In recent years, group signature techniques are widely used in constructingprivacy-preserving security schemes for various information systems. However,conventional techniques keep the schemes secure only in normal black-box attackcontexts. In other words, these schemes suppose that (the implementation of) thegroup signature generation algorithm is running in a platform that is perfectlyprotected from various intrusions and attacks. As a complementary to existingstudies, how to generate group signatures securely in a more austere securitycontext, such as a white-box attack context, is studied in this paper. We useobfuscation as an approach to acquire a higher level of security. Concretely, weintroduce a special group signature functionality-an encrypted group signature,and then provide an obfuscator for the proposed functionality. A series of newsecurity notions for both the functionality and its obfuscator has beenintroduced. The most important one is the average-case secure virtual black-boxproperty w.r.t. dependent oracles and restricted dependent oracles whichcaptures the requirement of protecting the output of the proposed obfuscatoragainst collision attacks from group members. The security notions fit for manyother specialized obfuscators, such as obfuscators for identity-basedsignatures, threshold signatures and key-insulated signatures. Finally, thecorrectness and security of the proposed obfuscator have been proven. Thereby,the obfuscated encrypted group signature functionality can be applied tovariants of privacy-preserving security schemes and enhance the security levelof these schemes. Group signature was proposed by Cham and Heyst , which iGroup signature schemes provide functionalities which are applicable in manypractical scenarios. In recent years, applications of group signature schemes inmany emerging technologies or privacy-sensitive applications are studied, such as insocial networks ,4, medicIn fact, we can find many typical WBACs, such as (1) a server or an endpoint forwhich a hacker has got the “root” or “admin” privilege;(2) a malicious host where mobile agents are running ,20; (3) In WBACs, obfuscating techniques should be used while implementing key-relatedcryptographic functionalities to protect privacy-preserving schemes or securityprotocols based on group signature schemes. Hence, we contribute to the securityrequirements as follows.A special obfuscatable group signature functionality, i.e., the encryptedgroup signature, is proposed with a concrete scheme, and then acorresponding obfuscator is provided.Security notions of the encrypted group signature functionality and notionsof the corresponding obfuscators are proposed. The most important one of thenew security notions is average-case secure virtual black-box property(ACVBP) w.r.t. Dependent Oracles and Restricted Dependent Oracles, whichdescribes the security requirement of protecting the output of the proposedobfuscator, i.e., the obfuscated implementation of encrypted group signaturefunctionality against collision attacks from group members. The securitynotions fit for many other application scenarios.The correctness and security of the proposed obfuscator are proven. Theefficiency of the proposed encrypted group signature functionality and itsobfuscator is analyzed.Besides the contributions to cryptography, the result is useful in many applications.For example, the proposed technique can be applied in cloud computing. In thisapplication, an inner user can upload a file anonymously on the private cloud of acompany. However, in the case that an investigation is needed, the group manager iscapable of opening the identity of the user. For another example, in aprivacy-preserving emergency call (PEC) scheme for mobile healthcare socialnetworks, the obfuscatable encrypted group signature scheme and its obfuscator canbe used to implement a decentralized emergency response system for a rapid responseof emergency care in the network. The application demonstrates that, with the helpof encrypted group signature technique, the PEC preserves users’ privacy byhiding their identities, and it avoids unnecessary disclosure of personal healthinformation. The details of these applications are provided in Section 6.1.1 and6.1.2 to demonstrate the applicability in concrete scenarios.Furthermore, we found that the proposed solution can be adapted to identity-basedcryptography and key-insulated cryptography. Therefore, how to transform theproposed obfuscatable encrypted group signature scheme into an obfuscatableencrypted identity-based signature scheme and an obfuscatable encryptedkey-insulated signature scheme are sketched out in Section 6.1.3.The remainder of this paper is organized as follows. The next section presentsbackground information about obfuscation and obfuscators. The section also providesa brief introduction on the complexity assumptions needed in this paper. Section 3first proposes an overall scheme that is a combination of a group signature schemeand an asymmetric encryption scheme, then an obfuscatable Encrypted Group Signature(EGS) functionality based on the overall scheme is provided. Section 4 presents anobfuscator for the proposed EGS functionality and proves the correctness andsecurity of the obfuscator. Moreover, a series of security notions of thefunctionality and the obfuscators is also introduced in Section 4. In Section 5, wecompare the results of the proposed scheme with the ones in other studies onobfuscation for cryptographic purpose. A discussion on possible applications andextensions of the proposed scheme and the obfuscator is provided in Section 6. Therationale behind the obfuscatable sign-then-encrypt functionalities and our maincontributions is also discussed in this section. Finally, the article concludes inSection 7 with future work.Informally, the goal of obfuscation is to make a computer program"unintelligible" while preserving its functionality, and an obfuscator is a“compiler” that performs such transformations ,30. As tDespite these positive results for theoretical study, obfuscators forcryptographic-related functionalities with acceptable runtime cost remainedelusive until the obfuscatable encrypted signature  and the We note that these positive results do not violate the general impossibilityresults. One of the reasons is that they are not general-purpose obfuscation.The other one is that they have used distinct weak security criteria such as theACVBP proposed by Hohenbergeret al.  or the sTwo general-purpose obfuscators ,44 wereBoth general-purpose obfuscators and specialized obfuscators for cryptographicfunctionalities usually set the input as a (probabilistic) circuit intheoretical analysis. A brief review on probabilistic circuits and circuitobfuscators is provided in the following subsection.As prerequisites of security analysis, we use the conventional definitions andnotations of probabilistic circuits and circuit obfuscators following those in,30,34,35λ) denote the set of all polynomials ofλ. Let ℂλbe a set of polynomial-size circuits with input lengthlInput(λ)∈ Poly(λ) and output lengthlOutput(λ)∈ Poly(λ). Usually, we use ℂ ={ℂλ}λ∈ℕto denote a class of such circuits, where there exists an associatedProbabilistic Polynomial Time (PPT) generation algorithm which takes as input1λ and generates a random circuitℂ ∈$ ℂλ. Instudies on obfuscation for cryptographic purpose, it usually corresponds to therandom generation of system parameters or cryptographic keys on the securityparameter 1λ. The generation process isdenoted by C ←ℂλ. When a circuitC is used as an input argument or an output result of analgorithm, it is assumed that a specification of the circuit is usedimplicitly.Let Poly is a probabilistic circuit.Given an arbitrary regular input para, we can considerC (orCpara(·)) as asampling algorithm for the distribution obtained by evaluating the output ofC on random variablerand. Given a couple of probabilistic circuits whoseregular inputs are of the same length, the two distributions produced byC0 andC1 are denotedby the pair (C0(para),C1(para)). The statisticaldifference between them is defined in (1)  denote the set ofgenerators of the “group”. The usage of thealgorithm is illustrated in The Setup algorithm takes a security parameter in unary as input. Thealgorithm outputs The algorithm proceeds as follows.AlgorithmSetupBeginp andq are prime numbers s.t.log2p =Θ(λ)>k∧log2q=Θ(λ)>k n←p·q builss a cyclic bilinear group Gof order n Gp be the cyclic subgroupsof G of order p LetGq be the cyclic subgroupsof G of order q Let    gω ∈G Ω←u,v',v1,…,vm∈ Gen[G] PP←∈ G × Gq ×Gm+3 ×GT  MK←∈ G ×Zn TK→q ∈ℕ output Endk<p. The enroll algorithm takespub , the master key MK, and the user’s identityID as input. It outputs a unique identifier sID∈ Zn and a corresponding private signingkey KID. The secret unique valuesID can be later used for tracing purposes.This value must be chosen so thatω+sID lies inThe algorithm Enroll serves for creating a signing key for a user whoseidentity is ID, where 0≤ID< 2The algorithm proceeds as follows.AlgorithmEnroll Begin g, h,u, v',v1,…,vm,Ω, A) ← PP ← MK , a group member’s private signing keyKID, and the message M = ∈{0,1}m as input. The algorithm outputs asignature  using the followingprocedure.The signing algorithm takes AlgorithmSignBegin g, h,u, v',v1,...,vm , Ω,A) ← PP ← M  Endpub, a signature σ from an unknown group member,and a message M as input. If the signatureσ is valid, it outputs 1, otherwise, outputs 0.Note that the verification algorithm outputs 1 only implies that thesignature is generated by a member of the given group, but does not revealthe identity of the original signer.The group signature verification algorithm Verify takesAlgorithmVerifyBeginσ1,σ2,σ3,σ4,π1,π2) ←σ  ← PP  ←M Beginσ1,σ2,σ3,σ4,π1,π2) ←σ  ← PP  as follows. Notethat for each user, the TA should provide a certification for the publicencryption key PKe.The encryption key generation algorithm EKGen takes the public parametersAlgorithmEKGen(pub)Begin g,h,u,v′,v1,…,vm,Ω,A)←PP ,SKe← output Endpub, the encryption keyPKe, and a (encoded)plaintext M ∈ G as input. Thealgorithm encrypts the plaintext and then outputs the ciphertext asfollows.The encryption algorithm Enc takes the public parametersPKe. Similarly, we useSKe.For simplicity, we use AlgorithmBegin g, h,u, v',v1,…,vm, Ω,A) ← PP  ←PKe  Output C EndThe decryption algorithm Dec works as follows.AlgorithmeSK(C)DecBegin C1,C2,C3) ←C  ←SKe  functionality AlgorithmBeginM = NULL) IF   Elseσ1,σ2,σ3,σ4, π1,π2) ←Sign  q.The algorithm Open can be done in constant time. Because the valueIn the next section, we introduce an obfuscator for the proposed EGSfunctionality, and prove the correctness and security of the obfuscator.In this section, we introduce a secure obfuscator for the special EGS functionality.The proposed obfuscator can either be deployed in a physically secure device or inthe signer’s host.EGSfor the In this subsection, we propose an obfuscator ObfPKe,0,PKe,1)←PKe←sk(M:Constructs and outputs an obfuscated circuit BeginM=NULL) IF   Elseμ1…μm)←M  ←PKe    For  For(   i≤3)   IF(        i=5)   Else IF  For behaves identically toC on all inputs with probability 1. Formally, this propertyis called “Preserving Functionality” as described in Definition 1and Pr[d′ = 1∧d = 1] in thesecurity game as follows.We compute Pr (M)//Compute the output directly  EndAlgorithm𝓓.Answer_OEGS(id)Begin IF (id = ID)𝓓.RR(C*)   Else  End𝓕 (and 𝓓) outputs 1when coin = 0 and 𝓕 queriesq′Obf times. Clearly, we have𝓕 (and 𝓓) outputs 1when coin = 1 and 𝓕 queriesq′Obf times. Let 𝓓 is a distinguisher of a simulator and areal obfuscator because: Hence, The following propositions are easy to verify, hence we omit the proofs.EGS is rerandomizablew.r.t. the dependent oracle The proposed obfuscator ObfEGS: FT w.r.t. the proposed EGS functionality.FA w.r.t. the proposed EGS functionality.EGS.FT w.r.t. the proposed obfuscatorObfEGS.FA w.r.t. the proposed obfuscatorObfThe group signature scheme in  satisfiegeneral-purposeobfuscation is impossible even under rather weak security definitions. This resultis extended in many publications, such as the impossibility of obfuscation withauxiliary input [The work of Barak et al. initiatery input , theimpry input , the impry input , and thery input . BecauseFortunately, some positive results were obtained besides these negative results. Itwas shown by Canetti that under a very strong Diffie-Hellman assumption, pointfunctions can be obfuscated . FurtherHowever, these positive results mainly serve as theoretical illustrations. Forinstance, because the speed of the encryption algorithm in an asymmetric encryptionscheme is usually much slower than that of a traditional block cipher, the stronglyobfuscatable symmetric encryption scheme  is not sBesides these positive results for theoretical study, some obfuscatable cryptographicfunctionalities and corresponding obfuscators for these cryptographicfunctionalities with acceptable runtime costs are introduced in recent years. Inand RDOs introduced in this