Otyłość u dzieci prowadzi do zaburzeń metabolicznych i zmian strukturalnych w naczyniach, które są przyczyną rozwoju chorób układu krążenia i cukrzycy typu 2.badania była ocena czynników ryzyka miażdżycy u dzieci otyłych badanych w Oddziale Hospitalizacji Jednego Dnia Instytutu Matki i Dziecka.Zbadano 75 dzieci w wieku 6-12 lat  z BMI >97 centyla, stanowiącą grupę badaną. Grupę kontrolną stanowiło:36 dzieci w wieku 5-10 lat  z BMI 75-90.Analizowano dane z wywiadu dotyczące otyłości, chorób układu krążenia i zaburzeń lipidowych w rodzinie, u badanych dzieci analizowano BMI, obwód pasa, lipidogram, stężenie insuliny, grubość kompleksu błony wewnętrznej i środkowej tętnicy szyjnej (IMT) w obu grupach.U 82,6% otyłych dzieci z grupy badanej stwierdzono w rodzinie otyłość, a u 72% choroby układu krążenia i zaburzenia przemiany lipidowej, u dzieci z grupy kontrolnej: u 34,2% była otyłość w rodzinie a u 36,8% choroby układu krążenia i zaburzenia lipidowe. Średni obwód pasa u otyłych dzieci wynosił 72,7 cm, w grupie kontrolnej 59,9 cm . Średnie stężenie cholesterolu, LDL, trójglicerydów było wyższe w grupie badanej . Poziom insuliny u otyłych był prawie dwukrotnie wyższy niż w grupie kontrolnej . Średnia grubość IMT u otyłych dzieci wynosiła 0,36 mm ; ±0,059 SD (strona prawa) i 0,37 mm; ±0,033 SD (strona lewa). W grupie kontrolnej: 0,323 mm; ±0,087 SD (strona prawa), 0,32mm; ±0,082 SD (strona lewa), różnice między grupami były istotne statystycznie. Znaleziono dodatnią korelację pomiędzy obwodem pasa i poziomem insuliny. Wnioski: Otyłość i zaburzenia lipidowe częściej dotyczą dzieci obciążonych rodzinnie otyłością i chorobami układu krążenia. Zaburzenia lipidowe statystycznie częściej występują u dzieci otyłych. IMT jest znamiennie większa u dzieci otyłych w porównaniu z grupą kontrolną co sugeruje, że zmiany miażdżycowe struktury naczyń szyjnych mogą wystąpić u dzieci otyłych już we wczesnym dzieciństwie. Pozwala je wykryć nieinwazyjna ultrasonograficzna metoda oceny IMT. Dzieci z otyłością, szczególnie trzewną mają wyższy poziom insuliny, co może przyczynić się do rozwoju insulinooporności.U rodziców brak jest dostatecznej wiedzy o skutkach otyłości i jego wpływu na występowanie miażdżycy i chorób układu krążenia. Nadwaga i otyłość stanowią narastający problem w medycynie rozwojowej na całym świecie. Według raportu opracowanego przez The International Obesity Task Force co piąte dziecko w Europie ma nadwagę lub jest otyłe . RównieżOtyłość jest jednym z najważniejszych czynników rozwoju miażdżycy, która jest procesem chorobowym, a jego istotą są zmiany zapalno-proliferacyjne prowadzące do uszkodzenia ściany tętnicy. Zmiany miażdżycowe mogą rozwijać się jednocześnie w różnych naczyniach. Podstawą ich rozwoju jest dysfunkcja śródbłonka naczyń , 7, 8. CMiażdżyca, prowadząca do uszkodzenia i zwężenia naczyń krwionośnych należy do głównych zdrowotnych zagrożeń cywilizacyjnych współczesnego świata. Procesy te zaczynają się już we wczesnym dzieciństwie i mogą prowadzić do rozwoju chorób układu krążenia w wieku średnim i starszym , 10, 11.Postępy w dziedzinie diagnostyki obrazowej pozwalają na mierzenie i ocenę grubości śródbłonka naczyń, który pierwszy ulega procesom patologicznym w dużych naczyniach obwodowych oraz w nerkach.tunica intima), błonę środkową (tunica media) oraz błonę zewnętrzną (tunica adventiva). Kompleksem intima-media nazywamy struktury ściany naczyniowej mierzone od linii granicznej przydanka–błona środkowa do linii granicznej błona wewnętrzna– światło naczynia. Pomiar grubości kompleksu błony wewnętrznej i środkowej tętnicy szyjnej wspólnej (IMT − intima-media thickness) pozwala na ocenę wczesnych zmian miażdżycowych stosując nieinwazyjną metodę ultrasonograficzną [W tętnicach możemy wyróżnić błonę wewnętrzną , które stanowiły grupę badaną i 36 dzieci z prawidłową masą ciała (BMI 75-90 centyl), które stanowiły grupę kontrolną. Wśród dzieci otyłych było 36 chłopców i 39 dziewczynek, w grupie kontrolnej 18 chłopców i 18 dziewczynek.Do wykonania badań zastosowano następujące metody:wywiad dotyczący występowania rodzinnych czynników ryzyka miażdżycy ,ocena wskaźnika BMI,pomiar obwodu pasa,badania biochemiczne: lipidogram, TSH , fT3, fT4, stężenie glukozy i insuliny we krwi na czczo,badanie usg szyi – pomiar grubości kompleksu błony wewnętrznej i środkowej ściany tętnicy szyjnej wspólnej (IMT).Badania krwi były wykonywane przy pomocy aparatu Cobas-Integra, usg szyi za pomocą aparatu usg firmy Philips U22. Pomiaru kompleksu błony wewnętrznej i środkowej tętnicy szyjnej dokonywano na ścianie dolnej tętnicy szyjnej, w pozycji typowej, w projekcji podłużnej, przy użyciu głowicy 7,5 MHz. Wykonywano dwa pomiary ze strony prawej i lewej, oznaczano średnią z pomiarów. Poziomy oznaczanych wskaźników lipidogramu porównywano do norm podanych przez NCEP  zależnych od wieku dzieci (za punkt odniesienia przyjęto wartości prawidłowe) .Wszystkie otyłe dzieci zostały skierowane na konsultacje dietetyczne.2, do badania korelacji – współczynnik korelacji rho-Spearmana.Do analizy statystycznej zastosowano: test Kołmogorowa-Smirnowa, test U Manna-Whitney’a, χ2(1)=26,53; p<0,001.Z danych z wywiadu wynika, że w grupie dzieci z otyłością (grupa badana) dodatni wywiad rodzinny w kierunku otyłości  dotyczył 82,6% dzieci. W grupie kontrolnej otyłość w rodzinie dotyczyła tylko 34,2% dzieci. Różnica jest istotna statystycznie: χW grupie badanej 32% dzieci miało otyłych obydwoje rodziców; 17,3% dzieci miało tylko otyłą matkę, natomiast 16% dzieci miało otyłego tylko ojca.2(1)=13,00; p<0,001.W tabeli I przedstawiono sumarycznie dane z wywiadu.W grupie badanej 72% miało obciążony wywiad rodzinny dotyczący również chorób układu krążenia i zaburzeń lipidowych, podczas gdy w grupie kontrolnej dotyczyło to 36,8% dzieci. Różnica między grupami jest istotna statystycznie: χDzieci z grupy badanej miały średnio 7,97 lat, zaś z grupy kontrolnej – 8,02 roku. Grupy nie różniły się istotnie pod względem wieku: t(110)=-0,15; p>0,05.Średnie BMI w grupie dzieci otyłych wynosiło − 24,3 , a w grupie kontrolnej z prawidłową masą ciała − 19,00 .Średni obwód pasa w grupie badanej wynosił 72,7 cm , w grupie kontrolnej 59,9 cm , różnica była istotna statystycznie, t(103)=8,171;p<0,001.Prawidłowe wartości składników lipidogramu wg NCEP dla dzieci z grupy kontrolnej, do których porównywano wyniki badań uzyskanych badanych przez nas dzieci były następujące: cholesterol całkowity <170 mg/dl, LDL<110 mg/dl, HDL>45 mg/dl, trójglicerydy (TG): do 9 r.ż. <75 mg/dl, 10-18 r.ż.<90 mg/dl.Dzieci z otyłością miały znacząco wyższe poziomy cholesterolu całkowitego i jego frakcji LDL oraz trójglicerydów. Średnie stężenie cholesterolu w tej grupie wynosiło 167 mg/dl , a w grupie kontrolnej 124 mg/dl . Różnica była znacząca statystycznie, t(111)=9,12; p<0,001. Wartości cholesterolu całkowitego powyżej 170 mg/dl dotyczyły 47% dzieci z otyłością (z grupy badanej).Średnie stężenie frakcji LDL cholesterolu w grupie badanej wynosiło 101 mg/ dl , a w grupie kontrolnej 72 mg/ dl , różnica była istotna statystycznie, t(110)=6,870;p<0.001. Wartości LDL>110 mg/ dl obserwowano u 33% dzieci otyłych.