"Heaven casts its gentlest influence over this place," "Bacchus has fulfilled all our wishes!" "Bacchus has fulfilled all our wishes!" "Flow, precious nectars, flow in abundance!" "You are the spirit of our pleasure." "You are the spirit of our pleasure!" "Flow, precious nectars, flow in abundance..." "You are the spirit of our pleasure..." "Hideous winter approaches in vain, love, increasing her power through your presence, brings back to our hearts the season of balmy zephyrs." "You kindle our passion and tender desires anew." "Flow, precious nectars, flow in abundance!" "You are the spirit of our pleasure!" "You are the spirit of our pleasure..." "What do I see?" "Is it Thespis?" "Yes, it is he slumbering there." "This sweet nectar has worked liked poppies upon his eyes." "Should he relinquish himself unto sleep on this great day, he who sings so well the praises of the great god of wine?" "Revive your drowsy senses, awaken and sing, most affable Thespis!" "Revive your drowsy senses, awaken, sing, affable Thespis!" "Sing, awaken, affable Thespis!" "Our thanks to Bacchus for the sleep he gives us." "How tranquil it is!" "How sweet it is!" "Thespis, sing, awaken!" "Let us sing, since you force me to, but remember that in Autumn," "I shall spare no one my songs." "Joyous Thespis, be not angry!" "I feel a sweet inspiration take hold and transport me." "Enchanting Bacchus, god of liberty, father of sincerity, you allow us to laugh at mortals." "...laugh at mortals." "You allow us to laugh at mortals." "...laugh at mortals." "My heart, full of truth, takes relief in telling it, although I will surely be misunderstood." "Enchanting Bacchus, god of liberty, father of sincerity, you allow us to laugh at mortals." "...laugh at mortals!" "Fair young Maenads, are you faithful to your lovers?" "It is not easy for us to believe." "Sleep again, Thespis!" "Worthy lovers of these young coquettes, invincible drinkers, you are all deceived, yet you do not love enough to feel jealousy." "Sleep again, Thespis!" "In the middle of an orgy, where licence prevails," "Maenads, your secrets are poorly guarded." "I have been told almost all." "Sleep again, Thespis..." "No, continue, Thespis, listen to Thalia!" "For the excercise of your fantasy, I give you my mask." "Spread your humour as far as you can!" "Naught can resist your songs and games." "I have come with Momus to perform a play, which will correct the shortcomings of mortals." "Is your satire limited to mortals alone?" "You could extend your empire as far as the gods." "I shall lend my support." "On Mount Olympus, reason has fallen out of fashion." "Ha!" "Who could stand being immortal and forever wise and well-behaved?" "...being immortal and forever wise and well-behaved?" "We will mock and tease in all places, let us laugh at heaven and earth..." "Let us wage an eternal war with absurdity!" "We will spare neither mortals nor gods!" "We will mock and tease in all places, let us laugh at heaven and earth!" "We will spare neither mortals nor gods..." "This is the place where Jupiter himself once took leave of his sobriety." "With a ludicrous stratagem he once cured his beloved spouse of jealousy and pride." "With Thespis I wish to recount the story, that Greece may remember well the celebrated occurrence." "What dares anyone undertake here below without the aid of Eros?" "Cast aside your reckless scheme!" "What play could give pleasure, were it not inspired by love?" "Come, Eros, guide our steps," "be forever our patron god." "Let our games and laughter mingle." "Would you criticise the passions I bring to life?" "When you are beset by them, for better or for worse, I alone govern them, as master." "You will see in the end that each will have his prize, when Eros reveals himself to all." "Momus, Eros, God of the vine, enchanting divinities," "by means of playful lessons, we will correct the faults of mortals." "And you, happy witnesses of this beauteous union, show what your zeal can do in its service." "We shall create a new entertainment." "The daughters of memory will sing the authors' eternal praises for such a fine project." "The daughters of memory will sing the authors' eternal praises..." "Bacchus, this is your victory..." "We shall create a new entertainment." "Bacchus, this is your victory!" "Let us abandon ourselves to drink, for this is the slope of Hippocrene..." "We shall create a new entertainment." "Bacchus, this is your victory!" "Let us abandon ourselves to drink for this is the slope of Hippocrene..." "For this... is the slope of Hippocrene." "Sing to Bacchus, sing to Momus, sing to Eros and his passions!" "Sing to Bacchus, sing to Momus, sing to Eros and his passions!" "May one soul after another be filled by these gods!" "May one soul after another be filled by these gods!" "Without wine, without rapture, tenderness is nothing but sadness." "Sing to Bacchus, sing to Momus, sing to Eros and his passions!" "May one soul after another be filled by these gods!" "Do you wish to laugh?" "Then turn to Bacchus." "To him Momus owes his finest transports of delight." "Sing to Bacchus, sing to Momus, sing to Eros and his passions!" "May one soul after another be filled by these gods!" "Gods, who hold the universe in your hands, see how the elements declare war on us!" "If there be any guilty mortals, punish them with thunder and restore to the earth the calm and the gentleness" "of its earliest destiny." "Restore to the earth the calm and the gentleness of its earliest destiny." "But..." "I see Mercury descending!" "Have my cries then been heard?" "Mercury, tell us with what new tribulations heaven will make us feel her vengeance or her hatred?" "The devouring blasts of violent and icy winds threaten our fields and vines without pause." "The Goddess of the winds yields herself to excesses in her cruel jealousy." "All efforts to calm the torments that have seized her heart are in vain." "Jupiter waxes impatient, and I am charged with seeking sweet amusement for him." "With some feigned ardour, some innocent ruse, can we not cure his spouse with ease?" "Should Juno prove implacable, he should feign preparations for a new marriage." "Soon he will cease to appear guilty, and soon their love will regain its former delights." "This plan is full of merriment." "Mercury promises you the amusement of the celestial court." "I expect a successful outcome." "It could well lead to a disaster." "It is sometimes dangerous to make pretence of amorous passion." "This sort of playfulness can lead us to expose our hearts to another's true passion." "It is all a question of the person..." " Propose one." "I will." "In a deep marsh, a vestige of that flood once seen by Deucalion, a nymph has taken refuge, at the foot of a gloomy valley..." "This ridiculous Naiad, for all time excluded from love, oblivious of her comical appearance, hopes each day that a thousand lovers will come one after another to worship her." "Jupiter should pretend to yield to her charms and offer his tender alliance." "Inform Juno of this, excite her anguish, we shall await the moment of clarification." "You may behold the object of his new love." "I must ascend to the heavens, where Jupiter has summoned me." "It is up to him to judge this charming object." "How delightful this place is!" "How pleasant it is!" "Ah, how favourable it is for one who wishes to surrender her liberty..." "How delightful this place is!" "How pleasant it is!" "Ah, how favourable it is for one who wishes to surrender her liberty for one who wishes to surrender her liberty..." "Tell me, my heart, tell me... have you considered well?" "Ah, my heart, how agitated you are!" "Ah, my heart, you leave me!" "Is it for Kithaeron?" "Does he truly deserve it?" "How delightful this place is!" "How pleasant it is!" "Ah, how favourable it is for one who wishes to surrender her liberty..." "What ground have you to hope that Kithaeron will yield to you?" "I see how he, often from afar... like a shy lover avoids my presence." "What?" "Thus become enamoured..." "Alas!" "I believe so!" "Of a mere mortal!" "One must make a choice!" "Where to direct my tenderness?" "Never does love touch the cold hearts of our gods..." "Eros, Eros is with me!" "My love is coming, I see him!" "Blessed denizens, neighbours in these groves, quit your gloomy marshes..." "Make haste, come swiftly and gather among the tender grasses!" "Even if you are seen by no one, your voices shall be heard... celebrating this happy moment!" "Let your voices applaud me, that the air resounds!" "Sing and cry out all, raise your voices in unison with these charming birds, whose songs are so sweet!" "...with these charming birds, whose songs are so sweet!" "So sweet!" "Let our voices applaud, that the air resounds!" "Let all sing and cry out raise your voices in unison with these charming birds, whose songs are so sweet..." "Let our voices applaud, that the air resounds!" "Let all sing and cry out... raise your voices in unison with these charming birds, whose songs are so sweet..." "Has some sweet anxiety brought you to this place?" "No." "I seek solitude." "One can find better." "The Dryads can be found, who come gladly to such deserted places." "And even the moist water nymphs, all sense it, all sense that you are worthy of it." "Could I dare to inspire the divinities?" "Respect prevents me from it." "It would be as good... to hear a more tender sentiment." "Words of compliment... are always gladly heard." "For a lover who knows how to please, there is no rank too high, even though he may have faults, or may be overly bold." "For a lover who knows how to please, there is no rank too high..." "Audacious love..." "Yours is circumspect." "It is true, I see it, that all adore you, and my profound respect..." "What?" "Respect again!" "How tedious it is, this respect!" "Alas!" "Alas!" "And how suspect..." "I am so moved." "Cruel one, you laugh!" "I see in your face that you have divined my secret!" "Ah, charming victor, you love me not..." "No, you disdain my heart!" "Were you so shy..." "No, you are untrue!" "No!" "Untrue to me!" "Untrue to me!" "Tell me then, tell me why?" "Why?" "Why?" "Why?" "Naiad, calm yourself at the sight of Mercury." "He descends from the heavens, I see him." "Mercury?" "Can it be?" "No doubt, and it shows that one of the Gods, filled with love..." "What?" "What?" "What?" "What?" "What?" "Goddess, who reigns over this superb marsh, over subjects without number, wandering among the grasses..." "Would you find unworthy of your shackles, the god who launches thunder?" "This god, drawn to earth by your beauty, wishes to cast at your feet his heart and the universe." "Can I believe it, fair Mercury, that a pure flame burns in your heart for me?" "Can I be sure enough of this, to breath a gentle sigh..." "Plataea is worthy of this dazzling glory..." "No longer will you blame an indifferent spirit for unachieved happiness." "All in you bore witness to the glorious fortune... with which the love of a great God would elevate you..." "All in you bore witness to the glorious fortune with which the love of a great God would elevate you..." "Plataea is worthy of this dazzling glory..." "But this God, so full of ardour, to lay siege to my heart, why tarries he so long?" "He will come, and soon, to be near you." "The darkening heavens gives me the portent." "The goddess of the winds signals her rage." "But nothing can stop her spouse!" "I have little fear of her anger, in my moist empire, such storms are welcome." "Let us announce this splendid day to the nymphs of my court..." "Leave, nymphs, quit..." "Quit your deep dwellings!" "A torrent of celestial waves will inundate our abode..." "Quit your deep dwellings..." "Quit your deep dwellings, a torrent of celestial waves will inundate our abode..." "A torrent of celestial waves will inundate our abode..." "And you, Juno, weep," "increase my realm!" "Weep..." "Weep, Juno..." "Increase my realm!" "Leave, nymphs, quit... quit your deep dwellings." "A torrent of celestial waves will inundate... our abode." "Dark rain clouds, gather together..." "Fall upon us..." "Fall upon us..." "Dark rain