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You are here: Home / Obituaries / Robert H. Aument
Robert H. Aument
Robert H. Aument, formerly of Millersville, died on June 27, 2020 at Brereton Manor, Washington Boro at the age of 98. He was the son of the late Loyd and Edith (Frey) Aument and the husband of the late E. Susan Hess Aument and Kathryn Alexander Baer Aument.
Bob graduated from Manor High School in 1939. He served in the 45th Infantry of the Army from 1942 to 1945 in Sicily, Anzio, Munich, South Africa, and France. Upon his return to the States, he was a self-employed builder and worked into his 90's. He was a life member of Grace United Methodist Church and served on Trustees, Administrative Board, Building Committee, and sang in the choir for over 50 years. He was a member of Millersville Boro Council and served on the Zoning Board and was also a member of the Lions Club. Bob was an avid reader, enjoyed cracking and donating black walnuts, woodworking, and, as a young man, enjoyed racing cars.
Surviving are his children J. Michael (Esther), Marietta; Thomas H. (Louise), Lancaster; and Susan E. (Dennis) Sneath, Mountville; 4 grandchildren Brandon (Michelle) and Cameron (Amanda) Aument, Emily Greider (Cem Ekinci) and Benjamin (Alysha) Greider; one great grandchild and a brother, D. Glenn Aument (Jeanie), Lancaster. He was predeceased by a son, John, and 4 brothers and 3 sisters.
Relatives and friends are invited to attend a Memorial Service at Millersville Community UM Church, Grace Campus, 163 W. Frederick St., Millersville, PA 17551 on Thursday, July 2, 2020 at 11:00 AM. Interment with full military honors will follow in the adjoining church cemetery.
In lieu of flowers, Bob has requested that memorials be sent to Lancaster Area Habitat for Humanity, 443 Fairview Ave. Lancaster, PA 17603 or Caring Hospice, 101 Good Drive, Lancaster, PA 17603.
Offer Condolence for the family of Robert H. Aument
Harry and Carol Stigelman
What a wonderful, long life he had. I have so many fond memories of him at Grace and in the choir. May all the beautiful memories of him surround you during this time of loss. Your family is in my prayers and peace to all.
Mary Lynn DiPaolo
Dear Mike and Esther,
Please accept my sincere condolences with your father's passing. He sure lived a wonderful long life.
Mary Lynn Hobday DiPaolo
Toni Bowman
Sue,
I am sorry to hear about your father. May he rest in peace. Keep him in your heart and he will live forever.
Toni (Tuna) Bowman
Sue Sneath
Toni, how sweet of you to remember us after so many years. Miss so many of you from my long ago Wild's days. Your message means the world to me as we did share so much as a work family.
Barbara Skelly
Dear Aument family,
Our caring, loving hearts go out to each one of you. Bob lived a meaningful, purpose filled life and was loved and respected by all. What a wonderful heritage he left each of you. With love and sympathy, Bill & Barb Skelly
Dann and Barb Poukish
Our thoughts and prayers are with you during this very difficult time. Bob has always been very special to us. The way he lived his life was truly an inspiration to all that knew him. We will miss him greatly!
TIMOTHY S RISSER
Bob will surely be missed – A loyal and maybe the first customer we had at J H Brubaker, beginning April 1st 1972 and was the favorite of almost all of our sales people. Have comfort in knowing Bob is in the presence of our Lord and Savior JESUS – Blessings to all the family members !
Tim Risser
William Dewan
On behalf of the staff of Lancaster Lebanon Habitat for Humanity, our deepest condolences go out to Bob's family and friends. You are in our prayers during this difficult time.
Keith and Mindy Aument
To all of Uncle Bob's family – please know you are in our thoughts and prayers. What a wonderful man and a wonderful and full life. We are saddened by his passing, but have many cherished memories of him especially with his brothers and sisters and the entire Aument clan. With our deepest sympathies –
Keith, Mindy, Andy and Tess Aument
Debra Aument Conrey
To family of Uncle Bob. Our family has lived in Texas and Georgia all of our lives, but visits to Pennsylvania to visit Grandma, Uncles, Aunts and the millions of cousins left a lasting impression on us all. I am so sorry for your loss. He was an amazing, talented man and I cannot believe he was 98. I lost my Dad in his 50's (Uncle Pete) and did not have all of these years of memories with him, God bless you all and know that you are in our families prayers
Fred R Hashinger III
The cool thing is that Uncle Bob and my Dad were in business together. So, we always had a good time together, family reunions and even after my Dad passed . . . Uncle Bob helped me work on my house on Chestnut Street. Mike and I were a few years apart and Tommy was younger. Susie was the youngest one. So sorry for your loss. Thinking of all of you at this time and remembering you all.
Jack and Ann Willett
Mr. Aument was so much more than a good neighbor! He enriched our lives with his stories of World War II and his varied life experiences. He was our local historian. His character was an example of hard work, responsibility, and self-discipline.
We are so grateful to have known him!


Photographs today by a 9 year old child or Janet. The above photo shows that our orchard is beautiful and our bees are active, but we do not have the photography smarts to get the bee in focus.
A wise market manager just said to us: "Enjoying the weather! Concerned for the climate." Ditto.
This weather is ideal for starting seeds, which we are doing in great abundance right now, but so far away from normal that our physical comfort is tempered with worry. Many people have been asking what this means for vegetables this spring: It means we may have more to harvest than usual during the above-average temps, late-winter weeks, but if the temps continue to be this high above average through the spring, it means cool-loving spring crops won't grow well. Broccoli won't head up. Kale will want to flower early. Stay tuned to see what Mother Nature, influenced by our activities, doles out.
Bread and vegetables are rolling out of here in good quantities this weekend. Catch all of our goods at the Farmers Market @ St. Stephens Saturday morning, 9-12, and find our greens at Union Market this weekend.
Want the best access to our vegetables, all season long? Grab one of the remaining shares of our harvest through our CSA. Join here!
We're celebrating our CSA members by drawing names for gift cards to some of our favorite partner businesses. Our most recent winner, of a gift card to Perk! Coffee & Lunchbox in Bon Air, has been contacted. All members who have signed up by March 15 will be entered to win a gift card to Good Health Herbs in Brandermill/Midlothian. Sign up today and you'll get to relish being part of the Broadfork Farm family.
Chervil, Microgreens, Salad Mix, Scallions, Spinach, Parsley, Pea Shoots, Pepper Jelly…and our Organic Hearth Baked Bread (Whole Wheat, Sunny Greens, Seed, & Raisin loaves for this weekend) PLUS new this week: You can purchase Certified Organic flour that we have freshly stone-ground in our Bakery. Whole Wheat & Whole Rye available, with a delicious recipe included with each purchase.
2017 Market Share CSA Members: Reserve your selection online to pick up Saturday, March 5th at our farm, at Good Health Herbs, or at the Farmers Market @ St. Stephens.
Previous Post CSA Sign Up Week!


