La, with PdII cations. By combining the concept of “coordinationsphere engineering”, relying on bulky quinoline donors, withreversible photoswitching between the ligand’s open (o-La) and closed (c-La) forms, a [Pd2(o-La)4] cage (o-C) and a [Pd2(c-La)3] bowl (c-B) were obtained,respectively. This structural rearrangement modulates the system’sguest uptake capabilities. Among three bis-sulfonate guests , the cage can encapsulateonly the smallest (G1), while the bowl binds all of them.Bowl c-B was further used to synthesizea series of heteroleptic cages, [Pd2LA3LB], representing a motif never reported before. Additionalligands (Lc-f), with shortor long arms, tune the cavity size, thus enabling or preventing guestuptake. Addition of Br–/Ag+ makes itpossible to change the overall charge, again triggering guest uptakeand release, as well as fourth ligand de-/recomplexation. In combination,site-selective introduction of functionality and application of externalstimuli lead to an intricate system of hosts with different guestpreferences. A high degree of complexity is achieved through cooperativitybetween only a few components.It is difficult toassemble multi-component metallo-supramoleculararchitectures in a non-statistical fashion, which limits their developmenttoward functional materials. Herein, we report a system of interconvertingbowls and cages that are able to respond to various selective stimuli, based on the self-assembly of a photochromicdithienylethene (DTE) ligand,  Increasing such a system’s complexityvia the introduction of multiple different ligands is challenging,since metal-mediated self-assembly under thermodynamic control easilyresults in a statistical mixture of products.7 In recent years, several strategies to combine distinguishable ligandsin a non-statistical fashion, leading to the clean assembly of heterolepticcages, have been reported. For example, the Yoshizawa group exploredthe use of a templating guest,8 Crowleyet al. took advantage of hydrogen-bonding between adjacent ligands,9 and Zhou,10a Fujita,10b Mukherjee,10d Chand,10f and our group10i utilized geometric principles to form heterolepticcages containing two different types of ligands. Recently, our groupset out to investigate a “coordination sphere engineering”approach based on the introduction of steric hindrance around themetal centers to increase the structural complexity in a series ofPdII-mediated assemblies. On one hand, banana-shaped ditopicligands with picoline donors allowed us to form cis-configured [Pd2LA2LB2]4+ cages,11 whilethe introduction of quinoline or acridine donor groups resulted inthe assembly of [Pd2L3X2] bowl-shapedstructures or [Pd2L2X4] rings, respectively.12 Despitethese few examples, rational multi-component assembly to give a singleproduct remains challenging.13 Even moreso, exploiting the multi-ligand character of heteroleptic cages tointroduce functional complexity is still largely unexplored, in particularwith respect to emergent properties resulting from an interplay ofdifferent components. Owing to their dynamic behavior and the presenceof an accessible cavity, coordination cages with stimuli-responsivefunctionality represent a promising platform to develop novel materials14 and adaptive host–guest systems.15Natural systems arecapable of fabricating supramolecularassemblies with multiple components in a precise and controlled manner.For example, ribosomes and virus capsids are assembled in a non-covalentfashion, starting from smaller building blocks. To mimic the functionand complexity of natural nanostructures, chemists have developedsupramolecular self-assembly, a field that has become a burgeoningarea of study.16 The addition of halideanions to positively charged hosts can trigger the uptake of neutralguest molecules.17 Guest uptake can alsobe tuned in redox-responsive systems.18 Sophisticated pH effects have recently been investigated19 and combined with light, leading to reversiblecage disassembly.20 Light as a waste-free,precisely administered external stimulus has gained high popularityin the control of supramolecular systems.21 We previously reported light-responsive cages in which photochromicdithienylethene (DTE) ligands made it possible to control structuralrearrangements as well as guest uptake and release in a fully reversiblefashion.22 Most reported examples of stimuli-responsive,metallo-supramolecular systems are based on homoleptic structures,though.23 Hence, the introduction of morethan one functionality or response to more than one stimulus is achallenge.It has been shown that interaction ofsuch cages with chemicalstimuli, e.g., additional ligands, allows structural rearrangementand guest release.2LA4] cage- and [Pd2LA3] bowl-shapedhosts, the former deriving from the open-form photoisomer (o-La) and the latterfrom the closed isomer , making it possible to differentiatea series of anionic guests and control their uptake and release.We herein demonstrate that multi-stimuli-responsivesystems canbe realized by combining three well-established trigger elements vianon-statistical, heteroleptic self-sorting. We combine a strategywe coined “coordination sphere engineering” in tandemwith light-responsive DTE backbones to achieve the self-assembly ofan interconvertible pair of  assemblies formedfrom banana-shaped bis-pyridine ligands having DTE backbones.21 In this work, we combine the effect of the photochromic backboneswith coordination sphere engineering by installing quinolin-3-yl donorgroups that introduce significant steric hindrance around the coordinatedPdII cations. We had shown previously that, in such quinoline-basedassemblies, a fine balance separates the formation of sterically lesscrowded [Pd2L3] bowl-shaped structures and moreconstrained [Pd2L4] cages.12 Here, we show how this balance can be tipped by photoisomerizingthe ligand backbones in one direction or the other. Ligand o-La was synthesizedby a Suzuki cross-coupling reaction of perfluoro-1,2-bis(2-iodo-5-methylthien-4-yl)cyclopenteneand 2 equiv of quinolin-3-yl boronic acid. Self-assembly of o-La, with the photoswitchin its open form, and [Pd(CH3CN)4](BF4)2 in a 2:1 ratio in CD3CN at 70 °C ledto the quantitative formation of cage compound [Pd2(o-La)4](BF4)4 . Cage o-C features a pair of PPPP/MMMM enantiomersin the crystal structure .24 Irradiation of a CD3CN solution of o-La using 313 nm lightallows conversion into c-La with >99% yield. Reacting c-La, featuring the photoswitch in itsclosed form, with [Pd(CH3CN)4](BF4)2 in a 3:2 ratio at 70 °C led to the formationof the bowl-shaped structure [Pd2(c-La)3(Solvent)2](BF4)4 (c-B) .The generaldesign of the system is based on previous work from our group on photochromic4+, [Pd2(c-La)3+BF4]3+, and [Pd2(c-La)3+2BF4]2+) , corresponding to one-third of the total amount. However,after addition of one-sixth more equivalent of [Pd(CH3CN)4](BF4)2, the free ligand was completelyconsumed and the bowl species c-B remainedquantitatively . Diffusionof Et2O into a CD3CN solution of c-B under strict exclusion of light gave a single crystalwhose X-ray diffraction analysis revealed the structure of cage [Pd2(c-La)4](BF4)4 (c-C) rather than the expected bowl c-B. While seemingly surprising, this result agrees with previousfindings from our group, where quinoline donor groups were shown tolead to [Pd2L3] bowls in solution but [Pd2L4] cages in the solid state.12a Gas-phase DFT studies indicate that energies for the formalcage/bowl transformation do not depend significantly on the photoisomericstate, further corroborating a fine energetic balance between solutionand solid-state behavior .The compound crystallizes in the P42/mcm space group, and the asymmetric unit contains both possibleenantiomers with 50:50 occupancy. The crystal structure further showedthat all ligands c-La are arranged without any twisting with respect to the Pd–Pdaxis (Supporting Information).Due to symmetry reasons, one set of signals is expected toderivefrom the middle ligand, while the second set can be assigned to thetwo ligands flanking the side of the bowl structure.solution S17. As csolution S17. As csolution S17. As co-C under UV light(313 nm) led to the formation of bowl c-B, accompanied by the release of free ligand c-La, as observed by 1H NMRanalysis . Addition of PdII cations (0.17 equiv) and further heating at 70 °C for1 h made it possible to convert the excess free ligand c-La into c-B quantitatively. The obtained broad 1H NMR spectrumindicated that c-B was formed as a mixtureof diastereomers. Vice versa, irradiation of c-B at room temperature with 617 nm light resultedin a broad 1H NMR spectrum, not showing any signs of freeligand . HR-ESI and even cold-sprayionization (CSI) mass spectrometry (−30 °C) could notdetect any peaks related to a tentative o-B species. However, a low-temperature (−35 °C) 1H NMR measurement resulted in much sharper signals, allowing us toidentify o-B . Heating this o-B solutionfor 1 h accelerates complete reassembly to the thermodynamic product o-C .UV–vis analysis of ligand and cage samples corroborated quantitativeswitching fidelity in both cases, with the ligand showing faster photoisomerizationas the cage, in accordance with our previous results for related systems.22 Cage–bowl interconversion was studiedover 10 cycles, showing very good fatigue resistance .Irradiation of o-C and c-B was investigated. To do this, we performed 1H NMR titrations with three different anionic bis-sulfonateguests, G1–G3 (with NBu4+ as counterion) , into a CD3CN solution of o-C led to the appearance of a new set of sharp 1H NMR signals and significant downfield shifts of the signalsassigned to inward-pointing protons . This indicated encapsulation and slow exchange of guest G1 . The formationof host–guest complex [G1@o-C]2+ was further confirmed by HR-ESI-MS spectrometry. For the slightly larger guests G2  and G3 ,however, no encapsulation into o-C couldbe detected . On thecontrary, due to its more open cavity, c-B was indeed found to be able to bind all three guest molecules, G1, G2, and G3, as confirmed byNMR spectroscopy and HR-ESI-MS . To better evaluate the 1H NMR signal shifting, enantiopure S-c-B was used in the experiments.After stepwise titration with G1, two new signals arosearound δ = 11.0 ppm, showing the formation of a new speciesin solution. Observation of a large peak assignable to [G1@c-B]2+ in the HR-ESI-MSspectrum further indicated binding with a 1:1 stoichiometry . After the addition of 3.0 equivof the guest, a crystal suitable for single-crystal X-ray diffractionwas obtained by leaving the sample overnight. Pleasingly, X-ray analysisconfirmed the assumed structure of [G1@c-B], where guest G1 is accommodated inthe concave well of the bowl between the two Pd atoms . Unfortunately, determination of associationconstants for the host–guest complexes from 1H NMRtitration data was not possible due to an onset of precipitation duringthe experiments. Notably, the stable interaction of G2 with bowl-shaped c-B inspired us to exploit this host–guestinteraction with the kinetic photoswitch product o-B at room temperature. After the addition of 1.0 equivof G2 into a freshly prepared solution of o-B, 1H NMR signals sharpened, allowing the characterization of the assembly. HR-ESI-MS furthersupported the formation of host–guest complex G2@o-B, showing a single peak assignedto [G2@Pd2(o-La)3]2+ . Moreover, G2@o-B and G2@c-B could be reversibly interconverted by irradiation with UVlight (313 nm) and red light (617 nm), respectively, as verified by 1H NMR spectroscopy .This photo-controlled reversibleinterconversion between cage and bowl makes it possible to tune thesize and accessibility of the inner cavity. Therefore, size-dependentguest encapsulation of nterion) 4a. Stepwguest G1 S61. The c-B, each PdII center is