Średnie stężenie trójglicerydów w grupie badanej było wyższe i wynosiło 86 mg/ dl  w porównaniu z grupą kontrolną, u której było na poziomie 63 mg/ dl , różnica była istotna statystycznie, t(100)-3,028; p=0,003. Stężenie trójglicerydów >75mg/dl dotyczyło 61% dzieci z otyłością.W grupie badanej (z otyłością) niższe stężenie frakcji HDL cholesterolu (<45 mg/ dl) obserwowano u 21% dzieci otyłych.Stężenie insuliny na czczo u dzieci z grupy badanej było prawie dwukrotnie wyższe i wynosiło 10,27 mU/ l, w grupie kontrolnej było równe 5,88 mU/l, t(109)=3,054; p<0,01. Wszystkie badane dzieci miały we krwi prawidłowy poziom hormonów tarczycy oraz poziom glukozy na czczo. Na podstawie badań obrazowych stwierdzono, że u dzieci otyłych średnia grubość kompleksu ściany tętnicy szyjnej wewnętrznej i środkowej po stronie prawej wynosiła 0,36 mm; ±0,059 , a po stronie lewej 0,37 mm; ±0,033 . Nie była to różnica istotna statystycznie.W grupie kontrolnej wartości te były następujące: po stronie prawej 0,32 mm; ±0,087 , po stronie lewej 0,32mm ; ±0,082 . Nie była to różnica istotna statystycznie. U dzieci otyłych, stanowiących grupę badaną, średnia grubość kompleksu ściany wewnętrznej i środkowej tętnicy szyjnej wspólnej była większa niż w grupie kontrolnej, szczególnie dotyczyło to tętnicy lewej, różnica była istotna statystycznie, t(111)=2,394,p <0,01 po stronie lewej, natomiast po stronie prawej - t(111)=2,609, p<0,05. Dane zebrano w tabeli II.Analizowano również korelacje pomiędzy badanymi parametrami w grupie dzieci otyłych. Nie znaleziono istotnej korelacji pomiędzy BMI, stężeniem cholesterolu i jego frakcjami oraz poziomem trójglicerydów a grubością kompleksu ściany tętnicy szyjnej wewnętrznej i środkowej. Znaleziono dodatnią korelację pomiędzy poziomem insuliny na czczo a obwodem pasa u dzieci otyłych, im większy był obwód pasa tym wyższy był poziom insuliny, r=0,313, p<0,05.Wszystkie badane dzieci otyłe zostały skierowane na konsultację dietetyczną, ale tylko 35/75 dzieci zgłosiło się na wizytę: pod stałą opieką poradni dietetycznej pozostało 14/35 dzieci, 11/35 nie zgłosiło się na kolejny wyznaczony termin wizyty, a 10/35 dzieci było tylko na jednej wizycie i nie zgłosiły się na kolejne. 29/75 dzieci z grupy badanej zgłosiło się na badanie kontrolne do Oddziału Hospitalizacji Jednego Dnia, 20/29 z nich zgłosiło się po roku od pierwszej wizyty, a jedynie 9/29 po 2 latach. Siedmioro z 29 dzieci było dwukrotnie na wizycie kontrolnej, po roku i po 2 latach.Należy podkreślić, że tylko połowa otyłych dzieci skorzystała z zaplanowanych konsultacji dietetycznych i tylko 18% pozostało pod stałą opieką dietetyka. Na wizyty kontrolne do Oddziału zgłaszała się mała liczba pacjentów pomimo zaproszeń listownych lub telefonicznych.Otyłość jest narastającym problemem u dzieci polskich. Najczęściej mamy do czynienia z otyłością prostą związaną z nadmierną podażą energii w diecie. Jedynie w wyjątkowych przypadkach jest to otyłość wtórna do innych chorób lub uwarunkowana genetycznie. Na rozwój otyłości pierwotnej (prostej) ma wpływ wiele czynników, ale przede wszystkim tryb życia, nawyki żywieniowe, mała aktywność <zyczna lub jej brak. Otyłość częściej dotyczy dzieci, których rodzice są otyli, co wykazano w badaniu własnym, jak i innych autorów , 17.Dzieci otyłe częściej mają zaburzenia gospodarki lipidowej, szczególnie dotyczy to stężenia trójglicerydów, cholesterolu całkowitego i jego frakcji LDL. Według ostatnich doniesień Polskiego Towarzystwa Lipidologicznego, to właśnie frakcja LDL cholesterolu całkowitego ma największe znaczenie dla rozwoju blaszki miażdżycowej i nagłych zdarzeń sercowo-naczyniowych u dorosłych.W badaniu własnym średnie stężenie cholesterolu i jego frakcji LDL oraz trójglicerydów było znamiennie wyższe u dzieci z otyłością, co jest zgodne z doniesieniami wielu innych autorów , 18, 19.Wiele danych wskazuje, że markery i czynniki ryzyka sercowo-naczyniowego wykrywane w dzieciństwie, często stwierdzane są również u tych samych osób w wieku dorosłym. Najczęściej mamy do czynienia z nadmierną masą ciała, zaburzeniami w zakresie gospodarki lipidowej czy węglowodanowej , 20, 21.Bogalusa Heart Study wykazały istotną korelację między czynnikami ryzyka rozwoju miażdżycy, takimi jak, nadciśnienie tętnicze i otyłość we wczesnym okresie życia, a zmianami miażdżycowymi w aorcie i w tętnicach wieńcowych w wieku dorosłym [Większość dzieci ze zdiagnozowanymi, ale nieleczonymi zaburzeniami lipidowymi będzie charakteryzowała się nimi także w późniejszym okresie życia. Obserwacje z dorosłym .Przełomowe znaczenie dla zrozumienia patogenezy schorzeń układu krążenia miało dokonane ponad 20 lat temu odkrycie, że śródbłonek naczyniowy jest organem endo- i parakrynnym. Jego czynność odgrywa ważną rolę w regulacji napięcia ściany naczynia, proliferacji komórek mięśni gładkich, adhezji monocytów i leukocytów oraz procesów krzepnięcia, poprzez uwalnianie szeregu mediatorów rozszerzających naczynia utrzymuje właściwe napięcie ściany tętnicy. Nieprawidłowa funkcja śródbłonka traktowana jest jako wstępny etap rozwoju blaszki miażdżycowej , 22. CelW badanej grupie dzieci w niniejszej pracy średnia grubość IMT u dzieci otyłych wynosiła: w lewej tętnicy 0,37 mm, w prawej 0,36 mm i była znamiennie statystycznie wyższa niż w grupie kontrolnej . Podobne wyniki uzyskali inni badacze. W pracy Leite i wsp., oceniającej grubość IMT w grupie 150 portugalskich otyłych dzieci , średnia grubość kompleksu u otyłych dzieci wynosiła – 0,472 mm, w grupie kontrolnej − 0,418 mm , w badanAnalizowano również korelacje pomiędzy badanymi parametrami w grupie dzieci otyłych i nie znaleziono istotnej korelacji pomiędzy BMI, stężeniem cholesterolu i jego frakcjami oraz poziomem trójglicerydów a grubością kompleksu ściany tętnicy szyjnej wewnętrznej i środkowej (IMT). Według wielu autorów taka korelacja istnieje. W cytowanej wcześniej pracy Litwina i wsp. autorzy dowiedli, że w kształtowaniu wartości grubości IMT, oprócz biochemicznych czynników ryzyka rozwoju chorób sercowonaczyniowych rolę predyktora uszkodzenia ściany naczyń pełni także odpowiednio wysoka wartość BMI . Do podoW badaniach własnych zwrócono uwagę na grupę dzieci, które zgłosiły się na badania kontrolne. Obserwowano następującą zależność: gdy BMI tych dzieci wzrastało to również wzrastała grubość IMT, natomiast w przypadku obniżenia się wartości BMI zmniejszała się grubość kompleksu. Z uwagi jednak na małą grupę dzieci, u których takie zależności obserwowano zjawisko to wymaga dalszych badaniach. Jest wysoce prawdopodobne, że obniżenie masy ciała u otyłych dzieci może zahamować, a nawet być może cofnąć rozpoczynający się proces miażdżycowy w naczyniach.Insulina jest hormonem bezpośrednio działającym na komórki naczyń poprzez: zmniejszenie produkcji tlenku azotu, zwiększenie apoptozy komórek śródbłonka, obniżenie wychwytu glukozy, dodatkowo ma wpływ na ekspresję czynników wzrostowych i adhezyjnych oraz wzrost i migrację komórek mięśni gładkich naczyń. U osób z otyłością brzuszną dochodzi do rozwoju insulinooporności, która jest jednym z czynników rozwoju choroby niedokrwiennej serca. Rola hiperinsulinizmu w patogenezie miażdżycy jest złożona i nie do końca poznana , 26, 30.W badanej grupie dzieci stężenie insuliny na czczo było prawie dwukrotnie wyższe u dzieci otyłych niż w grupie kontrolnej . Znaleziono dodatnią korelację pomiędzy poziomem insuliny na czczo a obwodem pasa u dzieci otyłych, im większy był obwód pasa tym wyższy był poziom insuliny . Podobne obserwacje przedstawiają inni autorzy , 33. OtyBrak dostatecznej wiedzy wśród rodziców otyłych dzieci dotyczącej konsekwencji zdrowotnych związanych z nadmierną masą ciała wymaga podejmowania bardziej skutecznych działań uświadamiających społeczeństwo o skutkach nieprawidłowego stylu życia.Otyłość i zaburzenia lipidowe częściej dotyczą dzieci obciążonych rodzinnie otyłością i chorobami układu krążenia.Zaburzenia lipidowe statystycznie częściej występują u dzieci otyłych niż u dzieci z prawidłową masą ciała.Grubość kompleksu ściany tętnicy szyjnej wewnętrznej i środkowej jest znamiennie większa u dzieci otyłych w porównaniu z grupą kontrolna co sugeruje, że zmiany miażdżycowe struktury naczyń szyjnych mogą wystąpić u dzieci otyłych już we wczesnym dzieciństwie.Wczesne zmiany miażdżycowe u dzieci pozwala wykryć nieinwazyjna ultrasonograficzna metoda oceny grubości kompleksu błon wewnętrznej i środkowej tętnicy szyjnej.Dzieci z otyłością, szczególnie trzewną mają wyższy poziom insuliny co może przyczynić się do rozwoju insulinooporności.Wczesne leczenie dzieci otyłych może zahamować proces rozwoju miażdżycy i zapobiec rozwojowi chorób układu krążenia u tych dzieci w przyszłości.Brak dostatecznej wiedzy wśród rodziców otyłych dzieci dotyczącej konsekwencji zdrowotnych związanych z nadmierną masą ciała wymaga podejmowania bardziej skutecznych działań uświadamiających społeczeństwo o skutkach nieprawidłowego stylu życia prowadzącego do otyłości i jej powikłań.