clouds, gather together..." "Dark rain clouds, gather together..." "Fall upon us..." "Flood our rivers..." "Flood our rivers, we welcome even your ravages..." "Flood our rivers, we welcome even your ravages..." "Sun, you shine in vain, the humid Naiads deny you your wish..." "The humid Naiads deny you your wish..." "We wish to compell the Hyads" "to extinguish forever your beams and your fire." "Extinguish forever your beams and your fire..." "Nymphs, the north winds comes to disturb the festivities." "I see Iris leading the way." "An impetuous wind agitates the reeds, return to the depths of the waters!" "I come from assuaging Juno in her wrath, with words she believed sincere." "Athens will become the object of her rage, and already she consoles herself with the hope of catching the nymph and her spouse together!" "See how she hurries on." "And now in all freedom Jupiter will appear." "Here he comes." "Let us hide here in these woods!" "We shall see all, yet remain unseen." "Audacious Aquilons, fear my rage, flee this place!" "To see the beautiful creature who is so dear to me, and to pay her homage both deep and sincere," "I have left my abode in the heavens." "Audacious Aquilons, fear my rage, flee this place!" "From the appearance of this cloud I cannot be mistaken." "I cannot be mistaken." "Jupiter can dare all." "But have I the courage to receive his homage, or to decline it..." "The cloud opens, I see movements," "I see movements, I believe my adorable suitor will be revealed!" "I believe, I believe, he will be revealed, my adorable suitor..." "What a metamorphosis!" "Should I approach?" "I dare not." "Surely it is a test, that Jupiter has prepared for my new passion..." "Come..." "I am faithful, whatever disguise you take on." "Teach me what love inspires in you, and what your heart desires." "You sigh... and I sigh." "A tender word suffices." "You tell me all without speaking to me." "Oh, how eloquent love is..." "What?" "What?" "What?" "You disappear!" "In what new plumage do you appear before me as the finest of owls..." "Birds of the forest..." "Come all..." "Come, come, come all..." "Sing..." "Sing!" "But what chatter is this?" "Birds, you are jealous!" "Change your tongue..." "Pay homage to the finest of owls..." "Pay homage to the finest of owls..." "Pay... homage... to the finest of owls." "Alas!" "He flies away!" "I see him no more." "Jupiter!" "Jupiter!" "My cries are in vain." "Jupiter!" "Jupiter!" "My cries are in vain." "My heart must then despair!" "Alas!" "He flies away!" "I see him no more..." "Heavens!" "What a terrible visitation!" "Enchanting object of my worthy love, no longer shall you be plagued by doubt." "On my help you can rely..." "I free my hands of the dreaded thunder!" "I have not come to alarm you." "With you, Jupiter becomes more gentle, seeking nothing but the pleasure of loving you..." "Are you insensitive to my tender avowals?" "I offer you my undying vows..." "You give no reply." "Forgive me," "I gasp for air... and sigh at the same time." "Whilst we await the preparation of our sweet nuptials, let all rejoice here in my new conquest!" "Momus, bring all your games together!" "Let the gaiety of the festivities be as boundless as my ardour!" "Myriad subjects, may high delight... forever reign in my court." "Come!" "Give glory on this day, to the God who inspires you!" "How comical she is!" "How beautiful she is..." "Who could not but yield to such charms..." "How comical she is!" "How beautiful she is..." "Jupiter sighs for her..." "Behold the enchanting object..." "Behold the enchanting object." "But a new harmony likely announces Terpsichore or Thalia." "You are mistaken, Momus, no, no!" "What do I see?" "Oh, heavens!" "It is I," "Folly... the one who has stolen Apollo's lyre." "Honour, honour to Folly, who bears Apollo's lyre who bears Apollo's lyre!" "Let us pour forth the most brilliant concerts!" "When Jupiter is enchained... by the incomparable Plataea," "I want to express the transports of his enchanted soul through my diverse songs." "Let all admire my celebrated art." "May my songs transform a sorrowful image into one of joy." "May my songs transform a sorrowful image into one of joy!" "Daphne refused Apollo's amorous longings..." "Daphne refused." "Upon her tomb, Eros extinguished the flame and transformed her..." "Upon her tomb," "Eros extinguished the flame and transformed her and transformed her..." "Thus has love always taken vengeance..." "How cruel love can be... when unrequited when unrequited..." "Daphne refused Apollo's amorous longings..." "Upon her tomb..." "Eros extinguished the flame and transformed her and transformed her..." "Honour, honour to Folly!" "She surpasses Polyhymnia!" "Honour to her divine words..." "Honour, honour to Folly!" "Judge by the simple beauty and touching sounds, how well I know the melody." "Listen well... above all to my symphony." "Delightful pleasures, walk alongside our steps," "careless pleasures, within your arms the flames of our ardour burn anew..." "If Zephyr did not tease," "Flora Flora would be less faithful to him..." "Careless pleasures..." "You admire my supreme art," "I can turn joy itself into sorrow... with my plaintive, pitying sound." "I will finish with a stroke of genius." "Assist me, I sense I can achieve a masterpiece of harmony..." "Hymen, God of Marriage," "Eros summons you!" "Hymen, God of Marriage," "Eros summons you!" "Hymen, God of Marriage," "Eros summons you!" "Prepare for Jupiter a new union..." "Prepare a new union." "Come and crown his new Juno!" "Good, good, good!" "May the flame that devours his soul set your torch alight!" "Good, good, good!" "Good, good, good!" "Set my torch alight!" "...may the flame that devours his soul... set your torch alight that devours his soul..." "Good, good, good!" "May the flame set your torch alight!" "Good, good, good!" "May the flame that devours his soul set your torch alight!" "May the flame..." "Good, good, good..." "Hatred, scorn, jealous rage, I give my heart over to you..." "Smother my love for a wanton husband, inspire me with your fury!" "Hatred, scorn, jealous rage," "I give my heart over to you." "Halt!" "Jupiter was not in Athens." "You tricked me!" "You frustrated my desire!" "What pleasure do you find in redoubling my suffering?" "No!" "Soon... you will have newfound joy." "Though I serve Jupiter, applaud my noble intentions still:" "They serve you more than your spouse." "Do not believe you can thus appease my wrath!" "I want to catch that faithless one!" " Alas!" "He desires no one but you!" "In this very place he will appear." "Await the right moment to make yourself known, and put aside your jealousy for a moment!" "Let us sing, let us celebrate today the power of Eros..." "Through love, the nymph can aspire to a union with the greatest god." "And the most glorious king can yield to a shepherdess." "Let us sing, let us celebrate today the power of Eros..." "Let us sing, let us celebrate the power of Eros!" "During these festivities, my heart prepares itself to receive in ardour the avowals of my lover..." "But we lack something at this moment, for my happiness and yours, the Gods of Marriage and Love, or at least one of the two the Gods of Marriage and Love, or at least one of the two." "Mercury, tell me, why do these little gods not follow me to this place?" "These little gods, as you know, assemble but rarely." "Chance alone brings them together, be it on land, upon the waves, or even in the skies..." "What?" "Must await them yet longer?" "My agitated heart grows impatient, aroused by the importunate gravity of Terpsichore's fair sons..." "What do I see?" "Is it Eros, coming with his weapons to pierce my heart with further darts?" "Since he has come expressly for me, let him approach..." "He cannot come close enough." "Let him approach..." "He cannot come close enough..." "It is Momus..." "Is he not just as charming as Eros?" "Almighty Eros, having business elsewhere," "cannot appear here in person." "He charged me with bringing you all his blessings." "Give, give, it will be the same." "I give tears..." "Fie on it!" "Sweet pains..." "Fie on it!" "Cries... and longing..." "Fie, fie!" "These are afflictions." "If I should love, then I wish sweet and gentle things." "Receive, at least, the flattery of hope!" "Your hope is only suffering, a true sign of pain..." "Your hope is only suffering..." "Shoot your arrows, Eros..." "Empty your quiver, bring to us... your victory!" "Bring to us your victory..." "Shoot your arrows, Eros..." "Empty your quiver!" "Shoot your arrows, bring to us your victory..." "Add fresh exploits to your glory..." "Shoot your arrows, empty your quiver..." "Shoot your arrows, bring to us your victory..." "What noise is this?" " Come, gentle Graces!" "So jealous is Eros of your glory, that he wishes them to follow your steps, and answer for you in all places." "I thought the Graces so dull... but their amorous capers..." "Admire the effect of my great genius!" "I make them lively at times, then innocent, naive..." "Always encouraging them to outdo themselves in charm." "But... who else is coming?" "Nymph, your conquest has created such a sensation, that in the all surrounding villages, the people are in a daze." "And my people, on such a great day, wish to join in the noble festivities." "Plataea has won the heart of the greatest of all immortals." "Embellish this festival in her honour and dedicate altars to her!" "Sing the praises of Plataea, rejoice, sing of the power of her charms..." "Sing, sing, sing of the power of her charms..." "Let us sing the praises of Plataea, let us rejoice..." "Let us sing of the power of her charms!" "Let us sing..." "The god who has yielded to her will shower us with his gifts." "Let us sing, dance, leap..." "Let us sing the praises of Plataea, let us rejoice..." "Let us sing, dance, leap..." "Let us sing of the power of her charms!" "Let us sing the praises of Plataea, let us rejoice..." "Behold the instant to end this pretence." "But Juno has yet to come." "She is near this place." "Let a solemn bond..." "But when this fear?" "You do not doubt the power of your eyes?" "I am thinking of your former wife." "Ha, there!" "What do you fear from her?" "She is, it is said... jealous." "We shall give free rein to her powerless rage." "To celebrate such a legitimate nuptial bond..." "I swear..." "I swear..." "I swear..." "Stop, you ingrate!" "You shall not carry out this horrible deed!" "Mighty in my wrath," "I seize my victim!" "What do I see?" "Oh, heavens!" "You see your mistake." "My astonishment is great." "What confusion follows my suffering!" "Do you still doubt my love for you?" "No." "You have restored calm in my heart." "Let us leave this place!" "Let us ascend to the home of thunder." "The earth has not the right to detain the sovereign of gods any longer." "Let us sing the praises of Plataea, let us rejoice..." "Let us sing of the power of her charms..." "Be silent!" "Be silent!" "Be silent, or I will punish you with death..." "Let us sing the praises of Plataea, let us rejoice..." "What?" "Is my wrath so little to be feared?" "I shall disturb everything," "I shall trouble the waves, and from my deep cavern I shall launch my blows!" "I shall trouble the waves, and from my deep cavern I shall I shall launch my blows!" "Let us sing the praises of Plataea, let us rejoice!" " Be silent!" "Let us sing..." " Be silent, or I will punish you with death!" "Be silent, be silent, or I will punish you with death." "You see my rage, tremble with fear!" "I accuse you alone for this outrage!" "You!" " Me?" " Yes, you!" "Accuse the ingrate who deceived you!" "You see my rage, tremble with fear!" "Yes, you!" "Accuse the ingrate who deceived you!" "I accuse you alone..." " Accuse the ingrate..." "You see my rage, tremble with fear..." " Accuse the ingrate..." " You..." "You alone!" " Me?" "I accuse you alone!" "Let us sing the praises of Plataea, let us rejoice..." " Be silent!" "Or I will punish you with death!" " Let us sing..." "What?" "What?" "What?" "You dare to defy my blows?" "Let us sing!" "Let us make haste... and assuage my rage!"