paper is thefirst security notion to fulfill the security requirement to protect a signaturerelated scheme against collusion attacks. This new security notion should also beused to capture the security requirements of obfuscation of identity-basedcryptosystems, forward-secure cryptosystem, key-insulated cryptosystem and thresholdcryptosystem.From As we have mentioned at the end of section 2.1, there are two general-purposeobfuscators in ,44,58) p,58 p44,5C0 andC1 that agree on all inputsC0(x) =C1(x), it should be hard todistinguish the obfuscation of C0 from that ofC1. The new security notion used in this paper, i.e.the ACVBP w.r.t. DOs and RDOs, is stronger than the indistinguishabilityobfuscation.The first one is constructed for indistinguishability obfuscation that supports allpolynomial-size circuits, which were given by Garg et al.  and streThe second general-purpose obfuscator is capable of obfuscating all polynomial sizecircuits . TheobfNC1 denotes the class of decision problems decidable by uniform boolean circuitswith a polynomial number of gates of at most two inputs and depth O(logn), orthe class of decision problems solvable in time O(logn) on a parallel computerwith a polynomial number of processors. Many cryptographic functionalities areout of this class.The impossibility results in ,30,32 doHence, although there have been some important advances in general-purposeobfuscation, such as in ,44,58, tIn this section, we first introduce possible applications and extensions of theproposed technique. Then, the rationale behind the obfuscatable sign-then-encryptfunctionalities are investigated. Finally, the contribution of our findings isdiscussed at the last subsection.Group signature schemes are applicable in many practical applications, such as insocial networks ,4, medicMoreover, the proposed technique can be adapted to identity-based cryptographyand key-insulated cryptography. These extensions are introduced in section6.1.3.Group signature technique is perfectly suited for privacy-preserving securityschemes in cloud computing. The proposed application is inspired by . The appThe second application is a privacy-preserving emergency call scheme formobile healthcare social networks. It is adapted from .As illustrated in In an emergency situation, a patient must reveal his/her information to thenearby users in order to ask for their instant help. However, with privacyconcerns, the patients would preserve the identity privacy and prevent theirtransactions being linked to their unique identities. On the other hand, aTA must be able to trace the emergency call and identify the correspondingpatient. In this way, any malicious attacker who has generated a bogusemergency call would be detected and punished. Details of the applicationare listed as follows and illustrated in (1) Initialization Phase:Setup to generate system parameters, public values, themaster enrollment key MK, and the group manager’stracing key TK.At the beginning, the Trusted Authority (TA) uses the algorithm(2) Registration Phase:Enroll to issue asecret signing key sk and delivers the key to the patientthrough a secure channel.When a patient who needs medical services for possible emergency situationsjoins the system, TA uses the algorithm (3) Obfuscation phase:EGS togenerate an obfuscated implementation of encrypted group signaturefunctionality The patient uses the obfuscator Obf(4) Emergency call generation phase:The emergency call generation is started by a detection of the abnormalphysiological condition from body sensors. This condition can bepre-implanted into the patient’s PDA with the instructions of themedical professionals.ni denote a user whohas an emergency situation. The patientni’s PDAgenerates an emergency call according to the following steps.Let patient Location (LOC): It contains the emergency location informationwhich can be measured by a global positioning system (GPS) ofthe patient PDA.Incident (INC): It contains a general description of theenvironment where the emergency occurs.Time (TIME): It contains the exact time when the emergencyoccurs.(4.1) General information collection phase: The PDA intelligently collectsthe following general information: (4.2) Encrypted Signature generation phase: The PDA uses the obfuscatedimplementation EGS to generate an encrypted group signature on“LOC||INC||TIME”. The encrypted group signature will be putinto the “EGS” component.ni’sphysiological condition (PC) and health record (HR).(4.3) Data encryption phase: The PDA encrypts the patient(4.4) Information dissemination phase: The PDA disseminates the EmergencyCall Message (5) Emergency call response phase:nj receives the ECM frompatient ni and executes thefollowing steps, where a PDA represents usernj ‘s PDA:User Verify. If the verification passes,user nj confirms theinformation“LOC||INC||TIME.”(5.1) Cryptographic preprocess: First, the PDA decrypts the encrypted part ofthe message  to report the emergency to thehospital/first-aid center. Then, the PDA executes the step (5.3).(5.2) Local response: As an emergency response, usernjreceiving it, is larger than the threshold value, then the emergency call isdiscarded. Otherwise, the PDA forwards the ECM to the neighboring users.(5.3) Information re-dissemination phase: The PDA checks the“TIME” component: If the time period from the emergencyoccurrence to the user In a traditional (certification-based) public-key cryptosystem, theassociation between a user’s identity and his/her public key isobtained through a digital certificate issued by a Certifying Authority(CA). The CA checks the credentials of a user before issuing a certificateto the user. If a signer wants to sign a message, first the signer obtains adigital certificate for his/her public key from a CA. The signer then signsthe message using the private signing key and sends the signed message alongwith his/her certificate to the receiver. The receiver (verifier) firstverifies the validity of the certificate by checking the certificaterevocation list published by the CA, then the receiver verifies thesignature using the public key in the certificate. If many CAs are involvedbetween the signer and the verifier, then the entire certificate path has tobe verified.user’s public key is his/her identity (or derived from theidentity).no requirement of public key directoriesthe verification process of a signature requires only thesigners’ identity Hence, the process of certificate management requires high computational andstorage efforts. To simplify the certificate management process, Shamir introducThese properties make identity-based cryptosystems advantageous over thetraditional certification-based cryptosystems, as key distribution is farsimplified. It needs a directory only for authenticated public systemparameters of the KGC (Key Generation Center), which is clearly lessburdensome than maintaining a public key directory for total users.As a by-product of the paper, we found that one could easily transform theproposed functionality to an obfuscatable encrypted identity-based signatureby applying the following steps.The KGC sets up the group for all users.The KGC broadcasts the tracing key as a public value.When generating a signature, the signer appends his/her identity atthe end of the signature.Merge the verification algorithm and the opening algorithmtogether.The obfuscators, and even the encryption key generation algorithm,the encryption algorithm and the decryption algorithm can be used inthe identity-based scenario without modification.Note that the main security definition (ACVBP w.r.t. DOs and RDOs) can bealmost directly obtained from Section 4. Moreover, the security proof can bededucted from this paper easily. So we omit the details in this paper.Furthermore, as suggested in , by idenHence, besides the encrypted group signature, this paper adds two more newitems in the list of obfuscatable cryptographic functionalities, i.e., theencrypted identity-based signature and the encrypted key-insulatedsignature.Intuitively, design a sign-then-encrypt functionality needs a signing algorithmand an asymmetric encryption algorithm which are “commutable”.Hence, the obfuscator can encrypt the private signing key by using theencryption algorithm with the public encryption key. In fact, all the workswhich are listed in 𝓢takes the secret signing key sk ∈𝕊SSk and a messageM ∈𝕊Msg to return asignature  σ ∈{0,1}*∪⊥. The algorithm may be randomized. We write𝓢 for theoperation of running Sign on inputs sk, M andletting σ be the signature returned.In a digital signature scheme, the signing algorithm 𝓔 takes the public encryption keyPK ∈𝕊PEK and aplaintext M ∈𝕊PT to return avalue called the ciphertext. The algorithm may be randomized. We writeC←𝓔 for the operation of running 𝓔on inputs PK, M and letting C∈ 𝕊CT be theciphertext returned.In an asymmetric encryption scheme, the encryption algorithm𝓔∘𝓢.Suppose the encryption is semantic secure (IND-CPA). If (34) holds, it isexpected that we can obtain a secure obfuscator ofRE and the set of randomvariables in the signing process asRS. Let As to the security requirement, the formal security analysis hinges on thesecurity model of the signature scheme. Because there are many variations ofdigital signature schemes , it isout of the scope of this paper.The paper has introduced a new secure obfuscator for encrypted group signatureand corresponding security notions.Theoretically, six new security notions of the encrypted group signaturefunctionality and its obfuscators are proposed. The notions are ACVBP w.r.t.w.r.t. Dependent Oracles (DOs) and Restricted Dependent Oracles (RDOs),rerandomizable w.r.t. dependent oracle set and restricted dependent oracle set,full-traceability w.r.t. EGS Functionality, full-anonymity w.r.t. EGSFunctionality, full-traceability w.r.t. EGS Obfuscator, and full-anonymityw.r.t. EGS Obfuscator.The most important one of the new security notions is ACVBP w.r.t. DOs and RDOswhich describes the security requirement of protecting the output of theproposed obfuscator, i.e., the obfuscated implementation of encrypted groupsignature functionality against collision attacks from group members. Thesecurity notions fit for many other cryptographic schemes which collisionattacks from users need to be considered, such as identity-based signatureschemes, ring signature schemes, attribute-based signature schemes andkey-insulated signature schemes.Practically, as it was discussed in Section 5, a generic obfuscator  is hard to find. Therefore, the obfuscatorsfor various sign then encrypt functionalities must be studied one by one. In, Hada prA special obfuscatable group signature functionality, i.e., the encrypted groupsignature, is proposed with a concrete scheme, and then a correspondingobfuscator is provided in this paper. The correctness and security of theproposed obfuscator are proven. Then the efficiency of the proposed encryptedgroup signature functionality and its obfuscator is analyzed. The results ofthis paper can be used as building blocks of privacy preserving securityprotocol of various emerging applications such as social networks, medicalinformation systems, Vehicular Ad hoc Networks (VANets), electronic voting,Wireless Sensor Networks (WSNs), electronic cash, and especially cloudcomputing.Finally, as by-products of this paper, besides the encrypted group signature, weadd two more new items in the list of obfuscatable cryptographicfunctionalities, i.e., the encrypted identity-based signature and the encryptedkey-insulated signature.Group signature technique is used in many privacy-preserving security schemes forsocial networks, cloud computing, VANets, WSNs, electronic voting and electroniccash. To provide a building block for these schemes in white-box attack contexts, wegive an obfuscatable EGS functionality, and then provide an obfuscator for theproposed EGS functionality. We also introduce a new security notion (ACVBP w.r.t.Dependent Oracles and Restricted Dependent Oracles) to capture the requirement ofprotecting the output of an obfuscator for EGS functionality against collisionattacks from group members. Moreover, five other new security notions are alsoprovided. We prove that the proposed obfuscator preserves EGS functionality andsatisfies the proposed security notions. As a byproduct of this study, ACVBP w.r.t.Dependent Oracles and Restricted Dependent Oracles fits for many types ofcryptosystems, such as identity-based cryptosystems, forward-secure cryptosystems,key-insulated cryptosystems and threshold cryptosystems. Finally, we have introducedtwo possible applications and two extensions of the proposed technique.In the future, we plan to adopt the obfuscatable EGS functionality in practicalsecurity solutions for validation. By using the proposed obfuscatable EGSfunctionality as a building block, we also consider exploring novel approaches fordesigning privacy-preserving security schemes. Furthermore, we will try to explorethe design strategy for generalized constructions of obfuscatable sign-then-encryptfunctionalities.


