The clinical implication of O6-methylguanine-DNA methyltransferase (MGMT) promoter status is ill-defined in elderly glioblastoma patients. Here we report a meta-analysis to seek valid evidence for its clinical relevance in this subpopulation.Literature were searched and reviewed in a systematic manner using the PubMed, EMBASE and Cochrane databases. Studies investigating the association between MGMT promoter status and survival data of elderly patients (≥65 years) were eligible for inclusion.Totally 16 studies were identified, with 13 studies included in the final analyses. The aggregate proportion of MGMT promoter methylation in elderly patients was 47% , which was similar to the value for younger patients. The analyses showed differential effects of MGMT status on overall survival (OS) of elderly patients according to assigned treatments: methylated vs. unmethylated: (1) temozolomide (TMZ)-containing therapies: hazard ratio [HR] 0.49, 95% CI 0.41–0.58; (2) TMZ-free therapies: HR 0.97, 95% CI 0.77–1.21. More importantly, a useful predictive value was observed by an interaction analysis: TMZ-containing therapies vs. RT alone: (1) methylated tumors: HR 0.48, 95% CI 0.36–0.65; (2) unmethylated tumors: HR 1.14; 95% CI 0.90–1.44.The meta-analysis reports an age-independent presence of MGMT promoter methylation. More importantly, the study encouraged routine testing of MGMT promoter status especially in elderly glioblastoma patients by indicating a direct linkage between biomarker test and individual treatment decision. Future studies are needed to justify the mandatory testing in younger patients. Glioblastoma is the most frequent brain malignancy and is invariably associated with very poor prognosis, despite the use of multiple treatment modalities including maximal tumor resection, radiotherapy (RT) and chemotherapy.The standard treatment for elderly glioblastoma patients remains suboptimal.MGMT promoter methylation status and survival data in elderly patients (≥ 65 years old) with newly diagnosed glioblastoma were eligible for inclusion. Different treatment modalities or schedules, different testing methods and different types of tumor samples were all included. Outcomes of interest included: 1) OS defined as the time interval from the date of diagnosis or randomization to the date of death or last follow-up; 2) progression-free survival (PFS), or the time interval from the date of diagnosis or randomization to the date of progression, which was defined by both clinical and radiologic criteria Studies investigating the association between A systematic literature search was conducted using the PubMed, EMBASE and Cochrane Library, and no restrictions regarding publication date and language were applied. The following search strings were used 1) “glioma”, “glioblastoma”, “malignant glioma”, “high grade glioma”; 2) “elderly”, “older patients”, “advanced age”; 3) “O-6-methylguanine-DNA methyltransferase”, “MGMT”, “prognostic”, “biomarker”, “prediction”, “predictive”, “predictor”. Reference lists from related articles were also reviewed.Study selection was independently performed by two reviewers (YAA and ZLH) who were not blinded to study identity  during eligibility assessment, and disputes were resolved through discussion. The newest publication of a same study was included. Data of interest were extracted using a piloted extraction sheet, as follows: authors, publication years, contact address, sample size, patients’ characteristics, types of specimens, assay methods, statistical methods and survival data.The risk of bias in each study was independently assessed by two reviewers (YAA and CJX) using a customized domain-based Newcastle Ottawa Scale (NOS). 2 test and I2 statistic (the percentage of the variability in effect estimates that is due to heterogeneity rather than random error), with Pheterogeneity<0.1 or I2>40% considered to be statistically significant.Time-to-event data  were analyzed using the hazard ratios (HRs) with a value less than one indicating favorable outcomes in elderly patients with methylated MGMT promoter or those with TMZ-containing therapies. If the HR was not directly reported, the value was calculated using the Kaplan-Meier survival curves or the methods reported by Tierney et al Publication bias was assessed by visual impression, and was confirmed by analytic methods such as Egger’s test Both a subgroups analysis of studies with similar treatment and an interaction analysis between treatment and MGMT promoter status were implemented. A sensitivity analysis based on the assessment of risk of bias was also performed.All analyses were done in R software v2.15.3  and Review Manager v5.2 .With the above eligibility criteria, 16 studies The cutoff age for elderly patients was defined variably across the studies, among which seven studies set the age cutoff at 65 years Three studies were To explain explicitly the clinical impact of MGMT promoter status, the definition of the terms “prognostic” and “predictive” was used as follows: 1) a prognostic factor is a clinical or biologic characteristic that provides information on the likely outcome of the cancer disease independent of treatment; and 2) a predictive factor is a clinical or biologic characteristic that provides information on the likely benefit from one specific treatment rather than another .Promoter methylation status of MGMT was assessed using gel-based methylation-specific PCR (MSP) assays in 9 studies 2 = 41.75, P<0.0001; I2 = 71%; subgroup analysisP <0.00001; 2 = 15.49, P = 0.16, I2 = 29%; 2 = 4.13, P = 0.39, I2 = 3%; 2 = 14.94, P = 0.06; I2 = 46%; A random-effect meta-analysis of the studies with all treatments suggested a significant prognostic impact of MGMT promoter methylation status in older glioblastoma patients  and highly reproducible in independent laboratories.The presented information should be interpreted carefully because some limitations existed. First, data on MGMT promoter status were only available for a selected patient subgroup of overall trial population which can induce selection bias in the analysis. Second, with limited data on PFS, the influence of salvage treatment at progression cannot be ruled out. Third, the predictive significance of MGMT status was not always validated in a controlled, prospective manner , which is the only way to demonstrate the predictive value of a biomarker, and its prognostic effect was not always being studied in a group of patients free from systematic adjuvant treatment. Finally, even in a non-optimal trial design, the distributions of patient prognostic variables were not always being adjusted  and MGMT endpoints were usually secondary outcome with statistical under-powering issues .The meta-analysis encouraged the mandatory testing of MGMT promoter status in routine practice in elderly glioblastoma patients due to the observation of a strong predictive but not prognostic value of this biomarker to TMZ chemotherapy and a direct association between MGMT testing and individual treatment choice.Future studies of MGMT molecular analysis are needed 1) to validate its predictive value for the comparison of combined RT/TMZ vs. RT alone in older patients and 2) to justify its therapeutic implications for younger patients. Furthermore to optimize the personalized treatment of elderly ones, other clinically relevant factors  are needed to further stratify the elderly with methylated tumors for treatment choice between combined RT/TMZ and TMZ alone. Finally, the modification and standardization of MGMT testing approaches are much needed in future.Figure S1Forest plot of comparison: outcome: OS; comparsion: methylated versus unmethylated: all treatments included.(TIF)Click here for additional data file.Figure S2Forest plot of comparison: outcome: PFS; comparsion: methylated versus unmethylated: all treatments included.(TIF)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.Table S1Criteria for judgment of risk of bias in the modified domain-based Newcastle-Ottawa Scale (NOS).(DOC)Click here for additional data file.Table S2Assessment of risk of bias of included studies.(DOC)Click here for additional data file.Table S3Egger’s test for publication bias.(DOC)Click here for additional data file.Table S4The results of the sensitivity analysis.(DOC)Click here for additional data file.


