminal calcium-dependent C-type lectin domain that shares sequence similarity with host immune cell receptors such as macrophage mannose receptor and CD23. The lectin domain was expressed in bacteria and antiserum to the purified recombinant protein was used to confirm that Tc-ctl-4 did encode the native TES-70 glycoprotein. TES-70 selectively bound to ligands on the surface of Madin-Darby Canine Kidney cells in vitro in a calcium-dependent manner, inhibitable by mammalian serum, indicating that a host glycan is the native ligand for this new parasite lectin.

Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa.

Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli. The purified P. aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method. A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P. aeruginosa DXP synthase. Fluoropyruvate is described as a first inhibitor of DXP synthase.

Recombinant expression and modification analysis of protein agno-1b encoded by avian polyomavirus BFDV.

Among two pairs of agnoproteins encoded in upstream positions in the late mRNAs of avian polyomavirus BFDV, either agno-1a or its splice derivative agno-1b are required for viral propagation. Out of the two proteins both of which consist of multiple electrophoretic subspecies, the smaller and less complex agno-1b has been cDNA-cloned into an influenza-virus /RNA-polymerase I expression system for production of higher amounts of this protein in infected chicken embryo fibroblasts. Fractional modification of agno-1b by phosphorylation at residues serine 51, serine 53, and threonine 73 is demonstrated through dephosphorylation by alkaline phosphatase, mass spectrometry of individual protein species isolated by strong anion exchange chromatography, and single or multiple alanine substitutions of serine or threonine residues in site-directed mutagenesis.

A family of secreted mucins from the parasitic nematode Toxocara canis bears diverse mucin domains but shares similar flanking six-cysteine repeat motifs.

Infective larvae of the parasitic nematode Toxocara canis secrete a family of mucin-like glycoproteins, which are implicated in parasite immune evasion. Analysis of T. canis expressed sequence tags identified a family of four mRNAs encoding distinct apomucins (Tc-muc-1-4), one of which had been previously identified in the TES-120 family of glycoproteins secreted by this parasite. The protein products of all four cDNAs contain signal peptides, a repetitive serine/threonine-rich tract, and varying numbers of 36-amino acid six-cysteine (SXC) domains. SXC domains are found in many nematode proteins and show similarity to cnidarian (sea anemone) toxins. Antibodies to the SXC domains of Tc-MUC-1 and Tc-MUC-3 recognize differently migrating members of TES-120. TES-120 proteins separated by chromatographic methods showed distinct amino acid composition, mass, and sequence information by both Edman degradation and matrix-assisted laser desorption ionization/time of flight mass spectrometry on peptide fragments. Tc-MUC-1, -2, and -3 were shown to be secreted mucins with real masses of 39.7, 47.8, and 45.0 kDa in contrast to their predicted peptide masses of 15.7, 16.2, and 26.0 kDa, respectively. The presence of SXC domains in all mucin products supports the suggestion that the SXC motif is required for mucin assembly or export. Homology modeling indicates that the six-cysteine domains of the T. canis mucins adopt a similar fold to the sea anemone potassium channel-blocking toxin BgK, forming three disulfide bonds within each subunit.

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

Juvenile female Litomosoides sigmodontis produce an excretory/secretory antigen (Juv-p120) highly modified with dimethylaminoethanol.

A 120 kDa antigen produced by juvenile female Litomosoides sigmodontis (Juv-p120) was isolated and purified. The amino acid composition of the molecule was determined. Juv-p120 was shown to be highly modified with N,N-dimethyl-aminoethanol (28.4 mol%). Treatment of Juv-p120 with potassium hydroxide (beta-elimination) or with sodium m-periodate leads to the destruction of epitopes recognized by antibodies immune affinity-purified with isolated Juv-p120. Juvenile L. sigmodontis were shown to release Juv-p120 into the pleural cavity of infected Mastomys coucha before the onset of patency.

Imaging soft x-rays using a microchannel plate.

The GOLDHELOX Project is a student run project to construct a robotic solar telescope that will be used to take images of the sun in the soft X-ray region (171–181Å) of the spectrum. The optical system uses a microchannel plate (MCP) detector. We tested the MCP to familiarize ourselves with and verify that MCP's can be used to image soft X-rays. Soft X-rays were created by a Manson source attached to a proportional counter to determine the amount of emitted X-rays detected by the MCP. The voltages on the MCP were varied to observe responses of varying voltage differences. Most of the observations were visible observations along with images made by a 35 mm camera with a telephoto lens. We found the 1000 V difference to produce the strongest and clearest images.

NASA G-133 "GoldHelox": A Project Update.

NASA G-133, also known as the "GoldHelox Project", is a fully autonomous, soft X-ray, solar telescope designed for use on board the space shuttle. Conceived, designed and built by students at Brigham Young University, it will image the sun with a spatial resolution of 2.5 arc-seconds with a temporal resolution of one second. The instrument will image X-rays with wavelengths between 171Å and 181Å coming from highly ionized Fe lines in the sun's corona. Data will consist of several hundred high resolution photographs that will help in understanding the initial phases of solar flares, and the relationship between solar flares and the physics of the coronal-chromospheric transition region. This paper briefly outlines the project's goals, gives a brief overview of the construction and operation of the instrument and addresses the unique aspects of running a predominantly undergraduate research project. It summarizes the lessons learned to date, and the current project status.