Agudelo et al. discover expanded clones of similar antibodies from the memory B cells of tick-borne encephalitis virus–infected and vaccinated donors; however, the most potent and broadly neutralizing antibodies derive from donors recovering from natural infection. 50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI–EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC Tick-borne flaviviruses are responsible for a series of emerging infectious diseases including fatal encephalitis. Like other flaviviruses, the tick-borne encephalitis virus (TBEV) envelope protein (E) is composed of three structural domains . There is no specific therapy for TBE, and treatment is limited to supportive care. For those individuals who survive, long-term sequelae are common .Although TBEV vaccines are available, immunity requires regular boosting, and vaccination is less effective in the young and elderly. Vaccination requires administration of three separate doses spaced over up to 2 yr, with booster doses recommended at intervals of 3–5 yr . BreakthRecovered individuals can produce potent serologic neutralizing activity against TBEV, and the serum can be cross-reactive against other tick-borne flaviviruses, but the nature of the antibodies that mediate these effects is not known . PostexpHere, we report on the molecular properties of human anti-TBEV antibodies with exceptional neutralizing potency and breadth that are effective in protection and early therapy in mice.We analyzed sera from 141 individuals hospitalized with TBE during the 2011 and 2018 outbreaks in the Czech Republic . Samples were obtained at the time of hospitalization during the encephalitic phase of disease . In agre5 dilution for neutralization using luciferase-expressing TBEV reporter virus particles  for the top 28 infected individuals varied from 0.37 to 6.7 × 106 . In total, 776 IgG antibody heavy and light chain gene pairs were amplified by RT-PCR and sequenced , Far Eastern (TBEVFE), and Siberian  ranging from 0.2 to 12 ng/ml  as low as 0.02 ng/ml (50 of 8.3 ng/ml). Seven antibodies, all isolated from infected donors, were potent neutralizers of TBEV RVPs with IC50s below 1 ng/ml  for binding to the EDIIIs of Langat virus (LGTV), LIV, OHFV, KFDV, and Powassan lineage I and II viruses  and light chain (LC) contacts to the EDI–EDIII hinge and the BC loop and light chain contacts to the DE loop of the EDIII (2 of surface area on the EDIII (333 Å2 by the VH and 265 Å2 by the VL). T025 inserts Asp100HC and Trp94LC into a cleft in the EDIII, making a salt bridge (Asp100HC–Lys311EDIII) and hydrogen bonds to the EDIII , consistent with 100% sequence conservation between these three strains of virus in the EDIII epitope residues  from the broad and potent antibody T025 (IGVH3–30/IGVK3–15) in complex with the EDIII domains of all three subtypes of TBEV . The strhe EDIII . The anthe EDIII . Crystalresidues .WE structure to a 3.9 Å cryo-EM structure of a mouse monoclonal antibody (19/1786) bound to the TBEV virion . All mice treated with the isotype control antibody died by day 10 (n = 6). In contrast, even the lowest dose of T025 was protective; all but 1 of the 24 mice receiving the antibody survived  24 h before challenge with 10 d later . All 12 Tick-borne flaviviruses can cause a fulminant encephalitis for which there is no effective therapy. This group of viruses are a growing public health concern in Europe, Asia, and North America. Among disease-causing tick-borne flaviviruses, TBEV is prevalent in Central Europe and Russia. Although there is a great deal of information on the polyclonal humoral immune response to TBEV , there iHuman neutralizing antibody responses to pathogens frequently converge on the same IGV genes. Examples include neutralizing antibodies to HIV-1, influenza, Zika, hepatitis B, and SARS-CoV-2 viruses . Antibod50s ranging from 12–1,180 ng/ml . VH1–69 antibodies are highly represented in the human repertoire and are also common among broadly neutralizing antibodies to influenza, hepatitis C, and HIV-1 viruses , and they were only found in convalescent individuals. The absence of this class of potent antibodies in the vaccinees examined is consistent with the lower levels of serum neutralizing potency in this group. Finally, VH3–48 antibodies are also potent neutralizers of several related tick-borne flaviviruses including KFDV, LGTV, LIV, and OHFV, with IC50s of 1–36 ng/ml.Among the neutralizing antibodies to TBEV, VH1–69 and VH3–48 were recurrent in multiple donors. VH1–69 was paired with a variety of light chain genes to produce neutralizing antibodies that were found among vaccinees and recovered individuals. This group of antibodies varied broadly in neutralizing activity with IC viruses . Anti-TBAntibodies to a number of different flaviviruses, including dengue and Zika virus, can be protective if administered before and even after infection . In RussAntibodies against EDIII are often among the most potent raised during flavivirus infection . The broWhile most antibodies to flavivirus EDIII are thought to be virus specific , T025 isAvailable TBEV vaccines were developed over 30 yr ago and consist of inactivated virus grown on chick embryo cells. Vaccination is TBEV specific, requires priming and two boosts, and results in 90–100% seroconversion depending on the vaccine used . AdditioSamples of peripheral blood were obtained upon consent from individuals previously hospitalized with confirmed TBEV infection or individuals previously vaccinated against TBEV in České Budějovice, Czech Republic, under protocols approved by the ethical committees of the Hospital in České Budějovice , the Biology Center of the Czech Academy of Sciences , and The Rockefeller University (IRB DRO-0984). Clinical data were obtained at the treating hospital, and severity of disease was evaluated according to the following scale: mild, flu-like symptoms with meningeal irritation defined as meningitis, characterized by fever, fatigue, nausea, headache, back pain, arthralgia/myalgia and neck or back stiffness; moderate, previous symptoms together with tremor, vertigo, somnolence, and photophobia defined as meningoencephalitis; severe, prolonged neurological consequences including ataxia, titubation, altered mental status, memory loss, quantitative disturbance of consciousness, and palsy revealed as encephalitis, encephalomyelitis, or encephalomyeloradiculitis .Peripheral blood mononuclear cells (PBMCs) were obtained by gradient centrifugation using Ficoll and stored in liquid nitrogen in freezing media (90% FCS and 10% DMSO). Prior to experiments, aliquots of sera  were heat-inactivated at 56°C for 1 h and then stored at 4°C.Escherichia coli and purified from inclusion bodies as previously reported  or 301–397 for strains Sofjin  and Vasilchenko  were used to produce untagged EDIII proteins or EDIII proteins containing a C-terminal 6XHis-Avitag. Constructs encoding untagged EDIIIs of other tick-borne flaviviruses were constructed similarly . Expression plasmids were transformed into BL21(DE3) E. coli and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The cells were lysed and the insoluble fraction containing inclusion bodies was solubilized and refolded in 400 mM L-arginine, 100 mM Tris-base, pH 8.0, 2 mM EDTA, 0.2 mM phenyl-methylsulfonyl fluoride, 5 mM reduced and 0.5 mM oxidized glutathione, and 10% glycerol at 4°C. Refolded protein was purified by size exclusion chromatography  in 20 mM Tris, pH 8.0, 150 mM NaCl, and 0.02% NaN3. EDIIIs were concentrated to 10–20 mg/ml.EDIII antigens were expressed in reported . Express3. Fabs were concentrated to ∼15 mg/ml.T025 Fabs for structural studies were produced and purified as described in previous studies . Brieflyhttps://github.com/stratust/igpipeline). Nucleotide somatic hypermutation and CDR3 length were also analyzed using in-house R and Perl scripts, as described previously , based on 776 IGH CDR3 sequences from this study and 22,654,256 IGH CDR3 sequences from public databases