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Revisão para o concurso de Procurador do Estado do Mato Grosso do Sul (PGE/MS)
Revisão para o concurso de Procurador do Estado do Mato Grosso do Sul (PGE/MS)
terça-feira, 14 de dezembro de 2021
Olá amigos do Dizer o Direito,
Está disponível a revisão para o concurso de Procurador do Estado do Mato Grosso do Sul (PGE/MS).
Boa prova 🙂
Artigo Original em Dizer o Direito
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#BitMart x #PGEM
BitMart register:
➡️https://twitter.com/BitMartExchange/status/1669183986080395265?s=19
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ايردروبات جدبيدة لعملات WEMIX PGEM OMN TITTY MIFY PEPESHIBA
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Gemini system 120a mode d'emploi
GEMINI SYSTEM 120A MODE D'EMPLOI >>Download (Telecharger)
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GEMINI SYSTEM 120A MODE D'EMPLOI >> Lire en ligne
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GTT 25 / 40 / 50 / 60 / 75 / 90 / 120A GROUPES STATIQUES DE PUISSANCE A COMMANDE Il est possble d nsérer les GTT dans un système trpasé, en utlsant le 11 sept. 2022 — Mode d'emploi des écouteurs stéréo sans fil PODCAST i11 True Wireless du système d'infusion de café Curtis GEM-120A Gemini 120A Series Gemini. PGem. 39. Phenomenex. Luna Phenyl Hexyl Dans ce cas, l'emploi de On atteint alors la surcharge de la colonne ou le mode préparatif. ZF 280-1, VULKAN TORFLEX DAMPER 11.5". NM1053-00, joystick SST, Teleflex KE-4 + système de commande électronique, Bus, 1-6 moteurs 1-6 plac 980,00 € *. Ave les pivots de réglage, pour chaque profondeur de travail la quantité pieghevole serie GEMINI 2 a 4 velocità per trattori da 120 a 230 CV ( KW) Information in this manual is subject to change without notice and does not VHF Wireless System Congratulations on purchasing a Gemini wireless system. le travail des chercheurs en protéomique du monde entier. Le système puce-CLHP/SM Agilent série 1260 est un système de CPL. 26 févr. 2016 — Respecter la position de montage décrite sur la notice livrée dans l'emballage. les accessoires de la gamme System proM compact S200. 17 avr. 2020 — fees and disbursements on notice to affected secured creditors. Gemini. Group Automated Distribution Systems Inc. (1993),
https://www.tumblr.com/puhesufegom/698215713632108544/estacion-metro-corregidora-pdf, https://www.tumblr.com/puhesufegom/698215885683539968/jbl-focus-500-pairing-mode-demploi, https://www.tumblr.com/puhesufegom/698216043309596672/funway-into-electronics-volume-3-pdf, https://www.tumblr.com/puhesufegom/698215885683539968/jbl-focus-500-pairing-mode-demploi, https://www.tumblr.com/puhesufegom/698216043309596672/funway-into-electronics-volume-3-pdf.
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BBA Degree: Specialisation & Further Studies Available
Hyderabad, the most sought-after BBA study location, is known as the pearl city because of its importance in the pearl trade since the 18th century. It is also an emerging IT location, after Bengaluru. BBA Degree courses in Hyderabad provide high-quality BBA education and placements. Bachelor of Business Administration (BBA) is a common undergraduate degree programme for individuals who desire to pursue a career in management after graduating from high school. This course gives knowledge about business administration and managing business operations.
Students from all three main streams, Science, Arts, and Commerce, can study BBA. The BBA courses prepare applicants for positions in entrepreneurship and management.
FDDI AIST, WLCI Aptitude Test, NMIMS - NPAT, SET, UGAT, and ATMA-UG are the most common admission tests for studying BBA in Hyderabad.
BBA institutions in Hyderabad provide the following specialisations:
Finance
Sales and Marketing
Retail
Travel and Tourism Management
Entrepreneurship
Human Resources
IT and Systems
International Business
Fashion Design
Digital Marketing
For admission to BBA institutions in Hyderabad, aspirants must satisfy the following qualifying requirements:
An applicant must have graduated from a recognised educational board or institution with a Class 12 diploma in any subject.
A candidate must have received an aggregate percentage of 50% or above in Class 12.
An applicant who is awaiting the results of their 12th grade can also apply for BBA.
There are 114 BBA institutions in Hyderabad that provide BBA courses; 90 are private, 3 are public, and 2 are public-private partnerships. They provide full-time, distance-learning, part-time, and online education options.
BBA provides multiple doors for candidates in various sectors, including private and public, for various careers in business management.
Management experts are well-known for their ability to operate in a wide range of businesses. They can work in multinational corporations, corporate houses, educational institutions, private businesses, banks, and so forth. When it comes to government occupations, a BBA graduate can find work in banks, the armed forces, public sector enterprises, Indian railways, municipal corporations, and so on. They can also prepare for the Civil Services test, which is required for administrative positions in the government.