coordinated by threequinoline-based ligands, while the fourth coordination site is occupiedby a solvent molecule (here acetonitrile or H2O) to complementthe square-planar coordination sphere.12 This encouraged us to synthesize a new series of banana-shaped bis-pyridineligands . While the pyridine groupis a stronger donor as compared to the solvent used, it is also lesssterically demanding than the quinoline donors on the other ligands,thus allowing it to replace the coordinated solvent as a fourth ligand,leading to the formation of unprecedented [Pd2LA3LB] heteroleptic cages with a 3:1 ligand stoichiometry3Lc-f](BF4)4 (c-B-Lc-f), which were fully characterized by NMR techniques and HR-ESI-MS. Again, enantiopure R/S-c-B was used in orderto obtain better resolved 1H NMR spectra. Taking c-B-Ld as an example, reaction of Ld with c-B resulted in the transformationof the 1H NMR spectrum in which neither signals of theparental bowl species nor the free ligand Ld are present (2(c-La)3Ld + nBF4]n)+(4– (n = 0–2). Moreover, a recorded 1H DOSYspectrum confirmed the clear formation of one single species . It is worth noting that, to thebest of our knowledge, this is the first example of a non-statisticalself-assembly of a [Pd2LA3LB] heteroleptic cage. In addition, we explored the possibility ofusing also the photoisomeric o-B asa platform for the formation of heteroleptic cages. For this purpose, Ld and Lf were tested as fourth ligands. After addition of 1.0 equivof the respective ligand to a solution of o-B at room temperature, 1H NMR spectra of the resultingcompounds were found to suffer from signal broadening, pointing ata highly dynamic behavior of the system. Interestingly, NMR signalsturned sharp, allowing clear assignment, after guest G1 was added and apparently encapsulated . HR-ESI-MS spectra revealed prominentpeaks consistent with the formula n)+(4– (n = 1–2), even in the absence of the guest, suggesting thatthe formation of heteroleptic cages o-B-Ld,f from o-B and Ld or Lf proceeds smoothly, despite thedifficult-to-analyze NMR spectra .In bowl species such as ds Lc-f, 1b, havin present 5b. HR-ES present 5d clearl species S35. It io-B-Ld and c-B-Ld . Irradiation of c-B-Ld under 617 nm light led tothe same 1H NMR spectrum as obtained for the self-assemblyof o-B-Ld from o-B and Ld. Conversely, irradiation of cage o-B-Ld with lightof 313 nm wavelength restored the original 1H NMR spectrum,showing full reversibility between the closed and open heterolepticcages. Despite numerous attempts, we were not able to obtain singlecrystals suitable for X-ray analysis for any of the guest-free heterolepticcages. To help understanding their structural features, we performedDFT (ωB97XD/LanL2DZ) geometry optimizations . The models reveal a notable enlargement of the cavityvolume for c-B-Lf as compared to c-B-Ld. We therefore assumed that thisshould lead to a distinguishable guest uptake behavior.Next, we investigated light-triggeredswitching between the twophotoisomeric heteroleptic assemblies, d c-B-Ld S111. IrrG1, G2, and G3, with these heterolepticcages was characterized by means of 1H NMR titration experiments.The addition of G1 to the smaller cage c-B-Ld resultedin shifting of proton signals of Hb and Hb′, pointing outside from the cage cavity, while the inward-lookingprotons did not experience any shifting . It is worth emphasizing that, asshown above, bowl c-B is able to bindall three guest molecules, while heteroleptic cage c-B-Ld does not.On the other hand, all three guests could be encapsulated inside cage c-B-Lf  allows the inner cavity to be sufficiently large to bind guestmolecules, as confirmed by 1H NMR and HR-ESI-MS experiments. Addition of more guest equivalents led to interaction with thecage’s outer side, as confirmed by shifting of the signalsof outward-pointing protons Hb and Hb′, followed by precipitation of the sample. The formation and stoichiometryof the host–guest complexes with G2 and G3 were further confirmed by ESI-MS . Finally, guest affinity was also investigatedfor cage c-B-Le, leading to results comparable to those obtained for c-B-Lf .As described above, the interaction of the three guest molecules, shifting 6b. Furthshifting 6d. The seriments6b,c. Aftappeared 6b. Furthdetected 6c,indico-B-L cages is different from that of theanalogous assemblies with the closed-form DTE photoswitch, presumablydue to the higher flexibility of the backbone in its open photoisomericform. Thus, addition of G1 to the small-cavity cage o-B-Ld led to the formation of a [G1@o-B-Ld] host–guestcompound. While the NMR spectrum of o-B-Ld is characterized by broadsignals, stepwise titration with G1 led to a new setof sharp signals, showing at the same time guest uptake and stabilizationof a more defined conformer of the host . This allowed a better characterization of the systemwith 2D NMR spectra , and therefore an indirect confirmation of the o-B-Ld heterolepticcage assembly. Further support came from ESI-MS analysis, with a prominentpeak assigned to [G1@o-B-Ld]2+, confirmingonce more the binding of G1 with a 1:1 stoichiometry. Photoswitching of G1@o-B-Ld triggers guest release to give c-B-Ld, for which NMR results indicateonly outside guest interaction . Titration of o-B-Ld with the bigger guests G2 and G3 showedno signs of encapsulation, comparable to the closed-ligand cage analogue. On the other hand,when using heteroleptic cage derivative o-B-Lf, again possessing a largercavity size, uptake of guest G1 was certainly anticipatedand indeed confirmed by 1H NMR titration and ESI-MS. Asfor the shorter analogue, the titration with G1 led toa new set of 1H NMR signals, converting the broad signalsof the empty cage into a better resolved NMR spectrum . HR-ESI-MS proved a 1:1 binding stoichiometry,showing only the peaks of [G1@o-B-Lf]2+ . Addition of an excess of guest (3equiv) led to precipitate formation, together with the growth of singlecrystals suitable for X-ray analysis , suggesting partialdecomposition of the cage. Titration with G3 led to decompositionof o-B-Lf right from the beginning of the guest addition .Unfortunately, experiments probing theuptake of the bigger guests II coordination sites (– or Br– (NBu4+ as countercation) was added to a c-B solution. The clean formation of [Pd2(c-La)3(Cl/Br)2](BF4)2 (c-B-Cl/Br) was confirmed by a combination of NMRand HR-ESI-MS experiments . Interestingly, this stimulus could be reversed by addition ofa Ag+ salt (AgBF4), leading to recovery of theinitial bowl compound . Hence,the two species can reversibly interconvert under chemical control:the addition and removal of halide ions trigger the formation of either c-B-Cl/Br or c-B, respectively. The same concept can be applied to the host–guestcomplexes G@c-B. As coordinationof the halide anions affects the overall charge of the supramolecularcompound, going from a +4 charged species to a +2 cationic assembly,the induction of guest release was the consequence . Subsequently, we applied this approachto the heteroleptic cages [Pd2LA3LB] and corresponding host–guest compounds. Rewardingly,a reversible structural conversion between the heteroleptic cagesand c-B-Br bowl is achieved by additionof bromide and silver ions , giving rise to c-B-Br and releasing free Le at the same time. In this case, a stoichiometricamount of Ag+ (2.0 equiv) was not enough to restore thestarting heteroleptic cage. However, adding an excess amount of silversalt (up to 10 equiv) into the mixture at room temperature allowedus to reassemble cage c-B-Le. This approach can be extended to an evenmore complex system, where four components  are assembled through integrative self-sortingto give the host–guest system G1@c-B-Le. Herein,the combination of guest uptake properties and chemical stimuli ledto a reversible on/off guest binding event (– to a G1@c-B-Le solution, the 1H NMR spectra showed the complete release of G1 andthe decomplexation of ligand Le, together with the formation of c-B-Br (+ allowsthe complex mixture to reassemble, forming selectively only the host–guestcomplex G1@c-B-Le following a non-statistical integrativeself-sorting process (Finally, we explored thepossibility to use halide anions as anexternal stimulus and competitor, either to the solvent moleculesor to the pyridine-based ligands occupying the fourth Pdcompound S115. Hencompound S115. Hencompound S115. Hencompound S115. Hencompound S115. HenLa, carryingbulky quinoline donor groups, allowed us to combine light-switchablebackbone flexibility with steric hindrance around a PdII coordination center, leading to a multi-stimuli-responsive system.Self-assembly of La photoisomerswith PdII ions leads to the controlled formation of a coordinationcage, o-C, or a bowl-shaped object, c-B, reversibly interconvertible by irradiationwith light of different wavelengths. Their structural diversity resultsin a different guest binding behavior: compared to the small pocketof cage o-C, bowl c-B has a larger cavity for guest uptake. Moreover, thebowl structure can act as platform to form an unprecedented type of[Pd2LA3LB] heterolepticcages in a non-statistical way. Proper design of the fourth ligand,alone or in combination with the light stimulus, allowed us to furthertune the guest binding affinity. Finally, the addition of halide andsilver ions led to a reversible, charge-modulating stimulus to triggeron/off guest binding. Herein, we report a new strategy to form heteroleptic,multi-functional cages, where the host–guest properties derivefrom the synergy of all contained building blocks: a photochromicligand backbone, a quinoline-based, congested coordination environment,and a fourth ligand coming in different sizes. This generates a multi-stimuli-responsivesystem where light, halides, and additional ligands act either individuallyor in a cooperative fashion.In summary, the appropriate design of aphotochromic ditopic banana-shapedDTE-based ligand,


We aimed to assess antibiotic usage and knowledge regarding antibiotics and antimicrobial resistance (AMR) among undergraduate students of the Universiti Brunei Darussalam (UBD), public university located in Brunei Darussalam. A cross-sectional study was performed using a self-administered questionnaire. The questionnaire was adapted from the World Health Organization’s (WHO) “Antibiotic resistance: Multi-country public awareness” survey distributed online. Students at the UBD were invited to participate in the online survey through internal email. The questionnaire consisted of five sections: demographic information, antibiotic usage, knowledge on antibiotics, antibiotic resistance (AMR), and use of antibiotics in agriculture. The data were analyzed descriptively and appropriate inferential statistics were used accordingly. A total of 130 students returned a completed questionnaire. The result of the study found that 51% (n = 66) of the students had good level of knowledge of antibiotic and antimicrobial resistance with a mean total knowledge score of nine out of 14. Of note, 76% (n = 99) of the respondents mistakenly believed that antibiotic resistance is the result of the body becoming resistant to antibiotics. Only 14% (n = 18) of the respondents were found to have poor knowledge on antibiotics and antimicrobial resistance in the study. Misconceptions in regards to the use of antibiotics for conditions related to viral illnesses like cold and flu  were noticed among the respondents in our study. Thus, improving knowledge on antibiotics is crucial to address these beliefs. The abuse and excessive utilization of antibiotics have led to the rise of antimicrobial resistance (AMR) which poses a major threat to individual health globally . At leasAntimicrobial drugs can be defined as a group of drugs that are effective against microorganisms and comprise