It pays to know the price

It might have taken more than 60 years, but the price of cannoli at Mike’s Pastry is a mystery no more.

Mike’s, an institution in Boston’s North End, has been attracting tourists and locals alike for decades. The cash-only bakery had held fast to a policy of not posting any prices, but now, a few pieces of plain paper visible over the counter list the different cannoli prices — not all cannoli are created equal — and the prices of some other items.

Mike’s General Manager Angelo Papa says posting the prices has been a benefit for customers and the shop. Now, he says, “My customers, when they’re waiting in line, have their money ready.” And, he adds, they can see prices for items that they might not have realized were for sale in the frequently crowded bakery.

Generally speaking, it’s a good idea to know how much something costs before you buy it. And, thankfully, the number of places engaging in this sort of surprise-the-consumer game appears to be dwindling.

Readers are irked about several different types of establishment that still keep you guessing — most notably restaurants. Why is it that they can get away with a drink menu devoid of prices? Or list “market price” for fish or shellfish and leave you wondering if you’re going to be dropping $20 or $50 when you order?

You could just ask. But that puts the burden on the consumer and some people like to quietly contemplate what they want to spend.

“Market prices are listed as such due to the continual fluctuation in prices of certain species and the cost involved in printing menus,” explained Massachusetts Restaurant Association president Peter Christie.

“Drink prices are often not listed due to the huge variables involving brands, costs and preferences.”

Barbara Anthony, who heads the state’s Consumer Affairs Office, said there is no specific law that addresses restaurant pricing. She acknowledges that can lead to frustration for consumers, but suggested using your consumer power to express your displeasure.

“There are some restaurants where there are no prices on the menu,” she said. “I don’t go to those restaurants.”

While she isn’t advocating for a new law, she said that informing customers of how much items cost before they buy is the right way to do business.

“The idea,” she said, “that you buy something and you don’t know the price is a totally alien concept to me as a consumer and as someone in consumer protection.”

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All relevant data are within the paper and its Supporting Information files.

Introduction {#sec001}
============

Schistosomiasis is a disease affecting more than 200 million individuals living mostly in underdeveloped tropical and subtropical regions (<http://www.who.int/mediacentre/factsheets/fs115/en/>). The disease is caused by infection with schistosome blood flukes which can survive, if untreated, for decades inside the vascular system of immune competent permissive hosts. Illness is primarily a consequence of immunopathology from schistosome eggs trapped in tissues (reviewed by \[[@pntd.0005306.ref001]\]). Despite the long term presence of adult worms in their vascular system, permissive hosts, by definition, do not typically develop immune responses directed at juvenile or adult worms \[[@pntd.0005306.ref002]\] that are capable of preventing new infections or eliminating all adult worms. This is not to imply that anti-worm immune responses are completely ineffective, as a measure of protective immunity following multiple rounds of reinfection has been documented \[[@pntd.0005306.ref003], [@pntd.0005306.ref004]\].

The humoral response to schistosome infection has been extensively characterized in efforts to identify antigens for use in the diagnosis of infection or that might elicit a response that protects vaccinated individuals from infection. To seek protective vaccine candidates, researchers have sought to identify antigens that are recognized more intensely by serum antibodies from animal models with demonstrated resistance to infection. Examples include comparisons in schistosome antigen recognition by: 1) non-permissive rats vs permissive mice models \[[@pntd.0005306.ref005], [@pntd.0005306.ref006]\]; 2) mice +/- vaccination by irradiated cercariae \[[@pntd.0005306.ref007]\], and; 3) humans displaying susceptibility vs putative resistance to infection \[[@pntd.0005306.ref008], [@pntd.0005306.ref009]\]. Through these efforts, dozens of schistosome antigens have been found to be recognized by antibodies in serum from infected humans or rodents (reviewed by \[[@pntd.0005306.ref010]--[@pntd.0005306.ref012]\]). While these efforts have identified antigens recognized by various infection sera, it remains unclear as to which antigens are most abundantly targeted by antibodies in these sera. This information could aid in understanding the nature of the normal humoral immune response to schistosome infection and provide important guidance to efforts seeking improved diagnostic tools and new vaccines.

In an effort to identify which proteins on larval and adult schistosomes are most abundantly recognized by antibodies in serum of infected rodents and humans, we began by probing western blots with these sera on total schistosome antigen extracts. To better preserve antigen conformation, we did not destroy protein disulfide bridges by chemical reduction prior to resolution on gels. Highly diluted sera from schistosome infected rats, mice and humans (1:2000) all, surprisingly, produced similar banding patterns on blots of total extracts of schistosomula and adults, revealing a remarkably small number of antigen bands that predominate. When the antigen samples were reduced, these major bands were no longer observed by the same diluted sera.

Based on a variety of inferences, explained below, we selected a set of candidate schistosome antigens which were then expressed within insect cells. Here we use extracts of these cells to identify two tetraspanins: SmTsp2 and Sm23 and three Ly6 family proteins: Sm29, SmLy6B, and SmLy6F, as the major schistosome antigens recognized by the antibodies in dilute serum of infected mice, rats and humans. All of these proteins are reported to be present within the host-interactive surface tegument of schistosomes, and two of the antigens, SmTsp2 and Sm29, are currently strong candidates as vaccine immunogens to prevent schistosomiasis \[[@pntd.0005306.ref008], [@pntd.0005306.ref013], [@pntd.0005306.ref014]\]. We show that the infection serum antibodies recognizing these two antigens can be largely blocked by two previously reported monoclonal single-chain Fv domain antibodies (scFvs Teg1 and Teg4 \[[@pntd.0005306.ref015]\]), which suggests that the serum primarily recognizes limited epitopes on these antigens. These findings have significant implications regarding both the mechanisms by which schistosomes evade immune damage and the development of schistosomiasis vaccines, which are discussed.

Results {#sec002}
=======

Western blots of total larval and adult *S*. *mansoni* extracts probed by sera from recently infected mice, rats or humans {#sec003}
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Total antigen extractions were performed on larval and adult schistosomes by boiling the freshly frozen parasites in an SDS gel loading buffer under non-reducing conditions. Serum was typically diluted to 1:2000 so as to preferentially reveal the antigens recognized by the more prevalent anti-schistosome antibodies. [Fig 1A](#pntd.0005306.g001){ref-type="fig"} shows that similar patterns are recognized on Western blots prepared from either juvenile (5 day cultured schistosomula) or adult worm extracts when probed with pooled sera prepared from *S*. *mansoni*-infected mice, rats and humans ('infection sera'). All three infection sera recognize antigens of the same apparent MW, though with differences in intensity, on both juvenile and adult worm extracts. The predominant staining species were estimated to be about 11, 15, 20, 29 and 37 kDa (arrows, [Fig 1A](#pntd.0005306.g001){ref-type="fig"}) by comparison to molecular weight standards.

![Serum from *S*. *mansoni* infected mice, rats and humans recognize a limited number of reduction-sensitive epitopes on western blots of total worm extracts.\
Total extracts from juvenile (5 day cultured schistosomula) or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under reducing (0.1% βME) or non-reducing conditions and transferred to filters. Filters were probed with serum from mice, rats or humans (1:2000) that were infected by *S*. *mansoni*. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. A. Filters from a non-reducing gel probed by pooled sera from ten chronically infected mice; pooled sera from five, twice-infected Fischer rats, or; pooled serum from 8 human patients identified as having schistosomiasis. B. Filters from a reducing gel probed with the same sera employed in A. C. Filters from a non-reducing gel probed with five different pools of human sera (NR, SR, I, RR, NI), each derived from eight patients displaying common apparent susceptibility or resistance to schistosome re-infection (for details on these sera, see Pinheiro et al, 2014 \[[@pntd.0005306.ref013]\]). Numbers represent molecular mass markers (kDa).](pntd.0005306.g001){#pntd.0005306.g001}

Surprisingly, recognition of these major bands is largely lost from the infection sera when the same schistosome extracts are resolved on western blots under reducing conditions at 0.1% β-mercaptoethanol (βME) ([Fig 1B](#pntd.0005306.g001){ref-type="fig"}). A similar dramatic reduction in western blot signals produced by mouse, rat or human infection sera was also observed when the (non-reducing gel) filters were pre-washed with 0.1% βME prior to serum incubation (not shown). These results demonstrate that the major epitopes on the dominant schistosome antigens present in both larval and adult schistosome extracts are reduction-sensitive, and thus likely to be conformational epitopes dependent on disulfide cross-linkages.

