The Arabidopsis oligopeptidases TOP1 and TOP2 are salicylic acid targets that modulate SA‐mediated signaling and the immune response

Salicylic acid (SA) is a small phenolic molecule with hormonal properties, and is an essential component of the immune response. SA exerts its functions by interacting with protein targets; however, the specific cellular components modulated by SA and critical for immune signal transduction are largely unknown. To uncover cellular activities targeted by SA, we probed Arabidopsis protein microarrays with a functional analog of SA. We demonstrate that thimet oligopeptidases (TOPs) constitute a class of SA‐binding enzymes. Biochemical evidence demonstrated that SA interacts with TOPs and inhibits their peptidase activities to various degrees both in vitro and in plant extracts. Functional characterization of mutants with altered TOP expression indicated that TOP1 and TOP2 mediate SA‐dependent signaling and are necessary for the immune response to avirulent pathogens. Our results support a model whereby TOP1 and TOP2 act in separate pathways to modulate SA‐mediated cellular processes.     

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin (Etx) is a pore‐forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx‐H149A), previously reported to have reduced, but not abolished, toxicity. The three‐dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx‐H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx‐H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx‐H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx‐H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx‐H149A identified a glycan (β‐octyl‐glucoside) binding site in domain III of Etx‐H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.     

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the 99mTc(CO)3+ core

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH₂) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)₃(H₂O)₃]⁺ (M = ^99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new ^99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.     

The two Tribolium E(spl) genes show evolutionarily conserved expression and function during embryonic neurogenesis

Tribolium castaneum is a well-characterised model insect, whose short germ-band mode of embryonic development is characteristic of many insect species and differs from the exhaustively studied Drosophila. Mechanisms of early neurogenesis, however, show significant conservation with Drosophila, as a characteristic pattern of neuroblasts arises from neuroectoderm proneural clusters in response to the bHLH activator Ash, a homologue of Achaete–Scute. Here we study the expression and function of two other bHLH proteins, the bHLH-O repressors E(spl)1 and E(spl)3. Their Drosophila homologues are expressed in response to Notch signalling and antagonize the activity of Achaete–Scute proteins, thus restricting the number of nascent neuroblasts. E(spl)1 and 3 are the only E(spl) homologues in Tribolium and both show expression in the cephalic and ventral neuroectoderm during embryonic neurogenesis, as well as a dynamic pattern of expression in other tissues. Their expression starts early, soon after Ash expression and is dependent on both Ash and Notch activities. They act redundantly, since a double E(spl) knockdown (but not single knockdowns) results in neurogenesis defects similar to those caused by Notch loss-of-function. A number of other activities have been evolutionarily conserved, most notably their ability to interact with proneural proteins Scute and Daughterless.     

Suicidal ideation and reported suicide attempts in Greece during the economic crisis

The financial crisis in Greece is largely impinging on the health and mental health of the population, raising concerns about a potential rise in suicide rates. The aim of this study was to explore changes in suicidal ideation and reported suicide attempts between 2009 and 2011 in a representative sample of the population and in several population subgroups. The socio‐economic predictors of suicidal ideation and suicide attempts in 2011 were also investigated. Two nationwide cross‐sectional telephone surveys were conducted in 2009 and 2011 using the same methodology. A random and representative sample of 2192 and 2256 people, respectively, took part in the surveys. Between 2009 and 2011, there was a substantial increase in the prevalence of suicidal ideation and reported suicide attempts. People suffering from depression, men, married individuals, people experiencing financial strain, people with low interpersonal trust, and individuals with a history of suicide attempts were particularly vulnerable.     

Prevalence of BRCA1 Mutations in Familial and Sporadic Greek Ovarian Cancer Cases

Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23–24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer.     

Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse

We report on transient membrane perforation of living cancer cells using plasmonic gold nanoparticles (AuNPs) enhanced single near infrared (NIR) femtosecond (fs) laser pulse. Under optimized laser energy fluence, single pulse treatment (τ = 45 fs, λ = 800 nm) resulted in 77% cell perforation efficiency and 90% cell viability. Using dark field and ultrafast imaging, we demonstrated that the generation of submicron bubbles around the AuNPs is the necessary condition for the cell membrane perforation. AuNP clustering increased drastically the bubble generation efficiency, thus enabling an effective laser treatment using low energy dose in the NIR optical therapeutical window.

Schematic representation of single femtosecond laser pulse plasmonic bubble generation in the vicinity of a cell.     

Alirocumab,a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes

BackgroundProprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.ConclusionThese results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry.     

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors

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