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In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth
Kilian Vogele1, Thomas Frank1, Lukas Gasser1, Marisa A. Goetzfried1, Mathias W. Hackl2, Stephan A. Sieber2, Friedrich C. Simmel1,3, Tobias Pirzer1
1Physics of Synthetic Systems - E14, Physics-Department and ZNN, Technische Universität München, 2Departmant of Chemistry, Center for Integrated Protein Science (CIPSM), Technische Universität München, 3Nanosystems Initiative Munich
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Published:	6/28/2019
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Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.
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Vogele, K., Frank, T., Gasser, L., Goetzfried, M. A., Hackl, M. W., Sieber, S. A., Simmel, F. C., Pirzer, T. In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth. J. Vis. Exp. (148), e59831, doi:10.3791/59831 (2019).
Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.
The creation of synthetic living cellular systems is usually approached from two different directions. In the top-down method, the genome of a bacterium is reduced to its essential components, ultimately leading to a minimal cell. In the bottom-up approach, artificial cells are assembled de novo from molecular components or cellular subsystems, which need to be functionally integrated into a consistent cell-like system.
In the de novo approach, compartmentalization of the necessary biochemical components is usually achieved using membranes made from phospholipids or fatty acids1,2,3,4. This is because "modern" cell membranes mainly consist of phospholipids, while fatty acids are regarded plausible candidates of prebiotic membrane enclosures5,6. For the formation of new membranes or to facilitate membrane growth, amphiphilic building blocks must be provided from the exterior7 or ideally through production within a membranous compartment using the corresponding anabolic processes4,8.
While lipid synthesis is a relatively complex metabolic process, peptides can be produced quite readily using cell-free gene expression reactions9,10. Hence, peptide membranes formed by amphiphilic peptides represent an interesting alternative to lipid membranes as enclosures for artificial cell mimics that are able to grow11.
Amphiphilic elastin-like di-block copolymers (ELPs) are an attractive class of peptides, which can serve as the building block for such membranes12. The basic amino acid sequence motif of ELPs is (GaGVP)n, where "a" can be any amino acid except for proline and "n" is the number of motif repeats13,14,15,16,17. ELPs have been created with a hydrophobic block containing mainly phenylalanine for a and a hydrophilic block mainly composed of glutamic acid11. Depending on a and solution parameters, such as pH and salt concentration, ELPs exhibit a so-called inverse temperature transition at temperature Tt, where the peptides undergo a fully reversible phase transition from a hydrophilic to hydrophobic state. The synthesis of the peptides can be easily implemented inside vesicles using the "TX-TL" bacterial cell extract11,18,19,20,21, which provides all necessary components for coupled transcription and translation reactions.
The TX-TL system was encapsulated together, with the DNA template encoding the ELPs into ELP vesicles utilizing dehydration-rehydration from glass beads as a solid support. The formation of vesicles occurs through rehydration of the dried peptides from the bead surface11. Other methods22 for vesicle formation can be used, which potentially show lower polydispersity and larger vesicle sizes (e.g., electro-formation, emulsion phase transfer, or microfluidics-based methods). To test the viability of the encapsulation method, transcription of the fluorogenic aptamer dBroccoli23 can alternatively be used11, which is less complex than gene expression with the TX-TL system.
Due to the expression of the membrane building blocks in vesiculo and their subsequent incorporation into the membrane, the vesicles start to grow11. Membrane incorporation of the ELPs can be demonstrated through a FRET assay. To this end, the ELPs used for formation of the initial vesicle population are be conjugated with fluorescent dyes in equal shares constituting a FRET pair. Upon expression of non-labeled ELPs in vesiculo and their incorporation into the membrane, the labeled ELPs in the membrane are diluted and consequently the FRET signal decreases11. As a versatile and common method for conjugation, copper catalyzed azide-alkyne cycloaddition is used. With the use of a stabilizing ligand such as tris(benzyltriazolylmethyl)-amine, the reaction can be carried out in an aqueous solution at a physiological pH without the hydrolysis of reactants11, which is appropriate for conjugation reactions involving peptides.
The following protocol presents a detailed description of the preparation for growing ELP-based peptidosomes. The expression of the peptides and vesicle formation using the glass beads method are described. Furthermore, it is described how to implement transcription of the fluorogenic dBroccoli aptamer and the transcription-translation reaction for protein expression inside the ELP vesicles. Finally, provided is a procedure for the conjugation of ELPs with fluorophores, which can be used to prove vesicle growth through a FRET assay11.
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1. Expression of Elastin-like Polypeptides
Day 1: Preparation of a starter culture and supplies for peptide expression
Prepare and autoclave expression culture flasks (4 x 2.5 L) and 3 L of LB medium. For 1 L of LB medium, add 25 g of LB powder to 1 L of ultrapure water.
Prepare a starter culture with 100 mL of LB medium, 50 µL of sterile-filtered (0.22 µm filter) chloramphenicol solution (25 mg/mL in EtOH), and 50 µL of sterile-filtered (0.22 µm filter) carbenicillin solution (100 mg/mL in 50% EtOH and 50% ultrapure water).
Add a small streak from a pre-made bacterial stock containing E. coli strain BL21(DE3)pLysS with the pET20b(+) expression vector encoding the polypeptide sequence MGHGVGVP((GEGVP)4(GVGVP))4((GFGVP)4(GVGVP))3(GFGVP)4GWP (abbreviated as EF) to a pre-warmed 100 mL starter culture and incubate at 37 °C for 16 h with 250 rpm in a shaking incubator (E. coli strains and vectors are available upon request).
Pre-warm the expression culture flasks and the LB media at 37 °C overnight so that they are prepared for the next day.
Day 2: Protein expression
Add 750 mL of LB medium to each expression culture flask, 375 µL of sterile-filtered (0.22 µm filter) chloramphenicol stock (25 mg/mL in EtOH), and 375 µL of sterile-filtered (0.22 µm filter) carbenicillin stock (100 mg/mL in 50% EtOH and 50% ultrapure water).
After agitation to distribute the antibiotics, take 2 mL of the media as a reference sample for the optical density measurement at 600 nm (OD600).
Add 7.5 mL of the starting culture to the expression flask and incubate for 1 h at 37 °C with 250 rpm.
After this step the OD600 of each flask will be checked every 20 min.
When the optical density reaches approximately 0.8 reduce the temperature to 16 °C and induce peptide expression by adding 750 µL of 1 M sterile-filtered β-isopropyl thiogalactoside (IPTG) in ultrapure water into each expression flask.
Incubate the expression flask with the induced bacteria at 16 °C for 16 h with 250 rpm.
Day 3: Extraction of the expressed polypeptide
Pre-weigh the centrifugation flask to enable determination of the mass of the cell pellet after harvesting.
Harvest all the bacteria from the expression flasks by centrifugation for 20 min using a pre-cooled centrifuge at 4,000 x g and 4 °C.
Pour out the supernatant and blot the centrifuge bottles on a sterile paper towel.
Weigh the centrifugation flasks and calculate the cell pellet mass.
Resuspend the cells, which are pelleted down from 750 mL of original cell culture, in 15 mL of phosphate-buffered saline (PBS, pH 7.4) by pipetting, and centrifuge at 4,000 x g for 20 min at 4 °C. Pour out the supernatant and blot the centrifuge bottles on a sterile paper towel.
Resuspend the cell pellet for the lysis step in 2 mL of buffer per 1 gram of cell pellet using PBS (pH 7.4) supplemented with lysozyme (1 mg/mL), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, and 0.5 U of DNase I.
Sonicate the cells for further lysis on ice with a sonicator at 8 W for 9 min with alternating 10 s sonication and 20 s pausing steps, followed by a 9 min pause during which the sample should be cooled on ice. Repeat the 9 min sonication step once more.
Add 2 mL of 10% (w/v) polyethyleneimine (PEI) per 1 L of original cell culture for nucleic acid precipitation.
Distribute the sample into 2 mL centrifuge tubes and incubate at 60 °C for 10 min followed by an incubation for 10 min at 4 °C.
Centrifuge the solution at 16,000 x g for 10 min at 4 °C and collect the supernatant containing the ELP.
Protein purification via inverse temperature cycling:
Adjust the supernatant to a pH of 2 to switch the hydrophilic part of the peptide to hydrophobic and induce aggregation. To adjust the pH, phosphoric acid and sodium hydroxide are used. pH stripes are sufficient to determine the pH level.
To improve the yield of the purification the sample, heat the sample to 60 °C and add sodium chloride up to 2 M.
Subsequently, centrifuge the sample at 16,000 x g for 10 min at room temperature (RT).
Discard the supernatant and re-dissolve the pellet in PBS (but only half of the initial volume is used compared to the previous step).
Adjust the pH to 7 with phosphoric acid and sodium hydroxide. pH stripes are sufficiently accurate to determine the pH.
Centrifuge the sample subsequently at 16,000 x g for 10 min at 4 °C.
Collect the supernatant and repeat steps 1.4.1–1.4.6 up to 3x total, except that in the last repetition of step 1.4.4, only water is added instead of PBS.
Measure the concentration of the peptides with an absorption spectrometer. Use an extinction coefficient of 5500/M/cm at 280 nm.
Adjust the concentration of the ELP to 700 µM.
2. Vesicle Production Using the Glass Beads Method
Concentrate the ELP solution to 1.1 mM using a centrifugal vacuum concentrator.
Mix 200 µL of the concentrated ELP solution with 1,250 µL of a 2:1 chloroform/methanol mixture followed by vortexing.
Add 1.5 g of spherical glass beads (212–300 µm in size) to a 10 mL round bottom flask.
Add the solution containing ELP and the chloroform/methanol mixture to the round bottom flask and mix by gentle shaking.
Connect the flask to a rotary evaporator. Adjust the speed to 150 rpm and regulate the pressure to -0.2 x 105 Pa (-200 mbar) for approximately 4 min until the liquid is evaporated at room temperature.
Place the round bottom flask into a desiccator for at least 1 h to ensure that remaining chloroform and methanol are evaporated. To avoid loss of the glass beads, loosely attached aluminum foil should be wrapped around the round bottom flask opening.
For a single experiment, mix 100 mg of the peptide covered glass beads with 60 µL of the swelling solution, such as PBS. Incubate this sample at 25 °C for 5 min.
Centrifuge the sample quickly with a table-top centrifuge to sediment the glass beads.
Use a pipette to collect the supernatant which contains the vesicles.
3. Transcription of RNA Aptamer dBroccoli Inside the Vesicles
Clean the lab bench with wipes containing RNase decontamination solution to create an RNase-free working environment.
Mix the ssDNA (5 µM) comprising the T7 promotor and dBroccoli sequence (GAGACGGTCGGGTCCATCTGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCAGATGTCGAGTAGAGTGTGGGCTC), as well as the noncoding strand (5 µM) in nuclease-free water supplemented with RNAPol reaction buffer [40 mM Tris-HCl (pH = 7.9), 6 mM MgCl2, 1 mM dithiothreitol (DTT), and 2 mM spermidine], to prepare the DNA template for the transcription reaction.
Incubate the solution at 90 °C for 5 min followed by a slow temperature decrease to 20 °C for 30 min.
Prepare a 1 mM (5Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one (DFHBI) solution by dissolving 1.26 mg of DFHBI in 5 mL of DMSO.
Prepare the transcription reaction by mixing RNAPol reaction buffer [40 mM Tris-HCl (pH = 7.9), 6 mM MgCl2, 1 mM dithiothreitol (DTT), and 2 mM spermidine] with 125 mM KCl, 15 mM MgCl2, 4 mM rNTP, 10 µM DFHBI, 200 nM DNA template, 0.5 U/µL RNase inhibitor murine, and water.
Right before the reaction is started, add 4 U/µL T7 polymerase.
Use this reaction mix as the swelling solution in step 2.7 and incubate at 37 °C for the duration of the experiment, which is typically 1 h.
4. Transcription-translation (TX-TL) Reaction
NOTE: For the transcription-translation reaction, a crude cell extract is required as well as reaction buffer and DNA. The crude cell extract is prepared as described in Sun et al.18. For a TX-TL reaction, use the following: 33% (v/v) of the crude E. coli extract, 42% (v/v) reaction buffer, and 25% (v/v) phenol-chloroform purified DNA plus additives. The final concentrations are approximately 9 mg/mL protein, 50 mM HEPES (pH = 8), 1.5 mM ATP, 1.5 mM GTP, 0.9 mM CTP, 0.9 mM UTP, 0.2 mg/mL tRNA, 26 mM coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 68 mM folinic acid, 1 mM spermidine, 30 mM PEP, 1 mM DTT, 2% PEG-8000, 13.3 mM maltose, 1 U of T7 RNA polymerase, and 50 nM plasmid DNA in ultrapure water.
Phenol-chloroform purification of the DNA template
Prepare six overnight cultures with 5 mL of LB medium and 5 µL of sterile-filtered (0.22 µm filter) carbenicillin solution (100 mg/mL in 50% EtOH and 50% ultrapure water).
Add a small streak from a pre-made bacterial stock containing DH5α E. coli with the pET20b(+) expression vector to each pre-warmed 5 mL overnight culture and incubate at 37 °C for 16 h with 250 rpm. Depending on which peptide (EF) or protein (YPet or mVenus) is to be expressed, a specific encoding plasmid is used.
Extract the plasmid with a mini-prep kit as described in the user manual or by following the protocol provided by Zhang et al.24.
Prepare 100 µL of the pre-purified plasmid DNA (concentration can range from 200–600 ng/µL) and mix with 100 µL of Roti-phenol/chloroform/isoamyl alcohol (pH = 7.5–8.0) in a microcentrifuge tube to enable better phase separation.
Gently invert the tube up to 6x and centrifuge for 5 min at 16,000 x g at RT.
Add 200 µL of chloroform to the upper phase of the tube and invert the tube up to 6x.
Centrifuge the sample at 16,000 x g for 5 min at RT.
Pipet the supernatant to a separate tube and add 10 µL of 3 M of sodium acetate for ethanol precipitation.
Add 1 mL of -80 °C cold ethanol and store the sample at -80 °C for 1 h.
Centrifuge the sample at 16,000 x g for 15 min at 4 °C.
Decant the supernatant and add 1 mL of -20 °C cold 70% (v/v) ethanol.
Centrifuge at 16,000 x g for 5 min at 4 °C.
Remove the liquid by pipetting. Be careful to not disturb the DNA pellet.
Store the sample at RT for approximately 15 min to evaporate the remaining ethanol.
Add ultrapure water to the sample to adjust the sample concentration to approximately 300 nM, which is measured by absorption at 260 nm.
Preparation of the TX-TL reaction
Thaw the prepared crude cell extract and the reaction buffer on ice.
For a 60 µL reaction mix, add the plasmid DNA (phenol-chloroform purified) to 37.5 µL of the reaction buffer [50 mM HEPES (pH = 8), 1.5 mM ATP, 1.5 mM GTP, 0.9 mM CTP, 0.9 mM UTP, 0.2 mg/mL tRNA, 26 mM coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 68 mM folinic acid, 1 mM spermidine, 30 mM PEP, 1 mM DTT, 2% PEG-8000, and 13.3 mM maltose), followed by the addition of 28.7 µL of crude cell extract. Fill to a final volume with ultrapure water to 58.8 µL.
Right before the reaction starts, add 1.2 µL of the T7 RNA polymerase solution and mix the sample by pipetting up and down.
Use this reaction mix as a swelling solution in step 2.7 and incubate at 29 °C for the duration of the experiment, which is typically 4–8 h.
5. Conjugation of Elastin-like Polypeptides with Fluorophores via Copper Catalyzed Azide-alkyne Huisgen Cycloaddition
Prepare a 20 mM NHS-azide linker (γ-azidobutyric acid N-hydroxysuccinimide ester) solution in DMSO.
Mix 250 µL of the ELP solution (600 µM) with PBS (8 g/L NaCl, 0.2 g/L KCl, 1.42 g/L Na2HPO4, 0.27 g/L K2HPO4, pH = 7.2) and NHS-azide (1.2 mM) and incubate for 12 h at RT.
Load the sample in a 10 kDa dialysis cassette and store it at 4 °C for 12 h in 1 L of ultrapure water to remove the salts and residual NHS-azide.
Mix the activated ELP (500 µM) with the alkyne-conjugated dye (500 µM), Cy5-alkyne, or Cy3-alkyne.
Mix this sample with 1 mM TBTA (tris(benzyltriazolylmethyl)amine), 10 mM TCEP (tris(2-carboxyethyl)-phosphine hydrochloride) and 10 mM CuSO4 and incubate at 4 °C for 12 h.
Load the sample in a 10 kDa dialysis cassette and store it at 4 °C for 12 h in 1 L of ultrapure water, to remove the salts and residual alkyne-conjugated dyes.
These dye-modified ELPs are used in a 1:1 mixture of the Cy3 labeled ELPs and the Cy5 labeled ELPs analogous to the peptides in part two.