Studies were also performed to test the consistency of the infection sera western blot data when using independent pools of serum, and to compare sera from human patients having different infection histories. First, during the course of our studies we generated a second pool of mouse infection sera and found antigen recognition within juvenile and adult schistosome extracts to be virtually indistinguishable and both sera were used interchangeably (not shown). Next we compared four different serum pools from patients with different schistosome infection histories and different apparent susceptibilities to infection (see \[[@pntd.0005306.ref013]\]). Two of the pools were obtained from patients that had active schistosome infections (I, infected; SR, susceptible to reinfection) and two pools from patients that had frequent exposures to cercariae and yet were currently uninfected (RR, resistant to reinfection; NR, natural resistant). Unlike the I and NR groups, the SR and RR patients had been recently treated with praziquantel. A fifth pool of serum (NI, noninfected) came from people having no history of schistosome exposure. Each group contained both male and female donors. As shown in [Fig 1C](#pntd.0005306.g001){ref-type="fig"}, while the pools produced variable signal intensities, all pools produced similar banding patterns. Non-infected patient sera completely lack any signal, demonstrating the absence of background under the conditions used. Overall, these results suggest that the schistosome antigen recognition patterns reported here with infection sera, when resolved on gels under non-reducing conditions, are almost certainly representative of infection sera from other rodent and human individuals, even those having differences in their infection histories. Thus, we conclude that the dominant recognition of these five schistosome antigen bands appear to be generally consistent across various host genetic backgrounds and parasite exposure histories.

We previously reported \[[@pntd.0005306.ref015]\] the isolation of four recombinant single-chain Fv proteins (scFvs) recognizing surface antigens on live schistosomes that we obtained from the B cells of schistosome infected rats, three of which recognize living schistosomula ([Table 1](#pntd.0005306.t001){ref-type="table"}). Two of the scFvs were shown to recognize known tegumental antigens; Teg1 recognizes the tetraspanin SmTsp2 \[[@pntd.0005306.ref016]\], Teg4 recognizes Sm29 \[[@pntd.0005306.ref017]\] (also called SmLy6D \[[@pntd.0005306.ref018]\]). A third scFv (Teg5) was shown to weakly recognize living schistosomes, and an scFv called S3 had been obtained earlier from the same source \[[@pntd.0005306.ref019]\] which recognizes the schistosome tetraspanin antigen, Sm23. When schistosome extracts resolved by non-reducing gels were probed by western blotting with these scFvs, several produced strong signals ([Fig 2](#pntd.0005306.g002){ref-type="fig"}). These signals were dramatically diminished or undetectable when the extracts were resolved on reducing gels (not shown). Of interest, each of the scFvs recognized antigens having the same mobility as antigens recognized by the various (mouse, rat, human) infection sera depicted in [Fig 1](#pntd.0005306.g001){ref-type="fig"}. Teg1 and S3 recognized their tetraspanin targets (Tsp2 and Sm23) as 20 and 37 kDa species ([Fig 2](#pntd.0005306.g002){ref-type="fig"}, arrows). The presence of the larger species suggests that the \~20 kDa monomers become partially cross-linked into \~37 kDa dimeric forms under the non-reducing conditions. Teg4 strongly recognized the 29 kDa Sm29 while Teg5 recognized a minor antigen of about 20 kDa.

![Anti-tegumental rat scFvs recognize distinct juvenile and adult *S*. *mansoni* antigens when resolved on non-reducing gels.\
Total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Filters were probed by one of four different anti-tegumental scFvs (Teg1, S3. Teg4 and Teg5, all at 1 μg/ml) previously obtained from *S*. *mansoni* infected rats. Detection was by HRP/anti-E-tag recognition of a peptide E-tag expressed with each scFv. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g002){#pntd.0005306.g002}

10.1371/journal.pntd.0005306.t001

###### Summary of anti-tegumental rat scFvs.

![](pntd.0005306.t001){#pntd.0005306.t001g}

  scFv name   *S*. *mansoni* antigen                         Binding to surface of live schistosomula
  ----------- ---------------------------------------------- ------------------------------------------
  S3          Sm23^\[^[@pntd.0005306.ref019]^\]^             No[^b^](#t001fn002){ref-type="table-fn"}
  Teg1        SmTsp2^\[^[@pntd.0005306.ref015]^\]^           Yes^\[^[@pntd.0005306.ref015]^\]^
  Teg4        Sm29^\[^[@pntd.0005306.ref015]^\]^             Yes^\[^[@pntd.0005306.ref015]^\]^
  Teg5        SmLy6C[^a^](#t001fn001){ref-type="table-fn"}   Yes^\[^[@pntd.0005306.ref015]^\]^

^a^Identified in this report ([Fig 4](#pntd.0005306.g004){ref-type="fig"}).

^b^No staining observed with this scFv.

Identification of the major antigens recognized by schistosome infection sera--candidate antigen selection {#sec004}
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To identify the reduction-sensitive schistosome antigens from larval and adult worm extracts that are recognized by dilute schistosome infection sera on Western blots, we sought to produce conformationally-native recombinant versions of a set of selected candidate antigens and test their ability to specifically block infection sera antibodies recognizing one or more of the major bands. Three obvious candidate antigens were SmTsp2, Sm23 and Sm29 based on the fact that our prior studies had identified them as targets of scFvs that were obtained from B cells of schistosome-infected rats \[[@pntd.0005306.ref015]\], and the fact that the gel mobility of their target antigens matched those of the three larger antigens (20, 29, 37 kDa) recognized by infection sera (Figs [1](#pntd.0005306.g001){ref-type="fig"} and [2](#pntd.0005306.g002){ref-type="fig"}).

The two other major bands recognized by schistosome infection sera had apparent MWs of 11 or 15 kDa. This small size is similar to that of most members of the SmLy6 family of membrane proteins \[[@pntd.0005306.ref018], [@pntd.0005306.ref020]\]. The Ly6 proteins are GPI-anchored proteins, some of which are known to be present in the schistosome tegument \[[@pntd.0005306.ref020], [@pntd.0005306.ref021]\], and all are expected to have conserved, reduction-sensitive, intra-chain disulfide linkages within their extracellular domains \[[@pntd.0005306.ref022]\]. Several of the smaller SmLy6 proteins are also known to be well-expressed by mammalian stage schistosomes \[[@pntd.0005306.ref020]\]. In fact, Sm29 is also a Ly6 family protein (SmLy6D) containing two linked Ly6 domains \[[@pntd.0005306.ref018]\]. Based on their small size, sensitivity to reduction and available expression data, we selected four additional members of the SmLy6 family: SmLy6A, SmLy6B, SmLy6C and SmLy6F (nomenclature from \[[@pntd.0005306.ref018]\]) as strong candidates to be major antigens recognized by schistosome infection sera. These four SmLy6 proteins were also called SmCD59.1, SmCD59.2, SmCD59.3 and SmCD59.4, respectively, in another recent report on putative schistosome membrane antigens \[[@pntd.0005306.ref020]\].

Recognition of selected schistosome membrane proteins by infection sera {#sec005}
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Early efforts to produce recombinant schistosome SmTsp2 and Sm29 proteins were performed in *E*. *coli* host cells transformed with expression vectors containing the extracellular regions of these proteins fused to *E*. *coli* thioredoxin (Trx) to facilitate protein folding. Western blots with these purified recombinant proteins were poorly recognized by rat infection sera or the scFvs, probably because the reduction-sensitive epitopes were not reproduced well when expressed in *E*. *coli* cytosol. We thus began employing the baculovirus expression system within insect cell hosts in an effort to generate recombinant membrane protein antigens that we expected would better retain the native conformations of these proteins on schistosomes.

The seven rationally selected schistosome membrane protein candidates (above) were each expressed in recombinant insect cells as described in Materials and Methods. The SmLy6 proteins (SmLy6A, SmLyB, SmLy6C and SmLy6F) were expressed with an insect secretory leader and each contained a FLAG-tag epitope (DYKDDDDK) that remained at their amino ends following signal processing. The tetraspanin proteins, SmTsp2 and Sm23 were expressed using their complete *S*. *mansoni* coding DNA. Sm29 was also expressed with its natural signal peptide and using full-size coding DNA. Recombinant insect cells expressing the schistosome antigens were harvested and the membrane proteins solubilized in a lysis buffer containing 1% Triton X-100 and no reducing agents.

Western blots were prepared from insect cell extracts containing the four small recombinant SmLy6 proteins (SmLy6A, B, C, F) and then probed with anti-FLAG antibody (Ab). As shown in [Fig 3A](#pntd.0005306.g003){ref-type="fig"}, under non-reducing conditions the insect cell extracts expressing SmLy6A, B and F contained FLAG-tagged proteins in the expected size range (\~12 kDa). For each of the extracts, one or two larger FLAG protein species were also present in the 15--20 kDa size range. Unexpectedly, insect SmLy6C extracts did not contain a FLAG protein of the expected size, but instead contained several bands clustered about 19 kDa. Higher molecular weight forms were visible with all four SmLy6 proteins. When the SmLy6 extracts were resolved on gels under reducing conditions, the lower MW bands remained while most of the 15--20 kDa bands and higher MW forms were no longer visible, suggesting that the higher MW forms are multimers stabilized by disulfide linkages.

