The brain can learn and detect mixed input signals masked by various types of noise, and spike-timing-dependent plasticity (STDP) is the candidate synaptic level mechanism. Because sensory inputs typically have spike correlation, and local circuits have dense feedback connections, input spikes cause the propagation of spike correlation in lateral circuits; however, it is largely unknown how this secondary correlation generated by lateral circuits influences learning processes through STDP, or whether it is beneficial to achieve efficient spike-based learning from uncertain stimuli. To explore the answers to these questions, we construct models of feedforward networks with lateral inhibitory circuits and study how propagated correlation influences STDP learning, and what kind of learning algorithm such circuits achieve. We derive analytical conditions at which neurons detect minor signals with STDP, and show that depending on the origin of the noise, different correlation timescales are useful for learning. In particular, we show that non-precise spike correlation is beneficial for learning in the presence of cross-talk noise. We also show that by considering excitatory and inhibitory STDP at lateral connections, the circuit can acquire a lateral structure optimal for signal detection. In addition, we demonstrate that the model performs blind source separation in a manner similar to the sequential sampling approximation of the Bayesian independent component analysis algorithm. Our results provide a basic understanding of STDP learning in feedback circuits by integrating analyses from both dynamical systems and information theory. In natural environments, although sensory inputs are often highly mixed with one another and obscured by noise, animals can detect and learn discrete signals from this mixture. For example, humans easily detect the mention of their names from across a noisy room, a phenomenon known as the cocktail party effect. Spike-timing-dependent plasticity (STDP) is a learning mechanism ubiquitously observed in the brain across various species and is considered to be the neural basis of such learning; however, it is still unclear how STDP enables efficient learning from uncertain stimuli and whether spike-based learning offers benefits beyond those provided by standard machine learning methods for signal decomposition. To begin to answer these questions, we conducted analytical and simulation studies examining the propagation of spike correlation in feedback neural circuits. We show that non-precise spike correlation is useful for handling noise during the learning process. Our results also suggest that neural circuits make use of stochastic membrane dynamics to approximate computationally complex Bayesian learning algorithms, progressing our understanding of the principles of stochastic computation by the brain. Neurons receive inputs from a large number of other neurons encoding a variety of information about various signals. Despite the diversity and variability of input spike trains, neurons can learn and represent specific information during developmental processes and according to specific task requirements. Spike-timing-dependent plasticity (STDP) ,2 is a cAmong neural interactions, lateral inhibition is a basic interaction widely observed in various regions, such as the olfactory bulb , visual Here, by considering a simple feedback network model of spiking neurons, we investigated the algorithm inherent to STDP in neural circuits containing feedback. We analyzed the propagation of spike correlations through inhibitory circuits, and revealed how such secondary correlations influence STDP learning at both feedforward and feedback connections. We discovered that the timescale of spike correlation preferable for learning depends on whether the noise is independent from any signal (random noise) or generated from the mixing of signals . We also found that excitatory and inhibitory STDP cooperatively shapes lateral circuit structure, making it suitable for signal detection. We further found a possible link between stochastic membrane dynamics and sampling process, which is necessary for neural approximation of learning algorithm of Bayesian independent component analysis (ICA). We applied our findings by demonstrating that STDP implements a spike-based solution in neural circuits for the cocktail party problem ,28,29.rio}. Subsequently, spike activity at the input layer projects to the output layer, which also receives inhibitory feedback from the lateral layer. Neurons in the lateral layers are excited by inputs from the output layer. We assumed that all neurons in the input layer and the output layer are excitatory, whereas lateral-layer neurons are assumed to be inhibitory. Although excitatory lateral interactions also exist in the sensory cortex, they are typically sparse [We constructed a network model with three feedforward layers as shown in y sparse  and weaky sparse  comparedy sparse , which ry sparse ,33.υoX (See Table i is given as sμ(t) and the response probability of the neuron qiμ, then common inputs from the external layer induce a temporal correlation proportional to(t) is a response kernel  and fp(w) are synaptic weight dependence of LTD/LTP (long-term depression/potentiation), respectively. By taking the average of above equation over time and ensemble  = Fd if s<0, else F = Fp, and εX is the EPSP curve of input neurons (see Eqs (G1X(w), we can separate the effect of the intrinsic network property χX1 and that of the input correlation h(τ) for STDP-based learning. Due to causality, LTP/LTD balance, and dendritic delay, w = woX), so that g1X takes positive values, which enables coincidence-based learning [D = 2dXd+dY+dZ, and εY and εZ are EPSP/IPSP curves of output/inhibitory neurons, respectively. This term primarily causes LTD as the sign flips through lateral inhibition υoX  for forυoX(see Eqs  and 31)υoX (Selearning . The secg2X/g1X = 0), all output neurons acquire the principal component of the response probability matrix Q, and the other information is neglected [qA = 0.6, qB = 0.5), so that input neurons projected by source A show higher correlations (cA = 0.36) than those receiving the output of source B (cB = 0.25). In the matrix form,t). We refer to this as the minor source detection task below. Here, for lateral connections, we assumed that both excitatory-to-inhibitory (E-to-I) and inhibitory-to-excitatory (I-to-E) connections are well organized such that inhibition only works mutually between two output neuron groups  shapes, and correlation structure, as shown in Eqs ..dwμνXdt≅μ = A or B. As a result, we found that when the mutual inhibition is weak (wI = 10), the system has only one stable point at which w1A is larger than w1B , which means that both output neuron groups are specialized for the major source A ; however, if the inhibition is moderately strong (wI = 21.5), two new stable fixed points and two unstable fixed points appear in the system . On the right-hand side, neuron group 1 selects source A while neuron group 2 selects source B . At the stable point in the middle, both groups detect source A . Note that because of the mutual inhibition, the synaptic weight from A-neuron is smaller when both groups learn A than it is when only group 1 learns A. For strong inhibition (wI = 40.0), the stable point in the middle disappears, and the system is stable only when two neuron groups detect different sources , only the S-states are stable, whereas T-states also become stable for slower IPSPs , the minor component is no longer detectable. On the other hand, at θt = 2.0, the minor component is detectable even if the strength of the induced correlation is less than half . At θt = 4.0, g1X becomes smaller so that even the major signal is not fully detectable.In the previous section, we revealed the effects of network properties for a fixed input correlation structure; however, actual neurons show various timescales for correlations depending on the brain region ,42 and c/θt/2θt3  left, anes of θt . Becauseimescale . This meimescale . When θtqS is the response probability to the preferred signal and qN is that to the non-preferred signal (qN = 0 and changed the spontaneous firing rate of the input neurons (rio) to modify the noise intensity, whereas in simulation of crosstalk noise we removed random noise  and changed qN. For random noise, a smaller θt enables better learning because a large g1X competes with the homeostatic force  is typically smaller for this kernel than for the kernel we used throughout this study . In other cases, neurons are often organized into two groups with different population sizes. In such cases, for evaluating performance, we measured average weights from source A on the output neurons receiving stronger inputs from A-neurons than from B-neurons or Background-neurons. For randomly connected lateral inhibition, learning performance dropped significantly in noisy source detection , that network outperforms the network with random lateral connections in both noisy source detection  withgZ1 is negative and ϕZ calculated from the simulation data tends to be large  is the sample based approximation of pik in the previous equation. This rule has spike-timing and weight dependence similar to those seen in STDP  is the spectrum of auditory source q (left panel of aql(t) is the temporal change of the sound pressure (black lines in aqh(f)}q, blind separation is possible as long as {aql(t)}q are independent and have sharp rising profiles sufficient to cause spike correlations. After learning, four output neuron groups successfully detected changes in the sound pressure of the four original auditory signals  ,37. We aSimultaneously recorded neurons in close proximity often show correlated spiking, yet the precision of these correlations varies across brain regions. Neurons in the lateral geniculate nucleus show strong spike correlations ,51, whilIt is known that both glutaminergic synapses on inhibitory neurons ,54 and GRecently, inhibitory neurons in the rodent hippocampus CA1 were shown to display context-dependent activity rate changes during a spatial learning task, in association with the activity rate changes in excitatory cells . In addiOur results indicated that STDP in a lateral inhibition circuit mimicked Bayesian ICA ,47. Firse matrix ,65. ThisRecently, it was shown that STDP can perform Bayesian optimal learning ,67. In tPrevious theoretical results suggest that STDP can modulate synaptic weights in a way that optimizes information transmission between pre- and postsynaptic neurons ,72. In tHumans and nonhuman animals can detect a specific auditory sequence from a mixed, noisy auditory stimulus, a phenomenon often called the cocktail party effect. The mechanism underlying the cocktail party effect remains elusive ,28,29, aνSo. If we define the Poisson process with rate r as μ at time t is written as i followsrio is the instantaneous firing rate defined with qiμ is the response probability for the hidden external source μ, and t is several milliseconds  = u and gI(u) = u.Output neurons are modeled with the Poisson neuron model ,38,45 inWX, with fixed lateral synaptic weights WY and WZ. When the timing of the spikes at the cell bodies of pre- and postsynaptic neurons is tpre and tpost, spike timings at the synaptic sites are daji and ddji. For every pair of tspre and tspost, synaptic weight change is given withFor most of this study, we focused on synaptic plasticity in the feedforward connection fp(w) = Cp and fd(w) = Cd), the mean-field equation typically does not have any stable fixed point. In addition, under the multiplicative STDP in which LTD has a linear rather than a logarithmic dependence on synaptic weight, strong correlation is often necessary to induce salient LTP [Cp = 1 and For the synaptic weight dependence of STDP, we considered a pairwise log-STDP  in whichibutions ,33 and iibutions . Analytiient LTP . The coewoY = 50.0 and woZ = 25.0, and bounded synaptic weights with wYmax = 100.0 and wZmax = 50.0. In this normalization, the total lateral inhibition takes the same value as that in the non-plastic model at the initial state. Time windows are defined as τpY = τdY = τpZ = τdZ = 20.0 ms.The STDP at E-to-I connections and I-to-E connections is similarly defined. For simplicity, we assume that synaptic delays are solely axonal , and if qiν = 0 for all ν (≠μ), then LTP at the connection qiμ caused by an output spike yk-k’μ = 1 for xik = 1 is written asIf yik = 0), an output spike yk-k’μ = 1 causes LTD in total In the absence of the input spike  ≡ 〈xi(t)xl(t-s)〉-〈xi(t)〉〈xl(t)〉, and cross-correlation among input neurons satisfiesCil(s) becomesBecause input neurons receive common inputs