#sigma 30 mm

[ozgurhayalim]

"Her şeyi zamana bıraktık, zamanımız var mı bilmeden..."

 30 sn - F/9 - 27 mm - Sigma  1/4 sn - F/9 - 27 mm - Sigma

 Sonbahardan başlayalım....

MustafaKemalPaşa, Bursa

[thelukashoell]

Berliner Dom - Berlin 22 Germany . . . . . . . . 📷 Sony Alpha 6000 - 30 mm 2.8 Sigma #sonyalpha #madewithlightroom . #streetsofberlin #berlin #berlinmitte #bestofberlin #instaberlin #unlimitedberlin #ihavethisthingwithberlin #officialfanofberlin . Attachments: #tones_of_berlin #berlin_de #visit_berlin #igers_of_berlin #best_of_berlin #dasechteberlin #berlinstravel #wonderlustberlin #srs_berlin #nabendberlin #urbanromantix #deceptive_cities @city__features #city_features #raw_germany (at Berlin, Germany) https://www.instagram.com/p/Cl4bSLhjbvt/?igshid=NGJjMDIxMWI=

[anonymouscheeses]

THANK UU FOR THE TAG

1. Well. Pablo and Cheese. Some kid called me Pablo while i was walking home and it stuck ever since. And i gave myself the name Cheese.

2. April 22nd 😎😎

3. Fuckn 2 yrs 😒

4. Ummm im trying to get baxk into resding so for now, Warrior cats. FOR NOW.

5. Nothn cuz im so cool 😜🙏 (HAIR. grr)

6. I am also lesbian.

7. Im basic asf sooo: Taylor Swift, Billie Eillish, Margot Robbie(HOW SPELL HER NAME??), Kylie Cantrall, and Jenna Ortega.

8. Um, Police Officer ig, i wna be one the good ones but ehhh i js like the idea of it.

9. Mmm. Being alive?? Like. Iuno i havent done mch. But uh, i did save a kid one time from bein ran overed 😜

10. I'm christian. Ok maybe a few of yall would believe it, but others... its complicated 🤧 I praise the lord despite the trials 🙏💜

11. My high was when i got high /j uh. My highest was not wanting to burn alive 😜

12. Mm.. not france. Umm. Maybe Oklahoma? I have family there and i wanna get back in touch with my Native roots!

13. I dont. Js kidding, i take pills for my anxiety!

14. Tiktok ana ibidispaint save my life 😫

15. Alive.

16. Girl, im mid and thats ok. But anyway, maybe my alpha sigma rizz can take a lady by surprise heh 😈🙏

17. Art, ofc

18. Um nothin, cz im cool lik tat (getting up in the mornings. Im currently in my bed still supposed to get ready for the day but i cant.)

19. That i was Bi, ok it wasnt a lie at the time, but now looking back im embarrassed how i thought i was attracted to men 🤮

20. Dogs lick your skin because it has a natural saltiness. Especially when you sweat, its like a salty feast. They just dont bite nd eat you because they dont want to hurt you.

21. Umm.... @jasongotdrip ofc...

22. My plushie. Ok look, im way too old for toys, but fuck u im keepign my large ass brown bunny that STILL smells like chocolate even after years.

23. The one i have with @sweetoval WHO NEVER GETS ON TUMBLR TO SEE MY TAGS. but like whatever 😒😒

24. Thr day i had my quinceñeara. God. Everything changed that day, and i hinestly didnt know growing up felt this shitty 🤮

25. Yep! I used to play volleyball, only cuz soccer wasnt available. But omg thay was the best day of my life because Volleyball will forever be apart of my childhood, i always got in the top 3 of every game. I think once or twice even 1st? I dont remember but SNIFF SNUFF IM EMOTIONAL. AAAAAAA.

26. Sleepy. Tired. Exhausted. Sad. EMOTIONAL. AAAA.

27. Both kinda. Like i can wake up early if i tried but nahh

28. No. Im kinda suspecting im demiromantic, but even then. Nahh.

29. "Good luck, Babe." Not cz i have emotional attachment, but because its such a good line to use on women who say "my les/bi phase was so embarrassing" STFU.

30. Um. Not being literal bones. Umm. Iuno, being happy.

31. Billie eillish, Renée rappe, Chappel Roan

32. Dates. Schedules.

33. When people eat food loudly. I cant escape tho.

34. BABIES AND LOVING PARENTS. just last night i had cried because this woman was so happy and was teaching her daughter how to walk ☹️

35. That im mean. PLEASE. IT KEEPS HAPPENING. IM SORRY. DONT THINK I AM JS CUZ I CALLED MY FRIEND A SLUR I CAN RECLAIM. PLEASEE 😭😭🙏🙏

36. Do u like frsh griled hman fingers 🤤

Have a gud day. Im not gna tag anyone cz i gota do my morning routine. Splash water on my face and tell myself motivational quotes in the mirror.

Question Game

Are we tired of these yet?

What is your nickname?

When is your birthday?

What was your longest relationship?

What is your favorite book?

What is something you're insecure about?

5 Male celebrity crushes

5 Female celebrity crushes

What is your dream job?

What do you consider your biggest accomplishment?

What is a fact about you that nobody would believe?

What were your highs and lows for this last month?

Where is somewhere you'd like to visit?

How do you de-stress?

What are your favorite apps besides tumblr?

Describe yourself in one sentence.

What do you think makes you attractive?

What is something you're really good at?

What is something you're really bad at?

A time that you told a lie.

What's a totally random and useless fact that you know?

Who knows you the best?

What is your most prized possession?

What is your longest friendship?

When did you first feel like an adult?

Do you/ Have you played any sports?

How are you feeling right now?

Are you an early bird or a night owl?

Do you believe in love at first sight?

Favorite song lyrics right now?

What does self care look like for you?

Describe yourself with 3 singers.

What makes you nervous?

What’s a pet peeve you have?

What will always make you cry?

What kind of first impression do you think you make on people?