In the article titled “Long-Term Effects of Goshajinkigan in Prevention of Diabetic Complications: A Randomized Open-Labeled Clinical Trial” , we have(1) The abbreviation “DN” in the third paragraph of Section 2.4 “Primary and Secondary Outcomes” did not mean “diabetic nephropathy” and should be corrected as “diabetic neuropathy.”n = 100) and to control group (n = 49).(2) Section 3.1 “Participants Registration, Allocation, Follow-Up, and Analysis” should be corrected as follows: A total of 332 patients were registered for this clinical trial , among which 183 patients were excluded because of being out of criteria. Main reason of the exclusion was the retinopathy. Even though, in the regular ophthalmological check, these patients matched the inclusion criteria, fundus photography revealed that these patients did not match the inclusion criteria. Another reason was out of goshajinkigan pattern. This was relatively difficult for the physician who contributed to this study. As a result, 149 patients were allocated to goshajinkigan (GJG) group .(3) Section 3.4 “Secondary Outcomes” should be corrected as follows: The hazard ratio was calculated as 0.436 (95% CI: 0.198–0.962), and the


Lolium perenne L.) produces high levels of fructans as a mixture of oligosaccharides and polysaccharides with different degrees of polymerization (DP). The present study describes the analysis of the compositional changes in the full spectrum of fructans, fructan distribution between above ground biomass (top) and the roots, and the transcription of candidate genes involved in fructan metabolism during cold acclimation in perennial ryegrass variety “Veyo” and ecotype “Falster” from distinct geographical origins. We observed changes in fructan composition and induction of low-DP fructans, especially DP = 4, in both the top and the roots of “Veyo” and “Falster” in response to low-temperature stress. The accumulation of DP > 50 fructans was only apparent in the top tissues where the Lp1-FFT expression is higher compared to the roots in both “Veyo” and “Falster.” Our results also show the accumulation and depolymerization of fructans with different DP, together with the induction of genes encoding fructosyltransferases and fructan exohydrolases in both “Veyo” and “Falster” during cold acclimation, supporting the hypothesis that fructan synthesis and depolymerization occurring simultaneously. The ecotype “Falster,” adapted to cold climates, increased total fructan content and produced more DP > 7 fructans in the roots than the variety “Veyo,” adapted to warmer climates. This indicates that high-DP fructan accumulation in roots may be an adaptive trait for plant recovery after abiotic stresses.Perennial ryegrass ( Changes in the content and composition of water-soluble carbohydrates in response to abiotic stresses are important metabolic re-adjustments in temperate grasses  contains the inulin series, inulin neoseries, and levan neoseries-type fructans with both β-(2→1)-linked and β-(2→6)-linked fructosyl residues , and linkage types between fructosyl residues. Five major types of fructans are known: inulin, levan, inulin neoseries, levan neoseries, and branched graminan-type fructans  induces morphological, physiological, and biochemical changes in both shoot and root tissues that are required for the acquisition of freezing tolerance in cold-tolerant plants  variety “Veyo” originating from Italy and adapted to warmer climates, and ecotype “Falster” originating from Denmark and adapted to cold climates , with 450 μmol photons m−2 s−1 light intensity and relative humidity of ~70% at 7°C. The chlorophyll fluorescence of leaves was measured, and root and above ground biomass (top) samples were harvested in three biological replicates at the onset of the daily photoperiod on d 0, 1, 3, 5, 9, 13, and 17 of cold acclimation at 7°C. The samples were frozen in liquid nitrogen immediately after harvesting and stored at -80°C, until the analysis of carbohydrates and gene expression levels was performed. After d 17 of cold acclimation, the “Veyo” and “Falster” plants were transferred to a freezing chamber at −10°C and frozen for 3 h. As a control, non-cold-acclimated “Veyo” and “Falster” plants were also transferred to the same freezing chamber and frozen for 3 h. The chlorophyll fluorescence and electrolyte leakage of leaves were measured before freezing, and after 30 and 120 min of freezing.Perennial ryegrass (PSII) was calculated, as previously described  . The measurements were taken from three biological replicates, from three mature leaves of each plant (3 cm above the leaf sheath). The quantum yield of photosystem II  for 1 h at 65°C. Extracts from each sample (2 ml) were hydrolyzed using an equal volume of 0.074 M H−1 at 25°C). Sample extracts (5 ml) were filtered, using Omnifix®-F syringes  and Q-Max® RR syringe filters , and then lyophilized to dryness and redissolved in 500 μl of MilliQ water, yielding samples 10 times more concentrated for LC-ESI-TOF-MS (Agilent Technologies) analysis.Ground plant (100 mg) material was transferred into a 33 ml stainless cell  for accelerated solvent extraction and mixed with 10 g of Ottawa sand . The top of the cell was then fitted with a cellulose filter  and filled with glass beads (previously baked at 400°C to remove possible contaminants). Samples were extracted with MilliQ water on the Accelerated Solvent Extractor Dionex ASE 350 system (Unity Lab Services) under the following conditions: one cycle of 5 min pre-heating, 5 min heating at 80°C, 3 min static time, 60 s purge, and 60% flush. Extracts were collected in 52 ml glass cells and weighed to determine the exact extract volume  and an acquisition rate of 0.66 spectrum s−1, yielding the maximum number of transients for TOF detection. Mass accuracy was maintained by the simultaneous infusion of a reference solution of analytical standard compounds through a dual ESI nebulizer. The reproducibility of peak intensities was validated by analysing a reference plant material for all sample batches.A total volume of 50 μl of concentrated samples were injected, and fructans were chromatographed on a 100 × 2.1 mm Hybercarb column  at 40°C using a flow rate of 0.3 ml minAcquired data were processed in MassHunter Qualitative Analysis software version B.05.00 (Agilent Technologies) extracting ion chromatograms for DP3 to DP70 using a personalized database, including exact masses and chromatographic retention times , and On-column DNAse I digestion (Qiagen) was performed to avoid genomic DNA contamination, according to the manufacturer's instructions. Total RNA quality was verified using an Agilent 2100 Bioanalyzer (RNA 6000 Nano Assay) and quantified by Quant-iT™ RiboGreen® RNA Reagent assay, according to the manufacturer's protocol. First-strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase kit (Invitrogen), as per manufacturer's instructions.L. perenne cDNA sequences with sequence similarity to fructan-related genes and internal control genes were identified for primer design, querying the L. perenne genomic database  with the Lp1-SST (AY245431.1)    , Oryza sativa elongation factor 1-alpha (OsEF1a) (D63583.1), Zea mays actin (ZmACT11) (AY107106.2), and eukaryotic initiation factor 4A (ZmeIF4a) sequences. PCR primers  standard curve method for absolute quantification was carried out, as previously described . In all cases, the level of significance was set at P < 0.0001). In “Veyo” top, the fructan content increased rapidly from approximately 50 mg/g (d 0–3 of cold acclimation) to 81.8 mg/g at d 5 and it peaked at 118.6 mg/g on d 9. However, in “Veyo” roots, the fructan content increased only slightly to 54 and 50.8 mg/g at d 5 and d 9 and subsequently it decreased to levels similar to the initial level of 35 mg/g. The fructan content increased in the top and roots of “Falster” from 17 and 5.7 mg/g, respectively to 48.1 and 36.6 mg/g on d 5 of cold acclimation, with the highest content recorded on d 17 of cold acclimation. Here, the fructan level increased approximately six-fold in “Falster” top and thirteen-fold in “Falster” roots, compared to the levels on d 0 of cold acclimation.Fructan accumulation in the top and roots of “Veyo” and “Falster” was induced by cold, however, accumulation over time and the distribution between top and root varied in the two types Figure . In bothPSII) fluorescence parameter, which measures the efficiency of photosystem II (PSII) photochemistry, indicating the efficiency of carbon fixation  and it appeared that “Veyo” and “Falster” responded differently (P = 0.07) to the freezing treatment after cold acclimation. In both “Veyo” and “Falster,” a greater reduction of ΦPSII was observed in the non-cold-acclimated, compared to cold-acclimated plants 120 min after the transfer to freezing . Only the cold-acclimated plants from “Veyo” and “Falster” showed a re-growth after 7 d of recovery with “Falster” showing a more vigorous re-growth than “Veyo”  DP = 3–8, (2) DP = 9–14, (3) DP = 15–20, (4) DP = 21–26, (5) DP = 27–32, (6) DP = 33–38, (7) DP = 39–44, (8) DP = 45–50, (9) DP = 51–56, (10) DP = 57–62, and (11) DP = 63–70. The composition of fructan DP changed in both the above-ground biomass and the roots of “Veyo” and “Falster” during cold acclimation  were accumulated over the 17 day period of cold acclimation. Overall, the composition of the fructan mixture of oligosaccharides and polysaccharides changed in the top and roots of both “Veyo” and “Falster” during cold acclimation.Analysis of fructans was carried out using high-resolution LC-ESI-TOF-MS in top and root samples collected on d 0, 9, 13, and 17. Fructans accumulated as a mixture of oligosaccharides and polysaccharides in “Veyo” and “Falster” and fructans up to n Figure . Fructan Figures , whereas Figures . The cha” Figure . The frus Figure , whereasLp1-SST, Lp1-FFT, Lp6GFFT, and Lp1-FEH with β-(2→1) linkage specificity and Lp6-FEH with β-(2→6) linkage specificity. The expression of Lp1-SST, Lp1-FFT, and Lp6G-FFT was induced after d 1 of cold acclimation in the top and roots of “Veyo” and “Falster.” Expression levels of Lp1-SST in the roots were greater than in the top for both “Veyo” and “Falster”  of fructans suggests that fructans might play a role in neutralizing ROS leading to carbon-centered radicals that can be re-utilized for non-enzymatic de novo synthesis of fructan oligosaccharides (Peshev et al., de novo synthesis of fructan oligosaccharides is to be further investigated.Changes in fructan composition and induction of low-DP fructans, especially Low-DP fructan induced during cold acclimation might be involved in protective mechanisms such as membrane stabilization. The effect of fructan DP on liposome stability has been studied using seven defined size classes of fructans from perennial ryegrass and oat, showing that the fructans with a DP4 is more protective than other fractions in liposome fusion (Hincha et al., Morphological, physiological, and biochemical changes in the root system during cold acclimation are important determinants of overwintering capacity and recovery of plants. Root system has been suggested as the site of perception of low-temperature stimulus (Goulas et al., Lp1-FFT expression in the roots than in green tissues (Figure “Veyo” originates in the Mediterranean region, and drought-induced summer dormancy has been reported to influence fructan quantity and DP (Volaire et al., s Figure . Apart fs Figure  might alFTs and FEHs during cold acclimation in two types of perennial ryegrass adapted to different climatic conditions. Our results suggest that accumulation of high-DP fructan in roots as an adaptive trait for plant recovery during cold. The effects of compositional changes of fructan in membrane stability and other protective mechanisms are to be further studied.Hydrolysis of fructan, especially the high-DP fructans in roots might increase osmolality of cell sap rapidly in response to low-temperature stresses. Fructan storage in roots as a carbon sink can be utilized for a rapid growth in early spring to compete with neighboring species as suggested by Van den Ende et al. . This miThe 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.


