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Male C57BL/6J wild-type (WT) mice (8–12 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Tau−/− knockout (Tau-KO) mice on the C57BL/6J background were obtained from Jackson Laboratories (#007251, USA). Male and female homozygous mice were bred to generate Tau−/− homozygous mice, and 8–12 week-old male homozygous Tau-KO mice and WT littermates (C57BL/6J) were used in this study. All mice were housed in the animal room on a standard 12-h light/12-h dark cycle with a constant temperature and food and water available ad libitum. All experimental procedures and use of the animals were conducted in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee of Sichuan University. All efforts were made to minimize the suffering of the mice.
Mice were acclimated for one week before experiments and habituated to handling for 2 d before each behavioral test. Conditioned place preference (CPP) and self-administration were used to assess cocaine-cue memory formation. The home cage CPP, food CPP and locomotor activity were employed as control behavioral tests. All behavioral experiments were performed in a double-blind manner.
Conditioned place preference (CPP)
The CPP test was conducted using a standard three-chambered apparatus equipped with two large conditioning compartments (black and white) that differed in their flooring (bar and grid) and a small middle chamber (grey, smooth PVC floor) that connected the two large compartments. Prior to each session, animals were habituated to the chambers for at least 10 min for a continuous 2 d.
Baseline preference was assessed by placing the mice in the middle chamber and allowing them to habituate the entire chambers freely for 15 min; an initial measurement of baseline preference was defined as the time spent in the black chamber subtracted from the time spent in the white chamber (Timepre−test). Animals were excluded from the test if they showed a strong unconditioned preference for either side chamber (chamber bias > 300 s).
Animals were randomly assigned to two groups and trained for 6 d with alternating injections of cocaine (20 mg/kg or 2.5 mg/kg, i.p) or saline injections in both compartments. They were confined to the conditioning or unconditioning chambers for 30 min after the injection and then returned to their home cages. On the test day, the animals were placed in the middle compartment and the time of spent in the two compartments was recorded for 15 min. We also defined as the time spent in the black chamber minus the time spent in the white chamber as Timeblack. The CPP scores were calculated as follows: if Timeblack was positive (the mouse preferred the black chamber), namely, Timeblack minus Timepre−test, positive scores indicated a reinforced preference, and negative scores indicated a reversed preference. If Timeblack was negative (the mouse preferred the white chamber), Timeblack minus Timepre−test, negative scores indicated a reversed preference and positive scores indicated a reinforced preference. Generally, cocaine strongly reverses preference and results in a positive CPP score, and the CPP score was defined as the extent of the shift in preference after the cocaine injection.
Home cage CPP
Mice were exposed to cocaine as described for consolidation training, and then they were not confined to the conditioning or unconditioning chambers, but were returned to their home cages without any context exposure to the test apparatus. On the test day, mice were placed in the middle connecting chamber, and the time spent in each chamber was calculated as described in previous experiments to evaluate the CPP score.
The food CPP test used a similar apparatus and methodology as described above. Mice were restricted from food for one week before the test, and their weight was maintained at 80% of their original body weight. During the food conditioning sessions, the food-induced CPP group was transferred to the food-paired chamber (2–3 g of food were placed in the chamber) for 30 min. In the non-food conditioning sessions, mice were assigned to the non-food-paired chamber for 30 min. After conditioning training for 3 h, animals were only fed once daily (1 h). The alternating sessions of conditioning were repeated 3 times (a total of 6 d). On the test day, mice were placed in the central chamber and allowed to freely explore both compartments for 15 min; the time spent in each chamber was recorded to calculate the CPP score.
CPP and extinction
Mice were exposed to the CPP protocol, and the test day course was repeated each day until the preference of the cocaine group for the conditioned compartment had returned to the habituation baseline.
Mice were anesthetized with sodium pentobarbital (60 mg/kg) and implanted with a single sterilized silastic catheter (0.51 ID x 0.94 mm OD, BB518-20, Scientific Commodities) into the right jugular vein. The distal end of the catheter was threaded through the skin on the back of the mice and exited the skin via a stainless steel guide cannula (RWD Life Science). After surgery, catheters were flushed with 0.1 ml of a saline solution containing penicillin (160000 U/ml) and heparin (30 U/ml) daily. One week after recovering from surgery, animals were trained to self-administer intravenous injections of cocaine (0.75 mg/kg/infusion) or saline during 2 h sessions daily over 10 d on a fixed ratio 1 (FR1) schedule in an operant chamber. The response to the active poke produced a cocaine injection and was accompanied with a blue light stimulus for 20 s and an audible tone for a 5-s timeout period. At the same time, the inactive poke failed to inject the drug and produce conditioning stimuli.
The locomotor activity was measured as the distance traveled. Animals were acclimated to the chambers (48 cm x 48 cm) equipped with a camera for 15 min once a day for a consecutive 2 d. Before the test, the baseline locomotor activity was not statistically different between groups. In the following week, animals were injected cocaine (20 mg/kg, i.p) or an equal volume of saline, immediately placed in the locomotor activity chamber, and allowed to explore for 15 min. The distance traveled was measured daily for one week, and automated tracking was performed with EthoVision 7.0 software (EthoVision 7.0; Noldus Information Technology, Leesburg, VA).
Cocaine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in saline. LY294002 (S1105, Selleck) were dissolved in saline containing 4% DMSO, 30% PEG300 and 5% Tween 80.
Stereotactic surgery and inhibitor administration
Animals were anesthetized with sodium pentobarbital (60 mg/kg) and mounted in a standard stereotaxic instrument (RWD Life Science). After shaving the hair and cleaning the incision site with medical-grade alcohol, the scalp was incised to expose the skull and permanent bilateral guide cannulas (RWD Life Science) were bilaterally implanted into the dorsal dentate gyrus (dDG) (AP, − 2.0 mm; ML, ± 1.4 mm; DV, − 2.2 mm) with stereotaxic instruments. Dental cement was used to anchor the guide cannula and the stainless steel stylet blocker was inserted into each cannula to prevent blockage and infection. All mice were used for subsequent training after one week of recovery from surgery.
LY294002, a specific inhibitor of the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, was also dissolved in saline containing 4% DMSO, 30% PEG300 and 5% Tween 80 at a final concentration of 4.6 µg/µl. LY294002 were administered bilaterally (1 µl/side, 0.5 µl/min) with a micro-injector, 15 min before cocaine or saline administration during the cocaine-associated memory formation.
Adeno-associated virus injection
The adeno-associated viruses (AAVs) AAV2/8-CMV-2N4R-3Flag (1.83 × 1013 vg/ml), AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag (8.53 × 1012 vg/ml) and AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag (2.83 × 1012 vg/ml) were purchased from Obio Technology Co. Ltd. (Shanghai, China). Point mutations of arginine 205, proline 208 and proline 210 to alanine were synthesized in the murine full-length Tau amino acid sequence, as these three residues are normally important for the interaction of PI3K-p85 SH3 domains, and their mutation has previously been shown to attenuate PI3K-p85 binding to human 2N4R Tau . The AAV2/8-CMV-2N4R-3Flag, AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag and AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag were constructed for use in animal studies.
The coding regions of 2N4R Tau were amplified from cDNAs obtained from C57BL/6J mice by PCR and cloned into pAAV-CMV-MCS-3Flag and pAAV-CMV-MCS-2A-mNeongreen-3Flag to produce AAV-2N4R Tau or mutant 2N4R Tau, respectively. The vector was confirmed by sequencing. The recombinant plasmids were then packaged into AAV2/8 particles together with the AAV helper plasmid and purified using an iodixanol step-gradient ultracentrifugation method. Recombinant AAVs were produced by transient transfection in HEK293T cells and the expression of 4R Tau was analyzed using western blotting.