Free Pass! (Ask any question you want that's not on the list)

[jcmarchi]

Archer Demonstrated in Ukraine - How Many of These Howitzers do Ukrainians Have? - Technology Org

New Post has been published on https://thedigitalinsider.com/archer-demonstrated-in-ukraine-how-many-of-these-howitzers-do-ukrainians-have-technology-org/

Archer Demonstrated in Ukraine - How Many of These Howitzers do Ukrainians Have? - Technology Org

The war in Ukraine had many artillery duels and many more are yet to come. Howitzers are extremely important for defence and offence. The defenders of Ukraine are using a wide variety of different systems, slowly transferring entirely to the NATO 155 mm standard. Now for the first time, the Swedish Archer Artillery System was shown to be in Ukraine.

Archer Artillery System has been in service since 2016. Ukraine is the third active operator of this system. Image credit: Stridsvagn122 via Wikimedia (CC BY-SA 4.0)

The Archer Artillery System, also known as Archer – FH77BW L52, or Artillerisystem 08, is a highly mobile 155 mm self-propelled howitzer, developed in 1995-2009 by the Swedish defence company Bofors and manufactured by BAE Systems AB. It is a large turret with a long 155 mm gun, mounted on a 6×6 wheeled platform.

The barrel of the howitzer is raised automatically once the gun comes to a position. Then the crew of 4, with the help of the autoloader, can fire a quick burst of shots. Once the firing is completed, the Archer drives away before the counter-battery fire has any chance of finding it.

The speed and accuracy of the Archer are amazing. It has a computerized fire control system with a Sigma 30 navigation and pointing system. Thanks to it, it can be parked in any position and the fun will still be easily turned towards the target.

In a rapid-fire mode, the Archer can fire 8 rounds per minute. The range is about what you’d expect from NATO 155 mm artillery – around 30 km with conventional rounds, but up to 50 km with the smart Excalibur munition.

On 16 March 2023, the Swedish government announced that it would send 8 Archer artillery systems to the Armed Forces of Ukraine. This is a significant donation, because just around 50 of these systems were ever made. The Archer entered service with the Swedish Army in 2016.

The British Army also has a few Archer howitzers, which came in October to replace the AS-90s donated to Ukraine. Now the Swedish Defense Minister Pål Jonson has confirmed that 8 Archer self-propelled howitzers have been delivered to Ukraine, making it the third active operator of this system.

The defenders of Ukraine filmed a short thank-you video:

The Armed Forces of Ukraine showed the Swedish supplied Archer artillery installation for the first time. It is in use by the 45th separate artillery brigade. pic.twitter.com/8tdvsyBoJ2

— NOELREPORTS 🇪🇺 🇺🇦 (@NOELreports) November 3, 2023

This is the first time the Archer Artillery System has been spotted in Ukraine.

Minister Pål Jonson also revealed that 4 more Archers will be delivered to Ukraine in the near future. Ukrainian artillery crews have been preparing for this for a few months so it is likely that these howitzers will start smashing Russian positions very soon.

The number of different self-propelled howitzers that Ukraine is now operating is unbelievable. From the Polish Krab and the German PzH 2000, to the British AS-90 and the Swedish Archer – they have an unbelievable number of different types. One just needs to hope that all of this artillery is easy to deal with and is effective at the front lines. 

Written by Povilas M.

Sources: NOELreports Twitter, Wikipedia

You can offer your link to a page which is relevant to the topic of this post.

[georgevp]

Personal portraits, Event Photo 2

First shot taken with TTartisan 50 mm 1.2 lens that I got in January. The camera was a bit difficult to hold with the weight of the flash, and it’s diffuser, so only used it for the first model. Next and last taken with the Sigma 30 mm 1.4 DC DN Contemporary. Photos a little darker because not really sure if they might be too bright like the last shots (they did seem brighter on my older MacBook…

[suryatchandra]

Sans titre par Mariusz Lemiecha Via Flickr : cargocollective ello instagram

[festivesmartchoice]

Image from Amazon.in

Sigma 30 mm f/1.4 DC DN Contemporary Lens for Sony E-Mount

[kon-igi]

in cosa è diverso il vaccino Johnson rispetto agli altri, per non necessitare di una seconda dose?

C’è bisogno di un piccolo preambolo.

La differenza tra la SCIENZA e la RELIGIONE è che alla prima ci credi perché ne comprendi i meccanismi mentre con la seconda sei costretto a farlo in modo fideistico.

Quindi decidi tu i vaccini in quale categoria ricadano.

(mi piacerebbe mettere il pulsante ‘salta tutto il testo citato’ ma non esiste e quindi la ‘spiegazione’ è da scrollare fino in fondo) 

Vaccine design

The S protein of SARS-CoV-2 corresponding to positions 21,536–25,384 in SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank accession number: MN908947) was codon-optimized for expression in human cell lines. S designs were either based on the native SP, replacement of the native SP by the tissue plasminogen activator (tPA) SP or a SP upstream of the native signal, resulting in a sequence encoding SARS-CoV-2 amino acids 2-1273 and tPA leader as described for MERS-CoV spike protein13,39. In some constructs the furin cleavage site was abolished by amino acid changes R682S and R685G, or proline substitutions (K986P, V987P) were introduced.

Protein expression and purification

A plasmid encoding the SARS-CoV2 S-2P protein12 with the wt SP and with the wt SP replaced by the tPA SP and with a C-tag for purification were codon-optimized and synthesized at GenScript. The constructs were cloned into pCDNA2004. The expression platform used was the Expi293F cells. The cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 6 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The supernatant was subsequently sterile filtered using a 0.22 µm vacuum filter and stored at 4 °C until use. The C-tagged SARS-CoV2 S trimers were purified using a two-step purification protocol by 5 mL CaptureSelect™ C-tag Affinity Matrix (ThermoFisher Scientific). Proteins were further purified by size-exclusion chromatography using a HiLoad Superdex 200 16/600column (GE Healthcare).

Antibodies and reagents

SAD-S35 was purchased from ACROBiosystems (cat# SAD-S35-100ug). ACE2-Fc (ACE2) was made according to Liu et al. 201840. For CR3022, CR301541, CR3046, and S30934 the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody. The antibodies were made by transfecting the IgG1 expression construct using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher) in Expi293F (ThermoFisher) cells according to the manufacturer specifications. Antibodies were purified from serum-free culture supernatants using mAb Select SuRe resin (GE Healthcare) followed by rapid desalting using a HiPrep 26/10 Desalting column (GE Healthcare). The final formulation buffer was 20 mM NaAc, 75 mM NaCl, 5% Sucrose pH 5.5. Convalescent serum (SER-0743-00001) was obtained from Sanquin, the Netherlands.