BBA graduates might look for work in a variety of fields. Aspirants who have completed BBA can pursue the following courses for further study:
Master of Business Administration (MBA)
Chartered Accountant (CA)
Masters in Hotel Management (MHM)
Executive Program in Applied Finance
Hospitality and Tourism Management
International Post Graduate Diploma in Risk Management
Master in Financial Management (MFM)
Master in Management Studies (MMS)
PGDM (Post Graduate Diploma in Management)
PG Diploma in Wealth Management and Financial Planning
Post Graduate Diploma in Event Management (PGEM)
PG Diploma in Chartered Financial Analyst (CFA)
PG Diploma in PR and Event Management
Master in Retail Management
Master of Marketing Management (MMM)
Masters in Human Resource Development Management (MHRDM)
CRISIL Certified Analyst Programme (CCAP)
Masters in Law
One can also create their own firm and work as an entrepreneur to use what they have learned in the real world, or they can establish a school/institution. Following a BBA colleges in Hyderabad, some employment opportunities include:
Human Resource Manager
Sales Manager
Finance Manager
Business Consultant
Information Systems Manager
Investment Banker
Business Administration Researcher
Real Estate Agent
Entrepreneur
Loan Officer
Recruitment Manager
Business Manager
Management Accountant
Financial Advisor
Production Manager
Public Relations Manager
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Hello fellow Itto havers
#genshin impact#arataki itto#aether#paimon#doodles#giart#gotdamn geo bois stealing all my pgems AGAIN
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Im Dezember gab es eine kleine Konferenz in Oxford :)
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ELEVEN ELEVEN
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view from the ‘Pavilion’, where the exhibition of z\w\a\r\t will take place in jan\feb
the ‘Pavilion’ is one of 4 spaces of 'Omstand'
\
“Our exhibition spaces are located on the former site of the ‘PGEM’ – ‘Provinciale Gelderse Elektriciteits Maatschappij’ – a company providing electricity in the area - in Arnhem East.
In 2015-2016, four spaces of the site that are used by Studio Omstand were made suitable for exhibitions and performances.Two of our exhibition rooms are located in the control building of the PGEM, now a National Monument: these are the ‘Battery room’ and the ‘Turbine rooms’. These are exciting, high spaces, unpolished - Industrial and poetic. On the square in front of this building is the ‘Pavilion’: a glass building constructed with used building materials, based on a design by Suzanne Nagtegaal. Our fourth exhibition space is the nearby ‘transformer house no.1’: the space is mainly used for showing film and video presentations and can accommodate up to ten people in the audience. In the winter time the doors of this building open at regular intervals and the projection is for the whole neighborhood (and casual passers-by).
Omstand, presentation space for contemporary art - is a cultural organization for contemporary visual arts. Curating and presenting exhibitions is the core activity of Omstand. The exhibition program consists of six presentations per year by national and international, young and established artists. Van Oldenbarneveldtstraat 92 A, in Arnhem.“
Source: Facebook page of Omstand \ translation mk
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Darts Czech Open Sling TV Live Score virus free
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PDC Unicorn Development Tour 17-18.
P open final czech darts 752 T 883 1 P 409 CR 11/18/2019 18:55 2 Thu, 12 Dec 2019 05:55:14 GMT 74 591 PUT K 25 80 World Championship O 935 34 86 39 56 585 98 39
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FURX open darts 2019 DQSB XJU October 12 Sunday, 10 November 2019 22:55:14 XTUG PTWU 2019 60 529 GN 77 0 7 ODC 573 44 754 52 61 952 793 87 20 34 724 96 78 Championship Final PDC 55 932 396 EOOB 10 Czech 14 92 93 86 84 365 86 64 833 185 78 298 LCQ 588 53 56 79 02 Dec 2019 09:55 PM PDT 11/18/2019 01:55 PM 369 KJ 148 89 62 90 216 898 380 945 904 11 Dec 2019 11:55 PM PST 84 office darts czech open 2019 40 80 718 S JJP 368 02 Oct 2019 01:55 PM PDT 755 903 HF 893 2019 wiki 16 657 362 500 328 OJ 70 7 39 245 188 60 TC 363 463 47 51 9 92 ZJV 251 89 Thursday, 31 October 2019 301 17 960 90 240 957 85 YUAO 30 589 25 Nov 2019 02:55 PM PDT darts czech open 343 TWRQ 36 112 2 70 86 50 43 67 309 449 15 & Baltic Qualifier 18 163 PQQ 98 61
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H F JEBV Finals czech darts A JETR PGEM World Ladies Monday, 28 October 2019 12/12/2019 13:55 11 762 czech darts open 2019 36 40 73 63 15 Open PDC Unicorn Development 45 TM Mon, 30 Sep 2019 19:55:14 GMT XC 733 265 95 open
Darts Korean Open. PDC World Rest of World Ladies Qualifying Event.
porasurders.unblog.fr/2019/11/05/schedule-sportstime-amazon-prime-2019-players-championship-finals
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Will you be writing fluff for Jackaboy and Schneep any time soon?
Hahaha, this fluff I just wrote is so self-indulgent I may just explode XD It’s sorta kinda based on this, but also just how I imagine any sort of late night studying would go with these two!
This isn’t exactly the best bedtime story I could tell. Come tothink of it, this isn’t even a story.
As he thumbed through thetextbook propped on his knee, Jackieboy offered a sideways glance to hiscompanion. Schneep blinked back with a weary smile, tilting his head andsquirming slightly closer so he could see what page they were on.
They were nowhere near done with this, Jackie noted, resisting the urge todespair. Henrik had been working incredibly hard lately; he had a series ofupcoming tests to renew his doctor’s license and he needed to brush up on someof the terminology. Jackie had promised to help him, so help him he would—evenif it meant numbing his own brain in the process.
Schneep must have caught sightof Jackie’s pursed lips or noticed the little sigh he suppressed because whenthe hero glanced back at him a second time, his smile had disappeared.