Representative Results
Vesicle production
Figure 1 shows transmission electron microscopy (TEM) images of vesicles prepared with different swelling solutions and the glass beads method (also see Vogele et al.11). For the sample in Figure 1A, only PBS was used as swelling solution to prove the formation of vesicles and to determine their size. When TX-TL was used as swelling solution (Figure 1B), the vesicles also formed. Dynamic light scattering (DLS) measurements were performed to show that the glass beads method has an effect on vesicle formation. Figure 2 depicts the measured intensity autocorrelation curves of an EF sample prepared without glass beads in PBS (blue) and an EF sample prepared with the glass beads method with PBS as the swelling solution (red). The sizes were calculated from a cumulant fit25 and resulted in a diameter of 134 nm with a polydispersity of 25% when the vesicles were prepared without the glass beads method. When the glass beads were used, the cumulant fit resulted in a diameter of 168 nm with a polydispersity of 21%.
In vesiculo transcription11
Figure 3 shows the fluorescence intensity profile over time for the transcription of the dBroccoli aptamer inside the EF vesicles (green). As a negative control, DNase I was added to the swelling solution and thus also incorporated during vesicle formation (blue). The measurements were performed in fluorescence plate reader with excitation at 480 nm and emission at 520 nm.
In vesiculo protein expression11
Figure 4 shows the fluorescence intensity of two fluorescent proteins which were expressed inside the ELP vesicles using a fluorescence plate reader. Excitation was carried out 500 nm and emission at 520 nm. Transcription-translation of mVenus (Figure 4A) was performed to investigate expression dynamics and expression level of proteins in the ELP vesicle. It is important to note that after vesicle formation, the contents of the vesicles and outer solution are the same. Hence, to suppress protein expression outside of the vesicle, the antibiotic kanamycin was added to the exterior solution. As a control, kanamycin was also added to the swelling solution, in which case protein expression inside of the vesicle was suppressed. This further indicates that the small molecule kanamycin stays inside the vesicle and suggests that the membrane is not permeable for this molecule over the time scale of these experiments. If kanamycin diffused through the membrane, the mVenus expression would not have been suppressed and at 5 h, the fluorescence would be higher. In the second experiment, the expression of YPet (Figure 4B) was carried out. Both proteins were chosen because they exhibit a faster maturation time than, for instance, GFP. Furthermore, the T7 promoter was used for mVenus transcription and a constitutive promoter used for YPet transcription to show that both inducible and continuous expression are possible.
FRET assay11
Figure 5A shows the results of the FRET assay performed to demonstrate ELP incorporation into the membrane. Therefore, the vesicles were produced using a mixture of two equally concentrated fluorophore-peptide constructs. These were Cy3-EF (donor) and Cy5-EF (acceptor). At time t = 0, the starting FRET level was measured. The signals of the donor and the FRET signal depend on the mean distance between the dyes in the membrane. Upon expression of the membrane ELPs, additional peptides incorporate into the membrane, which increases the average distance between the FRET pairs. The latter was measured through the increase in donor fluorescence and a decrease of the FRET signal at time t = 2.5 h. Figure 5B shows the negative control. Here, kanamycin was added to the swelling solution before vesicle formation. Kanamycin suppresses protein expression; therefore, no change in FRET was visible. It is important to note that this assay only shows EF incorporation.
Figure 1: Representative TEM images. (A) EF vesicles formed in the swelling solution PBS. (B) EF vesicles formed in the swelling solution TX-TL. Scale bars = 200 nm. Please click here to view a larger version of this figure.
Figure 2: Representative DLS data. Intensity autocorrelation curves measured by DLS. The blue curve depicts EF peptides in PBS prepared without the glass beads method. The red curve depicts an EF solution produced with the glass beads method and PBS as the swelling solution. Please click here to view a larger version of this figure.
Figure 3: dBroccoli transcription. Representative data of the transcription of the dBroccoli aptamer inside EF vesicles. The green curve depicts the fluorescence signal and the blue curve the control measurement with encapsulated DNase I. Please click here to view a larger version of this figure.
Figure 4: Protein expression inside peptide vesicles. (A) A plasmid encoding mVenus and the TX-TL expression mix were encapsulated in the ELP vesicles. After approximately 5 h, the fluorescence saturated. When the antibiotic kanamycin was encapsulated as well, no fluorescence was observed. (B) Similar results were obtained upon encapsulation of a YPet plasmid. Error bars represent the standard deviation of the measured values at the indicated time during a time frame of 15 min. Please click here to view a larger version of this figure.
Figure 5: FRET assay. (A) Starting fluorescence levels at time t = 0 for donor emission and FRET signal (acceptor emission). At t = 2.5 h, the donor showed increased emission, and the FRET signal decreased. (B) When only hydrophilic ELP were expressed inside the vesicles, the FRET signal and donor emission stayed constant. Error bars represent the standard deviation of the measured values at the indicated time during a time frame of 15 min. Please click here to view a larger version of this figure.
Supplemental File: Contains all plasmid sequences resp. gene sequences. Please click here to download this file.
Film rehydration is a common procedure for the creation of small unilamellar vesicles. The main source of failure is the wrong handling of the materials used in the procedure.
Initially, the ELPs are produced by E. coli cells. The yield after ELP purification can vary significantly depending on how carefully the protocol is conducted during its crucial steps. These are the inverse temperature cycling (ITC) step and the resuspension of the ELPs after precipitation. For purification, we triggered the hydrophobic collapse of the peptide through the addition of phosphoric acid, which changes the pH to acidic and leads to protonation of the glutamic acid side chains in the hydrophilic block. After this step, both blocks are hydrophobic at RT. It is preferable to use an acid whose salt is already in the solution to keep the ionic strength constant, but other acids such as hydrochloric acid can be used, as well. To increase the yield of ELP production, additional salts can be added to enhance the coacervation process, because salt decreases the transition temperature Tt and makes the ELP more hydrophobic. Usually sodium chloride is used, but kosmotropic salts such as sodium sulphate are far better, whereas the salt concentration can be close to its solution limit. Additional heating using a water bath can increase the yield even further.
Another critical step is the redissolution of the ELP pellet. For maximum ELP solubility, the solution should be pre-cooled, and a quick pH adjustment can help to dissolve the pellet faster. The pH may need to be readjusted during this process several times. For further improvement, the sample can be stored in the fridge at 4 °C and shaking of the sample should be avoided. To exchange salts, the described dialysis step at the end of the purification protocol should be preferred. In principle, ITC can be used to separate the peptides from the salt-containing supernatant. But depending on the previous salt concentration, residual salt can be still found in this pellet. Furthermore, dialysis can be used to concentrate the peptides needed for the experiments without losses as mentioned before.
The next crucial step is the evaporation of the chloroform/methanol mixture. During evaporation at reduced pressures, a delay in boiling can cause splatters or turbulences, which may result in large loss of glass beads. This can be prevented using either filters or tissues. The same problem arises when the desiccator is used, but aluminum foil at the opening of the round bottom flask can be used to prevent this. For the handling of the ELP-coated glass beads, it is recommended to use disposable antistatic micro-spatula, which also reduces losses and simplifies the procedure.
The swelling of the vesicles using various swelling solutions such as PBS and transcription mix is straightforward and less error prone. However, for the TX-TL system, variations from batch-to-batch of up to 10% can occur. To minimize this problem, all experiments should be performed with one batch of cell-free extract only, which can be used for up to 3,000 reactions. After rehydration, the vesicle content and outer solution are the swelling solution. A purification of the outside solution should not be performed, because this may change the osmotic pressure difference between vesicle inside and outside. The result would be an uncontrolled size change or even bursting of the vesicles. Therefore, possible reactions at the outside should be only suppressed. Depending on the reaction, this can be done, for instance, by the addition DNase I to digest present DNA or by adding antibiotics such as kanamycin, which inhibits translation.
Film rehydration from glass beads has several advantages when compared to other preparation methods. In contrast to solvent injection, emulsion phase transfer or microfluidics displacement of solvent is not required, since the initial solvent is completely evaporated, and the peptides are rehydrated in aqueous solution. Therefore, the glass beads method is particularly suitable for sensitive samples such as TX-TL, which can be significantly affected or even destroyed by residual organic solvents used in other methods. Furthermore, the protocol is straightforward, less error prone, and able to be easily scaled up. Because of the large surface to volume ratio only small amounts of glass beads are needed and allow a high degree of parallelization with high throughput.
As its main disadvantage, the glass beads method is only useful for the creation of small unilamellar vesicles, which cannot be observed via fluorescence microscopy. The described method is somewhat limited when microscopic observation of the dynamics of single vesicles is desired, which is sometimes necessary (e.g., when observing potential vesicle division processes). Furthermore, the small size of SUVs limits the encapsulation of low-concentrated components such as the peptide-encoding plasmid, which explains the relatively low expression of the peptides. The underlying encapsulation process is a Poisson process, and thus the concentration of any particular component must be at least 150 nM to guarantee an encapsulation probability of 95%. Therefore, it is quite remarkable that it is possible to observe such a large increase in vesicle size in these experiments.
The protocol presented here will enable researchers to create peptide-based vesicles from elastin-like polypeptides. It also opens up opportunities to produce artificial cell-like structures encapsulated by peptide membranes, which represent attractive alternatives to classical compartments made from fatty acids or phospholipids. Peptides are advantageous in that they can be easily expressed in a cell-free environment (for instance, in the TX-TL system used here), which allows coupling of membrane growth directly to a transcription-translation process inside the vesicle. Furthermore, ELPs can be designed and adjusted to specific physicochemical parameters such as contour length, hydrophobicity/hydrophilicity of the used deblock, and sensitivity to stimuli such as pH or ionic strength.
We gratefully acknowledge financial support through the DFG TRR 235 (Emergence of Life, project P15), the European Research Council (grant agreement no. 694410 AEDNA), and the TUM International Graduate School for Science and Engineering IGSSE (project no. 9.05). We thank E. Falgenhauer for her help with sample preparation. We thank A. Dupin and M. Schwarz-Schilling for their help with the TX-TL system and useful discussions. We thank N. B. Holland for useful discussions.
Name Company Catalog Number Comments
2xYT MP biomedicals 3012-032
3-PGA Sigma-Aldrich P8877
5PRIME Phase Lock GelTM tube VWR 733-2478
alkine-conjugated Cy3 Sigma-Aldrich 777331
ATP Sigma-Aldrich A8937
Benzamidin Carl Roth CN38.2
BL21 Rosetta 2 E. coli strain Novagen 71402
Bradford BSA Protein Assay Kit Bio-rad 500-0201
cAMP Sigma-Aldrich A9501
Carbenicillin Carl Roth 6344.2
Chloramphenicol Sigma-Aldrich C1919
Chloramphenicol Carl Roth 3886.3
Chloroform Carl Roth 4432.1
CoA Sigma-Aldrich C4282
CTP USB 14121
CuSO4 Carl Roth P024.1
DFHBI Lucerna Technologies 410
DMSO Carl Roth A994.1
DNase I NEB M0303S
DTT Sigma-Aldrich D0632
Ethanol Carl Roth 9065.2
Folinic acid Sigma-Aldrich F7878
Glass beads, acid-washed Sigma-Aldrich G1277
GTP USB 16800
HEPES Sigma-Aldrich H6147
IPTG (β-isopropyl thiogalactoside ) Sigma-Aldrich I6758
KCl Carl Roth P017.1
K-glutamate Sigma-Aldrich G1149
LB Broth Carl Roth X968.2
Lysozyme Sigma-Aldrich L6876
Methanol Carl Roth 82.2
MgCl2 Carl Roth KK36.3
Mg-glutamate Sigma-Aldrich 49605
Micro Bio-Spin Chromatography Columns Bio-Rad 732-6204
NAD Sigma-Aldrich N6522
NHS-azide linker (y-azidobutyric acid oxysuccinimide ester) Baseclick BCL-033-5
PEG-8000 Carl Roth 263.2
pH stripes Carl Roth 549.2
Phenylmethylsulfonyl fluoride Carl Roth 6367.2
Phosphate-buffered saline VWR 76180-684
Phosphoric acid Sigma-Aldrich W290017
Polyethyleneimine Sigma-Aldrich 408727
Potassium phosphate dibasic solution Sigma-Aldrich P8584
Potassium phosphate monobasic solution Sigma-Aldrich P8709
Qiagen Miniprep Kit Qiagen 27106
RNAPol reaction buffer NEB B9012
RNase inhibitor murine NEB M0314S
RNaseZap Wipes ThermoFisher AM9788
rNTP NEB N0466S
Roti-Phenol/Chloroform/Isoamyl alcohol Carlroth A156.1
RTS Amino Acid Sampler 5 Prime 2401530
Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit) Thermo-Scientific 66382
Sodium chloride Carl Roth 9265.1
Sodium hydroxide Carl Roth 8655.1
Spermidine Sigma-Aldrich 85558
Sterile-filtered (0.22 µm filter) Carl Roth XH76.1
T7 polymerase NEB M0251S
TBTA (tris(benzyltriazolylmethyl)amine) Sigma-Aldrich 678937
TCEP (tris(2-carboxyethyl)-phosphine hydrochloride) Sigma-Aldrich C4706
Tris base Fischer BP1521
tRNA (from E. coli) Roche Applied Science MRE600
UTP USB 23160
DOWNLOAD MATERIALS LIST
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MainAll NewsDefense/Security'Bibi Should've Supported Death Penalty'
'Bibi Should've Supported Death Penalty'
Ron Kehrmann, whose daughter was murdered in a suicide attack, says Netanyahu should "act against terrorism in his backyard".
Benny Toker, 16/07/15 06:42
Ron Kehrmann
Miriam Alster/Flash 90
Ron Kehrmann, whose 17-year-old daughter Tal was murdered in a suicide terror attack on a Haifa bus in 2003, on Wednesday said he regretted the fact that Prime Minister Binyamin Netanyahu chose not to support the bill proposed by Yisrael Beytenu MK Sharon Gal, which would impose the death penalty on convicted terrorist murders.
"This is an important statement of intent, even if in the end we do not execute terrorists," he told Arutz Sheva, speaking hours after the Knesset soundly rejected a preliminary reading of the bill.
"If the Prime Minister is so angry at the world about Iran, at the very least he should act against terrorism in his backyard, and the death penalty for terrorists sends an important message," added Kehrmann.
He said that the death penalty for terrorists would have prevented his daughters' murderers from roaming about abroad.
"The same group of terrorists who blew up Route 37 where my daughter was murdered, three of them are abroad, while my daughter is buried in Haifa," said Kehrmann.
"Death penalty for terrorists is the most appropriate punishment for those who shouted 'Allahu Akbar' before taking her life," he added.
Netanyahu on Sunday foiled a planned discussion of the bill by the Ministerial Committee for Legislation, announcing that he would instead establish a government commission to examine the issues pertaining to the proposed law. This led Gal to bringing the bill for a discussion in the Knesset, where it was rejected.
Gal reacted angrily to Netanyahu's political maneuvering on Sunday, announcing his rejection of the "Prime Minister's proposal to postpone the vote on the law of the death penalty against terrorists, and instead establish a committee that will in effect bury the law."
"The Prime Minister's decision is further proof that this non-nationalist government is failing to take action according to the principles of the national camp," Gal charged.
Tags:terrorism, Binyamin Netanyahu, Ron Kehrmann, death penalty