Recreational Water–Associated Disease Outbreaks — United States, 2009–2010,” an error occurred on page 8 in the § footnote of the table. The second and third sentences of that footnote should read as follows: “Microcystin was considered a confirmed etiology if water testing detected ≥20 μg/L microcystin toxin in water samples collected during or within 1 day of the outbreak exposure period. Microcystin was considered a suspected etiology if water testing detected <20 μg/L microcystin toxin in water samples collected during or within 1 day of the outbreak exposure period.”In the report, “Algal Bloom–Associated Disease Outbreaks Among Users of Freshwater Lakes — United States, 2009–2010,” an error occurred on page 14 in the third sentence of the first full paragraph. That sentence should read as follows: “Microcystin concentrations of ≥20 μg/L exceeded the WHO guideline for moderate health risks in four outbreaks (Table 3) (2).”In the report, “1, Michael Lowe, PhD1, Scott A. Norton, MD2, Tiana Garrett, PhD1, Lamia Soghier, MD3, Edward Weiss, MD4, June Hatfield, MS3, Jeffrey Lapinski, MS3, Steven Abrams, MD5, Wanda Barfield, MD6” (Author affiliations at end of text). The correct affiliations for the two authors are “5Texas Children’s Hospital, Houston, Texas and 6Div of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, CDC,” respectively.In the report, “Zinc Deficiency–Associated Dermatitis in Infants During a Nationwide Shortage of Injectable Zinc — Washington, DC, and Houston, Texas, 2012–2013,” on page 35, in the author list, the affiliation footnotes for two authors were incorrect. The author list should read, “Duke Ruktanonchai, MD


RNA-seq studies have an important role for both large-scale analysis of gene expression and for transcriptome reconstruction. However, the lack of software specifically developed for the analysis of the transcriptome structure in lower eukaryotes, has so far limited the comparative studies among different species and strains.S. cerevisiae strains in two different key steps of the fermentation process. This comparative analysis revealed differences in the UTR regions of transcripts. By extending the transcriptome analysis to yeast species belonging to the Saccharomyces genus, it was possible to examine the conservation level of unknown non-coding RNAs and their putative functional role.In order to fill this gap, an innovative software called ORA (Overlapped Reads Assembler) was developed. This software allows a simple and reliable analysis of the transcriptome structure in organisms with a low number of introns. It can also determine the size and the position of the untranslated regions (UTR) and of polycistronic transcripts. As a case study, we analyzed the transcriptional landscape of six S. cerevisiae strains. Finally, we propose a regulatory role for some non-coding transcripts conserved within the Saccharomyces genus and localized in the antisense strand to genes involved in meiosis and cell wall biosynthesis.By comparing the results obtained using ORA with previous studies and with the transcriptome structure determined with other software, it was proven that ORA has a remarkable reliability. The results obtained from the training set made it possible to detect the presence of transcripts with variable UTRs between The online version of this article (doi:10.1186/1471-2164-15-1045) contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae.Since the completion of its genome sequence in 1996, in (d) the 3’-UTR size and in (e) the chromosome. The worksheet “Transcriptome” reported the output of ORA in “gff” format, in column (a) the chromosome, (d) transcript start, (e) transcript end, (g) strand and in column (i) are resumed the ID of the gene encoding the transcript (obtained from the gff annotation file), the length of the transcript and its coverage (reported as “note=”). In column (i) the transcripts expressed in antisense to other annotated genes are reported as “ncRNA_anti_gene ID in antisense strand”, while genes in intergenic regions are reported as “ncRNAs_ID of the closest annotated gene”. (XLSX 509 KB)Additional file 1: Table S1: Transcriptome reconstruction of Naumovia castellii obtained using ORA. Histograms reporting the size distribution of the 5’ and 3’-UTR size of N. castellii (a-b) and Saccharomyces cerevisiae S288c (at 6 g/l) (c-d). Notice the small 5’-UTR size of N. castellii (54 bp on average) in comparison to S. cerevisiae (81 bp on average). Analysis of the data allowed the identification of numerous introns in the 5’-end of the genes (frequently in the 5’-UTR) (red rectangles). Some of these introns were not previously predicted. (e) (h) Analysis of the 5’-UTR size revealed some genes having an incorrect prediction of the start codon (red arrows). In (e-h) the coverage in the forward and reverse strands determined using RNA-seq are indicated with “cov. for” and “cov. rev”, the transcriptome prediction obtained using ORA is indicated with “ORA” and the gene prediction obtained from ncbi database is indicates as “genes”. (PDF 756 KB)Additional file 2: Figure S1: The transcriptome reconstruction of Additional file 3: Figure S2: Selected results of the transcript structure obtained for the six strains under analysis. Genes reported belong to the GO categories “sulfur compound metabolic process” and “sterol metabolic process” that are described in the main text. Genes YDR213W and YGL012W have highly conserved 5’-UTRs and were reported in order to show that gene expression level has little influence on the transcript structure prediction. The 5’-end of the transcript is on the left for genes encoded on the forward strand and on the right for genes encoded on the reverse strand. 5’-UTR is indicated by a small arrow. (PDF 7 MB)S. cerevisiae strain or a Saccharomyces sensu stricto species. The worksheets “S. cerevisiae_S288c_haploid_6gl”, “S. cerevisiae_S288c_haploid_45gl”, “S. cerevisiae_EC1118_6gl”, “S. cerevisiae_EC1118_45gl”, “S. cerevisiae_P283_6gl”, “S. cerevisiae_P283_45gl”, “S. cerevisiae_R008_6gl”, “S. cerevisiae_R008_45gl”, “S. cerevisiae_R103_6gl”, “S. cerevisiae_R103_45gl”, “S. cerevisiae_P301_6gl”, “S. cerevisiae_P301_45gl” report the transcriptome structures determined for the S. cerevisiae strains S288c, EC1118, P283, R008, R103, P301 in two points of the fermentation curve (6 and 45 g/l). The worksheets “S. cerevisiae_S288c_diploid_1”, “S. cerevisiae_S288c_diploid_2”, “S. bayanus”, “S. paradoxus” and “S. mikatae” report the transcriptome structure determined for the Saccharomyces sensu stricto species whose transcriptomes were sequenced by Busby and coll. [Additional file 4: Table S2: Transcriptome reconstruction performed using ORA software. Each worksheet reports the trascriptome structure of a Additional file 5: Table S3: Comparison between 5’-UTR length in the six strains determined using ORA software. Results were obtained analyzing data obtained at 6 g/l (worksheet “6gl_5 UTR”) and at 45 g/l (worksheet “45gl_5 UTR”). On each worksheet are reported: (a) the primary SGD ID, (b) the gene name, (c) the gene description, (d-i) 5’-UTR length, (j-o) coverage, (p) the number of pairwise comparisons where the difference in 5’-UTR length is higher than 50 bases. (XLSX 432 KB)ARO4 gene. (a) To verify if the RBS in ARO4 (YBR249c) promoter was differentially represented between oenological and laboratory strains, PATMAN software [https://bioinf.eva.mpg.de/patman/patman-1.2.html) was used. In Freeberg and colleagues [ARO4 promoter showing the predicted TATA box and the RBS. (PDF 249 KB)Additional file 6: Figure S3: Analysis of the 5’-UTR region of software   identified by Freeberg and colleagues  and the Additional file 8: Figure S4: Directional reads validation. Frequency of antisense ncRNAs (named SAUTs in the paper) in highly expressed genes was determined and results are reported in (a). Frequency of genes having SAUT was calculated with respect to the coverage of the corresponding genes. Some selected images of highly expressed genes are reported (b-e) to show the high strand-specificity of the library, while two examples (f-g) are reported to show genes with high antisense transcription, SAUTs are highlighted by red boxes. (PDF 483 KB)Additional file 9: Figure S5: Verification of bicistronic transcripts identified using ORA. Sixteen bicistronic transcripts were identified by ORA only in one of the two growth conditions (6 or 45 g/l) and were excluded from further investigation. This result highlights the importance of comparing transcriptome reconstruction in different growth conditions. The remaining three transcripts are reported in (a-c) and include three pairs of genes YFR033C-YFR034C, YFL057C-YFL056C and YIL165C-YIL164C. These transcripts were further verified considering two lines of evidence: the presence of polyadenylation sites previously determined (red boxes in the figure)  and the


In our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at previous failures to a Raltegravir containing regimen, in patients who are currently undetectable and candidates to simplification to a Dolutegravir containing regimen, in order to decide on once a day or twice a day dosing.We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29–53) Integrase resistance mutations in proviral DNA were studied.All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was of 1.3 log cp/ml (range 0–1.47) and 765.5 cells/μL . The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29–53) months. At Raltegravir failure, N155H was detected in four patients, and other secondary mutations were detected in five patients (71.4 %). In proviral DNA, N155H was detected by population sequencing in three patients (42.8 %), and UDS demonstrated a 9.77 % relative abundance of N155H in the remaining patient. Sanger sequencing correctly identified all the secondary mutations.This is a pilot study that demonstrates the possibility of properly identifying N155H and some secondary mutations 29–53 months after failure. For the last 6 years, Raltegravir (RAL), the first strand Integrase transfer inhibitor (INIs) approved, has played a relevant role on the treatment of HIV infection, both for naive and pretreated patients .Nowadays, Elvitegravir (EVG) and Dolutegravir (DTG), two new INIs have become available. EVG is a first generation drug and shares its resistance profile with RAL, whereas DTG is a second generation INI with a high antiviral effect, and excellent efficacy and safety profile. Although DTG has a high genetic barrier to resistance, its activity may be limited by certain combination of resistance mutations that may accumulate during failure to RAL . The ocuIn our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at failure to RAL, and we attempted to provide the proof of concept that testing proviral DNA may be used as a sentinel for RAL mutations, in patients who are currently undetectable and candidates to simplification to a DTG containing regimen.We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29–53), Integrase resistance mutations in proviral DNA were studied.6 leukocytes aliquots, that were used for DNA extraction with QIAamp DSP DNA Blood Mini Kit (QIAGEN). DNA was quantified by spectrophotometry .Peripheral Blood Mononuclear Cells (PBMC) were separated by Ficoll gradient centrifugation, washed, counted and pelleted to 5x10Integrase was amplified using a nested-PCR. For the first round of amplification, we used outer primers IN1F (5′-GGAAAAGGTCTACCTGTCATGGGT-3′) and IN1R (5′- GGAGAAAGAGACTGGCATTTGG-3′) and the following PCR profile: 94 °C/2′;  x35 cycles; 72 °C/7′; 4 °C. This profile