Cell-based ELISA

HEK293 cells were seeded at 2 × 105 cells/mL in appropriate medium in a flat-bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 h, transfection of the cells was performed with 300 ng DNA for each well and the plate was incubated for 48 h at 37 °C in 5% CO2. Two days post transfection, cells were washed with 100 μl/well of blocking buffer containing 1% (w/v) BSA (Sigma), 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM Tris pH 8.0 in 1× PBS (GIBCO). After washing, nonspecific binding was blocked, using 100 μl/well of blocking solution for 20 min at 4 °C. Subsequently, cells were incubated in 50 μl/well blocking buffer containing primary antibodies ACE2-Fc (5 µg/mL, 1 µg/mL and 0.2 µg/mL)(1 µg/mL for radar plot), S309 (1 µg/mL), SAD-S35 (1 µg/mL), CR3015 (5 µg/mL), CR3022 (5 µg/mL), CR3046 (5 µg/mL), and convalescent serum (1:400) for 1 hr at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well of washing buffer containing 1 mM MgCl2, 1.8 mM CaCl2 in 1× PBS and then incubated with 100 μl/well of the blocking buffer for 5 min at 4 °C. After blocking, the cells were incubated with 50 μl/well of secondary antibodies HRP conjugated Mouse Anti-Human IgG (Jackson, 1:2500) or HRP Conjugated goat anti-mouse IgG (Jackson, 1:2500) then incubated 40 min at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well washing buffer. 30 μl/well of BM Chemiluminescence ELISA substrate (Roche, 1:50) was added to the plate, and the luminosity was immediately measured using the Ensight Plate Reader.

Flow cytometry

MRC-5 cells (0.4 × 106 cells/well) were seeded in 6-well plates and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes at 5000 vp/cell for 48 h. Harvested cells were washed with PBS and stained with LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Invitrogen). For SARS-CoV-2 surface staining, cells were washed twice with PBS and then incubated with ACE2-Fc (1 μg/ml), convalescent serum (1:400), and mAbs S309, SAD-S35, CR3022, CR3015 and CR3046 (1 μg/ml) for 30 min in FACS buffer (PBS with 0.5% BSA). Cells were washed twice with FACS buffer and stained with goat anti-Human IgG Alexa Fluor 647 (Invitrogen) or goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min in FACS buffer. Cells were washed twice with FACS buffer and fixed with 1× BD CellFIX (BD Biosciences) for 15 min. Cells were washed once with FACS buffer and resuspended in FACS buffer before analysis on a FACS Canto instrument (BD Biosciences). Data were analyzed with FlowJoTM Software (Becton, Dickinson and Company) and plotted as median fluorescence intensity of the MRC-5 single, live cell population (Fig. S6).

BioLayer interferometry (BLI)

Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 μm vacuum filter and used as the analyte in the experiment. A solution of ACE2-Fc at a concentration of 10 μg/mL was used to immobilize the ligand on anti-hIgG (AHC) sensors (FortéBio cat#18-5060) in 1× kinetics buffer (FortéBio cat#18-1092) in 96-well black flat bottom polypropylene microplates (FortéBio cat#3694). The experiment was performed on an Octet HTX instrument (Pall-FortéBio) at 30 °C with a shaking speed of 1000 rpm. Activation was 60 s, immobilization of antibodies 600 s, followed by washing for 300 s and then binding the S proteins for 1200 s. Data analysis was performed using the FortéBio Data Analysis 8.1 software (FortéBio). Binding levels were plotted as nm shifts at 1200 s after S protein binding.

Differential scanning fluorometry (DSF)

0.2 mg of purified protein in 50 µl PBS pH 7.4 (Gibco) was mixed with 15 µl of 20 times diluted SYPRO orange fluorescent dye (5000 x stock, Invitrogen S6650) in a 96-well optical qPCR plate. A negative control sample containing the dye only was used for reference subtraction. The measurement was performed in a qPCR instrument (Applied Biosystems ViiA 7) using a temperature ramp from 25–95 °C with a rate of 0.015 °C per second. Data were retrieved continuously. The negative first derivative was plotted as a function of temperature. The melting temperature corresponds to the lowest point in the curve.

Mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine the N-terminus on either purified soluble protein or on a pull-down of the full-length S protein from cell membranes using either Mab CR3022 or ACE2-Fc. The purified soluble proteins were subjected to direct digest, whereas the membrane-bound spike protein samples were either subjected to direct digest or in gel digestion. The experiments were performed on C18 nRP-LC connected to a ESI-Q-orbitrap mass spectrometer. Data processing for the different proteins was performed using Biopharma Finder 3.1 (Thermo Scientific). Each data file was compared to its corresponding amino acid sequence. Peptides were filtered by mass accuracy, confidence and structural resolution and reported. In order to pick up low abundant peptides, the thresholds for the MS noise level and the S/N were lowered 100-fold in comparison to the normal processing method.

Cell–cell fusion assay

Cell–cell fusion assays were performed to ascertain the relative fusogenicity of the different S protein variants. For fluorescent readout, full-length wild-type SARS-CoV-2 S protein and variants thereof, human ACE2, human TMPRSS2 and GFP were co-expressed from pcDNA2004 plasmids in HEK293 cells using Trans-IT transfection reagent according to the manufacturer’s instructions. Transfections were performed on 70% confluent cell monolayers in 6-well plates. Transfected cells were incubated at 37 °C and 10% CO2 for 24 h before imaging on an EVOS cell imaging system (Thermofisher). Overlays between brightfield and GFP channels were made in ImageJ.

The fluorescent fusion assay was adapted to allow quantitative measurement of cell–cell fusion by leveraging the NanoBiT complementation system (Promega). Donor HEK293 cells were transfected with full-length S and the 11 S subunit in 96-well white flat bottom TC-treated microtest assay plates. Acceptor HEK293 cells were transfected in 6-well plates (Corning) with ACE2, TMPRSS2 and the PEP86 subunit, or just the PEP86 subunit (‘No spike’) as negative control. Eighteen hour after transfection, the acceptor cells were released by 0.1% trypsin/EDTA and added to the donor cells at a 1:1 ratio for 4 h. Luciferase complementation was measured by incubating with Nano-Glo® Live Cell Reagent for 3 min, followed by readout on an Ensight plate reader (PerkinElmer).