from external sources, we define cross-correlation among input neurons as The synaptic weight dynamics defined above can be rewritten asxi and post-spikes yj, synaptic weight dynamics can be analytically estimated. Because the spike probability linearly depends on the membrane potential in our model, cross-correlation followsTherefore, by calculating the cross-correlation between pre-spikes Since we define cross-correlation among input neurons asThis result is consistent with that in previous studies ,38,45. TdZ+dY and EPSP/IPSP rise times is accumulated so that the effect on correlation rapidly becomes small, especially when the original input cross-correlation C(t) is narrow; however, even if C(t) is broad, the effect for learning is bounded by the STDP time window. Therefore, higher order terms practically influence weight dynamics only through firing rates, so that by applying the approximationνnZ is not analytically calculable, but by considering the balanced condition, it can be estimated. Therefore, the second term is given asX1 and ΓX2, whereas the remainders are homeostatic terms. In all terms, synaptic weight dependence is primarily caused by wXji and not by other synapses.Theoretically, expansion over a lateral connection should be performed infinite times to obtain the exact solution, but at each expansion, the delay caused by synaptic delay X1 and ΓX2 can be rewritten ast″ = r+q+r′-s+2dxd+dz+dY. Note that G1X and G2X do not depend on any indexes of the neurons, except for synaptic weight dependency, and so the two values are considered basic constants that decide how correlation shapes learning.By inserting the explicit representation of correlation into the equation above, ΓA.B)ij = AijBij. Especially if we approximate G1X and G2X with g1X ≡ G1X(woX) and g2X ≡ G2X(woX) (or if weight dependence is negligible as in additive-STDP),If we ignore the homeostatic term, then the synaptic weight dynamic is written in the matrix form as χ1X was derived fromχ2X was calculated in a similar way.The correlation kernel C(s) is simply organized, further analytical consideration is possible. In the two-source model shown in If the correlation structure Therefore, the original L × M differential equations can be reduced into 2 × 2 equations of representative neurons asThe firing rates of inhibitory neurons can be approximated as1Z and ν2Z,Therefore, by solving the simultaneous equations for νThis analytical approach is applicable only when the synaptic weight change is sufficiently slow relative to the neural dynamics. Also, because we ignored the variance in the synaptic weights, numerically the accuracy is limited.gY2 and gY3 are negligibly smaller than gY1, because trajectories (ii) and (iii) are secondary correlations and also contain synaptic delays. In this approximation, we additionally assume thatUsing a similar calculation as above, synaptic weight development of the lateral connections is given asdiagrams . In the gY1 is positive, the E-to-I connections are more likely to be structured in a way that the inhibitory neurons become feature selective. On the other hand, if that value is negative, such structure may not be obtained. Note that  is not the principal eigenvector in this simple analysis, because the eigensystem of the matrix is {{AL+BL, AL+BL, AL-BL, AL-BL}; {1, 1, 0, 0}, {0, 0, 1, 1}, {1, -1, 0, 0}, {0, 0, 1, -1}}.Therefore, Similarly, for inhibitory connectionsg2Z = 0, then the synaptic weight change follows g1Z is positive, reciprocal connections are enhanced (or inhibitory connections to the neurons coding a similar feature are enhanced), whereas for negative g1I, inhibitory connections develop non-reciprocally .We approximated with only two terms because the third term is negligible . If we ag1Y and g1Z. We have restricted our consideration to Hebbian STDP, but the properties of STDP on E-to-I and I-to-E connections are still debatable ,59. AlthΔt = 10 ms, the activity of source μ is defined as ν is νY is a set of output neurons coding a source ν. For these, cross-correlation is defined asTc = 10 ms for the analysis. Correspondence between sources and output groups are arbitrary, and so the learned correlation should be given as p! number of combinations with function Ψ between sources and output groups. For example, when p = 2,We evaluated the performance by measuring the mean cross-correlation between the external sources and population activity of the output neurons. For time bin j as belonging to ΩμY ifth = 1.5, and the cross-correlation was calculated based on ΩμY.For the models with randomly connected lateral inhibition and (e+i) STDP, we defined output neuron sμk, yνk, we defined the binary variablesk can be defined as tof takes 1 if X is true, otherwise it takes 0, for the statement X. Therefore, mutual information can be defined asBased on the discretized hidden external source/output neuron activity S1 Fig(A) Synaptic weight developments at the feedforward connection. (B) Cross-correlation and mutual information calculated for various delays. Both values were calculated by averaging five independent simulation results. (C) Development of two values for the simulation shown in (A). (D) PSTH of the membrane potential calculated for gray areas in (A). (E) Peristimulus time histogram (PSTH) of the firing probability for the same simulation.(EPS)Click here for additional data file.S2 Fig Synaptic weight development when the number of external inputs is three (A) and four (B). Thick lines represent averages over all synapses, and thin lines represent individual synaptic weights. Colors represent detected sources for output neurons (left) and inhibitory neurons (middle right). (C) Relationship between the number of inhibitory neurons and the lateral structure. (D) Propagation of structure. i to iii correspond to lateral excitatory connections, and iv to vi correspond to feedback inhibitory connections. (E) Analytic results for various types of STDP.(EPS)Click here for additional data file.S3 Figt. Note that in t = 0.5, 2.0, 4.0 ms, while here we used θt = 1.0, 3.0, 5.0ms.  The correlation kernels ge1X, ge2X (B) and their ratio ge1X/ge2X (C) are shown for the kernels ge1X and ge2X that were calculated from (A) Cross-correlations among input neurons responding to the same source calculated from simulated data for three different correlation timescale parameters θ(EPS)Click here for additional data file.S1 Auditory File0’00”–0’43”: Independent auditory signals (10 s each); 0’44”–0’54”: Mixed auditory signal; 0’55”–1’38”: Decoded independent signals.(MP3)Click here for additional data file.


Up to 150 million North Americans currently use a groundwater system as their principal drinking water source. These systems are a potential source of exposure to enteric pathogens, contributing to the burden of waterborne disease. Waterborne disease outbreaks have been associated with US and Canadian groundwater systems over the past two decades. However, to date, this literature has not been reviewed in a comprehensive manner.A combined review and pooled-analysis approach was used to investigate groundwater contamination in Canada and the US from 1990 to 2013; fifty-five studies met eligibility criteria. Four study types were identified. It was found that study location affects study design, sample rate and studied pathogen category. Approximately 15% (316/2210) of samples from Canadian and US groundwater sources were positive for enteric pathogens, with no difference observed based on system type. Knowledge gaps exist, particularly in exposure assessment for attributing disease to groundwater supplies. Furthermore, there is a lack of consistency in risk factor reporting . The widespread use of fecal indicator organisms in reported studies does not inform the assessment of human health risks associated with groundwater supplies.This review illustrates how groundwater study design and location are critical for subsequent data interpretation and use. Knowledge gaps exist related to data on bacterial, viral and protozoan pathogen prevalence in Canadian and US groundwater systems, as well as a need for standardized approaches for reporting study design and results. Fecal indicators are examined as a surrogate for health risk assessments; caution is advised in their widespread use. Study findings may be useful during suspected waterborne outbreaks linked with a groundwater supply to identify the likely etiological agent and potential transport pathway. The diversity of North America’s hydrology, and by extension, hydrogeology, is a reflection of its bio-physical diversity including extremely variable physiography, climate, bedrock geology and quaternary geology. Accordingly, many regional hydrological issues exist related to microbial groundwater contamination Canada is comprised of nine hydrogeological regions, ranging from extensive areas of permafrost in the north to Cordillera, the Western Canadian sedimentary basin and the St Lawrence platform in the south Groundwater contamination occurs as a direct result of human or animal waste ingress to groundwater resources (aquifers or wells). Enteric pathogens are prevalent in both developing and developed countries, including the United States and Canada Campylobacter infection per irrigation season et al. Escherichia coli O157:H7 infection in the US Waterborne illness is a public health issue in North America and around the world. Sensitive sub-populations including the young, the elderly, pregnant women, and the immune-compromised are particularly susceptible to enteric infections Estimating Waterborne Disease Risks in the United States’ Craun et al. In their summation of a 2005 workshop entitled ‘The research question that guided the review was:What has been the prevalence of contamination by enteric pathogens and fecal indicator organisms in Canadian and US groundwater systems during the period 1990-present?A review protocol was developed in order to capture existing, published references to contamination events in Canadian and US groundwater systems . The revStudy screening 2 (citation abstract) was based upon explicit inclusion/exclusion criteria . No studStudy screening 3 was completed by two review teams who independently assessed each publication , based upon explicit eligibility criteria . ArticleRelevant data were extracted to a spreadsheet (MS Office Excel 2007) and verified. Three primary data fields were extracted and exported, including:Citation-specific data: Title, Author(s), Publication Year, Publication Type, Study Country.Groundwater contamination data: Fecal Indicator Organism (FIO) Data , Pathogen Data , system type , Detection Method, Outbreak Association, Sampling Period/Season.“Pathway” data: Livestock/Agriculture Associated, Wild Animal Associated, Septic Tank/Sewage Associated, Precipitation/Climate Associated, Geology Associated, Source Associated, Local (Hydro)Geological Characteristics.E. coli O157 and Cryptosporidium spp. within one study, two records were extracted. Study design was classified into four categories, including: repeat sampling (sources sampled more than once over extended time period), “snapshot” sampling (sources sampled on a “one off” basis), outbreak investigations and laboratory-based studies . Data pertaining to pathogen detection method were categorised into three groups, molecular diagnostics, microscopy and culture-based techniques. System type  was categorized based on explicit classification in included studies. Finally, pathogens were classified on the basis of being either potentially zoonotic or non-zoonotic, based on current CDC classification.Pathogen records were extracted from all included studies, with each record representing analysis for one pathogen type or strain . For example, if groundwater samples were analysed for both p<0.05) were not systematically dispersed among categories. Two-tailed Spearman’s’ rho was used to investigate correlation between continuous variables. Tests for variance and categorical homogeneity were undertaken prior to all analyses via both Welch and Brown-Forsythe robust tests for mean equality. A significance level of p<0.05 was used throughout the study All data analyses were performed with IBM SPSS Statistics 20. Chi-square tests were employed to examine significance between categorical variables, using R×L contingency tables and variable categories . Continuous variables were subjected to one-way ANOVA (both weighted and un-weighted) analyses for both between- and within-group differences. One-way ANOVA was undertaken based upon prior Levene tests for the assessment of both between group and within group equality of variance. Bonferroni “post-hoc” multiple comparisons analysis was utilized after all ANOVA analyses to