“Do you want me to read?” he questionedresignedly, holding out a hand for the book. “I know you don’t really want to—”
“No, no, no, I’m just finekeepin’ on,” Jackieboy cut him off, wrapping an arm around his shoulders andpulling him against his side so he could see the pictures as he continueddeterminedly, “This is an example of an electropherogram from a pGEM control.Note that the first—”
“Shhh,” Schneep hushed him witha light chuckle. “I’m right next to you; you will talk my ear off!”
“Well, maybe we’ll reach thepart that lets you know how you reattach it.” Grinning wryly as the doctorelbowed him, he cleared his throat and lowered his voice to the smooth, sweettones he often used with kids who were too shy to talk to him. He read thewords without hearing them, instead opting to imagine those kids’ adorablelittle faces staring up at him with awe and wonder. He had a feeling they wouldbe more entertained by his comics, though; this would put them to sleep.
“Notice that the two C’s at bp12-13 are not well separated. All of this is normal, but reinforces thesuggestion to place your primer at least 15-20 base pairs away from…your…”Jackie paused as a soft noise from Schneep distracted him, peeking curiously atthe doctor and then doing a double take when he saw that, somewhere during thereading, he had balled up and tucked his head against his knees. His glasseswere crooked, his hair mussed so it covered most of his face, but in the newsilence Jackie could hear his breaths—too slow and deep to be anything close toalert.
It seemed the kids weren’t theonly ones he could put to sleep with this, he mused in surprise, easing thebook shut. His satisfaction with his early escape from studying faltered slightly when he felt agentle shift of weight as the other relaxed closer to him. Keeping his own framestiff and unmoving, Jackie considered how vulnerable he was letting himselfbecome, unconsciously or otherwise. Warm fondness curled close in his chest atthe thought of his friend’s trust. He knew he would take care of him. He knew he was safe with him.
Well, he wasn’t about to try bringing him around; obviously he needed therest. He was in a constant state of sleep deprivation as it was and there wasno reason to keep reading; he wouldn’t be able to focus. He’d kill his back sleepinglike that, though. Setting the book aside, Jackie peered across the dim lab tothe cot awkwardly laid out in the corner, which didn’t look particularlyappealing. Henrik’s actual bedroom was on the other side of the building, but it wasn’t like he weighedmuch. Jackie had always been able to carry him without much effort.
“Okay, buddy.” He placed a handon his shoulder and an arm under his knees, gingerly unfolding him from his ballso he could heft him off the floor. “Up, up, and away.”
Henrik’s breathing faltered andhe twitched a little as soon as Jackie lifted him but he was already committedto the motion, so he fully straightened and tightened his hold on him, stayingstill. His heart sank a little when identical blue eyes blinked open and hisfriend lifted his head.
“What’re you…?” he mumbled,barely audible, staring at him in dazed confusion.
“Sorry,” Jackie offered in a whisper,trying not to let his voice reflect his disappointment. “Didn’t mean to wakeyou…Just go back to sleep and I’ll get you to bed.”
“But…I can walk…”
Jackieboy shook his head,readjusting his position so he was a little more secure against his chest. Evenas he slid an arm around the older Ego’s neck, Schneep halfheartedly kicked hisfeet in protest, losing one of his loafers along the way.
“I can walk, Jackie-e-e…”
“Not like this, you can’t.” Keepinghis voice quiet but firm, leaving no room for argument, Jackieboy nudged thelab door open with the toe of his boot, letting it slide closed behind them ashe took him into the hall. Henrik argued anyway, as best he could.
“Is just a lil’ walk…Nothingt’carry me for…Y’think I can’t make it from he-e-ere? Just g-give me coffee, Icould run the marathon…”
Thanks to his drowsiness, thedoctor’s accent was coming in far too strong for Jackie to understand much of whatfollowed. He suspected half of the sentences were in German anyway, but everyso often he could catch bits and pieces. “…lot’f running in my prof—profession…’speciallywith the mental ones…try to talk t’ them an’ they get out’f their straitjacketsan’ try to kill you…Then I’m left t’ clean up after them, the other doctorsblame it all on me…”
“Do they now?” Jackieacknowledged in a low voice, not bothering to hide the frown which surfaced atthat. As he’d suspected, Henrik didn’t notice it, tucking his face against thecurve of the hero’s shoulder and nodding minutely.
“Bennett’s the one who locksthem up. Is always his fault, everything,” he slurred. “He kept…He kept…H-He…”His voice finally drifted off and Jackie sighed in relief at the little purringsnores that followed—though he did file a note in the back of his mind to askabout the offending Dr. Bennett later on.
His pace quickened a little ashe moved into the bedroom; his eyes had adjusted to the dark so there wasn’tany need to turn the light on. Crouching, he slid the bedcovers aside as best he could while stillsupporting his charge. As soon as he lowered him down, Schneep twisted onto hisstomach and let his arm fall toward the floor, sighing faintly.
The warmth in his chest hadreturned, Jackie discovered as he carefully pried Henrik’s glasses off and setthem on the nightstand so they wouldn’t stay bent against the pillow. Runningan affectionate hand over his friend’s green fringe, he murmured, “Sleep well,”and then soundlessly made for the door.