Arboviral diseases are major global public health threats. Yet, our understanding of infection risk factors is, with a few exceptions, considerably limited. A crucial shortcoming is the widespread use of analytical methods generally not suited for observational data – particularly null hypothesis-testing (NHT) and step-wise regression (SWR). Using Mayaro virus (MAYV) as a case study, here we compare information theory-based multimodel inference (MMI) with conventional analyses for arboviral infection risk factor assessment.n = 270 subjects) in a central Amazon rural settlement. NHT suggested that residents of village-like household clusters and those using closed toilet/latrines were at higher risk, while living in non-village-like areas, using bednets, and owning fowl, pigs or dogs were protective. The “minimum adequate” SWR model retained only residence area and bednet use. Using MMI, we identified relevant covariates, quantified their relative importance, and estimated effect-sizes (β±SE) on which to base inference. Residence area  and bednet use (β = −0.95±0.28) were the most important factors, followed by crop-plot ownership (β = 0.39±0.22) and regular use of a closed toilet/latrine (β = 0.19±0.13); domestic animals had insignificant protective effects and were relatively unimportant. The SWR model ranked fifth among the 128 models in the final MMI set.A cross-sectional survey of anti-MAYV antibodies revealed 44% prevalence  or a habitat/habits shift ; any such ecological/adaptive novelty could increase the likelihood of MAYV emergence in Amazonia. Flaviviridae, Togaviridae or Bunyaviridae are also emerging worldwide Arboviral infections are a major global public health concern; dengue is the most widespread, but other viruses in the families Apart from overall neglect resulting in fewer epidemiological studies than would be needed a priori hypotheses, based on both their fit to the data and their complexity Among the major practical shortcomings of NHT is the fact that p-values provide no information on the size, direction, or precision of effect estimates; such estimates, in the form of, for instance, regression slope parameters or odds ratios , are central to inference HaemagogusAedes mosquitoes Here, we analyze data from a cross-sectional serological survey on Mayaro virus (MAYV) infection as a case-study to illustrate how MMI can enhance arbovirus infection risk factor analyses. MAYV is an alphavirus transmitted primarily by diurnal, canopy-dwelling mosquitoes of the genus This study was approved by the Fiocruz Institutional Review Board, Brazil (Protocol 384/07); all participants gave written informed consent. Laboratory procedures involving mice followed the guidelines of the Brazilian National Council for the Control of Animal Experimentation (CONCEA) and were approved by the Institutional Review Board for Animal Research of the School of Medicine, University of São Paulo at Ribeirão Preto, Brazil (Protocol 115/2008).N = 583 inhabitants in 158 households within a terra firme rain forest matrix) is located ∼150 km north of Manaus  and consists of two village-like household clusters (30×50 m plots for a house with courtyard) plus extensive upland and riverbank areas with scattered households (in ∼250×2000 m farm plots) and hence lower population density; old-growth forests comprise most of the ∼280 km2 of the settlement. Typical upland houses are located in large forest clearings used for farming, whereas most riverbank houses, which have only boat access, are located in smaller clearings within better-preserved forest. To the north and west, the settlement shares boundaries with a large indigenous reserve composed of pristine forests. Agriculture is the most important economic activity in the settlement; the main crops are banana, manioc, papaya, black beans, rice, maize, and the native cupuaçu (Theobroma grandiflorum) and pupunha (Bactris gasipaes). Most of the harvest is consumed within the community. Only a few settlers raise cattle for commercial purposes, but many families breed other domestic animals (mainly fowl and pigs) for their own use; dogs and cats are common. Forest extractivist products include timber, Brazil nuts (Bertholletia excelsa), and a variety of medicinal herbs. Non-commercial hunting and fishing are also relatively common.In the context of a study on infectious disease ecology in the central Brazilian Amazon, we conducted a cross-sectional serological survey see  in a rurAedes aegypti and Ae. albopictus, have never been collected in longitudinal mosquito studies conducted in this remote settlement. At the time of our survey, Culex (Culex) and Cx. (Melanoconion) were the dominant mosquito subgenera in the village-like clusters, although Psorophora, Anopheles, and Coquillettidia were also present. Haemagogus, Sabethes, Ochlerotatus, Wyeomyia, and Trichoprosopon were only recorded in forest sites, whereas Aedeomyia, Mansonia, and Uranotaenia occurred only in crop-plots; Anopheles, Culex, Psorophora, and Coquillettidia were present in all three environments .The allochthonous dengue vectors, n = 232). Sera were tested for anti-MAYV IgG antibodies using an enzyme-linked immunoassay with infected cultured cells as antigenic matrix (EIA-ICC), following procedures described elsewhere Aedes albopictus C6/36 cells were cultured in Leibovitz L-15 medium with bovine fetal serum (Invitrogen), penicillin, and streptomycin. Cells were infected with lyophilized baby mouse infected brain tissue resuspended in PBS; cells and virus suspension were incubated for 3–4 days at 28°C. Infected and uninfected (negative control) cells were transferred to TPP tissue culture plates (Sigma-Aldrich), which were incubated at 28°C for 24 h. A formalin solution was then added (18 h at 4°C) and wells washed with PBS. Non-specific binding sites were blocked for 1 h at 37°C with skim milk (5% plus 0.05% Tween-20 in PBS). Eluted serum samples (100 µL) were then added to each well pair ; mouse immune ascitic fluid was used as a positive control. Plates were then incubated  and washed; 100 µL of 1∶200 peroxidase-labeled anti-human (test wells) or anti-murine (positive control wells) IgG antibody (KPL Inc.) were added to each well. Plates were again incubated at 37°C for 1 h, washed, and 50 µL of ABTS peroxidase substrate (KPL Inc.) were added to each well; after 15 min at room temperature, enzyme activity was blocked with 50 µL H2SO4, and plates read in a spectrophotometer at 450 nm. In each plate, absorbance values (test well minus paired negative control well) were averaged (â) and the standard deviation (SD) calculated; the plate cut-off value for positivity was â+3SD.Finger-prick bloodspots from 270 randomly selected subjects  were collected onto filter paper in 2007; the sample was representative of the community and ensured 0.05 precision for an expected prevalence of 50% , gender , and regular bednet use (yes/no);roça and usually located in forest clearings (yes/no); andhousehold-level traits: residence area within the settlement ; basic sanitation ; adequate solid waste disposal ; and ownership of a crop-plot – locally known as whether or not domestic fowl, dogs, cats, or pigs, which may represent bloodmeal sources for female mosquito vectors, were reared or kept near the household (yes/no for each one).2 tests from bivariate logistic regression. At this stage, we also checked for correlation between potential predictor variables. If any pair of covariates proved to be highly correlated, we planned to retain only that for which a clear hypothetical relationship with MAYV transmission could be specified; however, all correlation coefficients were <0.30 (details not shown).We first screened the dataset for associations between anti-MAYV seropositivity and putative risk factors with NHT statistics, using either Fisher's exact tests or likelihood-ratio (LR) χa priori as those with bivariate p≤0.10), we removed, at each step, the covariate with the largest p-value (from LR tests) until all covariates in the final, ‘minimum adequate’ model We then adopted the SWR approach Finally, we implemented a MMI strategy  for predicting seropositivity status in each subset;specifying and fitting the complete set of additive logistic regression models for the selected covariates ;back-checking that none of the covariates excluded after step (ii) improved the performance of the models with substantial support from the data identified in step (iii); andestimating weighted mean effect-sizes (see below) and the relative importance of each covariate based on the final model set.α is the intercept and iβ represents the effect of covariate i (icov) on the  probability that a subject is MAYV-seropositive. Models were fit in JMP 9.0.0 (SAS Institute), with parameters estimated via maximum likelihood, and compared using Akaike's Information Criterion corrected for small sample size (AICc); AICc combines likelihood and information theories to identify, within a given set of models, those with a better compromise between fit and complexity, providing an estimate of Kullback-Leibler information loss. AICc is given bym is the likelihood of the data given each fitted model, K is the number of estimable parameters in each model, and n is sample size.Logistic regression models were of the simple general formi, we calculated the variation in AICc relative to the best-ranking (lowest AICc) model (ΔAICc = AICci−AICcmin); models with ΔAICc<2 are generally taken to be substantially supported by the data. The likelihood of each model given the data was estimated as L (model | data) = exp(−ΔAICc/2); these values were then used to compute Akaike weights (denoted iw), which are normalized model likelihoods, as:w) was estimated, within each model set, as the sum of Akaike weights over all models in which the covariate was present; covariates with w≤0.35 were considered unimportant. Weighted mean effect-sizes (βs) were estimated, for each covariate within the final model set, as the sum of model-specific effect sizes times model-specific Akaike weights. Finally, model fit was assessed using goodness-of-fit χ2 tests and scaled generalized R2 values 0 is the likelihood of the data given the intercept-only model, Lm is the likelihood of the data given the fitted model, and n is sample size.For each model Anti-MAYV antibodies were detected in 119 serum samples (44.1%). NHT suggested no departure from a random distribution of seropositivity in relation to sex or age; 36.8% of 19 toddlers fewer than three years old were seropositive, and there was very little variation across age classes . Surpris2 = 0.006, p = 0.939), fowl , closed toilet/latrine , and pig . The SWR ‘minimum adequate’ model therefore retained just two covariates: residence area within the settlement , and regular bednet use ; this same model was selected by standard forward SWR (details not shown). This model , which represents the hypothesis of no predictable variation in seropositivity; as expected, this model always proved to be relatively very poor at explaining the data . Note thw = 0.945) than age (w = 0.280) or gender (w = 0.270), whose effects were indistinguishable from zero. Therefore, only bednet use was retained for further analysis.The individual-covariate model set included seven models ; both thw values: residence area (w = 1.000), crop-plot ownership (w = 0.965), closed toilet/latrine (w = 0.498), and, to a lesser extent, solid waste disposal (w = 0.385).Next, we fitted all possible additive household-level models; this model set can be envisaged as representing hypotheses stating that in our study setting MAYV seropositivity simply varies among households with different traits, and comprises 15 models with all combinations of four candidate covariates . All thew = 0.999) and dogs (w = 0.735) were retained for testing, whereas cats (w = 0.267) and fowl (w = 0.337) were not considered any further.Third, we assessed all 15 possible model specifications including the four domestic animal covariates in our dataset . On accoBased on these results, we finally specified and compared the 128 models with all possible combinations of important individual-level (bednet use), household-level , and domestic animal covariates ; the complete model set is provided in R2 = 0.55 and a goodness-of-fit test χ2 = 10.21 ; the overall misclassification rate (model-predicted seropositivity status different from observed status) was just 0.19. These satisfactory fit metrics were similar for the rest of models in a posteriori addition of individual-level and domestic animal covariates removed in previous steps did neither improve the performance of any of the models with ΔAIC<2 nor change their relative positions (details not shown); therefore, we based inference on this 128-model set.The best-performing model within this set had a generalized w = 1.000; regular bednet use, w = 0.997; crop-plot ownership, w = 0.627; closed toilet/latrine, w = 0.501; solid waste disposal, w = 0.364; pig, w = 0.351; and dog, w = 0.308. βs) for each of these covariates; exp(β) values, which estimate adjusted odds ratios, are presented in When ranked according to their relative importance, the covariates considered in the final model set performed as follows: residence area, Arboviral infections are increasingly recognized as major public health threats. Geographic range expansions by dengue virus, West Nile virus, Japanese encephalitis virus, or, more recently, chikungunya virus have focused attention on their importance Here, we address this gap by presenting a case-study on MAYV; in addition to providing the first MMI-based assessment of risk factors for infection with this virus, we aimed at illustrating the caveats of conventional approaches often used to analyze observational data from cross-sectional surveys – and how MMI provides a powerful alternative. We discuss our results within the hypothetical framework outlined in the Alphavirus antibodies βOther seropositive = 0.52±0.18; odds ratio 1.68, 95% confidence interval 1.18–2.39), but adjusted covariate effect estimates were very similar to those derived from MMI . Even if some of these results are false-negatives, this suggests that pathogens other than Plasmodium are a major cause of acute febrile illness in the settlement. Our data, including the frequent mention by local residents of ‘joint pain’ as a typical feature of non-malarial febrile illness , suggest that MAYV is likely involved in generating this epidemiological scenario.We report an antibody prevalence in the middle-upper range of previous cross-sectional surveys But what factors modulate MAYV infection risk? Previous work is strongly suggestive of a pattern overtly dominated by forest transmission, with outbreaks sporadically recorded in rural communities embedded within rainforest environments Our results are in partial contrast with these findings – and, hence, partly at odds with the prevailing hypothesis of forest transmission and sporadic spillover. Notably, age- and sex-specific seroprevalence suggest that people of both sexes and all age classes were similarly exposed to MAYV in our study population of non-indigenous settlers. The youngest seropositive subject was a girl just under two years of age residing in one of the village-like clusters; seropositive <3-year-olds were living in the two village-like clusters and in two distinct upland sites. This suggests, at least for these cases, that transmission was relatively recent and geographically widespread at the scale we consider, and casts doubts on the forest-transmission-only scenario.n = 56) were excluded from the ‘best’ model in β = 0.74±0.40 vs. β = 0.54±0.33), suggesting that crop-plot work did increase risk-exposure.In addition, our data strongly suggest that residing in the more densely populated village-like clusters greatly increased MAYV infection risk, whereas living in the better-preserved, sparsely populated riverbanks was protective. This sharply contrasts with previous reports explicitly showing the opposite pattern The strong protective effect of regularly sleeping under a mosquito bednet  is also Haemagogus populations have shifted habitat and habits, adapting to densely populated landscapes and nocturnal biting. That MAYV can naturally infect Psorophora and MansoniaCulex and AedesCulex and Psorophora were the most abundant vectors in the village-like clusters of our study site, where non-native Aedes spp. have never been recorded. Haemagogus and Mansonia seem to prefer forest and crop-plot habitats; both could therefore be involved in more typical MAYV transmission cycles in forested landscapes, which would help explain why owning a crop-plot increases risk, particularly among >8-year-olds.Thus, when considered as a whole, which MMI allows us to do, the effects of residence area and bednet use, and the clear lack of effect of age and gender, all suggest the possibility that MAYV cycles other than the classical ones  might occur in our study settlement. Ongoing research examines two main hypotheses: (i) that an alternative, nocturnal, endophilic vector species is involved in transmission, and (ii) that some local We have presented the first MMI-based assessment of risk factors for MAYV infection. The results suggest that two different, possibly overlapping MAYV transmission cycles might co-occur in a typical settlement of the Amazon agricultural frontier. If transmission by synanthropic vectors is confirmed, it could ultimately increase the risk of MAYV emergence in non-forest settings – perhaps even urban or periurban. This might have serious public health consequences Table S1The complete set of 128 models; covariates, AICc and related metrics, and number of parameters are given for each model.(XLS)Click here for additional data file.Text S1STROBE statement.(PDF)Click here for additional data file.


The authors review the biometrics-based user authentication scheme proposed by An in 2012. The authors show that there exist loopholes in the scheme which are detrimental for its security. Therefore the authors propose an improved scheme eradicating the flaws of An's scheme. Then a detailed security analysis of the proposed scheme is presented followed by its efficiency comparison. The proposed scheme not only withstands security problems found in An's scheme but also provides some extra features with mere addition of only two hash operations. The proposed scheme allows user to freely change his password and also provides user anonymity with untraceability. In the last two decades, digital authentication has originated as a preferred method to authenticate remote users over insecure networks. After the first proposal of user authentication scheme by Lamport , consideIn 2010, Li and Hwang  proposedIn this paper, we review An's biometrics-based user authentication scheme. We show that An's scheme is vulnerable to the security problems to which Das's scheme is susceptible like online and offline password guessing attacks, user and server impersonation attacks, lack of mutual authentication, and lack of user anonymity. Besides, An's scheme lacks password change facility which is an important part of password-based user authentication schemes. We remove drawbacks from An's scheme by means of proposing an improved user authentication scheme. In addition, to resist various security threats, the proposed scheme incorporates features of password changing and user anonymity. The rest of this paper is arranged as follows. In Ci), the server (Si), and the registration centre (R), where R is assumed to be a trusted party. Details of each phase are given in the following subsections.The notations useful in this paper are summarized along with their description in R and the user Ci carry out this phase involving the following steps. Ci submits his identity IDi and information (PWi ⊕ Ki) containing password to R via a secure channel. Ci also submits information (Bi ⊕ Ki) containing his biometrics via the specific device to R; here Ki is a random number chosen by Ci.Rcomputes fi = h(Bi ⊕ Ki), ri = h(PWi ⊕ Ki) ⊕ fi, and ei = h(IDi||xs) ⊕ ri, where xs is a secret key generated and maintained by Si. Then R stores {IDi, fi, ei, h(·)} in a smart card SCi for user and provides it to Ci via a secure channel.i = {IDi, fi, ei, h(·)}, the user stores the random number Ki into SCi issued by R so that now SCi = {IDi, fi, ei, h(·)}.On receiving SCIn the beginning of scheme, the registration centre Ci wishes to login the server Si, the user and his smart card SCi perform the following steps.(1)Ci inserts his smart card into a card reader and inputs his biometrics information Bi on the specific device. SCi computes h(Bi ⊕ Ki) and verifies if fi = h(Bi ⊕ Ki) or not. If this biometrics information matches, Ci passes the biometrics verification.(2)Ci inputs his IDi and PWi; then SCi generates a random number Rc and computes the following equations:(3)Ci sends the login request = {IDi, M2, M3} to Si.When the user i, M2, M3} from Ci, the server Si and the user Ci perform the following steps to authenticate each other.(1)Si first checks the format of IDi. If IDi is valid, Si computes M4 = h(IDi||xs) and M5 = M2 ⊕ M4.(2)Si checks if M3 = h(M4||M5) or not. If both are equal, it generates a random number Rs and computes the following equations:Si sends the reply message = {M6, M7} for its authentication to Ci.(3)M6, M7} from Si, the user Ci computes M8 = M6 ⊕ M1 and checks if M7 = h(M1||M8) or not. If both are equal, Ci computes M9 = h(M1||Rc||M8) and sends the reply message {M9} for its authentication to Si.On receiving {(4)M9} from Ci, the server checks if M9 = h(M4||M5||Rs) or not. If both are equal, Si accepts the login request = {IDi, M2, M3} of Ci.On receiving {On receiving the request login = {IDUa can mount different types of attacks on the scheme. Independent researches by Kocher and Messerges  ⊕ h(PWa ⊕ Ki). Then Ua computes Ma2 = Ma1 ⊕ Rca and Ma3 = h(Ma1||Rca), where Rca is the random number generated by the system of Ua. He sends {IDi, Ma2, Ma3} as login request to Si. (3)Ua does not receive any response from Si then he repeats step (2) with some other guess for user's password. But if Ua receives response message from Si, then it implies that his guessed password PWa is correct.If If extracts , 29 the Ua can easily identify the login request corresponding to a smart card since both contain the identity of user. If Ua extracts  ⊕ h(PWa ⊕ Ki). (3)Ua computes Rca = M2 ⊕ Ma1 and Ma3 = h(Ma1||Rca), and finally compares Ma3 with M3. For Ma3 ≠ M3, he repeats from step (2) with some other guess for user's password. But if Ma3 = M3, then it provides Ua with the exact password PWi of Ci. In the scheme, extracts , 29 the Ua can guess a user's password if he obtains the smart card of user. It is noticeable that the successful process of password guessing (online or offline manner) also yields Ma1 = h(IDi||xs). In fact, h(IDi||xs) is the key value required to compute a valid login request or valid reply messages. Further, Ua has easy access to user's identity IDi from SCi = {IDi, fi, ei, Ki, h(·)} or from the login request = {IDi, M2, M3} of Ci. Having h(IDi||xs) and IDi in hand, Ua can impersonate the user Ci as explained below. (1)Ua generates a random number Rca in his system and computesUa sends the login request = {IDi, Ma2, Ma3} to Si. (2)i, Ma2, Ma3}, the server Si first checks the format of IDi. Clearly, Si would proceed further because IDi is the identity of a legitimate registered user and hence it is in valid format. On receiving {ID(3)Si computes M4 = h(IDi||xs) and M5 = Ma2 ⊕ M4 and checks if Ma3 = h(M4||M5); clearly it would hold. Therefore Si believes that the login request = {IDi, Ma2, Ma3} is from the legitimate user.