elucidate multi-criteria variance and exhibited association “directionality” (I–J) and to ensure highlighted differences  were Canadian studies, while the remaining 76.4% (n = 42) were from the United States . IncludeE. coli was the FIO analysed most frequently, while heterotrophic plate counts were employed least often  and Somatic) were all strongly correlated with each other (p<0.01). No significant correlation between the total percentage of sample wells with enteric pathogens present (regardless of pathogen type) and the percentage of wells with fecal indicators present  was found. Weak (−0.7< r <0.7) negative correlations were found between pathogen contamination percentage  and total coliforms   and intestinal enterococci . A weak positive correlation between bacterial pathogen presence and E. coli . Both protozoan  and viral pathogen presence  were correlated with the presence of intestinal enterococci.In total, 39 studies reported FIO data related to six organisms. Based on the definition of FIO as “non-pathogenic, exclusively intestinal in habit and therefore exclusively fecal in origin if found outside the intestine” st often . Two-taist often . The pre2(3) = 7.749, p = 0.05), with a higher prevalence of pathogen detection found among “snapshot” type studies (2(12) = 34.594, p = 0.001); almost 60% of outbreak investigations took place during autumn, while 17.8%, 21% and 3.5% occurred during spring, summer and winter, respectively. Moreover, 100% of lab-based studies (5/5) comprised year round repeat sampling. Study design and enteric pathogen type were also associated (χ2(6) = 24.162, p<0.001); all (6/6) lab-based records were associated with waterborne enteric viruses, as were the majority (26/32) of repeat sampling studies. Outbreak investigations were split with respect to pathogen type .Of 102 identified records, 74 (72.5%) reported confirmed pathogen presence, employing four main study designs. The most frequent study design focusing on enteric pathogen detection was (i) outbreak investigations , followed by (ii) repeat sampling based fieldwork , (iii) “snapshot” (i.e. “one-off”) sampling studies  and (iv) laboratory method-based studies . A significant association was noted between pathogen detection percentage (% of records in which enteric pathogens were positively found) and study design (χ studies . Source  studies . While n2(2) = 6.438, p = 0.04), which were most commonly analysed using molecular diagnostics (9/14). Similarly, molecular methods were favoured for the detection of protozoan pathogens (χ2(2) = 13.365, p = 0.001) (7/11). Studies reporting pathogen detection were approximately equally split between mono- (51.2%) and multi-pathogen (48.8%) detection. The presence of bacterial pathogens was analysed in 35.7% of studies, while protozoan and viral detection took place in 28.6% and 54.8% of studies, respectively. Two studies reported on all three enteric pathogen categories.The majority (72.5%) of pathogen detection was undertaken using molecular techniques . Microsc2(3) = 6.957, p = 0.074); a lower mean prevalence of enteric pathogen presence was found among community systems  focused on private domestic wells, with a furter 24.2% (15/55) focusing on municipal groundwater systems . Further systems . In acco2(3) = 9.644, p = 0.022); community systems were significantly more likely to be associated with waterborne epidemic conditions. No association was found between the instance of waterborne outbreaks and groundwater source type, nor between outbreak-related studies and pathogen strain or type. Viral presence was significantly higher than either bacterial or protozoan presence (χ2(2) = 9.505, p = 0.009); overall, viral pathogens were found in 85.7% of studies in which viral detection methods were employed  were associated with “non-outbreak” conditions, while the remaining were instigated as a direct result of a confirmed waterborne outbreak. A significant association was found between the prevalence of outbreak-related studies and groundwater system type (χemployed  (regardl2(1) = 5.362, p = 0.021) (2(1) = 6.031, p = 0.017). Conversely, potentially zoonotic pathogens were approximately 3 times less likely to be detected where septic tanks and/or municipal sewage systems were noted as the primary local pathogen source  = 6.574, p = 0.014). Potentially zoonotic pathogens were more likely to be detected in shallow hand-dug and spring sources than deeper boreholes (χ2(3) = 11.751, p = 0.008). Pathogens were categorised based upon pathogen type  and similar analysis undertaken with respect to pathogen presence and potential pathogen source. Only one significant association resulted, with bacterial presence 75% more likely in areas where livestock presence was recorded  = 4.714, p = 0.03).Data extraction was carried out for potential pathogen sources  within all waterborne pathogen records. Data were extracted based upon being a confirmed (e.g. use of tracer study) or likely (e.g. population cohort study) pathogen source. Septic tanks/sewage systems were the most frequently reported confirmed or likely pathogen source . No stat= 0.021) . Additio2(1) = 12.531, p<0.001). Overall, 59.8% of records included data pertaining to aquifer/bedrock type (n = 61) , geological pathways  and inadequate source design/construction  . It is i(n = 61) .2(1) = 7.184, p = 0.007). Likewise, inadequately designed/constructed wells were almost 3 times more likely to have enteric protozoa present  = 9.545, p = 0.004). Viral pathogen presence was less likely to be associated with precipitation patterns  = 9.722, p = 0.002), with no association noted regarding well design/construction or local geological characteristics.No association was noted between the presence of bacterial pathogens and precipitation patterns or geological pathways; however, 100% of sources with confirmed bacterial pathogen presence were noted as being poorly designed/constructed, indicating “source-specific” contaminant pathways  = 4.61, p = 0.032). Included Canadian studies differed from US studies with respect to study design (χ2(3) = 8.386, p = 0.039). For example, 100% of lab-based study records (6/6) were derived from the US, while a higher proportion of repeat sampling records were from Canadian studies (2(2) = 7.278, p = 0.026); while just over 12% of extracted pathogen records were derived from Canadian studies, 29% (n = 6) of protozoan records were associated with Canada.Some notable differences were found with respect to study country; while only 23% (n = 3) of Canadian studies noted local (hydro)geological characteristics. These parameters were reported in 57% of US studies (24/42) (χ studies . US stud studies . Studied2(1) = 19.153, p<0.001). Conversely, while only 16% of Canadian records mentioned septic tanks/municipal sewage systems with respect to pathogen presence, 58% of US studies (n = 43) reported this link (χ2(1) = 8.289, p = 0.008). Geology was cited as a likely contaminant pathway in only 8.3% (n = 1) of Canadian records and in 58% (n = 43) of US records (χ2(1) = 11.376, p = 0.001).Tests of independence were used to ascertain whether specific contaminant sources or pathways were equally reported based on study location. Both agricultural sources (point and diffuse) and septic tanks/municipal sewage treatment reporting differed significantly . While aA review of groundwater pathogen contamination studies in Canada and the US was conducted, locating 102 enteric pathogen records from 55 studies. The majority of studies were from the United States. While almost 24% of studies were Canadian, only 12% of enteric pathogen records were from Canadian studies. Typically, US studies were more likely to include “multi-pathogen” data (>1 enteric pathogen per study) and therefore >1 record, while Canadian studies were more likely to include FIO data with no accompanying enteric pathogen data. There is a paucity of studies focusing on direct pathogen detection in groundwater systems in both countries. Given concerns regarding the use of FIOs as surrogate organisms Just over 42% of extracted records in this review were for private domestic wells. Currently 34–45% of groundwater users in Canada and approximately 33% of groundwater users in the US employ a private water source In Canada, the focus within scientific studies in the reviewed literature has been on protozoan detection in groundwater, while in the US, it has been on bacterial and viral detection, with viruses being the most commonly studied pathogen in this review. Pathogenic viruses are a particular concern in groundwater systems as their size enables them to evade natural attenuation processes and enter the aquifer E. coli, the two most frequently employed FIOs for groundwater system surveillance. Moreover no association between coliphage detection and viral presence was observed in this review. A 1-year study of 12 municipal wells in Quebec, Canada reported similar results et al. No correlation was found between viral presence and either total coliforms or Geology was rarely recorded or presented as a potential contaminant pathway in Canadian studies, while 60% of US studies stated that local geological conditions were a potential cause for concern. Awareness of local geological and hydrogeological characteristics is critical in understanding likely contamination mechanisms or pathways, resource management and remediation and, by extension, human health risk assessment Bacterial pathogens accounted for 22.6% of records; all studies identified poorly designed or constructed well sources as a likely pathway. In contrast to the viral pathogen findings, this suggests localized, source-specific contamination as a predominant ingress mechanism e.g. groundwater contamination occurring at source due to poor wellhead protection as opposed to generalised contamination of all or part of the aquifer et al. The increased likelihood of potential zoonotic pathogen presence in agricultural areas was anticipated; low travel times in the subsurface make it unlikely to find these pathogens in areas without animals (livestock or wildlife) et al. Much of North America, particularly the United States, is characterised by unconsolidated aquifers Escherichia coli O157 (VTEC) as a confirmed etiological agent Based on this review, we note an inconsistency between studied groundwater pathogen (category) and reported groundwater-related human health burden in North America. A knowledge gap exists with respect to information on both prevalence and concentration of pathogen contamination of groundwater systems, particularly enteric bacteria. For example, it has been reported that 11% of waterborne outbreaks were caused by Cryptosporidium spp. to conventional chlorination Little efficacy was found with respect to the predictive ability of FIOs and protozoan or viral contamination of groundwater systems. The lack of association between protozoan presence and local hydrogeological characteristics  reinforces the importance of rapid by-pass mechanisms Results and conclusions presented here are based on the extracted data from published studies and are subject to the inherent biases of the original study objectives, designs and what was reported. Few Canadian studies referenced local hydrogeological characteristics as a potential contamination pathway when reporting on contamination events or groundwater-associated outbreaks, representing a limitation in study reporting. This lack of reporting may impact subsequent analyses of contamination pathways, for example, findings may reflect a lack of reporting or examination of these factors in a rigorous manner. Furthermore, only one included study occurred in winter, representing a potential limitation.Study design differed significantly between US and Canadian studies. The majority of lab-based studies and outbreak investigations were in the US, while a higher proportion of repeat sampling records were from Canada. Repeat sampling studies integrated low well numbers with multiple samples from each well (mean – 6 samples). Outbreak investigations and “snapshot” studies comprised significantly higher well numbers, with wells typically sampled once. Outbreak investigations are an essential epidemiological tool; however, they are reactive and therefore not suitable for assessing endemic groundwater risks. Numerous studies report that multiple samples are necessary to positively determine contamination of a groundwater source Enteric pathogens were present in 72.5% of records (74/102) and 16.1% of wells. Upon exclusion of outbreak investigation data, 77.1% of records (54/70) and 14.7% of wells  were positive for at least one enteric pathogen. These figures may provide a useful foundation for endemic groundwater-associated