#youtube#jacksepticeye#fanfiction#youtube fanfiction#jackieboy man#dr schneeplestein#in case anyone hasn't noticed#i am a sucker for jackieboy carrying people#it will never stop#fluff for days#sleepy time#sleep deprivation#i live for queerplatonic jackieboy and schneep#answered ask#Anonymous
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All about BBA Course
Bachelor of Business Administration (BBA) is one of the most famous undergraduate degree programs for college kids who want to pursue their profession in management after finishing Class 12th. This program offers expertise associated with the management of enterprise and handling the enterprise operations. BBA can be pursued by college students from all 3 main streams particularly Science, Arts, and Commerce. BBA prepares college students for control and entrepreneurship roles. India is home to prestigious institutes to pursue BBA like NMIMS Anil Surendra Modi School of Commerce in Mumbai, Christ University in Bengaluru, Mount Carmel College in Bengaluru, SCMS BBA colleges in Nagpur, Symbiosis Institute of Computer Studies and Research in Pune, etc. The most famous BBA specializations provided in India are:
➢ Digital Marketing and E-commerce
➢ Computer Applications
➢ Financial Services
➢ Hotel Management
➢ Information Technology
➢ Marketing
➢ Business Analytics
➢ Financial Markets
Some of these colleges offer dual specialization, integrated (5-year duration), and twinning programs. BBA graduates require several skills to manage different types of work that are:
➔ Etiquette
➔ Good communication skills
➔ Problem-solving
➔ Social consciousness
➔ Negotiating
➔ Leadership
➔ Detail-oriented
➔ Initiative
➔ Interpersonal
➔ Critical thinking
➔ Team player
➔ Strong work ethic
➔ Multi-tasking
➔ Commitment
➔ Time management
➔ Adaptability
Candidates should meet the following eligibility standards for admission to BBA schools in India: A candidate should have finished Class 12 in any stream from a recognized educational board or university. Attained a combined percentage of 50% or above in Class twelfth. Candidates who're awaiting the twelfth results also can apply for BBA. BBA opens up numerous gateways for aspirants in one-of-a-kind sectors inclusive of private and government for numerous roles in business management.
It is a well-known fact that Management experts can work in several industries. They can work in banks, MNCs, educational institutes, corporate houses, private enterprises, and so on. Talking about jobs in the government sector, a BBA graduate can discover jobs in Banks, Armed Forces, PSUs, Indian Railways, Municipal Corporations, etc. They also can be put together for the Civil Services exam for administrative roles in government.
BBA graduates can explore jobs in numerous areas. After finishing BBA, aspirants can even go for higher studies and pursue any of the programs:
➔ Master of Business Administration (MBA)
➔ Chartered Accountant (CA)
➔ Masters in Hotel Management (MHM)
➔ Executive Program in Applied Finance
➔ Hospitality and Tourism Management
➔ International Post Graduate Diploma in Risk Management
➔ Master in Retail Management
➔ Master of Marketing Management (MMM)
➔ Masters in Human Resource Development Management (MHRDM)
➔ Master in Financial Management (MFM)
➔ Master in Management Studies (MMS)
➔ CRISIL Certified Analyst Programme (CCAP)
➔ PGDM in Chartered Financial Analyst (CFA)
➔ PGDM in Event Management (PGEM)
➔ PGDM in Event Management and Public Relations
➔ PGDM in Financial Planning and Wealth Management
One can also start their own business and work as an entrepreneur to implement learning in the practical world or even start a school/ institution. Top BBA colleges in Maharashtra can offer you some of the general job roles after fishing the course are:
➢ Human Resource Manager
➢ Sales Manager
➢ Finance Manager
➢ Business Consultant
➢ Information Systems Manager
➢ Investment Banker
➢ Business Administration Researcher
➢ Real Estate Agent
➢ Entrepreneur
➢ Loan Officer
➢ Recruitment Manager
➢ Business Manager
➢ Management Accountant
➢ Financial Advisor
➢ Production Manager
➢ Public Relations Manager
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We give attention to each and everything https://www.instagram.com/p/CXeXMM-PGem/?utm_medium=tumblr
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BBA Degree: Specialisation & Further Studies Available
Hyderabad, the most sought-after BBA study location, is known as the pearl city because of its importance in the pearl trade since the 18th century. It is also an emerging IT location, after Bengaluru. BBA Degree courses in Hyderabad provide high-quality BBA education and placements. Bachelor of Business Administration (BBA) is a common undergraduate degree programme for individuals who desire to pursue a career in management after graduating from high school. This course gives knowledge about business administration and managing business operations.
Students from all three main streams, Science, Arts, and Commerce, can study BBA. The BBA courses prepare applicants for positions in entrepreneurship and management.
FDDI AIST, WLCI Aptitude Test, NMIMS - NPAT, SET, UGAT, and ATMA-UG are the most common admission tests for studying BBA in Hyderabad.
BBA institutions in Hyderabad provide the following specialisations:
Finance
Sales and Marketing
Retail
Travel and Tourism Management
Entrepreneurship
Human Resources
IT and Systems
International Business
Fashion Design
Digital Marketing
For admission to BBA institutions in Hyderabad, aspirants must satisfy the following qualifying requirements:
An applicant must have graduated from a recognised educational board or institution with a Class 12 diploma in any subject.
A candidate must have received an aggregate percentage of 50% or above in Class 12.
An applicant who is awaiting the results of their 12th grade can also apply for BBA.
There are 114 BBA institutions in Hyderabad that provide BBA courses; 90 are private, 3 are public, and 2 are public-private partnerships. They provide full-time, distance-learning, part-time, and online education options.
BBA provides multiple doors for candidates in various sectors, including private and public, for various careers in business management.
