The most dramatic progress in the restoration of hearing takes place in the first months after cochlear implantation. To map the brain activity underlying this process, we used positron emissiontomography at three time points: within 14 days, three months, and six months after switch-on. Fifteen recently implanted adult implant recipients listened to running speech or speech-like noise in four sequential PET sessions at each milestone. CI listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech-like stimuli, unlike CI listeners with prelingual hearing loss. Furthermore, Broca's area was activated as an effect of time, but only in CI listeners with postlingual hearing loss. The study demonstrates that adaptation to the cochlear implant is highly related to the history of hearing loss. Speech processing in patients whose hearing loss occurred after the acquisition of language involves brain areas associated with speech comprehension, which is not the case for patients whose hearing loss occurred before the acquisition of language. Finally, the findings confirm the key role of Broca's area in restoration of speech perception, but only in individuals in whom Broca's area has been active prior to the loss of hearing. The cochlear implant (CI) transforms acoustic signals from the environment into electric impulses, which are then used to stimulate intact fibers of the auditory nerve. With this treatment, individuals with profound hearing loss (HL) are given the opportunity to gain or regain the sense of hearing. Current technology and speech processing strategies allow many CI recipients to achieve impressive accuracy in open-set speech recognition, and the CI is arguably the most effective neural prosthesis ever developed –3. HowevPlasticity is a term used to describe the reorganization of the central nervous system by means of synaptic changes and rewiring of neural circuits. In cases of cochlear implantation, neural plasticity associated with deprivation of auditory input and adaptation to the absence of stimuli is of particular interest. Reduced input to the brain from impaired auditory pathways results in significant changes in the central auditory system  and is aIn normal-hearing (NH) adults, language processing is associated with extensive frontal activation in the left cerebral hemisphere, including the anterior (Brodmann's Areas (BA) 45 and 47) and posterior (BA 44 and 45) parts of the left inferior frontal gyrus (LIFG), the latter often referred to as Broca's area , 20. TraSome observed activations outside the classic language areas, including anterior cingulate, parietal regions, and left hippocampus, have been attributed to nonspecific attentional mechanisms and memory in CI users , 32. FurWith the present study, we tested the cortical mechanisms underlying the restoration of hearing and speech perception in the first six-month period following implant switch-on. We expected to see inactive neuronal pathways reactivated in CI recipients, within three to six months of switch-on, and engagement of cortical areas resembling those of normal-hearing control participants. Furthermore, in previous findings, notwithstanding, we expected to see Broca's area involved in speech perception. Finally, we expected to see a difference in the progress of adaptation and the involvement of cortical areas between CI users with postlingual hearing loss and CI users with prelingual hearing loss.Mage = 51.8, age, range: 21–73 years). All participants were unilaterally implanted. Four participants had a prelingual onset of HL, indicated by their estimated age at onset of deafness and main use of signed language as communicative strategy. The remaining 11 participants had a postlingual or progressive onset of HL, as indicated by their main use of residual hearing, supported by lip-reading. In accordance with local practice, all CI participants followed standard aural/oral therapy for six months in parallel with the study. The therapy program includes weekly one-hour individually adapted sessions and trains speech perception and articulation. Over the course of two years, patients who were approved for implantation were contacted by mail and invited to take part in the research project, including positron emission tomography (PET) and speech perception measures. From a total of 41 patients, 15 accepted and were included in the study  for a single PET/test session. All NH participants met the criteria for normal-hearing by passing a full audiometric test.To obtain a normal-hearing reference, we recruited a group of NH adults  and (2) the prelingual (PRE) HL subgroup (N = 4).NH participants underwent PET once, while CI participants were tested consecutively at three points of time: (1) within 14 days after switch-on of the implant , (2) after three-months , and (3) after six months . For purposes of analysis, two subgroups were identified as (1) the postlingual (POST) HL subgroup .MRI. A high-resolution T1-weighted MR scan was acquired prior to PET scanning. In the case of CI participants, this was performed preoperatively. Stimuli. All participants were examined in 2 conditions: (1) multitalker babble (BAB) from multiple simultaneous speakers with a complexity close to that of speech and perceived by the listeners as speech-like but devoid of meaning [http://audacity.sourceforge.net/) and delivered directly from the computer's headphone jack to the external input port of the implant speech processor. Bimodally aided participants removed their hearing aid and were fitted with an earplug in the nonimplanted ear during the tomography. These measures were taken to preclude background noise and possible cross-talk from the contralateral ear. The NH participants listened to the stimuli binaurally through a pair of headphones . All stimuli were presented at the most comfortable level. To define this level, participants were exposed to the two stimuli once before the tomography. In the tomograph, prior to bolus injection, participants had no information about the nature of the next stimulus, but they were instructed to listen attentively in all cases. After each of the four scans, participants were required to describe what they had heard and, if possible, review the content of the narration.  meaning  and (2)  meaning . The stiPET. Positron emission tomography (PET) is a molecular imaging method that yields brain activity, by means of detecting changes in regional cerebral blood flow (rCBF). This is done by computing and comparing the spatial distributions of the uptake of a blood flow tracer. PET measurements are generally limited with respect to spatial and temporal resolution and the invasiveness of the procedure, which requires injection of oxygen-15-labelled water. In this case, however, anatomical and temporal specificity could not have been improved by using functional magnetic resonance imaging (fMRI), as the auditory implants are not MRI compatible. In addition, PET is an almost noiseless imaging modality, which is useful for both CI participants and for the study of speech. Finally, because only the head of the participant is positioned in the tomograph, compared with the whole body imposition of fMRI, it is possible to communicate visually with the participant during tomography.2  15O oxygen-15-labeled water, which matches the distribution of cerebral blood flow (CBF), using an ECAT EXACT HR 47 Tomograph (Siemens/CTI). Emission scans were initiated at 60,000 true counts per second after repeated intravenous bolus injections of doses of tracer with an activity of 500 MBq (13.5 mCi). The tomography took place in a darkened room with participants' eyes closed.We measured raised or reduced cerebral activity as the change of the brain uptake of HThe babble and running speech conditions were duplicated, generating a total of four tomography sessions. The uptake lasted 90 seconds (single frame) at intervals of 10 min. Each frame registered 47 3.1 mm sections of the brain. After correction for scatter and measured attenuation, each PET frame was reconstructed with filtered back projection and smoothed with a postreconstruction 10 mm Gaussian filter resulting in a resolution of 11 mm full-width-at-half-maximum (FWHM).Rules of regulation mean that participants who volunteer for scientific experiments may receive a total maximum radiation of 6 millisieverts (mSv) within one year. Here, the total radiation dose administered over the three times of scanning was approximately 5.58 mSv. Due to these restrictions, no preoperative baseline scans could be acquired. Participants' MR images were co-registered to an MR template averaged across 85 individual MR scans in Talairach space , using ahttp://www.fil.ion.ucl.ac.uk/spm/)). Local maxima of activation clusters were identified using the Montreal Neurological Institute (MNI) coordinate system and then cross-referenced with a standard anatomical brain coordinate atlas [P < 0.05, family wise error (FWE) corrected for multiple comparisons. We tested the effect of side of implant and type of implant in a separate preanalysis. As we found neither main effects nor interactions with functional data involving these variables, we concluded that these factors had no significant effect on the results. They were thus not included in further analyses.All images were processed using Statistical Parametric Mapping 8  and possible interactions between these effects in the whole brain, across CI participants. This analysis was performed as a single SPM matrix in a factorial 3-way design with time, contrast, and subgroup as factors. To define a region-of-interest (ROI), we created a mask based on the main effect of contrast. Analysis  2. The second analysis identified possible main effects of contrast, time, and interactions between these factors in the inferior frontal gyrus (IFG), more specifically Broca's area (BA 44/45). This analysis was performed as two 2-way factorial analyses of the POST HL and the PRE HL subgroups separately. To define a region-of-interest (ROI), we created a mask based on bilateral inferior frontal gyri (Broca's region), including the putative Brodmann regions 44, 45, and 47 using the WFU pick-atlas [ck-atlas . Analysis  3. The third analysis investigated main effects of contrast and group (CI versus NH) at the CI baseline and possible interactions between these effects. This analysis was performed as a single SPM matrix in a factorial 2-way design with condition and group as factors. To define a ROI, we created a mask based on the main effect of contrast.F = 60.14). A t-test confirmed that the effect was driven by higher activity during running speech  = 20.42)  . A plot = 20.42) .t-test confirmed that the effect was driven by higher activity during running speech. Furthermore, we found a main effect of time in left IFG  = 14.19; P = 0.006, FWE corrected)  , with norrected) .t-tests showed that the superior temporal gyri bilaterally and the left middle temporal gyrus were more active during running speech, while the right inferior parietal lobule was more active during babble. We found a main effect of CI versus NH exclusively in the caudate nucleus. A t-test showed that this effect was due to higher activity of this area in the NH group than in the CI group. No interaction was found between the effect of contrast and the effect of group in whole-brain analysis  = 19.62, P < 0.001), but not in the PRE group. Post hoc tests showed that the effect was driven by the 51.3 percentage points endpoint gain . Furthermore, the POST group scored significantly higher than the PRE group at all three points of measurement . Ceiling performance (100% correct) was observed in all NH participants  were significantly more active when participants listened to running speech than when they listened to multi-talker babble. Thus, on average, the auditory brain regions, known to be involved in the processing of complex auditory stimuli –51 displFurthermore, the results showed a difference in the way CI recipients with postlingual hearing loss and recipients with prelingual hearing loss distinguish between speech and babble. The CI users with postlingual hearing loss displayed a greater activation during speech than during babble in BA 21 and 22 in the left superior temporal gyrus, indicating differential processing of the stimuli by the two subgroups. We speculate that the postlingually deaf listeners disengage attention when they are presented with the incomprehensible babble stimulus. This disengagement is then reflected in decreased temporal brain activity. In contrast, the prelingually deaf CI listeners may be equally attentive to the two stimuli, regardless of their nature, as reflected in undifferentiated activity.This difference between postlingual and prelingual deafness was mirrored in the behavioral measures. The postlingual subgroup not only possessed a moderate level of speech perception at baseline (within 14 days after switch-on of the implant), but also made significant gains in performance, the majority of which occurred in the first three month period. In contrast, the prelingual subgroup had no baseline speech perception and only modest, if any, progress during the study period. This finding is consistent with expectation and implies an association between behavioral performance and brain activity related to the history of hearing loss. In prelingual deafness, the neuronal connections of the auditory pathways  may not be established in the appropriate time window of opportunity –56. The We found a main effect of time exclusively in Broca's area, and only in the postlingual subgroup. This is in line with recent studies showing that, in CI users, the activation of Broca's area during speech processing is negatively correlated with the duration of deafness and positively correlated with the progress in the restoration of speech comprehension , 37, 59.Our whole-brain analysis revealed a significant activation of bilateral superior temporal gyri and left middle temporal gyrus during speech across CI participants at baseline and NH individuals. This is partly consistent with Naito et al. , who, inThe significant involvement of the right parietal lobule in the CI participants during babble suggests that, at this initial stage of the CI adaptation, CI listeners, unlike NH individuals, need to pay attention to the speech-like noise to determine its possible character . The obsIn contrast to the significantly higher right-lateralized activation observed in NH individuals in the present study, Giraud et al.  showed aGiraud and colleagues  consisteIn Scandinavia as in most European countries, cochlear implantation is administered by the public health care system and offered for free to all patients who meet the clinical implantation criteria (<40% open-set word-recognition scores). This includes patients with a prelingual hearing loss, despite recognition that these patients may have very limited linguistic benefit. As a result of this policy, the group of CI users in general is very heterogeneous, which was also the case in this study. While the difference in group size was far from optimal and may have confounded the results, we maintain that it is of high importance to also study cortical activity in prelingually deaf patients following cochlear implantation. In a previous study involving music training, we found that while little gain was achieved in speech perception, prelingual deafness did not preclude acquisition of some aspects of music perception . This imOur normal-hearing control group had a less optimal size, which may have made direct comparisons between groups less valid than desired. However, ethical restrictions limit the number of healthy participants in studies involving PET.As stated, the degree of deafness  varied across subjects and may have influenced the adaptation to the implant. Unfortunately, a correlation analysis between preoperative speech understanding and PET and behavioral results was not possible since such data were not available for all participants.The present PET study tested brain activation patterns in a group of recently implanted adult CI recipients and a group of normal-hearing controls, who listened to speech and nonspeech stimuli. CI listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech-like stimuli, unlike CI listeners with prelingual hearing loss. This group difference was also reflected in a behavioral advantage for patients with postlingual hearing loss. Furthermore, Broca's area was activated as an effect of time, but only in CI listeners with postlingual hearing loss. Comparison of the CI listeners and the normal-hearing controls revealed significantly higher activation of the caudate nucleus in the normal-hearing listeners. The study demonstrates that processing of the information provided by the cochlear implant is highly related to the history of hearing loss. Patients whose hearing loss occurred after the acquisition of language involve brain areas associated with speech comprehension, which is not the case for patients whose hearing loss occurred before the acquisition of language. Finally, the findings confirm the key role of Broca's area in restoration of speech perception, but only in individuals in whom Broca's area has been active prior to the loss of hearing.


Few have studied how personal activities of daily living (P-ADL) develop over time in nursing home residents with dementia. Thus, the aim was to study variables associated with the development of P-ADL functioning over a 52-month follow-up period, with a particular focus on the importance of the degree of dementia.In all, 932 nursing home residents with dementia  were included in a longitudinal study with four assessments of P-ADL functioning during 52 months. P-ADL was measured using the Lawton and Brody’s Physical Self-Maintenance Scale. Degree of dementia (CDR), neuropsychiatric symptoms and use of psychotropic medication were assessed at the same four time points. Demographic information and information about physical health was included at baseline. Linear regression models for longitudinal data were estimated.Follow-up time was positively associated with a decline in P-ADL functioning. Degree of dementia at baseline was associated with a decline in P-ADL functioning over time. The association between degree of dementia and P-ADL functioning was strongest at baseline, and then flattened over time. A higher level of neuropsychiatric symptoms such as agitation and apathy and no use of anxiolytics and antidementia medication were associated with a decline in P-ADL functioning at four time points. Higher physical co-morbidity at baseline was associated with a decline in P-ADL functioning.P-ADL functioning in nursing home patients with dementia worsened over time. The worsening was associated with more severe dementia, higher physical comorbidity, agitation, apathy and no use of anxiolytics and antidementia medication. Clinicians should pay attention to these variables (associates) in order to help the nursing home residents with dementia to maintain their level of functioning for as long as possible. Dementia is, in most cases, characterized by a progressive decline in cognitive function. In Europe, it is estimated that between six to ten million persons have dementia. The preIn general, dementia will negatively affect the person’s ability both to function adequately in everyday life and to perform personal activities of daily living (P-ADL), such as bathing, dressing, eating, grooming, ambulation/transferring and toileting-17.Dementia and the accompanying P-ADL impairment increase the risk of being admitted to nursing home care,19 as inUp to now, relatively few studies have explored how P-ADL functioning changes over time in nursing home residents with dementia. An earlA recent six-month follow-up study of long-term residents with dementia reported that a decline in P-ADL functioning was associated with the patients’ degree of dementia. HoweverA number of disease specific and generic P-ADL indexes have been developed, validated and found sensitive to small but significant changes in persons’ ability to perform P-ADL,38. EvenTo our knowledge no long-term follow-up of nursing home residents has been conducted including both a measure of cognition and physical co-morbidities. Thus, our aim was to study the association between degree of dementia, both at baseline and over more than four years, and the development in P-ADL functioning measured by the Physical Self-Maintenance Scale (P-ADL score), adjusting for a number of other variables know to have an influence on P-ADL functioning in nursing home residents with dementia. We hypothesize that P-ADL functioning in persons with dementia living in nursing homes will decline over time, and that this decline will be associated with a worsening of dementia, the neuropsychiatric symptom load, medication and physical co-morbidity.This was a 52 months prospective study with four assessments. Baseline assessment took place between November 2004 and January 2005. The follow-up assessments took place after 12, 31 and 52 months.All residents in 26 nursing homes in 18 municipalities and four counties in Norway with a stay of a minimum of 14 days were asked to participate in the study. The municipalities were chosen to make the sample representative in terms of municipality size. Thus, 9The Demographic information age, level of education, marital status and length of stay at inclusion, was collected from the medical records.th Revision (ICD-10). The diagnoses were sorted into diagnostic groups, i.e. stroke-, cardiovascular-, pulmonary-, musculoskeletal-, digestive-, endocrine-, neurological-, genito-urinal- and neoplasm diagnoses[The Medical health information was collected from the medical records with the use of diagnoses in the International Statistical Classification of Diseases and Related Health problems 10iagnoses. A co-moSensory loss was registered by two single items for vision and hearing impairment, respectively, i.e. perceived severely impaired or not. No specific measurement was used and the occurrence of vision and hearing impairment are based on the primary professional caregiver’s clinical judgement.Dementia was assessed using the Clinical Dementia Rating (CDR) scale which covers six domains  with five response categories ,43. The Neuropsychiatric symptoms were assessed using the Neuropsychiatric Inventory (12-item NPI) in a traPsychotropic medications were grouped according to the ATC code, into antipsychotics (N05A except lithium), antidepressants (N06A), anxiolytics (N05B), hypnotics/sedatives (N05C) and antidementia medication (N06D) (yes versus no). The information was collected from the medical record of each resident.Level of functioning in personal Activities of Daily Living (P-ADL), the dependent variable, was measured by the Physical Self-Maintenance Scale . High scPrior to the study, 16 research assistants  attended a two-day training program on how to conduct the interview. A one-day training program was carried out prior to each follow-up assessment. The project leader (GS) was available for consultation while the data were collected. The research assistants collected the data by means of a standardized