(4)Si generates a random number Rs and computes M6 = M4 ⊕ Rs and M7 = h(M4||Rs). Then Si transmits the reply message {M6, M7}.(5)M6, M7} from Si, the attacker Ua first obtains the random number Rs by computing Ma8 = M6 ⊕ Ma1. Next, it computes Ma9 = h(Ma1||Rca||M8) and sends {Ma9} to Si.On receiving {(6)M9}, the server Si checks if M9 = h(M4||M5||Rs) or not. Clearly, this would hold, so Si will accept the login request = {IDi, Ma2, Ma3}.On receiving {As just discussed in previous subsections, Ua can easily impersonate the legal server Si to cheat the user Ci whose information {IDi  and  Ma1 = h(IDi||xs)} he possesses as described in Si the attacker proceeds in the following manner.Ua can easily recognize the login request = {IDi, M2, M3} of Ci transmitted over open channel as he possesses the identity IDi of Ci. So when Ci sends his login request = {IDi, M2, M3} to Si, the attacker Ua intercepts and blocks it from reaching Si. Ua first obtains the random number Rc by computing Ma5 = M2 ⊕ Ma1. Next, he generates a random number Rsa in his system and computes Ma6 = Ma1 ⊕ Rsa and Ma7 = h(Ma1||Rsa). Then Ua transmits the reply message {Ma6, Ma7} to Ci. Ma6, Ma7}, the user Ci first obtains the random number Rsa by computing M8 = Ma6 ⊕ M1, where M1 = h(IDi||xs). Next, he checks if Ma7 = h(M1||M8) or not. Clearly, this equivalence will hold and hence Ci will believe that he is communicating with the intended server. However, it is the clever attacker Ua who is deceiving Ci. On receiving {Ua extracts values {IDi, fi, ei, Ki, h(·)} from the smart card SCi of user Ci and successfully obtains the secret value h(IDi||xs), then he can easily craft valid login request and reply messages so as to deceive the legal user or the legal server. Therefore, the scheme loses mutual authentication feature.Like Das's scheme , the enhCi sends {IDi, M2, M3} as his login request to Si through an insecure channel. User's identity IDi is openly available if an attacker Ua intercepts the login request of Ci from the open channel. Moreover, identity IDi is also stored inside user's smart card SCi. Having IDi in hand, it is easy for Ua to craft threats against Ci. To the worst, Ua may be able to compromise user's biometrics information which would result in serious consequences. Thus, the scheme does not provide user anonymity.In An's scheme, Ci), the server (Si), and the registration centre (R). In the proposed scheme, the server maintains two secret keys xs and ys. Details of each phase along with In this section, we propose a new user authentication scheme which is an improvement of An's scheme. In addition to resist the security problems found in An's scheme, it also provides password change phase with which user can change his password at his will. It has four phases: registration phase, login phase, authentication phase and password change phase. Registration phase, and password change phase are carried over a secure channel whereas login phase and authentication phase are carried over an insecure channel. It also consists of three participants, the user (R and the user Ci carry out this phase involving the following steps. (1)Ci submits his identity IDi and information (PWi ⊕ Ki) containing password to R via a secure channel. Ci also submits information (Bi ⊕ Ki) containing his biometrics via a specific device to R; here Ki is a random number chosen by Ci.(2)R computes the following values:R stores {ci, ei, h(·)} in a smart card SCi for user. Then R provides SCi = {ci, ei, h(·)} and fi to the user Ci via a secure channel.(3)i = {ci, ei, h(·)}  &  fi], the user computes the following values:Ci inserts gi and ji into SCi issued by R so that now SCi = {ci, ei, gi, ji, h(·)}.On receiving ). Then Ci sends the reply message {M12} for its authentication to Si.On receiving {(4)M12} from Ci, the server checks if M12 = h(M8||M7||Rs) or not. If both are equal, Si accepts the login request = {M3, M4, M5} of Ci.On receiving {On receiving the request login = {i, he invokes this phase. Details of the steps required to update the smart card SCi with new password (PWi)new are as follows.(1)Ci inserts his smart card into a card reader, keys in his identity IDi, and password PWi and inputs his biometrics information Bi on the specific device. (2)i retrieves fi ← (IDi||PWi) ⊕ gi and Ki ← (IDi||PWi) ⊕ ji. It then checks if fi = h(Bi ⊕ Ki) or not. If this biometrics information matches, Ci passes the biometrics verification, otherwise terminates the session. This process also verifies the correctness of inserted IDi and PWi. Then SCi allows the user to enter the new password (PWi)new.SC(3)i computes the following equations:SC(4)i replaces ei, gi, and ji with (ei)new, (gi)new and (ji)new, respectively.SCWhen the user wishes to change his old password PWUa extracts , he requires fi. But Ua cannot obtain fi from gi = (IDi||PWi) ⊕ fi as he does not know about user's identity IDi and password PWi. The attacker Ua can obtain fi ⊕ Ki by performing gi ⊕ ji = [(IDi||PWi) ⊕ fi]⊕[(IDi||PWi) ⊕ Ki]. Next, he can computeUa cannot compute forged Ma2  ( = h(IDi||xs)) = [ei ⊕ fi ⊕ Ki] ⊕ h(PWa ⊕ Ki) using a guessed password PWa because it requires knowledge of Ki. It is troublesome for Ua to obtain Ki because Ki is not stored in plaintext inside user's smart card but is stored securely in ji = (IDi||PWi) ⊕ Ki. Further Ua cannot obtain Ki from ji without knowing IDi and password PWi. Besides, Ua cannot compute Ma1  ( = h(xs||ys)) = (ci ⊕ fi) as he does not have access to fi. Moreover, Ua does not have IDi of Ci as IDi is not stored in plaintext inside user's smart card. Thus, Ua cannot compute a login request {Ma3, Ma4, Ma5} in a way so as to guess user's password in an online manner. Hence, the proposed scheme withstands online password guessing attack.On having access to user's smart card SC extract , 29 all Ua obtains the smart card of some user. Though Ua can intercept login message of any user from open channel, he cannot relate a user's smart card with its corresponding login request. This is due to the fact that, unlike An's scheme, in the proposed scheme user's identity in plaintext is neither stored inside user's smart card nor transmitted in login request. As a result, Ua cannot combine values extracted from a user's smart card with values of corresponding login request to guess user's password in an offline manner. If we consider the situation that Ua somehow happens to get the correct combination of user's smart card and login request, we show that still Ua cannot mount offline password guessing attack. To guess password of Ci and then verify the guess, Ua can use M5 = h(M2||Rc) provided that he possesses the values {[h(IDi||xs) ⊕ h(PWi ⊕ Ki) ⊕ Ki], Ki  and  Rc} in hand. As explained in Ua can obtain [h(IDi||xs) ⊕ h(PWi ⊕ Ki) ⊕ Ki] using {gi, ji  and  ei} extracted [i, but he cannot obtain the random number Ki. Besides, Ua cannot obtain the random number Rc using M3 = M1 ⊕ Rc without having M1  ( = h(xs||ys)) and Ua fails to obtain M1  ( = h(xs||ys)) as discussed in Ua cannot guess user's password in an offline manner. Suppose xtracted , 29 fromUa should possess M1 = h(xs||ys) and M2 = h(IDi||xs); otherwise he cannot compute a valid login request {Ma3, Ma4, Ma5} or a valid reply message {Ma12}. The value h(IDi||xs) is equally important if Ua wishes to masquerade as legal server. Unlike An's scheme, in the proposed scheme Ua is not able to obtain M2  ( = M8) = h(IDi||xs) while making attempts of guessing user's password. This is due to the fact that password guessing is not feasible as explained in Sections Ua cannot obtain M1 = h(xs||ys) (i) from M3 = M1 ⊕ Rc obtained by intercepting the login request of Ci because of not having random number Rc and (ii) from ci = h(xs||ys) ⊕ fi extracted from user's smart card without knowing fi. Thus, the proposed scheme resists impersonation attacks. To impersonate a legal user, Ua has many hurdles before him to act as a legal user or a legal server: (i) the secret keys xs and ys maintained by the server are unknown for Ua and (ii) Ua has no access to the identity IDi of user Ci. As a result, Ua cannot compute h(xs||ys) and h(IDi||xs) required to mount impersonation attacks. Besides, Ua has no method to retrieve these values either from the parameters extracted out of user's smart card or from the login request or using both. Therefore, the proposed scheme provides proper mutual authentication. The success of mutual authentication in the proposed scheme follows directly from resistance against user impersonation attack and server impersonation attack as described in i is completely out of scene; it is neither stored in user's smart card SCi nor sent in any of the login-authentication messages transmitted over insecure network. If Ua extracts [ci, ei, gi, ji, h(·)} from SCi, we explain in the following that he cannot obtain IDi of Ci. To guess IDi from gi = (IDi||PWi) ⊕ fi and from ji = (IDi||PWi) ⊕ Ki, the attacker must have the knowledge of {PWi, fi} and {PWi, Ki}, respectively. Ua cannot guess out IDi from ei = h(IDi||xs) ⊕ ri without knowing ri and xs. If Ua intercepts a login request {M3, M4, M5} or the reply message {M9, M10}/{M12}, he cannot guess out IDi using {M5, M10, M12} without the knowledge of {xs, Rc  and  Rs}. Besides, it is not feasible for Ua to retrieve IDi out of {ei, M5, M10, M12} due to one-way property of hash function. Moreover, each value {M3, M4, M5, M9, M10, M12} transmitted over insecure network is dynamic in nature by virtue of random numbers Rc and Rs which are different for each session. Thus, Ua can neither obtain user's identity IDi nor can he trace the legal user by means of observing and analyzing some fixed parameter in the login request or the reply messages. Hence, the scheme provides user anonymity as well as user untraceability.In the proposed scheme, user's plaintext identity IDextracts , 29 the i)new, the smart card verifies the correctness of identity IDi old password PWi along with verifying the biometrics information fi = h(Bi ⊕ Ki). Thus the proposed scheme provides secure and easy password changing facility.In An's scheme, once user chooses his password during registration phase, it is fixed forever as user cannot change his password at his will. Probably the author might have opined that in the presence of biometrics verification procedure there is no need of password change facility. Undoubtedly, it is very difficult to forge copy or compromise biometrics, but once compromised then biometrics cannot be changed like passwords. So we opine that if password is employed in user authentication scheme then there should be the provision to facilitate the user to freely change his password. The proposed scheme provides password changing facility with which a user can freely (without interacting with server) change his old password to a new one whenever he feels to do so. Before updating stored values with the new password (PWIn this section, we examine the proposed scheme by means of comparing its efficiency with Li-Hwang's scheme , Das's sThis paper shows that the recently proposed biometrics-based user authentication scheme by An is susceptible to many threats. Once an attacker obtains the smart card of a legal user, he can guess user's password and impersonate the user. Further, the attacker can also cheat the user by masquerading as the legal server. Consequently, the scheme fails to provide mutual authentication. Besides, the scheme also suffers from the restriction of static password. We have proposed a new scheme based on the design of An's scheme so as to fix the problems identified in An's scheme. In the proposed scheme an attacker cannot figure out the identity of user either from the smart card or by intercepting all login-authentication messages transmitted over insecure network. Analysis and comparison show improved performance of the proposed scheme.


