time = time(); $this->startSession(); } /** * startSession - Performs all the actions necessary to * initialize this session object. Tries to determine if the * the user has logged in already, and sets the variables * accordingly. Also takes advantage of this page load to * update the active visitors tables. */ function startSession(){ global $database; //The database connection session_start(); //Tell PHP to start the session /* Determine if user is logged in */ $this->logged_in = $this->checkLogin(); /** * Set guest value to users not logged in, and update * active guests table accordingly. */ if(!$this->logged_in){ $this->username = $_SESSION['username'] = GUEST_NAME; $this->userlevel = GUEST_LEVEL; $database->addActiveGuest($_SERVER['REMOTE_ADDR'], $this->time); } /* Update users last active timestamp */ else{ $database->addActiveUser($this->username, $this->time); } /* Remove inactive visitors from database */ $database->removeInactiveUsers(); $database->removeInactiveGuests(); /* Set referrer page */ if(isset($_SESSION['url'])){ $this->referrer = $_SESSION['url']; }else{ $this->referrer = "/"; } /* Set current url */ $this->url = $_SESSION['url'] = $_SERVER['PHP_SELF']; } /** * checkLogin - Checks if the user has already previously * logged in, and a session with the user has already been * established. Also checks to see if user has been remembered. * If so, the database is queried to make sure of the user's * authenticity. Returns true if the user has logged in. */ function checkLogin(){ global $database; //The database connection /* Check if user has been remembered */ if(isset($_COOKIE['cookname']) && isset($_COOKIE['cookid'])){ $this->username = $_SESSION['username'] = $_COOKIE['cookname']; $this->userid = $_SESSION['userid'] = $_COOKIE['cookid']; } /* Username and userid have been set and not guest */ if(isset($_SESSION['username']) && isset($_SESSION['userid']) && $_SESSION['username'] != GUEST_NAME){ /* Confirm that username and userid are valid */ if($database->confirmUserID($_SESSION['username'], $_SESSION['userid']) != 0){ /* Variables are incorrect, user not logged in */ unset($_SESSION['username']); unset($_SESSION['userid']); return false; } /* User is logged in, set class variables */ $this->userinfo = $database->getUserInfo($_SESSION['username']); $this->username = $this->userinfo['username']; $this->userid = $this->userinfo['userid']; $this->userlevel = $this->userinfo['userlevel']; return true; } /* User not logged in */ else{ return false; } } /** * login - The user has submitted his username and password * through the login form, this function checks the authenticity * of that information in the database and creates the session. * Effectively logging in the user if all goes well. */ function login($subuser, $subpass, $subremember){ global $database, $form; //The database and form object /* Username error checking */ $field = "user"; //Use field name for username if(!$subuser || strlen($subuser = trim($subuser)) == 0){ $form->setError($field, "* Username not entered"); } else{ /* Check if username is not alphanumeric */ if(!eregi("^([0-9a-z])*$", $subuser)){ $form->setError($field, "* Username not alphanumeric"); } } /* Password error checking */ $field = "pass"; //Use field name for password if(!$subpass){ $form->setError($field, "* Password not entered"); } /* Return if form errors exist */ if($form->num_errors > 0){ return false; } /* Checks that username is in database and password is correct */ $subuser = stripslashes($subuser); $result = $database->confirmUserPass($subuser, md5($subpass)); /* Check error codes */ if($result == 1){ $field = "user"; $form->setError($field, "* Username not found"); } else if($result == 2){ $field = "pass"; $form->setError($field, "* Invalid password"); } /* Return if form errors exist */ if($form->num_errors > 0){ return false; } /* Username and password correct, register session variables */ $this->userinfo = $database->getUserInfo($subuser); $this->username = $_SESSION['username'] = $this->userinfo['username']; $this->userid = $_SESSION['userid'] = $this->generateRandID(); $this->userlevel = $this->userinfo['userlevel']; /* Insert userid into database and update