interview with the primary professional caregiver and by extracting information from the medical records.Study information was given to the patient and their family members. An explicit consent was not required for enrollment, but the patients and their next of kin were informed in writing that they could refuse to participate at any stage of the study. The study, and the procedure for information and the right to decline participation were approved by the Regional Ethics Committee of Eastern Norway in 2004, the Data Inspectorate and the Directorate for Health and Social Affairs.Continuous socio-demographic and clinical characteristics were presented as means and standard deviations (SD), while frequencies and proportions were used for categorical characteristics. A linear regression model was estimated to assess the development in P-ADL throughout the follow-up period of 52 months. As P-ADL consists of repeated measurements, the assumption of independent observations required for ordinary linear regression was violated. Therefore, a linear mixed model containing both linear and second–order time components as fixed effects (growth model) as well as random effects for intercepts and slopes for time was estimated first. Such a model accounts for correlations due to repeated measurements and accommodates any degree of imbalance in longitudinal data. That is, the model is particularly suited for analysing data with missing values and drop-outs due to death, for instance. The SAS MIXED procedure was used to fit the model. The main independent variable, degree of dementia assessed with CDR sum of boxes, and interaction term between degree of dementia and time were then entered into the model as fixed effects. Two different models with respect to adjustment were further estimated. In both models, adjustment variables were demographic characteristics , physical health information , neuropsychiatric symptoms and use of psychotropic medications. Length of stay, age, and gender were defined to be the confounders, while the other adjustment variables were defined as secondary independent variables. The first model contained adjustment variables all measured at baseline. The second model included demographic characteristics, somatic health difficulties at baseline, while neuropsychiatric symptoms of dementia and use of psychotropic medications were entered as longitudinal variables. The following modelling strategy was employed. Firstly, a model containing all defined secondary independent variables was reduced by applying Akaike’s Information Criteria (AIC). The least significant secondary independent variables were removed from the model one at a time and AIC was calculated at each step. The model with the smallest value of AIC was chosen. Secondly, the CDR sum of boxes, interaction term between the CDR sum of boxes and time, and the three confounders were entered into the reduced model and a final model was estimated. Lastly, to assess eventual gender-specific differences, interactions between gender and CDR sum of boxes, age, and education were entered into the final model. However, none of them were significant and therefore excluded. The results of the regression analysis were tabulated as coefficients with the corresponding 95% confidence intervals (CI). Regression coefficients were used to estimate the P-ADL score for each time point from an unadjusted model as well as from both adjusted models at the median baseline CDR sum of boxes value and were presented graphically.Analyses were performed in SAS v9.2 and SPSS v20. P-values below 0.05 were considered statistically significant. All tests were two-sided.At baseline, the mean (SD) age and length of stay for the participants were 84.5 (7.5) years and 932 910.1) days, respectively (see Table 0.1 days,The importance of degree of dementia for P-ADL functioning over time was studied in two adjusted models. In the first adjusted model Table , the assTable In addition, longer length of stay, being married, having a higher comorbidity index score and having severely impaired vision at baseline were associated with worse P-ADL functioning through the observation period. Among the independent variables with assessments at all four time points included in the analysis, a higher agitation sub-syndrome score and apathy score, as well as not using anxiolytics and antidementia medication were associated with lower P-ADL functioning at the same time points.Figure This follow-up study of 932 nursing home residents with dementia found that degree of dementia at baseline and the course of dementia as measured by the CDR during follow-up was significantly associated with lower P-ADL functioning. The rate of decline in P-ADL functioning explained by the degree of dementia decreased during follow-up. Independently of the adjustments made, there is a decline in P-ADL function over time and the rate of decline due to time of follow-up decreased during follow-up.Other authors have pointed to a relationship between more severe dementia and worse P-ADL both among older hospitalized patients, communiInterestingly, we found that worse P-ADL functioning at each time point was associated with a higher level of neuropsychiatric symptoms at the same time points, i.e. higher agitation sub-syndrome or apathy symptoms. Our findings are in line with previous research. Previously, it has been reported that a higher baseline sum score of neuropsychiatric symptoms is associated with a higher tendency for falls the coming year and apatIn contrast to prospective studies which have not studied the importance of physical health in nursing home residents for the association between degree of dementia and P-ADL functioning or have excluded residents with co-morbid physical disorders,29-32, tEven if the study has a number of advantages, such as using a well-known, internationally accepted P-ADL measure, having a high number of baseline residents and adjustment for a high number of variables of potential importance to the outcome, such as health and demographic variables, there are some limitations that need to be addressed. Firstly, associations found in our study should be interpreted with caution since our design does not allow for inferences about causality.Secondly, assessments of P-ADL functioning of older persons in a longitudinal study with several follow-ups over a period of years are statistically challenging. Repeatedly measured P-ADL functioning for the same individuals implies dependency in data. Having a large number of drop-outs, due to death leads to a varying number of observations per individual and generates unbalanced longitudinal data. Furthermore, a number of the independent variables in the adjusted models are time-varying. However, the linear mixed model used is particularly flexible with respect to these issues. The model handles any degree of imbalance in data by including all available observations. It also accounts for the correlations among repeated measurements in a relatively parsimonious way.Thirdly, even if information about use of each category of psychotropic medications were available, we did not have the dosages of the psychotropic medications. Moreover, use of a well-known comorbidity index, f. ex. Charlson comorbidity index,58 ratheIn a large-scale longitudinal nursing home study among patients with dementia, using four assessments during a period of 52 months, we found that P-ADL functioning worsened over time. The worsening of P-ADL functioning was associated with a higher baseline degree of dementia and with an increasing degree of dementia during follow-up. The importance of degree of dementia and follow-up time for degree of decline in P-ADL functioning decreased during follow-up.Clinicians should pay attention to the associates of P-ADL functioning identified in our study in order to help the nursing home residents with dementia maintain their level of functioning as long as possible.The authors declare that they have no competing interests.GS and KE designed the study, and GS was responsible for the data collection and quality as e study results. JSB analyzed the majority of the data for this manuscript. ASH has analyzed sections of the data and drafted the manuscript. All authors participated in the interpretation of the study results and in editing the manuscript, and have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/14/45/prepub


Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs  were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568  in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.Genome sequence of  For the complete hydrolysis of chitin to GlcNAc, concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) is essential. Chitin was extracted as two allomorphs, namely α- and β- forms β-N-acetylglucosaminidases. Chitinase genes from bacteria have been cloned from both terrestrial and marine environments Chitin is a highly insoluble Sm CBP21 Sm CBP21 revealed that the protein has a “budded” fibronectin type 3-fold consisting of two β-sheets, arranged as a compact β-sheet sandwich-fold surface, having a at conserved region that binds chitin through interactions mediated mainly by polar amino acids Sm CBP21 was designated as “chitin oxidohydrolase” as it acts on the surface of crystalline chitin, to introduce chain breaks and generates oxidized chain ends, promoting further degradation by chitinases Efficient chitin degradation also depends on the action of a family 33 chitin binding proteins (CBPs). The CBPs bind to the insoluble crystalline chitin, leading to structural changes and increased accessibility of substrate. The function of family 33 CBPs was first demonstrated for S. proteamaculans 568, a member of family Enterobacteriaceae, was isolated as a root endophyte from Populus trichocarpahttp://www.cazy.org/) S. proteamaculans 568 has at least eight genes involved in chitin turnover, coding for four family 18 chitinases , three family 33 CBPs , and a family 20 N-acetylhexosaminidase (Sp CHB). The present study, describes the cloning and characterization of three CBPs from S. proteamaculans 568 and their synergy with Sp chitinases in degradation of natural chitin variants.Sp cbp genes were amplified using gene specific primers with gDNA of S. proteamaculans 568 as template. The three Sp CBPs were predicted to contain N-terminal leader peptide directing sec-dependent secretion. Signal peptide was predicted using the SignalP server . The genes were cloned without the signal peptide-encoding portion . The amplicons 0.51 kb of Sp cbp21, 0.76 kb of Sp cbp28 and 1.35 kb of Sp cbp50 were cloned in the Nco I and Xho I sites of pET 22b (+) and Eco RI and Xho I sites of pET- 28a (+), respectively.Three Sp CBPs were over expressed with a C-terminal His-tag in E. coli. The expressed Sp CBPs were separated either from periplasmic fraction (Sp CBP21 and Sp CBP28) or from whole cell lysate (Sp CBP50) as soluble proteins, and purified using Ni-NTA agarose chromatography. The PelB signal sequence in pET-22 b (+) directs the expressed Sp CBP21 and Sp CBP28 proteins towards periplasmic space. SDS-PAGE analysis of the purified Sp CBPs revealed approximate molecular weight of 18.6, 28.0 and 50.0 kDa, which correspond to Sp CBP21, Sp CBP28 and Sp CBP50, respectively . Both Sp CBP21 and Sp CBP50 bound equally high to β-chitin (86.2% and 77.0%), followed by colloidal chitin (68.8% and 65.6%), α-chitin (30.9% and 25.6%) and Avicel (25.9% and 19.3%) (The binding preference of d 19.3%) .Sp CBPs as a function of time to find the time required for the Sp CBPs to get saturated with natural chitin variants. After