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Everything for the making of a modern suburban myth was there. An autumn night in the Pacific Northwest that was very dark. An old grocery store that had been converted into an arcade and a club for all ages. A local band with a lead singer that was to be very famous very soon. This heavy metal band from England, having a few days to kill, were visiting Seattle for the first time. These were the ingredients in 1981 that corrupted a wonderful evening at a short lived club that was located in an old Treadwell market of the 1980's in downtown Kirkland known as The End Zone.
The store as well as the huge parking lot have been vacant for years, and are located across from the current location of the transit center and the library. In the flourishing neighborhood of Eastside in KIrkland there was one of the last large parcels of undeveloped real estate in the thriving neighborhood of Eastside. The memories of one magical night still remain, although, in order to make room for retail stores and condos, the building is about to be demolished.
A man who was born in the early 1960's named Brent Christensen, who grew up on the Eastside. The golden age of Heavy Metal music was present on the east of Lake Washington was during the i980'S. Bands that included Culpret and TKO played to fans with big hair, crowds of all ages at Lake Hills, Skating Rinks and at local bars.
Mr. Christensen was a rock and roller. He was also was main roadie for a Redmond band known as Myth. Mr. Christensen also hung out at The Endzone in Kirkland. The end Zone that formerly was in the old Treadwell Building between 1980 and 1981. It had the first Donkey Kong Game, modern video games, and old pinball machines.
Mr. Christensen appreciated the high supermarket ceilings in the old Treadwell building. The low headroom at Lake Hills took something away from the live concerts there. The idea came to home to construct the back wall of the cavernous building as a stage and start offering live music there. He convinced the management that building a stage would be profitable. Mr. Christensen even designed the stage himself, and it was ready to rock and roll by the last part of October. The fact of the matter is that his timing could not have been better.
Later that same year, the relatively new heavy metal band named Def Leppard was on their second tour in this country. Back then, MTV was a new station, and videos of the band help make them and their High n Dry album became very popular all of the nation.
The tour finally made it to the Northwest in 1981 and Def Leppard played the Moore Theatre on Halloween in Seattle. They were not supposed to be in Portland for their next show until November fourth, and they remained in Seattle. For some unknown reason band agreed to do a meet and greet in Kirkland at The End Zone, to sign autographs for fans.
An author named David Frick wrote a book about Def Leppad in 1987 titled Animal Instinct. Mr. Fricke penned that the bassist named Rick Savage told him that another guitarist named Steve Clark went on a drinking binge during a few days' stopover in Seattle, and that Savage thought the band might literally implode.
Reportedly, Mr. Savage told author David Fricke that this was the only time that he ever considered the possibility that the band was actually dying and almost finished. We were having many problems with Rick Allen who was just getting over his attitude thing and that he just couldn't see much life in us if Steve went over the edge. They were very close to success with their album was in the Top 40.
Apparently, Mr. Clark apologized after he sobered. However, in 1983, Pete Willis was dismissed from the band. Mr. Clark remained with Def Leppard until 1991, when he died of an overdose.
However, in 1981, back in Kirkland, it was time for Myth to take stage of The End Zone. Radio was the medium in those pre-social media and pre-Internet days that called the metalheads from the neighborhood of Eastside in Kirkland to the event. Mr. Christensen reported that at least a few hundred people showed up that night by the time it was all over. Myth brought down the house and played the four songs that they knew. In addition, the members of Def Leppard were significantly impressed. Radio helped to spread the word.
A lot changed within a year. Mr. Tate left Myth and became a member of what would become Queensryche. In order to make room for an antique mall, The End Zone was closed. Def Leppard went on to become one of the biggest selling bands of the 1980's, and, with some cast changes, continues to tour and record to this day. The rest, as they say, is history.


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Oh Berlin! Where to start? I guess I didn't quite expect to fall in love with you as fiercely as I did. I didn't expect to see so much beauty, so much greenery, so many cute vintage shops and cafes and ultimate hipster vibes... yes, I'm sold!
The purpose behind the trip was to meet up with my lovely friend Louisa, who I hadn't seen in four months since she left Melbourne to return to France. Obviously we had so much to catch up on, and it felt so great to be able to talk face to face again instead of our many hour long calls over the phone.
A super nice breakfast place if you're after healthy and nutritious food and good coffee! Everything we tried was insanely tasty.
AMAZING waffles! Like seriously amazing. Coffe was good too! They also do heaps of vegan pastries like apple pie and croissants. Bonus points for a super cute and cozy outdoor terrace.
Probably the best dinner I've had all summer! We literally skipped the talking and smashed our dinner in complete silence and the occasional "OMG" when tasting something new. We ordered a plate of hommus, pita bread, a baked eggplant and a whole roasted cauliflower with tahini sauce.
If you're up to super duper tasty vegan burgers, look no further. One of the best ones I've ever had in my life.
If you love artsy magazines and books, don't miss SODA BERLIN and Do You Read Me? I wish I'd had space in my luggage to buy something to take home! Next time I'll know to come prepared.
We did so many things during our three days in the city! Can't wait to be back here again one day.


text:
Markus Richter (* 3. Januar 1972 in Füssen) ist ein deutscher Roman-, Sach- und Kinderbuchautor.

Leben
Nach dem Abitur begann Richter ein Studium zum Hauptschullehramt an der Universität Augsburg. Mitte der 1990er Jahre erhielt er eine Anstellung in Neuschwanstein als mehrsprachiger Schlossführer. Von 1996 bis 2002 wohnte er in Schloss Neuschwanstein und wurde 2001 Stellvertretender Kastellan. Später übernahm Markus Richter als Kastellan die Liegenschaftsverwaltung von Neuschwanstein.
Während seiner Zeit auf Neuschwanstein führte er auch prominente Gäste durch das Schloss. 2012 kam es zu öffentlich ausgetragenen Betrugsvorwürfen um Schloss Neuschwanstein. Bei dem folgenden Gerichtsprozess rund um diese Vorgänge wurde Markus Richter von allen Vorwürfen freigesprochen.
Markus Richter verließ dann Neuschwanstein und wechselte für kurze Zeit zur Museumsabteilung der Bayerischen Schlösserverwaltung nach Schloss Nymphenburg. Heute arbeitet er bei einer Kreisverwaltungsbehörde und nebenberuflich als Autor.

Literarisches Schaffen
2006 erschien die Broschüre ""Neuschwanstein - Traumschloss des Märchenkönigs", bei der er als Co-Autor mitwirkte. 2007 folgte die Broschüre "Heldensagen von Neuschwanstein", die sich mit den Sagen beschäftigt, die Richard Wagner für einige seiner Opern als Vorlagen verwendete. 2010 entstand gemeinsam mit der Illustratorin Beate Speck-Kafkoulas das Kinderbuch "Ludwig und Poldi, das Schlossgespenst von Neuschwanstein". Im Herbst 2017 erschien sein erster historischer Roman "Ins Herz-Neuschwanstein Thriller". Zwei Jahre später folgte die Fortsetzung "Ohne Herz-Neuschwanstein Thriller".