Bacillus anthracis Incident, Kameido, Tokyo, 1993” by Hiroshi Takahashi, et al., errors occurred in the 4th paragraph under “Discussion” on page 119: mu symbols were inadvertently replaced by the letter “m.” The corrected sentences appear below:In the article “7). Kameido residents described a gelatinous substance, suggesting the suspension would be poorly dispersed and droplets would be too large to form particles <5 μm in diameter.The human respiratory infectious dose 50 (dose that will produce an infection in 50% of exposed persons) is unknown but has been estimated to be 8,000 to 10,000 spore-bearing particles <5 μm in diameter (In addition, the name of the lead author of this article is misspelled in the table of contents of this issue. In the table of contents, the article should be attributed to “H. Takahashi et al.”http://www.cdc.gov/ncidod/EID/vol10no1/03-0238.htmThe corrected article appears online at We regret any confusion these errors may have caused.


Trpv1) knockout (−/−) mice have a higher locomotor activity than their wild-type littermates (+/+). We have also found that, with age, Trpv1−/− mice become substantially heavier than Trpv1+/+ controls, thus forming a paradoxical association between locomotor hyperactivity and overweight. The present study solves this contradiction. By using two experimental paradigms, we show that aged Trpv1−/− mice have not an increased, but a decreased, locomotor activity, as compared to age-matched Trpv1+/+ controls. We also confirm that aged Trpv1−/− mice are overweight. We conclude that TRPV1 channels are involved in the regulation of both general locomotor activity and body mass in an age-dependent manner.We have recently found that, at a young age, transient receptor potential vanilloid-1 ( Trpv1−/−), we made two unexpected observations [Trpv1−/− mice expressed a higher locomotor activity in a thermogradient apparatus, especially during the light (inactive) phase, as compared to their wild-type littermates (+/+Trpv1). In this apparatus, each mouse could move freely inside a 200-cm-long chamber with a linear ambient-temperature gradient from 20 to 30°C. The magnitude of the hyperactivity of Trpv1−/− mice in this thermogradient apparatus was remarkable: at one time point during the inactive phase (3:00 P.M.), Trpv1−/− mice were moving with an average velocity of 85 cm/min (> 1.2 km/day), which was ~3 times higher than the average speed of wild-type mice at the same time of day.Recent studies have shown that transient receptor potential (TRP) channels, including the vanilloid-1 channel (TRPV1), are involved in the regulation of multiple physiological functions in health and disease . While srvations . First, Trpv1−/− mice of both sexes became heavier than their +/+Trpv1 counterparts. For example, at the age of ~36 wk, the body mass of male Trpv1−/− mice was 14% higher than that of the age-matched male controls: 49 ± 1 vs 43 ± 1 g (P < 1 × 10−4). The two heaviest mice in our colony were Trpv1−/− males: one had a body mass of 59 g at the age of 35 wk, and the other weighed 58 g at 61 wk. Even though we did not measure the amount of fat in aged Trpv1−/− mice, their phenotype was consistent with obesity. The animals had larger bellies, which suggested visceral fat accumulation, a generally accepted hallmark of aging [Trpv1−/− mice while performing surgeries for a different study , as well as for the experiments reported in the present paper.Our second unforeseen observation was that, with age, Trpv1−/− mice contradicts several studies showing that the deletion of the Trpv1 gene does not affect the locomotor activity [Trpv1−/− mice, some of them showed strong tendencies that would agree with our results [Trpv1−/− mice tended to have higher scores in several activity tests. An increased activity of Trpv1−/− mice was also observed by Marsch et al. [+/+ and Trpv1−/− animals [Trpv1−/− mice) and pharmacological tools suggest that TRPV1-mediated signals from the periphery tonically suppress general locomotor activity. Whether such suppression occurs due to a direct action on the locomotor system  or indirectly  remains to be elucidated.The finding of increased locomotor activity of young activity -8. Howev results . For exa results  found thh et al.  was unlih et al. . We hypo animals . Hence, Trpv1−/− mice to age-associated overweight — was also unexpected. It contradicted the studies by Davis et al. [Trpv1−/− mice compared to controls, when maintained on either a regular or high-fat diet. Our observations also contradicted the study by Motter and Ahern [Trpv1−/− mice were found less prone to become obese on a high-fat diet compared to wild-type controls. However, all abovementioned studies used young mice with the body mass in a 10-30 g range (on a regular diet). In this body mass range, the intergenotype difference in our study was also small, but with age (we observed the mice up to the age of 61 wk), the difference increased [Trpv1−/− mice was not reported previously, the obesity-protective role of TRPV1 channels was proposed [TRPV1 polymorphism: the subjects with the Val/Val and Val/Ile variants lost about twice as much abdominal fat as the study average, whereas Ile/Ile subjects lost none.Our second observation — the predisposition of s et al. , Rong ets et al. , and Zhas et al. , who allnd Ahern , in whicncreased . Furtherproposed , 13 and proposed  detectedproposed , 14-17, proposed , decreasproposed , and incCumulatively, the studies reviewed above, support the hypothesis that TRPV1 channels protect from aging-associated obesity.e.g., attention deficit hyperactivity disorder [Trpv1−/− mice  and five aged male +/+Trpv1 littermates . All surgeries were performed as in our recent study [Trpv1−/− mice are increased compared to their +/+Trpv1 littermates, whereas their mean daily body temperature is unchanged. This is the same phenotype that was found in young Trpv1−/− mice by Szelényi et al. [Trpv1−/− mice was suppressed ' not exaggerated ' throughout the day, as compared to +/+Trpv1controls .One paradox, however, remains unexplained. The association of locomotor hyperactivity (recorded at a younger age) with overweight (recorded at an older age) observed in our study  seems todisorder , but suci et al.  and, morTrpv1−/− and 14 +/+Trpv1, used in a different study . These data are plotted in Figure Trpv1−/− were > 12% heavier than +/+Trpv1 . The locomotor activity of these animals was measured for 3 h during the inactive phase (6:00-9:00 AM). This time period was chosen, because it corresponded to the maximum difference in the locomotor activity between aged Trpv1−/− and +/+Trpv1 mice .In addition to performing the measurements described above, we pooled together data from aged mice of both sexes, 14 Trpv1−/− mice are young, they show increased levels of activity, at least in some experimental paradigms [Trpv1−/− mice age, they become hypoactive (present observations) and overweight (compared to wild-type littermates), as has been shown in our previous study [Trpv1−/− animals are hyperactive at a young age, but then start moving less (and gain body mass) is unclear. A similar relationship, however, was reported for a group of healthy humans [Hence, the paradox of non-matching locomotor activity and body mass has now been solved. When aradigms ; at thisaradigms , 10-12. us study  and the y humans . In that


This accessory nicotinic receptor subunit alters the properties of high-affinity nicotinic receptors in layer VI pyramidal neurons in both development and adulthood. Studies investigating the consequences of genetic deletion of α5, as well as other disruptions to nicotinic receptors, find attention deficits together with altered cholinergic excitation of layer VI neurons and aberrant neuronal morphology. Nicotinic receptors in prefrontal layer VI neurons play an essential role in focusing attention under challenging circumstances. In this regard, they do not act in isolation, but rather in concert with cholinergic receptors in other parts of prefrontal circuitry. This review urges an intensification of focus on the cellular mechanisms and plasticity of prefrontal attention circuitry. Disruptions in attention are one of the greatest contributing factors to disease burden in psychiatric and neurological disorders, and enhancing attention may require different approaches in the normal and disordered prefrontal cortex.Cholinergic modulation of prefrontal cortex is essential for attention. In essence, it focuses the mind on relevant, transient stimuli in support of goal-directed behavior. The excitation of prefrontal layer VI neurons through nicotinic acetylcholine receptors optimizes local and top-down control of attention. Layer VI of prefrontal cortex is the origin of a dense feedback projection to the thalamus and is one of only a handful of brain regions that express the α5 nicotinic receptor subunit, encoded by the gene  Attention has been eloquently described as the ‘searchlight’ that focuses on relevant information in the midst of distraction in order to support goal-directed behavior . In partAcetylcholine has long been known to play a role in cognition –10. Non-nominata –26. Intrnominata . Bilaternominata , 18, 29.The importance of prefrontal cholinergic modulation was further suggested by microdialysis studies showing robust acetylcholine efflux within the prefrontal cortex during the performance of attention tasks –32, whicAcetylcholine optimizes prefrontal cortical circuitry for top-down control –41. Cortfocus thalamic and thalamocortical excitation [Layer VI corticothalamic neurons constitute the major source of excitatory afferents to the thalamus , where tcitation , in partcitation , 52–54. citation .The high percentage of layer VI neurons responding to acetylcholine  suggests+, K+, and Ca2+ ions [The neurotransmitter acetylcholine acts on two classes of receptors—the ionotropic nicotinic receptors, which are the main focus of this review, and the metabotropic muscarinic acetylcholine receptors, which are G-protein coupled. Nicotinic acetylcholine receptors are pentameric ligand-gated cation channels , 66, pera2+ ions , 67. Twoa2+ ions , 66, 68.a2+ ions , 66. Thea2+ ions , 69–72.The most widely expressed nicotinic acetylcholine receptors in the brain are the α4β2-containing receptors (α4β2*) , 73–75, 2(β2)3 and (α4)3(β2)2. In the relatively rare brain regions that express the accessory α5 nicotinic subunit, such as layer VI of prefrontal cortex [2(β2)2(α5) receptors (α4α5β2) [2+ permeability [The α4β2* receptors have high affinity for nicotinic agonists (including acetylcholine and nicotine) and desensitize slowly, on the timescale of seconds , 82–84. l cortex , these r(α4α5β2) , 85–88. (α4α5β2) , 79. How(α4α5β2) , 87, 88:(α4α5β2) , modulat(α4α5β2) , 91, inceability , 88.