exposure assessment, until more direct enteric pathogen data become available.et al. The lack of association between the incidence of outbreak investigations and municipal and private groundwater systems was not anticipated; however, as the majority of groundwater sampling was undertaken pre-treatment, this does not accurately reflect the public health burden posed by specific system types . Large municipal systems are more likely to be adequately maintained and appropriately treated Our review has highlighted a number of knowledge gaps and requirements relating to contamination of Canadian and US groundwater systems over the past two decades. Improved endemic risk assessment will be possible with increased direct enteric pathogen detection and reporting. Repeat-sampling studies represent a priority for the design of future studies for the collation of these data, as they are considered more representative with respect to human endemic risk. More data are required for groundwater-associated municipal systems, both pre- and post-treatment, as currently these systems are not adequately represented in the literature. Additionally, more data are required for bacterial pathogens, as these microorganisms are associated with a large public health burden. These data would also provide a foundation for improved risk management and communication via increased awareness and understanding among well owners, well users, system operators and policy makers.Within the literature the four study designs that were identified were of a proactive (pre-waterborne illness event) or reactive (post-waterborne illness event) nature, with the proactive designs being more suitable for background characterisation. Pooled analyses of reported data found that enteric pathogens were present in approximately 15% of sampled wells; this may be useful for future microbial risk assessments of human health relating to Canadian and US groundwater systems, although it should only be employed in the absence of area-specific data, and then, only with caution due to inherent limitations associated with the current study . Accordingly, the authors recommend future research be focused on addressing this current lack of temporal and spatial data.It is critically important to account for study design when presenting and interpreting results of a groundwater study, given that groundwater is not similar to surface water and many confounding factors will influence the presence of pathogens in one system versus another, as well as the occurrence of an associated outbreak.Few studies have been published on pathogen presence in municipal groundwater systems. By addressing this knowledge gap, evidence would be developed to help support local decision-making around appropriate treatment or other interventions to further promote the safety of the drinking water supply. It is recommended that future health-related studies, particularly those associated with sporadic waterborne infection and quantitative health risk assessments, focus on direct enteric pathogen detection where possible, given the limited efficacy of fecal indicator organisms for enteric pathogen presence in groundwater systems. These data will aid more focused, pathogen-specific exposure assessment and hazard characterisation, therefore providing more data for evidence-based groundwater and human-health protection measures.Local (hydro)geology is an important risk factor in the likelihood of pathogen presence in a groundwater supply and was not reported in a substantial proportion of study records included in this review. The availability and reporting of these data could be used in combination with land-use, source type and seasonality to more accurately inform outbreak investigators, epidemiologists and sanitary engineers.Bacterial and protozoan contamination appear to be associated with localised contamination mechanisms (well-specific), lending credence to the concept of “consumer control” . Viral contamination may be more generalised  but intervention at the source could be targeted to address all enteric pathogens (e.g. ultraviolet disinfection).Our findings further reflect the importance of frequent inspection and maintenance of both private and municipal groundwater supplies in Canada and the US. Additionally, it is important that potential system vulnerabilities including climate, local hydrogeological setting and adjacent hazard sources are considered during study design.Appendix S155 included studies in the review with extractable data.(DOCX)Click here for additional data file.Checklist S1PRISMA guideline checklist for reporting systematic reviews.(DOC)Click here for additional data file.


Scientific Reports6: Article number: 25609; 10.1038/srep25609 published online: 05122016; updated: 08122016.In this Article, the Data resources have been omitted from the Methods section. It should read:“All data used in the preparation of this paper are available from the University of Bristol’s research data repository, and can be accessed using the DOI 10.5523/bris.rz5m2a3q61851txlxs6eecuhx”.


Self-rated health (SRH) has been demonstrated as a valid and appropriate predictor of incident mortality and chronic morbidity. Associations between lifestyle, chronic diseases, and SRH have been reported by various population studies but few have included data from developing countries. The aim of this study was to determine the prevalence of poor SRH in Malaysia and its association with lifestyle factors and chronic diseases among Malaysian adults.This study was based on 18,184 adults aged 18 and above who participated in the 2011 National Health and Morbidity Survey (NHMS). The NHMS was a cross-sectional survey (two-stage stratified sample) designed to collect health information on a nationally representative sample of the Malaysian adult population. Data were obtained via face-to-face interviews using validated questionnaires. Two categories were used to measure SRH: “good” (very good and good) and “poor” . The association of lifestyle factors and chronic diseases with poor SRH was examined using univariate and multivariate logistic regression.Approximately one-fifth of the Malaysian adult population (20.1 %) rated their health as poor (men: 18.4 % and women: 21.7 %). Prevalence increases with age from 16.2 % (aged 18–29) to 32.0 % (aged ≥60). In the multivariate logistic regression analysis, lifestyle factors associated with poor SRH included: underweight , physical inactivity , former smoker , former drinker , and current drinker . Chronic diseases associated with poor SRH included: asthma , arthritis , hypertension , hypercholesterolemia , and heart disease .This study indicates that several unhealthy lifestyle behaviours and chronic diseases are significantly associated with poor SRH among Malaysian adults. Effective public health strategies are needed to promote healthy lifestyles, and disease prevention interventions should be enhanced at the community level to improve overall health.The online version of this article (doi:10.1186/s12889-015-2080-z) contains supplementary material, which is available to authorized users. A variety of lifestyle factors and health-related behaviours such as smoking, alcohol consumption, physical activity, and dietary habits can affect a person’s health. An unhealthy lifestyle often results in a higher risk of chronic diseases such as heart disease, hypertension, hypercholesterolemia, and diabetes , 2. PrevUnderstanding the correlates of SRH can help public health professionals prioritise health-promotion and disease-prevention interventions. To date, studies that have investigated the determinants of SRH have focused primarily on Western countries, although we have found a amall number of studies that examine the topic from the perspective of developing countries in the Asian regions. Previous studies from Singapore , PakistaIn Malaysia, few studies have examined the topic of SRH. A recent study by Cheah  reportedThe NHMS 2011 was a nationwide, cross-sectional, population-based survey that used a two-stage stratified sampling design to select a nationally representative sample. The Malaysian states constituted the primary strata, with further stratification by urban–rural residence. The primary sampling units were enumeration blocks (EBs) provided by the Malaysian Department of Statistics from the 2010 census. Living quarters (LQs) represent the secondary sampling units. A total of 794 EBs  were systematically selected from all the Malaysian EBs  via a probability-proportional-to-size sampling technique. Subsequently, 12 LQs were randomly selected from the approximately 80 to 120 LQs in each EB. Finally, all households and eligible household members in the selected LQs were included in the sample. The survey methodology is described in greater detail by Fadhli et al. .Data collection for the NHMS 2011 was carried out from April to July 2011. Information on SRH, socio-demographic characteristics, lifestyle, and self-reported medically-diagnosed chronic diseases was obtained by trained interviewers via face-to-face interviews. The interviewers were extensively trained and conducted mock interviews prior to actual data collection. The interviewing process was closely monitored by field supervisors, who were health care personnel. A total of 18,231 eligible adults aged 18 years and older, living in the sampled households, were interviewed. Of these, 18,184 responded to the SRH questionnaire section of the NHMS 2011, giving a response rate of 99.7 %. To ensure a high response rate, unoccupied households were revisited up to three times. All of the eligible respondents provided written consent to be interviewed for the survey. The study protocol was approved by the Malaysian Medical and Research Ethics Committee (MREC), Ministry of Health Malaysia (NMRR-10-757-6837).Self-rated health was measured based on the question: “In general, how would you rate your health today?” There were five response categories: “very good”, “good”, “moderate”, “not good” and “very bad”. For purposes of analysis, the response categories were combined into two groups: “Good”, which included “very good” and “good”, and “Poor” which included “moderate”, “not good” and “very bad”. “Good” SRH served as the reference category in the logistic regression analyses.Socio-demographic variables included sex, age, ethnicity, residential area , marital status , educational level , current work status (working/not working) and monthly household income. Age was categorised into five groups: 18–29 years, 30–39 years, 40–49 years, 50–59 years, 60 years and above. Ethnicity was categorised into five groups: Malays, Chinese, Indians, other Bumiputeras , and “Others” . Monthly household income was grouped into four levels: less than MYR1000, MYR1000–MYR1999, MYR2000–MYR2999, MYR3000 and above [1 Malaysian Ringgit (MYR) ≈ 0.29 US Dollar (USD)].2). According to the World Health Organization (WHO 2000) guidelines [Lifestyle variables included body mass index (BMI), consumption of fruits and vegetables, physical activity, smoking, and alcohol consumption. Respondents’ weight and height were measured using the Tanita 318 digital weighing scale  and the Seca 206 Bodymeter measuring tape , to the nearest 0.1 kg and 0.1 cm, respectively. BMI was calculated as weight divided by height in meters squared , and a half cup of cooked chopped eggplant or tomato or carrot or leafy green vegetables. According to the WHO STEPS criteria [Consumption of fruits and vegetables was measured based on four questions: i) “In a typical week, how many days do you eat fruits?” ii) “Usually on the day you eat fruits, how many servings of fruits did you eat in a day?” iii) “In a typical week, how many days do you eat vegetables”, and iv) “Usually on the day you eat vegetables, how many servings of vegetables did you eat in a day?” Photographs of foods were used to help respondents recall the serving sizes of the fruits and vegetables they had consumed. The photographs showed single servings of commonly consumed fruits and vegetables such as the following: one medium-sized apple, one medium-sized banana, one slice of papaya, one slice of watermelon, eight medium-sized grapes, one cup of chopped raw leafy green vegetables or criteria , responscriteria , consumpThe level of physical activity was determined based on the short form of the International Physical Activity Questionnaire (IPAQ), the reliability and validity of which have been established . The oriSmoking status was divided into three categories: “never smoker” (never smoked in lifetime), “former smoker” , and “current smoker” . Alcohol consumption status was divided into three categories: “non-drinker” , “former drinker”  and “current drinker” .For