was used for the nested PCR, using inner primers INR2F (5′- TGGAGGAAATGAACAAGTAGATAAATT-3′) and (INT2R: 5′-GGGTCTGCATACAGGAGAAA-3′).For Sanger sequencing, we used a bidirectional sequencing protocol and the Thermo Sequenase Dye Primer Manual Cycle Sequencing Kit (Affymetrix USB), which was optimized for the TruGene/OpenGene DNA sequencing system (SIEMENS); sequencing primers were P1F:5′- Cy5.5-GTAGCCAGCTGTGATAAATGTC-3′) and P2R (5′- Cy5-CTGCCATTTGTACTGCTGTCT-3′), allowing to sequence 414 nucleotides in the Integrase region, covering positions 40 to 178. The amplification profile was: 94 °C/5′;  x30 cicles; 70 °C/5′; 4 °C. The obtained sequences were aligned and combined using OpenGene Geneobjects™ software, and fasta sequences were interpreted Stanford HIV Database Versión 7.0 [Ultra Deep Sequencing (UDS) was done using an HIV-1 UDS prototype (Roche diagnostics) and the 454 GS Junior . As template for the UDS PCR, we used the same amplification products as for standard Sanger sequencing following from this point the manufacturer’s recommendations. Once UDS was performed, sequences were exported by AVA  software and interpreted by DeepChek®- VIH  software, using for interpretation the same Stanford version as for Sanger sequencing.This was a retrospective, non-interventional study, and patient information was anonymized and de-identified prior to analysis.All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was 1.3 log cp/ml (range 0–1.47) and 765.5 cells/μL . The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29–53) months. Detailed information on baseline characteristics is shown in Table Table Dolutegravir has shown excellent efficacy and safety in individuals infected by HIV both in naïve , and patProviral DNA may be a useful tool to investigate the presence of resistance mutations –14, espeIn our study, using Sanger sequencing of the Integrase region of proviral DNA, we could correctly identify failing selected mutations in 6/7 patients. Although for the remaining patient we could not demonstrate the failing mutation with Sanger sequencing, using a more sensitive test , resulteAlthough the sampling time in patients 2, 3 & 7 exceeded the half-life of the HIV-1 reservoir, this did not interfere in the correlation between the failing sample and proviral DNA testing. Despite some studies have demonstrated that the latent viral reservoirs half-life is from four to six months in patients who start therapy in the acute infection stage, there are other studies in chronically infected patients who have shown a half-time of 44 months , 17.Our study has certain limitations. First, only subtype B patients have been included, so the methodology needs to be validated for other subtypes. Secondly, only the N155H pathway was confirmed in some patients and it is possible that resistance pathways other than N155H, that could have emerged before N155H was established, may have been archived in the proviral DNA of the patients compromising DTG activity.In summary, despite the limitations of our study, which is just a pilot study that should be confirmed in further studies, we have shown the proof of concept that for patients who failed a Raltegravir containing regimen in the past, who are currently virologically suppressed, and lack the resistance information at failure, studying Integrase resistance in the proviral DNA accurately reflects the possibility of properly identifying N155H and some secondary mutations 29–53 months after failure.The Ethics Committee of the San Cecilio Hospital approved the study, and no consent information was required as patient information was anonymised and de-identified prior to analyses.Not applicable.All data supporting our findings is contained within the manuscript.


Young non-small cell lung cancer (NSCLC) patients under the age of 40 can further be categorized into different age subgroups. Whether they have homogeneous clinical features and survival outcomes remains unexplored.Information of 4623 NSCLC patients up to 40 years old from 1988 to 2012 was retrieved from the Surveillance, Epidemiology, and End Results (SEER) database. Clinicopathologic characteristics and survival outcomes were compared between patients diagnosed at 18–30 years old (younger group) and those at 31–40 years old (older group).P = 0.016), a lower proportion of large cell carcinoma (P = 0.008), a higher proportion of stage I disease (P = 0.002) and a lower proportion of stage III disease (P < 0.001). The younger patients had significantly better lung cancer-specific survival (LCSS) in the whole cohort (P < 0.001) and in the subgroup of patients with stage I (P = 0.038) or stage IV (P < 0.001) disease. Multivariate survival analysis showed that patients under 30 years old was an independent predictor of both better LCSS (P = 0.010) and overall survival (OS) (P = 0.018).The proportion of patients in the younger group among all lung cancer patients was stable between 1988 and 2012. However, the proportion of patients in the older group decreased from 1.2% to 0.5%. The younger patients had a higher proportion of adenocarcinoma (Adult NSCLC patients under 30 years old had distinctive clinicopathologic characteristics and survival outcomes compared to patients diagnosed at 31–40 years old. According to the recently released global cancer statistics , lung caThe proportion of lung cancer patients in age groups “18–30”, “31–40”, “41–50”, “51–60”, “61–70”, “71–80”, “81–85” and “85+” from 1988 to 2012 were demonstrated in Figure A total of 4623 young NSCLC patients (up to 40 years old) were identified from 1988 to 2012, of which 429 were 18–30 years (the younger group), and 4194 were 31–40 years (the older group) (Table P < 0.001). Gender distribution was comparable in the two age subgroups. There were a significantly higher proportion of African American in the older group compared to the younger group .A higher proportion of the younger group patients were diagnosed after 2000 . Large cell carcinoma was more common in the older group compared to the younger group . The proportions of squamous cell carcinoma and adenosquamous cell carcinoma were comparable between the two groups. The younger group had a significantly higher proportion of well differentiated tumors , whereas the older group had a significantly higher proportion of poorly differentiated tumors .Adenocarcinoma was the predominant histology in both groups. However, patients in the younger group had a higher proportion of this histology , whereas the older group had a higher proportion of stage III disease . Comparable proportions of patients in the two groups were diagnosed as stage II and stage IV.Stage distribution was different in the two groups. The younger group had a higher proportion of stage I disease , whereas the proportion of patients undergoing radiation therapy was significantly higher in the older group . There was a higher proportion of patients in the younger group receiving lymph nodes resections . A significantly higher number of positive lymph nodes was found in the younger group (P = 0.033).SEER database provided information of surgery and radiation. A higher proportion of patients in the younger group received surgery . We further categorized patients into four age subgroups: 18–25, 26–30, 31–35 and 36–40 .First, we compared the lung cancer-specific survival between the “18–30” and the “31–40” age subgroups Figure , and fou0 Figure . There wP = 0.038) and stage IV (P < 0.001) groups. There was a trend towards higher LCSS for the younger group with stage II or stage III disease, although statistical significance was not achieved  and better OS . We further performed multivariate survival analysis adjusted for race, sex, year of diagnosis, histology, tumor grade, AJCC stage, radiotherapy, lymph nodes removed and lymph nodes positive in patients undergoing surgery. Younger age at diagnosis was not an independent predictor of LCSS  or better OS . Moreover, in patients who had not received surgery, younger age was also not a significant prognostic factor  in multivariate analysis adjusted for race, sex, year of diagnosis, histology, tumor grade, AJCC stage and radiotherapy.Multivariate survival analysis adjusted for race, sex, year of diagnosis, histology, tumor grade, AJCC stage, surgery and radiotherapy showed that patients under 30 years old was an independent predictor of both better LCSS .A total of 281 (6.1%) young NSCLC patients died of second cancers other than lung cancer. Among them, 31 patients (7.2%) were in the younger group and 250 patients (6.0%) were in the older group  decreased from 1978 to 2010 [P < 0.001). Gender distribution was not significantly different in the two age subgroups, although previous studies found a higher proportion of female patients in the young NSCLC [P = 0.008).We observed a higher proportion of “18–30” years-old patients were diagnosed after 2000 compared to the “31–40” years-old group . Besides, we also observed a lower proportion of large cell carcinoma in patients under 30 years old . Young NSCLC patients were reported to have late disease stage at presentation [Previous studies reported that young NSCLC patients were more likely to have adenocarcinoma histology , 4, 6, 7entation , 4, 7. IP = 0.038) and stage IV (P < 0.001) groups than patients diagnosed at 31–40 years old. Although statistical significance was not reached, there was a trend towards higher LCSS for the younger group with stage II or stage III disease. This result suggested that the association between better LCSS and young patients under 30 was not likely to be confounded by disease stage. Multivariate survival analysis adjusted for race, sex, year of diagnosis, histology, AJCC stage, tumor grade, surgery and radiotherapy also demonstrated the independent role of younger age as a positive prognostic factor.As disease stage was usually the most important prognostic factor, we conducted stage-wise comparison of LCSS between the two age subgroups. Patients under 30 years old had significantly better LCSS in stage I . Informed consent of this study was not required. The Institutional Review Board of Shanghai Chest Hospital approved this study.This retrospective study was based on publicly available SEER database. Data were retrieved through online access using the SEER*Stat software Version 8.2.1  staging information, or sufficient survival data were excluded. Individual data retrieved for each case included age at diagnosis, gender, race, year of diagnosis, tumor histology, tumor grade, AJCC staging information, treatment modality (radiotherapy/surgery/lymph nodes removed/lymph nodes positive), cause-specific death classification, vital status and survival months. Lung cancer-specific survival (LCSS) was defined as time from diagnosis to death from lung cancer.2 test or Fisher's exact test was used to compare the clinicopathologic characteristics in different age subgroups. Kaplan-Meier method with log-rank tests were used for survival comparisons in univariate analysis. Multivariate survival analysis was performed using the Cox proportional hazards regression to identify independent prognostic factors. Hazard ratio (HR) and its 95% confidence interval (CI) were calculated. Statistical analysis was conducted in SPSS 16.0 . The yearly bar graph and the line chart showing the proportions of patients in the “18–30” and “31–40” age subgroups among all lung cancer patients were made by Excel. All tests were two tailed, and P < 0.05 was considered as statistically significant.In this study, young NSCLC patients were stratified into two (18–30 and 31–40) or four  subgroups according to age at diagnosis. Pearson's χ