Werke
 Isabella Schinzel, Markus Richter, Hassilo von Wissmann: Neuschwanstein – Traumschloss des Märchenkönigs. Top Spot Guide Verlag, Hamburg, 2006. ISBN 978-3-938722-16-9
 Markus Richter, Hassilo von Wissmann: Heldensagen von Neuschwanstein. Top Spot Guide Verlag, Hamburg, 2007. ISBN 978-3-938722-10-7
 Markus Richter, Beate Speck-Kafkoulas: Ludwig und Poldi, das Schlossgespenst von Neuschwanstein. Top Spot Guide Verlag, Hamburg, erschienen 2010, ISBN 978-3-938722-41-1
 Ins Herz – Neuschwanstein Thriller, edition tingeltangel, München, 2017. ISBN 978-3-944936-32-1
 Ohne Herz – Neuschwanstein Thriller, edition tingeltangel, München, 2019. ISBN 978-3-944936-53-6
 Die Königshütte am Ochsenälpeleskopf, in: Anthologie Mordsgipfel: Krimis aus den bayerischen Bergen. edition tingeltangel, München, 2021. ISBN 978-3-944936-57-4

Weblinks
 Offizielle Website
 Markus Richter Buchszene, abgerufen am 10. November 2021

Einzelnachweise

Autor
Kinderliteratur
Roman, Epik
Literatur (Deutsch)
Deutscher
Geboren 1972
Mann

redpajama_set_name: RedPajamaWikipedia


text:
Intitulé publication: Humanitarian Affairs Officer, NOC
Intitulé code d'emploi: Humanitarian Affairs Officer
Département / Bureau: Bureau de la coordination des affaires humanitaires
Lieu d'affectation: Dnipro
Période de candidature: 14 juin 2022 - 03 juillet 2022
No de l'appel á candidature: 22-Humanitarian Affairs-OCHA-183800-R- (X)
This position is located in the Operations and Advocacy Division (OAD), in the Office for the Coordination of Humanitarian Affairs (OCHA). OCHA is part of the United Nations Secretariat responsible for bringing together humanitarian actors to ensure a coherent response to emergencies. OCHA also ensures there is a framework within which each actor can contribute to the overall response efforts. OCHA's mission is to mobilize and coordinate effective and principled humanitarian action in partnership with national and international actors in order to alleviate human suffering in disasters and emergencies; advocate the rights of people in need; promote preparedness and prevention and facilitate sustainable solutions.
This position-specific job opening is being published for the position of Humanitarian Affairs Officer/Civil-Military Coordination in Dnipro (the duty station may be subject to change, as the operational situation in the country evolves), Ukraine. Under the overall supervision by the Senior Humanitarian Affairs Officer/Deputy Head of Office and the guidance by the Humanitarian Affairs Officer/Head of Sub-Office in Dnipro, the incumbent reports to the Humanitarian Affairs Officer/Civil Military Coordination, Head of Civil-Military Coordination and Access Unit.
Within limits of delegated authority and depending on location, Humanitarian Affairs Officer/Civil-Military Coordination at this level may be responsible for the following duties:
Humanitarian Civil-Military Coordination:
• Ensures the establishment of the necessary links with relevant interlocutors from armed forces and civilian entities.
• Monitors, analyzes and reports on any major accomplishments and progress, as well as identifies and follows up on any potential or actual friction points in relations between the humanitarian and military communities.
• Can serve as the primary focal point (in the absence of the Head of Unit) for all matters related to civil-military coordination including policy, in close consultation with the Head of Sub-Office.
• Prepares, or provides substantive input in the preparation of, policy documents, position papers and reports on civil-military coordination issues for presentation to various agencies throughout the UN system and IASC members. Contributes to the Office regular reporting on civil-military coordination issues.
• Supports and maintains the Humanitarian Notification System (HNS). Serves as HNS focal point. Disseminates the HNS SOPs, provides training and guidance to humanitarian partners on the HNS. Ensures all notifications are processed and followed-up according to the HNS SOP and guidance. Relays any feedback received from partners concerning the HNS to upgrade the system.
Humanitarian Access:
• Monitors, analyses and reports on humanitarian access issues and actions, including identification and follow-up on potential or actual friction points in relations between humanitarian and armed actors.
• Maintains contact with humanitarian actors in the area of responsibility, facilitates information sharing and advocates for humanitarian issues with relevant military, paramilitary organizations and Non-State Armed Groups for facilitating humanitarian access in coordination with other OCHA entities.
Policy and Advocacy:
• Supports the development of country-specific guidelines on civil-military relations, access, SOPs and position papers, based on the current "Guidelines on the Use of Military and Civil Defence Assets in Disaster Relief" and "Guidelines on the Use of Military and Civil Defence Assets to Support UN Humanitarian Activities in Complex Emergencies", as well as the "IASC Reference Paper on Civil-Military Relationship in Complex Emergencies", the IASC Discussion Paper and Non-Binding Guidelines on the "Use of Military or Armed Escorts for Humanitarian Convoys", applicable Security Council resolutions and other relevant instruments.
• Ensures that country-specific guidelines, and the generic guidelines mentioned above are properly disseminated and understood by both the humanitarian actors and the military forces present, as well as by local actors, as appropriate. Promotes and ensures adherence to the above Guidelines within the entire humanitarian community and advise on potential consequences if these principles are compromised.
• Organises, with relevant and balanced Humanitarian and Military representation, Workshops on Civil-Military relations, by advocating adherence to applicable guidelines and principles and focusing on positive examples, contributes to a positive working relationship between relevant international military and IASC members.
• Can act as an advocate for the promulgation of UN-CMCoord concepts, principles and activities, the promotion and protection of humanitarian principles and the dissemination of UN-CMCoord related guidelines.
Establish and maintain UN-CMCoord Network:
• Maintains dialogue and coordination with the military and security forces in the area of responsibility. Identify, establish and maintain contact with the appropriate armed forces counterparts and ensure mutual exchange of information about ongoing humanitarian activities and issues. Advises the military forces in the area of responsibility on humanitarian coordination mechanisms. These mechanisms may include the UN Country Team, UN Security or Disaster Management Teams, Humanitarian Clusters, etc.
• Maintains contact with humanitarian actors in the area of responsibility and serve as an information channel and advocate for their issues with relevant armed actors.
• In coordination with the Head of Sub-Office, leads or participates in working groups, meetings and consultations with other UN agencies and humanitarian partners; organize meetings on UN-CMCoord matters with Heads of Agencies and/or other appropriate entities.
• Maintains, if appropriate, an information exchange forum for stakeholders and interested parties in civil-military relations and participate in relevant meetings.
Performs other duties as required.
PROFESSIONALISM: Knowledge of a range of humanitarian assistance, emergency relief and related humanitarian issues, including humanitarian access and civil-military coordination approaches and techniques to address difficult problems. Analytical capacity and in particular the ability to analyze and articulate the humanitarian dimension of issues which require a coordinated UN response. Ability to identify issues and judgment in applying technical expertise to resolve a wide range of problems. Ability to conduct research, including ability to evaluate and integrate information from a variety of sources and assess impact on the humanitarian situation in assigned country/area. Ability to work under extreme pressure, on occasion in a highly stressful environment (e.g., civil strife, natural disasters and human misery); ability to provide guidance to new/junior staff. Shows pride in work and in achievements; demonstrates professional competence and mastery of subject matter; is conscientious and efficient in meeting commitments, observing deadlines and achieving results; is motivated by professional rather than personal concerns; shows persistence when faced with difficult problems or challenges; remains calm in stressful situations. Takes responsibility for incorporating gender perspectives and ensuring the equal participation of women and men in all areas of work.
PLANNING & ORGANIZING: Develops clear goals that are consistent with agreed strategies; identifies priority activities and assignments; adjusts priorities as required; allocates appropriate amount of time and resources for completing work; foresees risks and allows for contingencies when planning; monitors and adjusts plans and actions as necessary; uses time efficiently.
CLIENT ORIENTATION: Considers all those to whom services are provided to be "clients" and seeks to see things from clients' point of view; establishes and maintains productive partnerships with clients by gaining their trust and respect; identifies clients' needs and matches them to appropriate solutions; monitors ongoing developments inside and outside the clients' environment to keep informed and anticipate problems; keeps clients informed of progress or setbacks in projects; meets timeline for delivery of products or services to client.
An advanced university degree (Master's degree or equivalent) in political science, social science, international studies, public administration, economics, engineering, earth sciences or a related field is required. A first-level university degree in combination with an additional two (2) years of qualifying experience may be accepted in lieu of the advanced university degree.
A minimum of five (5) years of progressively responsible experience in humanitarian affairs, emergency preparedness, crisis/emergency relief management, military, or other related area is required.
Relevant experience in civil-military coordination, access negotiations, or experience in the military forces, dealing with major humanitarian operations is desirable.
Experience in emergency situations (complex emergency or natural disaster) is desirable.
Experience in a humanitarian context within the UN Common System or other comparable international organization is desirable.
French and English are the working languages of the UN Secretariat. For this position advertised, fluency in English and Ukrainian, (both oral and written) is required. Knowledge of Russian is desirable.
The evaluation of qualified candidates may include an assessment exercise, which may be followed by competency-based interview.
This position is funded for a finite period of one year. Extension of the appointment is subject to extension of the mandate and the availability of the funds. The contract will be administered by United Nations Development Programme (UNDP) on behalf of OCHA.
Appointment against this post is on a local basis. Candidates for the National Professional Officer category shall be of the nationality of the country, where this position is located (limited to Ukrainian nationals).
Staff members are subject to the authority of the Secretary-general and to assignment by him or her. In this context, all staff are expected to move periodically to new functions in their careers in accordance with established rules and procedures.
The United Nations Secretariat is committed to achieving 50/50 gender balance in its staff. Female candidates are strongly encouraged to apply for this position. An impeccable record for integrity and professional ethical standards is essential.
Please note that the duty station within Ukraine may be subject to change depending on the evolving operational situation in-country.