separating the protein bound to chitin, the decrease in concentration of the unbound protein (remaining in the supernatant) was monitored at different time points up to 24 h. The binding of Sp CBP21 to β-chitin occurred rapidly and reached equilibrium within 6 h, while Sp CBP50 reached equilibrium by 12 h. On the other hand, Sp CBP21 and Sp CBP50 have established binding equilibrium to α-chitin by 12 h (The time course of binding was monitored for both the  by 12 h .Sp CBP21 and Sp CBP50 towards α- and β-chitin were estimated and plotted with fixed concentration of substrate and varied concentrations of the CBPs. The dissociation binding constants (Kd) of α- and β-chitin were estimated from the non-linear regression function. The Kd value of the Sp CBP21 to α-chitin (5.31±1.03 µM ) was much lower than the Kd value of Sp CBP50 to α-chitin (9.34±1.67 µM ), whereas the Kd value of Sp CBP21 to β-chitin (2.22±0.45 µM) was slightly lower than the Kd value of Sp CBP50 to β-chitin (2.37±0.5 µM)  . Bindingbstrates .Sp CBP21, Sp CBP28 and Sp CBP50 with Sm CBP21 displayed 93%, 55% and 18% identity, respectively. The surface-located polar residues of Sm CBP21 Sp CBP21  and Sp CBP50 . Sp CBP28 had only one (Asp-176) matching residue . Sp CBP50 displayed only 55% of identity to Sm CBP21. The 3D structure models of Sp CBP21 and Sp CBP50 were generated using the template structure of Sm CBP21 (PDM ID: 2BEM)  of 0.182 Å and 0.164 Å for Sp CBP21 and Sp CBP50. Closer inspection of these structures revealed that the conformations of the several regions  were differing from the template structure  . We havetructure . SuperimSp chitinases  on natural chitin variants was estimated in the presence/absence of Sp CBP21/Sp CBP50 and/or an external electron donor (reduced glutathione). The results indicate that, upon addition of Sp CBP21/Sp CBP50, efficiency of hydrolysis of α- and β-chitin by all the four Sp chitinases increased, especially in the presence of a reduced glutathione. Combination of both enzymes and the external electron donor led to total solubilization of β-chitin within 24 h, whereas only small fraction of the α-chitin was solubilised under the same conditions. The reactions shown in Sp CBPs showed optimum binding at 35°C-40°C, while all four Sp chitinases were also optimally active at the same temperature range. So, the synergistic experiments were carried out at 37°C (data not shown).The hydrolytic efficiency of four Sp CBP50 with Sp chitinases was lower when compared to the Sp CBP21 in degrading natural chitin variants. This was almost compensated when reduced glutathione was supplemented to the Sp CBP50. The product formation efficiency by Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD increased by 0.38, 1.04, 0.99 and 5.75-fold, respectively on α-chitin in presence of Sp CBP21 and reduced glutathione  are known to occur as discrete domains in chitinases and also exist independently as CBPs grouped in families 14, 18, and 33. Families 14 and 18 constitute small anti-fungal proteins that share a structurally similar chitin-binding motif S. proteamaculans 568 revealed the presence of genes coding for three CBPs of family 33. The Sp CBP21 was designated according to its homology to the reported Sm CBP21. Sp CBP21 showed high sequence identity to Sm CBP21 (93%). There were no reports on the presence of additional CBPs in S. marcescens. The two additional CBPs of S. proteamaculans were designated as Sp CBP28 and Sp CBP50 according to the estimated molecular weights  of these two proteins. S. proteamaculans 568 codes for at least 3 CBPs, while others produce either one or two CBPs (Sp CBPs were arranged distantly in the genome of S. proteamaculans 568 and shares conserve 8 bp regions (5′-C(C/T/A) C(C/T) (T/G) G (C/A) (C/G)-3′) in the upstream sequences with other Sp chitinolytic genes (data not shown). Therefore, these Sp cbp genes might be co-ordinately controlled by the same regulatory protein(s) along with other Sp chitinolytic genes. Since S. proteamaculans 568 produced additional CBPs, characterization of CBPs in terms of their binding properties as well as synergism with Sp chitinases in chitin degradation was investigated.Analysis of genome sequence of two CBPs . The thrThe amino acid sequence of Sp CBP21 was BLAST at NCBI database to search for homologs. The result displayed 93% identity to Sm CBP21 (BAA31569), 57% to CBP from Bacillus cereus G9241 (EAL13960) and CBP from B. thuringiensis serovar tochigiensis BGSC 4Y1, 44% to Cbp21 from from B. anthracis str. CDC 684 (ACP12567), 41% to CHB2 from Streptomyces reticuli (EEM22267), 30% to CbpD from Stenotrophomonas sp. SKA14 (EED38588) and 27% to CbpD from Pseudomonas aeruginosa (AF196565). Sp CBP21 contained a signal peptidase site between amino acid residues Ala-27 and His-28. ChBD (Chitin binding domain) was present from His-28 through Asn-194.Sp CBP28 showed 36% identity to CBP1 from Pseudoalteromonas piscicida (BAB79619) and CBP from Vibrio cholerae NCTC 8457 (EAZ71564), 33% to CBP from B. thuringiensis BMB171 (ADH07314) and CBP from B. cereus ATCC 1457 (AAP09751), and 31% to CBP from Streptomyces sp. e14 (EFF90245). A signal peptidase site was located between amino acid residues Ala-22 and Gln-23 of Sp CBP28. ChBD was present from His-39 through Asn-273.The BLAST search for Sp CBP50 homologs displayed 62% identity to N-acetylglucosamine-binding protein A from Enterobacter cloacae subsp. cloacae ATCC 13047 (ADF60226), 57% to CBP21 from S. marcescens (BAA31569), 46% to CBP from Shewanella sp. HN-41 (ABK37291), 45% to CBP from Aeromonas hydrophila subsp. hydrophila ATCC 7966 (ABK37291), 40% to CHB2 from Streptomyces reticuli (CAA74695), and 24% to CbpD from Vibrio harveyi HY01 (EDL70242). Sp CBP50 contained a signal peptidase site between amino acid residues Ala-21 and His-22 and ChBD present from His-22 through Asp-188.Sp CBPs to chitin variants and cellulosic substrates revealed that Sp CBP28 did not to bind to the test substrates. Sp CBP21 and Sp CBP50 were similar to the Sm CBP21, with maximum binding to β-chitin followed by α-chitin, colloidal chitin and Avicel , and other family 33 CBPs have tryptophan e.g. Trp57 in CHB1 from St. olivaceoviridis, which has been shown to be important for the ability of CHB1 to bind α-chitin Sm CBP21. The closest homologue of LlCBP33A is ChbB from B. amyloliquefaciens (66% sequence identity), which binds both α- and β-chitin Sm CBP21 in the same two positions as LlCBP33A: Tyr54 is replaced by Asp62, and Glu55 is replaced by Asn63.The difference in substrate preference was mainly attributed to the difference in the amino acid sequence of respective CBPs. The only available three-dimensional structure of close to Sp CBPs with Sm CBP21revealed that all the amino acid residues that are important in chitin binding Sp CBP21  and Sp CBP50 , while Sp CBP28 showed only one conserved residue (Asp-176)  . MinimumSp CBP21 and Sp CBP50 to β-chitin occurred rapidly and reached equilibrium within 6 and 12 h, respectively. Sm CBP21 established binding equilibrium after 16 h of incubation Sp CBP21 and Sp CBP50 showed relatively slow binding to α-chitin and reached equilibrium by 12 h. LlCBP33A from Lactococcus lactis subsp. lactis established binding equilibrium by approximately 24 h of incubation with both α- and β-chitin Kd values of Sp CBP21 and Sp CBP50 indicate that both these CBPs have high binding strength towards the β-chitin in comparison with α-chitin. Sp CBP21 and Sp CBP50, Kd values towards the β-chitin were relatively higher, while Bmax values were lower when compared to the Kd and Bmax values of reported CBPs from S. marcescens and B. thuringiensis serovar konkukianSp CBPs bound to soluble substrates, as observed for Sm CBP21 by Vaaje-Kolstad et al., The binding of Sm CBP21 catalyzes cleavage of glycosidic bonds in crystalline chitin Sm CBP21. Vaaje-Kolstad et al., Sm chitinases . The Sm CBP21 strongly promoted hydrolysis of crystalline β-chitin by Sm ChiA and Sm ChiC, while Sm ChiB it was essential for complete degradation, and Sm CBP21 activity was boosted by external electron donor LlCBP33A and EfCBM33A increased the hydrolytic efficiency of LlChi18A and EfChi18A, respectively to both α- and β-chitin. These results show the general importance of CBPs in chitin turnover.Sp chitinases, Sp ChiA, Sp ChiB and Sp ChiC released chitobiose as major end product Sp ChiD released GlcNAc from chitin substrates (based on HPLC) (data not shown). Hydrolysis of natural chitin variants by four Sp chitinases in the presence of Sp CBP21 and Sp CBP50 showed that efficiency of all the four Sp chitinases increased with both α- and β-chitin (Sp chitinases were less active on α-chitin (data not shown) and Sp CBP21 and Sp CBP50 had only minor binding preference to α- chitin. Therefore, there was no significant increase in the product formation on α-chitin. The addition of Sp CBP21 and Sp CBP50 had only minor effect on hydrolysis of β-chitin by Sp ChiA and Sp ChiC, while efficiency of Sp ChiB and Sp ChiD increased significantly high. These results are in line with the earlier report on Sm ChiB Sm CBP21 in β-chitin degradation. Interestingly, in both the organisms, the cbp21 gene is located 1.5 kb downstream to the Sm chiB. It was even reported that Sm CBP21 was produced along with three chitinases, Sm ChiA, Sm ChiB and Sm ChiC Sp ChiD obtained major benefit from Sp CBP21 and Sp CBP50. Sp ChiD is a single GH18 domain enzyme that exhibited lower activity on both α- and β-chitin when compared to other Sp chitinases. CBP-mediated enhancement of substrate availability increased the efficiency of Sp ChiD. Sp CBP21 and Sp CBP50 are useful to convert the chitin biomass into production of chitooligosaccharides, which are useful in agriculture, food, and pharmaceutical industries. The uniqueness of Sp CBP28 is being investigated in terms of its importance in biology of S. proteamaculans 568.Among the four β-chitin . The Sp Pfu DNA polymerase were from MBI Fermentas . Primers were procured from Eurofins India . Isopropyl-β-D-thiogalactoside (IPTG), ampicillin, kanamycin, chloramphenicol and all other chemicals were purchased either from Sigma–Aldrich , or Merck , or Hi-media labs . The polymeric substrates α- and β-chitin were kindly provided by Mahtani Chitosan . Colloidal chitin (CC) was prepared according to Berger and Reynolds Restriction enzymes, T4 DNA ligase, and Serratia proteamaculans 568 was grown in Luria-Bertani (LB) broth at 28°C for 16 h for the extraction of gDNA . Plasmid vectors pET 22b(+), pET 28a(+), and Escherichia coli Rosetta-gami 2(DE3) were used as vector and host  for expression, respectively. To express CBP genes, E. coli Rosetta-gami 2(DE3) carrying pET 22b(+) or pET 28a(+) was grown in LB broth with ampicillin (100 µg/mL) and chloramphenicol (25 µg/mL) or kanamycin (50 µg/mL) and chloramphenicol (25 µg/mL), respectively.Sp cbp21, Sp cbp28 and Sp cbp50; GenBank accession no. ABV42576.1, ABV42205.1, and ABV43333.1, respectively) were amplified from the gDNA by referring to the annotated sequence of S. proteamaculans 568 at 55°C annealing temperature using gene specific forward and reverse primers listed in Nco I and Xho I (pET 22b(+), Sp cbp21 and Sp cbp28), and Eco RI and Xho I (pET 28a(+) and Sp cbp50), gel purified and ligated using T4 DNA ligase at 16°C for 16 h. The resultant plasmids were designated as pET 22b–Sp cbp21, pET 22b–Sp cbp28 and pET 28a–Sp cbp50 to express Sp CBP21, Sp CBP28 and Sp CBP50, respectively in E. coli.Three genes encoding CBPs . The cells were lysed by sonication at 20% amplitude with 30×15 s pulses (with 20 s delay between pulses) on ice, with a