of memory B cell receptor sequences  and conjugated to streptavidin-PE  and streptavidin-Alexa Fluor 647 . Ovalbumin  was biotinylated using the EZ Sulfo-NHS-LC-Biotinylation kit according to the manufacturer’s instructions  and conjugated to streptavidin BV711 . Biotinylation was confirmed by ELISA before use in flow cytometry.Avi-tagged TBEV−CD8−CD14−CD16−ZombieNIR−CD20+Ova−EDIII-PE+EDIII-AF647+ B cells were sorted using a FACS Aria III (Becton Dickinson) into individual wells of 96-well plates. Each well contained 4 µl of a lysis buffer comprising 0.5× PBS, 10 mM DTT, and 3,000 U/ml RNasin Ribonuclease Inhibitors . Sorted cells were snap-frozen on dry ice and then stored at −80°C. Antibody sequences are derived from memory B cells because they originate from small CD20+ cells, and the antibody genes were PCR amplified using IgG-specific primers.PBMCs from sample 111 were enriched for B cells via positive selection using CD19 microbeads . PBMCs from all other donors were enriched for B cells by negative selection . All selection protocols were performed according to the manufacturer’s instructions. Enriched B cells were incubated for 30 min on ice in FACS buffer  with fluorophore-labeled EDIII and ovalbumin, and in the presence of anti-human antibodies anti-CD3-APC-eFluro 780 , anti-CD8-APC-eFluro 780 , anti-CD14-APC-eFluro 780 , anti-CD16-APC-eFluro 780 , anti-CD20-PECy7 , and Zombie NIR . Single CD3RNA from single cells was reverse transcribed using SuperScript III Reverse transcription . The resulting cDNA was stored at −20°C until amplification of the variable Ig heavy (IGH), Ig light (IGL), and Ig kappa (IGK) genes by nested PCR followed by Sanger sequencing. Amplicons from the first PCR reaction were used as template for nested PCR-amplification followed by sequence- and ligation-independent cloning into antibody expression vectors as previously described . RecombiA West Nile virus subgenomic replicon-expressing plasmid encoding Renilla luciferase (pWNVII-Rep-REN-IB) and a ZIKV CprME expression plasmid had previously been obtained from Ted Pierson , was amplified with primers DFRp1532 (5′-GGAATTCGCGGCCGCCTCAGG-3′) and DFRp1533 (5′-GCGGCTCGAGTTAATTAA-3′) before cloning at the NotI and PacI sites of plasmid pPOWV-LB-CprME (see below), resulting in pTBEV-WE-CprME.Synthetic DNA with CprME coding sequence  corresponding to TBEV, Western European subtype strain Neudoerfl (GenBank accession no. L06436.1 with six synonymous changes (in lowercase and bold) to reduce complexity; 5′-CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTATCAACAAGGAATTCGCGGCCGCCAGGCTATGATGACCACTTCTAAAGGAAAGGGGGGCGGTCCCCCTAGGCGCAAGCTTAAAGTGACCGCAAATAAGTCGCGACCAGCAACGAGCCCAATGCCAAAGGGCTTCGTGCTGTCGCGCATGCTGGGGATTCTTTGGCACGCCGTGACAGGCACGGCCAGACCCCCAGTGCTGAAAATGTTCTGGAAAACGGTACCACTGCGCCAGGCGGAGGCTGTTCTGAAGAAGATAAAGAGAGTTATCGGGAACTTGATGCAGAGCCTTCACATGAGAGGGCGTCGCAGGTCAGGTGTGGACTGGACTTGGATTTTTTTGACGATGGCGTTGATGACCATGGCCATGGCAACCACCATCCACCGGGACAGGGAAGGATACATGGTTATGCGGGCCAGTGGAAGGGACGCTGCAAGCCAGGTCAGGGTACAAAACGGAACGTGCGTCATCCTGGCAACAGACATGGGAGAGTGGTGTGAAGATTCAATCACCTACTCTTGCGTCACGATTGACCAGGAGGAAGAACCCGTTGACGTGGACTGCTTCTGCCGAGGTGTTGATAGGGTTAAGTTAGAGTATGGACGCTGTGGAAGGCAAGCTGGATCTAGGGGGAAAAGGTCTGTGGTCATTCCAACACATGCACAAAAAGACATGGTCGGGCGAGGTCATGCATGGCTTAAAGGTGACAATATTCGAGATCATGTCACCCGAGTCGAGGGCTGGATGTGGAAGAACAAGCTTCTAACTGCCGCCATTGTGGCCTTGGCTTGGCTCATGGTTGATAGTTGGATGGCCAGAGTGACTGTCATCCTCTTGGCGTTGAGTCTAGGGCCAGTGTACGCCACGAGGTGCACGCATCTTGAGAACAGAGATTTTGTGACAGGAACTCAAGGGACCACCAGAGTGTCCCTAGTTTTGGAACTTGGAGGCTGCGTGACCATCACAGCTGAGGGCAAGCCATCCATTGATGTATGGCTCGAAGACATTTTTCAGGAAAGCCCGGCTGAAACCAGAGAATACTGCCTGCACGCCAAATTGACCAACACAAAAGTGGAGGCTCGCTGTCCAACCACTGGACCGGCGACACTTCCGGAGGAGCATCAGGCTAATATGGTGTGCAAGAGAGACCAAAGCGACCGTGGATGGGGAAACCACTGtGGaTTcTTcGGGAAGGGCAGTATAGTGGCTTGTGCAAAGTTTGAATGCGAGGAAGCAAAAAAAGCTGTGGGCCACGTCTATGACTCCACAAAGATCACGTATGTTGTCAAGGTTGAGCCCCACACAGGGGATTACTTGGCTGCAAATGAGACCAATTCAAACAGGAAATCAGCACAGTTTACGGTGGCATCCGAGAAAGTGATCCTGCGGCTCGGCGACTATGGAGATGTGTCGCTGACGTGTAAAGTGGCAAGTGGGATTGATGTCGCCCAAACTGTGGTGATGTCACTCGACAGCAGCAAGGACCACCTGCCTTCTGCATGGCAAGTGCACCGTGACTGGTTTGAGGACTTGGCGCTGCCCTGGAAACACAAGGACAACCAAGATTGGAACAGTGTGGAGAAACTTGTGGAATTTGGACCACCACATGCTGTGAAAATGGATGTTTTCAATCTGGGGGACCAGACGGCTGTGCTGCTCAAATCACTGGCAGGAGTTCCGCTGGCCAGTGTGGAGGGCCAGAAATACCACCTGAAAAGCGGCCATGTTACTTGTGATGTGGGACTGGAAAAGCTGAAACTGAAAGGCACAACCTACTCCATGTGTGACAAAGCAAAGTTCAAATGGAAGAGAGTTCCTGTGGACAGCGGCCATGACACAGTAGTCATGGAGGTATCATACACAGGAAGCGACAAGCCATGTCGGATCCCGGTGCGGGCTGTGGCACATGGTGTCCCAGCGGTTAATGTAGCCATGCTCATAACCCCCAATCCAACCATTGAAACAAATGGTGGCGGATTCATAGAAATGCAGCTGCCACCAGGGGATAACATCATCTATGTGGGAGACCTTAGCCAGCAGTGGTTTCAGAAAGGCAGTACCATTGGTAGAATGTTTGAAAAAACCCGCAGGGGATTGGAAAGGCTCTCTGTGGTTGGAGAACATGCATGGGACTTTGGCTCAGTAGGCGGGGTACTGTCTTCTGTGGGGAAGGCAATCCACACGGTGCTGGGGGGAGCTTTCAACACCCTTTTTGGtGGtGTTGGATTCATCCCTAAGATGCTGCTGGGGGTTGCTCTGGTCTGGTTGGGACTAAATGCCAGGAATCCAACGATGTCCATGACGTTTCTTGCTGTGGGGGCTTTGACACTGATGATGACAATGGGAGTTGGGGCATAATAGTTAATTAACTCGAGCCGCGGTTCGAAGGTAAGCCT-3′) was PCR amplified with primers DFRp1511 (5′-ATCTACGTATTAGTCATCGCTATTA-3′) and DFRp1514 (5′-ACCGCGGCTCGAGTTAATTAA-3′) and cloned at the Eco105I and SacII sites of plasmid pZIKV-HPF-CprME (Synthetic DNA containing the CprME sequence (underlined) of POWV-LB strain , resulting in a fragment fusing the CMV promoter with POWV-DTV C-encoding sequences (bolded in primer RU-O-26690). Using as template DTVp1 (ATGGTGACTACTTCTAAAGGAAAG-3′) and RU-O-26711 (5′-GTTTCCCCATCCTCTATCGCTCTG-3′), with bolded nucleotides indicating synonymous mutations introduced to ablate the SacII site. DNA was amplified using DTVp1 as template and oligos RU-O-26710  and RU-O-26688 (5′-TTCGAACCGCGGCTGGGTCCTATTATGCTCCGACTCCCATTGTCATCATC-3′) to generate a fragment overlapping the killed SacII site to the end of the E protein coding region followed by a SacII site. The three DNA fragments were annealed, extended and then PCR amplified using primers RU-O-24611 and RU-O-26688. The resulting DNA fragment was digested with SnaBI and SacII and cloned into similarly digested pZIKV-HPF-CprME to generate pPOWV-DTV-CprME.A three-piece assembly PCR strategy was used. DNA upstream of the CMV promoter in pZIKV-HFP-CprME to just downstream of the beginning of the C-encoding region was PCR amplified with primers RU-O-24611 (5′-CTTGACCGACAATTGCATGAAG-3′) and RU-O-26690 , was amplified with primers DFRp1532 and DFRp1533 before cloning at the NotI and PacI sites of plasmid pPOWV-LB-CprME (see above), resulting in pKFDV-W-377-CprME.Synthetic DNA with the CprME-flanked sequence of KFDV, strain W-377 (GenBank accession no. NC_003690 (A590G and A1893C).The CprME of LGTV, isolate TP21-636, was amplified from a plasmid kindly provided by Dr. Sonja Best  with primers DFRp1563 (5′-GGAATTCGCGGCCGCCTCAGGATGGCCGGGAAGGCCGTTCTA-3′) and DFRp1566 (5′-CCGCGGCTCGAGTTAATTAACTATTAGGCTCCAACCCCCAGAGTCAT-3′) before cloning at the NotI and PacI sites of plasmid pPOWV-LB-CprME, resulting in pLGTV-TP21-636-CprME. There are two nucleotide mutations from GenBank accession no. KP144331), was amplified with primers DFRp1532 and DFRp1533 before cloning at the NotI and PacI sites of plasmid pPOWV-LB-CprME, resulting in pLIV-LI3/1-CprME.Synthetic DNA with the CprME-flanked sequence of LIV, isolate LI3/1 (GenBank accession no. NC_005062), was amplified with primers DFRp1532 and DFRp1533 before cloning at the NotI and PacI sites of plasmid pPOWV-LB-CprME, resulting in pOHFV-CprME.Synthetic DNA with the CprME-flanked sequence of OHFV, strain Bogoluvovska  containing 20 mM Hepes and 10% FBS. For the next 72 h, in 24-h intervals, RVP-containing supernatants were harvested and filtered through a 0.45-μm filter and frozen at −80°C, and media were replaced with DMEM containing 20 mM Hepes and 10% FBS. Frozen RVPs were later thawed and titrated on Huh-7.5 cells to determine the dilution of RVPs at which cells express 106 relative light units in the absence of sera or antibody.RVPs were produced by cotransfecting 1 µg pWNVII-Rep-REN-IB plasmid with 3 µg of the flavivirus CprME plasmid of choice into the