Mice (8–12 weeks old) were anesthetized with sodium pentobarbital (60 mg/kg) and placed on a stereotaxic apparatus (RWD Life Science) to inject the virus into the dDG (AP, − 2.0 mm; ML, ± 1.4 mm; DV, − 2.2 mm). After shaving the hair and cleaning the incision site with medical-grade alcohol, the scalp was incised to expose the skull and the connective tissue was gently removed from the skull surface with cotton swabs. Small craniotomy holes were drilled with a skull rotor (RWD Life Science) for virus injection. The micro syringe needles were used to bilaterally infuse the tissue with AAV2/8-CMV-2N4R-3Flag (0.2 µl, 0.05 µl/min), AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag (0.4 µl, 0.05 µl/min) or AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag (1.2 µl, 0.05 µl/min). After each injection, the syringe was left in place for an additional 5 min and then slowly withdrawn to allow the virus to diffuse. Mice recovered for at least 3 weeks before behavioral tests.
Lentiviral vector construction
The lentiviral vectors (LV) pLenti-EF1a-CMV-2N4R-3Flag (3.39 × 108 TU/ml), pLenti-EF1a-CMV-mutant 2N4R-3Flag (3.41 × 108 TU/ml), pLenti-CMV-p85α-MYC-2A-mCherry-PGK-blasticidin (3.94 × 108 TU/ml) and pLKD-CMV-EGFP-2A-Puro-U6-shRNA Tau (5.44 × 108 TU/ml) were also purchased from Obio Technology Co. Ltd. (Shanghai, China). The pLenti-EF1a-CMV-2N4R-3Flag, pLenti-EF1a-CMV-mutant 2N4R-3Flag, pLenti-CMV-p85α-MYC-2A-mCherry-PGK-blasticidin and pLKD-CMV-EGFP-2A-Puro-U6-shRNA Tau were constructed for cell-based studies.
The coding regions of 2N4R Tau and PI3K-P85α were amplified from murine cDNAs using PCR and cloned into pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag and pLenti-CMV-MCS-MYC-2A-mCherry-PGK-blasticidin vectors to produce LV-2N4R Tau, LV-mutant 2N4R Tau and PI3K-P85α, respectively. The Tau shRNA was cloned into pLKD-CMV-EGFP-2A-Puro-U6-shRNA and confirmed by sequencing. The sequences of the scrambled control shRNA and Tau shRNA were 5’-TTCTCCGAACGTGTCACGT-3’ and 5’-GGAGTTTGACACAATGGAA-3’, respectively. Recombinant lentiviruses were also produced by transient transfection in the HEK293T cells and then the levels of 4R Tau and PI3K-p85 were analyzed using western blotting.
Mice were sacrificed by rapid decapitation at the end of the behavioral tests. The hippocampus or dentate gyrus (separated from the hippocampus) were removed from the brain, snap frozen in dry ice, and stored at -80 °C until the assay.
Brain tissues and cells were lysed and proteins were extracted using a mammalian cell and tissue extraction kit (K269-500, Biovision) containing phosphatase inhibitors (4906845001, Roche) according to the manufacturer’s protocols. The total protein concentration was analyzed with a Bradford assay kit (P0006, Beyotime). Twenty micrograms protein were loaded and separated on 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gels. After separation, the gels were then transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010, Millipore) in a mixed solution of Tris-glycine buffer and 20% (v/v) methanol. The membrane was blocked in TBST buffer containing 5% non-fat dry milk (9999, Cell Signaling Technology) for 1 h at room temperature, and then incubated and gently shaken overnight with the primary antibody at 4 °C. On the next day, after three washes with TBST for 15 min, the blots were incubated with the secondary antibody at room temperature for 2 h. Immunoreactivity was visualized using a chemiluminescence substrate (WBKLS0500, Millipore) with a chemiluminescence imagine system (CLINX, Shanghai, China). The optical density of each band was quantified using Chemi Analysis software (CLINX, Shanghai, China). The following antibodies were used for western blotting: mouse anti-Tau (1:1000, Thermo Scientific), mouse anti-3R Tau (1:1000, Millipore), mouse anti-4R Tau (1:1000, Millipore), rabbit anti-cAMP response element-binding protein (CREB; 1:1000, Cell Signaling Technology), rabbit anti-p-CREB (S 133) (1:1000, Cell Signaling Technology), rabbit anti-extracellular signal-regulated kinase (ERK)1/2 (1:1000, Cell Signaling Technology), rabbit anti-p-ERK1/2 (T 202/T 204) (1:1000, Cell Signaling Technology), rabbit anti-cofilin (1:1000, Abcam), rabbit anti-p-cofilin (S 3) (1:1000, Cell Signaling Technology), rabbit anti-c-fos (1:1000, Cell Signaling Technology), mouse anti-Flag (1:5000, Sigma), mouse anti-2A (1:1000, Millipore), rabbit anti-PI3K-p85 (1:1000, Cell Signaling Technology), rabbit anti-PI3K-p110α (1:1000, Abcam), rabbit anti-AKT (1:1000, Abcam), rabbit anti-p-AKT (S 473) (1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Abcam), rabbit anti-p-GSK-3β (S 9) (1:1000, Cell Signaling Technology), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-p-β-catenin (S 33/37/T 41) (1:1000, Cell Signaling Technology), rabbit anti-β-actin (1:1000, Cell Signaling Technology), mouse anti-GAPDH (1:2000, ZSGB-BIO), rabbit anti-lamin B1 (1:1000, Abcam), goat anti-mouse (HRP) (SAB), goat anti-rabbit (HRP) (SAB), goat anti-mouse light chain-specific (HRP), and mouse anti-rabbit light chain-specific (HRP) (Jackson ImmunoResearch).
Animals that had undergone behavioral training were deeply anesthetized with sodium pentobarbital (60 mg/kg) and perfused transcardially with phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains were carefully extracted from the skull, postfixed with 4% PFA overnight, and then dehydrated with 30% sucrose at 4 °C. Brains were sectioned into 45-µm coronal slices using a freezing microtome (Leica, Germany) and stored in 12-well plates filled with a cryoprotectant solution (glycerol, ethylene glycol and 0.1 M phosphate buffer, pH 7.4, 1:1:2 by volume) at -20 °C until processing for immunohistochemical staining.
The sections were washed with TBS 3 times (10 min each) and incubated with a blocking solution containing 0.3% TritonX-100 and 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in TBS for 2 h at room temperature. Sections were then incubated with primary antibodies in the blocking solution at 4 °C overnight. On the next day, the sections were also washed with TBS-0.3% TritonX-100 (TBST) 3 times, transferred to the blocking solution containing fluorescent dye-conjugated secondary antibodies, and then incubated for 1.5 hours at room temperature. After another three washes with TBST, sections were cover slipped with anti-fade mounting medium containing DAPI (H-1200, Vector), and confocal images were acquired with a laser confocal microscope (Nikon, Japan). The corresponding DG areas and cell numbers were determined using Image J software (US National Institutes of Health, Bethesda, MD, USA) to assess the cell densities. The following antibodies were used for immunostaining: rabbit anti-Tau (1:100, Protech), mouse anti-3R Tau (1:200, Millipore), mouse anti-4R Tau (1:50, Biolegend), rat anti-BrdU (1:100, Abcam), mouse anti-Ki67 (1:100, BD), goat anti-sex determining region Y-box 2 (SOX2; 1:100, RD), rabbit anti-glial fibrillary acidic protein (GFAP; 1:100, Abcam), mouse anti-c-fos (1:100, Abcam), rabbit anti-doublecortin (DCX; 1:100, Abcam), donkey anti-mouse (Alexa Fluor 488) (1:500, Invitrogen), donkey anti-rabbit (Alexa Fluor 488) (1:500, Invitrogen), donkey anti-goat (Alexa Fluor 488) (1:500, Invitrogen), donkey anti-rabbit (Alexa Fluor 568) (1:500, Invitrogen), and donkey anti-rat (Alexa Fluor 647) (1:500, Jackson ImmunoResearch).
For BrdU staining, DNA was denatured by incubating the section in 2 M hydrochloric acid for 30 min at 37 °C and then renatured with 0.05 M boric acid (28341, Thermo Scientific) at room temperature for 30 min, followed by TBS rinses for 10 min. Immunohistochemistry was then performed.
The cannula placements were confirmed in 5-µm thick coronal sections using hematoxylin eosin (HE) staining, and images were acquired using a light microscope. Mice with misplaced cannulas were excluded from the statistical analysis.
Cells cultured on glass coverslips coated with poly-D-lysine (P6407, Sigma) were washed 3 times with PBS and then fixed with 4% PFA for 10 min. After 3 washes with PBS, cells were blocked with blocking buffer (5% normal donkey serum and 0.3% TritonX-100 in PBS) for 1 h and then incubated with primary antibodies overnight. On the next day, coverslips were washed 3 times with PBS and incubated with the fluorescent dye-conjugated secondary antibodies for 1 h at room temperature. Coverslips were then washed 3 times with PBS, stained with Hoechst (H1399, Life Technologies) for 5 min, and mounted with anti-fade mounting medium (H-1000, Vector). The following antibodies were used for immunostaining: rabbit anti-PI3K-p85α (1:400, Abcam), mouse anti-4R Tau (1:100, Biolegend), donkey anti-mouse (Alexa Fluor 488) (1:500, Invitrogen), and donkey anti-rabbit (Alexa Fluor 568) (1:500, Invitrogen).