−/−) suggest that the vast majority of α4β2* nicotinic receptors of its layer VI neurons are affected by this subunit [Immunohistochemistry for YFP-tagged nicotinic α4 subunits in a knockin mouse suggests that high-affinity nicotinic receptors are densely expressed in layer VI of prefrontal cortex , where t subunit . As we w subunit .+, K+, and Ca2+ cations through the pore [−/− mice [−/− mice [During the performance of attention tasks, brief transients of acetylcholine are released in medial prefrontal cortex , 98. Popthe pore , 66, 83.the pore , 100, suthe pore , but the−/− mice , 59, whi+, K+, and Ca2+ ions through the nicotinic receptor pore can be examined electrophysiologically in voltage clamp  or in current clamp . Figure The nicotinic responses that result from the current carried by the flow of Nal cortex . Since cl cortex , 102, Kal cortex  were fur+ channel antagonist tetrodotoxin and to pharmacological inhibition of ionotropic and metabotropic glutamate receptors [−/−) [The excitatory nicotinic responses of layer VI pyramidal neurons are directly mediated by postsynaptic somatodendritic receptors since currents are resistant to blockade of synaptic transmission by the Naeceptors . Pharmaceceptors , 88. Theeceptors . Most cors [−/−) . Togethe−/− mice show deficits in accuracy on the 5-CSRTT when stimulus duration is brief, a condition that requires greater attentional demand, but perform normally under baseline training conditions, when stimulus duration is longer. Interestingly, equivalent deficits in attention performance in humans are highly disruptive to cognitive function [−/−) also show significant impairments on the 5-CSRTT, and these deficits can be rescued by lentiviral vector-mediated re-expression of β2-containing nicotinic receptors in the prefrontal cortex [−/− and β2−/− mice employed different training and testing approaches, which may explain subtle differences in the nature of the attention deficit observed [At the behavioral level, the α5 subunit is required for normal attention performance under challenging conditions . The fivfunction –107. Micl cortex . The 5-Cobserved , 38.−/− mice result completely from the impaired nicotinic stimulation of α4α5β2-containing nicotinic receptors within corticothalamic circuits of adult prefrontal cortex or whether the loss of this nicotinic stimulation leads to functional or structural alterations of attention circuitry. It is conceivable that plasticity in the cholinergic system might ameliorate attention deficits that might otherwise be more severe; for example, allowing α5−/− mice to perform at near-normal levels of accuracy when longer stimulus durations are used in the 5-CSRTT [The question arises whether the differences in attention performance observed in α5 5-CSRTT .−/− and β2−/−, respectively) leads to the compensatory upregulation of muscarinic acetylcholine receptor excitation [−/− mice to a greater degree than those from α5−/− mice [We have observed that cholinergic excitation of the layer VI pyramidal cells primarily involves nicotinic receptors in wild-type mice ; howevercitation . These Gcitation . A schem−/− mice . This un−/− mice .The maturation of executive function and attention requires the normal development of prefrontal cortex –110, andCholinergic innervation of the prefrontal cortex is well developed by the third week of postnatal life in rodents , 122, a −/− mice, without any further differences in overall cortical morphology [−/− mice show negligible developmental changes in nicotinic excitation [Developmental differences in nicotinic excitation and dendritic morphology coincide temporally with changes in α5 expression. The excitatory nicotinic currents of layer VI neurons exhibit a developmental profile, peaking within the first postnatal month . Nicotinrphology . Furthercitation . Thus, t−/− mice in development and adulthood. These differences are relevant to the deficits in attention performance seen in α5−/− mice in adulthood and are summarized in Table In summary, there are extensive differences between WT and α5Interestingly, there are also developmental sex differences in nicotinic excitation . PrefronAlthough nicotinic receptors located on pyramidal neurons in layer VI of the medial prefrontal cortex play a critical role in mediating attentional processes, they do not act in isolation. There are cholinergic receptors on other prefrontal neurons and on neurons in other brain regions that also contribute to attentional processing in prefrontal cortex. Relevant cholinergic receptors within prefrontal cortex itself include those on layer V neurons, on the terminals of thalamocortical projections, monoaminergic projections, and on cortical interneurons.Acetylcholine exerts layer-specific effects in the prefrontal cortex , and altNicotinic modulation of prefrontal GABAergic interneurons also likely contributes to attentional processing. Although α4β2*- and α7-containing nicotinic receptors excite only limited subpopulations of interneurons in the cerebral cortex , 147, maThe prefrontal cortex is a critical node in widespread and dynamic brain networks that sustain higher cognitive function in health and that perpetuate executive dysfunction in psychiatric illness , 156. ThA substantial body of work addresses the effects of acetylcholine on attention by manipulating endogenous levels of acetylcholine and by pharmacologically or genetically altering nicotinic acetylcholine receptors. Indeed, many genetic and pharmacological studies using both animal models and human subjects have found that nicotinic acetylcholine receptors are of particular importance for attention, as summarized in Table  5-CSRTT , 167, an 5-CSRTT –174, whi 5-CSRTT , 172. Ph 5-CSRTT –180, whe 5-CSRTT , 181. Ho 5-CSRTT  and on s 5-CSRTT , 183.The agonist nicotine is an interesting example since it is selective for nicotinic receptors and has been used in a large number of animal and human studies. Overall, the effects of nicotine in humans are far more complex and controversial, with inconsistent effects on attention performance , 185. Wh−/− mice. The low-affinity α7* nicotinic acetylcholine receptors, on the other hand, do not appear to desensitize at these concentrations [At the cellular level, nicotinic receptors are subject to desensitization; that is, they can become temporarily inactive in the continued presence of agonist, leading to a reduction in response , 84. Nicy et al.  reportedtrations .Deficits in attention have been reported in normal human aging  as well Layer VI nicotinic receptors are integral components of prefrontal attention circuitry in development and adulthood. Despite recent advances, there remains much to be understood about their effects on the maturation of the prefrontal cortex and the modulation of its neurons and networks. Fundamental questions about the regulation of nicotinic receptors in neurons of the living brain remain unanswered. An apparently large reserve of nicotinic receptors within layer VI prefrontal neurons , 92, for


However, the results were conflicting and controversial.Several studies evaluated the associations of Databases including PubMed, Embase, Web of Science, and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Data were extracted independently by two investigators. Crude odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated.TNF-α −308A/G polymorphism and pneumonia risk was observed for AA +AG vs. GG . In addition, TNF-α −308A/G polymorphism was not associated with pneumonia mortality . Furthermore, there was no association of TNF-α −238A/G polymorphism with the risk of pneumonia .Twelve case-control studies and one cohort study were included. Overall, no association between TNF-α −308A/G, −238A/G polymorphisms were not associated with pneumonia risk. Moreover, TNF-α −308A/G polymorphism did not play a role in the pneumonia mortality risk. Excessive TNF-α production contributed to lung injury in a variety of diseases TNF-α gene is located on chromosome 6, within the class III region of MHC. Several polymorphisms in the promoter region of TNF-α, such as −238A/G (rs361525), −308A/G (rs1800629), −857G/A (rs1799724), and −1031T/C (rs1799964), have been identified. These polymorphisms could alter the expression of TNF-α TNF-α polymorphisms and the risk and outcomes of pneumonia TNF-α polymorphisms were associated with an increased risk of pneumonia or higher pneumonia mortality. To our knowledge, this was the first meta-analysis of the associations between TNF-α polymorphisms and pneumonia risk and mortality.The electronic databases of PubMed, Embase, Web of Science, and China National Knowledge Infrastructure (CNKI) were searched. The following search terms were used:  and (tumor necrosis factor or TNF or tumor necrosis factor-α or TNF-α) and (polymorphism or mutation or variant). Last search was updated in January 23, 2013. Additional relevant references cited in searched articles were also retrieved. No language restrictions were applied.TNF-α polymorphisms and risk of pneumonia or pneumonia mortality, (2) using a case-control design or cohort design, (3) sufficient data for estimating an odds ratio (OR) and 95% confidence interval (CI). Studies were excluded if one of the following criteria existed: (1) not relevant to TNF-α polymorphisms, TNF-α, pneumonia, or pneumonia mortality, (2) non-clinical studies, (3) reviews or abstracts, and (4) not reported genotype frequencies or numbers. For the overlapping studies, only the one with the largest sample size was included in our study.The included studies should meet the following criteria: (1) evaluation of the Two investigators (Li and Nie) assessed the quality of each study independently. Any disagreement was resolved by consensus. The predetermined quality assessment criteria were modified from a previous review TNF-α polymorphisms, and genotype numbers in cases and controls. Two independent reviewers (Li and Nie) collected these data and any discrepancy was resolved by discussion. Authors of the included studies were contacted via E-mail if further study details were needed.The following data were collected from each study: the first author's surname, publication year, ethnicity, age group, type of pneumonia, severity of disease, outcome , sample size, genotyping method, TNF-α −308A/G, −238A/G polymorphisms and the risk of pneumonia and mortality. For this meta-analysis, we examined the dominant genetic model (AA+AG vs. GG) because data were most commonly either presented in this format or convertible to this format. Crude ORs with 95% CIs were computed to assess the strength of the associations. The statistical significance of OR was determined with Z test.Where the data from at least three similar studies were available, meta-analysis was performed. Thus, we evaluated the strength of associations between I2 statistic were used to assess the degree of heterogeneity among the studies included in the meta-analysis. A P value greater than 0.10 for the Q-test indicated a lack of heterogeneity among studies, so that the pooled OR estimate of each study was calculated by the fixed-effects model. Otherwise, the random-effects model was used. Subgroup analyses were carried out by ethnicity, type of pneumonia, and severity of disease. Sensitivity analysis was performed by excluding the low quality studies and excluding the study not in HWE. Publication bias was evaluated with the funnel plot and the linear regression asymmetry test by Egger et al. P value<0.05 was considered statistically significant. Bonferroni correction of critical P values was applied when performing a high number of comparisons. In order to avoid spurious positives, the alpha value was lowered to account for the number of comparisons being performed.Departure from Hardy-Weinberg equilibrium (HWE) in controls was tested by the Chi-square test. The Q statistic and the Our search strategy yielded 306 articles for review. TNF-α −308A/G polymorphism and pneumonia risk P = 0.07) (P = 0.0008) but not among Caucasians . Subgroup analysis was also performed by the type of pneumonia. No increased risk was found among CAP patients . In the subgroup analysis by the severity of pneumonia, significant increase of severe pneumonia risk was found among the A allele carriers . No significant association was found between TNF-α −308A/G polymorphism and non-severe pneumonia risk .Eleven studies determined the association between  = 0.07) Figure 2TNF-α −308A/G polymorphism and pneumonia mortality risk TNF-α −308A/G polymorphism and mortality with an OR of 1.96 . In the subgroup analysis by ethnicity, no association was found among Asians . Stratification by pneumonia type showed that CAP patients carrying A allele were not associated with mortality risk . Furthermore, there was no significant association between TNF-α −308A/G polymorphism and severe pneumonia mortality risk . Summary results of comparisons are listed in Seven studies identified the association between TNF-α −238A/G polymorphism and pneumonia risk P = 0.20) (Seven studies (2235 cases and 2673 controls) studied the association between  = 0.20) Figure 4In order to assess the stability of the results of the meta-analysis, sensitivity analyses were performed by omitting the low quality studies TNF-α −308A/G polymorphism and pneumonia risk, no significant between-study heterogeneity was observed in the dominant genetic model (Pheterogeneity = 0.13 and 2I = 33%). For TNF-α −308A/G polymorphism and mortality risk, there was statistically significant between-study heterogeneity in the dominant genetic model (Pheterogeneity = 0.01 and 2I = 64%). In the CAP subgroup, no significant heterogeneity was found (Pheterogeneity = 0.19 and 2I = 34%). For TNF-α −238A/G polymorphism and pneumonia risk, there was also statistically significant between-study heterogeneity (Pheterogeneity = 0.0009 and 2I = 74%). As shown in Pheterogeneity = 0.30 and 2I = 16%).For P = 0.191).Publication bias was examined by funnel plot qualitatively and estimated by Egger's test quantitatively. The shape of the funnel plot seemed almost symmetrical Figure 5TNF-α −308A/G, −238A/G polymorphisms and risk and mortality of pneumonia. We found that −308A/G polymorphism was not a risk factor for developing pneumonia in the overall study population. In the subgroup analysis, however, we noted that individuals carrying A allele had increased pneumonia risk in Asians, but not in Caucasians. This result indicated that interactions between different ethnicities and genetic variants may contribute to pneumonia risk. We also carried out subgroup analysis for type of pneumonia. No evidence supported the association between this polymorphism and risk of CAP. However, we found that patients with A allele had increased severe pneumonia risk, suggesting a possible role of −308A/G polymorphism in severe pneumonia pathogenesis. Although subgroup analyses found significant associations among Asians and patients with severe pneumonia, we should exercise great caution in interpreting apparent results. Chance is the explanation for spurious subgroup analyses. Thus, these results should be validated by future studies.This meta-analysis of 13 studies systematically evaluated the associations between A non-significant result was observed for the association of −308A/G polymorphism with pneumonia-related mortality. Teuffel and colleagues also did not find an association between this polymorphism and mortality from sepsis in their meta-analysis TNF-α −238A/G polymorphism may play a possible role in the pneumonia-related mortality. However, there were only two studies estimated this association and meta-analysis could not be conducted. This issue is needed to be further studied.Results from our meta-analysis showed the lack of association between −238A/G polymorphism and pneumonia risk. Subgroup analyses also did not detect significant association between this polymorphism and pneumonia risk. These results suggested that −238A/G polymorphism might have no role in the etiology of pneumonia. However, the study from Pappachan et al. Escherichia coli growth both in vitro and in vivo. In addition, systemic administration of TNF-α in rat worsened pneumonia by reducing alveolar neutrophil recruitment and bacterial clearance TNF-α gene could influence the expression of TNF-α Pneumonia is a complex inflammatory disease. Moderate degree of local inflammation is required to control infection; however, excessive release of inflammatory cytokines favours intracellular and extracellular bacterial growth and organ dysfunction TNF-α −238A showed a significantly decreased transcriptional activity. Furthermore, Huizinga and coworkers revealed that the multiple sclerosis patients with −238 GA produced lower TNF-α compared to those with −238 GG Kaluza et al. We should address the importance of heterogeneity and publication bias, which might influence the results of meta-analysis. In our meta-analysis, significant heterogeneity was observed in −308A/G and mortality risk and also in −238A/G and pneumonia risk. Subgroup analysis was used to explore the sources of heterogeneity. After subgroup analysis by the the type of pneumonia, the heterogeneity was effectively decreased in the CAP subgroup. Therefore, it could be speculated that the large heterogeneity mainly resulted from the type of pneumonia. In addition, funnel plot and Egger's test were performed to find potential publication bias. No significant publication bias was noted. Furthermore, we carried out the sensitivity analysis. Removal of the low quality studies or the HWE-violating study did not change the conclusions of meta-analysis. These procedures suggested that our results were stable and robust.Several limitations of this meta-analysis should be considered when interpreting results. First, the number of included studies was moderate. It was possible that some relevant published studies or unpublished studies with negative results were missed. Second, most of the studies were conducted in Caucasian and Asian populations. Therefore, our results may be applicable only to these ethnic groups. Third, because insufficient data could be extracted from the primary publications, we could not assess the effects of +488A/G, −1031T/C, −863C/A, −857C/T, and −376G/A polymorphisms on pneumonia risk and mortality risk. Fourth, the overall outcome was based on unadjusted data, whereas a more precise analysis could be performed if individual data were available to allow adjustment. Finally, pneumonia is a complex disease, and many genes are related to pneumonia TNF-α −308A/G polymorphism and −238A/G polymorphism were not associated with the risk of pneumonia in the overall population. In addition, TNF-α −308A/G polymorphism was not associated with pneumonia mortality. Future large sample size studies with more ethnic groups are needed to confirm our findings. Moreover, other TNF-α polymorphisms and gene-gene interactions should also be considered in future studies.In conclusion, our results suggested that


Vocal learning is a central functional constituent of human speech, andrecent studies showing that adult male mice emit ultrasonic sound sequencescharacterized as “songs” have suggested that the ultrasoniccourtship sounds of mice provide a mammalian model of vocal learning.We tested whether mouse songs are learned, by examining the relative role ofrearing environment in a cross-fostering experiment.We found that C57BL/6 and BALB/c males emit a clearly different pattern ofsongs with different frequency and syllable compositions; C57BL/6 malesshowed a higher peak frequency of syllables, shorter intervals betweensyllables, and more upward frequency modulations with jumps, whereas BALB/cmales produced more “chevron” and “harmonics”syllables. To establish the degree of environmental influences in mouse songdevelopment, sons of these two strains were cross-fostered to another strainof parents. Songs were recorded when these cross-fostered pups were fullydeveloped and their songs were compared with those of male mice reared bythe genetic parents. The cross-fostered animals sang songs with acousticcharacteristics - including syllable interval, peak frequency, andmodulation patterns - similar to those of their genetic parents. In additiontheir song elements retained sequential characteristics similar to those oftheir genetic parents' songs.These results do not support the hypothesis that mouse “song” islearned; we found no evidence for vocal learning of any sort under theconditions of this experiment. Our observation that the strain-specificcharacter of the song profile persisted even after changing thedevelopmental auditory environment suggests that the structure of thesecourtship sound sequences is under strong genetic control. Thus, theusefulness