Ad26 vectors

Replication-incompetent, E1/E3-deleted Ad26 vectors were engineered using the AdVac system42, here using a single plasmid technology containing the Ad26 vector genome including a transgene expression cassette. The codon-optimized SARS-COV-2 Spike genes (as described in vaccine design) were inserted into the E1-position of the Ad26 vector genomes under transcriptional control of the human cytomegalovirus promoter and the SV-40 polyadenylation sequence. Rescue and manufacturing of the Ad26 vectors was performed in the complementing cell line PER.C6 TetR43,44. Virus particle (vp) titers in the Ad26 vector preparations were quantified by measurement of optical density at 260 nm45 and infectivity was assessed by quantitative potency assay (QPA)46, using PER.C6 TetR cells. PCR including subsequent sequencing of PCR products has confirmed the identity and integrity of the SARS-COV-2 Spike genes (primers used are listed in Table S1). Ad26 vector-mediated expression of SARS-COV-2 Spike genes was confirmed by western blot analysis of cell-culture lysates from infected MRC-5 cells (Fig. 2d). Bioburden levels and endotoxin levels met the preset release criteria for animal experiments.

SDS-PAGE and western blotting

Twenty-four-well plates were seeded with MRC-5 cells (1.25 × 105 cells/well), and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes. Cell lysates were harvested 48 h post transduction and, after heating for 5 min at 85 °C, samples were loaded under non-reduced conditions on a precast 3–8% Tris-Acetate SDS-PAGE gel (Invitrogen). Proteins were transferred to a nitrocellulose membrane using an iBlot2 dry blotting system (Invitrogen), and membrane blocking was performed overnight at 4 °C in Tris-buffered saline (TBS) containing 0.2% Tween 20 (V/V) (TBST) and 5% (W/V) Blotting-Grade Blocker (Bio-Rad). Following overnight blocking, the membrane was incubated for 1 h with 2.8 µg/ml CR3046. in TBST-5% Blocker. CR3046 is a human monoclonal antibody directed against SARS-CoV-1 Spike. After incubation, the membrane was washed three times with TBST for 5 min and subsequently incubated for 1 h with 1:10,000 IRDye 800CW-conjugated goat anti-human secondary antibody (Li-COR) in TBST-5% Blocker. Finally, the PVDF membrane was washed three times with TBST for 5 min, and after drying developed using an ODYSSEY® CLx Infrared Imaging System (Li-COR). All samples derived from one experiment and were processed in parallel. The uncropped blot is provided in Fig S7.

Animals

Animal experiments were approved by the Central Authority for Scientific Procedures on Animals (Centrale Commissie Dierproeven) and conducted in accordance with the European guidelines (EU directive on animal testing 86/609/EEC) and local Dutch legislation on animal experiments.

Female BALB/c or C57BL6 mice (specific pathogen-free), aged 8–12 weeks at the start of the study were purchased from Charles River laboratories (Sulzfeld, Germany). Mice were immunized via the intramuscular (i.m.) route with 100 μl vaccine (50 μl per hind leg) under isoflurane anesthesia. Intermediate blood samples were collected via submandibular bleeding route. At the end of the experiment, under anesthesia, animals were exsanguinated by heart puncture and sacrificed by cervical dislocation. Blood was processed for serum isolation and spleens were collected. Spleens were processed into single cell suspensions for cellular assays (when applicable).

Virus neutralization assay

Neutralization assays against live SARS-CoV-2 were performed using the microneutralization assay previously described by Algaissi and Hashem47. Vero-E6 cells [CRL-1580, American Type Culture Collection (ATCC)] were grown in Eagle’s minimal essential medium (EMEM; Lonza) supplemented with 8% fetal calf serum (FCS; Bodinco BV), 1% penicillin-streptomycin (Sigma–Aldrich, P4458) and 2 mM L-glutamine (PAA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Clinical isolate SARS-CoV-2/human/NLD/Leiden-0001/2020 (Leiden L-0001) was isolated from a nasopharyngeal sample and its characterization will be described elsewhere (manuscript in preparation). The NGS-derived sequence of this virus isolate is available under GenBank accession number MT705205 and shows 1 mutation in the Leiden-0001 virus compared to the Wuhan sequence resulting in Asp614 > Gly at position 614 of the Spike protein. Isolate Leiden-0001 was propagated and titrated in Vero-E6 cells using the tissue culture infective dose 50 (TCID50) endpoint dilution method and the TCID50 was calculated by the Spearman-Kärber algorithm as described48. All work with live SARS-CoV-2 was performed in a biosafety level 3 facility at Leiden University Medical Center.

Vero-E6 cells were seeded at 12,000 cells/well in 96-well tissue culture plates 1 day prior to infection. Heat-inactivated (30 min at 56 °C) serum samples were analyzed in duplicate. The panel of sera were two-fold serially diluted in duplicate, with an initial dilution of 1:10 and a final dilution of 1:1280 in 60 μL EMEM medium supplemented with penicillin, streptomycin, 2 mM L-glutamine and 2% FCS. Diluted sera were mixed with equal volumes of 120 TCID50/60 µL Leiden -0001 virus and incubated for 1 h at 37 °C. The virus-serum mixtures were then added onto Vero-E6 cell monolayers and incubated at 37 °C in a humidified atmosphere with 5 % CO2. Cells either unexposed to the virus or mixed with 120 TCID50/60 µL SARS-CoV-2 were used as negative (uninfected) and positive (infected) controls, respectively. At 3 days post-infection, cells were fixed and inactivated with 40 µL 37% formaldehyde/PBS solution/well overnight at 4 °C. The fixative was removed from cells and the clusters were stained with 50 µL/well crystal violet solution, incubated for 10 min and rinsed with water. Dried plates were evaluated for viral cytopathic effect. Neutralization titer was calculated by dividing the number of positive wells with complete inhibition of the virus-induced cytopathogenic effect, by the number of replicates, and adding 2.5 to stabilize the calculated ratio. The neutralizing antibody titer was defined as the log2 reciprocal of this value. A SARS-CoV-2 back-titration was included with each assay run to confirm that the dose of the used inoculum was within the acceptable range of 30 to 300 TCID50.