Golgi staining was performed using the FD Rapid Golgi Stain Kit (PK401, FD Neurotechnologies) to label neurons in the dDG. Briefly, animals were deeply anesthetized with sodium pentobarbital (60 mg/kg), and the brains were immediately removed and rinsed with MilliQ water. Afterwards, the retrieved brains were immersed in equal parts of Solutions A and B (containing potassium dichromate, mercuric chloride, and potassium chromate) and stored at room temperature for 2 weeks in the dark. Tissues were subsequently rinsed, placed in the cryoprotectant solution, and stored at room temperature for at least 72 hours in the dark before cutting. The brain slices were cut in the coronal plane at approximately a 100-mm thickness with the freezing microtome (Leica, Germany). Tissue sections were placed on poly-D-lysine-coated slides and dried in the dark. After drying, sections were transferred to distilled water, subsequently stained with a developing solution, and then successively dehydrated with 50, 75, 95, and 100% ethanol. Finally, the sections were defatted with a xylene substitute and cover slipped with DPX mounting medium (06522, Sigma).
Images were acquired from prepared slides using a confocal microscope (Olympus, Japan). Each neuron was scanned at high magnification (100X, oil immersion) to ensure that all parts of the dendrites were intact. For each group, a minimum 5 neurons per slice were examined. At least 60 neurons were selected in one group. 3D neuronal reconstruction was also performed using a confocal microscope. The total length and dendrite spine density were measured using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).
Cells and tissues were harvested for the co-immunoprecipitation (co-IP) analysis using the simplified and reliable Pierce™ Crosslink Magnetic IP/co-IP Kit (88805, Thermo Scientific). Briefly, the Tau or PI3K-p85 primary antibody was bound to 50 µl of Protein A/G magnetic beads (B23201, Bimake) for 15 min and washed three times. After the incubation, the protein supernatants extracted using the mammalian cell and tissue extraction kit were collected, and the protein concentration was determined using a Bradford assay kit. The supernatant from each sample was incubated with the antibody-crosslinked beads overnight at 4 °C. On the next day, beads were washed two times with IP lysis/wash buffer and one time with ultrapure water. Elution buffer was used to elute the bound antigen, and neutralization buffer was used to neutralize the low pH. The supernatants were collected for western blot analysis.
Cell culture and transfection
Mouse neuroblastoma 2a (N2a) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, C1195500BT, Gibco) containing 10% fetal bovine serum (10099-141, Gibco) and grown in a 5% CO2 atmosphere at 37 °C.
N2a cells were grown in 6-well plates to generate 2N4R Tau overexpression and Tau knockdown-resistant cells. DMEM was replaced with serum-free DMEM and incubated for 2 h before transfection, follow by incubations with the lentiviruses contain the mouse 2N4R Tau, mutant 2N4R Tau, PI3K-p85, Tau-shRNA (5’- GGAGTTTGACACAATGGAA-3’) and the non-specific target oligonucleotides (5’-TTCTCCGAACGTGTCACGT-3’) and selection with 2.5 µg/ml puromycin or 10 µg/ml blasticidin. Antibiotic-resistant clones were picked and cultured in DMEM containing puromycin or blasticidin.
Cytoplasmic and nuclear fractionation
Harvested cells and tissues were extracted using the ProteoExtract® Subcellular Proteome Extraction Kit (539791, Millipore). The cytoplasmic and nuclear fractions were separated according to the manufacturer’s instructions.
Preparation of the hippocampal single-cell suspension
The brain tissue was dissected on ice and enzymatically digested using the Adult Brain Dissociation kit (130-107-677, Miltenyi). The single-cell suspension was prepared for the flow cytometry assay according to the manufacturer’s instructions.
Single-cell suspensions from each group were centrifuged and the pellet was resuspended with a viability dye Fixable Viability Stain 780 (1:1000; BD) for cell live/dead discrimination, followed by an incubation with blocking buffer containing CD16/CD32 (1:100; BD) for 10 min. Cells were then incubated with Foxp3/Transcription Factor Staining Buffer Set (00-5523, Invitrogen) at 4 °C for 30 min in the dark. Finally, samples were incubated with the antibody mixture at 4 °C for 30 min in the dark, washed, centrifuged, resuspended in staining buffer containing 2% fetal bovine serum (FBS) and then sorted using a FACSAria SORP (BD, USA). Gating parameters and data analysis were performed using FlowJo 10 software (Tree Star, USA). The following antibodies were used for flow cytometry: mouse anti-SOX2 (Alexa Fluor 647) (1:100, Biolegend), mouse anti-DCX (PE) (1:100, BD), mouse anti-GFAP (Alexa Fluor 405) (1:100, Novus), rat anti-Ki67 (PerCP/Cy5.5) (1:100, Biolegend), mouse anti-AKT (Alxa Fluor 350) (1:100, R&D Systems), and mouse anti-pAKT (S 473, BV421) (1:100, BD).
EdU or BrdU incorporation assays
For the analysis of newborn neuron proliferation in the hippocampus, the mice were injected with 50 mg/kg (i.p) bromodeoxyuridine (BrdU) (00-0103, Invitrogen) or 5-ethynyl-2’-deoxyuridine (Edu) (900584, Sigma) 3 times every 4 h on the last training day, and analyzed after the tests were complete . BrdU and EdU immunostaining were performed to assess the proliferation of newborn hippocampal cells during cocaine memory formation.
For the flow cytometry assay of proliferation, single-cell suspensions were prepared from mice injected intraperitoneally with EdU as described above. Cultured N2a cells were serum-starved overnight, exposed to the external stimuli, and then 10 µM EdU was added to the culture medium for 2 h before cells were collected.
For the flow cytometry detection of EdU expression, collected cells were transferred to 5-ml FACS tubes, fixed, washed with staining buffer and stained with the Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (C10634, Thermo Scientific) at 4 °C for 30 min in the dark. Finally, samples were incubated with the antibody mixture at 4 °C for 30 min in the dark and then evaluated using a FACSAria SORP (BD, USA). Data were analyzed as described above.
Quantification of the Tau-interacting proteome
The dDG was removed from the hippocampus for co-IP, and the co-IP method was employed to detect the Tau-interacting proteome. The proteins were extracted and the co-IP procedures were conducted as described above. The collected protein supernatants were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and then subjected to gel digestion followed by the LC-MS/MS analysis.
For in-gel tryptic digestion, gel pieces were destained in 50 mM NH4HCO3 in 50% acetonitrile (v/v) until they were clear. Gel pieces were dehydrated with 100 µl of 100% acetonitrile for 5 min, rehydrated in 10 mM dithiothreitol and incubated at 56 °C for 60 min. Gel pieces were again dehydrated in 100% acetonitrile and rehydrated with 55 mM iodoacetamide. Samples were incubated at room temperature for 45 min in the dark. Gel pieces were washed with 50 mM NH4HCO3 and dehydrated with 100% acetonitrile, followed by rehydration with 10 ng/µl trypsin and resuspension in 50 mM NH4HCO3 on ice for 1 h. The excess liquid was removed and gel pieces were digested with trypsin at 37 °C overnight. Peptides were extracted with 50% acetonitrile in 5% formic acid, followed by 100% acetonitrile. Finally, peptides were dried completely and resuspended in 2% acetonitrile in 0.1% formic acid.
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reverse-phase analytical column (15-cm length, 75-µm i.d.). The gradient was comprised of 6–23% solvent B (0.1% formic acid in 98% acetonitrile) in 16 min, 23–35% in 8 min, increasing to 80% in 3 min, and then maintained at 80% for the last 3 min with a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive™ Plus coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for the full scan, and the intact peptides were detected in the Orbitrap at a 70000 resolution. Peptides were then selected for MS/MS using NCE at a setting of 28%, and the fragments were detected in the Orbitrap at a resolution of 17500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion was performed. Automatic gain control (AGC) was set to 5E4.
The MS/MS data were processed using Proteome Discoverer 1.3. Tandem mass spectra were searched in the SwissProt Mouse database. Trypsin/P was specified as the cleavage enzyme, allowing up to 2 missing cleavages. The mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as the fixed modification and oxidation on Met was specified as the variable modification. Peptide confidence was set at high, and the peptide ion score was set to > 20.
Protein interaction networks
All interesting gene name identifiers (mainly proteins involved in regulating cell proliferation) were searched against the STRING database version 10.5 for protein-protein interactions. Only interactions between the proteins belonging to the searched dataset were selected, thereby excluding external candidates. STRING defines a metric called a confidence score to define the interaction confidence, and we retrieved all interactions with a confidence score ≥ 0.7 (high confidence). The interaction network identified using STRING was visualized with Cytoscape.
All data were analyzed with GraphPad Prism 7 software, presented as means ± SEM, and subjected to the Kolmogorov-Smirnov test to assess the normality of the distribution. For simple comparisons, an unpaired two-tailed Student’s t test was used. For multiple comparisons, one-way or repeated-measures two-way ANOVA test was utilized for each experiment. In all cases, n refers to the number of animals. For all results, statistical significance was defined as p < 0.05.