of mouse “song” as a model of mammalian vocallearning is limited, but mouse song has the potential to be an indispensablemodel to study genetic mechanisms for vocal patterning and behavioralsequences. Many animals, including humans, use vocal signals to communicate with conspecifics.Song is a long, complex vocalization of several acoustic elements arranged inspecific sequences Mus musculus, is a genetically and neurochemicallywell-described mammalian organism. Mice emit ultrasonic vocalizations withfrequencies higher than 30 kHz, which is far beyond the human audible range The mouse, The B6D2F1strain of male mice showed individual differences in syllable usage and thetemporal structure of their songs as reported by Holy and Guo To elucidate genetic and environmental effects on mouse songs, we conducted across-fostering study to understand the effects of the social experience during thejuvenile developmental period on song development. First, we compared 2 strains ofinbred C57BL/6 and BALB/c males and found that these 2 strains of male mice emitteda different pattern of songs with regard to frequency, inter-syllable intervals, andsyllable composition. C57BL/6 males showed a higher peak frequency of syllables andmore frequency-modulated syllables with 1 or multiple jumps and short- and upwardsyllables, whereas BALB/c males produced more chevron-, flat-, andharmonics-syllables. None of these strain-specific parameters were affected bycross-fostering. Therefore, developmental social environments appear to have nosignificant role in adult male songs of mice. In other words, mouse songs do notseem to involve imitative learning.When a male subject encountered a female, he emitted complex ultrasounds.Sound spectrograms demonstrated that B6 males showed a peak at 70–80kHz, and BALB males at 50–60 kHz  and S2. According to previous studies The sequential patterns of B6 and BALB mice songs are shown in Sound spectrograms demonstrated that B6-sons and B6-foster males showed apeak at 70–80 kHz, whereas BALB mice showed a peak at 50–60 kHz andS6.We compared songs between fostered groups, and found that the main straindifferences we quantified were not affected by fostering. BALBcross-fostered males still showed a lower peak frequency = 106.5, p<0.0001) and longerinter-syllable intervals  = 9.67, p<0.01)than B6-fostered males . The sylMANOVA revealed a significant effect of strain = 25.9, p<0.0001), but not of fostering = 0.91, p = 0.55) or aninteraction of strain and fostering  = 0.41,p = 0.89). Regardless of fostering experience, B6 miceproduced more “short,” “one jump,” and “morejumps” syllables than BALB mice , p<0.05.Regardless of fostering, B6 and BALB mice showed distinct transitionalpatterns of the song syllables, and these characteristics were displayed bycross-fostered males. MANOVA revealed a strain difference = 24.6, p<0.0001), but no fostering effect = 0.655, p = 0.687) andno interaction between these  = 1.56,p = 0.241). A Bonferonni post hoc test revealed thatsons of BALB mice showed a greater occurrence of B to B self-transitions, Bto Z and Z to B transitions as well as a lower occurrence of A to Aself-transitions, A to B, B to A, A to Z, and Z to A transitions compared tosons of B6 and B6-foster male mice p<0.05, . BALB-foIn the present study, we revealed that B6 and BALB male mice showed distinct patternsand sound profiles of songs when encountering a female. Our syllable categories aresimilar to those reported in earlier studies Studies of the natural history of mice have demonstrated that a pair of male andfemale mice lives in a nest together with their juveniles Several studies have demonstrated that female mice show attraction to male songsRecent studies have demonstrated that ultrasonic vocalization of mouse pups isaffected by genes related to neuropsychiatric disorders such as Autism Here we showed that imitative vocal learning is not involved in the strainspecificity of mouse songs. Vocal learning requires two independent processes.First, the animal must have voluntary control over the vocal output. Second, theanimal should be able to match its vocal output with the externally acquiredauditory memory. For the first process, the existence of the direct motor pathwayconnecting the oro-facial motor cortex and the medullar phonatory and respiratoryareas, including the nucleus ambiguus, has been suggested as an anatomical substrateresponsible for vocal plasticity Arriaga et al. reported singing-related gene expression in mice cingulated, motorcortex and basal ganglia Our results show that the auditory environment does not affect song phenotypes inmice, and, thus, vocal learning does not appear to be involved in mouse songs.Nevertheless, mouse song is a very complex behavior, with at least 10 categoriesof vocal tokens and complex note-to-note transition rules. Even if thisphenotype is largely controlled by genetic factors and only limited learning isinvolved, we can still pose interesting questions regarding the genetic encodingof acoustic categories and the neural mechanisms involved in sequencegeneration. Thus, the mouse song should remain an important model in which tostudy the biological basis of complex communicative behavior, including spokenhuman language.BALB/cAJcl (BALB) and C57BL/6JJcl (B6) mice were originally obtained from JapanClea Co. Ltd.  and bred in our laboratory. Food andwater were given ad libitum, and all the animals were kept at a constanttemperature (23±1°C) and humidity (40%±10%)under a 12-h light:dark cycle (light on at 0600). All experiments were conductedin accordance with the guideline of the "Policies Governing The Use of LiveVertebrate Animals" by Azabu University, and were approved by The EthicalCommittee for Vertebrate Experiments (ID# 070418).A male and a female mouse of the same strain were pair-housed in a cage (17.5 cm× 24.5 cm × 12.5 cm) for breeding. When the female was pregnant,delivery was examined every 6–8 hours. When newly born pups were found atthe same time in both strains of parents, a part of the litter was reciprocallycross-fostered to parents of the other strain of mice (B6-foster andBALB-foster). The control mice were handled in the same manner as fostered pupsbut returned to their own parents (B6-son and BALB-son). All litters were leftundisturbed until weaning  21). After PD21, they were housedwith males of the non-cross fostered controls of the different strain untilultrasound recording at 10–20 weeks of age . The numAll experiments were carried out in a soundproof chamber  under a red dim light, from 1300 to 1700 hours. Ultrasonic sounds weredetected using a condenser microphone  designed for recordings between 10 and 200 kHz.The microphone was connected to an A/D converter  with a sampling rate of 300 kHz and acousticsignals were transmitted to a sound analysis system . During the recording, a subject male mouse wasindividually housed in a test cage (12.5 cm × 20.0 cm × 11.0 cm) andkept there for at least 2 h for habituation. The test cage was placed in thesoundproof chamber, and a female mouse, devocalized by unilateral sectioning ofthe inferior laryngeal nerve Spectrograms were generated with an FFT-length of 1024 points and a time-windowoverlap of 75% . The spectrogram wasproduced at a frequency resolution of 488 Hz and a time resolution of 1 ms. Alower cut-off frequency of 20 kHz was used to reduce background noise outsidethe relevant frequency band. Parameters analyzed for each subject included thenumber of syllables, duration of syllables, and qualitative and quantitativeanalyses of sound frequencies measured in terms of frequency at the maximum ofthe spectrum.F = 0.13,p = 0.72). The occurrence of eachsyllable was compared between groups using MANOVA, followed by a Bonferonnipost-hoc test.Waveform patterns of calls collected from every group  were analyzed in detail. Eachsyllable was identified as 1 of 10 distinct categories, based on internal pitchchange, length, and shape, according to previously reported categories withminor modifications  [14]. Thhttp://sites.google.com/site/eurekawiki/). The occurrence ofeach transition type was compared between groups using MANOVA, followed by aBonferonni post-hoc test.The prevalence of a syllable type was defined as follows on the basis of aprevious study Figure S1Song syllable characteristics. Ten categories were defined asfollows. Upward: duration of 5–50 ms, frequency increaseof more than 5kHz from starting point to end. Downward: duration of 5–50 ms,frequency decrease of more than 5 kHz from starting point to end. Flat:duration of 5–35 ms, frequency difference of less than 5 kHz betweenstarting point and end. Short: duration of less than 5 ms, frequencydifference of less than 5 kHz between starting point and end. Chevron:duration of 15–80 ms, frequency increase of more than 5 kHz fromstarting point to frequency peak and frequency increase or decrease of morethan 5 kHz from frequency peak to end . Wave:duration of 15–100 ms, frequency increase or decrease of more than 5kHz from starting point to the first frequency peak (or bottom) andcontaining 1 frequency peak and 1 frequency bottom . Complex: duration of 30–150 ms, frequency increase ordecrease of more than 5 kHz from starting point to the first frequency peak(or bottom) and containing more than 3 frequency peaks and/or frequencybottoms that differ from each other by more than 5 kHz in frequency . One jump: duration of 10–50 ms andcontaining 1 frequency gap . More jumps: duration of 15–100 ms andcontaining more than 2 frequency gaps . Harmonics:duration of 10–100 ms and containing more than 2 Chevron, Wave,Complex, One jump, or More jumps syllables in parallel with a main syllablethat has the highest dB count.(TIF)Click here for additional data file.Figure S2Sequential analysis of syllable types in B6 and BALB mice. Thesequential analyses of 10 categories of syllables demonstrated a verycomplicated transition both in B6 (upper) and BALB (lower) mice. a: upward,b: downward, c: flat, d: short, e: chevron, f: wave, g: complex, h: onejump, i: more jumps, j: harmonics, Z: gap.(TIF)Click here for additional data file.Audio S1B6 male song.(WAV)Click here for additional data file.Audio S2BALB male song.(WAV)Click here for additional data file.Audio S3B6-son male song.(WAV)Click here for additional data file.Audio S4B6-foster male song.(WAV)Click here for additional data file.Audio S5BALB-son male song.(WAV)Click here for additional data file.Audio S6BALB-foster male song.(WAV)Click here for additional data file.