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Ancient Greece includes an interview with underwater archaeologist Faith Hentschel, a past grantee of the National Geographic Society.
The architecture of ancient Greece was produced by the ancient Greeks (Hellenes), whose culture flourished on the Greek mainland, the Aegean Islands and their colonies, for a period from about 900 BC until the 1st century AD, with the earliest remaining architectural works dating from around 600 BC.The most The most important were Arete of Cyrene, Aristoclea, Axiothea, Damo, Diotima, Elpinice, Hipparchia, Leontium, Perictyone, Theano, and Theoclea. Join us here at National Geographic Kids as we travel thousands of years back in time to discover ten fascinating facts about Ancient Greece.Navigate your way through ancient Greece, exploring unique artifacts and important individuals along the way.
From artistry to politics, ancient Greece left a considerable impression on world history.
Learn about how the ancient Greeks voted citizens—including political leaders—out of office.Explore the ruins of Greece and Turkey, on land and under sea.On Assignment project theme: Ancient Culture and Architecture.
Once these two levels are done, the 4D National Geographic App allows your child to.Ancient Greek civilization: Ancient Greek civilization, the period following Mycenaean civilization, which ended about 1200 BCE, to the death of Alexander the Great, in 323 BCE.TROY: BEYOND THE MOVIE National Geographic looks at the ancient myths and unsolved mysteries behind the epic tale.Name of National Geographic Trip: National Geographic Student Expedition to Italy and Greece.Hermes (Roman name: Mercury) was the ancient Greek god of trade, wealth, luck, fertility, animal husbandry, sleep, language, thieves, and travel.
Learn why Greek and Roman gods share so many similarities, how the alphabet got its name, and how the legacy of ancient Greece ha.
It marks the location of major and minor ancient cities, and places of interest.How did you decide to travel with National Geographic, and why did you choose your particular trip.National Geographic the Greeks: An Illustrated History (Hardcover),On the culture that brought us democracy, the Olympics, Socrates, and Alexander the Great, this Booktopia - National Geographic The Greeks, An Illustrated.
WORLD The discovery may shed light on the lost world of Odysseus and other heroes described in the epics of Homer. (Nat Geo News and New York Times) Use our resources to learn more about Odysseus and Ancient Greece.
Discover the secrets and history of ancient civilizations with this interactive jigsaw puzzle series from National Geographic including 3D replicas of all the major architectural wonders.The National Geographic publication was a childhood dream came true and the opportunity to tell the story, because after all, everything is about telling stories.Here, fishing boats cast colorful reflections in azure waters, whitewashed villages cascade down lavender-dusted slopes, and craggy headlands are punctuated by ancient ruins.
Students will investigate ancient Greek artifacts and artworks and will design museum exhibits to be placed in an Athens subway station.
Excellence as longed for by the heroes of Greece. Minoans. Legendary group of people who lived on the isle of Crete and were named after the legendary king Minos by Arthur Evans, the discoverer of the civilization. Mycenea. A fortified site in Greece.
The first layer is a historical jigsaw map, which consists of approximately 600 puzzle pieces.Learn how ancient Greeks viewed the success of the individual as the success of the community. Greece: Greece, the southernmost of the countries of the Balkan Peninsula.You will love learning with the National Geographic Ancient Greece 4D Puzzle.
Ancient Corinth was once one of the most powerful regions of the Greek empire. In 146 B.C., it was razed by the Romans and rebuilt a century later, serving as the capital of occupied Greece.The capital is Athens.The second layer contains model replica monuments and buildings, to showcase the landscape of the city in the ancient era.


