Metal Heptafluoroisopropyl (M-hfip) Complexes for Use as hfip Transfer Agents

New coinage-metal heptafluoroisopropyl (L_<i>n</i>M-hfip) complexes are synthesized from the metal fluoride and inexpensive hexafluoropropene (M = Ag, Cu; L = PPh₃, 2,2,6,6-tetramethylpiperidine (Htmp)). Reaction of the silver Htmp complex with a Ni dibromide complex led to efficient hfip transfer to afford L₂NiBr­(hfip) (L = 2-ethylpyridine). Treatment of the Ni-hfip complex with ZnPh₂ gave the corresponding L₂NiPh­(hfip) complexes, which were investigated for reductive elimination of PhCF­(CF₃)₂. Although the desired reductive elimination proved unsuccessful, addition of carbon monoxide to L₂NiPh­(hfip) effected an efficient heptafluoroisopropyl carbonylative cross-coupling. Further, while the silver complex does not undergo hfip transfer to organic electrophiles, the copper complex (phen)­(PPh₃)­Cu­(hfip) (<b>3b</b>) effectively transfers the hfip unit to various substrates. We investigated the scope of <b>3b</b> with acid chlorides toward the synthesis of perfluoroisopropyl aryl ketones. Additionally, reaction conditions for hfip transfer to <i>p</i>-fluorobenzyl bromide and <i>p</i>-fluorobenzaldehyde were identified. As a bonus, <b>3b</b> was easily generated on a gram scale using commercially available copper hydride by taking advantage of a rapid hydrodefluorination to generate “Cu–F” in situ. Aspects of the observed reactivity are supported by DFT calculations

Engineered FKBP** permits rapid, reversible and tunable regulation of PTEN function in cellular models.

<p><b>(A)</b> FKBP**-PTEN is reversibly regulated in response to Shld1 treatment in cells. U87MG cells stably expressing FKBP**-PTEN were treated with vehicle or with Shld1 (1μM) for 24 h. One set of cells was then washed thoroughly with DMEM and cultured in Shld1-free culture media for further 24hs. The expression level of FKBP**-PTEN was examined by immunofluorescence staining. <b>(B)</b> upper panel, U87MG cells stably expressing FKBP**-PTEN were treated with Shld1 (1 μM) for different time periods; lower panel, Shld1 (1 μM) was removed after 24 h treatment, and cultured in Shld1 free culture media for different time periods as indicated. <b>(C)</b> PTEN protein levels and activity can be maintained at specific concentrations over a prolonged period of time in dependence of Shld1. U87MG cells stably expressing FKBP**-PTEN were cultured in media containing indicated concentrations of Shld1 for 24 h. <b>(D)</b> Stable FKBP**-PTEN expressing U87MG cells were analyzed in cell invasion (left) and colony formation (right) assays. In the cell invasion assay, data are shown as the mean ±SEM of the number of cells migrated in three independent experiments. Plating efficiency refers to the ratio of the number of colonies to the number of cells seeded, shown as mean ±SEM of three independent experiments.</p

Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein.

<p>(<b>A)</b> Rationale of the DD-technology: Fusion of PTEN with a modified human FKBP variant harboring the F36V and L106P point mutations (FKBP*) leads to degradation of the fusion protein. Presence of the FKBP* ligand Shld1 confers stabilisation of FKBP*-PTEN and results in inhibition of PI3K/Akt signaling. (<b>B)</b> The fusion protein FKBP*-PTEN shows no PTEN activity. PTEN-deficient U87MG cells were transiently co-transfected with FKBP*-PTEN and AKT-GFP. After one day, cells were treated with Shld1 for 12 hours at 1μM before cell lysis and samples analyses by western blotting using indicated antibodies. <b>(C)</b> Consequence of linker optimization on PTEN activity of the FKBP*-PTEN fusion protein. Schematic representation of linker optimization. FKBP*-linker-PTEN variants with different linker regions (A-E) were generated. Linker A—short flexible linker; Linker B—a helix forming linker. Linker C—long flexible linker. Linker D—long flexible linker containing the PWR motif. Linker E—the original linker present in the enzymatic active GFP-PTEN. <b>(D)</b> Analyses of the effect of different linkers on PTEN activity in FKBP*-PTEN fusion variants in U87MG cells. Constructs expressing FKBP*-PTEN variants or GFP-PTEN were co-transfected with AKT-GFP into a PTEN null U87MG cells. Stabilization of FKBP*-PTEN or GFP-PTEN in presence or absence of Shld1 was monitored by PTEN antibody. The phosphorylation level of AKT-GFP at Ser473 served as a fast and effective experimental protocol to control for PTEN activity towards PIP3-dependent signaling in transfected cells, only. Only constructs 4 and 5, which contain proline in the linker sequence, showed moderate PTEN activity towards PI3K signaling.</p

Codon optimization of FKBP** establishes a conditional fine-tuning protein regulation system.