Pseudotyped virus neutralization assay

MLV pseudotyped with SARS-COV-2 S protein was produced as previously described49 with some minor changes. In short, Platinum-GP cells (cell Biolabs, Inc) were transfected with a plasmid encoding the codon-optimized SARS-COV-2 Spike gene from strain Wuhan-Hu-1 (GenBank: MN908947) carrying a 19-aa cytoplasmic tail truncation, a GAG-Pol packaging vector and an MLV vector encoding a luciferase reporter gene using Lipofectamine 3000 transfection reagent (Life Technologies) according to manufacturer’s protocol. Cells were incubated overnight at 37 °C 10% CO2 with OPTIMEM transfection medium. Next day, medium was replaced by OPTIMEM supplemented with 5% FBS and 1% PenStrep and incubated for 48 h prior to harvest. The harvested pseudotyped MLV particles were stored at −80 °C prior to use. In the neutralization assay, soluble ACE2-Fc and mAbs CR3015, CR3022, CR3046 and SAD-S35 (ACROBiosystems) were two-fold serial diluted (n = 3) in DMEM without phenol red supplemented with 1% FBS and 1% PenStrep and incubated with an equal volume of pseudotyped MLV. After 30 min incubation, the mixture was inoculated onto susceptible Vero-E6 cells in MW96 well plates. Luciferase activity was measured after 40 h of incubation by addition of an equal volume of substrate NeoLite (Perkin Elmer) followed by luminescence readout on the EnSight Multimode Plate Reader (Perkin Elmer). The percentage of infectivity was calculated as ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb.

Direct coat ELISAs

IgG binding to SARS-CoV-2 Spike antigen was measured by ELISA with the full-length in-house produced Spike protein COR200099. COR200099 is an in-house produced SARS-CoV-2 Spike protein, stabilized by two point mutations in the S1/S2 junction that knocks out the furin cleavage site, and by two newly introduced prolines in the hinge region in S2. In addition, the transmembrane and cytoplasmic regions have been replaced by a foldon domain for trimerization mutations, allowing the protein to be produced as soluble protein (see S.dTM.PP Fig. 3C). Briefly, 96-wells Perkin Elmer white ½ area plates were O/N coated with COR200099 protein. Next day plates were washed, blocked for 1 h and subsequently incubated for 1 h with 3-fold serially diluted serum samples in block buffer in duplicate. After washing, plates were incubated for 1 h with goat anti-mouse IgG-HRP in block buffer, washed again and developed using ECL substrate. Luminescence readout was performed using a BioTek Synergy Neo instrument.

For IgG1 and IgG2a ELISAs, a similar protocol was followed as described above, but respectively using Goat anti-Mouse IgG1-HRP and Goat anti-Mouse IgG2a-HRP as secondary antibodies.

ELISpot assay

IFN-γ ELISpot was performed on splenocytes of mice isolated after sacrifice using mouse IFN-γ ELISpot-plus kit (Mabtech). Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. IFN-γ ELISpot assay was performed by stimulating splenocytes from individual mice for 18 h with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 1 μg/peptide/mL. Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. PMA/ionomycin stimulation was used as a positive control (data not shown); medium was used as negative control and used to calculate the lower limit of detection. Stimulation was done overnight in duplicate wells containing 0.5–2.5 × 105 cells per well.

Multiplex ELISA

The Th1/Th2 multiplex ELISA assay was performed on splenocytes obtained after sacrifice. Splenocytes were stimulated by 48 h culturing in the presence of two Spike 15-mer peptide pools (pool 1 and pool 2). Resulting supernatants were diluted 4-fold and measured for the presence of Th1 (IFN-γ) and Th2 cytokines (IL-10, IL-4, and IL-5) using a 10-plex multiplex ELISA proinflammatory panel (mouse) kit (Meso Scale Discovery, V-PLEX Proinflammatory Panel 1 Mouse Kit cat# K15048D). Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. Ratios of Th1/Th2 cytokines (IFN-γ/IL-10, IFN-γ/IL-4, and IFN-γ/IL-5) were calculated on basis of cytokine measurements in supernatants by dividing the Th1 cytokine (IFN-γ) with the respective Th2 cytokines. Multiplex ELISA measurements were done on supernatant diluted either 2-fold or 4-fold.

ICS assay

The intracellular cytokine staining assay (ICS) was performed on splenocytes obtained two weeks after sacrifice. Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. ICS assay was performed by stimulating splenocytes from individual mice for 16 h, 106 cells per well, with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 0.2 μg/peptide/well. Stimulation with peptide pools was done at 37 °C, 10% CO2 for 1 h in presence of Rat-anti-Mouse CD49d (1:500, BD; cat #553153) and Hamster-anti-Mouse CD28 (1:500, BD; cat#553294). Protein transport was blocked (1:1000; BD GolgiPlugTM cat#51-230KZ) overnight at 37 °C, 10% CO2. Cells were washed twice with PBS and stained during 20 min at room temperature in the dark, according to manufacturers’ instructions, with a violet viability dye (1:5000; Invitrogen Violet ViD cat#L34955). Cells were washed twice with 0.5% BSA in PBS (PBA) and Fc receptors were blocked during 10 min in the fridge in the dark (1:50; BD Mouse Fc block cat# 553142). Cells were washed once with PBA and stained during 30 min in the fridge in the dark with anti-CD3e FITC (BD, cat#553062), anti-CD4 PerCP-Cy5.5 (BD, cat#550954) and anti-CD8α APC-H7 (BD, cat#557654). Cells were washed twice with PBA and permeabilized/fixated during 20 min in the fridge in the dark (BD Cytofix/CytopermTM cat#51-2090KZ/554722), after which 1× BD Perm/WashTM buffer (BD cat#51-2091KZ/554723) was added. Cells were stained during 30 mins in the fridge, in the dark with anti-IFN-γ PE (BD, cat#554412), anti-TNFa PE-Cy7 (557644) and anti-IL-2 APC (BD, cat#554429). Cells were washed twice with Perm/Wash buffer, resuspended in PBA, and fluorescence was measured on the BD FACSCantoTM II and analyzed with BD FACSDivaTM software: Flow Jo version 10.06.1. Cells were gated on single cells, excluding dead cells, and gated for lymphocytes (Fig S8a). CD8-CD4+ and CD8+ T cells were then gated on IFN-γ, IL-2, and TNF-α (Fig S8b).