domain: medical


text:
4 edition of A history of the Jefferson Medical College of Philadelphia found in the catalog.
A history of the Jefferson Medical College of Philadelphia
|Statement||by James F. Gayley, M.D. ; with biographical sketches of the early professors.|
|Series||Library of American civilization -- LAC 40061.|
|The Physical Object|
|Number of Pages||59|
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DR. STEPHEN KLASKO is a transformative leader and advocate for a revolution in our systems of healthcare and higher education. His book is titled, Bless This Mess: A Picture Story of Healthcare in America – an account of how the USA was elected to the Intergalactic Council of Cool Health Systems by As President of Philadelphia-based Thomas Jefferson University and CEO of Jefferson. Bl Pa Jefferson Medical College Phildelphia Yearbook Free Shipping Philadelphia - $ Philadelphia Pa Ward 8 Jefferson Medical Collegehospital Plat 10 Atlas Map.
The history of Jefferson College is a success story. It begins with Senate Bill Number Seven, presented by Senator Earl Blackwell of Hillsboro, in the spring of , that made possible the formation and financing of public junior colleges in several Jefferson County citizens began to recognize the need for a public junior college in their community, they met, organized and. Find information about and book an appointment with Dr. Eric Samuel Hager, MD in Monroeville, PA, Pittsburgh, PA. Specialties: Vascular Surgery.
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Titles of the Jefferson Medical College yearbook volumes vary: Class book ; Ganglion ; Class book ; Opsonin ; Autopsy. John H. Gibbon Jr., MD, focused international attention on Jefferson by ushering in a new era of cardiac Gibbon, Jefferson professor and chairman of surgery (), is renowned in medical history as the inventor of the heart-lung machine, which made possible the world's first successful open-heart operation at Jefferson in Thomas Jefferson University began as a medical school.
During the early 19th century, several attempts to create a second medical school in Philadelphia had been stymied, largely by University of Pennsylvania School of Medicine alumni.
In an attempt to circumvent that opposition, a group of Philadelphia physicians led by George McClellan sent an letter to the trustees of Jefferson College President: Stephen K.
Klasko. Other articles where Jefferson Medical College is discussed: Thomas Jefferson University: led by George McClellan created Jefferson Medical College in Philadelphia in It served as the medical department of Jefferson College (then located in Canonsburg) until the state granted the medical college an independent charter in In it opened one of the first teaching hospitals in.
The history of Washington & Jefferson College begins with three log cabin colleges established by three frontier clergymen in the s: John McMillan, Thaddeus Dod, and Joseph three men, all graduates from the College of New Jersey, came to present-day Washington County to plant churches and spread Presbyterianism to what was then the American frontier beyond the Appalachian Mountains.
A history of the Jefferson Medical College of Philadelphia: with biographical sketches of the early professors James Fyfe Gayley Wilson, - Medical colleges - 59 pages. Thomas Jefferson University, private, state-aided, coeducational institution of higher education in Philadelphia, Pa., has one of the largest independent medical schools in the United university comprises Jefferson Medical College, the College of Health Professions, the College of Graduate Studies, and a teaching hospital, the Thomas Jefferson University Hospital.
About Jefferson Medical College. Jefferson Medical College is a group practice with 1 location. Currently, Jefferson Medical College specializes in Psychiatry, General Surgery, Gynecology, Obstetrics & Gynecology and Other Specialty with 16 physicians.
From Jefferson Medical College to Sidney Kimmel Medical College, and Philadelphia Textile School to Philadelphia University – we have a rich legacy.
In fact, through unifying two widely acclaimed and highly honored legacies of innovation, education, research and professional excellence we’ve surpassed years of combined history. Philadelphia, PA Stay Connected Visit us on Twitter Visit us on Facebook Visit us on Instagram Visit us on Pinterest Visit us on LinkedIn Visit us on YouTube Email Us.
The Sidney Kimmel Medical College at Thomas Jefferson University (Kimmel) has an application deadline of Nov. The application fee at Thomas Jefferson University (Kimmel) is $ The Jefferson Medical College of Philadelphia, Benefactors, alumni, hospital etc.
Its founders, officers, instructors, A HISTORY (Volume 2) Thomas Jefferson University - A chronological history and alumni directory, -edited by Frederick B.
In addition, alumni have founded the Philadelphia Zoo, served as the last president of the Republic of Texas, and become best-selling authors of fictional works. It is a close-knit group, held together by the Alumni Association of Jefferson Medical College, the first such medical association of its kind.
Thomas Jefferson University Hospitals have major programs in a wide range of clinical specialties. Services are delivered at five primary locations — Thomas Jefferson University Hospital (our teaching hospital, which was established in ) and Jefferson Hospital for Neuroscience, both in Center City Philadelphia; Jefferson Methodist Hospital and Jefferson at the Navy Yard, both in South.
VIEW FULL TEXT. Full text online. Alberta Government Library Access from Gale (Sabin Americana ). Burman University Access from Gale (Sabin Americana ). Concordia University of Edmonton Access from Gale (Sabin Americana ).
Grande Prairie Regional College Access from Gale (Sabin Americana. The relatively new American Medical Association, founded inturned to Samuel Gross, an eminent Civil War surgeon and professor at Jefferson Medical College in Philadelphia, to examine and report on the best methods for organizing and running institutions that would formally train nurses.
A history of the Jefferson Medical College of Philadelphia: with biographical sketches of the early professors by Gayley, James F. (James Fyfe), Listen to oral history interviews with graduates from Thomas Jefferson University's Sidney Kimmel Medical College, College of Nursing, and College of Life Sciences to learn more about student life at Jefferson.
Use the dropdowns to filter by college and decade, and follow the links below to access each interview's audio file and written transcript. A history of the Jefferson Medical College of Philadelphia: with biographical sketches of the early professors Alternate Title(s): Album of the Jefferson Medical College of Pennsylvania.
Jefferson History Books by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: [email protected] Recommended Citation Gayley, James F., "History of the Jefferson Medical College of Philadelphia with Biographical Sketches of the Early Professors" ().
Jefferson History Books. s - InM.E.H. begins affiliation with Jefferson Medical College and agrees to make its ward facilities and staff available for clinical teaching; Jefferson provides additional house staff for M.E.H. Miss Richardson breaks the racial barriers as the first African American nurse to graduate from the School of Nursing in Planning.The Historical Medical Library of The College of Physicians of Philadelphia, established inis one of the world's premier research collections in the history of medicine.
The unique holdings of the Library include over incunables, an extensive collection of manuscripts and archives, and a comprehensive collection of 19th and early.As Philadelphia College of Textiles & Science the institution offered its first graduate degree, the Master of Business Administration, in With the purchase of properties in andthe size of the campus nearly doubled again and grew to include additional classrooms, research laboratories, student residences and athletic facilities.

domain: library


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Led by James Raath, Surrey-based Calibre-Elite believes it has developed a method of measuring business potential and assessing the competitive strength and sustainability of growth businesses. The idea, says Raath, ‘is to measure the entrepreneurial flair of an organisation. It’s not just about those at the helm but everything that firm does, even the way in which a receptionist answers the phone.’
Using anonymous questionnaires to assess the entrepreneurial spirit of individuals right throughout a business, Calibre-Elite then calculates an ‘E-Rating’, which is designed to ‘represent the potential of that venture to make money.’ A rating of 0.725 or above (the scale runs from 0-1) suggests a firm capable of achieving long-term sustainable growth. A score of 0.5 or below, meanwhile, indicates that existing revenue streams could be at risk.
Having initially trialled the system with several blue chip companies in his native South Africa, Raath is now looking for validation in the UK and has already teamed up with London Business School to this end. ‘Over the next two or three years were hoping to test the scale with around 200 companies. We started working with the business school in April and several AIM companies have now signed up as well.’
Whether the notion of E-Rating takes off long-term, remains to be seen.

domain: news


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Flat black and white Vector Illustration including .ai and .pdf. Shorts with slanted and straight side pockets, back pockets, belt loops and leg pockets. All including in an easy to use Adobe Illustrator file and grouped in layers.
This item is licensed under CC BY-NC-SA 4.0 - the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International license.
Just Shorts :)

domain: design


Some very sad news to wake up to on a Monday morning – Justin Townes Earle, americana singer-songwriter and son of Steve, has died at the age of 38. On a post on his Instagram page, it states: “It is with tremendous sadness that we inform you of the passing of our son, husband, father and friend Justin. So many of you have relied on his music and lyrics over the years and we hope that his music will continue to guide you on your journeys.”
He was named by his dad after songwriter Townes Van Zandt which he told RS last year wasn’t universally appreciated: “My mother hated Townes Van Zandt. My first name was supposed to be Townes, but my mother would not have it. She hated him because of the trouble that Dad and him got into but she still played his music.”
Earle like his father had issues with addiction throughout his life, but speaking in 2017 to podcast Walking the Floor, said that he was finally sober after finding a new path: “I got all my craziness out of the way as a coffeehouse musician and a roadie.”
He released eight studio albums across the course of his career, which saw him win twice at the Americana Music awards including for his best-known song, ‘Harlem River Blues’.
Reaction to his death has come from all over the world of entertainment, including the writer Stephen King:
It appears that Justin Townes Earle has died. I hope it's a hoax but fear it is not. What a loss.
— Stephen King (@StephenKing) August 24, 2020
AUK will publish its obituary shortly, and you can read our A-Z piece on Earle which we published last month here. In the meantime here’s a clip of Earle performing his best-known song on David Letterman back in 2011.