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redpajama_set_name: RedPajamaGithub


Embroiled in a criminal case over her legal residency, state Rep. Daisy Baez resigned from the Florida Legislature on Wednesday, the first step in a deal with prosecutors that will also require her to plead guilty to perjury.
Investigators believe Baez, a Coral Gables Democrat, lied when she filled out a voter-registration form changing her address days before the November 2016 election. She claimed to live in her district, but investigators concluded she never paid rent or slept in the apartment.
Facing a potential third-degree felony for lying on an official form, Baez agreed to plead guilty to a similar misdemeanor charge and resign.
She lasted less than a year in the Florida Legislature.
Baez sent House Speaker Richard Corcoran, R-Land O'Lakes, a one-sentence resignation letter Wednesday morning: "Effective immediately, I am resigning my office as a member of the Florida House of Representatives, HD 114."
She is the third lawmaker to resign in scandal in just over six months. Democratic Sen. Jeff Clemens of Atlantis, the incoming Senate Democratic leader, quit last Friday after Politico reported on his affair with a lobbyist. Republican Sen. Frank Artiles of Miami stepped down in April after the Herald revealed he used a slur in front of two black lawmakers and hired "political consultants" who were former Hooters calendar models.
The Herald revealed in May that Baez still did not appear to live in her district, as required by the Florida Constitution.
Under the terms of her pending plea agreement with Miami-Dade State Attorney Katherine Fernández Rundle's office, Baez will also pay a $1,000 fine, take an ethics course and serve one year of probation, during which she'll be banned from seeking public office.
"I want to thank the residents of Florida, Miami-Dade County and District 114 for giving me the opportunity to serve, it's been a great honor," Baez said in a statement late Tuesday to the Miami Herald. "When I began my service as a Representative last year, I vowed to serve the public interest to the best of my ability and I am confident I have done so. As I return to my life as a private citizen, I pledge to continue fighting for universal healthcare, empowering our teachers, and improving the quality of life for the youngest, most vulnerable Floridians."
Baez's resignation leaves House Democrats with only 40 members in the 120-member chamber. That leaves Republicans on the verge of a super majority going into the annual legislative session that begins Jan. 9. Republican Gov. Rick Scott must call a special election to fill Baez's swing seat, and he's under no obligation to do so before session.
"Daisy did what was right for herself, her family and the community, in her view," House Democratic Leader Kionne McGhee of Miami said. "The only thing we can do us support her in whatever decision she made."
On May 16, Baez told the Herald, "I have two residences."
She produced a lease that had her living in District 114 from Oct. 1-April 30. But investigators concluded Baez lived in the home she owns in District 112 -- and did not lease a third rental property in District 114 until May 26, nine days after the Herald published its story.
Baez voted by mail in District 112 in October 2016 before changing her voter address ahead of Election Day.
The controversy forced Baez to drop out of a high-profile race for state Senate District 40. She had been tapped by top Florida Democrats, who considered her a rising party star, even though she was a freshman representative with only one legislative session under her belt. Last November, she defeated Republican John Couriel by 2 percentage points -- a difference of 1,336 votes out of 67,268 cast.
Prosecutors questioned Baez earlier this month, shortly after a House committee found probable cause to investigate a citizen complaint in June over Baez's residency. That investigation could have resulted in Baez's expulsion from the chamber; the committee issued subpoenas last week to depose witnesses in the case, though the depositions were pushed back after the death of Baez's mother.
Following Baez's resignation, the House investigation will now be moot.


Join L'Usine and axa Studio at our Christmas Market on December 9 to learn how to customise canvas using the artful technique of printmaking. Guests will print their own designs on our exclusive holiday L'Usine shopping bags. Tinker away at your creation and enjoy a drink on us!
Note: Remember to write more content when transferring : "Full name - Workshop's name - Phone number".