antibiotics, antifungals, antivirals, and antiparasitic drugs, whereas an antibacterial agent is any agent that is directed against bacteria . BacteriAMR occurs when microorganisms such as bacteria develop resistance to an antimicrobial agent. Due to the emergence of resistance, the ability to treat various infectious diseases becomes less effective thus causing treatment failure, increasing health care cost, and even leading to death . The facAccording to the national statistics, the total population in Brunei Darussalam as of 2019 was 459,500 . Brunei The total antibiotic consumption in Brunei is 5.92 defined daily doses (DDD) per 1000 inhabitants per day, but this value is low due to the incomplete coverage and it only represents the use in public health . The mosIn addition, the WHOGAP on antimicrobial resistance was implemented in 2015, the main goals whereof are to ensure antimicrobial agents are used rationally and to prevent infectious diseases with the proper use of effective and safe medicines of guaranteed quality accessible to all who are in need .Multiple studies have been conducted regarding the knowledge of antibiotics and antimicrobial resistance among university students in other countries ,31,32,33To the best knowledge of the author, there are no documented Bruneian studies assessing the knowledge and perception of antibiotics. The Ministry of Health of Brunei often relies on the data of the neighboring countries as a yardstick in planning the public health campaign. Thus, this study is aimed to explore antibiotic usage and knowledge regarding antibiotics and antimicrobial resistance (AMR) among the Bruneian population, targeting specifically undergraduate students. This study could provide baseline data for future interventions and public health campaigning on antibiotic usage and antimicrobial resistance.This was a cross-sectional online survey study conducted from March to April 2020. The study involved undergraduate students from the Universiti Brunei Darussalam. The criteria of eligibility for the study were as follows: (i) adult (over 18 years old) and (ii) able to read and understand the Malay or English language. The students who were not willing to participate were excluded from the study.An e-mail invitation was initially mailed to the Academic Registrar at the Institute of Health Sciences, then it was forwarded to all the Registrars from various faculties at the Universiti Brunei Darussalam. The data were collected from an online survey questionnaire. E-mail invitations for participation containing the online survey link and the information sheet for participants were disseminated to the targeted participants. To increase the response rate, reminder e-mails were forwarded twice to the contact persons following a two weeks interval. Other than reminder emails, the survey was also distributed among the students through WhatsApp application as a reminder.Raosoft website was used to calculate the sample size with a confidence level set to 95% and the 5% margin of error . AssuminThe questionnaire was adapted from the questionnaire based on the World Health Organization’s (WHO) “Antibiotic resistance: Multi-country public awareness” survey . This quThe online questionnaire was displayed in English, which is the medium of instruction at the university, and the questions were mostly close-ended. The questionnaire consisted of the following 5 sections: A: basic demographic data of the participants, including age, gender, nationality, years of study, and faculty; B: antibiotic use: 3 questions on the prior use of antibiotics, advice received, and the place of obtaining them; C: knowledge of antibiotics: 4 questions about the treatment duration, knowledge of sharing of antibiotics, disease conditions which require antibiotics; D: knowledge of antibiotic resistance: 5 questions on the commonly used terms related to AMR; 8 true/false statements regarding knowledge of AMR; 8 statements related to AMR measured using a five-point “Likert scale” of agreement, where 1 denotes “Disagree strongly”, 2 stands for “Disagree slightly”, 3—for “Neither agree nor disagree”, 4—for “Agree slightly”, and 5—for “Agree strongly”; E: antibiotic use in the community: one question on antibiotic use in agriculture and food products. The questions from the section on the knowledge of antibiotics and the true/false statements from the “Knowledge of antibiotic resistance” section were scored as follows: 1 for each correct answer and 0 for an incorrect one or “Do not know”. The knowledge scores for the questionnaire comprised of 12 questions with 14 correct answers were then categorized into “Good” (≥10 correct answers), “Moderate” (6–9 correct answers), and “Poor” (<6 correct answers).The questionnaire underwent preliminary pilot testing with subsequent revisions conducted among 15 respondents for initial validation. Necessary adjustments were made based on the feedback received from the respondents before finalizing the questionnaire. Three questions about obtaining antibiotics from a physician or nurse, vaccination, and washing hands from the original World Health Organization’s (WHO) “Antibiotic resistance: Multi-country public awareness” survey were omitted, as most antibiotics can only be obtained from a medical practitioner and the latter were found to be out of the research topic. The face validity of the online questionnaire was also assessed for readability, length, and relevance of the questions by two pharmacists with postgraduate qualifications at the Institute of Health Sciences, UBD.The internal consistency of the survey questions was determined using the Cronbach’s alpha coefficient. The Cronbach’s alpha values of the section on antibiotic use and knowledge were 0.66 and 0.86, respectively, i.e., satisfactory.The data collected in the web-based survey’s database were exported into Microsoft Excel or other appropriate software. Data analysis was done using R statistical software (version 3.6) and Microsoft Excel. The data were summarized using descriptive statistics, which includes percentages, frequencies, and also means. Appropriate inferential statistics was also used according to the data distribution.The study received ethical approval from the Research Ethics Committee of Pengiran Anak Puteri Rashidah Sa’adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam. All the participants were notified about the aims of the study, all the data collected were to remain anonymous, and confidentiality was strictly maintained. A written consent form was provided in the survey by ticking an option placed at the end of the cover letter prior to the distribution of the questionnaire. The study was conducted on a voluntary basis and respondents had the freedom to not participate in the online survey.A total of 130 respondents returned a completed questionnaire yielding a response rate of 53%. The median age of the respondents was 21 years . The majority of the respondents were female  and Bruneian . The proportions of the students in relation to the year of study (year 1–year 4) were 26% (n = 34), 18% (n = 23), 28% (n = 36), and 28% (n = 37). The students were mostly from the Faculties of Science , Health Sciences , Arts and Social Sciences , and from the School of Business and Economics . The characteristics of the respondents are presented in The results obtained for antibiotic use  showed tp = 0.001) and faculty (p < 0.001) but not between gender, nationality, place of resident, and year of study (p = 0.001) and in Health Sciences faculty (p < 0.001).More than half of the students  showed a good level of knowledge (score more than 10) on antibiotics and antimicrobial resistance with a mean total knowledge score of 9 out of 14. Only 14% (n = 18) of the students scored below 6. Fisher exact test revealed that level of knowledge of the students was significantly different in relation to age , agreed that antibiotics should be taken as a full course as directed. The frequency and the percentage of students for the statements regarding antibiotic use are presented in A large number of students correctly identified that antibiotics are used to treat bladder or urinary infections (UTI) , skin or wound infections , and gonorrhea   35]. Mo. Mo35]. When asked about the terminology, the students recognized the terms related to antibiotic resistance , drug resistance , antibiotic-resistant bacteria , and antimicrobial resistance . However, the acronym AMR  which stands for “antimicrobial resistance” and superbugs  were not well-known among the students. The most common source of the terminology was media , followed by a family member or friend, including social media , physician , nurse , pharmacist ; other sources include pre-university and university lectures and secondary schools  .In terms of knowledge about antimicrobial resistance , 76%  reported that they were aware about the use of antibiotics in agriculture and food products in the country, and more than half  of them were unsure about it.This study assessed the knowledge of antibiotics and antimicrobial resistance of the students. To the extent of the author’s knowledge, this is the first study carried out among Bruneian university students.A quite high degree of antibiotic consumption was evident in the study. Sources of antibiotics were mostly public and private hospitals or health clinics, as all antimicrobials in Brunei are classified as prescription-only medicines. This is unlike many developing countries where antibiotics can be acquired without a valid prescription, which is commonly practiced among university students in China, India, and Nigeria ,36,37. TMoreover, there were only few respondents found who had not received advice on how to take antibiotics and the need to complete the full course. Those who did not obtain advice are more likely to stop taking antibiotics when they feel better, which eventually leads to the growth of resistance in the community. Respondents also reported obtaining antibiotics from the previous use, but the rate was lower than in the previous studies in Malaysia (46/204), Qatar (161/596), China , and Saudi Arabia (165/347) ,26,27,28Furthermore, most of the students demonstrated good knowledge of antibiotics and antimicrobial resistance. The majority correctly identified conditions that can be treated with antibiotics, namely, bladder or urinary infections, skin or wound infections, and gonorrhea as suggested by the WHO in the antibiotic resistance survey. However, the misconceptions regarding the use of antibiotics for viral infections were also noted in the study, which was consistent with findings from other studies. The proportion was lower than in Jordan (527/1158), and Jatinangor, Indonesia (145/250), but higher than in Italy (210/1050) ,32,33. SThis study has several limitations. The results of the study may not be extrapolated to other universities, since it was conducted at one university. Other limitations of the study include the risk of response bias, as the findings of the survey were solely based on the self-reported data.Although a good level of knowledge was found in this study, there are still gaps in areas of antibiotic use, especially the part on the appropriate antibiotic use for certain disease conditions, as some mistakenly considered antibiotic therapy for viral conditions. The current study findings provide baseline data for future research studies. However, further research is needed on this topic, especially to assess the attitudes and practice of antibiotic use among the students and not just focus on knowledge so that appropriate interventions can be carried out. Awareness of the proper use of antibiotics among the students is greatly required to correct their misconceptions and prevent the rise of antimicrobial resistance.


