Exome Enrichment and SOLiD Sequencing of Formalin Fixed Paraffin Embedded (FFPE) Prostate Cancer Tissue

Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study's aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.     

Minimally invasive coronary artery bypass grafting: port-access approach versus off-pump techniques

BACKGROUND: Within the past 5 years several surgical techniques have been developed for less invasive surgical treatment of coronary artery disease. The aim of this study was to define specific indications for the various minimally invasive coronary artery surgical procedures. METHODS: Minimally invasive direct coronary artery bypass grafting through a minithoracotomy was performed in 67 patients. The left internal mammary artery was anastomosed on the beating heart with the use of a pressure or suction stabilizer without the use of extracorporeal circulation. In 58 other patients with multivessel disease, the off-pump coronary artery bypass grafting technique through a sternotomy was applied with a left internal mammary artery to left anterior descending artery and additional vein grafts without extracorporeal circulation. In a third group, Port-Access (Heartport Inc, Redwood City, CA) coronary artery bypass grafting was performed through a left minithoracotomy with the use of an endovascular extracorporeal circulation system and cardioplegic arrest. Angiographic follow-up was complete in 64% of the patients. RESULTS: There was minimal perioperative or postoperative mortality (0.5%). The medium surgical procedure time for all minimally invasive and off-pump procedures was 2.5 hours; it was 4.5 hours for Port-Access procedures. The median postoperative intensive care unit stay was 1.0 days, and the median hospitalization was 5.0 days. Overall graft patency was 97.3%; in 8 patients (4.1%) a stenosis either at or distal to the graft anastomosis was dilated with coronary angioplasty. CONCLUSIONS: For single-vessel disease of the left anterior descending artery, the minimally invasive coronary artery bypass grafting procedure can be performed safely without the use of extracorporeal circulation. In case of hemodynamic instability or anatomic variation, the Port-Access procedure can be applied without additional necessity for sternotomy. For multivessel disease, the off-pump bypass grafting procedure with sternotomy can be recommended depending on the coronary arteries involved. In case of necessary grafts to the lateral marginal or circumflex branches, Port-Access grafting can be recommended and may play an important role in the future for the development of fully endoscopic robot-assisted coronary artery bypass grafting.     

Changes in the calpains and calpastatin during postmortem storage of bovine muscle

Changes in activity and protein status of micro-calpain, m-calpain, and calpastatin in bovine semimembranosus muscle during the first 7d of postmortem storage were monitored by using assays of proteolytic activity, SDS-polyacrylamide gel electrophoresis, and Western blot analysis. Extractable m-calpain activity changed slightly during the first 7d after death (decreased to 63% of at-death activity after 7d), whereas extractable calpastatin activity decreased substantially (to 60% of at-death activity after 1d and to 30% of at-death activity after 7d of postmortem storage) during this period. Extractable micro-calpain activity also decreased rapidly (to 20% of at-death activity at 1d and to less than 4% of its at-death activity at 7d after death) during postmortem storage. Western blot analysis showed that the 80-kDa subunit of m-calpain remained undegraded during the first 7d after death but that the 125- to 130-kDa calpastatin polypeptide was gone entirely at 7d after death. Hence, the calpastatin activity remaining at 7d originates from calpastatin polypeptides that are 42 kDa or smaller. The 80-kDa micro-calpain subunit was almost entirely in the 76-kDa autolyzed form at 7d after death; this form is proteolytically active in in vitro systems, and it is unclear why the postmortem, autolyzed micro-calpain is not active. Over 50% of total muscle micro-calpain is tightly bound to myofibrils 7d after death; this micro-calpain is also nearly inactive proteolytically. Unless postmortem muscle contains some factor that enables micro-calpain in this muscle to be proteolytically active, it is not clear whether micro-calpain could be responsible for any appreciable postmortem myofibrillar proteolysis.     

(R)-(-)- and (S)-(+)-Synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6. Reaction with N, N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative 7. Ammonolysis furnished (S)-(+)-synadenol (2a). Absolute configuration of 1a was established by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropanecarboxylic acid (10) was resolved by a modification of the described procedure. The R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides 14. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established the R-configuration of (-)-synadenol (1a). These results were confirmed by X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses. Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is less effective (EBV) or devoid of activity (HIV-1).     