text:
Experts@Syracuse Home
Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy
Sophie M. Cendrowski-Guillaume, Jennifer L. O'Loughlin, István Pelczer, James T. Spencer
In the low-temperature reaction of B10H14 with C5H5N, a new product, identified as arachno-6,6-(C5H5N)2B10H12, was formed in high yield and purity. The proposed 6,6-L2B10H12 compound represents the first known report of this decaborane substitution pattern. The formation of an asymmetric 6,6-(C5H5N)2B10H12 isomer was unexpected on the basis of literature precedent describing the synthesis and structural elucidation of numerous 6,9-L2B10H12 species (where L = Lewis base). The observed reaction sequence in the formation of the 6,6-(C5H5N)2B10H12 compound proceeded through an initially observed [H·C5H5N]+[B10H13]− intermediate. In addition to the formation of the 6,6-isomer, the synthesis of the 6,9-(C5H5N)2B10H12 isomer is also reported from the reflux of nido-B10H12 in pyridine. Refluxing the 6,6-(pyridine)2B10H12 isomer in pyridine was also found to convert this isomer into the 6,9-isomer. Both isomers were characterized by 11B NMR, FITR, UV-vis, mass spectroscopic, and elemental analyses. The structure of the 6,6-isomer was established by 2D 11B−11B COSY NMR data and by the first application of a pure phase 11B−11B 2Q correlation NMR (double-quantum) experiment to the elucidation of a borane cluster framework. This latter NMR technique was very successful in greatly simplifying the NMR assignments of the 6,6-substituted decaborane cluster species and should be a very powerful tool in cluster structure elucidation in general.
https://doi.org/10.1021/ic00119a015
10.1021/ic00119a015
Dive into the research topics of 'Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy'. Together they form a unique fingerprint.
pyridines Physics & Astronomy 100%
reactivity Physics & Astronomy 79%
isomers Physics & Astronomy 78%
synthesis Physics & Astronomy 57%
Pyridine Chemical Compounds 56%
Compound Isomer Chemical Compounds 54%
NMR Spectroscopy Chemical Compounds 53%
Cendrowski-Guillaume, S. M., O'Loughlin, J. L., Pelczer, I., & Spencer, J. T. (1995). Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy. Inorganic Chemistry, 34(15), 3935-3941. https://doi.org/10.1021/ic00119a015
Reactivity of Decaborane(14) with Pyridine : Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy. / Cendrowski-Guillaume, Sophie M.; O'Loughlin, Jennifer L.; Pelczer, István et al.
In: Inorganic Chemistry, Vol. 34, No. 15, 01.07.1995, p. 3935-3941.
Cendrowski-Guillaume, SM, O'Loughlin, JL, Pelczer, I & Spencer, JT 1995, 'Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy', Inorganic Chemistry, vol. 34, no. 15, pp. 3935-3941. https://doi.org/10.1021/ic00119a015
Cendrowski-Guillaume SM, O'Loughlin JL, Pelczer I, Spencer JT. Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy. Inorganic Chemistry. 1995 Jul 1;34(15):3935-3941. doi: 10.1021/ic00119a015
Cendrowski-Guillaume, Sophie M. ; O'Loughlin, Jennifer L. ; Pelczer, István et al. / Reactivity of Decaborane(14) with Pyridine : Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy. In: Inorganic Chemistry. 1995 ; Vol. 34, No. 15. pp. 3935-3941.
@article{d485ce4107a641e98a5625490002172f,
title = "Reactivity of Decaborane(14) with Pyridine: Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy",
abstract = "In the low-temperature reaction of B10H14 with C5H5N, a new product, identified as arachno-6,6-(C5H5N)2B10H12, was formed in high yield and purity. The proposed 6,6-L2B10H12 compound represents the first known report of this decaborane substitution pattern. The formation of an asymmetric 6,6-(C5H5N)2B10H12 isomer was unexpected on the basis of literature precedent describing the synthesis and structural elucidation of numerous 6,9-L2B10H12 species (where L = Lewis base). The observed reaction sequence in the formation of the 6,6-(C5H5N)2B10H12 compound proceeded through an initially observed [H·C5H5N]+[B10H13]− intermediate. In addition to the formation of the 6,6-isomer, the synthesis of the 6,9-(C5H5N)2B10H12 isomer is also reported from the reflux of nido-B10H12 in pyridine. Refluxing the 6,6-(pyridine)2B10H12 isomer in pyridine was also found to convert this isomer into the 6,9-isomer. Both isomers were characterized by 11B NMR, FITR, UV-vis, mass spectroscopic, and elemental analyses. The structure of the 6,6-isomer was established by 2D 11B−11B COSY NMR data and by the first application of a pure phase 11B−11B 2Q correlation NMR (double-quantum) experiment to the elucidation of a borane cluster framework. This latter NMR technique was very successful in greatly simplifying the NMR assignments of the 6,6-substituted decaborane cluster species and should be a very powerful tool in cluster structure elucidation in general.",
author = "Cendrowski-Guillaume, {Sophie M.} and O{\textquoteright}Loughlin, {Jennifer L.} and Istv{\'a}n Pelczer and Spencer, {James T.}",
doi = "10.1021/ic00119a015",
journal = "Inorganic Chemistry",
T1 - Reactivity of Decaborane(14) with Pyridine
T2 - Synthesis and Characterization of the First 6,6-Substituted Isomer of nido-BwHu, 6,6-(C5H5N)2B10H12, and Application of 11B−11B Double-Quantum NMR Spectroscopy
AU - Cendrowski-Guillaume, Sophie M.
AU - O'Loughlin, Jennifer L.
AU - Pelczer, István
AU - Spencer, James T.
N2 - In the low-temperature reaction of B10H14 with C5H5N, a new product, identified as arachno-6,6-(C5H5N)2B10H12, was formed in high yield and purity. The proposed 6,6-L2B10H12 compound represents the first known report of this decaborane substitution pattern. The formation of an asymmetric 6,6-(C5H5N)2B10H12 isomer was unexpected on the basis of literature precedent describing the synthesis and structural elucidation of numerous 6,9-L2B10H12 species (where L = Lewis base). The observed reaction sequence in the formation of the 6,6-(C5H5N)2B10H12 compound proceeded through an initially observed [H·C5H5N]+[B10H13]− intermediate. In addition to the formation of the 6,6-isomer, the synthesis of the 6,9-(C5H5N)2B10H12 isomer is also reported from the reflux of nido-B10H12 in pyridine. Refluxing the 6,6-(pyridine)2B10H12 isomer in pyridine was also found to convert this isomer into the 6,9-isomer. Both isomers were characterized by 11B NMR, FITR, UV-vis, mass spectroscopic, and elemental analyses. The structure of the 6,6-isomer was established by 2D 11B−11B COSY NMR data and by the first application of a pure phase 11B−11B 2Q correlation NMR (double-quantum) experiment to the elucidation of a borane cluster framework. This latter NMR technique was very successful in greatly simplifying the NMR assignments of the 6,6-substituted decaborane cluster species and should be a very powerful tool in cluster structure elucidation in general.
AB - In the low-temperature reaction of B10H14 with C5H5N, a new product, identified as arachno-6,6-(C5H5N)2B10H12, was formed in high yield and purity. The proposed 6,6-L2B10H12 compound represents the first known report of this decaborane substitution pattern. The formation of an asymmetric 6,6-(C5H5N)2B10H12 isomer was unexpected on the basis of literature precedent describing the synthesis and structural elucidation of numerous 6,9-L2B10H12 species (where L = Lewis base). The observed reaction sequence in the formation of the 6,6-(C5H5N)2B10H12 compound proceeded through an initially observed [H·C5H5N]+[B10H13]− intermediate. In addition to the formation of the 6,6-isomer, the synthesis of the 6,9-(C5H5N)2B10H12 isomer is also reported from the reflux of nido-B10H12 in pyridine. Refluxing the 6,6-(pyridine)2B10H12 isomer in pyridine was also found to convert this isomer into the 6,9-isomer. Both isomers were characterized by 11B NMR, FITR, UV-vis, mass spectroscopic, and elemental analyses. The structure of the 6,6-isomer was established by 2D 11B−11B COSY NMR data and by the first application of a pure phase 11B−11B 2Q correlation NMR (double-quantum) experiment to the elucidation of a borane cluster framework. This latter NMR technique was very successful in greatly simplifying the NMR assignments of the 6,6-substituted decaborane cluster species and should be a very powerful tool in cluster structure elucidation in general.
U2 - 10.1021/ic00119a015
DO - 10.1021/ic00119a015
JO - Inorganic Chemistry
JF - Inorganic Chemistry


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Q: How to import CSV-file with Microsoft Excel 2016? I try to import a CSV-file in Excel. The program has funny default properties. It puts the content of the rows all in one column including the commas. Lets say the file content is (test.csv):
1, 2, 3
4, 5, 6

What I expect Excel to do is to put each number into a separate field using the comma as separators. However, the excel table has only two fields each containing 3 numbers including the commas, something like this (not what I want):
"1, 2, 3"
"4, 5, 6"

What do I have to change in Excel settings so that it does this standard task correctly?

A: You are probably not importing the CSV but instead opening it either double clicking or using the open dialog.
One of the correct ways of importing a CSV into Excel is using Data | From Text/CSV menu. This way you will be able to select the delimiters, change data types if you need etc.

A: Importing CSV via Data | From Text is -- altough working -- rather cumbersome: You need to select the file, the wizard asks about "Delimited" or "Fixed width", then you need to select "Comma" as seperator and finally how to import the data. And when you want to reimport later on the whole procedure starts again and again and again ..
Instead use Data | New Query | From File | From CSV and select the file to import from. I still had to adjust the encoding to UTF-8 while other settings are correctly detected (delimited by comma).

The really great advantage is that you have to do this only once as the above procedure creates a so called "workbook query". To update or reimport just click "Refresh" in the workbook query's context menu.

A: Depending of the configuration of Excel, the CSV files can be separated with "," or ";". To open a CSV file it's better to go to file-->open file-->text. There Excel asks you for the way you want to separate your fields in CSV.
Regards