Accumulating studies implicate that the metformin (MET)- and oligofructose (OFS)-altered gut microbiota may play roles in the improvement of type 2 diabetes mellitus (T2DM) and obesity. However, whether the combined administration of OFS and MET could effectively affect the gut microbiota and improve metabolic profiles remains unknown. Here, we randomized diet-induced obesity (DIO) rats to OFS, MET, or MET+OFS for 8 weeks and demonstrated that the combined administration of OFS+MET possessed potentiated effects on the glycemia, body weight, and gut microbiome. In addition, fecal samples from the MET and MET+OFS group were exchanged and transferred to germ-free rats induced by antibiotics. Not surprisingly, the glucose tolerance and serum levels of endotoxin, free fatty acids (FFA), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and interleukin-6 (IL-6) were all sustainably improved among OFS+MET fecal microbiota-treated DIO rats while the MET fecal microbiota-treated ones presented a relatively reverse trend. Furthermore, transfer of fecal samples from the rats after 8 weeks of treatment to antibiotics-treated germ-free mice significantly improved metabolic profiles, including glucose tolerance and weight reduction in mice that received MET+OFS-altered microbiota. In conclusion, the present study illustrated that the effects of OFS and MET combined treatment on gut microbiota, especially for the MET-induced side effect-related ones, and host metabolism were of greater magnitude than individual OFS or MET treatment in obese rats and mice. Therefore, it is likely that combined administration of OFS and MET may offer a novel and promising strategy for reducing side effects induced by MET and improving metabolic outcomes, particularly glycemia control and weight reduction. T2DM and obesity are both representative chronic systemic disorders of overweight and hyperglycemia mainly resulting from the genetic and environmental risk factors, including the relative lack of insulin caused by insulin resistance, high-fat and high-sugar diet, and lack of exercise –4. ObesiMost recently, attention on the role of the gut microbiota in the development and maintenance of obesity and T2DM is growing rapidly. Changes in insulin sensitivity, energy balance, lipid metabolism, and secretion of gut hormone through altered gut microbiota have all been considered as probable mechanisms –20. Not The aim of the present study was to examine the combined effects of MET and OFS on gut microbiota and metabolic disorders, especially for obesity and T2DM, in an animal model. Here, we investigate how the combination of OFS (10% wt/wt) and MET (150 mg/kg) affects the metabolism of animals by using rats with diet-induced obesity for 8 consecutive weeks, and the glucose tolerance, metabolic parameters, and blood biochemistry indexes were carefully evaluated. After the treatment of antibiotics, the DIO rats treated with MET and MET+OFS were both divided into two groups receiving fecal microbiota from the two groups to investigate whether combination of MET and OFS could effectively reduce the side effect-related indexes and improved metabolic disorders. Further, we also transferred the fecal microbiota from DIO rats treated with OFS, MET, and OFS+MET, respectively, to antibiotics-induced germ-free mice so as to evaluate the effects of MET-altered microbiota on host glucose metabolism.The Animal Care Committee of Binzhou Medical University approved the protocol of animal experiments, with approval codes BAC18-115 and BAC19-056. Male Wistar rats and C57BL/6J mice were purchased from Shandong Laboratory Animal Center  and housed on a 12-h light–dark cycle, at a temperature of 24°C. Male DIO rats and mice were fed a diabetogenic diet, which is high-fat diet with 60% kcal from fat [high-fat diet (60%) diet D12492], for a minimum of 4 months, and the final experimental animals used were between the ages of 7 and 8 months. Wistar rats were randomly assigned to one of four groups:  MET Sig. ATM SkiAfter overnight fasting (12 h), 2 g/kg glucose was orally administered and blood was collected at 0, 15, 30, 60, 90, and 120 min. Blood glucose was measured immediately with a OneTouch blood glucose meter . The insulin level was detected by the rat insulin ELISA kit .Plasma amylin, ghrelin, PYY, CCK, glucose-dependent insulinotropic polypeptide (GIP), and plasma inflammation-associated indexes, including endotoxin, TNF-α, IL-2, and IL-6, were measured using rat ELISA kits . In mice, the levels of leptin, resistin, non-esterified fatty acid (NEFA), tumor necrosis factor-α (TNF-α), adiponectin, and IL-6 were also detected using ELISA kits purchased from Millipore.Stool samples from DIO rats were collected after 8 weeks of treatment, quickly frozen using liquid nitrogen, and stored in a freezer (−80°C). The analysis of 16S rRNA sequence was conducted at BGI Genomics . The extraction and quality of microbial genome DNA were conducted using the conventional method of TACB/SDS and measured on agarose gel (1%). V3–V4 region 16S rRNA genes were amplified using previous reported primers and methods . LibrariFigure 6, from day −56 to −1, DIO rats were randomly assigned to the four groups. At day 0, OGTT and other metabolic profile baseline of rats were measured. Then, the rats were conducted with gavage of antibiotic solution during days 0–7. Further, intragastric administration of 0.2 ml of fecal material was conducted from day 7 to day 21 (14 consecutive days) to pseudo-germ-free rats. The OGTT and hematological index detection was carefully performed at days 22 and 23, respectively. As shown in Figure 8A, after a 7-day acclimatization, the pseudo-germ-free mice model was successfully established by administrating a large dose of antibiotic for 10 consecutive days. In the next 14 days (day 1 to day 14), 0.2 ml of the obtained fecal microbiota from the rats was orally transplanted to pseudo-germ-free mice and OGTT tests were subsequently measured.Fecal samples were obtained and then stored immediately after defecation at −80°C for further transplantation. One gram of fecal sample was diluted in 10 ml of PBS and then suspended to obtain supernatant including fecal microbiota. Transfer of fecal microbiota was conducted on both DIO rats and mice treated with antibiotics. As shown in P < 0.05 was considered significant.All data are presented as the mean ± SEM. The comparison between three treatment groups to the control and normal groups were performed by one-way ANOVA test. P < 0.05) and OFS+MET (P < 0.01) groups; however, the effect was not significant, though the result had the same situation in the OFS group. Both MET- and MET+OFS-treated groups showed decrease in food intake compared to the control and OFS-only group. After the 8-week period of the experiment, significant differences in fat weight and fat/body (%) were observed in OFS+MET  compared to the control  and similar to the normal ones  OFS, and 10% (wt/wt) OFS+150 mg/kg of MET, respectively, for 8 consecutive weeks, using the healthy rats treated with saline as normal group. As a result, the effects of OFS, MET, and OFS+MET treatment on metabolic parameters in DIO rats are shown in mal ones . Adminismal ones . The othmal ones .The effects of OFS and MET treatment on fasting blood glucose, insulin levels, and HOMA value for insulin resistance (IR) in DIO rats are presented in To evaluate whether an 8-week treatment of OFS, MET, and OFS+MET helped to improve the glucose metabolism in DIO rat, OGTT tests were conducted before and during the chronic treatment . As is presented in We further evaluated whether and how the plasma levels of appetite-related hormones were affected by the administration of OFS, MET, and OFS+MET during the OGTT at week 8. As shown in To explore whether the 8-week treatments of OFS, MET, and OFS+MET affect the gut microbial composition of DIO rats or not, we detected 40 fecal samples from each group using the 16S rRNA gene sequencing-based method. As a result, the 16S rRNA sequencing yielded probably 83,000 clean reads (duplicate reads > 95%) in each sample after a strict process including quality control, denoising, and the removal of chimera. Then, the clean reads were clustered into operational taxonomic units (OTUs) and then carefully assigned to taxa from phylum to species level.Beta diversity analysis was performed by principal coordinates analysis (PCoA) of the unweighted UniFrac distances of gut microbiota samples to explore the similarity of bacterial community patterns among all the DIO rat groups. As a result, a clear separation of the microbial community is shown in the placebo-treated control group, while the OFS and OFS+MET group tended to be closer to the normal group as compared with the MET group . Alpha dBacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Verrucomicrobia at the phylum level, especially for Bacteroidetes and Firmicutes. Compared with the normal rats, abundance of Firmicutes in placebo-treated DIO rats was obviously increased, while the Bacteriodetes and Actinobacteria were decreased relatively. In addition, the ratio of Bacteriodetes/Firmicutes, a measure associated with obesity and increased host inflammation, was reduced in the control group. In contrast, OFS+MET treatment increased the abundance of Bacteriodetes and Actinobacteria, which not only partially rescue the declining ratio of Bacteriodetes/Firmicutes, but also substantially correct the loss in obesity, respectively. Notably, abundance of Verrucomicrobia, which was a kind of beneficial bacterium helping to improve insulin resistance and reduce weight, in the OFS group and OFS+MET group was similar to the normal ones, while these were significantly decreased in placebo-treated DIO rats.As shown in B. pseudolongum in normal rats, obesity induced an obvious decline in this species, while the treatment of OFS and MET led to a reverse trend. In addition, combination treatment of OFS and MET in DIO rats was associated with a more significant improvement. A similar pattern is shown in the abundance of Bifdobacterium species , which were associated with intestinal inflammation and obesity, were both significantly reduced under OFS or OFS+MET treatment , hypertension, and hyperlipidemia. The development of chronic diseases is usually affected by many factors