![*S*. *mansoni* infected rat sera recognize reduction-sensitive epitopes in recombinant insect cell extracts containing SmLy6 proteins.\
Insect cells engineered to express SmLy6A, SmLy6B, SmLy6C and SmLy6F were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing or reducing conditions, as indicated. Filters with insect cell extracts containing recombinant SmLy6A, B, C or F were probed by anti-FLAG (**A**) or rat infection sera (1:2000) (**B**). Detection employed HRP/anti-FLAG tag or HRP/anti-rat IgG. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g003){#pntd.0005306.g003}

The same four recombinant SmLy6 insect cell extracts were also probed with pooled *S*. *mansoni* rat infection sera ([Fig 3B](#pntd.0005306.g003){ref-type="fig"}). All four extracts contained a protein species recognized by the rat sera that was a subset of bands recognized by anti-FLAG, indicating that only some of the recombinant protein had folded to a conformation recognized by the infection sera. Surprisingly, for SmLy6A, C and F, the bands primarily recognized by immune rat sera were the MW species \~15--20 kDa. These bands are larger than expected and may be glycosylated. Only the SmLy6B extract contained a band of \~11 kDa, the expected size of these SmLy6 proteins based on coding sequence, which was clearly recognized by the rat infection sera. As when probing the schistosome extracts ([Fig 1](#pntd.0005306.g001){ref-type="fig"}), rat infection sera recognition of the four recombinant SmLy6 proteins was lost when the gel was run under reducing conditions ([Fig 3B](#pntd.0005306.g003){ref-type="fig"}, right panel).

Insect extracts expressing SmTsp2, Sm23 and Sm29, which lack a FLAG tag, were resolved on non-reducing or reducing gels and probed with the appropriate scFvs (Teg1, S3, Teg4 respectively) or with rat infection sera ([Fig 4](#pntd.0005306.g004){ref-type="fig"}). As expected, the three scFvs recognized their insect cell expressed target antigens. Rat infection sera produced a virtually identical recognition pattern as the scFvs on replicate non-reducing blots, indicating, as has been previously reported, that schistosome infection sera contain antibodies recognizing SmTsp2, Sm23 and Sm29 \[[@pntd.0005306.ref008], [@pntd.0005306.ref009], [@pntd.0005306.ref023]\]. Most or all recognition of these proteins by the scFvs or the infection serum was lost when these recombinant insect extracts were resolved under reducing conditions.

![*S*. *mansoni* infected rat sera recognize reduction-sensitive epitopes in recombinant insect cell extracts containing SmTsp2, Sm23 or Sm29.\
Insect cells engineered to express selected SmTsp2, Sm23 or Sm29 were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing (-) or reducing (+) conditions. **A**. Filters containing insect cell extracts containing SmTsp2 were probed with Teg1 scFv or rat infection sera (1:2000). Detection employed HRP/anti-E-tag for Teg1 scFv, or HRP/anti-rat IgG. **B**. Filters containing insect cell extracts containing Sm23 were probed with S3 scFv or rat infection sera (1:2000). Detection employed HRP/anti-E-tag for S3 scFv, or HRP/anti-rat IgG. **C**. Filters containing insect cell extracts containing Sm29 were probed with Teg4 scFv or rat infection sera (1:2000). Detection employed HRP/anti-E-tag for Teg4 scFv, or HRP/anti-rat IgG. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g004){#pntd.0005306.g004}

We also tested Teg5 scFv for recognition of the insect cell extracts containing the various *S*. *mansoni* membrane proteins. As shown in [Fig 5](#pntd.0005306.g005){ref-type="fig"}, Teg5 clearly recognizes a reduction-sensitive epitope on SmLy6C. Since Teg5 was shown to recognize living worms \[[@pntd.0005306.ref015]\], these results shows that SmLy6C can be expressed on the outside surface of larval and adult schistosomes. Thus Teg5, like Teg1 and Teg4, recognize conformational epitopes on schistosome tegumental antigen targets, consistent with the finding that rat infection sera predominantly recognize reduction-sensitive epitopes on schistosome antigens.

![Anti-*S*. *mansoni* tegument scFv, Teg5, recognizes reduction-sensitive epitopes on SmLy6C.\
Insect cells engineered to express recombinant SmLy6A, B, C or F were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing or reducing conditions and transferred to filters. Filters were probed by Teg5 scFv and recognition detected with HRP/anti-E-tag. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g005){#pntd.0005306.g005}

Each of the insect extracts expressing the selected schistosome membrane proteins were characterized by non-reducing Western blots for their recognition by serum from infected rats, mice and humans. Blots with the five SmLy6 proteins are shown in [Fig 6A](#pntd.0005306.g006){ref-type="fig"} and reveal that rat, mouse and human infection sera recognize each of the proteins, though with variable intensity. In general, mice and rats recognize SmLy6A and SmLy6B more intensely than SmLy6C and SmLy6F, while the reverse was true with the human infection serum pool. All infection sera recognized Sm29 to a similar extent. Results with the two tetraspanin proteins, SmTsp2 and Sm23, were more variable ([Fig 6B](#pntd.0005306.g006){ref-type="fig"}). When quantified by imaging, the rat infection serum pools recognized both proteins about equally, while the mouse pool recognized Sm23 with \>10x greater intensity than SmTsp2 and the human serum pool recognized SmTsp2 with \>10x greater intensity than Sm23.

![*S*. *mansoni* infected mouse, rat and human sera recognition of selected *S*. *mansoni* antigens expressed as recombinant proteins by insect cells.\
Insect cells engineered to express selected *S*. *mansoni* membrane antigens were extracted in a mild detergent buffer and resolved by SDS-PAGE under non-reducing conditions. **A.** Replicate filters containing SmLy6A, SmLy6B, SmLy6C, SmLy6F and Sm29 were probed by mouse, rat or human infection sera (1:2000). **B**. Replicate filters containing SmTsp2 and Sm23 were probed by mouse, rat or human infection sera. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g006){#pntd.0005306.g006}

Identification of major *S*. *mansoni* antigens by infection sera pre-incubation with recombinant proteins {#sec006}
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Mild detergent extracts of insect cells expressing a single recombinant schistosome antigen were next tested for their ability to block recognition of specific bands produced by various infection sera on western blots of SDS-PAGE resolved juvenile and adult schistosome extracts (Figs [7](#pntd.0005306.g007){ref-type="fig"}--[10](#pntd.0005306.g010){ref-type="fig"}). The insect cell extracts from all of the seven rationally selected schistosome membrane antigens were separately incubated with mouse, rat or human infection sera prior to and during the incubation of the sera with western blots of total extracts of juvenile and adult schistosomes. All the results shown were repeated at least three times, and compared two different pools of mouse infection sera and four different pools of human infection sera over the course of these studies, and yet produced consistent results. In some cases, we were unable to completely block recognition of a specific band through pre-incubation with an insect cell extract, and it is possible in these cases that the insect cell recombinant protein lacks a conformer of the target antigen which is present in schistosome extracts, or that a second, unidentified minor antigen having the same gel mobility co-migrates with the major antigen species.

![Insect extracts containing recombinant SmLy6B block recognition of a major 11 kDa reduction-sensitive *S*. *mansoni* antigen on western blots by mouse and rat infection sera.\
Total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer (-) or lysates of insect cells expressing SmLy6B (+) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of SmLy6B. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g007){#pntd.0005306.g007}

![Insect extracts containing recombinant SmLy6F block recognition of a major 15 kDa reduction-sensitive *S*. *mansoni* antigen on western blots by mouse, rat and human infection sera.\
Total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer (-) or lysates of insect cells expressing SmLy6F (+) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of SmLy6F. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g008){#pntd.0005306.g008}

![Insect extracts containing recombinant Sm29 block recognition of a major 29 kDa reduction-sensitive *S*. *mansoni* antigen on western blots by mouse, rat and human infection sera.\
Total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer (-) or lysates of insect cells expressing Sm29 (+) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of Sm29. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g009){#pntd.0005306.g009}

![Insect extracts containing recombinant tetraspanins, SmTsp2 and Sm23 block recognition of a major 20 kDa reduction-sensitive *S*. *mansoni* antigen on western blots by mouse, rat and human infection sera.\
Total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Pooled mouse, rat or human infection sera (1:2000) were pre-incubated for 1 hr with control lysate buffer or lysates of insect cells expressing SmTsp2 or Sm23 (as indicated) prior to overnight incubation with the filter strips. Detection employed the appropriate HRP/anti-mouse, -rat or -human IgG reagent. The arrow indicates the position of migration of SmTsp2 and Sm23. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g010){#pntd.0005306.g010}

Figs [7](#pntd.0005306.g007){ref-type="fig"} and [8](#pntd.0005306.g008){ref-type="fig"} show that pre-incubation of rodent or human infection serum pools with insect cell extracts containing recombinant SmLy6B or SmLy6F specifically absorbed antibodies that recognize the 11 kDa or 15 kDa bands, respectively. Insect SmLy6B blocked the strong recognition of an 11 kDa band by infection sera from both of the rodent sera ([Fig 7](#pntd.0005306.g007){ref-type="fig"}). A band at 11 kDa was also weakly recognized by human infection sera and this band was reproducibly blocked by SmLy6B in similar blots, though it is not clear in the example shown. Insect extracts containing SmLy6F consistently blocked recognition of the 15 kDa band that is well recognized by rodent and human infection sera ([Fig 8](#pntd.0005306.g008){ref-type="fig"}). This antigen was not expressed well by insect cells and the blocking studies were less robust, yet we consistently observed reduction in the 15 kDa signals produced by all the infection sera. These results show that 11 kDa and 15 kDa schistosome antigens prominently recognized by infection sera on western blots of schistosome total extracts are SmLy6B and SmLy6F.

We were repeatedly unable to observe a reduction in the recognition of any of the antigens when the three infection sera pools were pre-incubated with extracts containing SmLy6A or SmLy6C (not shown). Negative results in these studies though are inconclusive due to uncertainty as to whether sufficient insect antigen, in its properly folded state, is present in the insect cell extracts to successfully block the infection serum antibodies. Nevertheless, since the rat infection sera recognize recombinant SmLy6A and SmLy6C ([Fig 3B](#pntd.0005306.g003){ref-type="fig"}), these results suggest that these antigens may not be sufficiently well expressed by larval or adult schistosomes to produce major bands on Western blots of total worm extracts.

Serum blocking studies with Sm29, shown in [Fig 9](#pntd.0005306.g009){ref-type="fig"}, were particularly dramatic and robust. The major 29 kDa antigen, which is present in both schistosomula and adult schistosomes and recognized by mouse, rat and human infection sera, is almost quantitatively quenched when the sera are pre-incubated with recombinant Sm29 extracts. Antibody recognition of Sm29 has proven to be extremely sensitive to reduction and other denaturing conditions (not shown), yet when care is taken in the preparation of schistosome extracts, Sm29 has consistently been observed as one of the two or three most dominant antigens recognized by infection sera from mice, rats and humans.