chronic disease status, presence or absence of chronic diseases was based on self-reported information on chronic conditions diagnosed by a doctor or health care professional. Respondents were asked: “Have you ever been told by a doctor or Medical Assistant that you have __________?” followed by a list of chronic diseases included asthma, arthritis, diabetes mellitus, hypertension, hypercholesterolemia, and heart disease, to which the answer is “yes” or “no”.p = <0.25 in the univariate analysis were retained in the multivariate logistic regression model [p = <0.05 was considered to be statistically significant. The statistical analyses were performed using SPSS for Windows (version 20.0) statistical software . All analyses were conducted using a complex sampling design to ensure that sample weights and study design were taken into consideration.Frequency distributions of socio-demographic, lifestyle, and chronic disease variables were analysed by self-rated health status. Descriptive statistics were used to calculate the overall prevalence of poor SRH among Malaysian adults and the prevalence of poor SRH by socio-demographic, lifestyle and chronic disease variables. Associations of the measured factors with poor SRH were tested using chi-square tests. Univariate and multivariate logistic regression analyses were used to investigate the association between poor SRH, socio-demographic, lifestyle, and chronic disease variables, with “Good” SRH as the reference category. The results are presented as crude and adjusted odds ratios (ORs) with 95 % confidence intervals (CIs). Variables that were associated with poor SRH at n = 18,184). The sample included more women (53.4 %) than men (46.6 %). Among the 18,184 respondents, the distribution of self-rated health was as follow: “very good” 3,468 (19.4 %); “good” 11,031 (60.4 %); “moderate” 3,349 (18.2 %); “not good” 316 (1.8 %); and “very bad” 20 (0.1 %)  of participants reporting good SRH, and 20.1 % (n = 3685) reporting poor SRH. A higher proportion of women (57.5 %) than men (42.5 %) reported poor SRH. Similarly, a higher proportion of older adults (aged 60 years and above) reported poor SRH than younger adults (Table Table p = <0.001). Regarding lifestyle variables, individuals who were underweight, physically inactive, and former smokers had higher prevalence of poor SRH. Survey participants who had chronic diseases such as asthma, arthritis, diabetes mellitus, hypertension, hypercholesterolemia, and heart disease were found to have a higher prevalence of poor SRH, compared with their counterparts who did not have these diseases.Table p = <0.25 except for “Others” ethnic group, monthly household income, overweight BMI group, and current drinkers. In the final multivariate logistic regression model, we included all variables that were statistically significant in univariate analysis (p = <0.25) except for the “monthly household income” variable. After adjusting for all selected factors in the multivariate analysis, the odds of poor SRH were significantly higher for women compared with men, among older people (aged ≥60 years) compared with younger people (aged 18–29 years), and among people living in rural areas compared with urban areas. Malays, other Bumiputeras and “Others” ethnic groups had higher odds of poor SRH compared with the Chinese. Respondents who were underweight (p = <0.05), physically inactive (p = <0.001), former smokers (p = <0.01), current drinkers (p = <0.01), and former drinkers (p = <0.05) reported significantly higher odds of poor SRH compared with their respective counterparts. Self-reported medically-diagnosed chronic diseases that were significantly associated with poor SRH were asthma, arthritis, hypertension, hypercholesterolemia, and heart disease (p = <0.001 for each). Individuals with diabetes mellitus were not significantly associated with poor SRH in our study.Table The prevalence of poor SRH in Malaysia (20.1 %) is similar to that reported in neighbouring Singapore (23.2 %) , which mThe present study found a significant association between specific lifestyle factors and SRH. Individuals who are underweight, physically inactive, former smokers, and current drinkers were significantly associated with poor SRH. Previous studies have reported that obesity and underweight are significantly associated with poor SRH , 32, 33;total physical activity including different forms of physical activity . A recent study of physical activity using the same national survey data (NHMS 2011) reported that about two in five Malaysian adults were physically inactive, particularly women and the elderly [Previous studies have consistently shown that physical activity and exercise have positive effects on SRH –43. Our  elderly . In the In contrast to findings from the 2007 Behavioural Risk Factor Surveillance System (BRFSS) survey conducted in the United States  and the In our study, no statistically significant difference was found in poor SRH for current smokers compared with never smokers. This finding is contrary to previous studies , 13, 48,In line with previous studies , 53, 54 There is substantial evidence supporting the negative impact of chronic diseases on SRH , 34, 58.While this study emphasised the associations between lifestyle, chronic diseases, and SRH, several socio-demographic factors such as age, sex, ethnicity, and residence  were shown to have statistically significant influences on SRH in Malaysia. Older people were more likely to report poor SRH than younger people, which has been shown in other studies , 13, 35.The major strengths of this study are the representativeness and large sample size. The results can be extrapolated accurately and generalised to the Malaysian adult population. Furthermore, the participants were interviewed face-to-face by trained interviewers and the response rate was higher than reported for other studies. However, the study has several limitations that should be noted. The main limitation is the cross-sectional design of the study, which does not allow for determination of causal effect relationships. Another limitation is the possibility of recall bias associated with self-reported data on smoking, alcohol consumption, physical activity, and consumption of fruits and vegetables. Third, the “social desirability” of certain answer categories is a concern in face-to-face interviews. To project a favourable image, respondents may give answers that are deemed more “socially accepted” than their “true” answers. Finally, several other factors found to be associated with SRH in previous studies  were not investigated in this study.The prevalence of poor SRH among Malaysian adults is 20.1 %, which increases with age. Several negative lifestyle behaviours and chronic diseases were significantly associated with poor SRH among Malaysian adults. Additional public health strategies are needed to promote healthy lifestyles and disease prevention interventions should be implemented to improve personal and community health. More preventive measures and health care services targeting chronic diseases are urgently needed, particularly in rural communities. Further research that takes into account the influences of other potential confounders such as psychososial and environmental factors is needed to fully understand the correlates and determinants of SRH.


Campylobacter jejuni is the one of the leading causes of bacterial diarrheal illness worldwide. This study aims to design specific epitopes for the utility of designing peptide vaccine(s) against C. jejuni by targeting invasive, virulent and membrane associated proteins like FlaA, Cia, CadF, PEB1, PEB3 and MOMP.C. jejuni. The tools include Bepipred, ABCpred, Immune Epitope databse (IEDB) resource portal, Autodock vina etc.In the present study, various immunoinformatics approaches have been applied to design a potential epitope based vaccine against C jejuni with varying proportions of population coverage ranging from 22–60 %. Further authentication of these peptide epitopes as probable vaccine candidate was mediated by their binding to specific HLA alleles using in silico docking technique.Peptides “EINKN”, “TGSRLN”, “KSNPDI”, “LDENGCE” respectively from FlaA, MOMP, PEB3, CadF proteins were found to be the most potential B cell epitopes while peptides “FRINTNVAA”, “NYFEGNLDM”, “YKYSPKLNF”, “YQDAIGLLV”, “FRNNIVAFV” and “LIMPVFHEL” respectively from Fla, CadF, MOMP, PEB1A, PEB3 and Cia might elicit cell mediated immunity and “IFYTTGSRL” from MOMP protein might elicit both humoral and cell-mediated immunity. All these potential peptidic epitopes showed almost 80–100 % conservancy in different strains of C. jejuni bacteria and thus, could be validated in model hosts to verify their efficacy as vaccine.Based on the present study, it could be concluded that these predicted epitopes might be used to design a vaccine against The online version of this article (doi:10.1186/s40203-016-0020-y) contains supplementary material, which is available to authorized users. Campylobacter jejuni, a gram-negative bacillus, is considered to be a commensal organism of chicken gut. It causes gastroenteritis in humans  in FASTA format.Sequences of flaA, CadF, Cia, PEB1, PEB3 and MOMP of strain 11168 of www.imtech.res.in/raghava/abcpred/)  with 0.5 as threshold  (tools.immuneepitope.org) which provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments  binding to MHC1 molecules. For all the alleles, peptide length was set to 9 amino acids prior to the prediction. The parameters for detection of immunogenicity  were normalized on a scale of 0 to 1 and were given a weighted score to prioritize the parameters  transport efficiency and proteasomal cleavage efficiency was involved to predict overall scores for each peptide's intrinsic potential of being a T cell epitope. The Stabilized Matrix Base Method (SMM) was used to calculate IChttp://tools.immuneepitope.org/mhci/). The peptides which interacted with highest number of alleles were selected.The nonamers were given as input in MHC class l binding prediction tool available in the Immune Epitope Database (IEDB) server (http://tools.immuneepitope.org/mhcii/) since MHC class II can accommodate much longer peptides – possibly even whole proteins. The Stabilized Matrix Base Method (SMM) was used to calculate IC50 values of peptide binding to MHCll molecules. The peptides (containing 15 amino acid residues) that interacted with highest number of alleles were again selected.On the other hand, the whole protein sequences were submitted in the IEDB MHC class II binding prediction tool  were finally selected to predict epitope conservancy and population coverage.In both cases, SMM-align method was employed to find out good binders and the cut-off value of IChttp://tools.immuneepitope.org/tools/population/iedb_input) by maintaining the default analysis parameters. This server employs the most comprehensive database allelefrequencies.net for their coverage calculation.To become a good vaccine candidate, prediction of T cell epitope is not enough. The predicted peptide(s) should effectively cover human population. To find out the population coverage of the individual epitopes, predicted epitopic sequences with the corresponding Class l HLA alleles were submitted to the population coverage analysis tool of IEDB  along with 1000 bootstrap value and default parameters. The aligned sequences from each protein that generated the highest number of identical amino acid with no gap were considered as the conserved regions for respective proteins. In the present study, the minimum length of the conserved regions was set at 15 as binding predictions for MHC class II alleles use to create 15mers. The identified largest conserved regions were further analyzed using similar steps as done in case of full protein sequence analysis.