Vibra cell Ultrasonic Processor, converter model CV33, equipped with a 3 mm probe . To pellet the insoluble cell debris, sonicate was centrifuged at 15,200× g for 10 min at 4°C. The expressed protein was purified using Ni-NTA column as the expressed protein having C-terminal His-tag. After purification, the Sp CBPs were buffer exchanged with 50 mM sodium phosphate buffer pH 7.0 using Macrosep Centrifugal Devices , and stored at 4°C until use.Expression and purification of Sp CBPs were quantified by BCA (bicinchonic acid) protein assay kit  using a standard calibration curve constructed from BSA . For the chitin binding assay, protein concentration was measured from the absorption at 280 nm using the molar extinction coefficients (ε) calculated from the amino acid composition of the protein as described by Pace et al., Purified Sp CBPs was done as described by Vaaje-Kolstad et al., Insoluble substrate binding of Sp CBP21 and Sp CBP50 (described above) was getting saturated with natural chitin variants (α- and β-chitin) was assessed at different time points up to 24 h.To study the time at which the binding of Sp CBP21 and Sp CBP50, towards α- and β-chitin, were obtained as described by Vaaje-Kolstad et al., Sp CBP, up to 10.0 µM, was incubated with α-/β-chitin for different saturation periods: 12 h for α-chitin and 6 h for β-chitin with Sp CBP21, and 12 h for both α- and β-chitin with Sp CBP50.Adsorption isotherms of Sp CBPs and non-interacting BSA) were electrophoresed in 8.0% polyacrylamide gels impregnated with substrates (glycol chitin or laminarin or CM-cellulose) under non-denaturing conditions at 4°C. The gels were visualized by staining with Coomassie blue G-250.Binding of Sp CBPs to soluble polysaccharides  was evaluated by affinity electrophoresis as described by Hardt and Laine Sp CBPs from S. proteamaculans were aligned with Sm CBP21 using clustalw2 . A 3D structure models of Sp CBP21 and Sp CBP50 were generated using the template structure of Sm CBP21 (PDM ID: 2BEM) by Modeller9v8 . About 40 models and corresponding Ramachandran plots were generated for each protein to check the protein structure quality using PROCHECK. The figures were prepared using PyMOL (http://www.pymol.org/).All three Sp CBP21 and Sp CBP50 with Sp chitinases in chitin degradation.Synergistic effect of Sp chitinase (unless stated otherwise)  were amplified using gene specific primers (Sp cbps (data not shown) and 1.0 mM reduced glutathione. Reaction mixtures were incubated in triplicates at 37°C for 7 days at 1000 rpm in a thermomixer. After every 24 h, 100 µL of reaction mixture was transferred and mixed with 100 µL of 0.02 N NaOH was added to stop the reaction and stored at −20°C until products quantification. Products were quantified by standard chitinase assay as described by Neeraja et al. .Chitin degradation assay was performed as described by Vaaje-Kolstad et al.,  primers  and clonFigure S1Ni-NTA agarose purification of Sp CBPs. Recombinant Sp CBP21, Sp CBP28 and Sp CBP50 were purified using Ni-NTA agarose column chromatography. Elution buffer containing 250 mM imidazole was used to elute Sp CBPs from the column and loaded on 12% SDS-PAGE followed by staining with Coomassie brilliant blue G-250. Lane 1: Protein standards size in kDa indicated to the left, lane 2–4: Purified Sp CBP21, Sp CBP28 and Sp CBP50, respectively.(DOCX)Click here for additional data file.Figure S2Binding of Sp CBPs towards soluble polymeric substrates. Affinity non-denaturing gel electrophoresis was performed at 4°C by preparing 8% polyacrylamide gels. Ten micrograms of Sp CBPs and BSA were electrophoresed without (A), or with 0.1% (w/v) substrates glycol chitin (B), laminarin (C), and CM-cellulose (D). Proteins were visualized by Coomassie blue G-250 staining after electrophoresis. Lane 1: BSA, lane 2–4: Sp CBP21, Sp CBP28 and Sp CBP50.(DOCX)Click here for additional data file.Figure S3Dose-response effects for Sp CBP21 and Sp CBP50 in degradation of β-chitin. Reaction mixture containing 0.25 mg/mL of chitin substrates (α-/β-chitin), 1.0 µM Sp ChiD incubated with different concentrations of Sp CBP21/Sp CBP50 (0.05–0.40 µM) as indicated in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments. (A and B) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP21 and reduced glutathione (RG), (C and D) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP50 and reduced glutathione (RG). Sp CBP21+RG or Sp CBP50+RG: Sp CBP21/Sp CBP50 with reduced glutathione, Sp CBP21+ or Sp CBP50+: only Sp CBP21/Sp CBP50 without reduced gluthathione, Sp CBP21- or Sp CBP50 -: without Sp CBP21/ Sp CBP50 and reduced glutathione.(DOCX)Click here for additional data file.


Current human and experimental studies are indicating an association between stress and hearing problems; however potential risk factors have not been established. Hearing problems are projected to become among the top ten disabilities according to the WHO in the near future. Therefore a better understanding of the relationships between stress and hearing is warranted. Here we describe the prevalence of two common hearing problems, i.e. hearing complaints and tinnitus, in relation to different work-and health-related stressors.A total of 18,734 individuals were invited to participate in the study, out of which 9,756 (52%) enrolled.The results demonstrate a clear and mostly linear relationship between higher prevalence of hearing problems (tinnitus or hearing loss or both) and different stressors, e.g. occupational, poorer self-rated health, long-term illness, poorer sleep quality, and higher burnout scores.The present study unambiguously demonstrates associations between hearing problems and various stressors that have not been previously described for the auditory system. These findings will open new avenues for future investigations. In 2002ear 2030 ,3 and tiear 2030 ,5 respecear 2030 . The stuear 2030 -9, so thWhile the deleterious effects of mechanical stress (i.e. noise) on hearing have been studied extensively in both animal models  and humaHuman and animal studies are providing more evidence of an association between stress and hearing problems. It also needs be considered that long-term hearing problems can be stressful. Regarding the high prevalence of hearing problems in human populations and the negative future projections of the WHO, further investigations of the relationship between stress and hearing problems are warranted. Therefore, the aim of this study is to assess the prevalence of two common hearing problems, i.e. hearing complaints and tinnitus, in relation to different work- and health-related stressors. More specifically, we will study if prevalence differs with poor self-rated health, poorer sleep, higher burnout scores (work-related), and more symptoms of long-term stress and higher levels of performance-based self-esteem.The Swedish Work Environment Survey (SWES) is conducted biennially by Statistics Sweden (SCB) and consists of subsamples of gainfully employed people, aged 16-64 years, from the Labor Force Survey (LSF). These individuals were first sampled into the LFS through stratification by county of birth, sex, citizenship, and inferred employment status. The respondents to SWES 2003 and 2005 were invited to enroll in the Swedish Longitudinal Occupational Survey of Health (SLOSH) , which wApart from socioeconomic status and demographic factors, it also included approximately 120 questions about psychosocial and physical work-environment, lifestyle, as well as physical and mental health.Tinnitus. Have you during the most recent time experienced sound in any of the ears, without there being an external source  lasting more than five minutes? . Tinnitus severity. How much do you feel that the tinnitus sounds worry, bother or upset you? . The questions about tinnitus were adapted from Davis [Hearing complaints. How difficult is it for you to (without hearing aid) hear what is said in a conversation between several persons? . In this study, hearing complaints reflects difficulties in communicating. The questions about hearing complaints were derived and adapted from Statistics Sweden and have been used in population studies for several years.Hearing problems were assessed with three questions. om Davis  and Palmom Davis . HearingA new variable, "hearing problems", was computed based on the existence or non-existence of either tinnitus or hearing complaints or both. This consequently yielded three groups; those without hearing problems, those with either tinnitus or hearing complaints or those suffering from both. The cut-off for tinnitus was "yes, sometimes" or more often, and for hearing complaints "quite difficult" or "very difficult".Work-related stressors/threats. Risks of being moved to another work/job against ones will, threats of getting fired were derived from the Swedish Labor Force Survey (LFS). Threats of bankruptcy were constructed for SLOSH 2008 to assess a threat particularly important for self-employed, a group who contacted the research group and expressed feelings of neglect in the SLOSH 2006 survey. The question was formulated: "Are you subjected to any of the following risks or threats in your work?" Response alternatives were yes/no.Self-rated health was assessed with the single item "How would you rate your general state of health?" This question has been widely used in research [research -30 and tLong-term illness, inconvenience after an accident, any handicap or other weakness. One question was asked about long-term illness, inconvenience after an accident or other weakness: "Do you have any prolonged sickness, accident-related complaints, a disability or other weakness?" This question was derived from the WOLFF  [llow-up) ,32 questSleep quality. Sleep quality was assessed by the single item: "How is your sleep quality in general?" This item was derived from the Karolinska Sleep Questionnaire [ionnaire . ResponsBurnout was assessed with the Maslach Burnout Inventory general survey (MBI-GS) using the emotional exhaustion subscale [subscale . The scasubscale . The indLong lasting stress (LLS) was assessed with 11 items reflecting stress arousal symptoms but not stress reactions. The participants were asked how they felt during the last three months with regard to both physiological  and cognitive-behavioral symptoms . The four response alternatives reached from "Not at all" to "Nearly all the time". The scale was introduced in the 2008 SLOSH questionnaire and is currently being validated (data not yet published). A factor analysis yielded one factor of interest. This factor included 7 of the 11 items and only the cognitive-behavioral symptoms. Factor loadings ranged from .675 ± 798 and a Chronbach's α of .863. The 7 included items were :A) I have days when I feel geared up all the time (FL = .675). B) I have days when I feel very pressured, on the verge of what I can handle (FL = .737). C) I find it hard to relax during my leisure time (FL = .787). D) I often feel tense (FL = .798). E) I often have disturbing thoughts (FL = .768). F) I often feel restless (FL = .720). G) I do not feel rested after taking it easy for a few days (FL = .699). The correlation between LLS and MBI-GS is r = .64, p < .0001, two-tailed. The index was calculated on the basis that 6 out of 7 items had to be answered in order be included in the index.Performance based self-esteem [f-esteem ,37 was aThe programs SPSS 18.0 and SAS 9.2 were used for statistical analyses. Prevalence was calculated via frequency plots and crosstabs were used for calculation of χ, Kendall's tau-b, and specific prevalence within different groups. Kendall's