Recommended for: Any maximum-effort race or street GT500 that is producing increased power levels over stock. Can easily support 1000 HP or more!
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text:
A lady walks into a fancy jewellery store. She browses around, spots a beautiful diamond bracelet and walks over to inspect it. As she bends over to look more closely she inadvertently breaks wind. Very embarrassed, she looks around nervously to see if anyone has noticed her little accident and prays that a sales person doesn't pop up right now. As she turns around, her worst nightmare materializes in the form of a salesman standing right behind her. Cool as a cucumber and displaying complete professionalism, the salesman greets the lady with, "Good day, Madam How may we help you today?" Very uncomfortably, but hoping that the salesman may not have been there at the time of her little "accident!" she asks, "Sir, what is the price of this lovely bracelet?" He answers, "Madam, if you farted just looking at it, you're going to shit when I tell you the price."
Joke has 85.26 % from 709 votes. More jokes about: customer service, dirty, money
A woman decided to have a face lift for her birthday. She spent $5000 and felt really good about the results. On her way home she stopped at a dress shop to look around. As she was leaving, she said to the sales clerk, "I hope you don't mind me asking, but how old do you think I am?" "About 35,"he replied. "I'm actually 47," the woman said, feeling really happy. After that she went into McDonald's for lunch and asked the order taker the same question. He replied, "Oh, you look about 29." "I am actually 47!" she said, feeling really good. While standing at the bus stop she asked an old man the same question. He replied, "I am 85 years old and my eyesight is going. But when I was young there was a sure way of telling a woman's age. If I put my hand up your skirt I will be able to tell your exact age." There was no one around, so the woman said, "What the hell?" and let him slip his hand up her skirt. After feeling around for a while, the old man said, "OK, You are 47." Stunned, the woman said, "That was brilliant! How did you do that?" The old man replied, "I was behind you in line at McDonald's."
Joke has 85.63 % from 2547 votes. More jokes about: age, birthday, dirty, food, money
A guy walks into a pub and sees a sign hanging over the bar that reads: CHEESEBURGER: $1.50 CHICKEN SANDWICH: $2.50 HAND JOB: $10.00 He walks up to the bar and beckons one of the three exceptionally attractive blondes serving drinks. "Can I help you?" she asks. "I was wondering," whispers the man. "Are you the one who gives the hand jobs?" "Yes," she purrs. "I am." The man replies, "Well, wash your hands. I want a cheeseburger."
Joke has 85.45 % from 1740 votes. More jokes about: blonde, dirty, food, money
A psychiatrist was conducting a group therapy session with three young mothers and their small children. "You all have obsessions," he observed. To the first mother, he said, "You are obsessed with eating. You've even named your daughter Candy." He turned to the second mom. "Your obsession is money. Again, it manifests itself in your child's name, Penny." At this point, the third mother got up, took her little boy by the hand and whispered, "Come on, Dick, let's go."
Joke has 85.38 % from 2809 votes. More jokes about: dirty, doctor, food, kids, money
A woman goes into a store to buy a fishing rod and reel. She doesn't know which one to get so she just grabs one and goes over to the register. There is a store employee standing there with dark shades on. She says, "Excuse me sir... can you tell me anything about this rod and reel?" He says , "Ma'am I'm blind but if you drop it on the counter I can tell you everything you need to know about it from the sound that it makes." She didn't believe him, but dropped it on the counter anyway. He said "That's a 6′ graphite rod with Zebco 202 reel and 10 lb. test line... It's a good all around rod and reel and it's $20.00." She says, "That's amazing that you can tell all that just by the sound of it dropping on the counter. I think it's what I'm looking for so I'll take it." He walks behind the counter to the register. And in the meantime the woman farts. At first she is embarrassed but then realizes that there is no way he could tell it was her... being blind he wouldn't know that she was the only person around. He rings up the sale and says, "That will be $25.50." She says, "But didn't you say it was $20.00?" He says, "Yes ma'am, the rod and reel is $20.00, the duck call is $3.00, and the catfish stink bait is $2.50!"
Joke has 85.27 % from 206 votes. More jokes about: customer service, fish, money, women
Harry and his wife are having hard financial times, so they decide that she'll become a hooker. She's not quite sure what to do, so Harry says, "Stand in front of that bar and pick up a guy. Tell him that you charge a hundred bucks. If you got a question, I'll be parked around the corner." She's standing there for 5 minutes when a guy pulls up and asks, "How much?" She says, "A hundred dollars." He says, "All I got is thirty". She says, "Hold on," and runs back to Harry and asks, "What can he get for thirty?" "A hand job", Harry reply. She runs back and tells the guy all he gets for thirty dollar is a hand job. He agrees. She gets in the car. He unzips his pants, and out pops this HUGE... She stares at it for a minute, and then says, "I'll be right back." She runs back to Harry, and asks, "Can you loan this guy seventy bucks?"
Joke has 85.24 % from 1814 votes. More jokes about: dirty, money, wife
A young man and his date were parked on a back road some distance from town. They were about to have sex when the girl stopped. "I really should have mentioned this earlier, but I'm actually a hooker and I charge $20 for sex." The man reluctantly paid her, and they did their thing. After a cigarette, the man just sat in the driver's seat looking out the window. "Why aren't we going anywhere?" asked the girl. "Well, I should have mentioned this before, but I'm actually a taxi driver, and the fare back to town is $25…"
Joke has 85.13 % from 1913 votes. More jokes about: dating, dirty, money, sex
A doctor was having an affair with his nurse. Shortly afterward, she told him she was pregnant. Not wanting his wife to know, he gave the nurse a sum of money and asked her to go to Italy and have the baby there. "But how will I let you know the baby is born?" she asked. He replied, "Just send me a postcard and write 'spaghetti' on the back. I'll take care of expenses." Not knowing what else to do, the nurse took the money and flew to Italy. Six months went by and then one day the doctor's wife called him at the office and explained, "Dear, you received a very strange postcard in the mail today from Europe, and I don't understand what it means." The doctor said, "Just wait until I get home and I will explain it to you." Later that evening, the doctor came home, read the postcard, fell to the floor with a heart attack. Paramedics rushed him to the ER. The lead medic stayed back to comfort the wife. He asked what trauma had precipitated the cardiac arrest. So the wife picked up the card and read, "'Spaghetti, Spaghetti, Spaghetti, Spaghetti - Two with sausage and meatballs, two without.'"
Joke has 82.86 % from 564 votes. More jokes about: dirty, doctor, money, nurse, travel
A man and a woman meet in an elevator. "Where are you heading today?" the man asks. "I'm going down to give blood." "How much do you get paid for giving blood?" "About $20." "Wow," says the man, "I'm going up to donate sperm, and the sperm bank pays $100." The woman angrily gets off the elevator. The next day, the man and woman meet in the elevator again. "Fancy meeting you again. Where you off to today?" "Sperm bank," she says with her mouth full.
Joke has 82.47 % from 263 votes. More jokes about: dirty, money
A guy walks into a bar and sits on a stool. In front of him he see's a big jar full of change and a little card that reads: "Hello, if you would like to win all of this money you have to make the horse at the end of the bar laugh. COST $5" So, he puts in five dollars and takes the horse into the bathroom. Two minutes later they come out and the horse is laughing so hard that he pissed on the floor. So the guy takes the money and leaves. The next day the same guy walks in the bar again and see's the horse and the jar, this time it says: "You can win all of this if you make the horse cry. COST $10" So he puts in 10 dollars and takes the horse into the bathroom. Four minutes later they come out and the horse is crying like no body ever had. So the guy takes the jar but before he could leave the bartender asks "How did you do that?" The guy says "The first time I told him my dick was bigger than his and the second time I showed him!"
Joke has 82.15 % from 156 votes. More jokes about: bar, dirty, horse, money
I was in Venice Beach in January and there was a homeless man with a sign that said "1 dollar for dirty joke." Seemed like a good investment to me so I gladly handed over a dollar. Homeless man: "Alright sir whats your name?" Me: "John" Homeless man: "So Johny, there is black rooster alright? How many legs does that chicken have." Me: "Two?" Homeless man: "Right, now how many wings this black rooster got?" Me: "Two?" Homeless man: "Right, now how many eyes this black rooster got?" Me: "Two?" Homeless man: "Right again, now there is this white cat walking around how many hairs are on that white cat?" Me: "I don't know? A lot?" Homeless man: "Well Johny, why do you know so much about black cock and not enough about white pussy."
Joke has 81.96 % from 305 votes. More jokes about: animal, dirty, life, money