redpajama_set_name: RedPajamaStackExchange


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Wind to energy in Far North QLD
Laura Harvey, May 21, 2018 May 29, 2018 , Electricity, News, Projects, Renewables, Sustainability, Wind, 0
The Queensland Government has approved a wind farm project for up to 29 turbines as part of Neoen's planned green power hub at Kaban 80km southwest of Cairns.
Minister for State Development, Manufacturing, Infrastructure and Planning, Cameron Dick, said the development approval for the wind farm project would bring substantial investment to the region and is expected to create around 150 jobs during construction.
"The proposed development at Kaban is for a wind farm for up to 29 turbines and additional infrastructure, including a substation and battery storage facility," Mr Dick said.
"This means jobs for the region over the 12 month estimated construction period and more clean energy for the region to tap into.
"The green power hub, which will incorporate the wind farm, is a $300 million project, planned to operate for at least 30 years and generate enough power to supply 57,000 homes.
"The Queensland Government welcomes private sector investment of this kind in support of the state's renewable energy policy."
Minister for Energy, Anthony Lynham, said the new wind farm would complement more than two dozen large-scale projects that were currently committed or under construction throughout Queensland, including the recently announced $200 million Lakeland wind farm, also in the Far North.
"When complete, these projects will more than double Queensland's renewable energy output and produce enough electricity to power close to one million homes," Dr Lynham said.
"Our 50 per cent renewable energy generation target by 2030 has boosted the level of renewable energy investment, bringing significant projects to regions right across the state.
"This is yet another project to join our $4.3 billion pipeline of renewable energy projects underway or financially committed."
Mr Dick said each turbine would have an electrical output of up to 5.5MW. It was expected that the total generation capacity would be up to 160MW. The turbines would have a maximum height of 240m.
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Sec16A defines the site for vesicle budding from the endoplasmic reticulum on exit from mitosis
Helen Hughes, David J. Stephens
Journal of Cell Science 2010 123: 4032-4038; doi: 10.1242/jcs.076000
David J. Stephens
For correspondence: david.stephens@bristol.ac.uk
Supp info
Mitotic inhibition of COPII-dependent export of proteins from the endoplasmic reticulum results in disassembly of the Golgi complex. This ensures ordered inheritance of organelles by the two daughter cells. Reassembly of the Golgi is intimately linked to the re-initiation of ER export on exit from mitosis. Here, we show that unlike all other COPII components, which are cytosolic during metaphase, Sec16A remains associated with ER exit sites throughout mitosis, and thereby could provide a template for the rapid assembly of functional export domains in anaphase. Full assembly of COPII at exit sites precedes reassembly of the Golgi in telophase.
The inheritance of organelles during mitosis is essential to ensure the proper organisation and function of successive cell generations. The Golgi is a single copy organelle and as such is dispersed throughout the cytoplasm before inheritance to ensure distribution between both daughter cells (Wei and Seemann, 2009). This is achieved through two means, the dispersion of structural components of the Golgi complex to a peripheral distribution (Lucocq and Warren, 1987; Shima et al., 1997; Seemann et al., 2002) and absorption of other components with the ER [such as Golgi glycosylation enzymes (Storrie et al., 1998; Zaal et al., 1999)]. Many resident glycosylation enzymes of the Golgi complex cycle slowly through the ER during interphase (Storrie et al., 1998). Export of secretory cargo from the ER is inhibited during mitosis (Prescott et al., 2001) and therefore these enzymes accumulate in the ER and are inherited via equal partitioning of this compartment. In other cases, separation of the ER and Golgi through mitosis is essential to ensure segregation of key signalling components that would become activated if localised together (Bartz et al., 2008).
The integrity of the Golgi complex depends on ongoing transport from the ER (Ward et al., 2001). ER export (for a review, see Hughes and Stephens, 2008) is mediated by the COPII coat, which assembles at sites on the ER membrane known as transitional ER (tER). COPII assembly is directed by the Sar1 GTPase, which, following activation by Sec12, directs sequential recruitment of Sec23-Sec24 and Sec13-Sec31. Together, these components form a vesicle coat that selects cargo for export, deforms the membrane, and ultimately generates coated transport vesicles following budding. An additional component, Sec16, potentiates budding (Supek et al., 2002) and appears to mark the transitional ER membrane and define the site of COPII vesicle budding (Connerly et al., 2005; Ivan et al., 2008; Hughes et al., 2009). Inhibition of vesicle budding from the ER during prometaphase (Farmaki et al., 1999) correlates with the loss of COPII proteins (Sec23-Sec24 and Sec13-31) from the ERES membrane (Farmaki et al., 1999; Hammond and Glick, 2000; Prescott et al., 2001; Stephens, 2003; Altan-Bonnet et al., 2006). This pre-budding arrest of COPII-dependent trafficking results in Golgi disassembly and can be induced experimentally in interphase through inhibition of COPII function [for example, by blocking the GTPase cycle of Sar1 (Kuge et al., 1994)]. In telophase, the Golgi reassembles through re-initiation of ER export activity. This results in re-formation of the Golgi stack and repopulation with Golgi-resident proteins (Souter et al., 1993; Altan-Bonnet et al., 2006).
Because ER export activity directs Golgi reassembly, it is important to understand the spatial coordination of the onset of ER export on exit from mitosis. In Trypanosoma brucei, centrin2 localises to a bi-lobed structure, one lobe lying adjacent to the old Golgi with the other defining the localisation of the new Golgi during duplication (He et al., 2005). Although the Golgi in interphase mammalian cells lies adjacent to the centrosome, it is not obvious how a centrosomal protein could reorganise peripherally dispersed Golgi elements on exit from mitosis. We have previously shown that the principle human orthologue of Sec16, Sec16A (Watson et al., 2006), localises to the transitional ER membrane independently of other COPII proteins (Hughes et al., 2009). Sec16A interacts with COPII proteins [at least Sec23 (Bhattacharyya and Glick, 2007; Iinuma et al., 2007) and Sec13 (Hughes et al., 2009)] and could therefore act in directing the assembly of COPII on the transitional ER membrane before vesicle formation. In interphase, this probably serves to optimise COPII assembly at ERES. Computational modelling of COPII assembly has shown that such directed recruitment of Sar1, Sec23–Sec24 and Sec13–Sec31 (i.e. biased binding to a predefined site) more accurately describes the organisation of ERES seen in mammalian cells (Heinzer et al., 2008). Notably, this model does not include Sec16 itself, but intriguingly, the model initiates from a naked membrane with no prior membrane localisation of COPII proteins. This situation is analogous to that occurring when cells exit mitosis with COPII proteins Sar1, Sec23–Sec24, and Sec13–Sec31 localised to the cytosol.
Using quantitative 4D imaging of cells, we show that Sec16A remains associated with the ER membrane throughout mitosis and defines the localisation for the re-initiation of COPII-dependent budding on exit from mitosis.
We generated a stable HeLa cell line expressing Venus–Sec16AΔN, a construct that lacks the N-terminal 1008 amino acids of Sec16A, yet behaves in an indistinguishable manner to transiently transfected fluorescent protein (FP)-tagged Sec16A or to endogenous Sec16A (supplementary material Fig. S1). We were unable to generate stable cell lines expressing FP-Sec16A by plasmid transfection. Lentiviral transduction of FP–Sec16A was precluded by persistent recombination of the parent vector during cloning. Importantly, Venus–Sec16AΔN includes both the central conserved domain and upstream sequences shown to be necessary for membrane association and targeting to ERES (Ivan et al., 2008; Hughes et al., 2009). Specifically, it colocalises exactly with endogenous Sec16A and localises adjacent to Sec24C and Sec31A labelling, as is seen for endogenous Sec16A (Hughes et al., 2009); its expression does not alter the expression of endogenous Sec16A, and it rescues the phenotypes of decreased ERES number and Golgi fragmentation caused by suppression of endogenous Sec16A (supplementary material Fig. S1). We visualised Venus–Sec16AΔN-expressing HeLa cells throughout mitosis in 4D using spinning disk confocal microscopy. This approach provides excellent 3D spatial information over time without significant photobleaching. Fig. 1A (enlarged in Fig. 1B) shows that Venus–Sec16AΔN remained associated with defined puncta throughout mitosis [fluorescence and differential interference contrast (DIC) images, see also supplementary material Movie 1]. During fluorescence recovery after photobleaching (FRAP) experiments, Venus–Sec16AΔN showed similar dynamics to full-length GFP–Sec16A (Hughes et al., 2009) with t1/2=~8 seconds (Fig. 1C). Images and further quantification of these experiments is shown in supplementary material Fig. S2. The rate of cycling was not significantly affected by the transition from interphase to mitosis. This suggests that membrane association of Sec16 is not subject to mitotic regulation. However, it does not preclude mitotic inhibition of interactions of Sec16 with downstream COPII components (Altan-Bonnet et al., 2006). Although the mechanisms underlying this inhibition remain unknown, these data also reinforce previous data showing that Sar1, Sec23–Sec24, Sec13–Sec31 all lie downstream of Sec16 (Ivan et al., 2008; Hughes et al., 2009).