tau-b is a correlation analysis that illustrates the direction and magnitude of association between two variables. Multivariate analyses, proportional odds model , were used to calculate possible interacting or confounding effects of age, gender and SES. Comparisons were made between those having no hearing problems compared to those with either tinnitus or hearing loss or both tinnitus and hearing loss. Statistical significance was set at p < 0.05 level.The regional ethics committee in Stockholm approved the research project and all participants gave their informed consent to participate.In a previous analysis of the present data it was shown that 31% of the working population report either hearing complaints or tinnitus (i.e. 25%) or both i.e. 6%) and the prevalence of hearing problems increased with age, was higher among men and in persons with lower self-rated SES, and co-varied with exposure to noise at work [% and theOverall, the results describe an association between hearing problems and work- and health-related stressors. All the results were controlled for possible confounding effect of age, gender or SES with multivariate analyses. These analyses showed no confounding effect of these variables. The demographics of the population in the present study are as follows: 4,462 (46%) men and 5,294 (54%) women. The mean age was 48.6 (± 10.8) for men and 48.2 (± 10.5) for women. Age distribution was: under 40 years 1,148 (26%) men and 1,314 (25%) women; 41 ± 51 years 1,202 (27%) men and 1,520 (29%) women; 51-60 years 1,415 (32%) men and 1,754 (33%) women; 60 years or older 697 (26%) men and 706 (13%) women. Marital status: married 2,491 (56%) men and 2,946 (56%) women; unmarried 1,494 (34%) men and 1,516 (29%) women; divorced 444 (10%) men and 726 (14%) women; widow 33 (1%) and 106 (2%) women. With regard to highest completed educational level, 1,016 (10%) had no gymnasium, 4,510 (46%) had gymnasium, 619 (6%) had undergraduate studies of two years or less, 3,472 (36%) had undergraduate studies of three years or more and 134 (1%) hade post graduate studies.2 = 54.704df = 2, p < 0.0001) and threats of getting fired . There was however no statistically significant difference between those who were exposed to threats of bankruptcy compared to those who were not.Table The proportional odds model did not exhibit any differences in odds ratios when age and socioeconomic status were included in models for neither women nor men. For instance, the unadjusted odds ratio of having hearing problems when exposed to a threat of being moved to another job against ones will (yes vs. no) were 1.39 for men (p < 0.001) compared to the adjusted odds ratios which were 1.43 (p < 0.001). For women, the corresponding values were 1.7 (p < 0.001) and when adjusted the value was 1.74 (p < 0.001).2 = 262.522df = 6, p < 0.0001, for women χ2 = 141.535df = 6, p < 0.0001, for men: χ2 = 123.306df = 6, p < 0.0001). Figure There was a statistically significant difference in the prevalence of hearing problems between individuals with different levels of self-rated health  compared to the adjusted odds ratios which were 3.29 (p < 0.001). For women, the corresponding values were 3.81 (p < 0.001) and when adjusted the value was 3.49 (p < 0.001).2 = 181.650df = 2, p < 0.0001). Those with long-term handicaps and illnesses reported more hearing problems  were 1.92 for men (p < 0.001) compared to the adjusted odds ratios which were 1.76 (p < 0.001). For women, the corresponding values were 1.72 (p < 0.001) and when adjusted the value was 1.64 (p < 0.001).2 = 169.875df = 8, p < 0.0001, for women χ2 = 112.509df = 8, p < 0.0001, for men: χ2 = 75.068df = 8, p < 0.0001). The association was negative  and Figure 2 = 126.884df = 4, p < 0.0001, Kendall's τ-b = -0.106 p < 0.0001) compared to those reporting hearing complaints  compared to the adjusted odds ratios which were 2.67 (p < 0.001). For women, the corresponding values were 3.51 (p < 0.001) and when adjusted the value was 3.24 (p < 0.001).2 = 214.473df = 6, p < 0.0001, for women χ2 = 159.205df = 6, p < 0.0001, for men: χ2 = 98.935df = 6, p < 0.0001). Hearing problems were significantly more prevalent among those with higher burnout scores. Multivariate analyses showed no age, gender or SES related differences in prevalence increases with increasing burnout scores. The association was positive  and Figure There was a statistically significant difference in the prevalence of hearing problems between those with higher burnout scores compared to those with lower scores  compared to the adjusted odds ratios which were 2.63 (p < 0.001). For women, the corresponding values were 2.80 (p < 0.001) and when adjusted the value was 2.79 (p < 0.001).2 = 196.855df = 6, p < 0.0001, for women χ2 = 145.608df = 6, p < 0.0001, for men: χ2 = 90.613df = 6, p < 0.0001). Hearing problems were more prevalent among those with more symptoms of long-lasting stress. Similarly to the pattern for burnout, the prevalence increase was higher for women than for men, even if it was less pronounced for this variable. The association was positive  and Figure There was a statistically significant difference in the prevalence of hearing problems between those with more symptoms of long-lasting stress scores compared to those with less (χNo major differences in odds ratios were found when age and socioeconomic status were included in models for women or men. The unadjusted odds ratio of having hearing problems when being in the highest vs. lowest long-lasting stress quartile were 2.06 for men (p < 0.001) compared to the adjusted odds ratios which were 2.42 (p < 0.001). For women, the corresponding values were 2.61 (p < 0.001) and when adjusted the value was 2.79 (p < 0.001).2 = 39.946df = 6, p < 0.0001, for women χ2 = 36.410df = 6, p < 0.0001, for men: χ2 = 15.181df = 6, p < 0.05). Hearing problems were more prevalent among those with higher levels of PBS. The association was positive and linear and more pronounced for women than for men  and Figure A statistically significant difference was found in the prevalence of hearing problems between those with higher and lower levels of PBS (χNo major differences in odds ratios were found when age and socioeconomic status were included in models for women or men. The unadjusted odds ratio of having hearing problems when being in the highest vs. lowest PBS quartile were 1.26 for men (p < 0.001) compared to the adjusted odds ratios which were 1.41 (p < 0.001). For women, the corresponding values were 1.64 (p < 0.001) and when adjusted the value was 1.80 (p < 0.001).The aim of the present study was to assess the prevalence of two common hearing problems, i.e. hearing complaints and tinnitus, in relation to different work-, life- and health-related stressors. The results demonstrate a clear and mostly linear relationship between higher prevalence of hearing problems (tinnitus or hearing complaints or both) and different stressors, symptoms of ill health and stress as well as poor sleep. Thus, the salient features of the present study illustrate that occupational stressors, poorer self-rated health, long-term illness, poorer sleep quality, higher burnout scores, more symptoms of long-lasting stress, and higher performance-based self-esteem are statistically significantly associated with a higher prevalence of hearing problems. To our knowledge, this is the first time that such vivid and systematic associations have been found for hearing problems (hearing complaints and tinnitus) in an extensive human population.The findings were consistent for both males and females, and men showed a slightly higher prevalence of hearing problems in absolute levels for all items analyzed except for performance-based self-esteem. Previously it has been found that the prevalence of hearing problems is higher among men and that prevalence increases with age . The preIt is also noteworthy to mention that in the present study we have assessed two different factors for hearing problems. First we analyzed the prevalence of hearing problems either for tinnitus or hearing complaints and secondly for both tinnitus and hearing complaints. This assessment is more realistic since it is common for individuals who have hearing complaints to have tinnitus and vice versa. In a previous study, the prevalence of tinnitus was higher for men than women . When anHearing problems were also more common among individuals exposed to occupational stressors, i.e. employment-related stress such as risk of being moved to another work/job against ones will and threats of getting fired. Threats of bankruptcy were not significantly associated with a higher prevalence of hearing problems. These problems include difficulties in communicating, lower awareness for important surrounding sounds such as telephone calls or warning signals or oversensitivity to sounds (hyperacusis) and even reduce productivity due to emotional exhaustion. These burdens become exaggerated for individuals with long-lasting stress symptoms and high burnout scores, as demonstrated in the present study. This is important for many reasons including the public awareness and the clarification that long-lasting stress and burnout symptoms are risk factors correlated with hearing problems.Self-rated health (SRH) is one of the most widely used single measures of perceived current health status . There iIt has been proposed that SRH represents an individual's general perception of health, including biological, psychological and social dimensions. Therefore SRH might be more sensitive in health monitoring than external measures of health . FurtherA poorer self-rated health was correlated to hearing problems. Poor self-rated health can be influenced by several factors including social and marital status , type of employment , sleep qPoor sleep quality was found to be associated with a higher prevalence of hearing problems in both men and women. In a study aimed at evaluating age specific prevalence of general symptoms it was found that five symptoms increased with increasing age . These sIn a previous study we found that individuals with hearing problems have a poorer ability to unwind or activate their parasympathetic system . The preIt is by now well established that stress-related disorders, such as burnout, are associated with several forms of co-morbidity -62, e.g.The relation between long-term stress and tinnitus has been previously explored and tinnitus sufferers often report enhanced problems by stress and fatigue . It remaIn the present study there was a higher prevalence of hearing problems among those with higher PBS. High PBS is associated with high but vulnerable engagement, as well as higher prevalence of sickness presenteeism. When combined with high scores on a burnout scale it is a strong predictor of burnout and increased risk  for long-term sick leave . This fiOne of the major strengths with this study is the large sample size and that the sample is representative for the general Swedish working population. One weakness is the cross-sectional design, which does not allow conclusions about causality. Accordingly, prospective studies, which are forthcoming from our group, will be needed to illuminate the extent to which the observed associations are causal. Also, the study population is typical of a post-industrial country, with a high proportion of participants having a high educational level. It is possible that associations would be different in a population with a higher proportion of blue collar workers.The assessment of hearing complaints and tinnitus via questionnaires has its advantages and disadvantages and there is no consensus as to which method is most valid. For example, the subjective evaluation of hearing problems may be difficult to interpret when there is a mild hearing loss. However, individuals having constant difficulties in understanding speech  are usually well aware of their problem. It must be pointed out that an individual who has difficulties in understanding speech in background noise can have a completely normal audiogram. Thus, there are also limitations to audiological testing. This is not to say that an audiological test would not complement our subjective ratings, but we argue that addressing the question if there are "hearing problems" is acceptable. In fact, rating scales are commonly utilized to assess hearing problems ,66 and iIn conclusion, the present study unambiguously demonstrates associations between hearing problems and occupational stressors, poorer self-rated health, higher burnout scores, more symptoms of long-lasting stress, long-term illness and poorer sleep quality. The interaction between the prevalence of hearing problems and the above mentioned features have not been previously described for the auditory system. These findings also indicate that hearing problems are multidimensional, which warrants further investigations of possible predictors.The authors declare that they have no competing interests.DH conducted the statistical analyses and drafted the manuscript together with BC, MBW, with substantial and essential input from CL and TT. TT was PI of the project and CL data manager. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/130/prepub