Statistical analysis

Statistical differences between immunization regimens were evaluated two-sided for S-specific binding antibodies as measured by ELISA, NAb titers as measured by VNA, IFN-γ producing cells as measured by ELISpot and cytokine production by MSD and ICS assays. Comparisons between Ad26.S, Ad26.tPA.S, Ad26.tPA.SS, Ad26.tPA.WT.S, Ad26.S.PP, adjuvanted S protein, tPA.S, tPA.S.PP, S and S.PP groups were made using the exact Wilcoxon rank-sum test, Cochran–Mantel–Haenszel test, Mann–Whitney U test, t-test from ANOVA, or z-test from Tobit ANOVA. Results were corrected for multiple comparisons by Bonferroni correction; 3-fold Bonferroni correction Fig. 5a, b, 2-fold Bonferroni correction Fig. 5c, d.

Lo ammetto... SONO STATO STRONZO ma era per farti capire che la mia è la semplificazione di una semplificazione di una semplificazione.

Molto sinteticamente, il vaccino Johnson & Johnson sfrutta una tecnologia specifica di ancoraggio dell’encoding della proteina S (Ad26) a un vettore virale modificato (Adenovirus) simile a quello del vaccino AstraZeneca ma, evidentemente, questa metodica è più efficace nello stimolare una risposta anticorpale che da subito raggiunge livelli stimati tra il 64% e il 72% senza bisogno di una seconda dose di boost.

Perché non te l’ho detto subito?

Perché è bene non dimenticare che anche dietro a una banale compressa di paracetamolo ci sono una serie di azioni, interazioni e reazioni ‘scientifiche’ che in un mondo ideale sarebbe bello che tutti conoscessero ma che allo stato attuale funzionano come la comunione con l’ostia benedetta...

Credeteci e verrete salvati.

[juniperpublishersajop]

A New Composition of High Heat General Purpose Polystyrene (High Heat GPPS) Resin

Abstract

A new composition of high heat general-purpose polystyrene (High heat GPPS) resin synthesis is discussed. The new composition of resin involves addition of cross-linker/comonomer in very low concentration during synthesis process. Commercially available vinyl cross-linkers like divinylbenzene (DVB), Ethylene dimethacrylate (EDM), Tricycle (5.2.1.02,6) decanedimethanol diacrylate (TDDDA), Dicyclopentadiene (DCPD) and vinyl comonomer like maleic anhydride, α-methylstyrene have been evaluated via bulk polymerization method. DVB showed enhancement in the heat distortion temperature (HDT), vicat softening temperature (VST) & melt flow index (MFI) compared to the benchmark grade of GPPS, whereas other mechanical and impact properties remained same or better than benchmark grade under identical methods of measurement.

Keywords: Divinylbenzene; Cross-linked; Branching; Polymerization; Heat Distortion Temperature (HDT); Melt Flow Index (MFI)

Abbreviations: HDT: Heat Distortion Temperature; MFI: Melt Flow Index; DVB: Divinylbenzene; EDM: Ethylene Dimethacrylate; TDDDA: Tricycle (5.2.1.02,6) Decanedimethanol Diacrylate; DCPD: Dicyclopentadiene; VST: Vicat Softening Temperature; High Heat GPPS: High Heat General- Purpose Polystyrene

Introduction

Polystyrene (PS) is a multipurpose polymer used in varied applications in rigid and foamed forms. Based on orientation of phenyl group on the polymer backbone, polystyrene can be classified into isotactic, syndiotactic and atactic. Isotactic or syndiotactic polystyrene prepared by metallocene catalyst can give HDT/VST around 250 oC and shows lowest specific gravity compared to any other engineering plastics in the market [1]. XAREC is world’s first syndiotactic polystyrene commercialized by Idemitsu Corporation, used in electronic component molding [2]. The first two are commercially not important due to complexity in the synthesis and processability compared to atactic polystyrene. Atactic polystyrene is known as generalpurpose polystyrene (GPPS). It is produced by simple thermal initiated radical bulk polymerization [3]. GPPS is clear, hard and can be used in packaging, household items, and electronics. The excellent physical and processing properties make GPPS suitable for many applications as compared to any other plastics, but its glass transition temperature (Tg) is only 100 oC that leads to limit its use in certain applications. Tg dictates the continuous service temperature that is a very important factor for polymer to evaluate its processing and application performance. Therefore, there is a demand for high Tg GPPS. Some efforts have been reported in this direction by copolymerizing styrene with maleimide and imide, which act as hydrogen-bond interaction site in the copolymer [4]. Although there is no direct relation between Tg and HDT in polymers, but in amorphous polymer like GPPS, HDT is close to Tg [5].

HDT can be improved by increasing the interaction of chains or by restricting the chain mobility at elevated temperature. Although GPPS synthesis process is well established and matured, the market requirement and customer application always demand to develop new process or improve the efficiency of the process. Vinyl based anhydrides, amides, maleimides and methylstyrenes have been explored as comonomers in different loading to enhance the heat resistance of polystyrene [4-5]. This is generally achieved by introducing heteroatom containing monomer in the polystyrene chain, which leads chemical irregularity or steric hindrance in the polystyrene chain. GPPS has been widely used in food packaging containers, disposable containers, kitchenware & cutleries owing to its excellent transparency, water resistance and colorability. Molded articles prepared by GPPS with lower HDT/VST can undergo structural deformation especially when they are used in hot filled applications and lower HDT can affect the production rate during molding due to the long cycle time. Good thermal properties like higher HDT/VST along with flow are important requirement for the product performance with better heat stability & shorter cycle time in the production line. Therefore, our research was focused in first instant to increase the Tg and HDT/VST, without affecting flow properties.

Experimental Section

Material and Methods

All the chemicals that were used in the process procured from Sigma-Aldrich and used as such.