including genetic, physiological, and behavioral factors . Due to In this study, we investigate the combination effects of OFS (10% wt/wt) and MET (150 mg/kg) on the metabolism of animals using rats with diet-induced obesity for 8 consecutive weeks. Results showed that OFS+MET treatment, but not TG and CHOL decrease, had obvious improvement on fat weight, %fat/body and %HbA1c , weight To determine whether and how the combination of MET+OFS affects the gut microbiome, we performed 16s rRNA analysis of the obtained 40 fecal samples and further analysis of Beta and Alpha diversity. As a result, microbial community was clearly separated in the placebo-treated control group, while the OFS and OFS+MET group tended to be closer to the normal group as compared with the MET group . CompareBacteriodetes/Firmicutes, a measure associated with obesity and increased host inflammation, and substantially corrected the loss in obesity. Similarly, the abundance of Verrucomicrobia that helps to improve insulin resistance and reduce weight in the OFS+MET group was similar to the normal ones, while it decreased in the control group. In addition, obesity induced a significant decline of the abundance of B. pseudolongum and Bifdobacterium, which are reportedly related with positive health effects, such as decreasing inflammation, while the OFS+MET-treated group showed a more obvious reverse trend compared with individual treatments, indicating that the potentiated effects were indeed exist. Furthermore, the increased abundance of Shewanella and Allobaculum, which were associated with improved metabolic endotoxemia, are shown in the MET treatment group. Obvious changes in the OFS group were not observed yet, which indicated that MET treatment partially accounted for the effects of OFS on this beneficial bacterium. A similar pattern was shown in the abundance of Peptococcaceae rc4-4 sp. and Peptostreptococcaceae sp. related to diet-induced obesity and intestinal inflammation. Both OFS and OFS+MET treatment significantly reduced these two bacteria. Interestingly, MET treatment increased and maintained the abundance of Peptococcaceae rc4-4 sp. and Peptostreptococcaceae sp., respectively, indicating that OFS may be an effective complement to MET. In addition, Intestinibacter species was previously reported to improve the resistance to oxidative stress, be able to degrade fucose, and possess the genetic potential for sulfite reduction, including part of an assimilatory sulfate reduction pathway, and, in the present study, this species displayed an obvious increase in abundance under OFS treatment and the addition of OFS also reversed the down-regulatory effect on the abundance of Intestinibacter under only MET treatment. Furthermore, similar improvement of OFS on function of MET is also shown in the alteration of the abundance of Escherichia species, which was reportedly related to side effects of MET treatment including enrichment of virulence factors and gas metabolism genes. There is an increasing influence of gut microbiota on energy metabolism, and it will be critical to evaluate not only the effects of dietary factors known to alter gut microbiota but also the effects of pharmacological agents such as MET, OFS, or MET+OFS that are used to treat metabolic disease. It is likely that combination of MET- and OFS-altered gut microbiota might be associated with improved weight reduction, hyperglycemia, and insulin resistance. Not only that, some inflammation reactions and side effects induced by MET were also ameliorated due to the supplement of OFS.As shown in In this study, the combination of MET and OFS was found to show potentiated effects on hyperglycemia, insulin resistance, and side effects related to gut microbiota compared with individual action. It is likely that MET+OFS-altered gut microbiota plays a role in these improvements. In order to further determine whether the OFS+MET-altered microbiota could effectively contribute to the improvement of side effects related to factors and other metabolic profiles induced by MET, the DIO rats treated with MET and MET+OFS were divided into two groups receiving fecal microbiota from the abovementioned two groups after the treatment of antibiotics for consecutive 12 days . Not surFurther, the transplantation of the fecal microbiota from DIO rats treated with OFS, MET, and OFS+MET, respectively, to antibiotics-induced germ-free mice showed that the OFS+MET-altered fecal microbiota could still improve glucose tolerance, weight reduction, Lee's index, and obesity-related disease risk factors . InteresEscherichia and other related species. In the present study, OFS is found to have potential to be a supplement for the most widely used antidiabetic treatment of MET, especially in terms of enhanced positive effects and improved negative effects. In addition, the potential molecular mechanism of these outcomes will be deeply explored in future research.In conclusion, our results suggest that the combination of MET and OFS induced gut microbial mediation of more perfect therapeutic effects, including improved glucose tolerance, HA1bc, weight reduction, and other blood biochemical indexes, compared with administration of individual MET or OFS. Based on previous reports , 32, sighttp://www.ncbi.nlm.nih.gov/bioproject/596853).The datasets generated for this study can be found in NCBI accession number PRJNA596853 (The animal study was reviewed and approved by The Animal Care Committee of Binzhou Medical University. Written informed consent was obtained from the owners for the participation of their animals in this study.QL and RH: design and analysis of ideas. FZ: methodology. JZ: resources. SL: writing—original draft preparation. HL: writing—review and editing. QL: methodology and experimental operation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Information integration theory has been developed to quantify consciousness. Since conscious thought requires the integration of information, the degree of this integration can be used as a neural correlate ( Information Integration Theory ,2 definets parts . At the ts parts , but we Information integration has been proposed to be a neural correlate for conscious experience  under thiousness  and thusUnfortunately, we do not yet have the ability to thoroughly measure research ,9,10,11 research   that are genetically encoded and subject to evolution ,18,19,20feedback gates to the repertoire of gates from which Markov Brains can be built. For a detailed description, see Sheneman and Hintze 2017 [Markov Brains built from deterministic and probabilistic logic gates can perform complex computations and can control animats in virtual environments. They can use past experiences to inform future decisions, and form representations about their environment . Howevertze 2017 .In essence, feedback gates have two additional inputs for positive or negative reinforcement. At each update, a feedback gate records which output was mapped to the current input. For positive feedback, the probability that leads to this mapping becomes increased; in the case of negative feedback, this probability becomes decreased. After that, the probability table is normalized so that each row of the probability table sums to decomposable) versions of all the gates, including feedback gates (see the Methods for a detailed description of decomposable gates). In addition to facilitating Unfortunately, feedback gates and probabilistic logic gates violate a critical requirement of information integration theory 3.0 . To accumposable . Decompomple, in  it took A key part of MB evolution is that their interaction with their environment requires them to be embodied . These lIn previous work, we evolved animats who learn to solve a navigation task see . We now ra paths  to the gThis presents a very easy task and would not require animats to remember or learn anything. To improve the complexity of the task and to require animats to remember and learn, the animats were not allowed to directly choose to move or turn but instead had to choose from a set actions associated with results. An animat would choose one of four actions , which were each associated with a different one of four results: turning 90Using animats evolved in this environment, we can now measure the amount of  of ΦMax  seems ton (nodes), which scales super exponentially. Instead, using Fortunately, Equation ) is a comic, see ).(1)Φatt). Each i as t to Therefore, for any time step i, and Thus, ariables ,27,28,29With this “more than the sum of its parts” system information, and spatial integration of nonindependence, X) at every time point (t), and one needs all of these t we can compute all possible state-to-state transitions the animat’s brain could theoretically experience. This is done by computing the state-to-state transition probability matrix (TPM) at time point t assuming It has also been shown that ons of Φ ,8, makineriments ,8, ΦatomAnimats were evolved to perform a navigation task for which they had to use environmental clues to determine the correct motor outputs, and thus the correct behavior. Given the difficulty of the task, only 13 of all the 200 replicate experiments resulted in animats that performed well at the end of evolution. Specifically, all animats were tested on all possible 24 variations of the environment, one with learning , and one without learning. After 200,000 generations, only those animats that reached the goal two-fold more with learning than without were considered “elite.” From these 13 elite performers, all animats on the line of decent  were evaluated in the same environment to analyze this see . We furtOver the course of evolution, animats optimized their ability to navigate the environment, and to learn how the possible choices map to their movements. As expected, animats learn to move more deliberately through the environment, by reducing the number of unnecessary turns, and idle behavior, while moving forward more often see . These cDecomposable feedback gates must change their probability matrix to allow for lifetime learning. The mutual information that these gates convey between their inputs and outputs should increase over the lifetime, and we expect that the delta of this change also increases over evolutionary time, as observed before  for