The potential risks of GMTs, the fate of recombinant material and the potential relevance of recombinant genes on plantÂ´s omics are other main aspects to be considered and compared to similar processes with endogenous genes in conventional breeding. The group involves experts from public research, government and independent regulatory sectors across COST and non-COST member countries. The activities of this group have been organized into three Task Groups focusing on (1) risk assessment studies and guidance documents, (2) the monitoring of the transgenes and recombinant plant material, and (3) the impact of GMTs on exposed ecosystems. As a first step of WG2 activity, a database of guidance documents from national and transnational sources dealing with impacts and risk assessments of GMTs is being created to identify common and case-specific issues on biosafety. It is expected that the information gained will facilitate (1) a science-based understanding of the impacts of GMTs on the environment in comparison with that of traditionally tree breeding, and (2) future socio-economic and cost/benefits analyses of GMTs in plantations.Transgenic biotechnology can assist forest tree improvement programs but it may also raise environmental safety concerns. The environmental effects of genetically modified transgenic trees (GMTs) have been studied in many countries during the last 15 years. Today there is an urgent need of putting together this scattered knowledge to build-up a European knowledge platform for addressing GMTs in plantations. The main aims of Working Group 2 (WG2) of the European Cooperation in Science and Technology (COST) Action FP0905 “Biosafety of transgenic forest trees” (


Nardostachys jatamansi (NJ) exhibits anti-inflammatory properties against lipopolysaccharide (LPS) challenges. However, the potency of NJ constituents against LPS-induced inflammatory responses has not been examined. In this present study, we determined which NJ extract fractions exhibit inhibitory effects against LPS-induced inflammatory responses. Among the NJ fractions, NJ-1, NJ-3, NJ-4, and NJ-6 inhibited LPS-induced production of NO. The NJ-3, NJ-4, and NJ-6 fractions also inhibited the production of cytokines, such as IL-1β, IL-6, and TNF-α. However, NJ-1, NJ-3, NJ-4, and NJ-6 showed differential inhibitory mechanisms against LPS-induced inflammatory responses. NJ-1, NJ-3, and NJ-4 inhibited LPS-induced activation of c-jun NH2-terminal kinase (JNK) and p38 but did not affect activation of extracellular signal-regulated kinase (ERK) or NF-κB. On the other hand, NJ-6 inhibited activation of MAPKs and NF-κB. In addition, in vivo experiments revealed that administration of NJ-1, NJ-3, NJ-4, and NJ-6 reduced LPS-induced endotoxin shock, with NJ-6 especially showing a marked protective effect. Taken together, these results provide the evidence for the potential of selective NJ fractions against LPS-induced inflammation. Thus, it will be advantageous to further isolate and determine single effective compounds from these potent fractions.It has been previously shown that  Infecti (NF-κB) . Subsequ (TNF-α) .Nardostachys jatamansi (NJ) is widely used as a bitter tonic and antispasmodic [ in vitro and in vivo analyses to examine whether the fractions of NJ have potential inhibitory effects against LPS-induced inflammation in murine peritoneal macrophages and in an animal model of LPS-induced endotoxin shock. Moreover, to elucidate a potential molecular anti-inflammatory mechanism, we examined the activation of MAPKs and NF-κB.pasmodic . It has pasmodic –10, partpasmodic . In partpasmodic . Howeverβ, IL-6, and TNF-α were purchased from R&D Systems . LPS from Escherichia coli 055:B5 was purchased from Sigma-Aldrich Chemical . Antibodies against total and phospho-specific MAPKs  were obtained from Cell Signaling Technology . Iκ-Bα monoclonal antibody and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. . Prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis markers were obtained from Bio-Rad . Trizol reagent and polymerase chain reaction (PCR) kits were purchased from Invitrogen Corporation .RPMI-1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL . Enzyme-linked immunosorbent assay (ELISA) antibodies for detection of mouse IL-1The roots of NJ were purchased from a standard commercial source . The herb's identity was confirmed at Wonkwang University. Voucher specimens (number 02-03-29) were deposited at the College of Oriental Medicine Herbarium of Wonkwang University. The NJ roots were prepared by decocting the dried prescription of herbs (100 g) with boiling distilled water (1 L) for approximately 2 h. The water extract was frozen at −80°C and freeze dried to be powdered .μm particle size) chromatography , yielding 6 fractions .The water extract (3.8 g) was subjected to octadecyl functionalized silica gel flash column  was used. The mobile phase was composed of (A) water and (B) acetonitrile, with an applied gradient of 10% B increasing to 60% B in 50 min and 60% B increasing to 100% B in 2 min, and then the system was equilibrated for 8 min with the 100% B conditions . Detection of the peaks was performed at 254 nm and sensitivity was set at 0.5 AUFs. The injection volume was 20 μL, and the flow rate was 1.0 mL/min. A standard solution was prepared by dissolution in distilled methanol (10 μg/10 mL). The solution of biological active fraction was filtered through a 0.45 μm membrane filter and subjected to HPLC , a UV detector , and an autosampler . A hydrosphere C to HPLC .Thioglycollate (TG) elicited peritoneal macrophages were harvested 4 d after intraperitoneal injection of 3 mL TG. Peritoneal lavage was performed using 8 mL of RPMI supplemented with 10% heat-inactivated FBS. After incubation for 3 h, nonadherent cells were removed and the medium was changed for adherent cells. Cells were then treated with LPS in the absence or presence of NJ fractions.2PO4, 25 mM NaHCO3, 1 M CaCl2, and 1 M MgCl2). Fifty microliters of the mixture was added to each well. After 30 min of incubation at 37°C, the suspension was removed and formazan crystals were dissolved in 200 μL of dimethyl sulfoxide. Aliquots of cells were seeded in wells of a 96-well plate in duplicate and assayed at 540 nm using a micro-ELISA plate reader. The number of NJ fraction-treated viable cells was expressed as a percentage of the control (untreated cells) maintained for the same time period.Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert the tetrazolium compound 3--2,5-diphenyl tetrazolium bromide (MTT) into purple formazan crystals. MTT (5 mg/mL) was dissolved in Krebs-Henseleit buffer  were pretreated with NJ fractions for 1 h and then stimulated with LPS (500 ng/mL) for 24 h. To measure NO concentration, 100 μL aliquots were removed from conditioned media and incubated with an equal volume of Griess reagent at room temperature for 10 min. The absorbance at 540 nm was then measured.Murine peritoneal macrophages (2 × 106 cells/well) were stimulated with 500 ng/mL of LPS and/or various concentrations of NJ fraction for 24 h. Culture supernatants were collected and stored at −80°C until use. Cytokine levels in supernatants were determined using a commercial system (R&D Systems) according to the manufacturer's instructions. The ELISA was devised by coating 96-well plates with antibodies specific for IL-1β, IL-6, and TNF-α. Coated plates were washed with PBS containing 0.05% Tween-20. All reagents used in this assay were incubated overnight at 4°C. Recombinant IL-1β, IL-6, and TNF-α were diluted and used as a standard. Serial dilutions starting at 20 ng/mL were used to establish the standard curve. Assay plates were exposed sequentially to biotinylated mouse IL-1β, IL-6, and TNF-α, avidin peroxidase, and a substrate solution of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing 30% hydrogen peroxide. The plates were read at 405 nm.Murine peritoneal macrophages (1 × 10μg) was converted to cDNA by 200 units of reverse transcriptase and 500 ng of oligo-dT primer in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 1 mM dNTPs at 42°C for 1 h. The reaction was stopped by heating at 70°C for 15 min.Total cellular RNA was isolated using Trizol  according to manufacturer's instructions. Total RNA . For each sample, triplicate test reactions and a control reaction lacking reverse transcriptase were analyzed for expression of the gene of interest and results were normalized to those for the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT). Arbitrary expression units were calculated by dividing expression of the gene of interest by ribosomal protein HPRT mRNA expression. PCR was performed at an annealing temperature of 60°C with 40 amplification cycles. The commercial forward, reverse, and probe oligonucleotide primers for multiplex real-time TaqMan PCR were purchased from ABI .