active users table */ $database->updateUserField($this->username, "userid", $this->userid); $database->addActiveUser($this->username, $this->time); $database->removeActiveGuest($_SERVER['REMOTE_ADDR']); /** * This is the cool part: the user has requested that we remember that * he's logged in, so we set two cookies. One to hold his username, * and one to hold his random value userid. It expires by the time * specified in constants.php. Now, next time he comes to our site, we will * log him in automatically, but only if he didn't log out before he left. */ if($subremember){ setcookie("cookname", $this->username, time()+COOKIE_EXPIRE, COOKIE_PATH); setcookie("cookid", $this->userid, time()+COOKIE_EXPIRE, COOKIE_PATH); } /* Login completed successfully */ return true; } /** * logout - Gets called when the user wants to be logged out of the * website. It deletes any cookies that were stored on the users * computer as a result of him wanting to be remembered, and also * unsets session variables and demotes his user level to guest. */ function logout(){ global $database; //The database connection /** * Delete cookies - the time must be in the past, * so just negate what you added when creating the * cookie. */ if(isset($_COOKIE['cookname']) && isset($_COOKIE['cookid'])){ setcookie("cookname", "", time()-COOKIE_EXPIRE, COOKIE_PATH); setcookie("cookid", "", time()-COOKIE_EXPIRE, COOKIE_PATH); } /* Unset PHP session variables */ unset($_SESSION['username']); unset($_SESSION['userid']); /* Reflect fact that user has logged out */ $this->logged_in = false; /** * Remove from active users table and add to * active guests tables. */ $database->removeActiveUser($this->username); $database->addActiveGuest($_SERVER['REMOTE_ADDR'], $this->time); /* Set user level to guest */ $this->username = GUEST_NAME; $this->userlevel = GUEST_LEVEL; } /** * register - Gets called when the user has just submitted the * registration form. Determines if there were any errors with * the entry fields, if so, it records the errors and returns * 1. If no errors were found, it registers the new user and * returns 0. Returns 2 if registration failed. */ function register($subuser, $subpass, $subemail){ global $database, $form, $mailer; //The database, form and mailer object /* Username error checking */ $field = "user"; //Use field name for username if(!$subuser || strlen($subuser = trim($subuser)) == 0){ $form->setError($field, "* Username not entered"); } else{ /* Spruce up username, check length */ $subuser = stripslashes($subuser); if(strlen($subuser) < 5){ $form->setError($field, "* Username below 5 characters"); } else if(strlen($subuser) > 30){ $form->setError($field, "* Username above 30 characters"); } /* Check if username is not alphanumeric */ else if(!eregi("^([0-9a-z])+$", $subuser)){ $form->setError($field, "* Username not alphanumeric"); } /* Check if username is reserved */ else if(strcasecmp($subuser, GUEST_NAME) == 0){ $form->setError($field, "* Username reserved word"); } /* Check if username is already in use */ else if($database->usernameTaken($subuser)){ $form->setError($field, "* Username already in use"); } /* Check if username is banned */ else if($database->usernameBanned($subuser)){ $form->setError($field, "* Username banned"); } } /* Password error checking */ $field = "pass"; //Use field name for password if(!$subpass){ $form->setError($field, "* Password not entered"); } else{ /* Spruce up password and check length*/ $subpass = stripslashes($subpass); if(strlen($subpass) < 4){ $form->setError($field, "* Password too short"); } /* Check if password is not alphanumeric */ else if(!eregi("^([0-9a-z])+$", ($subpass = trim($subpass)))){ $form->setError($field, "* Password not alphanumeric"); } /** * Note: I trimmed the password only after I checked the length * because if you fill the password field up with spaces * it looks like a lot more characters than 4, so it looks * kind of stupid to report "password too short". */ } /* Email error checking */ $field = "email"; //Use field name for email if(!$subemail || strlen($subemail = trim($subemail)) == 0){ $form->setError($field, "* Email not entered"); } else{ /* Check if valid email address */ $regex = "^[_+a-z0-9-]+(\.