Gel-permeation chromatography (GPC)

The molecular weight of intermediate samples and final polymer was measured by GPC at 40 oC (Make-Shimadzu, Class-VP). THF was used as mobile and diluent solvent, sample concentration 2 mg/ml, column specification- PLgel 5 μm MIXED-C, 300 x 7.5 mm, Detector- UV @254nm, standards- Monodispersed polystyrene standards.

Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) of the samples was done with TA Instruments/DSC Q 1000 by heating the samples from room temperature to 160 ºC and heating rate always maintained at 10 ºC/min in nitrogen atmosphere.

High performance liquid chromatography (HPLC)

Unreacted residual styrene monomer was detected and quantified by high performance liquid chromatography (HPLC) Agilent HPLC-1260 series. The chromatographic condition are as follows, column- Agilent Zorbax,-C18, (4.6X150mm), 5μ; Mobile phase: Reservoir A -0.02% Orth phosphoric acid in Milli-Q water, Reservoir B-Methanol, Reservoir C - Acetonitrile at different gradients. Flow rate 1 ml/min, detector-diode array detector, wave lenth-254 nm. Column temperatue-40 oC.

Melt flow index (MFI)

Melt flow index was measured at 230 oC & 2.16 kg load as per the ASTM D 1238, in GOTTFERT MFI machine (model-MI-4).

Heat distortion temperature (HDT)

Heat distortion temperature was measured as per the ASTM D648, method B at 0.45 MPa load, on the CEAST machine. All the samples were annealed for 70 hrs. at 70 oC prior to test.

Vicat softening temperature (VST)

Vicat softening temperature was also measured in CEAST machine for annealed samples as per ASTM D1525. All the samples were annealed for 70 hrs. at 70 oC prior to test.

Mechanical property (Tensile and Izod Impact notched)

Injection molded specimens are conditioned for 48 h at 23 oC & relative humidity at 50%. Tensile strength was measured were accordance to ASTM D638, on Zwick 2.5 RTI-UTM machine. Izod impact notched was measured as per the ASTM D256.

Polymerization and Processing

Comonomer/cross linker screening

The following comonomers/cross linkers were screened in 100 g batch scale, the concentration of comonomer was maintained at 0-5 wt. %.

Styrene with comonomer was transferred to the pre-heated (128 oC) glass reactor, the residence time/reaction time and temperature were increased at four different time intervals to make a continuous process. Intermediate samples were collected to monitor molecular weight, Tg and conversions at every 1 h interval. Unreacted styrene monomer or residual monomer was removed by precipitating polymer solution (in THF) in hexane and then measured Tg.

Scale-up (1kg) in lab glass reactor setup

Following three formulation were shortlisted for scale-up in 1kg scale in the lab glass reactor setup (Figure 1 SI) based on the screening experimental results and evaluated thermal, flow & mechanical properties. The reaction conditions were kept as in Table 1. The unreacted styrene was removed by applying high vacuum (0-30 mbar) for 10-12 min at the end of the polymerization.

a. High molecular weight GPPS (Mw > 300 kg mol-1)

b. Evaluation of cross linker DVB at 100 ppm loading

c. Evaluation of comonomer MA at 5 wt. % loading

GPPS process and compounding

To evaluate the final properties of the synthesized polystyrene and copolymers were processed in different steps as shown in the Figure 1.

Results and Discussion

Different approaches to develop high heat GPPS

Based on literature search and our expertise three different approaches had been identified to achieve the targeted properties (CTQ’s).

a. High molecular weight GPPS (Mw > 300 kg mol-1)

b. Evaluation of cross linkers & comonomers

High molecular weight GPPS (Mw >300 kg mol-1)

This approach is simple and straightforward; this has been achieved by optimizing reaction time and temperature of the synthesis process as shown in the Table 2. In general, polystyrene molecular weight (Mw) can grow up to 340-350 kg mol-1 in radical bulk polymerization technique at 3-3.5 h reaction time (pre-poly), but later decreased due to thermal degradation and long residential time. In order to achieve Mw ≥300 kg mol-1, the polymerization was stopped after 4 h reaction time, with ~ 80 % conversion. At final stage high vacuum was applied for 10 min to remove 17-18 % unreacted styrene, after double extrusion the final residual was 1500 ppm.

From Table 3, MFI data shows that as molecular weight increased the flow decreased, but HDT and VST increased by 2 oC. Other mechanical properties like impact and tensile properties are better or equal to the baseline material PS160. Due to the low MFI, high molecular weight approach may have negative effect on processability and cycle time during molding. Figure 2, the GPC chromatograms shows that decreased molecular weight due to thermal degradation at different stages of processing.

Evaluation of cross linkers & comonomers

Following cross linkers and comonomers were screened at different loading for high heat GPPS (Table 4) at 100 g scale. All the cross linkers were screened in the range of 50-500 ppm to avoid gelling in the reactor, whereas comonomers were tested in 1-5 wt. % loading. DVB was shortlisted for scale-up in 1kg scale and evaluated for thermal and mechanical properties as it showed better Tg (107 oC, when residual styrene ~100 ppm). Moreover, it is commercially available in bulk quantities, FDA approved and reactivity is almost similar to the styrene monomer [6-7]. The same formulation was scaled up to 5 kg scale in batch reactor in similar reaction conditions. Tricyclo (5.2.1.02,6) decane dimethanol diacrylate and N,N-Hexamethylenebis (methacrylamide) as crosslinkers did not show any improvement in Tg in the final polymer. MA showed best Tg among comonomers at 4-5 wt. % loading and was scaled up to 1 kg in lab scale. DVB loading was optimized to 100 ppm, higher loading showed low MFI (< 3.0 g/10 min) because of high molecular weight and more crosslinking. Intermediate sample analysis showed that molecular weight increased up to 3rd hour and then started decreasing (Figure 3). This is may be due to thermal degradation under dynamic reaction conditions. Styrene-DVB copolymer always gave broader PDI (≥3.0) due to partial crosslinking or chain extension/branching, which leads to higher MFI. The chain extension was envisaged by lower slope/slower relaxation in the lower frequency region in rotational rheometer study as shown in Figure 4.