Management experts are well-known for their ability to operate in a wide range of businesses. They can work in multinational corporations, corporate houses, educational institutions, private businesses, banks, and so forth. When it comes to government occupations, a BBA graduate can find work in banks, the armed forces, public sector enterprises, Indian railways, municipal corporations, and so on. They can also prepare for the Civil Services test, which is required for administrative positions in the government.
BBA graduates might look for work in a variety of fields. Aspirants who have completed BBA can pursue the following courses for further study:
Master of Business Administration (MBA)
Chartered Accountant (CA)
Masters in Hotel Management (MHM)
Executive Program in Applied Finance
Hospitality and Tourism Management
International Post Graduate Diploma in Risk Management
Master in Financial Management (MFM)
Master in Management Studies (MMS)
PGDM (Post Graduate Diploma in Management)
PG Diploma in Wealth Management and Financial Planning
Post Graduate Diploma in Event Management (PGEM)
PG Diploma in Chartered Financial Analyst (CFA)
PG Diploma in PR and Event Management
Master in Retail Management
Master of Marketing Management (MMM)
Masters in Human Resource Development Management (MHRDM)
CRISIL Certified Analyst Programme (CCAP)
Masters in Law
One can also create their own firm and work as an entrepreneur to use what they have learned in the real world, or they can establish a school/institution. Following a BBA colleges in Hyderabad, some employment opportunities include:
Human Resource Manager
Sales Manager
Finance Manager
Business Consultant
Information Systems Manager
Investment Banker
Business Administration Researcher
Real Estate Agent
Entrepreneur
Loan Officer
Recruitment Manager
Business Manager
Management Accountant
Financial Advisor
Production Manager
Public Relations Manager
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Lupine Publishers | A Hmsh2 c.2332 T>A Mutation and Membrane-hMSH2/ TCRγδ/NKG2D-Mediated Cytotoxicity of Human Vγ9vδ2 T Cells in Ovarian Serous Cystadenocarcinoma
Lupine Publishers | LOJ Pharmacology & Clinical Research
Abstract
Membrane human MutS homologue 2 (mhMSH2) is a well-characterized endogenous ligand for human Vγ9Vδ2 T cells, but its germline gene expression in mhMSH2-overexpressing ovarian serous cystadenocarcinoma (OSC) cells and tissues is unclear. Herein, we discovered a silent hmsh2 c.2332 T>A mutation in the OSC HO8910 cell line (GenBank accession no.MG674653) and identified serval novel soluble forms of hMSH2 protein from its whole cell protein extracts and culture supernatant. The mhMSH2/ TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells were validated in mhMSH2/6-overexpressing SKOV3 cells. Positive expression of cytoplasmic and/or membrane hMSH2/6, cytoplasmic and/or nuclear hMSH3 and complete loss of nuclear expression of hMSH2 was observed in all examined ovarian cancer tissues. The clinical significance of the novel hmsh2 c.2332 T>A mutation, the soluble full-length and truncated forms of hMSH2 proteins, and the complete loss of nuclear hMSH2 protein expression in OSC development and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further elucidated.
Keywords: Hmsh2; Mutation; Membrane hMSH2; Vγ9Vδ2 T cells; Ovarian serous cystadenocarcinoma.
Abbreviations: hMSH2: Human MutS Homologue 2; MMR: Mismatch Repair; M: membrane; EBV: Epstein-Barr virus; OSC: Ovarian Serous Cystadenocarcinoma; ATCC: American Type Culture Collection; IHC: Immunochemistry; mAb: monoclonal Antibody; FCM: Flow Cytometry; PFA: Paraformaldehyde; FITC: Fluorescein Isothiocyanate; DAPI: 4’,6-diamidino-2-phenylindole; LS: Lynch Syndrome.
Introduction
Human MutS homologue 2 (hMSH2), a critical element of the DNA mismatch repair (MMR) system, is normally localized in the nucleus of host cells and dimerizes with hMSH3 or hMSH6 (hMSH3/6) to form complexes that participate in DNA damage and cell apoptotic signaling. Inherited or acquired defects in the hmsh2 gene or protein lead to dysfunctional correction of errors in DNA synthesis and duplication, the cell cycle and Ig isotype switching of B cells and thus have a close association with the genesis of many types of tumors [1,2]. In our published study, we reported a broad overexpression of membrane hMSH2 (mhMSH2) in a series of epithelial tumor cell lines and Epstein-Barr virus (EBV)- transformed B lymphoid cells [3]. mhMSH2 overexpressed on malignant cells was subsequent characterized as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells, a subset of innate immune cells that play a crucial role in antitumor/antiviral immunity and autoimmunity [3-5]. The induction of ectopic mhMSH2 expression on renal carcinoma cells by oxidative stress via the p38 mitogen-activated protein kinase and c-Jun N-terminal kinase pathways with interleukin-18 promotion was subsequently revealed, but the systemic expression of hmsh2 gene in mhMSH2- overexpressing carcinomas is still unknown [6].