The National Instruments cFP-DO-410 module features eight sourcing digital output channels. Each channel is electronically fused to protect against short circuits and wiring errors. Over-current errors light an LED on the module and create an alarm that can be monitored in software. Each channel is compatible with voltages from 5 to 30 VDC and can source up to 1 A per channel. Each channel has an LED to indicate the channel on/off state. The module features 2300 V transient isolation between the output channels and the backplane. To make installation and setup easier the NI cFP-DO-410 features HotPnP (plug and play) operation and is automatically detected and identified by the configuration software.
National Instruments designs and manufactures all products to minimize failures, however unexpected failures can still occur. Extended warranties provide a fixed economical price at the time of system purchase, covering any repair costs for up to three years. In addition, they offer the following benefits:
Significant cost savings compared to individual repair incidents
Fault location, diagnostics, and repair by NI any time the system product fails
All parts and labor costs covered as well as any adjustments needed to restore the hardware to manufacturing specifications
NI recognizes the need to maintain properly calibrated devices for high-accuracy measurements. NI provides manual calibration procedures, services to recalibrate your products, and automated calibration software to calibrate many NI measurement products.
Return your registered product under warranty at no additional labor and parts cost. NI offers fault location, diagnostics, and repair any time the system fails as well as any adjustments needed to restore the hardware to manufacturing specifications.


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This is a poem about the function of keeping people and things connected. It is a constant battle to keep up the connections we have. Let’s appreciate or enjoy the work it takes to hold the fabric of our societies together.
- Post author:admin
- Post published:April 20, 2023
- Post category:Poetry
- Post comments:0 Comments

domain: artistic


Bring your family out for a day of fun!
Join D.C.’s creative community for the city’s first ever, Sound of Family Fun on Saturday, September 28, from 12-4 p.m. at 2241 MLK Jr. Avenue SE (Between W and Chicago Streets, SE).
Come enjoy live performances, food, family fun and support the city’s most remarkable creatives for a free event as they bring their stunning artwork and merchandise made in D.C. to stage.
The Sound of Family Fun is one of many celebrations apart of 202Creates, a month-long campaign led by the DC Office of Cable Television, Film, Music, and Entertainment (OCTFME) and the Office of the Deputy Mayor for Planning and Economic Development (DMPED) to enrich D.C. communities through events, activities and resources.
202Creates was launched in September 2016 by D.C. Mayor Muriel Bowser as a way to celebrate the city’s diverse and vibrant creative community with food, fun, festivities and family throughout the entire month of September.
202Creates strives to do three important things, “to promote and amplify the District’s creative economy and creative residents through digital media marketing, content creation and original television programming; to connect District residents with government resources, educational opportunities, and space to support their creative work and grow their creative businesses; and to build an interdisciplinary community by connecting creative District residents with each other through events and networking activities.”
For more updates on upcoming events check out the 202Creates website.
Come celebrate D.C.’s creative culture!
photo by Dean Clark From WMATA: “As part of the investigation into the Blue Line derailment, Metro is holding out of service all of its 7000-series railcars, which is nearly…
Connecticut and Windom Place, NW Thanks to Endrit for sending Saturday night around 10:15pm: “Some unintended pyrotechnics in Van Ness tonight. Hope nobody got hurt.”
Updates/if when more is known/official reports are released. A reader reports: “Shooting at 3 am. By Nats Park across from Mission. Victim was responsive as they loaded him on stretcher…
Florida Ave, NW 4:49pm Readers report: “Heard 8-12 gunshots at 4:30ish at R and Florida.” “8-12 shots at least one hit at playground on Fla Ave/1st in Bloomingdale – fla…


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Chesapeake Wellness Center has been committed to providing exceptional medical care and individualized treatment programs for over 20 years. Our patients are our #1 priority. Whether it’s routine care through physicals, expert management of chronic conditions we focus on excellence, honesty and the quality of our health care services. At Chesapeake Wellness Center you will be supported by an outstanding medical team. You will be treated with the respect, dignity and personalized attention you deserve.
Encore (encore never compromises on results and expectations) was established several years ago to provide high level consulting and advisory services primarily but not exclusively to healthcare and related enterprises. The firm specializes in interim management, executive search, corporate governance and reorganization, financial and operational feasibility services.
We provide diagnostic testing with a patient-centered approach to solutions for hearing loss. We offer the latest hearing technology available and our staff is ready to work with you to ensure you receive the most from your hearing.
Seidenberg Protzko Eye Associates was founded in 1998 and specializes in general ophthalmology, which includes cataract, refractive, and pediatric surgery, as well as therapeutic optometry. Seidenberg Protzko Eye Associates has offices in Havre de Grace, Bel Air, Elkton, and Chestertown MD. Seidenberg Protzko Eye Associates regularly provides lectures, screenings, and on-site education and is involved in many community activities.
The University of Maryland Shore Regional Health System provides state-of-the-art healthcare to residents of Maryland’s five county Mid-Shore Region. It includes Shore Medical Center at Chestertown, Shore Medical Center at Easton, Shore Medical Center at Dorchester, Shore Emergency Center at Queenstown.

domain: health


The American Trucking Associations (ATA), the Colorado Motor Carriers Association (CMCA) and Penske Truck Leasing are among nine recipients of the 2017 SmartWay affiliate challenge award from the Environmental Protection Agency (EPA).
“We take tremendous pride in receiving this award for the seventh time and for the sixth consecutive year. It is a testament to the remarkable progress our industry has made toward energy efficiency and environmental sustainability,” noted Chris Spear, ATA’s president and CEO, in a statement.
“Our federation is fully committed to reducing fuel use and emissions, setting our industry on a road to a sustainable future that shrinks our environmental footprint and boosts our bottom line,” he added.
EPA praised ATA in a statement for helping to advance freight efficiency policies through its work with state partners, outside stakeholders and international groups such as the Japanese Trucking Association.
“EPA commends the SmartWay Affiliate Challenge honorees for their extraordinary level of commitment and enthusiasm in supporting more efficient and sustainable business practices in moving goods,” noted Christopher Grundler, director of EPA’s office of transportation and air quality.
“These organizations represent diverse industry sectors and stakeholders who are showing that American prosperity and protecting the environment can go hand in hand,” he said.
EPA honored CMCA for using SmartWay criteria in a statewide awards program, offering assistance to small fleets interested in becoming SmartWay partners and educating state legislators on the benefits of developing a preference in contract awards for freight companies that demonstrate sustainability practices.
Penske was singled out for enrolling three new carriers into the SmartWay program, helping customers establish fuel economy benchmarks and completing a SmartWay tractor logo campaign for a customer.
"Being recognized as a five-time honoree is quite an accomplishment," stated Drew Cullen, Penske Truck Leasing’s senior vice president of fuels and facility services. "We appreciate the opportunity to consistently provide our customers with the means to enhance, or to create, quality sustainability programs that support their business models."
The other winners are the Environmental Defense Fund (EDF), International Sanitary Supply Association (ISSA), Mid-Atlantic Regional Air Management Association (MARAMA), North Central Texas Council of Governments, the Specialty Graphic Imaging Association and Wisconsin Clean Cities.
The voluntary SmartWay program, launched in 2004, is credited for saving more than $25 billion in fuel costs and cutting carbon emissions by 73 million metric tons according to EPA data.