permissive cell line Lenti-X 293T using Lipofectamine 2000  according to the manufacturer’s instructions. Cells were seeded 24 h previously at 1050 and IC50 were determined by nonlinear regression analysis using Prism software (GraphPad). In the cross-neutralization screening against the panel of flavivirus RVPs, recombinant antibodies were assayed at 1 µg/ml final concentration using the protocol described above, and the results were compared with no antibody control.96-well plates were seeded with 7,500 Huh-7.5 cells/well in 50 µl DMEM (Gibco) supplemented with 10% FBS and 1% nonessential amino acids. After 24 h, 100 µl diluted RVPs were combined with 100 µl diluted sera or antibody, incubated for 1 h at 37°C, and then 50 µl of the mix were added in triplicate to the plated cells. RVPs are diluted appropriately in BA-1 diluent (Medium 199 [Lonza] supplemented with 1% BSA and 100 units/ml penicillin/streptomycin) to achieve the desired relative light unit expression. After an additional 24 h of incubation at 37°C, media was aspirated off the cells, replaced with 35 µl lysis buffer , and the plates were frozen at −80°C. 15 µl of the subsequently thawed lysis buffer was used for Renilla luciferase expression measurement using the Renilla Luciferase Assay System . Sera were either diluted to 1:600,000 final concentration for TBEV RVP neutralization screening or serially diluted to generate curves. Recombinant monoclonal antibodies were used at 10 µg/ml final concentration and serially diluted 1:3 for neutralization assays. NT2 fragments conjugated to HRP  were diluted 1:5,000 in PBS-T, added to the plates, and incubated again for 1 h at room temperature. Between each step, the plates were washed with PBS-T four times. Plates were finally developed using TMB substrate ; the reaction was stopped using 1 M sulfuric acid and the plates read at 450 nm. Sera were screened for binding at 1:500 dilution. Recombinant monoclonal antibodies were diluted to 10 µg/ml and serially diluted 1:3; the half effective concentration (EC50) was determined by nonlinear regression analysis using Prism 8 (GraphPad). For cross-binding assays, recombinant antibodies were assayed at 1 µg/ml according to the protocol described above using the panel of flavivirus EDIII proteins. The anti-HIV monoclonal antibody 10–1074 was used as isotype control  were coated overnight with 250 ng of the EDIII protein in PBS per well at room temperature; plates were then blocked with 0.1 mM EDTA, 0.05% Tween, and 2% BSA in PBS for 2 h at room temperature. Samples were diluted in PBS-T, added to plates, and incubated for an additional 1 h at room temperature. Secondary goat anti-human-IgG Fab′ control . Antibod control  were conhttp://www.arboviruscollection.cz/index.php?lang=en). The virus was originally isolated from the blood of a diseased 10-yr-old child in Brno, Czech Republic (formerly Czechoslovakia), in 1953. Prior to the use in in vitro and in vivo experiments, the virus was propagated in suckling mouse brains and/or BHK-21 cells.The low-passage TBEV strain Hypr was provided by the Collection of Arboviruses, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic (PS (porcine kidney stable) cells  were cul5 cells per well) were added to 24-well tissue culture plates. After 4 h of incubation at 37°C with 0.5% CO2, each well was overlaid with carboxymethylcellulose (1.5% in L-15 medium). After a 5-d incubation at 37°C and 0.5% CO2, the cell monolayers were visualized using naphthalene black. Viral titers were expressed as PFU per milliliter.To determine virus titer, plaque assays were performed as previously described , with sl4 PS cells were added per well. After 4-d incubation (37°C), the cytopathic effect was monitored microscopically, and cell viability was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies) according to the manufacturer’s instructions. IC50 was calculated from two independent experiments done in octuplicates using GraphPad Prism .The virus neutralization test was performed as described previously , with seVirus neutralization was also assayed using a fluorescence-based virus neutralization test. Monoclonal antibodies  were incubated with TBEV, and PS cells were infected as described above. The cells were incubated for 48 h at 37°C. The cell monolayers were fixed with cold acetone/methanol (1:1), blocked with 10% FBS, and incubated with mouse anti-flavivirus antibody . After washing, the cells were labeled with secondary goat anti-mouse antibody conjugated to FITC  and counterstained with DAPI (diluted to 1 µg/ml) to visualize cell nuclei. The fluorescence signal was recorded with an Olympus IX71 epifluorescence microscope and processed by ImageJ software.Data were analyzed using Mann–Whitney tests or ANOVA and Tukey’s multiple comparison tests as specified and comparison of survival curves was analyzed by log-rank (Mantel–Cox) test, calculated in GraphPad Prism (version 8.4.3). P values < 0.05 were considered significant.WE EDIII-His-Avitag complex  were obtained by combining 0.2 µl crystallization complex with 0.2 µl of 0.1 M sodium citrate tribasic dihydrate, pH 5.0, 10% PEG 6000 in sitting drops at 22°C. Crystals of T025 Fab–TBEVFE EDIII-His-Avitag complex  were obtained by combining 0.2 µl crystallization complex with 0.2 µl of 0.1 M sodium citrate tribasic dihydrate, pH 5.0, 10% PEG 6000 in sitting drops at 22°C. Crystals of T025 Fab–TBEVSi EDIII complex  were obtained by combining 0.2 µl crystallization complex with 0.2 µl of 5% (±)-2-methyl-2,4-pentanediol, 0.1 M Hepes, pH 7.5, 10% PEG 10,000 in sitting drops at 22°C. Crystals were cryoprotected with 25% glycerol before being cryopreserved in liquid nitrogen.Complexes for crystallization were produced by mixing Fab and antigen at a 1:1 molar ratio and incubating at room temperature for 1–2 h. Crystals of T025 Fab–TBEVWE EDIII complex structure was solved by molecular replacement using the VHVL domains from PDB accession no. 2GHW, the CHCL domains from PDB accession no. 4OGX, and TBEV EDIII from PDB accession no. 6J5F as search models in PHASER . The protocol was approved by the Committee on the Ethics of Animal Experimentation of the Institute of Parasitology and the Departmental Expert Committee for the Approval of Projects of Experiments on Animals of the Czech Academy of Sciences (permit no. 4253/2019).Specific pathogen–free BALB/c mice were obtained from ENVIGO RMS. Sterilized pellet diet and water were supplied ad libitum. In all experiments, female mice aged 6–8 wk were used. Mice were housed in individually ventilated plastic cages (Techniplast) with wood-chip bedding, with a constant temperature of 22°C, a relative humidity of 65%, and under a 12-h light/dark cycle. Three mice per group were used in experiments. Mice were inoculated i.p. 1 d before or 1, 3, or 5 d after infection with monoclonal antibodies T025 or 10–1074 in 200 μl PBS and infected subcutaneously with 100 PFU TBEV-Hypr (propagated eight times in suckling mouse brains). Mice were monitored for symptoms and survival over time and euthanized when reaching a humane endpoint.FE and TBEVSi by ELISA, screening results for antibody binding and neutralization of related tick-borne flaviviruses, and neutralization curves for selected antibodies against related tick-borne flaviviruses. 50 and IC50 values for all antibodies tested. Table S1lists clinical data from TBEV-infected and vaccinated donors.Click here for additional data file.Table S2lists sequences of anti-TBEV-EDIII IgG antibodies.Click here for additional data file.Table S3lists highly similar antibody sequences shared across individuals.Click here for additional data file.Table S4lists sequences of cloned recombinant monoclonal antibodies.Click here for additional data file.Table S5lists effective and inhibitory concentrations of recombinantly expressed monoclonal antibodies.Click here for additional data file.Table S6lists data collection and refinement statistics for the crystal structures.Click here for additional data file.