[_+a-z0-9-]+)*" ."@[a-z0-9-]+(\.[a-z0-9-]{1,})*" ."\.([a-z]{2,}){1}$"; if(!eregi($regex,$subemail)){ $form->setError($field, "* Email invalid"); } $subemail = stripslashes($subemail); } /* Errors exist, have user correct them */ if($form->num_errors > 0){ return 1; //Errors with form } /* No errors, add the new account to the */ else{ if($database->addNewUser($subuser, md5($subpass), $subemail)){ if(EMAIL_WELCOME){ $mailer->sendWelcome($subuser,$subemail,$subpass); } return 0; //New user added succesfully }else{ return 2; //Registration attempt failed } } } /** * editAccount - Attempts to edit the user's account information * including the password, which it first makes sure is correct * if entered, if so and the new password is in the right * format, the change is made. All other fields are changed * automatically. */ function editAccount($subcurpass, $subnewpass, $subemail){ global $database, $form; //The database and form object /* New password entered */ if($subnewpass){ /* Current Password error checking */ $field = "curpass"; //Use field name for current password if(!$subcurpass){ $form->setError($field, "* Current Password not entered"); } else{ /* Check if password too short or is not alphanumeric */ $subcurpass = stripslashes($subcurpass); if(strlen($subcurpass) < 4 || !eregi("^([0-9a-z])+$", ($subcurpass = trim($subcurpass)))){ $form->setError($field, "* Current Password incorrect"); } /* Password entered is incorrect */ if($database->confirmUserPass($this->username,md5($subcurpass)) != 0){ $form->setError($field, "* Current Password incorrect"); } } /* New Password error checking */ $field = "newpass"; //Use field name for new password /* Spruce up password and check length*/ $subpass = stripslashes($subnewpass); if(strlen($subnewpass) < 4){ $form->setError($field, "* New Password too short"); } /* Check if password is not alphanumeric */ else if(!eregi("^([0-9a-z])+$", ($subnewpass = trim($subnewpass)))){ $form->setError($field, "* New Password not alphanumeric"); } } /* Change password attempted */ else if($subcurpass){ /* New Password error reporting */ $field = "newpass"; //Use field name for new password $form->setError($field, "* New Password not entered"); } /* Email error checking */ $field = "email"; //Use field name for email if($subemail && strlen($subemail = trim($subemail)) > 0){ /* Check if valid email address */ $regex = "^[_+a-z0-9-]+(\.[_+a-z0-9-]+)*" ."@[a-z0-9-]+(\.[a-z0-9-]{1,})*" ."\.([a-z]{2,}){1}$"; if(!eregi($regex,$subemail)){ $form->setError($field, "* Email invalid"); } $subemail = stripslashes($subemail); } /* Errors exist, have user correct them */ if($form->num_errors > 0){ return false; //Errors with form } /* Update password since there were no errors */ if($subcurpass && $subnewpass){ $database->updateUserField($this->username,"password",md5($subnewpass)); } /* Change Email */ if($subemail){ $database->updateUserField($this->username,"email",$subemail); } /* Success! */ return true; } /** * isAdmin - Returns true if currently logged in user is * an administrator, false otherwise. */ function isAdmin(){ return ($this->userlevel == ADMIN_LEVEL || $this->username == ADMIN_NAME); } /** * generateRandID - Generates a string made up of randomized * letters (lower and upper case) and digits and returns * the md5 hash of it to be used as a userid. */ function generateRandID(){ return md5($this->generateRandStr(16)); } /** * generateRandStr - Generates a string made up of randomized * letters (lower and upper case) and digits, the length * is a specified parameter. */ function generateRandStr($length){ $randstr = ""; for($i=0; $i<$length; $i++){ $randnum = mt_rand(0,61); if($randnum < 10){ $randstr .= chr($randnum+48); }else if($randnum < 36){ $randstr .= chr($randnum+55); }else{ $randstr .= chr($randnum+61); } } return $randstr; } }; /** * Initialize session object - This must be initialized before * the form object because the form uses session variables, * which cannot be accessed unless the session has started. */ $session = new Session; /* Initialize form object */ $form = new Form; ?> RMgmDB - Rodent Malaria genetically modified Parasites - P. berghei