JJay Alix thought he was doing Dominic Barton, the man then leading McKinsey & Co., a favor.
It was the summer of 2014, and Alix had recently discovered that the powerful management consulting firm had entered the bankruptcy advisory business that he — as founder of AlixPartners — had helped launch more than 30 years earlier.
The problem, Alix had come to believe, was that McKinsey was acting illegally.
“I thought if I were him and somebody were doing this in my company, I would want to know about it and I would want to stop it immediately. It’s a federal offense,” he says. “Of course he’ll want to do the right thing if I just tell him.”
Alix phoned Barton, then McKinsey’s global managing partner — the firm’s equivalent of CEO — and told him the two needed to meet.
An Oxford graduate who had risen through the ranks to head McKinsey, Barton, after much pleading by Alix, finally agreed to a one-on-one with the bankruptcy expert at Alix’s law firm in midtown Manhattan that September.
Barton, no doubt, had a lot on his mind. McKinsey was still reeling from the scandal that erupted when Rajat Gupta, a former managing partner, was hauled off to prison for insider trading. And in the years to come, it would continue to endure a series of scandals that encompassed everything from alleged corruption in South Africa — what The New York Times called the “biggest mistake in McKinsey’s nine-decade history” — to a Massachusetts lawsuit over its role in advising opioid producers on how to “turbocharge” sales.
But Alix’s allegations didn’t involve former partners, faraway lands, or odious advice. Instead, he claimed something potentially more serious and certainly closer to home: that McKinsey itself was engaged in repeated efforts to flout U.S. bankruptcy law.
Read my entire story at Institutional Investor here:
GREAT article! ________________________________


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Johnny Laguardia’s Trinitron Mixtape is an experimental play that haunts the viewer as a live movie, radio play and dance piece in one performance hybrid. The show explores the personal narrative of Blanche Barrow, one time member and longest survivor of the Bonnie and Clyde gang, to stir sensational imagery of manifest destiny, gendered violence, southern longevity and longing. Mixtape is a tribute to analog dreamscapes, sculptures of early video art, the voice behind the microphone, punk carpentry, den carpet television overload, sleuthing a live feed and a performative / collaborative installation.
Reviews from Fusebox 2017:
“This show hit me between the eyes and shook my soul a bit. It was hard-edged and raw and yet balletic and quiet…..I needed this and I didn’t even know. I needed deep Southerness and complicated femininity It was beautiful and sometimes hard to watch. The script was cutting and brilliant. We sat in silence as the last song of the show played, stunned. Still drinking in the experience, not ready to applaud, and bring it to an end.” Aaron Sanders
“In Trinitron Mixtape we watch two lovers onstage; they are in the same theater, though they are not in the same worlds onstage. One is in prison, the other we assume is in her home; in their own way they are both incarcerated. We as the audience are able to witness them from a variety of perspectives (another wonderful metaphor for life, especially in the digital age). Though I did not actually count, what seems like half a dozen cameras are situated around the space, allowing the actors to be filmed as they speak in real time, and allowing the audience to witness their experience from a variety of gorgeously crafted stage pictures, which are simultaneously transmitted to a wall of screens. Like in the movies, what’s been staged for the screen to capture lacks a sense of genuineness; like in theater, there’s no hiding what we are actually witnessing.” Lindsey Greer Sikes
This event begins at 8pm; doors at 7pm. The event is free and open to the public, with a suggested donation of 10$ and an artist talk following the October 20th performance.

domain: entertainment


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Tennis legend battles cancer
Chris Evert: “The death of my sister saved my life”
Chris Evert (67) had to endure six chemotherapies, now the tennis legend has defeated ovarian cancer for the time being. Sister Jeanne, who died from the same disease, played an important role in this.
Published: 12:03 p.m
Updated: 12:18 p.m
It was news that shocked the tennis world: Chris Evert (67) has ovarian cancer. In January, the American made her illness public. “Good Morning America” now accompanied her to her sixth and hopefully last chemotherapy session.
“In tennis, you can control a lot. Not such things,” says the 18-time Grand Slam winner. “Thousands of people have cancer. I’m like everyone else.” Fortunately, the cancer was diagnosed at stage one, the earliest possible stage, so treatment was promising.
Sister died in 2020
It is thanks to her sister that she was diagnosed with the tumor so early – in 70 to 80 percent of cases it is not discovered until stage three or four. Jeanne Evert, also a former tennis player, died of ovarian cancer in February 2020 at the age of 62. The doctor immediately advised Chris Evert to have himself examined, since ovarian cancer can be genetic. And he was right.
“The death of my sister saved my life,” says the 67-year-old. Her younger sister was therefore also her motivation in the treatment sessions. “When I have chemo, it is my inspiration. I always think of her. She will carry me through it, »she said at the beginning of her therapy.
Now the chemo is no longer necessary for the time being, the doctors give her a 90 percent chance that the cancer will not return. As luck would have it, Evert’s tennis win rate at the end of her career was 90 percent. A good omen? Hopefully. (che)

domain: celebrity


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Dame Deborah James passed away yesterday after a six-year-long battle with bowel cancer. She was just 40 years old.
Back in May, Deborah revealed that she only had weeks left to live. However, that didn’t stop her from making the most of her final days.
Dame Deborah James’ final days
Deborah was incredibly busy during her final weeks, with achievements and incredible milestones being smashed up until her death.
Perhaps the biggest thing that happened to Deborah before her death was when she received a damehood.
Prince William himself hand-delivered Deborah’s damehood to her at her parents’ home.
The damehood was awarded to the star after she raised millions of pounds for Cancer Research UK.
Prince William later paid tribute to Deborah, branding her “incredible”, and he had been “honoured” to meet her.
View this post on Instagram
Deborah James’ t-shirt collection
One of the ways in which Deborah helped raise money in her final days was by launching her own t-shirt range.
Her t-shirts, which had ‘Rebellious Hope’ written on them, helped raise even more money for Cancer Research UK.
Deborah James’ memories with family
It wasn’t just charity work that Deborah got involved in during her final days.
She also spent a lot of time with her family too. Back in May, the 40-year-old made “new memories” with her daughter and her friends with a sleepover.
Speaking about the slumber party, Deborah wrote: “I went from staying in my wheelchair to ending up everyone helping to get me into an actually Tee pee to watch Cinderella with the gang and sit there like a 5-year-old with a huge Cheshire Cat smile on my face next to my daughter and sister.”
As well as making memories with her kids, Deborah also attended a number of events in her final weeks despite having end-of-life care.
After her trip to the races, she said: “I find myself living in limbo-land. Not really knowing what the future holds and for how long.
“So despite the unnerving tears, I look at the sunshine, smile, and think, wow, life is a funny thing, isn’t it!! Better enjoy it!”
Deborah also found out that her brother was engaged to his partner before she died, and even managed to celebrate raising a million pounds with her t-shirt range.
Just earlier this week, Tesco pledged £300k to Deborah’s Bowel Babe fund – something she and her family celebrated too.
Her legacy will definitely live on.
Leave us a comment on our Facebook page @EntertainmentDailyFix and let us know what you think of this story.


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LAS VEGAS: The increasing willingness of vendors to work with security researchers is making the process of reporting vulnerabilities easier, according to a panel of Black Hat presenters.
The researchers, all of whom were presenting their findings under the "Breaking things" track of the conference, said that many firms have grown more receptive to vulnerability reports and are more willing to work with researchers.
"Things have definitely gotten a lot faster, some comps even have deadlines to turn around a fix," said Chris Rholf, a consultant with Leaf Security Research.
"I think vendors have gotten a lot better, and bounties are proof of that."
James Forshaw, a consultant with Context Information Security, discovered a high-profile .NET vulnerability which Microsoft patched earlier this year. He said that the true range and scope of the flaw was only realised when Microsoft conducted its own investigation into the issue.
"I did not realise how much of an issue it was until it had gone through a few months in Microsoft's vetting process, it turned out to be considerably more troublesome than they thought," he said.
"To a degree it pleased me in some ways to find something that is that troublesome in a product."
Not all vendors are as accommodating and willing to work with researchers. Fermin Serna, an information security engineer with Google, who uncovered a vulnerability in Internet Explorer, said that firms such as Microsoft, Facebook and Google tend to be more accommodating of vulnerability reports.
"Whenever they find a vulnerability they are super responsive," he said.
"The smaller vendors are not as good as the big ones."
Found by calculating the strength of the material deep inside the crust of neutron stars
Can highlight in real-time the relevant regions of an image being described
Double legal trouble for Musk as he also faces civil lawsuit over renewed British pot-holer 'paedo' claims
Battery development could help boost performance of smartphones