<p><b>(A)</b> The transcription of the destabilization cassette FKBP**-PTEN was subcloned downstream of LoxP-Stop-LoxP; transcription was driven by the ubiquitous CMV-enhanced chicken beta actin promoter (CAG). Presence of Cre induces FKBP**-PTEN gene transcription; however, the translated FKBP**-PTEN will be rapidly degraded. Addition of Shld1 confers FKBP**-PTEN stabilization in a tunable and reversible manner. <b>(B)</b> Cre-mediated cleavage of LoxP-Stop-LoxP (LSL) created a truncated FKBP**-PTEN fusion protein. Mouse forebrain neurons were nucleofected with LSL-FKBP**-PTEN and Cre-IresRFP (Cre (+)), or RFP (Cre (-)). Cells were treated with Shld1 (or control vehicle) overnight, before analyses of cell lysates using indicated antibodies. <b>(C)</b> Sequence alignment of FKBP** and LoxP. The third ATG codon region sequence (TATGCTA) of FKBP** (M3L^cta) is identical to the linker region of the LoxP stem-loop sequence (TATGCTA). Codon optimization of CTA with TTG (both code for amino acid Leu) will destroy the potential pseudo-cleavage site of Cre on FKBP**. <b>(D)</b> Codon optimization of M3L^cta to M3L^ttg in FKBP** abolished the Cre-dependent FKBP**-PTEN truncation and produced a Shld1 dependent PTEN fusion protein. The M3L^ttg modified LSL-FKBP**-PTEN construct was co-expressed with Cre-IresRFP (Cre (+)), or RFP (Cre (-)) in mouse forebrain neurons as before. <b>(E)</b> Combinatorial use of the Cre-LoxP system with the FKBP**-PTEN/Shld1 chemical-genetic protein control system in PTEN^flox/flox cells. Constructs were nucleofected into <i>PTEN</i>^{<i>loxp/loxp</i>} mouse primary neurons as before. Note that nucleofection of primary cells occurs with efficiencies at approx. 80%, and result in a residual endogenous PTEN signals detected by western blotting. <b>(F)</b> The generated system is able to couple PTEN-loss directly with the expression of tunable FKBP**-PTEN. Upon Cre-mediated recombination, <i>PTEN</i>^{<i>loxp/loxp</i>} cells will lose PTEN and, at the same time, activate expression of FKBP**-PTEN. In essence, endogenous PTEN is replaced by tuneable PTEN, which will enable to test whether PTEN-loss induced phenotypes can be rescued at different time-points (<i>t</i>_Shld1) and/or at different PTEN-concentrations (<i>d</i>_Shld1).</p

Engineered FKBP** permits regulation of PTEN function in the zebrafish.

<p><b>(A)</b> The migration and growth of stable FKBP**-PTEN expressing U87MG cell clones injected into zebrafish embryos can be inhibited by Shld1. Prior to microinjection into the perivitelline cavity of 2 days post fertilization (dpf) zebrafish embryos, cells were pre-stained with DiI. Embryos were then transferred into fish water with (n = 11) or without (n = 13) Shld1 (4 μM) and maintained for 24h. Images were taken immediately after injections (0 days post injections, dpi) and after 24 h (1dpi). <b>(B)</b> The migration indices and the increase in tumor mass size of transplanted cells were quantified. ** p<0.01; ***p<0.001.</p

FKBP surface engineering restores PTEN activity of FKBP**-PTEN fusion protein.

<p><b>(A)</b> Electrostatic potential map of PTEN (PDB ID: 1d5r) and FKBP (PDB ID: 1BL4). Positively charged binding pocket of the PTEN PIP3 substrate is circled with a white dashed line; the negatively charged lump formed by E31/D32 residues on the FKBP surface is circled by a black dashed line. <b>(B)</b> Schematic representation of amino acid residue substitutions of FKBP* protein surface at E31/D32. <b>(C)</b> Different FKBP*-PTEN mutants were transiently co-tansfected with AKT-GFP into U87MG cells, the expression of fusion protein and pAKT were examined by Western blotting. <b>(D)</b> E31S/D32S amino acid substitutions on FKBP* (FKBP**) restores PTEN activity of the FKBP**-PTEN fusion protein. FKBP**-PTEN (or GFP/GFP-PTEN) was transiently co-expressed with AKT-GFP in U87MG cells. Cells were treated with Shld1 and analyzed as before.</p

Additional file 2: Figure S1. of Dietary supplementation with bovine-derived milk fat globule membrane lipids promotes neuromuscular development in growing rats

Real time RT‐PCR analysis of Agrn and Z+ Agrn expression in rat soleus muscle. Figure S2. Real time RT‐PCR analysis of synaptophysin expression in rat soleus muscle. A (PDF 369 kb

High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett’s Esophagus

<div><p>Purpose</p><p>Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients.</p><p>Experimental Design</p><p>Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with ≥ 1 GC, and the proportion of crypts with ≥1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length.</p><p>Results</p><p>High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with ≥1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively).</p><p>Conclusions</p><p>The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities.</p></div

Results of univariate analysis regarding goblet cell parameters in patients who did or did not progress to cancer.

<p>NC = No cancer</p><p>C = Cancer</p><p>^ǂ p-value: test for the risk difference between absent/present of the feature</p><p>Results of univariate analysis regarding goblet cell parameters in patients who did or did not progress to cancer.</p

Goblet Cell Counts and Risk of Adenocarcinoma, Per Patient Analysis (Kaplan-Meier Curves).

<p>In a per patient analysis, the Kaplan-Meier curves show an inverse association between risk of adenocarcinoma and averaged GC parameters in non-dysplastic biopsies in each patient: # GC (A), mean #GC/crypt (B), # crypts with ≥ 1 GC (C) and proportion of crypts with ≥ 1GC (D). P-values are from log-rank tests and have been adjusted for multiple comparison using the Benjamini & Hochberg method.</p