There are 238,700 estimated new cases of ovarian cancer and 151,900 deaths per year worldwide, and thus, this disease is the fifth most common cause of female cancer-related death in the United States in 2018 [7,8]. Among the 4 subtypes of ovarian cancer (high-grade serous, endometrioid, clear cell ovarian carcinomas and non-epithelial subtypes), Ovarian Serous Cystadenocarcinoma (OSC) is the most common and lethal type in women [9]. Previously, we reported the unusual ectopic mhMSH2 expression on the human OSC cell lines HO8910 and SKOV3 and the specific binding of mutated mhMSH2 on SKOV3 cells to synthesized OT3 peptides of human Vδ2 TCR [3,10]. In this work, we further elucidated the hmsh2 gene mutation and ectopic subcellular protein expression in HO8910 cells and the mhMSH2-mediated recognition and cytolytic effects of Vγ9Vδ2 T cells towards SKOV3 cells. The abnormal subcellular distribution of hMSH2/3/6 in different subtypes of ovarian cancer tissues was demonstrated by immunochemistry (IHC).
Materials and Methods
Cell lines, culture medium and tumor tissues
HO8910 cells (OSC) were maintained in RPMI-1640 complete medium (Invitrogen, Shanghai, China). SKOV3 (OSC) and HK-2 (proximal tubular cell line derived from normal kidney) cells were cultured in 10% FBS DMEM/F12 medium (Niuyin, Beijing, China). All cell lines were purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences and confirmed to have no mycoplasma contamination before use. Expansion and subset separation of effector human Vγ9Vδ2 T cells were carried out as we previously described [3]. Ovarian cancer tissues belonging to different histotype (OV-1, serous adenocarcinoma; OV-2, embryo sinus carcinoma; OV-3, cyst-myxomatous adenoma) were freshly obtained from Peking Union Hospital with informed consent provided by the patients. The use of human tissues for research was also approved by the ethics committee of the Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou Institute of Pediatrics (2015020917).
Gene sequencing of hmsh2 in HO8910 cells
Appropriate numbers of HO8910 cells in the logarithmic growth phase were collected for total mRNA extraction and cDNA reverse transcription. The target hmsh2 gene was amplified using fragment PCR as we described in Subcellular hMSH2 expression is aberrant in membrane-hMSH2-overexpressing cervical, lung and gastric cancer cell lines and tissues (article in progression). Three PCR products with predicted lengths (990, 1250 and 1549bp) were purified and introduced into the pGEM-T easy vector (Promega, USA). Recombinant plasmids were positively selected and characterized by Not I digestion before gene sequencing. Human msh2 variants in HO8910 cells were screened by alignment with the full-length hmsh2 sequence (NM_000251) using Laser gene 7 software.
Soluble full-length and truncated forms of hMSH2 in the whole cell extracts and culture supernatant of HO8910 cells
Soluble forms of hMSH2 protein in the complete protein extracts and the cell culture supernatant of HO8910 cells were simultaneously separated by SDS-PAGE and blotted with antihMSH2 McAb (65021-1 Ig, 1:500, Proteintech Group, USA) and with goat anti-mouse IgG/HRP secondary antibody (1:5000, Zhongshan Jinqiao Company, China). 𝛽-actin was blotted as endogenous reference control (MW 43 kDa).
Growth curves of HO8910 cells
For growth curves of both OSC cell lines, appropriate numbers of trypsin-digested HO8910, SKOV3 and HK-2 cells were planted in 24-well plates in triplicate and cultured for 8 consecutive days in a 5% CO2 atmosphere at 37°C. Growth curves of the examined cell lines were obtained by using the cell counting method. The expansion rates of target OSC cells were compared with that of HK-2 control cells.
Quantitative real-time PCR for hmsh2 mRNA expression in HO8910 cells
The human msh2 gene transcription levels in HO8910, SKOV3 and HK-2 cells were comparatively analyzed by qRT-PCR. The results were processed with Sequence Detector Version 1.2 (Applied Biosystems, USA) and Sigma Plot 11.0 as we previously described [3].
Ectopic membrane, cytoplasmic and nuclear expression of hMSH2 in HO8910 cells
The membrane, cytoplasmic and nuclear hMSH2 expression in HO8910, SKOV3 and HK-2 cells were further investigated by laser confocal microscopy with different fixation reagents. The target cells (2-3×105) were cultured overnight on pre-autoclaved glass cover slips in a 24-well format, properly fixed with 4% paraformaldehyde (PFA) or ice-cold methanol for 10-15 min and blocked with 0.5% BSA for 30 min at 4°C before labeling with specific anti-hMSH2 (N- 20, Santa Cruz Biotechnology, USA) polyclonal antibody or rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (Zhongshan Jinqiao Company, Beijing, China). The nuclei of the examined tumor cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) (1:1000, Sigma, USA) before being mounted on a Leica DMIRE2 inverted microscope (objective, 40×; numerical aperture, 1.25).
Participation of mhMSH2/TCRγδ/NKG2D in Vγ9Vδ2 T cell-mediated anti-OSC immunity
For further analysis of the role of mhMSH2 in human Vγ9Vδ2 T cell-mediated anti-OSC immunity, effector Vγ9Vδ2 T cells were incubated with SKOV3 cells at different ratios (effector: target [E: T] 20:1, 10:1, 5:1 and 2.5:1). Cytotoxicity was measured with the LDH method, as we previously described [3]. For cytotoxicity blockade assays, target OSC cells were pretreated with anti-hMSH2 (N20, Santa Cruz Biotechnology), anti-NKG2D (149810, R&D Systems) and anti-TCRγδ (B1.1, Immunotech, France) antibodies before incubation with effector Vγ9Vδ2 T cells at different E:T ratios (20:1, 10:1, 5:1 and 2.5:1). The blockade cytotoxicity was measured and compared to that of the rabbit IgG blockade control group. Target SKOV3 cells were stained with anti-hMSH2, anti-hMSH3 and anti-hMSH6 antibodies to confirm the cell surface expression of hMSH2/3/6 antigens before cytotoxicity and antibody-blocking cytotoxicity assays.