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Home » China » Men Seeking Vasectomies in China Denied by Government as Birth Rate Continues to Decline
Men Seeking Vasectomies in China Denied by Government as Birth Rate Continues to Decline
Children in China sit on the roof with their legs dangling through the handrails. (Image: TED) (Image: Secret China)
When first-time mother Zhao Zihuan and her husband decided to have a vasectomy after welcoming a son last year, they were denied at a hospital in Jinan, the capital of China's Shandong Province. Zhao said she had previously suffered two miscarriages before giving birth to the boy.
Frustrated by the country's rising cost of living and steep child care expenses, the 32-year-old and her husband decided that they did not want any more children. Yet when they inquired about a vasectomy at two local hospitals in April this year, they were surprised when medical staff turned them away. Doctors told Zhao's husband that the surgery was no longer allowed under the country's new "family-planning rules."
"I was frightened and angry at the same time," Zhao, who works in publishing, told the Washington Post. "What if we accidentally get pregnant? We won't have a choice but to have the child. The burden will be too great."
In October 2015, the Chinese Communist Party (CCP) rolled out the slogan "all families should have two children." The two-child policy was promulgated in 2016, and this May, was further broadened to allow three children per couple. As the government loosened family-planning rules and encouraged more births, the number of vasectomies performed fell from 149,432 in 2015 to 4,742 in 2019, according to official data.
When the one-child policy was implemented in 1980, for over three decades, men and women were forced to undergo sterilization in an effort to control the country's booming population. The policy was so stringenly upheld that many women were forced to have late-term abortions if they were found to be "illegaly" pregnant. Chinese authorities boasted that they prevented approximately 400 million births.
Now, the Chinese government is desperately trying to reverse a plummeting birth rate as more couples opt to have only one child or remain childless altogether.
China Is Ending Its Population Controls, But Many No Longer Want to Have Children
Doctors in fear of punishment refuse to perform vasectomies altogether
A director of a hospital in central China's Hubei Province told reporters that, "Even though it's a rather simple surgery in theory, public hospitals will almost always turn patients away because we are aware of the risks involved in doing something that's not explicitly okayed by the government," the source who chose to use the name of Yang, in fear of repercussion, told the Washington Post on Dec. 9. "The fundamental policy is that China needs more childbirths," Yang added.
Zhou Muyun, a 23-year-old copywriter from Guangzhou said he and his girlfriend Han Feifei were choosing to maintain a "DINK" lifestyle, which means: double income, no kids. Zhou said he unsuccessfully tried to get a vasectomy this year but was turned away by two local hospitals who said the procedure was no longer allowed.
Doctors told Zhao that he was too young and would regret the decision later on. "Having a child or not is our choice to make and our fundamental right. We don't need anyone to tell us how to live," he said.
Similarly, another man who used the pseudonym of Jiang in fear of repercussions for criticizing government policy said he visited six hospitals in his home province of Fujian before finally finding a hospital in Chengdu that would perform the surgery. Jiang said he traveled more than 1,200 miles in order to receive the vasectomy and said the doctor there apprehensively agreed to the surgery.
Jiang, 30, works in customer service at an Internet company and said he had the surgery done in March. He also posted the clinic's details in an online forum hoping to help other men searching for a facility that would perform the surgery. However, Jiang said he heard shortly after from another user that the hospital he went to had since stopped offering the surgery.
"I felt like I had finally gotten rid of this huge burden," Jiang said. "Those around me who are married and have kids have nothing that makes me envious."
China Is Considering a Move That Effectively Scraps Population Control
China's Delayed Census Points to Severe Population Decline
Reducing medically unnecessary abortions
In September, the Chinese government also announced plans to reduce the number of abortions performed in the country, prompting concerns from some women over the prospect of reduced access to the procedure.
Health authorities in Beijing mentioned the goal was to decrease "non-medical abortions" in a policy report on women's health released on Sep. 8. Authorities have yet to clarify whether they plan to reduce abortions via an increase in the use and access of contraceptions, or if access to the procedure will be restricted altogether.