In the column headed “GSH cohort: Controls (n = 71)” and in the row “PUT, more affected”, the value was incorrectly stated as “9.07 ± 1.27”, where the correct value is “8.07 ± 1.27”. The original article has been corrected.In Table 1 of this article, the value in the column headed “GSH cohort: Controls (


Page 11: In Table 1, columns headed “Resistance phenotype” and “Antibiotic resistance gene(s),” all instances of “Amp” and “blaTEM-1B”, respectively, should not be boldface.Volume 6, no. 4, e00729-21, 2021,


We look at fractional Langevin equations (FLEs) with generalized proportional Hadamard–Caputo derivative of different orders. Moreover, nonlocal integrals and nonperiodic boundary conditions are considered in this paper. For the proposed equations, the Hyres–Ulam (HU) stability, existence, and uniqueness (EU) of the solution are defined and investigated. In implementing our results, we rely on two important theories that are Krasnoselskii fixed point theorem and Banach contraction principle. Also, an application example is given to bolster the accuracy of the acquired results. In recent years, fractional calculus has gained great importance by numerous renowned mathematicians. The most essential feature of this topic is that it allows us to execute integrations and differentiations in any order, not necessarily integer ones Such a benefit has been encouraged by the applications in various areas, conceivably including fractal phenomena which appear in many sciences such as physics and engineering. Also, discovering the fractional derivatives and their generalizations was done by well-known mathematicians such as Riemann, Caputo, Hadamard, Euler, Liouville, Laplace, Laurent, and Fourier and was constantly the major path of research in the fractional calculus area. Moreover, these derivatives will provide us new chances to get generalized solutions of fractional differential equations –6. It isAccording to varied literature, like –17, it hOur paper arranged as follows. In 𝔸ℂr to refer to all absolutely continuous functions ξ such that its derivative of order (r − 1) is absolutely continuous on , where r ∈ ℕ and e is the renowned Euler's number.We use the set ξ : +1 and q > 0.The qth-order fractional HCD is given byThe q.Rahman et al.  recentlyqth-order generalized proportional Hadamard fractional integral is given byq > 1 and ϱ ∈ , and χ ∈ C.The qth-order of the one side of a broader range of fractional proportional derivative is given bym=[q]+1, ϱ ∈ , and q > 0.The qth-order of the one side of Caputo-broader range of fractional proportional derivative is given bym=[q]+1, ϱ ∈ , and q > 0.The χ(t)=ln(t) in Definitions Obviously, if we put By using Proposition 3.1, Proposition 4.1, and Remark 1.1 in , we obtaq, p ∈ ℂ such that Re(p) > 0 and Re(q) > 0. Then, for any ϱ ∈  and m=[Re(q)]+1, we haveq,ϱet((ϱ − 1)/ϱ)ln (ln t)p−1)(ϖ)=(Γ(p)/ϱqΓ(p+q))eϖ((ϱ − 1)/ϱ)ln (ln ϖ)q+p−1/ϱ)ln (ln t)p−1)(ϖ)=(ϱqΓ(p)/Γ(p − q))eϖ((ϱ − 1)/ϱ)ln (ln ϖ)p−q−1 > 0, ϱ ∈ , m=[Re(q)]+1, ξ ∈ L1, and (ϖ) ∈ 𝔸ℂm. Then,Let Ml=/Γ(l+1)ϱl).Also, the fractional differential equationAlso,m − r by l has been modified. Hence, the solution of the FDE (By applying equation  in 12),,12), we  the FDE  is givenThe solution of the following fractional Langevin equation in the integral formq,ϱ to equation =0⇒M4=0, z′(1)=0⇒M3=0, and z″(1)=0⇒M2=0.From Therefore,From the conditions,t=1 in t=e in  is a Bananch space and its norm is defined by ‖𝔣‖=supt∈𝔣(𝔱) such that, for each 𝔣 ∈ 𝒞, it implies that 𝔣 : ⟶ℝ is continuous.Suppose that the space )| ≤ k(t)∀ ) ∈  × ℝ and k(t) ∈ , with supt|k(t)|=‖k‖|ξ) − ξ)| ≤ χ|z(t) − z1(t)|∀ t ∈ , z, z1 ∈ 𝒞 and χ > 0|Let us imagine that the continuous function e] ifThen, the FLEs  has a mi𝔯=(ϒ1‖k‖/(1 − ϒ2)), whereChoose Define the closed ball By combining the operators e].Keep this in mind First, we will show that et((ϱ − 1)/ϱ)ln  ≤ et − ln u)((ϱ − 1)/ϱ) such thatSecond, we will demonstrate that et((ϱ − 1)/ϱ)ln  ≤ et − ln u)((ϱ − 1)/ϱ)((ϱ − 1)/ϱ)ln( < et1/u))((ϱ − 1)/ϱ))((ϱ − 1)/ϱ) − 𝔍1z(t2)|⟶0.Taking the limit as 𝔍1 is an equicontinuous as a result of this. We may conclude that 𝔍1 compacts on z so that z=𝔍1z. As a consequence, equation , (II) andℵ=supt∈|ξ| < ∞  (I3) Let us imagine that the continuous function e].Ifthe FLEs  have a u𝔯1 > (ϒ1ℵ/(1 − ϒ1 − ϒ2)), to define the closed ball Choose 𝔍 is contractive.u ∈  and For each Therefore,et2/u)((ϱ − 1)/ϱ)ln( < et1/u))((ϱ − 1)/ϱ))((ϱ − 1)/ϱ) which satisfiesIt is said that the integral equation  is HU stξ :  × ℝ⟶ℝ satisfies inferences (I) and (II), then equation , (I2), and (I3) are fulfilled.χ=(1/4), we get ϒ3=0.372486 < 1, ϒ2=0.0565441, and ϒ1=1.98731. Hence, all assumptions of By putting χϒ1+ϒ2=0.553373 < 1, this implies that On the other hand, Fractional Langevin equations have a considerable role in modeling varied physical phenomena. For instance, they have been employed for describing single-file prevalence  and the


Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins  have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 + 3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 amino acids), and the H. pylori ‘26695 type’ (insertion ~46 amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.  •2.06 Å resolution X-ray structure of LabA shares L-type fold with SabA and BabA, despite low sequence homology.•Avoidance of multiple N-terminal truncations of LabA expressed in E. coli periplasm after redesign of expression construct.•ESI-MS suggests very low binding affinity for lacDiNac or related carbohydrates, calling into question current ligand.•Sequence analysis across different strains indicates 2 types of LabA (‘J99- vs 26695-type’) with high overall conservation. H. pylori establishes chronic infection in the stomach despite the hostile environment with its acidic conditions and the abundance of proteolytic enzymes. As a result of this adaptation, approximately half the world's population is infected with the bacterium  is a carbohydrate structure presented on the gastric mucous cells, carried by the mucin MUC5AC, which has been suggested as a receptor for H. pylori adherence -2-acetamido-2-deoxy-β-D-glucopyranoside, 530.52 Da) was purchased from Carbosynth . Chitotriose (β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc, MW 627.59 Da), chitotetraose (β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc, MW 830.79 Da) and chitohexaose (β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc-(1 → 4)-β-D-GlcNAc, MW 1237.17 Da) were purchased from Dextra . HMO1 (α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 4)-β-D-Glc, MW 488.17 Da), HMO2 -[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 488.17 Da), HMO11 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 853.31 Da), HMO12 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 853.31 Da), HMO18 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 999.36 Da), HMO19 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1072.38 Da), HMO21 (α-D-Neu5Ac-(2 → 3)-β-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1144.40 Da), HMO22 (α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 3)-[α-D-Neu5Ac-(2 → 6)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 11440.40 Da), HMO25 -[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1364.50 Da), HMO26 -[α-L-Fuc-(1 → 2)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 691.25 Da), and HMO27 -[α-L-Fuc-(1 → 2)]-β-D-Gal-(1 → 3)-β-D-GlcNAc(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1056.39 Da) were purchased from Elicityl SA ; HMO3 (α-D-Neu5Ac-(2 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 633.21 Da), HMO4 (α-D-Neu5Ac-(2 → 6)-β-D-Gal-(1 → 4)-β-D-Glc, MW 633.21 Da), HMO5 (α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 634.23 Da), HMO6 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 707.25 Da), HMO7 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 707.25 Da), HMO8 (α- L-Fuc-(1 → 2)-β-D-Gal-(1 → 3)-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 853.31 Da), HMO9 -[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 853.31 Da), HMO10 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 853.31 Da), HMO13 (α-D-Neu5Ac-(2 → 3)-β-D-Gal-(1 → 3)-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 998.34 Da), HMO14 (α-D-Neu5Ac-(2 → 6)-[β-D-Gal-(1 → 3)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)- β-D-Glc, MW 998.34 Da), HMO15 (α-D-Neu5Ac-(2 → 6)-β-D-Gal-(1 → 4)-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 998.34 Da), HMO16 (α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 999.36 Da), HMO24 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 6)-[α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 3)-β-D-GlcNAc-(1 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 1144.40 Da), HMO32 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 6)-[β-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 1364.50 Da), and HMO35 --[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 6)-[α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 1510.55 Da) were purchased from IsoSep ; HMO17 -[α-L-Fuc-(1 → 4)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-β-D-Glc, MW 999.36 Da), HMO20 -β-D-GlcNAc-(1 → 6)-[β-D-Gal-(1 → 4)-β-D-GlcNAc-(1 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 1072.38 Da), HMO28 (α-D-Neu5Ac-(2 → 3)-β-D-Gal-(1 → 4)-β-D-GlcNAc, MW 674.24 Da), HMO29 (α-D-Neu5Ac-(2 → 6)-β-D-Gal-(1 → 4)-β-D-GlcNAc, MW 674.24 Da), HMO30 -[α-L-Fuc-(1 → 2)]-β-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 4)]-β-D-GlcNAc(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 674.24 Da), HMO31 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 6)-[β-D-Gal-(1 → 3)-β-D-GlcNAc-(1 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc, MW 1218.44 Da), and HMO33 -[α-L-Fuc-(1 → 3)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-[α-L-Fuc-(1 → 3)]-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1364.50 Da) were purchased from Dextra ; HMO23 (α-D-Neu5Ac-(2 → 3)-β-D-Gal-(1 → 3)-[α-D-Neu5Ac-(2 → 6)]-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1289.44 Da), and HMO34 -β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-GlcNAc-(1 → 3)-β-D-Gal-(1 → 4)-β-D-Glc, MW 1072.38 Da) were purchased from CarboSynth . A 1.0 mM stock solution of each glycan was prepared in deionized