Mechanisms of V600EBRAF-Induced Tumour Development

BRAF is a serine/threonine protein kinase that functions as a component of the RAS/RAF/MEK/ERK signalling pathway. Gain-of-function BRAF mutations have been found in ~50%-70% of malignant melanomas and in a variety of other cancer types, including thyroid, colon, and lung cancers. The activating V600EBRAF mutation is the most frequent mutation comprising a single amino acid substitution (V600E) that results in constitutive activation of BRAF kinase activity. Here, the CreERT system is used to induce endogenous V600EBRaf expression from a single knockin allele after 4-hydroxytamoxifen treatment in MEFs. V600EBRaf induced a faster G1/S phase progression by a significant upregulation of Cyclin D1 at the transcriptional level and was reversed by MEK inhibition. V600EBRaf also suppressed apoptosis at the premitochondrial level following serum withdrawal by a drastic downregulation in BimEL expression at the post-transcriptional level which was rescued by MEK inhibition and proteasomal inhibition. In vivo, expression of endogenous V600EBRaf in mice induced senescence and autophagy in lung adenomas and this prevented lung tumour progression. Senescence was primarily mediated by the p53/p21Cip1 and p16INK4a pathways while autophagy was regulated by inhibition of the mTOR pathway. Mass spectrometry was used to screen for components of the senescence-messaging secretome induced by V600EBRaf in the lung. The cholesterol binding protein Npc2 was identified as being significantly induced by V600EBRaf expression. The role of Npc2 in tumour suppression was investigated and it was found that this protein induced upregulation of the p53/p21Cip1 pathway and triggered autophagy in a paracrine/autocrine manner. Knockout of Npc2 abrogated senescence and promoted growth of lung tumours by downregulation of the p53/p21Cip1 pathway in vivo, but did not prevent the induction of autophagy

The impact of HA2-47 on the thermal stability of H1N1pdm vaccine viruses.

<p>The 6∶2 <i>ca</i> viruses with equalized HA titers were incubated at the indicated temperatures. Following either a 20 minute incubation at the indicated high temperatures (A) or a 4 hour time-course at 57.5°C (B), the HA titers were re-measured. The 6∶2 <i>ca</i> viruses were purified and re-suspended in 1× SP-cGAG. Duplicate aliquots of virus were maintained within a controlled chamber at either 26°C for 35 days (C) or 4°C for 14 weeks (D). At the indicated time points, the infectivity of viruses was examined by TCID₅₀ assay in MDCK cells.</p

The impact of the HA2-47 residue on wt H1N1pdm infectivity in ferrets.

<p>Groups of ferrets were inoculated with the indicated amount of wt A/California/7/09 (Cal/09 HA2-E47), the E47K mutant (Cal/09 HA2-K47), wt A/Brisbane/10/2010 (Bris/10 HA2-K47) or the K47E mutant (Bris/10 HA2-E47). The number of infected ferrets in each group (N = 4 or 7) and geometric mean titers of the nasal wash samples from the infected ferrets at day 3 post-infection determined by TCID₅₀ are indicated. PFU: plaque forming units. FID₅₀: 50% ferret infection dose; SD: standard deviation.</p

Growth kinetics of H1N1pdm viruses with HA2-E47 or K47 in MDCK and Vero cells.

<p>MDCK (A, B) and Vero (C, D) cells were infected with recombinant wt Bris/10 (HA2-K47) and the HA2-E47 mutant (A, C), or wt Cal/09 (HA2-E47) and the HA2-K47 mutant (B, D) at an MOI of 0.004. At the indicated time of post infection, virus titers were determined by TCID₅₀ in MDCK cells.</p

Mapping the HA residues that affect the threshold pH for membrane fusion.

<p>The different amino acids between the HA of Cal/09 and Bris/10 are listed. Single amino acid substitution was introduced into the Cal/09 HA plasmid (-: no change). The threshold pH for fusion was determined by the HA/GFP co-expression fusion assay.</p

Cal/09 and Bris/10 HA differ in the threshold pH for membrane fusion.