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Q: Using iloc to replace a range of rows in the dataframe is not saved when assigning that index range to the original dataframe I have a dataframe with the following structure:
date    position    quantity    Unit
0   2015-01-01 00:00:00 1   720 MAW

Based on the conditions I set I wish to reorder dates during the daylight savings time in October (1 hour gets added here) based on the "position" column. So I subseted the dataframe and got the index values back:
indexData = data[(data.date.dt.hour >= 2) & (data.date.dt.hour < 3) & (data.date.dt.day == 25) & (data.date.dt.month == 10) & (data.date.dt.year == 2015)].index

So when I do this I can see that the range is ordered correctly (but not yet replaced in the dataframe):
data.iloc[indexData[0]:(indexData[-1])+1].sort_values("position")

date    position    quantity    Unit
28521   2015-10-25 02:00:00 25305   420 MAW
28523   2015-10-25 02:15:00 25306   418 MAW
28524   2015-10-25 02:30:00 25307   415 MAW
28526   2015-10-25 02:45:00 25308   415 MAW
28520   2015-10-25 02:00:00 25309   410 MAW
28522   2015-10-25 02:15:00 25310   407 MAW
28525   2015-10-25 02:30:00 25311   399 MAW
28527   2015-10-25 02:45:00 25312   378 MAW

BUT when I try to assign this range to the original data drame it does not change:
data.iloc[indexData[0]:(indexData[-1])+1] = data.iloc[indexData[0]:(indexData[-1])+1].sort_values("position")

# here I check the range if updated
data[(data.date.dt.hour >= 2) & (data.date.dt.hour < 3) & (data.date.dt.day == 25) & (data.date.dt.month == 10) & (data.date.dt.year == 2015)]

date    position    quantity    Unit
28520   2015-10-25 02:00:00 25309   410 MAW
28521   2015-10-25 02:00:00 25305   420 MAW
28522   2015-10-25 02:15:00 25310   407 MAW
28523   2015-10-25 02:15:00 25306   418 MAW
28524   2015-10-25 02:30:00 25307   415 MAW
28525   2015-10-25 02:30:00 25311   399 MAW
28526   2015-10-25 02:45:00 25308   415 MAW
28527   2015-10-25 02:45:00 25312   378 MAW

Any ideas where I am going wrong or should try?

A: Let's assume we have data like the following and we want to sort by position rows only for October (basically we want to swap lines 4 and 5):
date    position    quantity    Unit
0   2015-09-25 02:15:00 25310   407 MAW
1   2015-09-25 02:00:00 25309   410 MAW
2   2015-09-25 02:30:00 25311   399 MAW
3   2015-09-25 02:45:00 25312   378 MAW
4   2015-10-25 02:15:00 25306   418 MAW
5   2015-10-25 02:00:00 25305   420 MAW
6   2015-10-25 02:30:00 25307   415 MAW
7   2015-10-25 02:45:00 25308   415 MAW

Choosing October with some other filters you applied (basically using your logic here):
subset_index = data[(data.date.dt.hour >= 2) & (data.date.dt.hour < 3) & (data.date.dt.day == 25) & (data.date.dt.month == 10) & (data.date.dt.year == 2015)].index.values

subset_index is array([4, 5, 6, 7]) - these are rows we want to sort.
Having extracted index values to sort, you have two options:
Option 1: keep the index and reassign values in the right order(right index).
Just assign the values(numpy array) in the correct order to a dataframe subset:
data.loc[subset_index] = data.loc[subset_index].sort_values("position").values

The result is
date    position    quantity    Unit
0   2015-09-25 02:15:00 25310   407 MAW
1   2015-09-25 02:00:00 25309   410 MAW
2   2015-09-25 02:30:00 25311   399 MAW
3   2015-09-25 02:45:00 25312   378 MAW
4   2015-10-25 02:00:00 25305   420 MAW
5   2015-10-25 02:15:00 25306   418 MAW
6   2015-10-25 02:30:00 25307   415 MAW
7   2015-10-25 02:45:00 25308   415 MAW

As you can see only October rows have been sorted by position, September rows 0 and 1 were left unsorted.
Option 2: sort the index in the right order and reindex the dataframe.
Create a new sorted index order for this subset
reordered_subset_index = data.loc[subset_index].sort_values("position").index.values

reordered_subset_index is array([5, 4, 6, 7]). Now we need to create a correct index order for the whole dataframe:
new_index = data.index.values
new_index[subset_index] = new_index[reordered_subset_index]

new_index is array([0, 1, 2, 3, 5, 4, 6, 7]). This index defines the correct order of rows in our initial dataframe.
So now we can reindex our dataframe and just sort it by our new index:
data.reindex(new_index).sort_index()

The result is the same as in Option 1.

redpajama_set_name: RedPajamaStackExchange


text:
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<androidx.constraintlayout.widget.ConstraintLayout xmlns:android="http://schemas.android.com/apk/res/android"
    xmlns:app="http://schemas.android.com/apk/res-auto"
    android:id="@+id/class_dialog"
    android:layout_width="match_parent"
    android:layout_height="wrap_content"
    android:gravity="center"
    android:orientation="vertical">