“PEPFOLD” server was used to predict the three dimensional structure of selected peptide “FRLSDSLAL” and “NYFEGNLDM” from CadF  which was deposited as a complex with nonamer peptide m9. M9 is a model synthetic peptide that has the sequence of (GRFAAAIAK)  and visualized in PyMOL molecular Graphics system. The same procedure was also followed for the binding of “NYFEGNLDM” nonamer to HLA-C*07:02 allele. In that case, the grid/space box center was set at -13.677, 2.561 and 30.904 Å in the x-, y- and z-axes respectively to allow the epitope to bind to the groove of the HLA-C*07:02. The size was set at 30, 46 and 50 Å in the x, y, and z dimensions, respectively.These structures were then further prepared for running docking in AutoDock tool by adding polar hydrogens and water molecules. For the docking study both the HLA allele and ligand files were converted into PDBQT format. In case of HLA-B*27:05, the grid/space box center was set at 22.241, 15.002 and 21.475 Å in the The best output was selected on the basis of the best binding energy. To compare the binding of the predicted folded structure of the nonamer (“FRLSDSLAL”) to the binding groove of the class I MHC allele, the removed nonamer m9 was also allowed to dock as control. The control nonamer was docked by considering similar parameters.The 3D structure of MHC class I H-2Kb molecule complexed with octapeptide PKB1 (“KVITFIDL”) was retrieved from Protein Data Bank Database (ID: IKJ3) and visualized using PyMOL Graphics. The octapeptide was excluded before applying the structure of H-2Kb for comparing the validated data obtained for predicted structure of HLA-C*07:02.Also, to assess HLA-C*07:02-epitope docking results, octapeptide PKB1 (“KVITFIDL”) was used as the control. This peptide was docked with HLAs, HLA-C*07:02, and H-2Kb. The test epitope(s) and the control peptide were docked by setting similar parameters for each trial and successful binding of this peptide to these HLAs was demonstrated. Finally, H-2Kb - KVITFIDL docking result was used as control to compare with the test docking results of HLA-C*07:02 complexed with selected epitopes.Campylobacter jejuni strain 11168 several epitopes were predicted. However, only those peptides that were found fully overlapping between Bepipred and ABCpred prediction tools  alleles are known and different HLA types are expressed at different frequencies in different ethnicities. Population coverage by the most probable epitopes varied between 20 % – 65 % when MHC class I alleles were considered  and UniProt (www.uniprot.org/). The strains included 81-176; ATCC_700819/NCTC_11168; ATCC_BAA-1458/RM4099/269.97; 260.94; jejuni_CG8421; CF93-6; jejuni_IA3902; RM1221; CJM1cam; jejuni_M1; NCTC_11168-BN148 and jejuni_81116. The retrieved sequences were then aligned using ClustalW tool that uses Gonnet matrix in MEGA software (v 5.2). The conserved regions from each protein with the highest length were then used for T cell epitope prediction. They were subjected to IEDB and SYFPEITHI MHC class I and class II binding prediction tool. It was found out “FRLSDSLAL” epitope from CadF protein which stood out as the best potential vaccine candidate from that protein  to its specific HLA molecules were observed using AutoDock Vina. Epitope “FRLSDSLAL” bound to the binding groove of HLA-B*27:05 with the binding energy -6.1 kcal/mol and “NYFEGNLDM” bound with HLA-C*07:02 with the binding energy -6.3 kcal/mol Fig.  and c. OCampylobacter jejuni is recognized as a major risk factor for the onset of Guillain‐Barré syndrome, which is a serious post‐infection complication characterized by acute and progressive neuromuscular paralysis  that could be used to design a vaccine that would be universally applicable. Interestingly, “FRLSDSLAL” epitope was revealed as the best potential vaccine candidate from CadF protein which was also common among the most probable epitopes from CadF during whole protein sequence analysis. The epitope showed 37.98 % population coverage. Another important finding was that the most probable predicted B cell epitope from MOMP protein “TGSRLN” peptide overlapped with the predicted T cell epitope “IFYTTGSRL” in this study. So this epitope based vaccine would be able to elicit both humoral and cell mediated immunity.Campylobacter jejuni which could be tested for their efficacy in eliciting immunity through humoral and cell mediated immune responses. The results of our study provide computational data for the identification and screening of epitopes, and may be used for the development of epitope vaccines that have an enhanced safety and efficacy. This may result in the provision of improved regimens for the prevention of GBS. Our findings are based on sequence analysis and computational predictions. However, to prove the effectiveness of mounting an immune response, both in vitro and in vivo studies are required along with this in silico study.In this study, we made an attempt to design epitope based vaccines against


Annually, around 7.9 million children are born with birth defects and the contribution of congenital malformations to neonatal mortality is generally high. Congenital malformations in children born to mothers with hypertensive disorders during pregnancy has marginally been explored.Country incidence of congenital malformations was estimated using data on the 310 401 livebirths of the WHO Multicountry Survey which reported information from 359 facilities across 29 countries. A random-effect logistic regression model was utilized to explore the associations between six broad categories of congenital malformations and the four maternal hypertensive disorders “Chronic Hypertension”, “Preeclampsia” and “Eclampsia” and “Chronic hypertension with superimposed preeclampsia”.The occupied territories of Palestine presented the highest rates in all groups of malformation except for the “Lip/Cleft/Palate” category. Newborns of women with chronic maternal hypertension were associated with a 3.7 (95 % CI 1.3–10.7), 3.9 (95 % CI 1.7–9.0) and 4.2 (95 % CI 1.5–11.6) times increase in odds of renal, limb and lip/cleft/palate malformations respectively. Chronic hypertension with superimposed preeclampsia was associated with a 4.3 (95 % CI 1.3–14.4), 8.7 (95 % CI 2.5–30.2), 7.1 (95 % CI 2.1–23.5) and 8.2 (95 % CI 2.0–34.3) times increase in odds of neural tube/central nervous system, renal, limb and Lip/Cleft/Palate malformations.This study shows that chronic hypertension in the maternal period exposes newborns to a significant risk of developing renal, limb and lip/cleft/palate congenital malformations, and the risk is further exacerbate by superimposing eclampsia. Additional research is needed to identify shared pathways of maternal hypertensive disorders and congenital malformations. Annually, around 7.9 million children are born with birth defects . At leasEclampsia/pre-eclampsia syndrome consists of a state of excessive systemic inflammation causing new-onset proteinuria and hypertension during the second half of pregnancy . Pre-eclOnly a few studies have explored the associations between pre-eclampsia and malformations providing inconclusive results: one reported an increased risk of renal dysgenesis , esophageal atresia/stenosis  and rectal/anal stenosis  in the offspring of pregnant women who developed preeclampsia with superimposed chronic hypertension  whilst aThe study population and data collection methods used in this survey are described in detailed elsewhere . In brieThe Study involved five WHO regions: African Region ; Region of the Americas ; Eastern Mediterranean Region ; South-East Asia Region ; Western Pacific Region .The hospitals with a minimum of 1000 deliveries per year were identified. Within each country, the capital city was included, along with two randomly selected provinces with probability proportional to their population size. In each province and in the capital city, seven hospitals with over 1000 deliveries per year and the capacity to perform caesarean sections were selected using a multi-stage cluster sampling method.Participants in the study were all women giving birth during the data collection period in the participating hospitals together with their respective newborns, all maternal near-miss cases and all maternal deaths taking place in the participating hospitals up to seven postpartum/postabortion days, discharge or death (whichever came first), regardless of the gestational age and delivery status.Data were collected from hospital medical records through daily visits to the obstetrical/postpartum ward, gynecologic/abortion care unit, delivery room and intensive care unit for the duration of 2 months if the hospital had 6000 deliveries/year or and three months if the health facility had less than 6000 deliveries/year .In the WHOMCS, ascertainment of malformations was made by obstetricians and pediatrician and the inclusion criteria were major congenital anomalies among live births and stillbirths.n = 310 401) during the data collection were included to explore the frequency of congenital malformation.All live births  or number of fetuses (n = 60) were excluded. Individuals with missing values on congenital malformations (n = 20) and maternal hypertensive disorders (n = 9) were also excluded in the analysis leaving a final study population of 302 135 women. See flowchart showing the selection of study participants in Fig. Mothers and newborns with missing information on status at birth n = 426 or nuIn the WHOMCS, data collectors performed daily visits to the obstetrical/postpartum ward, gynaecologic/abortion care unit, delivery room and intensive care unit to review medical records and extract data from these into individual data forms.Six categories of congenital malformations were defined for data collector to extract data from the medical records in line with the International Clearinghouse for Birth Defects Monitoring Systems classification .The six groups consisted of: “Neural tube/Central Nervous System”  “Cardiac” , “Renal” , “Limb” , “Lip/Cleft/Palate”  and “Chromosomal” .Hypertensive disorders information was collected from delivery records and was categorized into “Chronic Hypertension”, “Preeclampsia” and “Eclampsia”. The study definition for pre-eclampsia was the presence of hypertension (blood pressure >140/90 mmHg) associated with proteinuria in women known to be previously normotensive. Eclampsia was defined as the occurrence of convulsions and/or coma unrelated to other cerebral conditions in women with signs and symptoms of pre-eclampsia. Seizures are of grand mal type and may first appear before labour, during labour or up to 48 h postpartum. Chronic hypertension was defined as a blood pressure >140/90 mmHg diagnosed prior to the onset of pregnancy or before the 20th week of gestation; all women with chronic hypertension and who developed preeclampsia were grouped and classified under the new variable “Chronic hypertension with superimposed preeclampsia”.We determined the frequency of newborns with congenital malformations per 1000 live births for each of the 29 countries participating in the WHOMCS stratified by WHO Region and by Human Development Index (HDI) . WhereasP < 0.05 was regarded as significant.We used frequencies to describe the congenital malformation categories by maternal age, marital status, woman education and number of previous births; as health facilities were the primary sampling unit of the WHOMCS, individual-level analyses may be affected by clustering. After excluding missing data, all estimates of association (chi-square tests) were corrected for the cluster effects  and Crude and adjusted ORs with 95 % CI were evaluated using a multi-category logistic regression models for the occurrence of any congenital malformation; the adjusted logistic regression model considered potential maternal confounders: age, marital status, woman education and number of previous births. Because of the possible cluster effect of individual analysis  we generStata 13 was used for statistical analyses and p-values <0.05 was regarded as significant.The WHOMCS was approved by the WHO Ethical Review Committee and the relevant ethical clearance bodies in participating countries and facilities. Written consent from individual participants was not required.In the WHOMCS, overall 1706 newborns with congenital malformations were recorded, distributed as follows: 322 (1.04 per 1000 livebirths) “Neural tube/Central Nervous System”, 494 (1.59 per 1000 livebirths) “Cardiac”, 136 (0.44 per 1000 livebirths) “Renal”, 367 (1.18 per 1000 livebirths) “Limb”, 212 (0.68 per 1000 livebirths) “Lip/Cleft/Palate” and 175 (0.56 per 1000 livebirths) “Chromosomal”.By country, the occupied territories of Palestine presented the highest rates in all malformation except for “Lip/Cleft/Palate” where Japan was highest. In the occupied territories of Palestine, it was found a prevalence of 10.2 cases of “Neural tube/Central Nervous System”, 24.6 cases of “Cardiac”, 11.3 cases for “Renal”, 8.2 cases for “Limb” and 3.1 cases for “Chromosomal”  followed countries with medium HDI (4.3 per 1000 livebirths), and finally countries with low HDI (2.7 per 1000 livebirths) , limb  and lip/cleft/palate malformations  while preeclampsia held significant associations with “Cardiac” . Eclampsia was not associated to the malformation for which data were available .The multivariable analysis strengthened the association between the chronic hypertension with superimposed preeclampsia and “neural tube/central nervous system” , “Renal” , “Limb”  and “Lip/Cleft/Palate”  malformations  and hypertensive disorders that have been identified in other studies –14. AnotFor the exposure variables  time of onset or duration of exposure is not specified in WHOMCS database. This is important since many congenital malformations occur before 8 weeks of gestation and risk lies in pre-conception period. In addition, the WHOMCS did not collect data on antihypertensive medication exposure and other risk factors associated with a number of specific birth defects. Many other major risk factors  were not available to give adjusted OR. ORs were calculated only for age, education, previous births and marital Status.Disparities in the ascertainment of congenital malformations in high and low HDI countries is clearly apparent with very high and high HDI countries reporting a majority of congenital malformations even with lesser number of live births.To our knowledge this is one of the few analyses reporting estimate the prevalence of congenital malformation in facilities in middle and low income countries where more than 90 % of all infants with a serious birth defect are born and where many of them will die young because of lack of adequate health care services . A conceIn view of findings and characteristics of our study we also suggest additional research on associations and possible common pathogenesis pathways in maternal hypertensive disorders and congenital defects, given their roles in maternal and child morbidity and mortality.AFR, African Region; AMR, American Region; EMR, Eastern Mediterranean Region; HDI, human development index; MCS, multi-country survey; SEAR, South-East Asian Region; WHO, World Health Organization; WPR, Western Pacific Region