water and stored at −20 °C until used.2.2E. coli XL10-Gold which had previously been developed and optimized for BabA J99 (AAD05605.1), as described in (AE000511.1) (constructs see 21–517K 26695 into pOPE101: FOR PvuII: 5′-CAGTAGCAGCTGGAAGACAACGGCTTTTTTGTG-3′ and REV (BamHI) 5′-GCTGCTGGATCCCTTCTTCTTCTTCTTCTTGAGTTCTTGACTCCTAGATTG-3’ (restriction sites underlined). The reverse primer introduces a hexalysine tag at the C-terminus of the recombinant protein. All oligonucleotide primers were from Merck, UK. The sequences of all recombinant plasmids were confirmed by Sanger sequencing . The only modification applied was the use of 0.2 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) for induction of protein expression, instead of 0.1 mM IPTG used previously. The proteins were expressed in 6 L of bacterial culture prior to harvesting and reconstituted with 600 mL of each of the two different cell lysis buffers as described in (47-496 J99 was obtained by gene synthesis by ThermoScientific Fisher (GeneArt) (sequence available in supplementary data), and subcloned into pOPE101.The pOPE101 plasmid  construct and protocol for periplasmic expression in BabA J99  was alsoribed in  and LabAribed in . In totaribed in B with a 2.32.3.1The combined periplasmic extracts were incubated for a maximum of 2 h with 5 mL of Ni Sepharose 6 Fast Flow resin  at 4 °C and then loaded on a gravity Econo-Column® chromatography column. The flowthrough was collected and the column was washed with ten column volumes (CV) of washing buffer, consisting of 20 mM Tris-Cl, pH 7.4 and 300 mM NaCl. Finally, the protein was consecutively eluted from the column with 3–5x CV of 20, 40, 100 and 200 mM and 10x CV of 500 mM imidazole in washing buffer. The protein content of the different fractions was analyzed with electrophoresis; the proteins were separated on NuPAGE 4–12% Bis-Tris protein gels  and the gels were stained with InstantBlue™ .2.3.2Approximately 30 mg of IMAC purified protein, at a concentration 1 mg/mL, were mixed with 100 μL of 3 mg/mL Tobacco Etch Virus (TEV) protease (in-house AstraZeneca product). The cleavage reaction was left to happen overnight at 4 °C in a dialysis setup. The reaction mixture was inserted in Spectrum™ Spectra/Por™ 1 RC dialysis membrane tubing made of regenerated cellulose dialysis with a MWCO of 6000–8000 Da , and was dialysed against 5 L of 20 mM Tris-Cl pH 7.4, 300 mM NaCl, in order to remove any residual imidazole from the IMAC elution fractions.2.3.3The overnight reaction mixture was incubated for 1 h at 4 °C with 1 mL of Ni Sepharose 6 Fast Flow resin and then loaded on a gravity column. The flow through was collected and the column was washed again with 10 CV. The contents of the column were then eluted with increasing imidazole step gradient, at 20, 40, 250 and 500 mM imidazole. Each eluted fraction was 5 CV. The collected fractions were analyzed with electrophoresis followed by InstantBlue staining, as previously.2.3.4The purest IMAC fractions were concentrated to 5 mL, using a Vivaspin sample concentrator with a molecular weight cut-off of 30,000 Da . The concentrated protein samples were loaded onto a HiLoad 16/60 Superdex 75 (120 mL) gel filtration column , previously equilibrated with buffer containing 25 mM Bicine, pH 8.4, and 150 mM NaCl, connected to an ÄKTA purifier system . The buffer was chosen based on buffer optimization results (data not shown). The flow rate was set at 1 mL/min. The fractions containing protein were analyzed with electrophoresis and InstantBlue staining, as previously.2.4The molecular weight of purified proteins was determined by liquid chromatography (LC) – time-of-flight (ToF) mass spectrometry (MS). Approximately 5 μg of purified protein sample was loaded onto an Agilent 1100 Series LC  which was coupled to a time-of-flight Q-ToF Premier mass spectrometer , equipped with an electron spray ionizer for acquisition in a positive ionization mode. The software MassLynx  was used to analyse the data.2.547-496 J99 grew in 28% poly(ethylene glycol) methyl ether 2000 and 0.1 M Bis-Tris (pH 6.5). Crystals appeared within one week of incubation at 20 °C.Protein samples were concentrated to 20 mg/mL and centrifuged for the removal of aggregated protein, before dispensing the crystal plates. Crystallization was performed using the sitting drop vapour diffusion method in 96-well MRC crystallization plates  and dispensed with the assistance of the Mosquito® Robot . Crystallization trials used commercial and proprietary sparse-matrix screens. Each droplet contained protein sample in 25 mM Bicine, pH = 8.4, and 150 mM NaCl mixed with a precipitant solution, at a volume ratio 1:1, and was equilibrated against 50 μL of the precipitant solution at 4 and 20 °C. Bipyramidal crystals for LabA2.6AIMLESS (4ZH7) as a search model in the program phaser  and were indexed and integrated using the XDS package . AnisotrAIMLESS . Phases m phaser . The modm phaser  and recim phaser  and Bustm phaser . RMSD va2.72.7.1a, HSA-Leb, HSA-Ley, HSA-sialyl-Lex  and HSA-LacdiNAc , were immobilized at a concentration of 5.0 μg/mL on Maxisorp plates  and incubated with LabA21-496 and LabA21-496-6 K at a concentration range from 5 to 20 μg/mL. After washing off unbound glycans and proteins, the wells were sequentially incubated, with three washes between the steps, with a mouse-anti-c-Myc biotinylated antibody  and the conjugate streptavidin-HRP , both at a 1:2000 dilution, in order to complex with bound recombinant LabA and enable tetramethylbenzidine chromogenic detection. Absorbance was measured at 450 nm on a Spark® 10 M multimode microplate reader .For enzyme-linked immunosorbent assay (ELISA), HSA , HSA-Le2.7.2ref) were each buffer-exchanged into 200 mM aqueous ammonium acetate (pH 6.8) using a 10 kDa MW cutoff Amicon Ultra-4 centrifugal filter . Protein concentrations were estimated by UV absorption (280 nm). These stock solutions were stored at −20 °C until used.LabA 26695 and a single chain antibody (which served as Pref) was loaded into the nanoESI tip. To perform ESI, a voltage of ~1 kV was applied to a platinum wire in contact with the solution. Mass spectra were acquired using a sampling cone voltage of 30 V and an extraction cone voltage of 2 V. The source pressure was 3.2 mbar and the temperature was 60 °C. The source wave velocity and wave height were 200 m s−1 and 0.2 V, respectively. Gas flow rates were 2 mL min−1 in Trap, 180 mL min−1 in helium cell and 90 mL min−1 in ion mobility cell. Ions were transmitted through the Trap and Transfer ion guides using voltages of 5 V and 2 V, respectively. At least 150 scans were collected for every acquisition. Data acquisition and processing were carried out using MassLynx (v 4.1).All ESI-MS measurements were performed in positive ion mode using a Synapt G2 ESI-quadrupole-ion mobility separation-time-of-flight mass spectrometer , equipped with a nanoflow ESI source. The nanoESI tips were produced from borosilicate capillaries  pulled to ~5 μm outer-diameter using a P-1000 micropipette puller . For each measurement, approximately 10 μL of sample solution  for the interactions between LabA 26695 and the glycan ligands were quantified using the direct ESI-MS assay  of the ligand-bound (PL) to free protein (P) ions, after correction for nonspecific ligand binding (0) and ligand ([L]0), eq Association constants  with the following codes: 6GMM. All remaining data are contained within the article or the supporting information.33.121-496 from H. pylori strain J99 was produced by periplasmic expression in E. coli as recently described (21-496 J99 (47-496) after TEV cleavage.LabAescribed , with C-escribed  and sizeescribed . Althoug-496 J99 . The mai-496 J99  E and F.-496 J99  A and B,47-496  added by the cloning and left after restriction with TEV . However, the ratio of the main  product to the secondary product  was increased and the purity of the protein sample used for protein crystallization screening was enhanced.Following recombinant expression, the protein was cleaved with TEV-protease. The two-step purification was repeated for the removal of the enzyme and the protein was analyzed C–E. TEV 47-496 F reveale3.247-496 construct were performed in 384 different sets of conditions in sparse-matrix screens. Within seven days, protein crystals were obtained in a crystallization drop containing 28% poly-(ethylene glycol) methyl ether 2000 and 100 mM Bis-Tris at a pH 6.5 and in the absence of salt, on a plate stored at 20 °C. The crystals displayed a bipyramidal morphology with maximum dimensions of 60 × 150 μm . The crystal parameters, as well as data processing and structure refinement statistics are shown in The crystals belonged to the space group Coot  formed an antiparallel coil bundle, similar to a tetratricopeptide repeat motif, at a near perpendicular angle to the ‘handle’ region, creating a kinked (or L-shaped) tertiary structure. The connecting features between the α-helices were: (i) A 48-amino acid segment between the α-2 and α-3 helices; this connecting segment, which extended out of the core of the head region, contained two small antiparallel β-sheet strands (β-1 and β-2); (ii) A 42-amino acid segment between the α-3 and α-4 helices; this segment contained the crown region, consisting of a two-strand antiparallel β-sheet (β-3 and β-4) between T204 and N230, and a disordered loop within the crown, between N211 and E224; (iii) A 28-amino acid loop between the α-4 and α-5 helices, with two short α-helices; (iv) A 21-amino acid loop between the α-5 and α-6 helices; (v) A 28-amino acid segment between the α-7 and α-8 helices, containing a short α-helix and a disordered loop between G380 and D388. LabA has three disulphide bonds , all three located in the head region Coot  is 0.921 Å; without pruning, the RMSD across all 402 pairs rises to 5.493 Å. Using the central (‘head’) part of the molecule (ranging from S57 to V462 in BabA) results in a low RMSD of 0.874 Å for 208 pruned atom pairs (without pruning: 4.959 Å). The pruned atoms in the ‘head’ region correspond to the inserted ‘crown’ region , as the crown is much smaller and contains an unstructured region in LabA. Without pruning, the RMSD for AA 57–462 rises to 4.780 Å. Matching the handle regions D27-P56 and V461–K528 gives RMSD values of 0.465 and 1.147 Å, respectively. Overall, these results show that the handle and head regions are very similar between LabA and BabA, but are connected at slightly different angles, with the largest discrepancies between the two structures found in the crown region.Despite the relatively low sequence identity, all these proteins share high conformational similarity in their head regions, where a 4 + 3 α-helix bundle was present; however, significant differences were observed in the crown region, which may be related to each protein's ligand binding specificity. The ‘crown’ regions of LabA and HopQ  compris3.3H. pylori to human gastric mucins. To verify this binding ability, a sandwich-type ELISA was carried out in order to test the binding of the recombinantly expressed LabA21-496 and LabA21-496-6 K  in the absence and presence of LacdiNAc. A reference protein  was added to the latter solution to correct the mass spectrum for the occurrence of nonspecific ligand-protein binding during the ESI process  was initially performed on aqueous ammonium acetate  solutions containing LabA process . However47-496 26695 with LacdiNAc was measured. 