Listeria monocytogenes, a foodborne pathogen is ubiquitous to different environments including the agroecosystem. The organism poses serious public health problem. Therefore, an attempt has been made to gain further insight to their antibiotic susceptibility, serotypes and the virulence genes.L. monocytogenes. The prevalence of the pathogen in the respective rhizosphere soil samples was 5%. Noticeably, L. monocytogenes was absent from only cabbage, broccoli, palak and cowpea, and also the respective rhizospheric soils. The 30 isolates + ve for pathogenicity, belonged to serogroup 4b, 4d or 4e, and all were positive for inlA, inlC, inlJ, plcA, prfA, actA, hlyA and iap gene except one (VC3) among the vegetable isolates that lacked the plcA gene. ERIC- and REP-PCR collectively revealed that isolates from vegetables and their respective rhizospheric soils had distinct PCR fingerprints.Out of the 10 vegetables selected, 6 , 20 isolates (10%) tested positive for L. monocytogenes in the selected agricultural farm samples. The increase in the number of strains resistant to ciprofloxacin and/or cefoxitin seems to pose serious public health consequences.The study demonstrates the prevalence of pathogenic  Listeria monocytogenes, the foodborne pathogen, causes listeriosis with high mortality rates (~30%), and presently considered to pose serious public health problem , and incubated for 30 min (37°C). Proteinase K (25 μl) and 200 μl Buffer AL (without ethanol) were added, mixed by vortexing and the mixture re-incubated at 56°C (30 min). Thereafter, 4 μl RNase A (100 mg/ml) was added and incubated (2 min) at room temperature. Pure ethanol (200 μl) was added to the sample, and mixed by vortexing. The DNA was eluted in AE Buffer, and the concentration and purity determined with the help of Eppendorf spectrophotometer at 260 and 280 nm, respectively.Chromosomal DNA was extracted from inlA, inlC and inlJ), virulence-associated genes  and serogroup  was determined by multiplex PCR as described by Liu et al., [et al., [et al., [et al., [The presence of internalin genes ( et al., , Noterma[et al.,  and Doum[et al., , respect[et al., . The PCRet al., [et al., [ERIC- and REP- PCR were performed as described by Rivera et al.,  and Vers[et al., , respect[et al., . The fin


In situ soil respiration was monitored during 2010–2012 under various conditions  in a 30-year-old artificial black locust plantation (Robinia pseudoacacia L.) on the Loess Plateau. Litter respiration with normal and double litter treatments exhibited similar seasonal variation, with the maximum value obtained in summer  and the minimum in spring . On average, annual cumulative litter respiration was 115 and 300 g C m−2 y−1 under normal and double litter conditions, respectively. Using a soil temperature of 17°C as the critical point, the relationship between litter respiration and soil moisture was found to follow quadratic functions well, whereas the determination coefficient was much greater at high soil temperature than at low soil temperature (33–35% vs. 22–24%). Litter respiration was significantly higher in 2010 and 2012 than in 2011 under both normal litter (132–165 g C m−2 y−1 vs. 48 g C m−2 y−1) and double litter (389–418 g C m−2 y−1 vs. 93 g C m−2 y−1) conditions. Such significant interannual variations were largely ascribed to the differences in summer rainfall. Our study demonstrates that, apart from soil temperature, moisture also has significant influence on litter respiration in semi-arid regions.Understanding the response mechanisms of litter respiration to soil moisture in water-limited semi-arid regions is of vital importance to better understanding the interplay between ecological processes and the local carbon cycle.  It is an arid and semi-arid region, with a continental monsoon climate. In addition, the loess region is particularly susceptible to soil erosion because of its fractured and steep terrain, aggravated by improper land use. To address this problem, the black locust (Robinia pseudoacacia L.) has been widely planted in the region since 1980 to control soil erosion (due to its high drought resistance), now covering an area within the region of 70,000 ha. Moreover, in degraded dryland forest ecosystems such as this, above-ground litter decomposition has important practical implications for ecological rehabilitation The Loess Plateau of northwest China covers an area of 640,000 kmin situ soil respiration and soil microclimate (temperature and moisture) in a 30-year-old artificial black locust plantation (Robinia pseudoacacia L.) within a small watershed; monitoring was conducted with different above-ground litter treatments over the course of three years (2010−2012). Litter treatments included normal litter, double litter, and no litter. The objectives of the study were: 1) to describe the dynamics of litter respiration with normal and double litter, and 2) to investigate the effect of soil moisture on litter respiration with normal and double litter in the semi-arid Loess Plateau.Given the above, in this study we monitored There were no specific permits required for the described field studies. We confirmed that the site was not privately owned or protected in any way. The field studies did not involve endangered or protected species.–2, and a frost-free period of 171 days.The study site was located on the southern Loess Plateau, at Wang-donggou watershed, Changwu, Shaanxi, China, and forms part of the Changwu State Key Agro-Ecological Experimental Station, Chinese Academy of Sciences . The study site has a continental monsoon climate, with mean annual rainfall of 580 mm and mean annual air temperature of 9.4°C. Rainfall variation occurs both seasonally and annually, and more than 50% of rainfall falls in summer (between June and August). Air temperature variations occur only seasonally, as the monthly mean temperature in summer is much higher than in other seasons. Site characteristics include the following: ≥10°C accumulated temperature of 3029°C, annual sunshine duration of 2230 h, annual total radiation of 484 kJ cm−1, total nitrogen (TN) of 0.66 g·kg−1, and an initial NaHCO3-extractable soil phosphorus content of 5 mg P kg−1.Soils of interest were derived from wind-deposited loess and form part of the loessial soil group, according to the soil classification system of the Food and Agriculture Organization and the United Nations Educational Scientific and Cultural Organization (FAO-UNESCO). The parent material was relatively uniform calcareous loess dominated by loam. Soil collected in 2009 had a depth of 0–20 cm, a pH of 8.3, a clay content (<0.002 mm) of 24%, a field capacity of 22.4%, a permanent wilting point of 9.0%, an SOC level of 6.80 g·kg−1 and was established approximately 30 years ago to control soil erosion and improve the ecological environment of the region. Approximately 64% of the study site is covered with trees, with these having a total area of 0.68 ha. Shrubs cover 24%, and the remaining 12% is covered by grass and bare soil. The plantations are located on a ridge with a gentle slope (<15°) on the west of the Loess plateau. Tree canopy height (H) and diameter at breast height  were recorded for trees with DBH>1 cm. Mean H was 6.8±1.6 m, mean DBH was 6.4±2.6 cm, and the canopy area was 55%. The main understory shrub was Rubus parvifolius L., with a mean H of 62.8±11.8 cm and a canopy area of 55%. The main understory herb was Bothriochloa ischaemum (L.) Keng, with a mean H of 42.5±11.9 cm and a canopy area of 75%.The black locust plantation has a tree density of 1213 stems ha−2.Slope tillage was converted to woodland with the implementation of integrated management of small watersheds in the 1980s to control soil erosion, because local people collected forest above-ground litter for heating and cooking. In March 2009, we chose a relatively homogeneous black locust plantation as a study site, and established an experiment with three litter treatments  at the site. For the normal litter treatment, litter inputs were allowed. For the no litter treatment, litter inputs were prevented using a sheet of nylon mesh screen (pore size ∼1.7 mm) over the exposed soil area. The protective screen was temporarily removed to allow for soil respiration measurements but otherwise left intact over the study period. For the double litter treatment, above-ground litter inputs were doubled by adding litter from no litter plots four to five times per year. Large branches that fell on screens were discarded. Five 1 m×1 m nylon meshes, with a mesh size of 1 mm×1 mm, were placed in the black locust plantation from 2010 to 2012 to collect above-ground litter. Litter was collected seasonally and dried at 70°C for 72 h in an oven. Mean annual above-ground litter content was 474 g mLitter respiration under normal litter conditions was determined as the difference in soil respiration between normal and no litter treatments, while litter respiration with double litter was defined as the difference in soil respiration between double litter and no litter treatments.The litter contribution rate (%) was determined by dividing litter respiration under normal litter by soil respiration under normal litter.2 flux system equipped with a portable chamber . Approximately one day before the first measurement, a polyvinyl chloride (PVC) collar (20 cm in diameter by 12 cm in height) was inserted 2 cm into each plot, where it remained for the entire experimental period (from 2010 to 2012). Before measurements were taken, all visible living flora and fauna were removed. If necessary, one or more additional measurements were taken until the variation between two consecutive measurements was less than 15% for a given collar. Final soil respiration values for a given collar were calculated as the mean values of two consecutive satisfactory measurements, with a 30 s delay between measurements. The measurement time for each collar was 150 s, which included 30 s pre-purge, 30 s post-purge, and a 90 s observation period. Field measurements were conducted between 09∶00 and 11∶00 a.m. from March 2010 to November 2012 . A total of 12, 15, and 19 soil respiration measurements were taken in 2010, 2011, and 2012, respectively.Soil respiration was measured using an automated closed soil COSoil temperature (three measurements per collar) and moisture (four measurements per collar) were measured 10 cm away from the collar chamber; soil respiration was measured at the same time. Soil temperature and moisture at 5 cm depth were measured using a Li-Cor thermocouple probe and a Theta Probe ML2X with an HH2 moisture meter , respectively. The soil water-filled pore space (WFPS) was calculated using the following equation: WFPS (%) = (volumetric water content/100×[2.65–soil bulk density]/2.65).P = 0.05) between different litter treatments. An exponential function was used to describe the relationship between soil respiration and soil temperature θ is soil moisture at 0–5 cm depth, and β0, β1, and β2 fitted parameters.Respiration rate, soil temperature, and soil moisture data were analyzed using the GLM procedure in SAS to detect any differences , and then summing mean daily respiration for a given year.P>0.05). The patterns of soil temperature variation followed variations in air temperature, with the lowest soil temperatures recorded in the spring and autumn and the highest in summer   . Mean soP<0.05) .