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The Valley Forge Tourism & Convention Board (VFTCB) is a nonprofit, membership-based sales and marketing organization that actively promotes Valley Forge and Montgomery County, PA as a convention site and leisure visitor destination by encouraging patronage of its 500+ member hotels, restaurants, attractions, and services. The award-winning website links visitors, meeting planners, tour operators, and residents to a diverse portfolio of brands focused on reasons to visit the region.
VFTCB Launches Month-long Date Night Marketing Campaign, Activation
Activation photos: https://www.dropbox.com/scl/fo/428c2etvtthjvguyypat6/h?rlkey=ez3o4r4e13hgd4fj8j2e9leon&dl=0
MONTGOMERY COUNTY, PA – During the month of February, a traditionally slower travel period in the tourism sector, the Valley Forge Tourism & Convention Board (VFTCB) is targeting locals and visitors with a consumer marketing program promoting easy-to-plan date ideas in Montgomery County, PA.
“February here in Montgomery County is all about having the most fun during the shortest month of the year,” said VFTCB’s AVP of Marketing Justine Garbarino. “From dinner dates to play dates, there are so many ways to enjoy a date night with significant others, family, and even friends in Montco.”
In partnership with Madden Media, the campaign strategy will target the Philadelphia DMA via digital and social media advertising, radio, and more. Campaign advertising is expected to drive users to the campaign landing page which outlines date night events and itineraries, 50+ creative date ideas, hotel deals, and more.
New this year, a consumer activation will help generate buzz for the campaign. Between February 1-11, a campaign-branded glass truck will drive throughout Montgomery County and Philadelphia (schedule attached) bringing dinner dates to life while encouraging people to make a reservation in Montco. WMMR’s on-air host and Preston and Steve Show producer, Marisa Magnatta, will document a Montco date night, catered by Harvest Seasonal Grill, live from inside the truck on her social channels.
Additionally, for the duration of the month-long campaign, the VFTCB will host giveaways via email marketing and social media for gift cards to local restaurants and dining experiences.
With this initiative, the VFTCB is hoping to take the hassle out of travel planning, and with more than 80 hotels in Montgomery County, the campaign makes it easy to turn Valentine’s Day celebrations into the perfect staycation.
“There’s an extra day this February to spice things up and enjoy a date in Montco,” continued Garbarino. “We know that time together is special for a lot of people and look forward to welcoming them to Montgomery County.”
More information regarding the campaign can be found at www.valleyforge.org/date-nights-montco and by following along on social media at @VisitValleyForge.