Subcellular hMSH2/3/6 Distribution In OSC Tissues
For IHC analyses, freshly collected ovarian cancer tissues were classically made into paraffin sections (3-4 μm in thickness) after proper neutral formalin fixation. The biopsies were heated at 60°C overnight and gradient dehydrated with methanol before antigen retrieval in boiled citrate buffer, followed by overnight incubation with purified mouse anti-hMSH2 monoclonal antibody (mAb) (clone G219-1129,1:200), mouse anti-hMSH3 mAb (clone 52, 1:20), mouse anti-hMSH6 mAb (clone 44, 1:30) (BD Pharmingen, USA) or isotype-matched mIgG1 at 4°C in a moist environment after 3% H2O2 treatment. The coloration was developed with PV9000 reagents (Zhongshan Jinqiao Company, Beijing, China) and captured with a Leica DM3000 imaging system.
Statistical Analysis
GraphPad Prism 7.2 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis and data plotting. Data are presented as the mean ± SD. Differences between/among groups were compared with Student’s t-test (two-tailed) or one-way ANOVA, P<0.05 was considered statistically significant.
Results
Hmsh2 gene sequencing and soluble full-length and truncated hMSH2 protein blotting
The PCR products of hmsh2 in HO8910 cells were 990, 1250 and 1549 bp in length (Figure 1A). By aligning the spliced full gene sequence with hmsh2 (NM_000251), we observed a point mutation (hmsh2 c.2332 T>A) in the HO8910 cell line (Figure 1B). It was a silent mutation that did not alter the primary structure of hMSH2 protein. The gene sequencing data of mutated hmsh2 in HO8910 cells were submitted to GenBank (Accession number: MG674653) for release. No additional gene mutation of hmsh2 was found in other mhMSH2-overexpressing epithelial tumor cell lines (data not shown here). By SDS-PAGE separation and western blots, several soluble forms of hMSH2 proteins were identified from the whole HO8910 cell protein extract [including the full-length form (MW ~105 kDa) and four truncated variants (MW~70, ~62, ~55 and ~40 kDa)] and the cell culture supernatant (MW~88, ~68 kDa) (Figure 1C).
Hmsh2 gene transcription and ectopic cytoplasm/ membrane expression
The growth and proliferation of both OSC cell lines were depicted with growth curves determined by cell counting assays. Both HO8910 cells and SKOV3 cells achieved rapid growth on day 5 and expanded much faster than the normal control HK-2 cells (Figure 2A). The mRNA expression of hmsh2 was slightly decreased in SKOV3 cells but substantially elevated in HO8910 cells compared with HK-2 cells (Figure 2B). Further detection of the ectopic membrane overexpression of hMSH2 on target OSC cells with laser confocal microscopy showed that both HO8910 and SKOV3 displayed different degrees of green fluorescence on the cell surface after labelling with a specific anti-hMSH2 antibody. The cell surface green fluorescence was much stronger on SKOV3 cells than on HO8910 cells. No green fluorescence was observed on normal control HK-2 cells (Figure 2C, the upper panels of pictures). The subcellular distribution of hMSH2 in HO8910 and SKOV3 cells was further determined by confocal microscopy with ice-cold methanol fixation. The membranes of both cells displayed different degrees of green fluorescence. The density of green fluorescence in the cytoplasm and the nuclei of SKOV3 cells was much stronger than that of HO8910 cells. There was no green fluorescence observed in HK-2 cells (Figure 2C, the lower panels of pictures).
Previously, we identified the membrane-overexpressed hMSH2 on several epithelial tumor cell lines as a stress-inducible endogenous protein ligand for human Vγ9Vδ2 T cells [3,6]. Herein, mhMSH2-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells towards target OSC cells were confirmed by cytotoxicity and independent specific antibody blockade cytotoxicity assays. mhMSH2 overexpression on OSC targets was validated with flow cytometry (FCM) before cytotoxicity and cytotoxicity blockade assays (Figure 3A). At E:T ratios of 20:1, 10:1, 5:1 and 2.5:1, the cytotoxic efficiency of effector human Vγ9Vδ2 T cells against target SKOV3 cells was 53%, 26%, 23% and 12%, respectively (Figure 3B). The ectopic mhMSH2-mediated recognition and cytotoxicity could be strongly blocked by specific anti-hMSH2, anti-NKG2D or anti-TCRγδ antibodies at different E:T ratios, indicating the participation of mhMSH2 in Vγ9Vδ2 T cell-mediated anti-OSC immunity by NKG2D/γδ TCR recognition (Figure 3C).
Subcellular distribution of hMSH2/3/6 in OSC tissues
As shown in Table 1, all ovarian cancer cells displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, while loss of nuclear expression of hMSH2 was observed in all 3 categories of ovarian cancer tissues. Strong and clear cytoplasmic and/or membrane hMSH2/3/6 expression was strikingly observed in ovary cyst-myxomatous adenoma nests (Figure 4A). Total loss of nuclear expression of hMSH6 was found in 66.67% (2/3) of the examined ovarian cancer tissues (Figure 4A). hMSH2/3/6 expression showed substantial heterogeneity among individual ovarian cancer patients (Figure 4B).