Similar blocking studies were also performed with insect extracts expressing SmTsp2 or Sm23 ([Fig 10](#pntd.0005306.g010){ref-type="fig"}). The major 20 kDa band recognized by all three infection sera was variably blocked by the presence of these extracts, likely because of the large variation in the titers of anti-SmTsp2 and anti-Sm23 antibodies in mouse vs human infection sera ([Fig 6B](#pntd.0005306.g006){ref-type="fig"}). While both extracts seemed to consistently diminish the 20 kDa signal produced by rat infection sera, only the Sm23 extracts had this effect on mouse infection sera. The human infection serum recognition of the 20 kDa band was largely and consistently blocked by incubation with SmTsp2 extracts. Sm23 extracts were not tested due to the negligible titer of Sm23 antibodies in human sera ([Fig 6B](#pntd.0005306.g006){ref-type="fig"}). We conclude that the major 20 kDa species recognized by the three infection sera is likely a mixture of both SmTsp2 and Sm23, and perhaps other proteins of this size such as other known tetraspanins \[[@pntd.0005306.ref024]\].

Schistosome infection sera primarily recognize a single conformational epitope on SmTsp2 and Sm29 {#sec007}
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We next tested whether our four anti-schistosome scFvs (Teg1, S3, Teg4 and Teg5) could effectively compete with antibodies in infection sera for binding to their antigen targets. In [Fig 11A](#pntd.0005306.g011){ref-type="fig"}, we individually tested our monoclonal scFvs for their ability to interfere with rat infection sera binding to their targets expressed by insect cells. Surprisingly, Teg5, Teg4, and Teg1, were able to almost completely block rat infection sera recognition of SmLy6C, SmTsp2 and Sm29 respectively, while pre-incubation of the filters with a different scFv had no effect. Only S3 scFv was unable to appreciably diminish recognition of its target, Sm23, by the rat sera.

![*S*. *mansoni* infected rat sera recognition of SmLy6C, SmTsp2 and Sm29 can be substantially suppressed by a single monoclonal scFv antibody.\
Detergent extracts of recombinant insect cells (A), or total extracts from juvenile or adult mammalian-stage *Schistosoma mansoni* worms (B) were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Replicate filter strips containing the indicated extracts were pre-incubated with the indicated scFv (Teg1, Teg4, Teg5 or S3, all at 10 μg/ml) or with buffer alone for 1 hr with *S*. *mansoni* rat infection sera (1:2000) prior to overnight incubation with the filter strips. Detection employed HRP/anti-rat IgG. Arrow indicates the position of migration of Sm29 and SmTsp2.Sm23. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g011){#pntd.0005306.g011}

We next tested the ability of the scFvs to block rat infection sera recognition of specific bands within the juvenile and adult schistosome total extract ([Fig 11B](#pntd.0005306.g011){ref-type="fig"}). The Teg1 and S3 scFvs both appeared to diminish recognition of the 20 kDa species in juvenile schistosomula, while only Teg1 had this effect in adult extracts. This is consistent with the results in [Fig 2](#pntd.0005306.g002){ref-type="fig"} showing that Sm23 is much more abundant than SmTsp2 in schistosomula than in adults. The results suggest that the Teg1 monoclonal scFv blocks most or all rat infection sera recognition of SmTsp2. This blocking effect was even more obvious with Teg4 pre-incubation which produced a major reduction in the rat sera recognition of the 29 kDa band identified as Sm29. Together these results suggest that the Teg1 and Teg4 scFvs bind epitopes that represent the dominant epitope for rat infection sera binding to either SmTsp2 or Sm29.

[Fig 12](#pntd.0005306.g012){ref-type="fig"} shows a Western blot comparing rat infection sera recognition of adult worm total extracts with an adult worm extract that is highly enriched in tegumental proteins \[[@pntd.0005306.ref015], [@pntd.0005306.ref025]\]. The figure summarizes the identities of the major antigens in total worm extracts recognized by infection sera (Figs [7](#pntd.0005306.g007){ref-type="fig"}--[10](#pntd.0005306.g010){ref-type="fig"}). The identities of these proteins in tegumental extracts were confirmed by antigen blocking studies (not shown). These results show that Sm29 and SmTsp2 (both monomer and putative dimer) are particularly enriched in the tegumental extracts. In contrast, SmLy6F, and particularly SmLy6B, are less enriched in the tegument extracts. This result is consistent with localization studies showing that SmLy6B is widely distributed throughout schistosomes, including the tegument (SmCD59.2 \[[@pntd.0005306.ref020]\]).

![*S*. *mansoni* infected rat sera recognition of total vs tegumental extracts of *S*. *mansoni* worms.\
Total extracts or tegumental extracts of adult mammalian-stage *S*. *mansoni* worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Filters were probed with rat infection sera. Major bands are identified (arrows) based on studies reported in this manuscript. Numbers indicate the positions of migration of molecular mass markers (kDa).](pntd.0005306.g012){#pntd.0005306.g012}

Discussion {#sec008}
==========

The use of non-reducing SDS-PAGE for the resolution of schistosome antigens substantially changes the nature of western blot data generated on filters probed by the sera of infected mice, rats and humans when compared to standard reducing gels. Studies reported here reveal that a major portion of serum antibodies from *S*. *mansoni* infected mice, rats and humans recognize reduction sensitive epitopes on a limited number of antigens in total extracts of schistosomula and adult worms. Using recombinant schistosome antigens expressed in eukaryotic insect host cells, we unambiguously identified dominant antigens as three Ly6 family proteins- SmLy6B, SmLy6F and Sm29; and two tetraspanins- Sm23 and SmTsp2. Of course it remains possible that other major conformational antigens are present in the schistosome extracts, but not observed on western blots because their epitopes are denatured by SDS-PAGE, perhaps because their conformations are not stabilized by the presence of disulfide linkages. While many other antigens not observed in our studies are known to be recognized by schistosome infection sera, the titers of these antibodies must be relatively low and/or the antigens present in low abundance in total worm extracts such that their signals are low or undetectable under the conditions we used. The parasite may even benefit by somehow biasing the host response away from some antigens, for example nutrient transporters or sensing receptors, against which a robust antibody response could be harmful to parasite survival.

All of the major antigens identified in this study are predicted to be membrane proteins and all except SmLy6F have been reported to be associated with the schistosome tegument in proteomic studies \[[@pntd.0005306.ref026], [@pntd.0005306.ref027]\]. Notably, SmLy6F has not been identified in any schistosome fractions through proteomics despite its predicted abundance \[[@pntd.0005306.ref020]\], perhaps because it is small, with several predicted N-linked glycosylation sites. Two of these antigens, SmTsp2 and Sm29, are currently strong candidates as vaccine immunogens to prevent schistosomiasis \[[@pntd.0005306.ref008], [@pntd.0005306.ref013], [@pntd.0005306.ref014]\] and we previously showed that both of these antigens have epitopes exposed on the surface of living schistosomes \[[@pntd.0005306.ref015]\]. SmLy6B was shown to be a surface tegumental GPI-anchored protein due to its susceptibility to worm surface cleavage by phospholipase C \[[@pntd.0005306.ref021]\]. These observations seem to suggest that host antibody responses during schistosome infection are targeted largely to a limited number of conformational epitopes present on tegumental membrane proteins, particularly those present at the host/parasite interface. The presence of glycosylation on some of these antigens may help limit epitope availability.

Finding two tetraspanins, Sm23 and SmTsp2, as major antigens recognized by *S*. *mansoni* infection serum, was not surprising. Both of these proteins have long been identified as important schistosome antigens \[[@pntd.0005306.ref008], [@pntd.0005306.ref028]\], and SmTsp2 is a particularly promising vaccine antigen \[[@pntd.0005306.ref008]\]. More surprising was the observation that the prominent epitopes are reduction-sensitive and infection sera recognition of SmTsp2 can be largely blocked with a single scFv (Teg4). These results suggest that tetraspanin recognition by infection sera involves a limited number of conformational epitopes. The functions of Sm23 and SmTsp2 remain unknown, although a role for SmTsp2 in tegumental turnover has been inferred \[[@pntd.0005306.ref029]\].

The identification of three Ly6 family proteins as dominant schistosome antigens was unexpected as, prior to this study, only Sm29 had been identified as an antigen during schistosome infections \[[@pntd.0005306.ref009]\]. The two other Ly6 family proteins identified as major antigens in these studies are SmLy6B and SmLy6F. Expression of both proteins is dramatically upregulated following transformation of cercariae to schistosomula \[[@pntd.0005306.ref020], [@pntd.0005306.ref030]\]. These proteins are both related to the complement inhibiting protein CD59, though SmLy6B was tested and shown not to possess this activity when expressed on recombinant mammalian cells \[[@pntd.0005306.ref020]\], while SmLy6D remains untested. To date, no evidence as to the functions of these proteins has been experimentally demonstrated despite their immunogenicity during schistosome infection and the identification of at least eight other schistosome Ly6 proteins encoded in the genome \[[@pntd.0005306.ref018]\].

The results of this study, and our previous study \[[@pntd.0005306.ref015]\], clearly show that schistosome infections result in a high titer of antibodies capable of binding to the living schistosomes. In the prior study, we found that infection sera from both mouse and rat clearly stained the surface of living schistosomula and lung worms. The staining was apparent only with infection sera, not pre-infection sera, showing that this staining was not non-specific binding to the antibody Fc domain \[[@pntd.0005306.ref031]\]. In this study, we find high titers of antibodies that recognize known tegumental surface antigens, SmLy6B, SmTsp2 and Sm29, and recognition of SmTsp2 and Sm29 can be largely blocked by pre-treatment with Teg1 and Teg4, scFvs known to recognize living schistosomes \[[@pntd.0005306.ref015]\]. These findings seem to contradict earlier reports that schistosomes bound little host antibody \[[@pntd.0005306.ref032], [@pntd.0005306.ref033]\], although results would be affected by any denaturants that destroy conformational epitopes. Our findings also seem inconsistent with multiple literature reports showing that humoral responses to tegumental antigens can be protective \[[@pntd.0005306.ref005], [@pntd.0005306.ref006], [@pntd.0005306.ref033]--[@pntd.0005306.ref035]\]. We find that a very similar host humoral response to the schistosome tegument occurs in both permissive (humans and mice) and non-permissive hosts (rats), suggesting that the presence of these anti-tegument antibodies have little demonstrable impact on the progression of schistosomiasis. We conclude that differences in susceptibility between rats, mice and humans are unlikely due to differences in their humoral responses to major tegumental antigens although differences in isotype production in different species might be important. In addition, we cannot exclude that humoral responses to less abundant surface antigens could be protective, particularly neutralizing antibodies targeting essential parasite functions.