Live cell imaging showing Sec16 present as membrane-bound puncta throughout mitosis. (A) Bright-field (greyscale) and fluorescence (green) movie stills showing HeLa Venus–Sec16ΔN stable cell line progression through mitosis. Stills are taken from a 180 minute movie of 1 image per minute. (B) Enlarged images of the two cells labelled a and b in the first frame of A. (C) FRAP of Venus–Sec16ΔN in interphase versus dividing cells showing no significant change in rate of turnover. FRAP was performed using spinning disk confocal microscopy. (D,E) Venus–Sec16ΔN stable cell line in metaphase immunolabelled for (D) Sec24C or (E) Sec31A, showing proportionality of Sec16 punctae relative to COPII subunits. Scale bars: 10 μm (A,D,E).
In fixed cells expressing Venus–Sec16AΔN, other COPII proteins, Sec24C (Fig. 1D) and Sec31A (Fig. 1E) were largely redistributed to the cytosol, as has been described previously (Farmaki et al., 1999; Stephens, 2003). Several Sec16A-positive puncta were apparent that do not show adjacent labelling of Sec24C (Fig. 1D) or Sec31A (Fig. 1E). We sought to validate these findings by accumulating cells in mitosis using a series of small molecule inhibitors. Monastrol is a cell-permeable inhibitor of the mitotic kinesin Eg5 (Mayer et al., 1999); its mode of action results in the accumulation of cells with monopolar spindles in mitosis. BI2536 is an inhibitor of polo-like kinase 1, which also arrests cells in mitosis (Steegmaier et al., 2007). UA62784 inhibits the function of CENP-E, thereby preventing proper chromosome congression in metaphase (Henderson et al., 2009). Application of each of these three inhibitors led to accumulation of cells in mitosis that showed clear localisation of Venus–Sec16AΔN to puncta, but loss of Sec24C and Sec31A to a cytosolic distribution (supplementary material Fig. S3). Labelling for other markers [giantin and galactosyltransferase (GalT)] confirmed Golgi dispersal. These data confirm our findings from unperturbed cells during mitosis.
Similarly to Venus–Sec16AΔN, endogenous Sec16A localises to defined puncta throughout mitosis (Fig. 2). This is in stark contrast to previous findings using other COPII proteins, which redistribute to the cytosol during metaphase (Farmaki et al., 1999; Stephens, 2003; Altan-Bonnet et al., 2006). Double labelling with antibodies specific for the Sec31A subunit of the COPII coat (Fig. 2) showed that, as has been previously described, Sec31A remained localised to puncta during interphase and prometaphase. At metaphase (when chromosome are tightly aligned on the metaphase plate) Sec31A was largely lost from discrete puncta (Fig. 2, see boxed enlargements); however, as we have described previously (Stephens, 2003), fixation of cells for immunolabelling enhances the appearance of punctate labelling. Notably these remaining puncta of Sec31A often do not align adjacent to Sec16A and therefore we would not characterise these as functional ERES (see Hughes et al., 2009).
To validate our findings in living cells without potential artefacts from cell fixation, we imaged cells stably expressing both Venus–Sec16AΔN and mRuby–Sec23A. A lentivirus expressing mRuby–Sec23A was constructed by switching the fluorescent protein tag from EYFP–Sec23A, which we have previously successfully used for similar experiments (Stephens, 2003). Control experiments showed that mRuby–Sec23A localised as expected to puncta perfectly aligned with Sec24C and Sec31A but offset from endogenous Sec16A (data not shown). Furthermore, in cells either singly expressing mRuby–Sec23A or doubly transfected cells coexpressing both Venus–Sec16AΔN and mRuby–Sec23A the structure of the Golgi (giantin labelling) and the localisation of GalT (as a marker of the efficiency of ER-to-Golgi transport) in cells were indistinguishable from that of non-transfected cells (data not shown). The rate of mitotic progression was also unaffected by stable transfection of either mRuby–Sec23A alone or of both Venus–Sec16AΔN and mRuby–Sec23A (data not shown). We observed that although Venus–Sec16AΔN remained associated with tER membranes throughout mitosis (Fig. 3A), mRuby–Sec23A dissociated from ERES during metaphase (Fig. 3B and supplementary material Movie 2). Contrast enhancement showed some detectable punctate localisation of mRuby–Sec23A, which could not easily be distinguished from background (Fig. 3C, arrow). These objects did not lie adjacent to Venus–Sec16AΔN-labelled puncta and therefore would not be defined as bona fide ERES (Hughes et al., 2009). Furthermore, FRAP analysis showed that, unlike during interphase, these mRuby–Sec23A-positive structures in metaphase did not recover fluorescence after photobleaching (HH and DJS unpublished observations) indicating that they are not functionally the same as ERES. They might represent clusters of Sec13p-labelled membrane tubules as previously observed by immunoelectron microscopy (Farmaki et al., 1999). We speculate that the punctae are more likely to be vesicular-tubular remnants resulting from juxtanuclear ERES clustering and Golgi breakdown.
Endogenous Sec16 and COPII subunit localisation during cell division. Fixed cells were labelled to detect endogenous Sec16A and the COPII subunit Sec31A at various stages of cell division. DAPI labelling of DNA was used to define the mitotic state of cells as indicated and 3D stacks of images were acquired to image the entire depth of the cells. Scale bars: 10 μm.
Quantification of Venus–Sec16ΔN versus mRuby–Sec23A during mitosis. (A,B,C) Cells expressing both Venus–Sec16ΔN and mRuby–Sec23A imaged through cell division. 1 z-stack (22 slices with 1 μm spacing shown as a maximum intensity projection) was acquired every 2 minutes over a total of 180 minutes. Scale bars: 5 μm. Enlargements of the boxed regions from A and B are shown in C. (D) Automated quantification of the relative number of puncta present within cells expressing both Venus–Sec16AΔN (green) and mRuby–Sec23A (red) that were undergoing mitosis (quantification of five cells from three independent experiments). (E) Error bars for the data in D show 1 s.d., the blue bar indicates those time point for which there is a statistically detectable (t-test, P<0.05) difference between the two channels [Venus–Sec16AΔN (green) and mRuby–Sec23A (red)]. (F,G) Quantification of every tenth frame from two individual examples of cells going through mitosis. Blue bars highlight those time points where there are no detectable mRuby–Sec23A-labelled puncta despite the persistence of Venus–Sec16AΔN-labelled puncta.
(A) mRuby–Sec23A-positive ERES emerge from Venus–Sec16ΔN-positive tER sites on exit from metaphase (arrows). Images show enlargements of cells expressing both markers where 3D stacks (22 z-slices with 1 μm spacing shown as a maximum intensity projection) of cells exiting metaphase were acquired at 2 time points per minute. Arrows indicate Venus–Sec16ΔN-positive punctae from which mRuby–Sec23A punctae appear to emerge. (B) Imaging of Golgi–RFP (red) and Venus–Sec16ΔN (green) on exit from mitosis, shows that the Golgi (arrows) forms at a juxtanuclear region adjacent to clustered ERES. Images show maximum intensity projections of 21 z-slices acquired with 1 μm spacing. Scale bars: 5 μm (A) and 10 μm (B).
Automated quantification of multiple experiments (automatically detecting and counting all ERES in total of 15 dividing cells) confirmed these observations (Fig. 3D). The number of discrete objects labelled with Venus–Sec16AΔN decreased by around 70% as cells enter metaphase. The loss of mRuby–Sec23A from discrete puncta was more pronounced, with <10% detectable objects per cell remaining. Our quantification method was based purely on intensity and so will include any sufficiently bright object within the field of view including mitotic ERES remnants (Farmaki et al., 1999). This, along with the increase in ERES association of Sec23A in cells expressing Venus–Sec16AΔN means that these data overestimate the number of discrete mRuby–Sec23A-labelled objects. The standard deviations of five averaged time sequences (Fig. 3E) further validated these observations and revealed a clear statistically detectable difference in numbers of Venus–Sec16AΔN and mRuby–Sec23A puncta. Frame-by-frame quantification (Fig. 3F,G) showed that we frequently did not observe any visible Sec23A-labelled puncta in metaphase cells (as indicated by the blue bars on these graphs). By contrast, we have never observed cells during metaphase that do not contain obvious Venus–Sec16AΔN puncta. Automated quantification of the intensity of objects revealed that although they are smaller, the mean intensity of individual spots was unchanged through mitosis (not shown).
The number of Venus–Sec16AΔN puncta increased on exit from metaphase (around 100 minutes from the start of the time series) and preceded an increase in mRuby–Sec23A-labelled objects. This is consistent with the idea that Sec16A precedes Sec23A during COPII assembly. Indeed, on exit from mitosis, we observed that mRuby–Sec23A-labelled puncta emerged in close apposition to Venus–Sec16AΔN-labelled structures (Fig. 4A, arrows highlight close juxtaposition of Venus–Sec16AΔN and mRuby–Sec23A, see supplementary material Movie 2). This also preceded re-formation of the Golgi, which occurs in late telophase (Fig. 4B, arrows highlight reforming Golgi clusters, see supplementary material Movie 3). Golgi structures appeared to emerge in the vicinity of the centrosomes, consistent with a role for microtubules in directing the spatial organisation of the reforming of the Golgi ribbon (Ho et al., 1989; Corthesy-Theulaz et al., 1992) as well as increasing the efficiency of the ER export and ER-to-Golgi transport (Watson et al., 2005; Palmer et al., 2009).