    <TextView
        android:id="@+id/class_dialog_title"
        android:layout_width="wrap_content"
        android:layout_height="wrap_content"
        android:layout_gravity="center"
        android:layout_margin="15dp"
        android:text="@string/choose"
        android:textColor="#000000"
        android:textSize="22sp"
        app:layout_constraintStart_toEndOf="parent"
        app:layout_constraintStart_toStartOf="parent"
        app:layout_constraintTop_toTopOf="parent" />

    <TextView
        android:id="@+id/class_class_tv"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:text="@string/choose_class"
        android:textAlignment="center"
        android:textSize="25sp"
        app:layout_constraintTop_toBottomOf="@id/class_dialog_title" />

    <Spinner
        android:id="@+id/class_class_spinner"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:textAlignment="center"
        app:layout_constraintTop_toBottomOf="@id/class_class_tv" />

    <TextView
        android:id="@+id/class_country_tv"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:text="@string/choose_country"
        android:textAlignment="center"
        android:textSize="25sp"
        app:layout_constraintTop_toBottomOf="@id/class_class_spinner" />

    <Spinner
        android:id="@+id/class_country_spinner"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:textAlignment="center"
        app:layout_constraintTop_toBottomOf="@id/class_country_tv" />

    <TextView
        android:id="@+id/class_boat_tv"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:text="@string/choose_boat"
        android:textAlignment="center"
        android:textSize="25sp"
        app:layout_constraintTop_toBottomOf="@id/class_country_spinner" />

    <Spinner
        android:id="@+id/class_boat_spinner"
        android:layout_width="0dp"
        android:layout_height="wrap_content"
        android:gravity="center"
        android:textAlignment="center"
        app:layout_constraintEnd_toStartOf="@+id/class_add_yacht_button"
        app:layout_constraintStart_toStartOf="parent"
        app:layout_constraintTop_toBottomOf="@id/class_boat_tv" />

    <ImageButton
        android:id="@+id/class_add_yacht_button"
        android:layout_width="wrap_content"
        android:layout_height="wrap_content"
        android:src="@drawable/ic_add_circle_black_24dp"
        android:background="@null"
        app:layout_constraintBottom_toBottomOf="@id/class_boat_spinner"
        app:layout_constraintEnd_toEndOf="parent"
        app:layout_constraintTop_toTopOf="@id/class_boat_spinner" />

    <ListView
        android:id="@+id/class_yacht_list"
        android:layout_width="0dp"
        android:layout_height="150dp"
        app:layout_constraintBottom_toTopOf="@id/distance_finish_b"
        app:layout_constraintEnd_toEndOf="parent"
        app:layout_constraintStart_toStartOf="parent"
        app:layout_constraintTop_toBottomOf="@id/class_boat_spinner" />

    <Button
        android:id="@+id/distance_finish_b"
        android:layout_width="match_parent"
        android:layout_height="wrap_content"
        android:layout_gravity="center_horizontal"
        android:background="@color/cmark_blue_light"
        android:gravity="center"
        android:text="@string/done"
        android:textAlignment="center"
        android:textColor="@color/cmark_orange_lighter"
        android:textSize="25sp"
        app:layout_constraintBottom_toBottomOf="parent"
        app:layout_constraintEnd_toEndOf="parent"
        app:layout_constraintStart_toStartOf="parent" />
</androidx.constraintlayout.widget.ConstraintLayout>

redpajama_set_name: RedPajamaGithub


text:
Q: How to deal with password security when sending credentials to a REST authentication endpoint? My question arises from the assumption that it's best practice not to handle passwords as String in a JVM environment. Android actually handles text inside EditTexts as array of char and provides a getChars method to get such array.
Given that I can handle a password this way, what happens when I have to serialize it to put it in a request body? Should I receive it on my server endpoint as char[]? Even with this option, client-side it will be eventually serialized to something like ['p','w','d'] which is again, a String.
To try to figure this out, I observed outbound traffic from a device to Amazon's authentication endpoints and there's actually my password in plain sight in a request body; so my question may even turn into: how big of a concern is this whole passwords vs Strings matter (at least in Android)?
Edit: an explanation of why I have this concern.

A: You can convert it using hash function while sending it on your API.


text:
Home Charles Barkley Charles Barkley: Lakers Are Behind Spurs And Not Contenders Despite LeBron James Signing
Charles Barkley: Lakers Are Behind Spurs And Not Contenders Despite LeBron James Signing
Lakers News: Michael Beasley Growing More Comfortable In New Setting
Dan Duangdao
Throughout the 2018-19 NBA season, there will be plenty of critiques about LeBron James and the roster the Los Angeles Lakers constructed around him.
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With James accompanied by veterans and a developing young core, the Lakers are projected to finish anywhere from the second seed to not even making the playoffs in the Western Conference.
Whether it is fair or not, having the best player in the league today join one of the greatest franchises will result in championship expectations in Year 1.
As for Charles Barkley, he does not believe the Lakers are even contenders despite signing James, via ESPN:
"I think obviously he's the greatest basketball player in the world today, but the problem is that team is not very good. The Lakers are not a contender. They're probably a five or sixth seed in the West, but they're going to be getting a lot of attention. … In my opinion, they're not even as good as the San Antonio Spurs. I think the Spurs, with DeMar DeRozan and LaMarcus healthy all year, the Spurs are better. I think Oklahoma City is probably better now that they got rid of Carmelo. And the Rockets are better. … I think the Rockets are the second-best team in the West, and then I think it's a toss up between Oklahoma City and the Spurs. Listen, no disrespect to the Lakers, but I'm not even sure they're better than the Portland Trail Blazers. I mean, LeBron is great, but it's going to take those young guys at least a year to adjust to playing with LeBron. Those guys have all played with the ball their entire career. LeBron is a scorer who has to have the ball. It's not just going to click right away. So I think the Lakers are going to have some growing pains, and by the time they get through the season, they're going to be the fifth or sixth seed."
James has shown he can win regular season games by himself but it will take plenty of support if the Lakers are to make a deep playoff run.
With James signing a four-year, $154 million deal, there is an understanding it will take time for the Lakers to truly challenge the Golden State Warriors. As part of their two-year window, president of basketball operations Magic Johnson and general manager Rob Pelinka have already created $38 million in salary cap space for 2019 free agency.
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text:
Electrical Brain Stimulation Releases Opiate-Like Painkiller
Researchers use new approach to treat severe facial pain (Jan. 2)
Researchers at the University of Michigan have used electricity on certain regions in the brain of a patient with chronic, severe facial pain to release an opiate-like substance that's considered one of the body's most powerful painkillers.
The findings expand on previous work in which the investigators delivered electricity through sensors on the skulls of patients with chronic migraine, and found a decrease in the intensity and pain of their headache attacks. However, the researchers couldn't completely explain how that was accomplished.
The new findings — published in Frontiers in Psychiatry — help explain what happens in the brain that decreases pain during the brief sessions of electricity.
The researchers administered an intravenous radiotracer that reached important brain areas in a patient with trigeminal neuropathic pain (TNP) — a type of chronic, severe facial pain. They applied the electrodes and electrically stimulated the skull directly above the motor cortex of the patient for 20 minutes during a positron emission tomography (PET) scan. This technique is known as transcranial direct current stimulation (tDCS).
The radiotracer was designed to measure indirectly the local brain release of mu-opioid — a natural substance that alters pain perception. For an opiate drug to function, it must bind to this receptor.
"This is arguably the main resource in the brain to reduce pain," said Dr. Alexandre DaSilva. "We're stimulating the release of our [body's] own resources to provide analgesia. Instead of giving more pharmaceutical opiates, we are directly targeting and activating the same areas in the brain on which they work. [Therefore,] we can increase the power of this pain-killing effect and even decrease the use of opiates in general, and consequently avoid their side effects, including addiction."
Most pharmaceutical opiates, especially morphine, target mu-opioid receptors in the brain, DaSilva said.
Electroconvulsive therapy (ECT), which is used to treat depression and other psychiatric disorders, uses amperage in the brain ranging from 200 to 1,600 milliamperes (mA). The tDCS protocol used in the new study delivered 2 mA — considerably lower than ECT.
One session of treatment immediately improved the patient's threshold for cold pain by 36%, but not the patient's clinical TNP pain. This suggests that repetitive electrical stimulation over several sessions is required to have a lasting effect on clinical pain, as shown in the previous migraine study, DaSilva said.
Source: University of Michigan; January 2, 2013.
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Fast-acting insulin aspart may simplify mealtime dosing


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