non-To obtain an additional impression of how animats behave and how their ability to learn changes over evolutionary times and over their lifetime, see the Earlier experiments with ary time ,8,9,11. alue see .However, this result can be explained. Random animats can have high values of n before ,8 and weThe addition of feedback gates to MBs allows these brains to change from generation to generation and during their lifetime. This allows us to test how these lifetime changes (learning) affect Beyond this apparent reduction of mats see .Specifically, we find that the amount of tion see .In addition, we characterize the reduction of mers see . Both tymers see  show thaIn this work, we measured how lifetime learning influences an animat’s ability to integrate information. We had no intuition of how information integration would respond to learning: increase, decrease, or remain. Because such measurement is currently prohibitively difficult with natural organisms—let alone human beings—we use evolved animats as the next best substitute. Markov Brains controlled these animats, because they were a proven study system used for similar work and thus allow for direct comparisons. The biggest constraint on this type of assessment for any continuous or probabilistic computational system is the necessity to be computationally decomposable. To date, only decomposable probabilistic logic gates  have hadInstead of measuring To study how information integration changes over an organism’s lifetime, we created a computational model in which animats are required to navigate in a changing environment. Animats needed to learn how to properly move within this environment. The brain of each animate was a probabilistic finite state machine that consisted of sensor nodes (input), memory nodes (hidden), and action nodes (output). This was implemented by a Markov Brain (MB) ,12. The Populations of 100 animats were evolved in 200 replicates to perform a spatial navigation task  describe0,0,0,1.Animats’ brains consisted of 16 states: 4 input sensors, 2 output motors, and 10 hidden states. Each input sensor was assigned a direction to detect: sensor 0 fired when the animat was told to stay put, 1 indicated turn right, 2 go forward, and 3 turn left. As described above, the environment was composed of tiles with arrows that point towards the shortest path to the goal. Sensors could read the arrow of the tile that the animat was currently on. When the sensor detects its given direction, the state was set to 1, otherwise the state was 0. For example, if the map indicated the animat’s next closest step was to the left, its first four states would then be: The LoD for an experiment was determined by choosing a random animat in the final generation and tracing its lineage to the first generation . Throughi, which results in output o is defined by a probability that has the potential to change with each update due to the feedback received. Each time these numbers change, so does the amount of predictive information shared between nodes, resulting in a change to The key feature of feedback gates is that every input  Φatomic . It is i0 0 with probability 0.36, 0 1 with 0.16, 1 0 with 0.16, and 1 1 with 0.04, it is possible to compute the independent probabilities for each of the two bits a and b of 0.4 and 0.4. 0 0 (both bits off) = 0 1 = 1 0 = 1 1 = Decomposable feedback gates superset includes features from feedback gates, decomposable gates, and probabilistic gates. A probabilistic gate relies on a probabilistic mapping between input and output patterns. However, each input pattern is associated with a separate set of probabilities over all output patterns. Decomposable gates are based on the design of probabilistic gates. To ensure decomposability of outputs for a single input pattern, the output patterns must have probabilities such that the probabilities of single outputs are independent. In other words, if an input pattern could produce output birth (construction) of the organism, and derive their probabilities directly from the genome. For a decomposable gate, the genome provides the factors for each input pattern that are then multiplied together to produce a full row of output pattern probabilities. In this way, each input pattern will probabilistically produce any of the possible output patterns such that the probability of individual output-bit activations within each pattern is independent across all patterns associated with that input.The probabilistic gates for an animat are constructed upon the t of the time point To allow learning via feedback over a lifetime, we add to the design of each gate a positive feedback connection to one of the states , and a negative feedback connection to one of the states. When the input bit for positive feedback is activated, the probability for the previous input–output activation is increased by a small amount, and one of the other output pattern probabilities randomly chosen for that input is decreased by the same delta. Negative feedback happens in a similar way when the input bit wired for negative feedback is activated. This configuration allows feedback to time From earlier experiments, we understood how Over the course of the animat’s life, The second interesting observation is that the reduction of information integration becomes further minimized over evolutionary time. The more evolved an animat becomes, the less it must reduce its information integration due to learning. This suggests that the learning process itself becomes optimized to reduce the change to information integration. This seems surprising. Because learning reduces alue see  instead Therefore, we hypothesize that the behavior of It is entirely possible that the phenomenon we observe is dependent on the specific task, type of evolutionary optimization, and the life time learning mechanism implemented here. Other forms of learning for other tasks, particularly those where time is not of the essence, may behave differently. Investigating the changes to orcement ) present


A striking drop in interstitial plasma cell infiltrate as well as normalization of IgG4/IgG-positive plasma cells was observed at repeat biopsy. Both clinical and immunological improvement persisted over a 4-year follow-up. Treating these patients who were affected by aggressive IgG4-RD with renal involvement in an effort to induce a prolonged B cells depletion with IgG4 and cytokine production decrease resulted in a considerable rise in eGFR, with IgG4-RD RI normalization and a noteworthy improvement in clinical and histological features. Furthermore, the TIN subgroup was shown not to need for any maintenance therapy.IgG4-related disease (IgG4-RD) is an immune-mediated disorder often showing elevated serum IgG4 concentrations, dense T and B lymphocyte infiltration, and IgG4-positive plasma cells and storiform fibrosis. We prospectively evaluated for 4 years 5 patients with histologically proven IgG4-RD of whom 3 had tubulointerstitial nephritis (TIN) and 2 had retroperitoneal fibrosis (RPF). They received an intensive B depletion therapy with rituximab. The estimated glomerular filtration rate of TIN patients after 1 year increased from 9 to 24 ml/min per 1.73 m2. IgG/IgG4 dropped from 3236/665 to 706/51 mg/dl, C3/C4 went up from 49/6 to 99/27 mg/dl, and the IgG4-RD responder index fell from 10 to 1. CD20 In detail, the latest include IgG4hlebitis , 2.Men in their 50s and above are most often affected and generally display non-severe clinical presentation, potentially mimicking several neoplastic, infectious, and inflammatory diseases .Most common laboratory features are high serum concentration of IgG4 levels (observed in 50–70% cases) (> 135 mg/dl) often associated with hypergammaglobulinemia (80–90% of cases). Almost half of the patients also present with high serum IgE levels, peripheral eosinophilia, and hypocomplementemia. Less frequently antinuclear antibodies and rheumatoid factor can be observed .+ T cells and activated B cells including plasmablasts, and secrete cytokines such as IFN-γ and TGF-β, chemokines, growth factors, and enzymes, which recruit and activate fibroblasts thus being partially responsible for inflammation and fibrosis [Both T and B lymphocytes are involved in disease pathogenesis. Indeed, IgG4-RD lesions abound with activated clonally expanded cytotoxic CD4fibrosis –13. Clinfibrosis . About 1fibrosis , 11. IgGfibrosis , 16. IgGfibrosis , 15–18.18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) are useful tools for diagnostic, staging, and monitoring purposes [In patients with IgG4-related TIN, progressive kidney failure, proteinuria (often in sub-nephrotic range), leukocyturia, and mild hematuria can be observed , 19–22, purposes . The IgGpurposes .Patients usually respond favorably to the administration of glucocorticoids which are the mainstay of treatment for IgG4-RD , 22, 26 We have already described the encouraging outcome of a group of patients with severe systemic lupus erythematosus (SLE) who underwent intensive B cell depletion therapy (IBCDT) with a combination of 4 plus 2 infusions of RTX, cyclophosphamide, and methylprednisolone pulses . Using tThe cohort of patients with IgG4-related kidney disease examined in this study Table  includesPatient N.1 had been diagnosed with a Sjogren-like syndrome that was being treated with small doses of oral prednisone. We admitted him to our unit 12 months later due to rapidly progressive renal failure. Kidney biopsy was in line with IgG4-related TIN.Sixteen months prior to coming to our attention, patient N.2 had undergone mononephrectomy elsewhere since a neoplasm was suspected. Final histological diagnosis reported xanthogranulomatous pyelonephritis. Due to rapidly progressive renal failure, he was admitted to our unit where he underwent renal biopsy which revealed typical IgG4-related TIN.On account of a malignant tumor, patient N.3 had been subjected to colon resection 20 years earlier. Admission to our unit was due to acute renal failure overlapping chronic impairment. Renal biopsy showed IgG4-related TIN.TIN patients  were investigated with ultrasonography at baseline and after 12 and 24 months. Contrast-enhanced CT was not performed due to the high risk of contrast-induced renal failure.Patient N.4 was referred to our unit due to acute