−2 s−1, 0.62–4.43 μmol m−2 s−1, and 0.73–8.84 μmol m−2 s−1 over three years (2010–2012), respectively.Soil respiration across the three treatments exhibited similar seasonal variation, with patterns of variation corresponding to variations in temperature; the maximum value occurred in summer and the minimum value was recorded in the spring or autumn  and 4. M−2 y−1, 657 g C m−2 y−1, and 967 g C m−2 y−1 under normal litter, no litter, and double litter treatments, respectively  for forest ecosystems −2 s−1 g) was much lower than in the deciduous forests of the northern hemisphere temperate regions (3.5 µmol m−2 s−1) −2 y−1, 529 to 766 g C m−2 y−1, and 724 to 1151 g C m−2 y−1, respectively. Inter-annual variation may be explained by differences in summer precipitation  −2 y−1 for annual cumulative litter respiration with normal and double litter treatments) are much lower than values measured in forest systems elsewhere having adequate rainfall −2 y−1 vs. 627 g m−2 y−1) also contributed to this difference −2 y−1, and 93–418 g C m−2 y−1, respectively; this inter-annual variation may be ascribed to differences in summer precipitation . Similar results were also noted in DIRT  experiments conducted at the H.J. Andrews Experimental Forest, Oregon R2) was lower at lower soil temperatures than at higher soil temperatures   . Similareatment) . Similar−2 y−1 but 300 g C m−2 y−1 with the double litter treatment); thus the priming effect cannot be ignored Soil respiration increased by 24% due to addition of litter, but decreased by 18% as a result of removing litter. Similar results were are also found in the previous studies −2 y−1, which was 15% of the total soil respiration. On the other hand, the carbon input into the soil through littering was 213 g C m−2 y−1 , implying that approximately 50% of organic carbon remained in the system. Apparently, the accumulated litter can not only effectively alleviate soil erosion, but can also progressively improve the ecological environment in the degraded and eroded loess regions Mean annual cumulative litter respiration  in our sites was 115 g C mUnderstanding the effect of soil moisture on litter respiration is of prime importance in water-limited regions in order to better understand the carbon cycle. Our study demonstrated that litter management had significant impacts on soil respiration, showing 24% increase but 18% decrease under litter addition and removal, respectively. Litter respiration with normal and double litter treatments exhibited similar seasonal variation, being controlled mainly by soil temperature; however, soil moisture also contributed to larger variations. Significant inter-annual variation in litter respiration was ascribed to differences in summer rainfall. Our study demonstrates that both soil temperature and moisture have significant influences on litter respiration in semi-arid regions.


Angiogenesis is also implicated in pathological processes such as retinopathy and tumor growth. Anti-angiogenic agents have been demonstrated to have promising therapeutic potential for inhibiting tumour growth. Earlier studies suggested that β3 integrin (i.e. αVβ3 that is formed by β3 subunit and αV subunit) expressed on angiogenic endothelial cells (ECs), is required for angiogenesis ; however−/− mice . It is tFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder that occurs when maternal alloantibodies cross the placenta and target paternally derived antigens, especially GPIIbIIIa (integrin αIIbβ3) and GPIbα, on fetal/neonatal platelets. In contrast to αVβ3, integrin αIIbβ3 is composed of a β3 subunit and αIIb subunit, which are almost exclusively expressed on platelets and megakaryocytes. Half of the polymorphisms known to cause FNAIT are located on the β3 subunit. Approximately 80-90% of reported FNAIT cases are caused by antibodies targeting Human-Platelet-Antigen-1a (HPA-1a) on β3 subunit. Antibodies targeting the HPA-2a on GPIbα have also been reported. Intracranial hemorrhage (ICH), which occurs in 10-20% of affected fetuses/neonates, is a major clinical complication of FNAIT, leading to neurological impairment and death. Unfortunately, the mechanism responsible for ICH, has only been inferred but not adequately explored.Thrombocytopenia was considered to be the cause of bleeding in FNAIT. Interestingly, mice deficient in transcription factor NF-E2, which lack circulating platelets, did not develop significant bleeding disorders in utero. More strikingly, the murine fetuses with combined deficiencies in NF-E2 and fibrinogen genes exhibited normal embryonic development and were morphologically indistinguishable from their wild-type control counterparts at 18.5 days postcoitum . Therefoin vitro. Importantly, anti-HPA-1a IgG purified from mothers who gave birth to neonates with FNAIT, also showed similar effects on HUVECs in vitro. This study suggests that anti-platelet β3 antibodies also cross-react with αVβ3 on ECs, thereby impairing angiogenesis and contributing to ICH. The impairment of retinal vascular development may explain why some FNAIT untreated siblings suffered visual impairment in adulthood. Our study may also provide insights into the mechanisms of intrauterine growth restriction (IUGR) in FNAIT as well as the possible pathogenic role of αVβ3 integrin in tumorigenesis.In a recent publication , we repoClinical management of such devastating bleeding diatheses in fetuses/neonate remains a formidable challenge. Since our findings indicate that impairment of angiogenesis but not thrombocytopenia may be the major cause of ICH, invasive fetal platelet transfusion may not be an effective prenatal therapy. Our data demonstrated that antenatal administration of intravenous immunoglobulin (IVIG) decreased EC apoptosis, rescued brain and retinal vessel development, increased platelet counts, and ameliorated ICH, suggesting that IVIG may be an effective therapy . In addiAside from its expression on platelets and ECs, β3 integrin is also expressed on highly invasive trophoblasts, which are essential for normal placental vascular remodeling and development. Therefore, further studies on the effects of anti-β3 antibodies on trophoblast proliferation, differentiation, migration, and invasion, may provide insights into the mechanism of IUGR and miscarriage in FNAIT, as well as aid in the development of new therapies to manage these disorders.


A great number of studies have investigated changes induced by morphine exposure in gene expression using several experimental models. In this study, we examined gene expression changes during chronic exposure to morphine during maturation and differentiation of zebrafish CNS.grb2, copb2, otpb, magi1b, grik-l, bnip4 and sox19b) have been detected for the first time in an experimental animal model treated with morphine. We have also identified a subset of genes  differentially expressed by chronic morphine exposure whose expression is related to mu opioid receptor gene expression. Altered expression of copb2, bnip4, sox19b, otpb, dao.1, grik-l and wls is indicative of modified neuronal development, CNS patterning processes, differentiation and dopaminergic neurotransmission, serotonergic signaling pathway, and glutamatergic neurotransmission. The deregulation of camk1γb signaling genes suggests an activation of axonogenesis and dendritogenesis.Microarray analysis showed 254 genes whose expression was identified as different by at least 1.3 fold change following chronic morphine exposure as compared to controls. Of these, several novel genes  contains supplementary material, which is available to authorized users. Morphine, the most widely known opioid agent, has been largely used for pain management. Besides non-analgesic uses of this drug, including experimental depression treatments as a control for opium addiction, has been developed , 2. DespOpioids modify the expression profile of certain mRNAs in many tissues, including central nervous system (CNS), the main target of drugs of abuse . With thDanio rerio) has been used in research related to drugs of abuse, studying the changes of gene expression produced by nicotine , neuron differentiation , somitogenesis  and glutamine biosynthetic process .For our study, the different categories within Gene Ontology Biological Process can be classified in other “functional” categories according to databases and consulted publications (see Discussion section). As shown in Figure RT-qPCR analysis was used to confirm a set of gene expression changes observed in the microarray analysis. Chosen genes for RT-qPCR confirmation were mainly selected based on ontological categories with potential roles in the nervous system.β-actin (β-act), ribosomal protein L13a (rpl13a), β-2-microglobulin (β2 mg) and elongation factor-1α (ef1α)] were selected according to their expression levels detected in the microarray studied. The expression of these four genes was also measured by RT-qPCR. The NormFinder software. As mentioned before, the expression of several morphine-induced genes identified in previous studies  to knock-down (KD)-oprm1 at the one-to-four-cell stage in the yolk  in the ur group –56.oprm1-MO injection plus exposure to morphine (oprm1-MO + morphine group).To establish that changes observed after MO microinjections were produced by MO action on their target, we also employed two more experimental groups  was decreased with respect to the Control group, indicating that oprm1 is important in the expression of these genes , grb2 expression was recovered and similar values were reached with respect to Control groups, including MO Control group  increased the expression of bnip4, camkγ1b and sox19b genes , sox19b expression was recovered and similar values were reached with respect to the Control groups  mechanism; however, it required the expression of oprm1 to exert its influence on the expression of dao.1, wls, bnip4 and camkγ1b. In other words, these four genes represent a putative collection of genes whose expression is related to oprm1 expression.In conclusion, we found that morphine deregulated (increased or decreased) the expression of Most of the published data addressing addictive drugs have focused on adults or neonates in mammalian models , 57, 58;In the present study, we used zebrafish embryos, since in this model external development can be observed, which is advantageous for the study of the actions of drugs in a specific embryonic stage oprm1 expression: (i) Immediate early genes (IEG) and other genes related to transcription; (ii) genes involved in monoaminergic neuromodulation; (iii) genes involved in glutamatergic neurotransmission; and (iv) others genes involved in neuronal function.We described four groups of genes regulated by chronic morphine treatment in zebrafish embryos. Each group contains both, genes already known to be related to morphine activity, and new genes that have not been related to morphine until now. We have also described some genes whose expression is related to fos and jun families, which have important roles in processes such as brain development, learning and long-term neuronal plasticity  * 100. All amplifications had an E value of 100 ± 10% the E value close to 100% being an indicator of efficient amplification. The relative gene expression value (FC) for each transcript was calculated according to the equation 2 −(ΔCt “condition 1” − ΔCt “condition 2”), where “condition 1” corresponds to experimental samples (“treatment with morphine”), “condition 2” to samples of control animals and ΔCt of each “condition” is Ct “experimental gene” − Ct “endogenous gene”[t-test for each gene, testing that |FC| > 1 is significant (p < 0.05).The comparative Ct method was used for presenting quantitative data . Followius gene”. A stand2, 0.33 