domain: travel


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Visual rehabilitation in aphakic children encounters major obstacles as a result of amblyopia, especially in monocular cataract patients. Postoperative management problems may interfere with adequate optical treatment in aphakic children wearing contact lenses. At various periods of time, the child might not wear his contact lenses. During that time, amblyopia treatment is withheld and regression of visual acuity may occur.
Implanting an intraocular lens (IOL) provides the child with an optimal permanent correction of the aphakia, thus enabling proper development of the child's visual acuity. Also, in the child with unilateral aphakia, the IOL prevents aniseikonia.
One of the main objections to IOL implantation in children, if not the most important, is the frequent and extreme changes in the child's ocular refraction during his first period of life. It has been a matter of dispute regarding which IOL power the child should receive. Some suggest >'2 that a high-power IOL should be used to achieve emmetropia or slight myopia during the child's first years. However, we still lack data about the child's ocular refraction later in Ufe following the implantation of an IOL, which results in emmetropia at the time of implantation.
Surgical Data of Eight Pseudophakie Infants (10 eyes)
Others have suggested implanting a standard adultpower lens so that the child will be emmetropic later in life.3'5 Dahan and colleagues6 undercorrect their patients because of the anticipated increase in axial length. However, the follow-up time has not been long enough to analyze the changes in the refractive status of the Pseudophakie eyes; therefore, no specific IOL power was suggested. It remains unclear whether emmetropia is preferred during the child's first years of life or later. We present our data in infants where a high-power IOL was implanted, with long-term follow up.
SUBJECTS AND METHODS
Ten eyes in eight infants were operated and followed up for at least 5 years. Clinical estimation of the cataract disturbance to vision was made by using slit lamp examination (opacity blocking at least 3 mm of the lens center) and distortion and blurring of the indirect ophthalmoscopic view of the fundus. All infants underwent extracapsular cataract extraction, posterior capsulotomy and anterior vitrectomy, and an intraocular lens (IOL) implantation.
The surgery was performed by a limbal approach. The lens material was removed using the Anis aspirating cannula (Storz, St. Louis, Mo) with the aid of an anterior chamber maintainer (Katena, Denville, NJ). A posterior capsulotomy and anterior vitrectomy was performed with the Ocutome (Coopervision, Fairport, NY). In infants where it was difficult to maintain a deep anterior chamber with the anterior chamber maintainer, sodium hyaluronate (Healon, Pharmacia, Kalamazoo, MI) was used during IOL implantation. It was our aim to fixate the IOL in the bag, however, we could not be sure it was not placed, at least in some cases, in the sulcus.
Except for cataract, all the infants were otherwise healthy, as they did not suffer from any systemic disease or congenital syndrome. Also, no other ocular syndrome was found. No biometrie calculation of IOL power was used before IOL implantation. Postoperatively, all the children underwent retinoscopy on a regular basis, and eyeglasses were prescribed accordingly. After 3 years of age bifocals were added. Patching of the good eye to treat amblyopia was recommended, although cooperation was poor in some of the children (eyes No. 1, 7, and 8). Postoperative examination of the children was performed every 3 to 6 months.
Age at surgery ranged from 2 months to 3 years. The IOL power implanted was between 27.0 D and 30.0 D in seven eyes, and 19.0 D and 23.0 D in three eyes. The follow-up period was between 5 and 11 years (median; 6.25 years). Seven eyes underwent a second surgery due to secondary cataract or posterior synechia secondary to excessive fibrin reaction in the anterior chamber (Table 1). In children with a high-power IOL, the resultant refraction after 5 to 7 years was between -5.50 and -12.0 D, and in children with lower IOL power refraction after 6 to 11 years, the resultant refraction was between -2.5 and +9.0 D (Table 2). The results were plotted on the graph: refraction (D) versus IOL power (D). Arrangement of these points in a graph (Fig) resulted in a line with a correlation coefficient of 0.885. The crossing point between this line and the X-axis shows the IOL power that may result in emmetropia when the child is older. This point is 23.2 D.
Fig: Linear refraction as function of intraocular lens power after 5 Ito 11 years' follow up (correlation coefficient is 0.885).
The change in refraction during the first 3 years of age for each child is detailed in Table 2. Emmetropization of the eyes was achieved at the following ages: In eyes No. 1 and 2 at younger than 6 months of age; in eye No. 4 at 12 months of age; in eye No. 7 at 2 years of age. Visual acuity ranged from 6/6 to 6/60. Good visual acuity (better than 6/12) was achieved in eyes No. 3, 5 and 7 (Table 2).
Studies show7 that emmetropization of the developing eye is dependent on normal visual stimulation during ocular development. Disruption of pattern vision early in life may result in myopia.6 In that study, it was also demonstrated that in aphakic patients a shift toward myopia was found in congenital cataract patients as compared with juvenile cataract patients.6 This fact also emphasizes the role of clear vision early in life on the development of normal refraction of the eye. Implanting an IOL would provide the developing eye with better conditions for emmetropization.
Implanting IOLs in infants propounds a dilemma: what IOL power should be implanted? Calculation of IOL power according to the ocular axial lens and corneal curvature is accurate and definite in adults. In contrast, in young children, a change in the refractive state of the eye is induced by the growth of the eye. Thus, implanting IOLs in infants results in only a temporary refractive state. Therefore, the decision regarding which IOL power to implant in a baby's eye is, to a certain degree, an arbitrary one. One cannot rely on the ocular biometrie parameters, because they will certainly change later in Ufe.
The main reason for early implantation of IOLs is to provide infants with an optimal visual acuity during the first 2 years of Ufe, which are of the utmost importance for vision development. The first 2 years of life constitute the main part of the critical period of vision development in humans.8 This creates an urgent need for achieving the best quality aphakic correction feasible during this period of time. Therefore, most of our children had a high power IOL implanted. Those children were emmetropic or mildly myopic during their first years of life.
Emmetropization of eyes No. 3 and 7 of our series, which developed good vision, occurred between 1 and 2 years of age. Emmetropia at that young age might have had a beneficial effect on the development of visual acuity. However, eyes No. 4 and 6 were also emmetropic at that young age, and visual acuity remained only 6/60 in those eyes. The number of eyes is too small for statistical analysis, therefore, we cannot conclude from the data whether emmetropization at that young age is crucial for visual development.
A relatively high rate of additional surgery was needed in our patients. In our opinion, this could have resulted from the implantation of an IOL. The posterior surface of the IOL may act as a site that is amenable to epitheUal cell proUferation.
In animals, anomalous patterned visual experience is considered to be a relevant factor for producing myopia experimentally. Various methods of attenuating patterned vision have been shown to induce myopia in a variety of animals.913 von Noorden and Lewis14 demonstrated that visual deprivation results in an elongated antero-posterior axis of the eye in monkeys; however, human outcome was less predictable. The axis was longer in most eyes, but shorter or unchanged in others.
Eye elongation due to visual deprivation is related to amblyopia and poor vision, but not to aphakia.18 This points to a possible role of visual perception modulating eye growth during infancy. However, two studies show that there is no significant difference in postoperative increase in axial length in phakic versus aphakic or Pseudophakie eyes in children,1617 Our results also do not support the theory of IOL-induced emmetropization with the potential for a clear retinal image being provided with IOL implantation. Ocular growth is probably regulated and maintained according to its genetic predetermined course.
During follow up, we found that implanting a highpower IOL (between 27.0 D and 30.0 D) results in high myopia (between -5.5 D and -12.0 D) after 5 to 7 years. On the other hand, implanting a lower power IOL (19.0 D to 23.0 D) results, after 6 to 11 years, in low myopia or hypermetropia. The results indicated that implanting a +23.2 D IOL and using it even at a very young age will result in an older child or adult with emmetropia. This choice may be preferred, because it avoids high refractive errors in adulthood. In our next series of patients, we are using 23.0 D IOLs. This will, however, necessitate additional refractive correction during the first years of the child's Ufe. This refractive error will diminish as the child grows older. A comparison between the visual acuity and rate of amblyopia in these two groups, such as children with high power IOLs and those with 23.0 D IOLs, will indicate to us which IOL power is preferable for implantation in infants.
Refraction and Visual Acuity in Eight Pseudophakic Infants (10 eyes)
Although no definite postoperative follow up was mentioned, a myopic shift was also found in Pseudophakie children.4 Gimbel and colleagues4 found a mean spherical equivalent of -2.45 D in the younger age group (4.7 years at surgery), as compared with -0.76 D in the older group of patients (8.4 years at surgery). In the normal infant, mild hypermetropia is usually present. The hypermetropia does not change, or slightly increases, in the first 6 or 7 years of life.18 This results from simultaneous and compensatory changes in the ocular axial length, lens power, and corneal curvature.
It was shown that the mean lens power in the full-term newborn eye is 34.4 D, which changes to about 19.0 D by age 7.19 In the Pseudophakie young infant, the eye growth- as well as corneal changes- are genetically controlled, in the presence of a fixed power aphakic correction. As a result of it, implanting a high power IOL (28.0 to 30.0 D) in the first few months of life creates emmetropia or mild myopia during their first 2 years of Ufe, and high myopia after 6 or 7 years (Table 2). Our results show that implanting an IOL with the power of 27 D to 30 D between 2 months and 3 years of age causes very high myopia (mean -8.25 D) at age 5 to 9.
According to the resultant refractions in 10 eyes, a line was drawn (correlation coefficient of 0.885). The cut point of this Une with the X-axis (Fig) indicates the lens power required to achieve emmetropia later in the child's Ufe. This cut point is 23.2 D. In this case, hypermetropic correction would be necessary during the first years of the child's life. Gordon and colleagues19 reached the same conclusion by analyzing direct measurements and data obtained in 148 normal eyes. They recommended that a 23- D to 24-D IOL should be used to create emmetropia in the adult Pseudophakie patient.
Preoperative keratometry and axial length measurements should be taken, and if the biometrie analysis falls within the age-matched normal limite, a 23-D IOL is recommended. If the measurement falls outside the normal physiological range, adjustment of the IOL power should be considered. Because of the large range of myopic shift over time in our patients (between 2.5 D and 12 D), implanting a 23-D IOL does not guarantee emmetropia in the future. However, based on the results of this series, it is the nearest estimation that can be achieved.
1 Aron JJ, Aron-Rosa D. Intraocular lens implantation in unilateral congenital cataract: a preliminary report. J Am Intraocular Implant Soc. 1983;9:306-308.
2. BenEzra D, Paez JH. Congenital cataract and intraocular lenses. Am J Ophthalmol. 1983;96:311-314.
3. Hiles DA. Intraocular lens implantation in children with monocular cataracts, 1974-1983. Ophthalmology. 1984;91:1231-1237.
4. Gimbel HV, Ferensowicz M, Raanan M, De-Luca M. Implantation in children. J Pediatr Ophthalmol Strabismus. 1993;30:69-79.
5. Burke JP, Willshaw HE, Young JDH. Intraocular lens implants for uniocular cataracts in childhood. J3r J Ophthalmol. 1989;73:860-864.
6. Dahan E, Welsh NH, Salmenson BD. Posterior chamber implants in unilateral congenital and developmental cataracts. Eur J Implant Refract Surg. 1990;2:295-302.
7. Rabin J, Van Sluyters RC, Malach R. Emmetropization: a vision-dependent phenomenon. Invest Ophthalmol Vis Sci. 1981;20:561-564.
8. Vaegan J, Taylor D. Criticai period for deprivation amblyopia in children. Trans Ophthal Soc UK. 1979;99:432-439.
9. Wiesel TN, Raviola E. Myopia and eye enlargement after neonatal lid fusion in monkeys. Nature. 1977;266:66-67.
10. Sherman SM, Norton TT, Casagrande VA. Myopia in the lidsutured tree shrew (Tupaia glis). Brain Res. 1977;124:154157.
11. Wilson JR, Sherman SM. Differential effects of early monocular deprivation on binocular and monocular segments of cat striate cortex. J Neurophysiol. 1977;40:891-903.
12. Wallman J, Türkei J, Trachtman J. Extreme myopia produced by modest change in early visual experience. Science. 1978;201:1249-1251.
13. Wiesel TN, Raviola E. Increase in axial length of the macaque monkey after corneal opacification. Invest Ophthalmol Vis Sci. 1979;18:1232-1236.
14. von Noorden GK, Lewis RA. Ocular axial length in unilateral congenital cataract and blepharoptosis. Invest Ophthalmol Vis Sci. 1987;28:750-752.
15. Rasoohy R, BenEzra D. Congenital and traumatic cataract. The effect on ocular axial length. ArcA Ophthalmol. 1988;106:1066-1068.
16. Kova Y, Shimizu K, Inatani M, Fukado Y, Ozana T. Eye growth after cataract extraction and intraocular lens implantation in children. Ophthalmic Surg. 1993;24:467-475.
17. Niederreiter P. Kiemen TJM, Leitner J. Zur ultrashall-biometrie in kindesalter (Ultrasound biometry in children). Klin Monatsb Augenheilkd. 1987;191: 355-357.
18. Duke-Elder S. System of Ophthalmology. London, England:Henry Kimpton; 1970:229.
19. Gordon RA, Donzis PB. Refractive development of the human eye. Arch Ophthalmol. 1985;103:785-789.
Surgical Data of Eight Pseudophakie Infants (10 eyes)
Refraction and Visual Acuity in Eight Pseudophakic Infants (10 eyes)

domain: literature


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Store bought pizza crusts have become my best friend. The last few weeks have been hectic and the next few days will be impossible. So I decided to make different kinds of pizza. This is my first experiment.
I wanted to make pizzas that were “different” from the traditional red sauce, mozzarella and pepperoni. So, I started thinking of combinations of flavors my family enjoys. This was my first attempt.
I now plan on ditching plain old pizza! This was flavorful and fresh tasting. My family ate it all. I mean I could not get them to stop. There were no leftovers for the next day.
- 2 pre-made pizza crusts (I used one GF and one regular thin crust)
- 1/4 to 1/2 cup of prepared jarred pesto sauce
- 1/2 cup of Parmesan cheese, grated
- 8 oz. mozzarella cheese, grated
- 1 lb. country sausage, removed from casings and cooked thoroughly
- Pre heat oven to 400 degrees.
- Spread about 1/4 cup of pesto over top of each prepared crust. You may use more or less pesto depending on how much your family likes. Be careful to leave about 1/2″ of crust without toppings around the edge so your pizza does not drip and burn in your oven.
- Sprinkle 1/4 cup of Parmesan cheese over the pesto on each crust.
- Sprinkle 1/2 of the mozzarella over the top of each pizza.
- Place 1/2 of the sausage over the top of the cheeses on each crust. Spread it around to evenly cover the entire top.
- Bake 10-12 minutes. Remove pizza from oven when it is cooked to your liking. Let rest for 5 minutes before slicing.

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