The results reported here have significant implications for schistosomiasis vaccine development. Most obviously, the results imply that induction of an antibody response to the major surface tegument proteins through vaccination may not be protective on its own, because a response of this type is already present following natural infection within permissive hosts such as mice and humans. A similar humoral response to these major conformational antigens was found among human patients that display evidence of both susceptibility and resistance to schistosome infection, suggesting that these antibodies provide no obvious protective benefit to the host. Yet several reports show that two of the major antigens we find to be recognized by serum antibodies following schistosome infection, SmTsp2 and Sm29 \[[@pntd.0005306.ref008], [@pntd.0005306.ref013], [@pntd.0005306.ref014]\], are also among the most promising vaccine antigens. One likely explanation is that these vaccines are effective because they elicit a cell-based immune effector response targeting the host-interactive schistosome tegument. These vaccine immunogens are typically produced in prokaryotic hosts which often do not reproduce the conformation of the native proteins and thus the humoral response they elicit is likely poor at binding to live schistosome *in vivo*. These same vaccine immunogens, though, are known to be capable of eliciting a Th1 response that can be harmful to schistosomes \[[@pntd.0005306.ref013], [@pntd.0005306.ref014]\], and it is likely that this is primarily responsible for the reduction in parasite load following vaccination. It will be interesting to compare the potency of SmTsp2 or Sm29 vaccines prepared in prokaryotic host cells vs conformationally native antigens produced in a eukaryotic host.

The results reported here seem to suggest that efforts to identify schistosome vaccine candidates that promote antibody-mediated damage to worms should focus on antigens that are not among the five antigens we identify as targets of major conformation-dependent antibodies within infection sera. These findings also suggest that antibody binding to the tegument is, in itself, insufficient to result in host immune damage to the parasites and protection from infections. Thus, our findings seem to best support the use of an 'Achilles Heel' (also called \"Waksman\'s postulate\") approach \[[@pntd.0005306.ref036], [@pntd.0005306.ref037]\] in which the vaccine target would be a host-exposed functional antigen, essential to worm survival, that is not naturally immunogenic. Antibodies generated to such targets through vaccination should block the function of this antigen, thereby damaging the parasite and protecting the host from future schistosome infections.

A striking finding of these studies is the surprising predominance of antibodies in schistosome infection serum recognizing a limited set of reduction-sensitive epitopes, particularly those on SmTsp2 and Sm29. These epitopes proved to be poorly reproduced when these antigens were expressed by *E*. *coli*. For example, Supplemental [S1 Fig](#pntd.0005306.s001){ref-type="supplementary-material"} shows that the major antigens in schistosome extracts generate strong signals on western blots with rat infection serum despite loadings too low for detection by conventional protein staining. In contrast, a recombinant version of SmLy6-2 produces a barely detectable, reduction-sensitive band on an infection serum blot despite an easily detected protein band. Similar results were obtained with the other SmLy6 proteins, including Sm29, and the recombinant proteins themselves were found to largely self-aggregate (not shown). An exception to this was SmTsp2 in which the *E*. *coli* recombinant protein did produce a strong, reduction-sensitive band on a western blot with infection serum. As another example, we attempted to express Sm29 in insect cells by replacing the schistosome leader sequence with a leader sequence from insect melittin, and even this minor change resulted in a protein that was unrecognized by the Teg4 scFv and much more poorly recognized by infection sera (not shown). These results highlight the importance of using conformationally-native immunogens when preparing antibodies for detection of schistosome tegumental antigens under natural conditions, such as for *in situ* localization or live worm staining.

It is interesting to speculate whether our surprising finding of a strong apparent bias to a limited subset of reduction sensitive tegumental epitopes in the humoral response during schistosome infection has implications with regard to schistosome host immune evasion. The subject of immune evasion by schistosomes has been extensively investigated (reviewed by \[[@pntd.0005306.ref038]\]) and the studies have suggested that schistosomes employ evasion strategies such as minimal protein exposure at the tegument surface, host antigen masking, epitope concealment by carbohydrates, poor immunogenicity of essential antigens, induction of blocking antibodies, secretion of immune modulators and rapid tegument turnover. Our results suggest that schistosomes may somehow elicit a strong antibody response to a limited set of surface antigens on which antibody binding is well-tolerated and perhaps even somehow beneficial to the parasite. For example, these anti-tegumental antibodies could serve as 'blocking Abs' \[[@pntd.0005306.ref039]\] that prevent induction of a damaging antibody response. It is worth noting that any agent that induces a minor conformational change to the epitope targeted by these anti-tegumental antibodies will result in the dissociation of the antibody, and schistosomes may somehow exploit this feature to evade immune damage.

Materials and Methods {#sec009}
=====================

Ethics statement {#sec010}
----------------

All studies followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) under Protocol \# G2015-113. This protocol adheres to the National Institutes of Health's *Public Health Service Policy on Humane Care and Use of Laboratory Animals*. The protocol for the use of human sera was previously approved by the Ethical Committee of the Federal University of Minas Gerais and the patients or their legal guardians provided written informed consent after receiving an explanation of the protocol.

Reagents {#sec011}
--------

Horseradish peroxidase HRP-conjugated anti-E-tag antibodies were purchased from Bethyl Labs; HRP-conjugated anti-rat IgG, HRP-conjugated anti-mouse IgG, and HRP-conjugated anti-human IgG were purchased from Santa Cruz.

Schistosome infection sera {#sec012}
--------------------------

The rat infection sera were obtained by performing two *S*. *mansoni* infections of non-permissive Fischer rats, each with 1,000 cercariae, performed 8 weeks apart as previously described \[[@pntd.0005306.ref015]\], and the serum was drawn four weeks after the second infection. Two preparations of mouse infection sera were employed, each obtained from two groups of 10 Swiss-Webster mice infected with 120 cercariae nine weeks earlier. All of the mice survived and contained adult worms at the time the serum was obtained. The pools of serum were prepared by mixing equal amounts of serum from each infected mouse. All human infection sera were obtained from individuals living in two different endemic areas for schistosomiasis ("Melquiades" and "Côrrego do Onça", Minas Gerais, Brazil). Four different pools of serum (previously reported in \[[@pntd.0005306.ref013]\]) were prepared that each included serum from eight patients. Patients in the pools included sixteen that had egg counts at the time of infection (eight that were recently treated with praziquantel), and sixteen that were stool negative despite known exposure to water contaminated with cercariae (eight there were recently treated with praziquantel). These infected individuals were examined for *S*. *mansoni* infection using the Kato--Katz technique and were negative for other helminthic infections as previously described \[[@pntd.0005306.ref013]\].

Schistosome extracts {#sec013}
--------------------

Five day old schistosomula were obtained from the Biomedical Research Institute (Rockville, MD, USA). The schistosomula were produced by in vitro transformation of cercariae and five days of tissue culture following the procedure of \[[@pntd.0005306.ref040]\]. Schistosomula were homogenized in PBS, briefly sonicated to shear the DNA, and diluted in non-reducing SDS buffer. Adult worms were recovered by perfusing infected Swiss Webster mice at 6--7 weeks post infection. Parasites' extracts were prepared by homogenizing the adult parasites in PBS on ice. Extracts were briefly sonicated to shear the DNA then diluted in non-reducing SDS buffer. Preliminary Western blot experiments were run to determine the appropriate amount of extract to be used in subsequent experiments.

Production of recombinant proteins in *E*. *coli* {#sec014}
-------------------------------------------------

The rat scFv proteins \[[@pntd.0005306.ref015], [@pntd.0005306.ref019]\] were expressed in *E*. *coli* host cells as fusions with an amino terminal *E*. *coli* thioredoxin to facilitate folding, with a hexa-histidine tag for purification and a carboxyl terminal E-tag epitope for detection. The expression, purification and quantification were performed as previously described for VHH antibody proteins \[[@pntd.0005306.ref041]\].

Production of recombinant schistosome membrane proteins in insect cells {#sec015}
-----------------------------------------------------------------------

Synthetic DNA encoding SmLy6A, B, C, F, SmTsp-2, Sm23 and Sm29 was ordered from GenScript Inc in which the codon usage is optimized by the manufacturer for insect expression. Restriction sites were included to facilitate simple ligation into the pFastBac vector (Invitrogen). For SmLy6 proteins, the natural schistosome signal sequence (predicted by SignalP) was replaced with the insect melittin signal sequence (MKFLVNVALVFMVVYISYIYA). Immediately downstream from the signal sequence, we included the following spacer coding sequence (AADYKDDDDKGGGGS) which includes a Not1 cloning site, FLAG epitope and an enterokinase cleavage site. The schistosome mature protein coding sequences followed through the carboxyl terminus. For SmTsp-2, Sm23 and Sm29, the synthetic coding DNA included the complete schistosome protein including leader sequence. We made an additional construction in which the natural signal peptide for Sm29 was replaced with the melittin signal sequence and the spacer sequence described above.

The synthetic coding DNAs were ligated into pFastBac and engineered for insect cell expression using the Bac-to-Bac system (Invitrogen) following the procedures recommended by the manufacturer. Typically, the first virus supernatant was used to infect Sf9 cells at an estimated MOI of 0.1 and cells were harvested 3--5 days post-infection. Recombinant insect cell pellets were either solubilized directly into non-reducing SDS Laemmli buffer (BioRad), or for use in western blot competition studies, we employed a non-reducing insect cell lysis buffer (1% Triton-X-100, 10mM Tris pH 8.0, 140 mM NaCl, 1x Sigma protease inhibitor cocktail).