Our data show that Sec16A marks the transitional ER throughout mitosis and is coincident with the reappearance of COPII puncta on exit from metaphase. These data are entirely consistent with our previous findings that siRNA-mediated depletion of Sec16A results in a decrease in the number of ERES within a cell (Watson et al., 2006). By directing ERES assembly, Sec16A ensures that intracellular architecture of the cell is restored quickly and effectively on exit from mitosis. Two major outstanding mechanisms remain to be defined – how Sec16A itself is targeted to the ERES membrane and how Sec16A is uncoupled from other COPII proteins during mitosis.
Generation of cDNA constructs and generation of stable cell lines
cDNAs encoding both Venus fluorescent protein (Nagai et al., 2002) and Sec16ΔN were amplified by PCR and subcloned via pGEM-T vector. Venus was excised with BglII-SbfI for insertion into a modified HIV-1-based self-inactivating pSEW sin lentiviral vector backbone lacking the WPRE region (kindly provided by Giles Cory, University of Exeter, Exeter, UK) via BamHI-SbfI. pHRSINcPPT-SEW-Venus was further digested via BamHI-SbfI for insertion of Sec16ΔN (excised from pGEM via BglII-SbfI). Virus preparation was undertaken using HEK293T cells in a 10 cm culture dish at 80% confluence. 40 μg vector construct, alongside packaging vectors pMDG 2 (10 μg) and p8.91 (30 μg) were combined in 5 ml Optimem. A 1:1 solution of DNA:PEI (2 mM in Optimem) was incubated for 20 minutes before adding drop-wise to washed cells. The cells were incubated at 37°C, 5% CO2 for 4 hours before replacing with complete DMEM. 48 hours after the transfection, viral particles were harvested and added to HeLa cells at 70% confluence. Cells were left to grow for 2 weeks post-confluence, Venus-expressing colonies were determined by light microscopy, picked and replated to produce a clonal Venus-Sec16ΔN stable cell line. The cDNA sequence encoding mRuby (Kredel et al., 2009) underwent codon usage adaptation before gene synthesis (MWG Eurofins) and cloning into pBluescript II SK(+). mRuby was excised with SalI-EcoRI for insertion into pLVX-Puro (Clontech) to generate pLVX-Puro–mRuby. A cDNA encoding human Sec23A was amplified by PCR, gel purified and subcloned via pGEM-T vector (excised with ApaI-XbaI) and cloned into pLVX-Puro–mRuby. Lentivirus preparation followed the Clontech Lenti-X. mRuby–Sec23A-expressing cells were selected using puromycin.
Cell synchronisation
Cell synchronisation was achieved using a double thymidine block. Cells grown to 40% confluency were subjected to 2 mM thymidine for 16 hours. Cells were washed three times with PBS, and incubated for 8 hours in complete medium, before repeating the block and wash-out. Cells undergo mitosis 12 hours after second wash-out.
Mitotic inhibitors
Inhibitors were incubated in complete medium at the following concentrations; 100 μM Monastrol, 100 nM BI 2536, 500 nM UA 62784 for 6 hours after the second wash-out before PFA fixation.
Imaging of fixed cells
PFA-fixed immunolabelled cells were imaged using Leica TCS-SP4 AOBS scanning confocal microscope and processed using Photoshop 6.0 (Adobe, Uxbridge, UK) and montages generated using Adobe Illustrator (Adobe). To provide the highest degree of accuracy, Nyquist sampling theorem was applied to ascertain the correct voxel size for sampling the image. This translates to 11.2 samples per μm or a maximum distance of 89 nm pixel width. To adjust to this criteria, a higher pixel number (1024×1024) and zoom factor (5.4×) was used. For Venus–Sec16AΔN, the calculated pixel size used was 43×43×130 (x, y, z in nm). Antibodies were sourced as follows: mouse monoclonal Sec31A and p150Glued (Transduction labs, BD Biosciences, Oxford, UK), giantin (Covance, Princeton, NJ), GalT (CellMab, Gothenburg, SE), α-tubulin (clone DM1A, Millipore, Watford, UK), Sec16A (Bethyl Labs, Montgomery, TX), rabbit polyclonal antibodies against Sec24C and Sec31A (Townley et al., 2008).
Cells grown on live cell dishes (MatTek, Ashland, MA) were imaged using a Perkin Elmer (Seer Green, UK) Ultraview ERS spinning disk confocal microscope with Photokinesis add-on running Volocity (version 5.3.2 build 0). The systems was attached to a Leica DMI6000 inverted microscope (Leica Microsystems, Milton Keynes, UK) with a Solent Scientific (Portsmouth, UK) incubator set to 37°C and a Leica HCX PL APO 63× 1.3 N.A. glycerol immersion lens mounted on a Piezo focus drive. Images were acquired with a Hamamatsu (Welwyn Garden City, UK) 9100-50 electron multiplying CCD. Cells were imaged in MEM without Phenol Red, supplemented with 30 mM HEPES, pH 7.4, 0.5 g l−1 sodium bicarbonate and 10% fetal calf serum. Venus-Sec16ΔN was imaged using a 514 nm laser with 405/440/514/640 nm band-pass filter and 587 nm emission filter. An exposure time of 400 mseconds and sensitivity of 165 was used. For mRuby–Sec23A a 568 nm laser was using with a 405/488/568/640 nm band-pass filter and 455/615 nm emission filter. Electron multiplier sensitivity was set to 165. The system was set to emission discrimination mode. For Fig. 1, 22×0.5 μm z-slices were acquired every minute for 90 time points (3 hours total imaging, 3330 images per time sequence). For Figs 3 and 4, 22×1 μm z-slices were acquired every 2 minutes for 90 time points (3 hours total imaging, 1980 images per time sequence). Shutters were managed for maximum sample protection.
For quantitative FRAP measurements a 63× 1.3 NA Plan-Apochromat objective was used. Photobleaching of Venus-Sec16AΔN was performed with a ~500 nm diameter circular region. Pre-bleach images were collected for 5 seconds and post-bleach images were collected for 60 seconds using 30% acousto-optical tunable filter power and low laser intensity. Background correction was performed by subtraction of a cytosolic region of interest within the cell periphery. ERES that moved out of focus or moved over 500 nm during the photobleaching series could not be analysed. Fluorescence recovery in the bleached region during the time series was quantified using Volocity 5 (Improvision) FRAP analysis software and exported for analysis to GraphPad Prism 4.02. Recycling kinetics were obtained by curve fitting to a one phase exponential f(t)=A•(1–e−kt) + B, Here, A is the mobile fraction, B is the fluorescence directly after photobleaching (%), and k is the rate of fluorescence recovery from which t1/2 is determined [t1/2=ln(2)/k]. Statistical significance was determined using s.d. and the Student's unpaired t-test.
Quantification of image data
Image sequences were analysed using Volocity Quantitation to determine the number of puncta labelled with either Venus–Sec16AΔN or mRuby–Sec23A. Two methods were used, the first an automated count of all frames of multiple sequences (total 15 cells, >2000 individual puncta per time point from four different imaging experiments i.e. performed on different days). All slices in each image sequence were compressed to a single slice using maximum intensity. Cells were marked outlined using the region of interest (ROI) tool and all objects within that ROI of intensity between 9 and 100% with a size >0.1 μm but <5 μm (counted for each channel separately). For the graph shown in Fig. 3D these data were then averaged from 5 cells. It is important to note that this approach includes all objects conforming to these criteria. One can see clearly within such time sequences that many such objects do not in fact conform to our typical definition of an ERES in that brighter spots of mRuby–Sec23A labelling are frequently seen some distance from that of Venus–Sec16AΔN; this is not consistent with a bone fide functional ERES (Hughes et al., 2009). This automated approach, although unbiased, allows for inclusion of non-dividing cells impinging on the initially drawn region of interest during the time sequence. These factors combined lead to an overestimation of the number of defined puncta. Consequently, we also took a second approach applying slightly more stringent thresholding criteria (10–100% intensity, >0.1 μm, <0.5 μm) but manually drawing a region of interest around the dividing cell on a frame by frame basis. This was done for every tenth frame of two representative sequences (one of which is shown in Fig. 3A,B). This approach removed objects from outside the dividing cell and provides absolute quantification of bona fide ERES because of the requirement for increased intensity. This approach is in our opinion the more representative of the two because it does not detect transient accumulation of fluorescent signals that do not conform to our definition of an ERES.
Statistical differences between two groups of data were analysed with a two-tailed unpaired Student's t-test.
We are very grateful to Atsushi Miyawaki for Venus fluorescent protein, Mark Jepson and Alan Leard for assistance with confocal microscopy, Jon Lane, Pete Cullen, and members of the Stephens lab for helpful discussions and critical input to the manuscript. This work was funded by a Non-Clinical Senior Research Fellowship from the MRC (to D.J.S.) and a BBSRC PhD studentship (to H.H.). Deposited in PMC for release after 6 months.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.076000/-/DC1
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JCS and COVID-19
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About Journal of Cell Science