D) and perfusion-related diffusion parameters  differ in healthy livers and fibrotic livers through intra-voxel incoherent motion (IVIM) MR imaging.This study was aimed to determine whether pure molecular-based diffusion coefficient  were included. Liver MR imaging was performed at 1.5-T. IVIM diffusion weighted imaging sequence was based on standard single-shot DW spin echo-planar imaging, with ten D, f, and D* in healthy volunteer livers and patient livers were 1.096±0.155 vs 0.917±0.152 , 0.164±0.021 vs 0.123±0.029 (p<0.0001), and 13.085±2.943 vs 9.423±1.737  respectively, all significantly lower in fibrotic livers. As the fibrosis severity progressed, D, f, and D* values decreased, with a trend significant for f and D*.f and D* in the liver is significantly associated liver fibrosis severity.Fibrotic liver is associated with lower pure molecular diffusion, lower perfusion volume fraction, and lower perfusion-related diffusion. The decrease of  Chronic liver disease is a major public health problem worldwide b values encompassing both low b values (<200 sec/mm2) and high b values (>200 sec/mm2) to a bi-exponential decay model. These three IVIM parameters have shown potentials for better tissue characterization than the apparent diffusion coefficient (ADC) derived from the normal mono-exponential diffusion weighted imaging (DWI) model for many clinical applications D) and perfusion-related diffusion parameters  differ in healthy livers and fibrotic livers through intra-voxel incoherent motion (IVIM) MR imaging.The principle of intra-voxel incoherent motion (IVIM) imaging is a method initially proposed by Le Bihan et al This prospective study was approved by The Shenzhen No. 3 People's Hospital Research and Ethics Committee and written informed consent for all participants was obtained. The consent form was also approved by the local Research and Ethics Committee and hard copy was archived in the Department of Radiology of the Shenzhen No. 3 People's Hospital. 17 healthy volunteers  and 34 consecutively patients  with confirmed liver fibrosis (stage 1 - stage 4) were included. All the patients had hepatitis B virus infection. Liver biopsy and MRI were performed with less than one month interval , with biopsy performed ahead of MRI. All liver MR imaging examinations were reviewed by two radiologists. Besides liver fibrosis, no patient had liver focal lesions. Biopsy specimen of 10 mm length was taken from each patient and Hematoxylin and Eosin (HE) staining was used. The histopathology results were read in consensus by two pathologists with>10 years' experience. Histopathologically All MR imaging examinations were performed with a 1.5-T MR imaging system . The liver MR imaging protocol included transverse T1-weighted gradient-echo sequence, transverse T2-weighted turbo spin-echo sequence, and the transverse IVIM DW imaging sequence. T1-weighted gradient-echo sequence included TR  = 11 ms, TE  = 6.9 ms, Matrix  = 252×151, FOV  = 375 mm×304 mm, slice thickness: 7 mm, slice gap  = 1 mm, Slices 24, NEX  = 1. T2-weighted gradient-echo sequence included TR  = 421 ms, TE  = 80 ms, Matrix  = 268×184, FOV  = 375 mm×297 mm, Slice thickness  = 7 mm, slice gap  = 1 mm, slices  = 24, NEX  = 1.b values of 10, 20, 40, 60, 80, 100, 150, 200, 400, 800 sec/mm2 respectively. SPIR technique  was used for fat suppression. Respiratory triggering was performed using an air-filled pressure sensor fixed around the upper part of abdomen for each subject. Respiration waveform was detected and monitored on a gating control screen. The DWI acquisition window was only limited to the expiratory phase while dummied to the inpiratory phase. The respiratory-gating resulted in an average TR of 1500 msec, and the TE was 63 msec. Other parameters included slice thickness  = 7 mm, matrix: 124×97, FOV  = 375 mm×302 mm, NEX  = 2. The IVIM DW imaging was centered around the liver and the number of slices  = 6. The data acquisition time was 6∶42 minutes.The IVIM DW imaging sequence was based on a single-shot DW spin-echo type echo-planar imaging sequence, with ten SI(b) and SI0 denote the signal intensity acquired with the b-factor value of b and 0, respectively. f is the fraction of the pseudo-diffusion linked to microcirculation, D is the true diffusion coefficient representing the pure molecular diffusion (slow component of diffusion), and D* is the pseudo-diffusion coefficient representing the incoherent microcirculation within the voxel .The IVIM signal attenuation is modeled according to the Equation D* is significantly greater than D due to the much faster capillary blood flow velocity than the water thermal diffusion, its influence on signal decay could be neglected for the b factors greater than 200 sec/mm2D was obtained by a least-squares linear fitting of the logarithmized image intensity at the b values greater than 200 sec/mm2 to a linear equation. The fitted curve was then extrapolated to obtain an intercept at b = 0. The ratio between this intercept and the SI0, gave an estimate of the perfusion fraction f. Finally, the obtained D and f were substituted into Eq. D* using the Levenberg-Marquardt algorithm. When the function failed to converge, pixel values were automatically discarded. To ensure the quantification reliability, the calculated IVIM parameter values for those pixels with the goodness of fit R2<0.7 were excluded for the subsequent analysis as well 2 was defined as IVIM parameter was quantified using the asymptotic fitting method f, D, and D*. All regions-of-interest (ROIs) were manually positioned on the b = 0 DW images by a single author who had 5 years' experience reading abdominal MR images. ROIs were positioned to cover a large portion of liver parenchyma while avoiding large vessels , which allowed the extraction of parametric maps of  vessels , with cop value <0.05 was considered significant.Statistical analysis was performed using software SPSS . The mean value of IVIM parameters as well as standard deviation  of each groups are presented. Mann Whitney U test was used for comparison between the IVIM parameter values obtained from healthy volunteer and patients. One-way analysis of variance (ANOVA) and Bonferroni adjustment for multiple comparisons were used in evaluating the IVIM values from different grades of liver fibrosis. For the comparison between different liver fibrosis stages, stage-3 subjects and stage-4 subjects were grouped together for analysis, due to the limited patient number of stage-4. A IVIM DW imaging data were acquired in all subjects. No data suffered from severe motion induced displacements or artifacts hampered IVIM quantification in this study. Curves of IVIM signal-intensity decrease demonstrated biexponential type decay as previously reported D, f, and D* (×10−3 mm2/s) values measured in healthy volunteers and patients liver were 1.096±0.155 vs 0.917±0.152 , 0.164±0.021 vs 0.123±0.029 (p<0.0001), and 13.085±2.943 vs 9.423±1.737  respectively , also its imaging sequence is readily available on all commercial clinical MRI scanners.In patients with early liver fibrosis, the liver parenchyma usually has a normal appearance or may exhibit only subtle, nonspecific heterogeneity on conventional MR images, though later-stage fibrotic liver develops characteristic morphologic alterations such as surface nodularity, widening of fissures, expansion of the gallbladder fossa, notching of the right lobe, atrophy of the right lobe, and relative enlargement of the lateral segments of the left lobe and caudate lobe b-values , and did not calculate pseudo-diffusion values D values, indicating a high contribution of perfusion f to the ADCs. With IVIM techniques, Luciani et al reported that D* was significantly reduced in the liver fibrosis compared with those in the healthy liver group, but there was no significant difference between D and f measurements in the healthy liver (n = 25) and the liver fibrosis (n = 12) groups et al reported that D and D* were significantly lower in steatotic compared with nonsteatotic livers. However, f was significantly higher in steatotic compared with nonsteatotic livers et al reported that f, D and D* in liver cirrhosis were lower than non-cirrhosis liver, however, no further grading was performed within their liver cirrhosis subjects as only 3 patients (out of 14 patients) had histopathology data et al reported that f values decreased significantly with the increasing fibrosis level; but D was poorly correlated with fibrosis level et al reported that as liver fibrosis progressed, D and D* decreased, however there was no change in fet al reported that f was significantly lower in rabbits with nonalcoholic fatty liver disease than in those with a normal liver, and it decreased further as severity of nonalcoholic fatty liver disease increased, on the other hand, D and D* did not differ significantly between the nonalcoholic fatty liver disease severity groups Yamada et al were the first to assess IVIM MRI in abdominal organs, although they used only a limited number of et al showed that the signal decay with the IVIM DW imaging sequence was monoexponential, thus suggesting that the biexponential signal decay encountered in vivo cannot be attributed to a systematic error due to the imaging sequence D f, and D* values were lower in the fibrotic livers compared with the healthy livers, and progressively so as the severity of liver fibrosis increased. To our knowledge, this is the first study such effects are demonstrated in biopsy confirmed human livers in a prospective clinical trial. Compared with the previous patient based studies, our study is characterized by that the coefficient of variation is relatively small, being 0.22 and 0.18 for healthy volunteers and patients respectively, while the patient number was relatively large. Different f and D* values have been reported in literature f and D* values In the phantom part of the study Luciani f and D*. This technical issue was compensated by the NEX of two for acquisition as well as the carefully optimized asymptotic IVIM fitting b-values used and measurement accuracy at low b-value There are a few limitations of this study. Only hepatitis B virus related liver fibrosis were included in this study. The study subject number was small for the stage 4 fibrosis group. For this reason, the ability of IVIM DW imaging to help provide a distinction between patients with scores of stage 3 to stage 4 could not be reliably assessed. Statistical power could be further increased with access to larger sample sizes in future studies. This study was carried out using a 1.5 T scanner. Although the image artifacts, e.g., susceptibility induced artifacts, at 1.5 T are considered less than at 3 T, the relatively low signal-to-noise ratio (SNR) may compromise the precision of the calculated IVIM parameters, particularly for In conclusion, our IVIM MR study shows lower pure molecular diffusion, lower perfusion volume fraction, and lower perfusion-related diffusion in fibrotic livers. Through employing multiple b-values, IVIM analysis has the potential to quantify diffusion and perfusion parameters in liver without the use of contrast agents.