Production of recombinant schistosome membrane proteins in *E*. *coli* {#sec016}
----------------------------------------------------------------------

DNA encoding the predicted extracellular domains of SmLy6B, C, F, SmTsp-2, and Sm29 was amplified by PCR and ligated into a pET32 E. coli expression plasmid in frame with the *E*. *coli* thioredoxin (Trx) coding DNA and containing hexahistidine and epitope tags. Coding DNA cloning, expression and purification of these recombinant schistosome proteins were all performed by standard methods as previously reported \[[@pntd.0005306.ref041]\].

Western blotting {#sec017}
----------------

Proteins were resolved by SDS-PAGE using AnyKd mini-PROTEAN TGX gels from BioRad under reducing (1.5% βME unless otherwise specified) or non-reducing conditions. Proteins were transferred from gels to Immobilon-P PVDF membranes (Fisher) using a BioRad semi-dry transfer apparatus. NEB Color Prestained Protein Standard, Broad Range (11--245 kDa), which contains only trace reducing agent, was used for estimating molecular weight and to guide the excision of filter strips. Membranes were blocked with 5% non-fat dry milk, 0.1% Tween-20 in PBS (mPBS) for 30 minutes at room temperature or overnight at 4°C. Primary antibody incubations (dilutions indicated in figure legends) were overnight at 4°C in fresh mPBS, and secondary antibody incubations (as recommended by manufacturers) were for one hour at room temperature, all followed by 3 washes, 5--10 minutes each, in PBS, 0.1% Tween-20 (PBST). In some cases, the filters were incubated for 1 hour at room temperature in mPBS 0.1% β-mercaptoethanol. The resulting immunoblots were developed using the ECL Western Blotting detection system from GE Healthcare, as recommended by the manufacturer, and imaged on a BioRad ChemiDoc Touch instrument.

Supporting Information {#sec018}
======================

###### Comparison of rat infection serum recognition of schistosome extracts and schistosome antigens expressed in recombinant *E*. *coli*.

Total extract of adult mammalian-stage *Schistosoma mansoni* worms, or purified recombinant proteins representing the extracellular domains of either schistosome antigen SmLy6-2 or SmTsp2 expressed by *E*. *coli* host cells (with an *E*. *coli* thioredoxin (Trx) fusion partner), were resolved by SDS-PAGE under non-reducing or reducing conditions. The same loadings of each antigen preparation were run in replicate, then either stained with Coomassie Blue to identify protein species (Protein) or transferred to filters and probed with 1:2000 rat infection sera (Rat sera). Numbers indicate the positions of migration of molecular mass markers (kDa).

(TIF)

######

Click here for additional data file.

We greatly appreciate the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) Biomedical Research Institute for providing the 5 day cultured schistosomula used in these studies.

[^1]: The authors have declared that no competing interests exist.

[^2]: **Conceptualization:** GKP CBS.**Data curation:** CBS.**Funding acquisition:** PJS CBS.**Investigation:** GKP MD JMT AAD.**Methodology:** GKP MD JMT SCO AAD PJS CBS.**Project administration:** CBS.**Resources:** SCO AAD PJS CBS.**Supervision:** CBS.**Visualization:** CBS.**Writing -- original draft:** CBS.**Writing -- review & editing:** GKP MD JMT SCO AAD PJK CBS.


Aga Khan Mzizima Secondary School

The Aga Khan Mzizima Secondary School, commonly referred to as Mzizima, is a co-educational non-government school in Dar es Salaam, Tanzania. It was founded in 1967 and is operated by the Aga Khan Education Services, an agency of the Aga Khan Development Network. The school's motto is "Better Always Better" and it is one of Tanzania's leading non-government schools.  For three consecutive academic years (2000-2001, 2001-2002 and 2002-2003), Mzizima was awarded the Certificate of Excellence (First Grade) from the Minister of Education and Culture and Inspectorate for exemplary performance at the Tanzanian National Examinations.

It adopted the International Baccalaureate system before the 2005/2006 academic year began.
Aga Khan Mzizima has been successful on the sports front, winning numerous tournaments, especially in basketball and football. The school holds an Annual Fiesta towards Mid-May open to all youths of the community which leads to achieving their sole purpose which is bonding.

Notable alumni
 Asanterabi Malima, researcher

See also
Aga Khan Education Services
Aga Khan Development Network

Dar es Salaam Mzizima
Category:Private schools in Tanzania
Category:International Baccalaureate schools in Tanzania
Category:Educational institutions established in 1967
Category:Education in Dar es Salaam
Category:1960s establishments in Tanzania


ORCHARD PARK, N.Y. -- Bills offensive lineman Cyrus Kouandjio remained in a Buffalo-area hospital Thursday afternoon after a bizarre encounter with police Wednesday.

The Buffalo News reported Thursday, citing a police source, that Erie County Sheriff's deputies found Kouandjio partially clothed in a field after he'd climbed over an electric fence. Kouandjio had abandoned his vehicle on a Buffalo-area highway.

Buffalo offensive lineman Cyrus Kouandjio remained hospitalized Tuesday, two days after he climbed over an electric fence and was found by police in a field. Rich Graessle/Icon Sportswire

Kouandjio was taken to Erie County Medical Center.

"We're going through the process right now of gathering the information, and that's all I can tell you at this time," Bills coach Sean McDermott said Thursday.

The Bills' second-round pick in 2014, Kouandjio had been recovering from hip surgery resulting from a fall at home, the team announced in January. He is expected to be ready for training camp.

Kouandjio started five games last season in place of injured left tackle Cordy Glenn. In 25 career games, Kouandjio has made seven starts.


A multifaceted investigation of the link between mental fatigue and task disengagement.
Mental fatigue is often characterized by reduced motivation for effortful activity and impaired task performance. We used subjective, behavioral (performance), and psychophysiological (P3, pupil diameter) measures during an n-back task to investigate the link between mental fatigue and task disengagement. After 2 h, we manipulated the rewards to examine a possible reengagement effect. Analyses showed that, with increasing fatigue and time-on-task, performance, P3 amplitude, and pupil diameter decreased. After increasing the rewards, all measures reverted to higher levels. Multilevel analysis revealed positive correlations between the used measures with time-on-task. We interpret these results as support for a strong link between task disengagement and mental fatigue.


Regional chromosomal localisation of APOA2 to 1q21-1q23.
Using in situ hybridisation, we have mapped APOA2 to the 1q21-1q23 region of chromosome 1. DNA hybridisation to somatic cell hybrids made from cells carrying a balanced translocation between X and 1 confirms the localisation as proximal to 1q23. This was further confirmed by the presence of two polymorphic alleles in a cell line carrying a deletion of 1q25-1q32.


India shuts down internet once again, this time in Assam and Meghalaya - bigpumpkin
https://techcrunch.com/2019/12/13/internet-shutdown-india-assam-meghalaya/
======
csense
> the Indian government sent in troops and shut down the internet

How does this work on a practical level?

I have so many questions. Are there a bunch of armed squads kicking in
datacenter doors with AK-47's and flashbangs? A group of (the Indian
equivalent of) FBI agents in a room making phone calls to the CEO's of the
ISP's, telling them to go dark or go to jail? Or does the Indian government
have a killswitch that just turns it off whenever they feel like it?

What if someone's operating a node that continues forwarding traffic from the
quarantine zone to outside? How do they locate it?

Also, I always thought India's a relatively free country, it's not like Iran
or China where the government can just stomp on the people as hard as it
wants. What does the Indian court system think about this?

------
ganzuul
I once heard about a mob killing a foregin engineer doing volunteer work at a
rural school because a rumor spread on some IM platform that he was abducting
children.

India sure is different. This action might have a very different context than
what we Westerners assume. Or not. Maybe a local can explain if there is a
difference.

~~~
rhnmhdv
Nope, its more or less in the same vein. There are big protests happening in
India and they're trying to control/silence it as much as possible.

------
IXxXI
India continues to push a cashless society paradigm. What the article neglects
to mention is how these types of heavy handed obstructionist activities
undermine faith in electronic finance which cannot continue normally with
internet blackouts. Forcing people to return to utilizing the paper money
which governments apparently do not want.


THE PRESIDENT: Hello, everybody! (Applause.) Well, how’s the food? (Cheers and applause.) We could not have a more beautiful day for this annual picnic, and we are just thrilled that all of you had a chance to get over. I know that folks were running a little bit late, many of you because of votes. But, you know, the fact of the matter is, is that we have had as tough a year and a half as America has seen in a couple of generations, and that's meant that Congress has had to do more on a whole range of issues.

Right now obviously we’re looking at what’s happening in the Gulf with deep concern, and that's going to put more additional pressure on Congress to work with states and the administration to help deal with this tragedy and this crisis.

It is important, though, given the incredible sacrifices that so many of you have made and frankly, from my perspective, just as important are the sacrifices that your family makes -- you being away, you missing family events -- that every once in a while we have an opportunity to get together and for me to say thank you to you for your incredible devotion to the country, regardless of party, and for both Michelle and I to say to your families how grateful we are for your service, and hopefully to have a little bit of time for fun and for fellowship and to remind ourselves of what’s so important in life.

So I’m thrilled to see all the young people here who are here today. And we hope that you have a wonderful time.

Band, you guys are great as always. (Applause.) Chefs, I think people are all voting with their mouths around here. (Applause.) I suspect there’s not going to be a lot of leftovers.

And on behalf of Michelle and myself, we hope that you enjoy the evening, and we are grateful to all of you for being here.


Role of Hydrogen Bonding on Transport of Coadsorbed Gases in Metal-Organic Frameworks Materials.
Coadsorption of multicomponents in metal-organic framework (MOF) materials can lead to a number of cooperative effects, such as modification of adsorption sites or during transport. In this work, we explore the incorporation of NH3 and H2O into MOFs preloaded with small molecules such as CO, CO2, and SO2. We find that NH3 (or H2O) first displaces a certain amount of preadsorbed molecules in the outer portion of MOF crystallites, and then substantially hinders diffusion. Combining in situ spectroscopy with first-principles calculations, we show that hydrogen bonding between NH3 (or H2O) is responsible for an increase of a factor of 7 and 8 in diffusion barrier of CO and CO2 through the MOF channels. Understanding such cooperative effects is important for designing new strategies to enhance adsorption in nanoporous materials.


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Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.
Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.