47-496 26695 (2.5 μM), Pref (3.2 μM) and LacdiNAc (15 μM). Only one isoform of LabA was detected, with a MW of 51,277 Da. After correction of nonspecific binding using the reference protein method (−1 (mean ± s.d.). We next decided to investigate binding to chito-oligosaccharides due to the structural similarities to LacdiNAc. The affinities of chitotriose, chitotetraose and chitohexaose (structures see ref (3.2 μM) and three chito-glycans (each 48 μM). The affinities for the three oligosaccharides ranged from 1000 M−1 to 2000 M−1.Binding of this newly produced LabAn method , the aff47-496 26695 and no binding at all of LabA21-496 J99 in ELISA-like format (−1).Having established only weak binding affinity of LabAe format  for Lacde format , all of e format . A repre4While our initial attempts to crystallize LabA protein were unsuccessful, removal of the C-terminal tags and addition of an N-terminal TEV cleavage site resulted in suitable crystals. It has been reported that excessive solubility can play a role in making protein crystallization more challenging. In particular, highly soluble protein versions bearing a C-terminal pentalysine tag have been found to yield small needle-shaped crystals, similar to those obtained by us here S inapproAt the same time, limiting the proportion of unstructured regions in the protein was also considered during the design of the new expression construct. It was known from the crystal structure of BabA J99 that the three C-terminal tags, used for the enhancement of solubility, detection by Western Blotting and purification with IMAC, were not visible in the crystal structure. This confirmed the conformational flexibility of these sequences. We removed the C-terminal hexalysine and c-Myc-tags and moved the hexahistidine tag needed for IMAC to the N-terminus, upstream of the TEV cleavage site. This approach resulted in the new recombinant LabA variant which yielded protein crystals sufficient for X-ray crystallography during initial screening already, without the requirement for further optimization.H. pylori.Overall, the protein adopts a similar structure to the previously described adhesins of H. pylori adhesins  adopt a three-dimensional L-shape, suggesting that this structure may fulfil some yet to be discovered functional role. The highest similarity of the LabA amino acid sequence to that of BabA was corroborated by the superimposition of their crystal structures and the obtained low RMSD values. The similarity among all adhesins was most pronounced in the handle and head and particularly low in the crown region, where the glycan binding site of BabA is found. While this explains the lack of binding affinity of LabA for Leb, it does not provide any information about the actual binding site of this protein. In the absence of a ligand, the crown appears partially disordered in most adhesin structures.Despite low amino acid sequence similarity, all four crystallized H. pylori ‘J99-type’, with an overall total length of approx. 686–691 amino acids and a shorter insertion domain of approx. 31 amino acids, and the H. pylori ‘26695 type’, with an approximate total length of 702–711 amino acids and a longer insertion domain of approx. 46 amino acids. In the absence of a ligand co-crystal structure, the exact significance of these structural variations between strains remains unclear. While BabA also shows strain variation in the crown region/insertion domain, the variations in this region are stronger in BabA, which has led to the identification of two hypervariable/diversity loops   in whichloops (N  (Moonensloops (N ). In conloops  and SabA , and in line with the obtained structure for LabA (6GMM), the N-terminal region is located close to the membrane insertion domain, which is buried deep in the outer membrane of H. pylori, and is a very unlikely binding site, as it would not be able to access its putative ligand on epithelial cells.However, our ESI-MS screening indicated that LabA only weakly binds to Lac/LacNAc-containing oligosaccharides, without any clear specificity. Furthermore, we were not able to detect any binding to LacdiNAc-HSA in an ELISA-type assay. We do not believe that the omission of the first 46 N-terminal amino acids in the improved construct can be responsible for the lack of LacdiNAc binding for two reasons. First, the longer LabAa values summarized in Because of our inability to detect any binding to LacdiNAc, we extended our study to the binding of 35 HMOs with similar structures and to three chito-oligosaccharides of different lengths . The latH. pylori J99 or 26695 strain  using a variety of methods, including whole bacteria binding assays in microtiter plates. This was not due to a lack of expression or translation of LabA protein, as this could be identified by LC-MS/MS. The complete lack of binding of H. pylori bacteria to LacdiNAc, together with our inability to detect specific and high affinity binding of the recombinant LabA protein, suggests that LacdiNAc might not be the physiological ligand for this adhesin, and that the glycan preference of LabA remains to be elucidated. Alternatively, the function of LabA in promoting binding may be accessory, rather than direct, supporting the structure and/or binding activity of another adhesin with specificity for LacdiNAc as determined by This lack of binding to LacdiNAc is in good agreement with the recent observation by Mthembu and co-workers . The aut10.13039/501100000266EPSRC (Grant EP/L01646X), 10.13039/100004325AstraZeneca R&D and the 10.13039/501100000837University of Nottingham. Y.C., L.N. and J.S.K. acknowledge funding from the 10.13039/501100003178Alberta Glycomics Centre. Ross Overman is currently employed by Leaf Expression Systems. FHF is currently fully funded by a grant from the LOEWE DRUID .This research has been supported through joint funding from Vasiliki Paraskevopoulou: Investigation, Methodology, Validation, Writing - original draft, Writing - review & editing. Marianne Schimpl: Investigation, Formal analysis, Methodology, Validation, Writing - review & editing. Ross C. Overman: Methodology, Conceptualization, Writing - review & editing. Snow Stolnik: Funding acquisition, Supervision, Writing - review & editing. Yajie Chen: Investigation, Methodology, Writing - review & editing. Linh Nguyen: Investigation, Methodology, Writing - review & editing. G. Sebastiaan Winkler: Funding acquisition, Supervision, Writing - review & editing. Paul Gellert: Methodology, Conceptualization, Writing - review & editing, Funding acquisition, Supervision. John S. Klassen: Conceptualization, Methodology, Writing - review & editing. Franco H. Falcone: Funding acquisition, Project administration, Supervision, Writing - original draft, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Nature Communications 10.1038/S41467-020-20875-W, published online 10 February 2021.Correction to: t test were reported with the incorrect degrees of freedom, incorrectly written as “t18858 = −20.915, p < 0.001.” The correct version replaced “t18858” with “t18558.” This has been corrected in both the PDF and HTML versions of the Article.The original version of this Article contained an error in the “Methods” section, under the “Decision-making task” heading. The results of a The original version of this Article also contained an error in the “Methods” section titled “Behavioural modelling of value-guided decisions” in which Eqs. 5 and 6 inadvertently swapped during production. The incorrect version statedu(m) and w(p), for reward magnitudes and probabilities, Eqs. 5 and 6, respectively)56.Since it is known that humans do not weigh magnitudes and probabilities in a statistically optimal way, we considered systematic distortions in the weighting of reward information in our models (pO are the objective reward probabilities and γ is a free parameter used to fit subjective reward probabilities. Subjective magnitudes were estimated by:where The correct version statesw(pO) and u(mO) for reward probabilities and magnitudes, Eqs. 5 and 6, respectively)56.Since it is known that humans do not weigh magnitudes and probabilities in a statistically optimal way, we considered systematic distortions in the weighting of reward information in our models (pO are the objective reward probabilities and γ is a free parameter used to fit subjective reward probabilities. Subjective magnitudes were estimated by:where The original version of this Article also contained an error in the Methods section under the heading “MRS data acquisition”, in which the terms “rM1 and lM1” were incorrectly ordered. The incorrect version statedx = −28.97 ± 0.82, y = −18.48 ± 0.92, z = 51.86 ± 0.59 and MNI x = 31.90 ± 0.71, y = −14.76 ± 1.06, z = 49.76 ± 0.88 for rM1 and lM1, respectively.Average M1 voxel centroids in standard space were estimated at MNI The correct version statesx = −28.97 ± 0.82, y = −18.48 ± 0.92, z = 51.86 ± 0.59 and MNI x = 31.90 ± 0.71, y = −14.76 ± 1.06, z = 49.76 ± 0.88 for lM1 and rM1, respectively.Average M1 voxel centroids in standard space were estimated at MNI