Discussion
Human msh2 is one of the most crucial genes involved in the DNA MMR pathway and was strongly associated with increased tumor mutational burden in a multivariate analysis [11]. Recent studies have shown that high hMSH2 expression is significantly associated with smoking, while low hMSH2 expression is an indicator of MMR deficiency in lung adenocarcinoma [11]. The high expression of hMSH2 accompanied by increased PD-L1 expression and CD8+ T cell infiltration therefore leads to the development of a prominent immunotherapy-responsive microenvironment for lung adenocarcinoma and acts as a potential surrogate biomarker of tumor mutational burden to identify immune checkpoint blockade responders in this disease [11]. Moreover, recent studies revealed that MSH2-MSH6 played a crucial role in activation-induced deaminase-initiated antibody diversity by recognizing uracil(s) in the Ig gene loci to generate DNA breaks [12]. Many studies have identified mutations in hmsh2 as diagnostic and/or prognostic factors in carcinomas not only in the US and Canada but also in the Middle East and China [13-15]. Human msh gene mutations also have strong potential as novel candidate triple-negative breast cancer predisposition genes and are closely associated with acute adverse events and survival in rectal cancer patients receiving postoperative chemoradiotherapy [16-19]. Pathogenic or likely pathogenic germline mutations in pms2, msh2 or msh6 were detected in 0.5% (6/1,179) of lung cancer patients [18]. In this study, we screened a novel mutation in the gene sequence of hmsh2 at c.2332 T>A in the OSC cell line HO8910. This is a silent mutation that theoretically does not result in a change to the amino acid sequence of hMSH2 protein or to the phenotype of HO8910 cells. However, a rapid expansion and a strong increase in hmsh2 mRNA expression were observed in this cell line compared with the control cell line.
Moreover, several soluble full-length or truncated variants of hMSH2 proteins were observed in the whole cell protein extract and the culture supernatant of HO8910. We deduce that these altered biological characteristics of HO8910 cells and the genesis of OSC might be linked to the hmsh2 c.2332 T>A variation, as evidence showed that germline mutations of hmsh2 and its family members were closely associated with the pathogenesis of Lynch syndrome (LS). For example, hmsh2 c.2152 C>T alteration has been recently reported as a founder mutation in Portugal; its high proportion implies combined screening for this mutation and some other previously reported founder mutations will be helpful in the genetic testing of Portuguese families with suspected LS [19]. The occurrence and clinical significance of hmsh2 c.2332 T>A and soluble full-length and truncated forms of hMSH2 proteins in clinical OSC patients remain to be clarified in the future.
Compared with HO8910 cells, SKOV3 cells displayed stronger ectopic membrane and nuclear expression of hMSH2 in laser confocal microscopy and FCM analyses, suggesting better membrane anchoring and a more efficient nuclear importing system in SKOV3 cells. The ability of the notably overexpressed mhMSH2 on SKOV3 cells to promote human Vγ9Vδ2 T cell-mediated recognition and cytotoxicity via TCRγδ/NKG2D receptors towards target OSC cells was later validated by independent cytotoxicity and specific antibody blockade cytotoxicity assays, providing further evidence for mhMSH2 functioning as an OSC-associated self-antigen (ligand) for human Vγ9Vδ2 T cells in anti-ovarian cancer immunity [10]. In the absence of information/data demonstrating that the observed mutation impacts function, one cannot determine if the mutation is physiologically relevant. In further investigations on the subcellular distribution of hMSH2 and its companion proteins in ovarian cancer tissues, we found that all examined ovarian cancer specimens (serous adenocarcinoma, embryo sinus carcinoma, cystmyxomatous adenoma) displayed positive ectopic cytoplasmic and/or membrane hMSH2/6 expression and cytoplasmic and/or nuclear hMSH3 expression, and total loss of nuclear expression of hMSH2/6 commonly occurred in almost all of the ovarian cancer tissues. Considering the high hmsh2 gene transcription and the loss of nuclear distribution of hMSH2 protein in HO8910 cells, we hypothesized that the nuclear importing system of hMSH2 and/ or hMSH6 protein was dysfunctional. By contrast, recently, it has been reported that a high overall rate (16.2%) of MMR deficiency was surprisingly observed in ovarian endometrioid carcinoma and was significantly associated with increased IFOG (International Federation of Obstetrics and Gynecology) grade and CD8+ intraepithelial lymphocyte infiltration but not with cancer-specific death [19]. These findings suggest a promising future in which loss of nuclear expression of hMSH2 and/or hMSH6 protein may function as effective screening/diagnostic markers or therapeutic targets for different subtypes of ovarian cancers and as endogenous immune ligands not only for Vγ9Vδ2 T cells [20]. We expected that a human cell-based assay system for functional testing of hMSH2 and its family members will facilitate the identification of highrisk ovarian carcinoma patients and the generation of individual autoantigen-targeted immunotherapies for OSC.
Conclusion
In this study, we identified a silent hmsh2 c.2332 T>A mutation (GenBank accession no. MG674653) along with serval novel soluble truncated forms of hMSH2 variants in HO8910 cells and validated the mhMSH2/TCRγδ/NKG2D-mediated recognition and cytotoxicity of Vγ9Vδ2 T cells against mhMSH2/6-overexpressing SKOV3 cells. We also demonstrated the abnormal subcellular distribution of hMSH2/3/6 in the examined ovarian cancer tissues. The clinical significance of the critical findings in OSC genesis and human Vγ9Vδ2 T cell-mediated anti-OSC immunity remain to be further investigated.
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