The natural environment is increasingly recognized as an effective counter to urban stress, and “Forest Therapy” has recently attracted attention as a relaxation and stress management activity with demonstrated clinical efficacy. The present study assessed the physiological and psychological effects of a forest therapy program on middle-aged females. Seventeen Japanese females  participated in this experiment. Pulse rate, salivary cortisol level, and psychological indices were measured on the day before forest therapy and on the forest therapy day. Pulse rate and salivary cortisol were significantly lower than baseline following forest therapy, indicating that subjects were in a physiologically relaxed state. Subjects reported feeling significantly more “comfortable,” “relaxed,” and “natural” according to the semantic differential (SD) method. The Profile of Mood State (POMS) negative mood subscale score for “tension–anxiety” was significantly lower, while that for “vigor” was significantly higher following forest therapy. Our study revealed that forest therapy elicited a significant (1) decrease in pulse rate, (2) decrease in salivary cortisol levels, (3) increase in positive feelings, and (4) decrease in negative feelings. In conclusion, there are substantial physiological and psychological benefits of forest therapy on middle-aged females. The term “forest bathing” was proposed in Japan in 1982, and penetrated as words to express for enjoying the comfort of the forest. However, there was little information regarding “What is so psychologically comforting about the forest?” and “What specific psychological and physiological changes are taking place in a body in the forest?” The elucidation of the phenomenon rapidly advanced around the past 10 years, and it developed into the term “forest therapy” programs. Indeed, “forest therapy” is now increasingly recognized as an effective relaxation and stress management activity with demonstrated a preventive medical effect and increased healthy effect among healthy Japanese adults [et al. reported that a brief nature experience decreased both self-reported rumination and neural activity in the subgenual prefrontal cortex (sgPFC) [Several studies have shown that time spent in a forest can decrease blood pressure (BP) ,3,4,5,6, (sgPFC) . These s (sgPFC) .It was also shown that a forest therapy trip can increase human natural killer (NK) cell activity and improve immunity in both males and females, and these effects were found to last for at least 7 days ,15,16,17et al. reported relaxation and stress-management effects of forest environments using several questionnaire-based metrics, in addition to improved mood [Park ved mood . In psycved mood ,10,11,12ved mood ,20,21; hved mood .et al. reported physiological and psychological relaxation effects on hypertensive individuals after a brief walk in the forest [Most field experiments on forest therapy have enrolled only healthy young adults as subjects, while those who need these benefits the most may be older adults at a higher risk of stress- and lifestyle-related diseases such as high BP, diabetes, and depression. Song e forest ; howevere forest  and founSeventeen Japanese females ranging in age from 40 to 73 years  were recruited from the Health Promotion Center in Agematsu, Nagano Prefecture. Inclusion criteria were female aged 40 years or older. Candidates who thought it may be difficult to walk in hot weather were excluded. Six subjects were on medication for hypertension, which was well controlled. All participants were free from other diseases and psychological disorder. Body mass index (BMI) ,23 was cThe forest therapy phase was conducted in Akasawa Shizen Kyuyourin , Agematsu, Nagano Prefecture  on 30 August 2014. The distance from the health promotion center to the forest was 14.6 km, and it took 42 min to drive by car. The weather was cloudy on the forest therapy day, with a mean temperature of 21.5 °C (18.2 °C–27.5 °C) and humidity of 81% (49%–96%).Both systolic and diastolic BP levels and pulse rate readings were obtained from the right arm using a portable digital sphygmomanometer .These procedures were performed between 15:09 and 15:22 on the day before forest therapy and between 14:44 and 14:56 after forest therapy to control for circadian effects.Salivary cortisol, which shows a reliable increase under stress, was measured as an index of endocrine activity. Saliva samples were collected using a saliva collection aid  between 15:28 and 15:35 on the day before forest therapy and between 14:57 and 15:05 after forest therapy. Saliva samples collected at the field site were immediately placed in a freezer and sent to a laboratory  for analysis.The semantic differential (SD) method and a short form of the Profile of Mood State (POMS) were used to evaluate psychological responses to forest therapy. These questionnaires were completed by subjects between 15:00 and 15:20 on the day before forest therapy and between 14:44 and 14:56 after forest therapy. The SD method uses three pairs of adjectives anchoring seven-point scales: “comfortable to uncomfortable,” “relaxed to awakening,” and “natural to artificial” . The shoThe subjects spent the previous night in their respective homes. On the morning of the forest therapy day, the subjects gathered in the same meeting room at 9:00 and participated in the forest therapy program as a group with a guide. They were not permitted to carry cell phones. The program consisted of multiple timed activities over 4 h and 41min  led by aEnergy expenditure was assessed for each activity using Lifecorder GS4 . Tobacco and all drinks  were prohibited during forest therapy. The subjects ate the same lunch made from local ingredients at the same time (11:34–12:24). After the subjects completed the program, they returned to a waiting room for post-treatment measurements and to complete the questionnaires. These results were then compared with those obtained on the previous day.We aimed to compare the physiological and psychological effects of forest therapy with everyday life activities on a normal day. Physiological and psychological inspections were performed at approximately the same time on the day before and on the day of the therapy.t-tests to compare physiological indices and the Wilcoxon signed-rank test to compare psychological test results obtained before and immediately after forest therapy. All statistical analyses were performed using SPSS 20.0 . Data are expressed as the mean ± standard error (mean ± SE). For all tests, p < 0.05 (one sided) was considered statistically significant.We used paired vs. 73.1 ± 2.5 beats/min; t(16) = 4.67, p < 0.01 by paired t-test)  = 2.63, p < 0.05 by paired t-test) (Pulse rate was significantly lower after forest therapy than on the day before forest therapy (baseline) in middle-aged females (69.1 ± 2.7  t-test) . Similar t-test) .vs. 0.71 ± 0.06 kcal/min; t(15) = 1.88, p < 0.10 by unpaired t-test). The mean salivary cortisol level was reduced in the BMI < 25 group after forest therapy  = 3.19, p < 0.01 by paired t-test), but it actually increased slightly in the BMI ≥ 25 group  = 4.01, p < 0.05 by paired t-test).The total energy expenditure during forest therapy was compared between subjects with BMI ≥ 25 (N = 4) and those with BMI < 25 (N = 13). A marginally significant difference was observed between groups, with 24% greater expenditure in the BMI ≥ 25 group compared with the BMI < 25 group (0.88 ± 0.08 p < 0.01), “relaxed” (p < 0.01), and “natural” (p < 0.01) after forest therapy than on the day before forest therapy (p < 0.01) and those for the positive subscale “vigor” (p < 0.01) being significantly higher after forest therapy.Significantly higher SD scores were observed for the adjectives “comfortable”  has been used to diagnose obesity in clinical practice and obesity research and to structure programs and goals for weight loss interventions [For several decades, BMI  in an urban area setting. However, this would be difficult to implement in practice because it involves activities such as “lying down” in an urban area. So the control experiments of the same activities completed indoors are thought to be necessary in future. Interestingly, differences in effect were observed with varying BMI. However, the limited number of subjects in this study decreased the significance of the analysis. Future studies should include a larger number of subjects. Furthermore, forest therapy has not yet been shown to actually reduce the risk of disease independent of the general effects of exercise. It is now necessary to design experiments that test whether forest therapy can reduce disease risk in vulnerable populations through these demonstrated physiological and psychological benefits.Our study revealed that forest therapy elicited a significant (1) decrease in pulse rate, (2) decrease in salivary cortisol levels, (3) increase in “comfortable,” “natural,” and “relaxed” feelings as assessed by the modified SD method, (4) decrease in the POMS negative subscale “tension–anxiety,” and (5) increase in feelings of “vigor” in middle-aged females. In conclusion, walking in a forest according to a standard “forest therapy” program induced physiological and psychological relaxation. These results clarified the physiological effects of the forest therapy program and suggested a possibility of clinical use.