<p>(<b>A</b>) Membrane fusion in 293T cells expressing Cal/09 or Bris/10 HA proteins. 293T cells were transfected with a GFP expression plasmid (−), a dual expression plasmid co-expressing GFP and Cal/09 HA or GFP and Bris/10 HA. Membrane fusion was observed after incubation with the indicated pH buffers. “+” indicates the fused cells. (<b>B</b>) Expression of the HA proteins in the transfected 293T cells. Both cleaved (HA1 and HA2) and uncleaved (HA0) forms of the HA proteins were detected by western blot using a sheep anti-HA polyclonal antibody. Values below the HA0 bands (−) trypsin represent the relative amount of Cal/09 and Bris/10 HA proteins. Values below the HA1 and HA2 bands (+) trypsin indicate the percentage of HA1 and HA2 to total HA (HA0+HA1+HA2). All the values are the average of three independent experiments. (<b>C</b>) Membrane fusion in Vero cells infected with Cal/09 or Bris/10 viruses. Vero cells were either mock-infected (−) or infected with Cal/09 or Bris/10 viruses at an MOI of 4.0. At 14 hours post infection, cell membrane fusion was triggered as above. “+” indicates syncytia formation. (<b>D</b>) Viral infectivity titers of wt Cal/09 and Bris/10 viruses (HA2-E47 or HA2-K47) after treatment with the indicated pH buffers.</p

An inter-monomer interaction between the HA residues HA1-21 and HA2-47 determines the threshold pH for fusion.

<p>(<b>A</b>) The Cal/09 HA trimer structure (PDB file: 3LZG) was analyzed using Py-Mol modeling software. The six amino acid differences between the Cal/09 and Bris/10 HA proteins are highlighted in orange. The conserved amino acid HA1-21 is highlighted in green. The locations of the three HA1-21 and HA2-47 residues in each monomer are indicated. Only the location in one monomer is shown for the other residues. (<b>B</b>) Structure modeling of residues HA1-21 and HA2-47, salt bridges between HA1-E21 and HA2-K47 (Bris/10), or HA1-K21 and HA2-E47 (Cal/09 E21K) are indicated. The threshold fusion pH of each combination, as determined by the GFP fusion assay, is indicated adjacent to each model.</p

sj-xlsx-1-sgo-10.1177_21582440231210428 – Supplemental material for R&D Investment, Executive Incentive, and Financial Performance in GEM: Based on Three-Stage Least Squares Method

Supplemental material, sj-xlsx-1-sgo-10.1177_21582440231210428 for R&D Investment, Executive Incentive, and Financial Performance in GEM: Based on Three-Stage Least Squares Method by Luxing Liu, Keyu Lei, Hong Jin and Yin-Pei Teng in SAGE Open</p

Seasonal LAIV on NL03 L-S vaccine virus induced immune responses and protection against a heterologous virus challenge infection.

<p><b>A</b>. Ferrets were immunized with the indicated viruses and serum antibody titers on day 14 post the second vaccine were determined by the HI assay or MN assay with NL03 L-S vaccine virus. The data are expressed as log₂ HI or neutralizing antibody titers, respectively. <b>B</b>. HA- or virus-specific IFN-γ secreting cells in PBMC on day 8 post vaccination. The solid lines represent geometric mean ± SE for the group. <b>C</b>. The ferrets were challenged with BC04 wt 28 days post the second dose. Challenge virus titer in NT and lung tissues at 3 days post challenge was expressed as log ₁₀EID₅₀/g (GMT ± SE). The dashed line indicates limit of detection. * indicates statistically significant (P<0.05) difference by <i>t</i> test.</p

Glycan binding specificity of the H7 vaccine viruses to biotinylated 3'S-Di-LN-LC-LC (A) or 6'S-Di-LN-LC-LC (B).

<p>The binding ability of the 2-fold serial diluted virus in HA unit to immobilized glycan was detected by ELISA assay using NL03 HA specific sheep antiserum. The data are presented as geometric mean of optical density (OD) at 450 nm ± standard error (SE) of two independent samples.</p