kidney injury. He also complained about lower back pain. Both ultrasound and CT imaging confirmed bilateral hydroureteronephrosis caused by retroperitoneal and periaortic abnormal tissue. Tissue specimens obtained by exploratory laparotomy revealed features of IgG4-related RPF.Ten months prior to admission to our unit, patient N.5 had undergone radical hysteroannessiectomy on account of suspected uterine neoplasia with associated, at first monolateral, hydroureteronephrosis. Ureteral stents were implanted because of recurrent hydronephrosis caused by the presence of high metabolic activity in pathologic pelvic and periureteral tissue as detected at FDG-PET/CT. Surgical specimen re-examination following admission to our center confirmed IgG4-related RPF.All patients underwent baseline and follow-up evaluation of the IgG4-RD RI, estimated GFR using the CKD-EPI formula, blood count, CRP, IgG/IgG4, C3/C4, CD20+ count, and urinalysis.The 3 TIN patients had repeat percutaneous needle biopsies 12 months after the initial histological diagnosis. The two RPF patients underwent contrast-enhanced CT or MRI and FDG-PET/CT at baseline and during follow-up.EDTA were stained with monoclonal antibodies against CD45 , CD3 , CD4 , CD19 , CD20 , CD25 , and FOXP3 .Circulating B and T cells in the peripheral blood were analyzed by flow cytometry. Whole blood samples collected in Patients N.1, N.2, and N.3 underwent real-time ultrasound-guided percutaneous needle renal biopsies. Histological confirmation was acquired by open biopsy of the mass lesion in patient N.4 and by surgical hysteroannessiectomy in patient N.5. All tissue specimens were examined by light and immunofluorescence microscopy. Histological confirmation was obtained by immunostaining with anti-IgG and anti-IgG4 antibodies in every case.plus  2 pulses of 500–750 mg cyclophosphamide (on days 1 and 15) plus 4 weekly rituximab administrations (375 mg/m2) were delivered. Two more doses of rituximab were given 1 and 2 months after the last weekly infusion.The IBCDT protocol we previously described for the treatment of severe cases of SLE with nephritis was followed for the treatment of , 39. ThrWe did not administer cyclophosphamide to patient N.3 on account of his prior history of colon cancer.Patient N.5 received a maintenance regimen consisting of RTX 500 mg every 6 months for 18 months, followed by RTX 500 mg after 12 months.Patients N.2 and N.5 received isoniazid prophylaxis on account of positive QuantiFERON-TB Gold test results.t test was used for normally distributed parameters and the non-parametric Mann-Whitney test for non-normally distributed parameters. For these analyses, the SPSS  software was used. P < 0.05 was considered statistically significant.For the comparison of variables at baseline and follow-up, Student’s Characteristics at inclusion and during the 48 month follow-up are presented in Table 2. This trend toward amelioration continued at 24 months , at 36 months , and at 48 months . Patient N.4 also showed an improvement in eGFR values at 12, 24, 36, and 48 months (from 48 to 66–75 ml/min per 1.73 m2). The eGFR values of patient N.5  remained stable 3 years after the stents had been explanted.Kidney function substantially improved in the 3 TIN patients  who underwent IBCDT. eGFR at 12 months had increased from 9  to 24  ml/min per 1.73 mDefinitive improvement of the immunologic parameters was observed for TIN patients , and especially with regard to total serum IgG, which dropped from 3,2  to 706  mg/dl at 1 year, and to 986  mg/dl at 4 years. IgG4 subclass also decreased from 665  to 51  mg/dl at 1 year and to 117  mg/dl at 4 years. Complement mean level rose to normal. C3 increased from 49  to 99  mg/dl at 1 year and to 95  mg/dl at 4 years, while C4 went from 6  to 27  mg/dl at 1 year to 24  mg/dl at 4 years. A drop in total IgG and IgG4 levels was also seen in our RPF patients (N.4 and N.5) on account of immunosuppressive treatment. C3 and C4 levels, which were not deranged at baseline, remained unchanged throughout follow-up. Urinalysis revealed mild proteinuria and mild microscopic hematuria at baseline, all of which rapidly and persistently improved after treatment (data not shown). All five patients had isolated kidney involvement requiring immediate treatment, except patient N.1 who presented also moderate salivary gland disease. The 3 TIN patients had increased serum IgG4 concentration at baseline. The IgG4-RD RI decreased from 8.4  to 1 at 1 year and remained stable during follow-up. The exceptions were patient N.1 whose RI rose to 4 after 36 months related to a moderate rise in IgG4 levels and patient N.4 whose RI increased to 3 after 24 and 48 months because of disease flares.+ B cell mean values dropped from 8.7  to 0% within 7 days of the 4th infusion (data not shown), mildly increased to 1.2%  12–15 months later, and rose to 2.2%  after 2 years.Pre-treatment CD20A repeat renal biopsy was carried out on TIN patients N.1, N.2, and N.3) 1 year after the first procedure. Representative results  are presented in Figs. , and N.3+ plasma cells (> 30/HPF).Light microscopy and immunohistochemistry results of the surgical periaortic tissue biopsy carried out on patient N.4 are shown in Fig. Ultrasonography in patients N.1, N.2, and N.3 showed no definite atrophic change after 12 and 24 months..Changes in the retroperitoneal, periaortic tissue of patient N.4 are shown in Fig. 52 every 4 months for 12 months, and 500 mg every 6 months for further 12 months. MRI and PET/CT scans revealed a gradual decrease in retroperitoneal tissue. Twelve months following the last RTX infusion, MRI and PET/CT studies highlighted left hydronephrosis caused by a pelvic fibro-inflammatory mass. The patient received intravenous steroids and RTX 375 mg/m2 every 6 months, which resulted in complete remission.Patient N.4 relapsed 16 months later concomitantly with a circulating CD20-positive cells increase, and was treated with oral steroids (tapered over the next 3 months), as well as RTX 375 mg/m. Retroperitoneal and perivascular fibro-inflammatory tissue on CT scan completely disappeared although residual minimal pelvic metabolic activity was still visible on PET/CT scans. The ureteral stents were explanted after 14 months, and no evidence of hydronephrosis was seen in the following annual MRI.Radiologic imaging carried out on patient N.5 prior to beginning the IBCDT protocol and again at 6 months is depicted in Fig. 6Transient insulin therapy was administered to patients N.1 and N.2 in whom a steroid-induced diabetes mellitus occurred. Insulin administration was discontinued after the treatment.While on immunosuppressive therapy, patient N.4 developed mild, acute bronchopneumonia which was quickly and thoroughly brought under control with oral antibiotics.Glucocorticoids are the mainstay of treatment for both IgG4-RD , 14, 26 An initial prednisolone dose of 0.6–1.0 mg/kg/daily, gradually tapered over 2–4 weeks based on clinical response, is the standard approach for IgG4-RD patients, especially those with type 1 autoimmune pancreatitis. Improvements in both clinical and laboratory findings are often quick, though there are remarkable variations depending on which organ is affected and the degree of fibrosis . DespiteThe efficacy of RTX in ameliorating the clinical and serological features of IgG4-RD patients with active inflammation , 32, 33,Most patients with IgG4-related TIN , 27, 35 Recommendations for managing IgG4-RD were recently drawn up by an international panel of experts. They confirmed the use of glucocorticoids as first-line drugs for remission induction in all patients with active, untreated disease (94% agreement). On the contrary, there was less agreement (46%) with regard to the administration of steroid-sparing agents such as RTX . This maMore attention should be reserved to the most severe cases who are highly susceptible to getting worst outcome. These cases specifically include IgG4-related TIN (in which RTX might be indicated as a single course) and especially RPF .We applied the RTX-based regimen that we follow in patients with severe SLE nephritis , 39. TheIgG4-related TIN patients displaying greatly reduced eGFR and extremely high IgG4-RD RI at presentation  responded remarkably well with regard to both renal function and immunologic parameters including the normalization of circulating regulatory T cells at the end of therapy. This observation suggests a potential role for these cells in the disease pathogenesis  and coulRegarding radiological findings, even if we could not properly investigate these patients with contrast-enhanced CT due to their severe renal failure, it is not excluded that also the development of renal atrophy reported with steroid therapy  could haConcerning IgG4-related RPF, characterized by a high recurrence rate , 36, 50,This study has a main limitation in the number of subjects. Furthermore, patient N.3 did not receive cyclophosphamide, and patient N.5 received RTX as maintenance treatment for the aforementioned reasons, so the treatment protocol was not the same for all the patients. However, severe renal involvement is quite unusual in IgG4-RD (which itself is per se a very rare disease), and the treatment should be personalized to each patient, thus making the feasibility of a randomized controlled study, at least in this restricted sample of patients, unrealistic.The main strengths of this study consist in the long follow-up and, most importantly, the histological monitoring through repeat renal biopsies in TIN patients. Ultrasonography imaging after 12 and 24 months in our patients was unchanged; thus, it could not adequately inform about the actual disease evolution. Re-examining both histology and therapy 1 year post-diagnosis proved to be useful for directing further therapeutic decisions, providing insight into possible disease progression, and, as with our subjects, supporting therapy discontinuation.In conclusion, while if untreated IgG4-RKD can lead to end-stage renal failure and steroid treatment may not suffice when dealing with the most serious forms of IgG4-TIN and is constrained by numerous side effects, a single course of IBCDT was found to be safe and effective in the long term in IgG4-TIN. Subjects with IgG-RPF need maintenance treatment and close follow-up.