Summary

RMgm-391
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0302500; Gene model (P.falciparum): PF3D7_0204700; Gene product: hexose transporter (hexose transporter 1; PfHT1, PfHT, PbHT)
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 23 January 2011, 12:38
  *RMgm-391
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20132450
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherK. Slavic; R. Tewari; S. Krishna
Name Group/DepartmentCentre for Infection, Cellular and Molecular Medicine
Name InstituteSt. George's University of London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-391
Principal namepbht-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGFP-expression in all blood stages. Localization of PbHT-GFP in the plasma membrane of early and late blood stages (see also 'Additional information')
Gametocyte/GameteGFP expression in female gametes and ookinetes. Colocalization of PbHT-GFP with protein P28 (PB000865.00.0), a surface marker of the ookinete, female gamete and zygote
Fertilization and ookineteGFP expression in female gametes and ookinetes. Colocalization of PbHT-GFP with protein P28 (PB000865.00.0), a surface marker of the ookinete, female gamete and zygote
OocystAnalysis of mosquito midgut oocysts, 11 days post infection, showed internal foci of GFP-fluorescence of developing sporoblasts and lower intensity membrane fluorescence signal. Twenty-one days post infection, strong fluorescence signal was detectable from sporulating oocysts and oocyst-derived sporozoites. Similarly, sporozoites released from dissected salivary glands 21 days post infection showed expression of PbHT-GFP.
SporozoiteTwenty-one days post infection, strong GFP-fluorescence signal was detectable from sporulating oocysts and oocyst-derived sporozoites. Similarly, sporozoites released from dissected salivary glands 21 days post infection showed expression of PbHT-GFP.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of the hexose transporter (HT)

Protein (function)
HT is a facilitative hexose transporter in the Major Facilitator Superfamily of integral membrane proteins that mediates the uptake of glucose and fructose by the parasite. HTis a single copy gene with no close paralogues. In P. falciparum three other proteins, PFI0955w, PFI0785c and PFE1455w have been annotated with putative sugar transport function, although they have diverged considerably from typical sugar transporters (21%, 13% and 7% amino acid sequence identity compared with PfHT, respectively). Unlike PfHT, PFI0955w and PFI0785c are expressed only late in the asexual cycle.
Unsuccessful attempts to disrupt the ht gene in P. berghei and P. falciparum indicate an essential function during asexual blood stage growth (see RMgm-390).
See also RMgm-603 for reports of unsuccessful attempts to disrupt pbht (pbht1) and analyses of a mutant that expresses a HA-tagged form of PbHT (PbHT1).
See also mutant RMgm-604, in which the endogenous hexose transporter 1 gene (pbht1) is replaced with the P. falciparum ortholog, pfht1.

Phenotype
All blood stages, female gametes, ookinetes, oocysts and sporozoites express PbHT-GFP as determined by GFP-fluorescence analysis.
Unsuccessful attempts to disrupt Pbht indicate an essential function during asexual blood stage growth (see RMgm-390). In the same paper, unsuccessful attempts are reported to disrupt the hexose transporter (Pfht) in P. falciparum.
See also RMgm-603 for analyses of a mutant that expresses a HA-tagged form of PbHT (PbHT1).

Additional information
Some of the observed fluorescence signal in blood stage parasites is internal and may be associated with perinuclear localization as well as the food vacuole. Observed internal PbHT-GFP signal could be associated with developing PbHT in its trafficking pathway or may come from a small proportion of PbHT-GFP protein that has been mislocalized.
Expression of PbHT-GFP in parasites from all analysed developmental stages was confirmed by Western blotting using an anti-GFP monoclonal antibody

Other mutants
RMgm-390: Unsuccessful attempts to disrupt the Pbht gene
RMgm-603: A mutant in which the endogenous hexose transporter 1 gene (pbht1) is replaced with a HA-tagged copy of pbht1.
RMgm-604: a mutant in which the endogenous hexose transporter 1 gene (pbht1) is replaced with the P. falciparum ortholog, pfht1


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0302500
Gene Model P. falciparum ortholog PF3D7_0204700
Gene producthexose transporter
Gene product: Alternative namehexose transporter 1; PfHT1, PfHT, PbHT
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: taggingThe chosen integration strategy for the construct results in integration at the target locus by a single cross-over homologous recombination event.
Commercial source of tag-antibodiesRoche
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid BstXI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1catGGTACCTAACATTTGGAATATTTGTTGCAGTTTTATTGGG
Additional information primer 1Pb10 (KpnI); PbHT-GFP fragment
Sequence Primer 2caaGGGCCCAACTCTTGATTTGCTTATATGTTTTTGTCTTTCTTC
Additional information primer 2Pb11 (ApaI); PbHT-GFP fragment
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6