mM MgSO4 in ddH2O). To inhibit oprm1 mRNA translation we used the following MOR-MO sequence: AATGTTGCCAGTGTTTTCCATCATG. The efficacy and specificity of MO employed was demonstrated by Sanchez-Simon et al. [Antisense MO oligonucleotides used were provided by Gene Tools . MO injection was performed according to the methodology developed by Nasevicius and Ekker , for whin et al. .Table listing the probe-sets of genes expressed differentially by chronic morphine treatment in zebrafish embryos. Significance level for each probe-set are presented as d-, p- and q-value. The expression changes between Morphine vs. Control are showed as R fold. List of known genes up-, down-, and up-/down-regulated. (XLSX 199 KB)Additional file 1:Figure presenting gene ontology analysis of the zebrafish gene collection. Genes were categorized with the Biological Process domain. Significantly enriched GO terms have a probability lower than 0.01  and include at least three genes. GO terms are shown if they are significantly enriched in at least one of them. Significantly enriched GO terms are indicated as black bars whereas non-significantly enriched terms are displayed as empty bars. Bars represent the number of genes assigned with the corresponding GO term. For more details, see also Additional file Additional file 2:Data file providing the detailed description of Gene Ontology analysis presented in Figure  and Table . List of probe-sets and gene names classified for each GO category. (XLSX 70 KB)Additional file 3:Table listing genes differentially expressed by morphine and included in each functional category presented in Figure  and Table . The neuronal function category is emphasized in two excel sheet tabs . (XLSX 98 KB)Additional file 4:Table listing genes differentially expressed by morphine validation by ourin silicoanalysis.(XLSX 34 KB)Additional file 5:Table listing the primers used for RT-qPCR.(PDF 35 KB)Additional file 6:


Objectives. This study assessed the correlation between performance intelligence and the postoperative cochlear implant (CI) outcome in Korean-speaking children. In addition, the relationship between the performance intelligence subscales and the post-CI speech outcome was evaluated. Materials and Methods. Thirteen pediatric CI users  years; median age at intelligence test 9.3 (range 5–16) years) who were tested using the Korean Educational Development Institute-Wechsler Intelligence Scale for children were studied. The correlations between the intelligence scores and 1-2 years postoperative Categories of Auditory Performance (CAP) scores and between subscales of performance and 1-2 years postoperative CAP scores were analyzed. Results. There was no correlation between the categories of verbal intelligence quotient (IQ) and performance IQ for “mentally retarded” and “average,” respectively . There was a strong correlation between performance IQ and the postoperative CAP scale . “Picture arrangement” and “picture completion,” reflecting social cognition, were strongly correlated with the postoperative CAP scales. Conclusion. Performance intelligence, especially social cognition, was strongly related to the postoperative CI outcome of cochlear implant users. Therefore, auditory rehabilitation, including social rehabilitation, should maximize the postoperative CI outcomes. Cochlear implant (CI) is a standard treatment option for children with profound hearing loss. However, the outcome of CI varies over a wide range among pediatric patients. Some prelingually deafened children show outstanding behavioral performance, such as the rapid acquisition of spoken language and the production of intelligible speech after years of CI-assisted rehabilitative effort, while other children develop awareness of environmental or speech sounds but never catch up with normal age-appropriate auditory language .Therefore, it is relevant, both scientifically and clinically, to unravel the factors underlying the wide variability in CI outcome. Researchers have repeatedly suggested that demographic factors—such as age at the onset of severe-to-profound hearing loss, the duration of the severe-to-profound hearing loss, age at CI, and absence or presence of linguistic experience—are factors underlying the CI outcome variability –4. OtherIn addition to these factors, recent studies have emphasized the role of the cognitive function of the subjects on the CI outcome . CognitiTherefore, we assessed the correlation between the performance IQ and postoperative CI outcome in prelingually deafened pediatric CI users. We also determined the performance IQ subscales that are most relevant to the CI outcome in Korean-speaking CI users using the Korean Educational Development Institute-Wechsler Intelligence Scale for Children (KEDI-WISC) .Of the children who underwent CI at the Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Children's Hospital, 18 prelingually deafened children who were subjected to the KEDI-WISC to evaluate their verbal and performance IQs between 16 April 2009 and 5 August 2013 were initially included in this study. Children who had severe inner ear anomalies, cochlear nerve aplasia/hypoplasia, or severe bony cochlear nerve canal narrowing on temporal bone computed tomography (CT) or internal auditory canal magnetic resonance imaging (MRI) were excluded. Of the 18 initially included children, 5 with full-scale IQs classified as “severe mental retardation ” were excluded because their verbal and performance IQ scores were unreliable. This study was approved by the Institutional Review Board of Seoul National University Hospital (number 1409-088-609).The 13 children ultimately included comprised five males and eight females. Their median ages at CI and the KEDI-WISC test were 6.2 (range 1.3–14.2) and 9.3 (range 5–16) years, respectively. All of the children had profound sensorineural hearing loss (>90 dB HL on pure-tone audiometry or auditory brainstem response) in both ears. Depending on the age and compliance of the subject, the auditory brain stem response or auditory steady-state response were evaluated, or play audiometry or visual reinforcement audiometry was performed. The demographic characteristics of the included children are summarized in We administered the KEDI-WISC to assess the intellectual function of the 13 children. The KEDI-WISC, a modified version of the Wechsler Intelligence Scale for children, is an intelligence test for Korean-speaking children between the ages of 5 and 15 years . This teThe verbal IQ test evaluates “information,” “similarities,” “arithmetic,” “vocabulary,” and “comprehension,” while the performance IQ test evaluates “picture completion,” “picture arrangement,” “block design,” “object assembly,” and “coding” . Of the Using the verbal and performance IQ, intelligence is divided into seven categories: mentally retarded (IQ ≤ 69), borderline (70–79), low average (80–89), average (90–109), high average (110–119), superior (120–129), and very superior (≥130). Of note, an IQ ≤ 40 is categorized as “severely mentally retarded.” An IQ of 80 separates the intelligence categories into borderline and low average .The post-CI outcome was measured using the postoperative Categories of Auditory Performance (CAP) scale 1-2  years postoperatively. The CAP scores indicate the following: CAP 0 , 1 , 2 (response to speech sounds), 3 , 4 (discrimination of some speech sounds without lip-reading), 5 (understanding of common phrases without lip-reading), 6 (understanding of conversation without lip-reading), and 7 (use of a telephone with a known listener) . U-test was used to compare pairs of subscales. Spearman's rank correlation test was performed to examine the correlations between the verbal and performance IQs, between the performance IQ and postoperative CAP scores, and between each performance subscale and the postoperative CAP scores. Statistical significance was set at P < 0.05. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) 21.0 .To compare the scores of the five performance IQ subscales, the Kruskal-Wallis test was performed. The Mann-WhitneyThe mean full-scale IQ of the 13 subjects was 74.5 ± 19.0 (range 47–113), falling in the category “borderline.” When the full-scale IQ was subdivided into verbal and performance IQs, the mean verbal and performance IQs were 65.2 ± 21.2 (range 48–111) and 91.9 ± 17.5 (range 55–118), respectively. The categories of verbal and performance IQ were in the “mentally retarded” and “average” categories, respectively.P = 0.010, Kruskal-Wallis test). Post hoc individual Mann-Whitney U-tests revealed significant differences between “picture completion” and “block design” (P = 0.009), “picture completion” and “object assembly” (P = 0.002), “picture arrangement” and “block design” (P = 0.043), and “picture arrangement” and “object assembly” (P = 0.018). That is, the scores of the “picture completion” and “picture arrangement” subscales, reflecting social cognition, were significantly lower than those of “block design” and “object assembly,” reflecting visual motor coordination.The mean scores for the five subsets of the performance IQ test are shown in P = 0.15) ( = 0.15) .P = 0.0008)  .We evaluated the correlation between each subset of the performance IQ and the postoperative CAP scores. The “picture arrangement” subset had the highest correlation with the postoperative CAP scores, followed by “picture completion.” As mentioned above, “picture completion” and “picture arrangement” reflect social cognition. Therefore, we inferred that social cognition correlates well with the postoperative CAP scores .To our knowledge, this is the first study to assess the verbal and performance IQs of Korean-speaking CI users using the KEDI-WISC test. The mean performance IQ of our subjects fell in the category “average,” which is in agreement with previous reports –22.There were discrepancies between the verbal and performance IQs in our CI subjects. Profiles between the verbal and performance IQs are often found in children with developmental disorders, such as autism  –26, and Verbal IQ reflects crystallized intelligence or knowledge coming from prior learning and past experiences . SituatiConsistent with other research, we found that social cognition, as measured by “picture completion” and “picture arrangement,” is relatively poor in CI users . In addiSocial cognition focuses on how people process, store, and apply information about other people and social situations . It focuIn this study, the postoperative CI outcomes were assessed using the CAP score. Many studies have evaluated CI outcomes using open-set phonetically balanced word recognition tests or sentence tests . These tLimitations of this study must be mentioned. First, because this study presents preliminary results obtained from a small sample, a future study of a larger group of CI subjects is mandatory to draw generalized conclusions. Second, as the developmental stages of the subjects differed, a longer follow-up and longitudinal evaluation are needed. Third, a more reliable result might be achieved if patients with the same total performance IQ were included to compare the performance IQ subscales. Finally, to explore the causal relationship between the performance IQ and CI speech outcome, a prospective study that includes a preoperative performance IQ evaluation should be conducted.Performance intelligence, especially social cognition, is correlated with the postoperative speech outcome in CI users. Therefore, postoperative rehabilitation—including a social rehabilitation program—might help to maximize the postoperative CI outcome.