The synthesis and reactivity of 3 beta-(2-alkynylsulfonyl)- and 3 beta-(2-alkynylsulfonylmethyl) androst-5-en-17-ones as inhibitors of glucose-6-phosphate dehydrogenase

3 beta-(Hexadec-2-ynylsulfonyl)androst-5-en-17-one, 2c, was designed as an analog of dehydroepiandrosterone sulfatide 1c, a potent, natural inhibitor of glucose-6-phosphate dehydrogenase (G6PDH). Nucleophilic substitution of 1-bromo hexadec-2-yne 11 with 3 beta-mercaptoandrost-5-en-17-one followed by oxidation afforded 2c. The propargylic sulfone 2c may tautomerize to the electrophilic allenic sulfone 3a and thus function as a masked affinity label of the steroidal binding site of G6PDH. Since 2c demonstrated low potency as an inhibitor of G6PDH, a sulfonylmethyl analog 4b was also designed and synthesized. Synthesis of 4b began by methylenation of androst-5-en-3,17-dione 17-ketal 6 with the Tebbe reagent, to yield the 3-methyleneandrost-5-ene 7. Hydroboration, followed by oxidation, gave a mixture of 3 alpha- and 3 beta-hydroxymethyl isomers 8a and 8b, respectively. The 3 beta alcohol 8b was converted to the thiol 10. Alkylation of 10 with 1-bromo-2-hexadecyne 11, followed by selective oxidation, gave the desired acetylenic sulfone 4b. Insertion of the methylene in 4a and 4b significantly increased their G6PDH inhibitory properties over the initial compounds, 2b and 2c.     

Systemic lupus erythematosus is associated with increased auto-antibody titers against calreticulin and grp94, but calreticulin is not the Ro/SS-A antigen

Auto-antibodies against purified human calreticulin were determined by an ELISA in sera from patients with systemic lupus erythematosus (SLE) and from healthy persons or patients without an autoimmune disease. More than 80% of patients with SLE had titers exceeding the highest value obtained in the group without SLE. Almost 30% of the patients had also elevated auto-antibody titers against purified rat grp94, another resident ER-protein of the KDEL-protein family, but not against rat ERp72 (CaBP2), an ER-resident protein of the proteindisulfide isomerase family. It could, however, be excluded that calreticulin is the Ro/SS-A antigen on the basis of the following observations: 1) Calreticulin purified from rat, bovine or human liver contained far less than 1 mol of phosphate per mol of calreticulin, showed an E280/E260-absorption ratio of about 2.0, and did not contain extractable RNA; 2) Sera from patients with SLE did not react with or precipitate endogenous calreticulin from Hep G2 cells; they did, however, precipitate hY-RNA from these cells; 3) Sera from SLE-patients, but not anti-calreticulin antisera precipitated [32P]-hY-RNA from [32P]-labelled Hep G2 cells.     

The electric resistivity of compacts of graphite intercalation compounds with antimony pentafluoride and with antimony pentachloride

The electric resistivities were measured for textured compacts of the intercalation compounds of graphite with SbF₅ and with SbCl₅. Starting materials were coarse flakes of natural graphite and graphite foils (0.35 and 2 mm thick). First stage compounds and mixtures of first and second stage compounds were studied. The resistivities of the compacts from flakes were strongly pressure dependent, maximum conductivity was not yet reached at 600 bar. The resistivities of preparations from graphite foils were much less pressure dependent. The resistivity of the foils decreased to 36 × 10^?8 Ωm, that is 3–4% of the resistivity of the parent foils. In general, the intercalation compounds with SbF₅ showed somewhat higher resistivities than the SbCl₅ compounds. This seems to be caused by formation of an insulating layer on the surface of the particles due to partial fluorination of carbon atoms with SbF₅. CF bonds were detected by IR spectroscopy and by XPS. The first stage SbF₅ compound is formed already at room temperature, contrary to literature reports, provided sufficient time is given.     

Data processing and law enforcement access to information systems at EU level

EU Agencies and Information Systems collect and exchange personal data with Member States but also between each other. In this context, law enforcement agencies such as Europol increasingly get access to data originally serving other purposes. Europol already has access to the Visa-Information System; plans to allow the access to Eurodac also exist. The rules regulating this exchange are so far not harmonised. The article illustrates the existing exchange and access possibilities in light of the EU data protection reform.     

Groundwater discharge: potential association with fecal indicator bacteria in the surf zone

Short-lived radium isotopes (223Ra and 224Ra) are used to investigate the potential association between groundwater discharge and microbial pollution at Huntington Beach, CA. We establish the tidally driven exchange of groundwater from the surficial beach aquifer across the beach face. Groundwater is found to be a source of nutrients (silica, inorganic nitrogen, and orthophosphate) to the surf zone, and these nutrients could possibly provide an environment for enhanced growth or increased persistence of fecal indicator bacteria (FIB). Ammonium and ortho-phosphate explain up to 12-20% of the variance in FIB levels in the surf zone. Elevated levels of FIB were only found in 1 of the 26 groundwater samples. However, FIB in the surf zone covary with radium at fortnightly, diurnal, and semi-diurnal tidal periods. In addition, radium accounts for up to 38% of the variance in log-FIB levels in the surf zone. A column experiment illustrates that Enterococcus suspended in Huntington Beach saline groundwater is not significantly filtered by sand collected from the field. This work establishes a mechanism for the subterranean delivery of FIB pollution to the surf zone from the surficial aquifer and presents evidence that supports an association between groundwater discharge and FIB.