SKIPS MARINE SERVICES PTE LTD provides a complete range of integrated marine services. Our services include ship management for offshore supply vessels in particular; however we are also capable to manage other type of vessels.
Our management team are equipped with a background in sea-going experiences and had been working for many years in this field. Above all else, our professional staff are dedicated to managing ships safely.
Closely associated with the practice of analyzing statistics, our specialists are also capable of making recommendations from information and data collected from the ship and compare it against industry standards for operational readiness.
If you need a customer oriented, fast acting and innovative Ship Management Company to manage your vessels or even your fleet of ships, give SKIPS MARINE SERVICES PTE LTD a call now to discuss your ship management needs.


text:
The Creek War
Began as a civil war within the Creek Nation but later included the U.S. and Chickasaws
The war began as a civil war within the Creek Nation over American encroachment and escalated a month later when Creek Red Sticks destroyed Fort Mims, north of Mobile, Alabama, killing more than 250 settlers and soldiers and turning this civil war into the southern front of the War of 1812.
The U.S. Army and various tribal nations, including Chickasaws, joined the battle. Chickasaw leader George Colbert and his brothers commanded about 230 warriors in 1814 to fight in present-day Alabama and then joined the U.S. Army for the remainder of the war.
On March 27, 1814, General Andrew Jackson at the Battle of Horseshoe Bend, destroyed the Creek Red Sticks as a military power. Close to 900 Native Americans were killed, more than in any other battle in American history.
The subsequent Treaty of Fort Jackson forced the Creeks to cede over 23 million acres of land in Georgia and Alabama to the United States and cleared the way for an influx of non-native settlement in the neighboring Mississippi Territory. This cession was uncomfortably close to the Chickasaw Homeland.
Setting the Stage for Removal


BSCAH's farm located in Far Rockaway is home to the National Grid Foundation Healing Garden, an urban green space uniting local agriculture, community development and economic development.
The idea is simple: Take a small cohort of young people of color, without college degrees, and teach them to be software developers, so that they ultimately can have access to real, high-growth, career-building jobs. Bring them "up to code" and transform them into engineers.


No Vuvuzelas at Wacken 2010!
Many of you might have a Vuvuzela , suitable for the World Championship in South Africa. Vuvuzelas officially have a sound level of 135 Decibel, which is 10 dB louder than a starting jet plane so we have decided to bann them completely from the festival.
To avoid that the live performances of the artists will be disturbed by the nerve strucking trumpet noises, our security will check the whole audience at the entrance to the festival area. If someone doesn´t abide to this rule, the security is advised to take theses instruments away.


Lowell, MA - January 13, 2016 - It's that time of the year again when millions of people vow to turn over a new leaf by making their New Year's resolutions. Global research* conducted by Jabra has revealed that more than 40% of people are resolving to make their bodies stronger and fitter in 2016, rather than opting for more traditional resolutions of just focusing on weight loss.
With over half of people surveyed (52%) admitting that they have never made a fitness related New Year's resolution before, 2016 looks to be the year of first-time resolutions with people focusing their attentions on a healthier and fitter lifestyle. 2015 has seen a healthy shift away from the traditional 'thin' celebrity images depicted in magazines, to the more favorable 'fitspiration', 'real body' trends led by social media channels such as Instagram.
This refreshing shift in attitude reveals an increased emphasis on strength and fitness training and dispels the notion that most people either won't make a resolution, or will simply fail to reach their goals. Teamed with growing confidence, 67% of people feel completely confident that they will stick to their fitness resolution.
A determination to succeed is clearly evident with 45% admitting while they may take a few breaks, they will still ultimately accomplish their goals.
Finding and sticking to a New Year's resolution is no easy task, motivation is clearly the key to success. Over 35% are motivated by music with another 34% saying that the buzz they feel post-workout is what inspires them most. To succeed, 68% revealed they would almost certainly consider investing in some sort of technology to keep them on track. 57% would most likely purchase new sports ear phones and 39% would invest in a fitness tracker or GPS device.
Jabra has conducted this research to prove just how much sound and hearable technology play a part in people's fitness and training goals. With motivation being the key to success, an overwhelming 82% of people surveyed reveal that they currently use some form of technology to assist them when they work out. The majority (69%) utilize music through in-ear buds to spur them on, 33% use some form of fitness tracker/GPS device, 26% use a heart rate monitor and 27% use a running or training app.
Jabra's wireless sports earbud range offers superior, state-of-the-art hearables and has been developed using Jabra's heritage and unrivalled R&D expertise in delivering audio innovation. It is designed specifically to deliver the combination of intelligent in-ear coaching or training tips and music to sports enthusiasts.
Features such as heart rate monitors (Jabra Sport Pulse Wireless) and motion sensors (Jabra Sport Coach Wireless) combined with the Jabra Sport Life app, can significantly enhance any workout. The free app which has been developed by Jabra, offers the ultimate all-in-one solution and contains optimized tools to help plan and monitor your fitness. Whether you're a casual gym goer or a seasoned triathlete, there is a headset to cater for your workout needs. Designed to US military standards and leading the market in audio fidelity, Jabra's innovation enables you to hear more, do more and be more.
As a result of this research, Jabra has initiated a new online community (the Jabra Fitness Community) to further support those that have set a fitness or training goal for the New Year. In addition to their portfolio of products, Jabra will be offering motivational and inspiring tips for success from fitness and training professionals, as well as highlighting "real" updates from those in the Community who share their progress and highs/lows. The Jabra Fitness Community can be found at: http://thisiswherewestart.jabra.com/ and posts can be made to #thisiswhereitstarts.
*Research conducted by Enalyzer for Jabra on December 2015. Global survey polling 1580 people across USA, Malaysia, Australia, Canada, Japan, Brazil, UK, The Netherlands, Singapore, Germany, New Zealand, Denmark, Romania, Trinidad and Tobago, Sweden, France, Puerto Rico, Norway, Switzerland, Hong Kong, Poland, Israel, India, Mexico, China and Nigeria.
Jabra Sport Pace is available now at a suggested retail price of USD 99.00.
Jabra Sport Coach is available now at a suggested retail price of USD 149.99.