Solid content and viscosity at prepoly stage was almost similar to the homopolymer (Table 5). From Figure 7 it is very clear that residual styrene can affect Tg, that is higher the residual, lower the Tg, hence HDT/VST can also change accordingly (Table 6). It is required to maintain optimum level of (< 500 ppm, more preferably < 200 ppm as in example 2) residual in the final polymer to hit HDT > 102 oC or VST >106 oC as showed in table 6, example 2-4. The mechanical properties as if tensile strength & impact were better than the baseline PS160 (Table 6). There is no change in the thermal stability of styrene-DVB copolymer at 100 ppm DVB loading in compared to baseline GPPS PS160 (Figure 2 SI). Comonomers were evaluated in 1-5wt.% loading along with styrene, under similar reaction conditions as mentioned in the Table 2. Maleic anhydride (MA) showed HDT-102 oC, and MFI ~ 10-12 at 5 wt. % loading, but imparts haziness to the final polymer and more brittle than baseline PS 160. MA cannot homopolymerize, however in the presence of styrene it always forms alternative copolymer. This is may be due to higher activity of MA (0.02 time higher than styrene) [8].

Since, we are restricted ourselves to MA content to 5 wt. % maximum in the formulation, MA was added slowly over 1 h time in the prepoly stage and anticipated fair distribution throughout the chain. As MA content increased in the formulation the copolymer molecular weight decreased below critical molecular weight (~240 kg mol-1) as showed in the Table 7 & Figure 9. As 5 wt. % loading showed Tg~ 108 oC (Figure 10), the same was scaled up to 1kg in the lab reactor and evaluated for the mechanical and thermal properties. In styrene-MA copolymerization, the propagating chains were styrinic in nature because the maleic anhydride radicals undergo chain transfer reaction in this highly reactive system [9]. The styrene-MA system was highly exothermic compared to styrene homopolymerization and styrene-DVB system. This is because hydrogen abstraction from the maleic anhydride radical is 40 kJ/ mol more exothermic than that of styrene radical. This finally leads to inferior mechanical properties compared to the baseline as shown in the Table 7. Other comonomers like alpha-methylstyrene and DCPD did not show considerable improvement in thermal or flow properties up to 5 wt. % loading.

Conclusion

Divinylbenzene (DVB) as a crossliker at 100 ppm loading showed balance of thermal and flow properties as compared to baseline PS160. The controlled crosslinking of polystyrene chains by DVB play major role in broad distribution of molecular weight and hence better flow (MFI). There is an increment in the HDT/ VST by 2-3 oC and MFI from 3.3 g/10 min to 7 g/10 min without compromising any other properties of GPPS.

For more Open Access Journals in Juniper Publishers please click on: https://juniperpublishersblog.wordpress.com/

For more articles in  Academic Journal of Polymer Science please click on: https://juniperpublishers.com/ajop/index.php

[yummyummy-404]

Geisha by lhernandezs Performing Arts

[thelukashoell]

“Ber..πųʨɧ…What?“ - Siegessäule - Berlin 22 Germany . It's harder than it looks...😅 . . . . . . . 📷 Sony Alpha 6000 - 30 mm 2.8 Sigma #sonyalpha #madewithlightroom . #streetsofberlin #berlin #berlinmitte #bestofberlin #instaberlin #unlimitedberlin #ihavethisthingwithberlin #officialfanofberlin . Attachments: #tones_of_berlin #berlin_de #visit_berlin #igers_of_berlin #best_of_berlin #dasechteberlin #berlinstravel #wonderlustberlin #srs_berlin #nabendberlin #urbanromantix #deceptive_cities #city_features #raw_germany (at Berlin, Germany) https://www.instagram.com/p/Cl7AnLyDko8/?igshid=NGJjMDIxMWI=

[swedishlandscapes]

Hej! Jag ville bara säga att du tar otroligt fina bilder. Vad har du för kamera och objektiv? :)

Hej! Tack för dom snälla orden. Jag använder en sony a6500. Objektiven jag använder är följande: sigma 16mm, sigma 30 mm, sony 18-135mm, sony 70-200mm och samyang 100mm  macro.

[georgevp]

Personal Portrait, Event photography.

Recently started doing personal profile event photography for a new meetup noticed recently. The shoot happened in an apartment party room. The organizer’s camera was a Hasselblad with the 110 mm lens, with a few light stands with a LED light behind a square softbox and a battery Flashpoint monolite. I with my Sony a6000 with Sigma 30 mm f1.4 and Nissin i40 with a Gary Fong lightsphere that I got…

[fabian-huesser]

CLASSIC MOUNTAIN VIEW Aus meiner Berner-Oberland-Tour, zeige ich nun einen Klassiker. Mit einer unspektakuläre Komposition, dafür mit einer Explosion am Himmel. Schon oft so gesehen, aber dennoch immer wieder traumhaft. 🤩 Die Bilder entstanden als „Zeitvertreib“, bis hoffentlich später die Wolken verschwinden und die Milchstrasse zu sehen ist. Das 1. Bild entstand zum Sonnenuntergang und das 2. kurz vor oder bereits während der blauen Stunde - weiss ich nicht mehr so genau. Camera: Panasonic LUMIX DC S5 Lens: SIGMA 14-24mm F2.8 DG DN Art 019 Settings: f/11 | 1,5s | ISO 100 | 24 mm | RAW | single shot | with tripod Camera: Panasonic LUMIX DC S5 Lens: SIGMA 14-24mm F2.8 DG DN Art 019 Settings: f/11 | 30s | ISO 100 | 20mm | RAW | dual shot | with tripod #fabianhüsser #lumix #lumix_ch #lumixS5 #amazing_shots #splendid_shots #splendid_nature #earth_shotz #loves_united_switzerland #splendid_nature #blickheimat #nature_perfection #switzerland #ig_switzerland #pictascollective #igpodium #got__greatshots #suissebook #super_switzerland #naturephotography #king_shots #rosenlaui #switzerland_vacations #landscapephotography #switzerlandwonderland #1000schweizerorte #mountainviews #landschaftsfotografie #longexposurephotography #swissphotographer @lumix @loves_united_switzerland @blickheimat @suissebook @super_switzerland @got__greatshots @igpodium @pictascollective @loves_united_switzerland @amazing_shots @lumix_de @haslital.berneroberland @berneroberland (at Berner Oberland) https://www.instagram.com/p/Chz79EbM8hk/?igshid=NGJjMDIxMWI=