IgG4-related disease (IgG4-RD) is a systemic disease that can involve various organs and is characterized by the infiltrations of IgG4-positive plasma cells and lymphocytes, fibrosis, and elevated serum IgG4 levels. IgG4-related sclerosing cholangitis (IgG4-RSC) is a subtype of IgG4-RD. No certain relationship between IgG4-RSC and cholangiocarcinoma has been established as yet, and there have been few reports of the simultaneous diagnosis of IgG4-RSC and cholangiocarcinoma.A 76-year-old male visited our gastroenterology department due to the recent occurrence of pruritus and jaundice.Computed tomography (CT) scan showed ductal wall swelling and enhancement from both intrahepatic duct confluence to the common bile duct, upper biliary dilatation, and accompanying autoimmune pancreatitis (a sub type of IgG4-RD). Biopsy of the distal common bile duct by endoscopic retrograde cholangiopancreatography (ERCP) resulted in a diagnosis of IgG4-RSC. Subsequently, adenocarcinoma was identified by repeated cytology of bile juice. Finally, Klatskin tumor type IIIA and IgG4-RSC were concurrently diagnosed.IgG4-RSC was treated with steroid and Klatskin tumors by gemcitabine + cisplatin chemotherapy.The jaundice had improved and CT showed substantial improvement of the intrahepatic duct dilatation.IgG4-RSC and cholangiocarcinoma are easily confused, but their treatments are quite different, and thus, care must be taken during diagnosis. Furthermore, these 2 diseases may co-exist. Therefore, even if IgG4-RSC is diagnosed first, the possibility of accompanying cholangiocarcinoma should be thoroughly investigated. In the majority of cases, IgG4-RDs respond well to steroids, although immunosuppressants can be used when response is inadequate or steroids are unavailable.IgG4-related disease (IgG4-RD) is characterized by the infiltrations of IgG4-positive plasma cells and lymphocytes, fibrosis, and an elevated serum IgG4 level. IgG4-RD is a relatively recently recognized disease, and the mechanism responsible for its pathogenesis has not been established.,4 Interestingly, few cases of IgG4-RSC and cholangiocarcinoma co-existence have been reported.–7 The relationship between IgG4-RD and the malignancy is not yet clear. Although several studies suggest that IgG4-RD may be a risk factor for malignancy,–11 the mechanism has not been revealed. Here, we report such a case of coexistent IgG4-RSC and Klatskin tumor in a 76-year-old male and discuss the relationship between the 2 diseases.IgG4-related sclerosing cholangitis (IgG4-RSC) occurs when IgG4-RD invades the biliary tract and can be confused with other benign or malignant diseases such as primary sclerosing cholangitis (PSC) or cholangiocarcinoma. However, their treatments are quite different, and thus, care must be taken during diagnosis. In fact, many case reports have mentioned cases in which IgG4-RSC and cholangiocarcinoma were confused.2Patient has provided informed consent for publication of the case. A 76-year-old male visited our gastroenterology department due to a recent deterioration in systemic pruritus and jaundice. The patient had diabetes mellitus, had been treated for autoimmune pancreatitis (a subtype of IgG4-RD) 3 years previously, and had undergone cholecystectomy and stent insertion 2 years previously due to cholecystitis and intrahepatic duct stricture. The patient had an acute ill-looking appearance and systemic jaundice. Vital signs were as follows; blood pressure 140/80, pulse rate 73/minute, respiratory rate 20/minute, and body temperature 36.6°C.The patient was admitted and laboratory tests showed; total bilirubin was increased to 9.5 mg/dL, direct bilirubin to 8.0 mg/dL, aspartate aminotransferase to 74 IU/L, alanine aminotransferase to 136 IU/L, alkaline phosphatase to 506 IU/L, gamma glutamyl transpeptidase to 605 IU/L, and C-reactive protein to 0.72 mg/dL. Amylase and lipase were normal, but CA19–9 (a tumor marker) was high at 328 U/mL. Computed tomography (CT) revealed ductal wall swelling and enhancement from both intrahepatic duct confluence to the common bile duct, and upper biliary dilatation. In addition, the pancreas was swollen and displayed low attenuation in its periphery , lower bile duct stenosis, and bile duct wall thickening in areas without stenosis strongly suggest IgG4-RSC. However, obstructive jaundice, elevated serum CA 19–9, and obstruction of the hilar bile duct by ERCP suggest cholangiocarcinoma. These features can help to differentiate the 2 diseases, but they do not always apply and there are cases where both diseases coexist. Therefore, it is critical that clinical findings, blood tests, imaging tests, and biopsies be judiciously assessed, and if necessary, repeat biopsy be performed.IgG4-RSC can be easily confused with PSC or cholangiocarcinoma, and thus, it is difficult to distinguish between these diseases. However, the treatment and prognosis of these diseases are very different, and thus, it is important that a differential diagnosis be made. Many case reports have mentioned cases in which IgG4-RSC and cholangiocarcinoma were confused.–7 Since IgG4-RSC and cholangiocarcinoma may coexist, a final diagnosis should not be made prematurely even if IgG4-RSC is suspected and repeat biopsies should be undertaken to exclude the possibility of accompanying cholangiocarcinoma. In our patient, no malignancy was found by the first biopsy and brushing cytology test. However, to exclude cholangiocarcinoma, an additional biopsy and brushing cytology test was performed by ERCP. Finally, brushing cytology testing of the common hepatic duct proved to be positive for adenocarcinoma.In the described case, IgG4-RSC and cholangiocarcinoma were not confused but diagnosed simultaneously, and such cases have rarely been reported.,11 In a study by Yamamoto et al, in which 106 patients diagnosed with IgG4-RD were analyzed, malignancies were observed in 11 patients (10.4%), either at diagnosis or during a follow-up period of 3 years. The standardized incidence ratio for these malignancies in IgG4-RD was 383.0, which was higher than that for the general population. In a prospective UK cohort study conducted by Huggett et al, in which 115 patients with autoimmune pancreatitis or IgG4-RSC were followed for an average of 33 months, malignancy occurred in 11% shortly before or after autoimmune pancreatitis or IgG4-RSC was diagnosed. The risk of any cancer at diagnosis or during follow-up when compared with matched national statistics was increased (odds ratio = 2.25). Indeed, cases in which IgG4-RSC may have acted as a risk factor of cholangiocarcinoma development have been reported. Similarly, cases where autoimmune pancreatitis may have acted as a risk factor of malignancy development have also been reported.The relationship between IgG4-RSC and cholangiocarcinoma has not been clearified. But, several recent studies have suggested that IgG4-RSC may be a risk factor for cholangiocarcinoma. Much research has addressed the possibility that IgG4-RDs, such as IgG4-RSC, are a risk factor for malignancy. Second, the immunological environments associated with IgG4-RD can interfere with immune responses that inhibit cancer development. The immunologic characteristic of IgG4-RD is the activation of type 2 helper T (Th2) cells and regulatory T (Treg) cells. Autoimmunity or infection may act as an immunologic trigger in IgG4-RD. Interleukins 4, 5, 10, and 13 and transforming growth factor β (TGF-β) are overexpressed through an immune reaction in which Th2 cells predominate, followed by activation of Treg cells. As a result, serum IgG4 increases and IgG4-positive plasma cells and lymphocytes infiltrate and damage organs. Furthermore, TGF-β may play an important role in the promotion of fibrosis. Treg cells which are activated in IgG4-RD suppress immune responses and interleukin-10 is a regulatory cytokine that broadly functions as an immune inhibitory cytokine to support tumor growth. This suggests that Treg cells play a role in the progression and metastasis of various malignant tumors. In a study of pancreatic ductal adenocarcinoma by Hiraoka et al, the prevalence of Treg cells in CD4+ T cells was significantly increased in the ductal adenocarcinomas compared with that in the stroma of nonneoplastic inflammation. And the prevalence of Treg cells in CD4+ T cells increased significantly during the progression of premalignant lesions. Furthermore, a better prognosis was observed in patients with a low prevalence of Treg cells in CD4+ T cells. These findings suggest that Treg cells play a role in controlling the immune response against pancreatic ductal carcinoma from the premalignant stage to established cancer. Based on these findings, it is thought that IgG4-RSC may promote the development of cholangiocarcinoma.It is not clear whether IgG4-RSC is a risk factor for cholangiocarcinoma, but there are 2 main reasons why it might be. First, sustained inflammatory conditions associated with IgG4-RD are considered to create immunological environments favorable for cancer development.In the described case, it was difficult to evaluate the causal relationship between IgG4-RSC and cholangiocarcinoma because they were diagnosed at the same time. However, considerations of a medical history of diagnosed autoimmune pancreatitis three years previously suggest the progress of IgG4-RSC may have sustained that long, and sustained IgG4-RSC may have been responsible for the development of cholangiocarcinoma. Given that IgG4-RSC may be a risk factor for cholangiocarcinoma, it is important that IgG4-RSC be diagnosed early and treated appropriately in the prevention of cholangiocarcinoma.4We report a case of concurrent IgG4-RSC and cholangiocarcinoma, which were diagnosed simultaneously. IgG4-RSC is a subtype of IgG4-RD and can be confused with PSC and cholangiocarcinoma, but its treatment and prognosis are quite different, and thus differential diagnosis is important. In addition, many studies have shown that IgG4-RSC may be a risk factor for cholangiocarcinoma, and since IgG4-RSC and cholangiocarcinoma may coexist, even if IgG4-RSC is diagnosed first, it is important that repeated tests be conducted to ensure that it is not accompanied by cholangiocarcinoma. IgG4-RD was only recognized as a disease entity relatively recently and its mechanism is unclear. Accordingly, we suggest that the mechanism responsible for the development of IgG4-RD and the relationship between this disease and malignancy be further researched.Conceptualization: Ha Won Hwang, Jin-Seok Park.Investigation: Ha Won Hwang.Resources: Seok Jeong, Don Haeng Lee, Suk Jin Choi.Supervision: Jin-Seok Park.Visualization: Suk Jin Choi.Writing – original draft: Ha Won Hwang.Writing – review & editing: Jin-Seok Park, Seok Jeong.