κ-Bα. After washing three times, each blot was incubated with a secondary antibody for 1 h, and immunoreactive bands were visualized using an enhanced chemiluminescence detection system  according to the manufacturer's recommended protocol.Whole cell lysates were prepared by boiling peritoneal macrophages in a sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol). Proteins in the cell lysates were then separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in PBS-Tween-20 for 2 h at room temperature and incubated overnight with antibodies against phosphorylated ERK 1/2, phosphorylated JNK, phosphorylated p38, and IκB p65 antibody . The cells were then washed and incubated with a 1 : 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG  for 2 h in a dark room. For nuclear staining, the cells were incubated with a 1 : 1000 dilution of DAPI for 5 min. The slide was finally washed and mounted for microscopic examination. Staining with NF-κB p65 antibody exhibited red fluorescence, which was detected by fluorescence microscopy.Peritoneal macrophages were plated in a chamber slide and incubated with 500 ng/mL of LPS for 1 h at 37°C. The cells were fixed in 4% paraformaldehyde for 30 min at RT and washed three times with PBS. The cells were treated with 0.1% Triton X-100 for 15 min at room temperature. After washing, the cells were incubated with a blocking serum for 1 h and incubated overnight with a 1 : 100 dilution of primary NF- E. coli serotype O55:B5, 37.5 mg/kg). At 1 h after NJ fraction administration, LPS (37.5 mg·kg−1) was injected intraperitoneally. Survival was monitored for 120 h. Animal use and relevant experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All experiments were approved by the Animal Care Committee of Wonkwang University.Endotoxin shock was induced in 6- to 8-week-old female C57BL/6 mice by intraperitoneal (i.p.) injection of bacterial endotoxin , and cell viability was evaluated by MTT assay. NJ-1, NJ-2, NJ-3, and NJ-6 did not demonstrate any significant cytotoxic effects on peritoneal macrophages. However, at a relatively higher dose, NJ-4 and NJ-5 showed significant cytotoxicity (#P < 0.001) (μg/mL) and NJ-5 (≥50 μg/mL) could affect cellular metabolism, we excluded data of NJ-4 and NJ-5 at these concentrations.The first approach to study the biological activity of any compound or plant extract is to ensure lack of effect on cellular metabolism. To determine whether fraction of NJ affects cell viability, peritoneal macrophages were incubated for 24 h with varying concentrations of extract (< 0.001) . TherefoNext, to examine the anti-inflammatory effect of NJ fractions, we measured NO production. Murine peritoneal macrophages were pretreated with the indicated concentrations of NJ fractions for 1 h and then stimulated with LPS (500 ng/mL) for 24 h. As shown in β, IL-6, and TNF-α were increased by LPS stimulation. However, pretreatment with NJ-3, NJ-4, and NJ-6 inhibited the production of IL-1β, IL-6, and TNF-α significantly but not with NJ-1 fraction , NJ-3 (100 μg/mL), NJ-4 (50 μg/mL), and NJ-6 (100 μg/mL) for 1 h and then stimulated with 500 ng/mL of LPS for 0, 15, 30, or 60 min. Pretreatment with NJ-1, NJ-3, or NJ-4 significantly inhibited the LPS-induced activation of JNK and p38, but not ERK 1/2  of NJ fractions on production of NO and cytokines, MAPK and NF- Figures . In addi Figures . To dete Figures . However Figures . in vivo, we used a model LPS-induced endotoxin shock. NJ fractions  were injected at the indicated doses. After 3 h, a lethal dose of LPS (37.5 mg/kg) was administered intraperitoneally. The survival rate was recorded every 12 h after LPS treatment. Generally, LPS-injected septic mice died within 48 h. Consistent with in vitro experiments, NJ fractions exhibited strong anti-inflammatory activities in vivo, as shown by significant inhibition of LPS-induced death in the treated mice (To verify effects of NJ fractionsted mice . In partted mice .Recently, our laboratory reported the beneficial effects of NJ on various diseases, such as endotoxin shock, pancreatitis, and diabetes –10. Partα, IL-1β, IL-6, and IL-8 are the most frequently altered cytokines [Generally, sepsis is a life threatening condition caused by the body's response to bacterial products, such as endotoxin, which is often present with severe fever, shock, and respiratory damage . Partialytokines . When inytokines , fractioκB as possible regulatory mechanisms. Generally, it has been reported that the production of proinflammatory mediators is modulated by the MAPKs and NF-κB pathways. Three major groups of MAPKs include the ERK, JNK, and p38, which are all activated by phosphorylation [κB activation occurs mainly through the degradation of IκB, thereby leading to subsequent release of NF-κB dimers [κB dimers translocate from the cytoplasm to the nucleus and activate the transcription of multiple κB-dependent target genes [κ-Bα. NJ-1, NJ-3, and NJ-4 inhibited the phosphorylation and activation of JNK and p38 but not ERK1/2. However, NJ-6 inhibited not only the activation of ERK1/2, JNK, and p38 but also degradation of Iκ-Bα. These results suggest that inhibition of NO and cytokine production by NJ fractions is primarily through MAPKs or NF-κB pathways.Using the fractions that were found to be effective against LPS, we examined activation of MAPKs and NF-rylation –16. NF-κB dimers . Subsequet genes . In thisκ-Bα and translocation of NF-κB p65, which was not regulated by the NJ total extract. Further isolation of the NJ-4 and NJ-6 fractions could potentially yield a single compound demonstrating inhibitory activity resembling that of NJ total extract.In this study, we isolated two beneficial fractions of NJ. The first one was NJ-4, which could show similar effects to NJ total extract. NJ-4 inhibited cytokine production and LPS-induced endotoxin shock similar to NJ total extract . FurtherIn conclusion, this study demonstrated which fractions of NJ exhibit inhibitory activity against LPS-induced inflammation. Among the 6 fractions isolated, NJ-1, NJ-3, NJ-4, and NJ-6 showed the inhibition of NO, and NJ-3, NJ-4, and NJ-6 showed the inhibition of cytokines. In addition, NJ-4 and NJ-6 exhibited dramatic prevention against LPS-endotoxin shock. These results suggest that NJ-4 and NJ-6 may be effective and beneficial candidates to ameliorate LPS-induced inflammation.


Background: Meiotic genes are very important candidates for genes contributing to female and male infertility. Mammalian MutL homologues have dual roles in DNA mismatch repair (MMR) after replication errors and meiotic reciprocal recombination. The MutL homologs, MLH1 and MLH3, are crucial for meiotic reciprocal recombination and human fertility. In this study the functional polymorphisms of MLH3 C2531T was investigated in Iranian women with unexplained infertility.Objective: Investigating the association between a common SNP (single nucleotide polymorphism) C2531T in the MLH3 gene and female infertility.Materials and Methods: In total, 105 women with unexplained infertility as case group and 100 women with at least one child and no history of infertility or abortion as controls were recruited for this association study. The MLH3 C2531T polymorphism was tested by tetra-amplification refractory mutation system-PCR (4P-ARMS-PCR) method.Results: The MLH3 2531C and T alleles frequencies were 43.33% and 56.67% among infertile patients, and 61.5% and 38.5% among normal controls, respectively. In the patient and control subjects the CC (Pro 844 Pro) genotype frequency of MLH3 C2531T was 4.76% and 25%, the CT (Pro 844 Leu) genotype was 77.15% and 73%, and the TT (Leu 844 Leu) genotype was 19% and 2%, respectively (p=0.0001).Conclusion: The presence of the polymorphic allele T leads to an increased risk of 2.09 times  for developing infertility in relation to the control group. Therefore, our data suggest that the MLH3 C2531T polymorphism can be associated with the risk of unexplained infertility in Iranian women. Infertility is described as the inability of a sexually active couple, not using any birth control to get pregnant after one year of trying. It is estimated that about 15% of couples in the world suffer from infertility  contributes to maintenance of genomic integrity and for correcting DNA mismatch after DNA replication in somatic cells while it participates in crossing over between homologous chromosomes in meiosis. A meiosis-specific mismatch protein, MSH4, heterodimerises with MSH5 appears at recombination sites at zygonema . At mid-The understanding of the mechanisms of meiosis and fertility has mainly benefitted from knockout and transgenic mouse models that exclusively have reproduction failure. Both male and female Mlh1-/- and Mlh3-/- mice are infertile. Males of both genotypes arrest in the metaphase I. Mlh1-/- oocytes fail to complete the meiosis II after fertilization with normal sperms, whereas a few numbers of abnormal Mlh3-/- oocytes extrude both the first and second polar body -13. MLH3 was first identified in 2000. The human MLH3 gene is located on the long arm of the chromosome 14 (14q24.3) with a coding length of 4.3 kb, and has 12 exons. Exon 1 of this gene is 3.3 kb, accounting for 75% of the coding region  method , and 200 ng of DNA.Genomic DNA was isolated from peripheral blood samples using the standard salting-out procedure. ) method . The pri) method . The PCRoC for 5 min; followed by 20 cycles of 94oC for 30 s, annealing temperature starting at 65oC for 30 s (decreasing 0.5oC per cycle), and 72oC for 30s for extension. This step was followed by 10 cycles of 94oC for 30 s, 55oC for 30 s, 72oC for 30 s, and a final elongation step of 5 min at 72oC. The PCR product was visualized on 3.0% agarose gel containing ethidium bromide under UV light. The C allele generated a 178 bp band, and the T allele generated a 220 bp band, although they had a common 320 bp band amplified by outer primers (MLH3 F1 and MLH3 R1)  .Statistical analysisMLH3 gene between case and control groups were evaluated by χ2 test with odds ratio (OR).The allele and genotype frequency of the infertile patients and normal controls were calculated by counting. The chi-square test was used to compare allele and genotype frequencies between patient and normal groups and to estimate the Hardy-Weinberg equilibrium. Statistical tests of significance and χ2 analysis were performed using SPSS statistic software version16.0.The differences in allelic and genotypic frequencies of C2531T locus in the MLH3 C2531T polymorphism are showed in MLH3 2531C and T alleles frequencies were 43.33% and 56.67% among infertile patients, and 61.5% and 38.5% among normal controls. Tests for Hardy-Weinberg equilibrium for the studied SNP was performed, and the null hypothesis of Hardy-Weinberg equilibrium was not rejected (p=0.0001). The genotype frequency of MLH3 C2531T polymorphisms in our study subjects were significantly different between the patients with unexplained infertility and normal controls (p=0.0001). In specific, in the patient and control subjects the CC (Pro 844 Pro) genotype frequency of MLH3 C2531T was 4.76% and25%, the CT (Pro 844 Leu) genotype was 77.15% and 73% and the TT (Leu 844 Leu) genotype was 19% and 2%, respectively. In the present study, there was a statistically significant difference between the groups of infertile women compared to the control group (p=0.0001) with regard to the MLH3 C2531T polymorphism, suggesting that this polymorphism might be related to women infertility. The presence of the polymorphic allele T leads to an increased risk of 2.09 times  to develop infertility in relation to the control group.The genotype and allele frequencies of MLH3 C2531T polymorphism was associated with increased risk for the female infertility. In other studies it was shown that both male and female of Mlh1-/- and Mlh3-/- mice are sterile. Males of both genotypes arrest in the metaphase of meiosis I. While Mlh1-/- oocytes fail to complete the meiosis II, whereas a small proportion of abnormal Mlh3-/- oocytes extrude both the first and second polar body  in ertility . Altoget13 cases . TherefoMLH3 gene and female unexplained infertility in blood samples, suggesting that this mutation may be an important genetic risk factor for female infertility in Iran. The MMR (Mismatch repair) system plays a crucial role in all organisms because it maintains the genome integrity during repeated duplication. In the present study, we found an association between the SNP C2531T (P844L) in the MMR system is composed of several highly-conserved proteins whose functions in repair of mismatched DNA after replication have been demonstrated. Multiple functions, in addition to mismatch repair during replication, have been identified for MMR proteins, such as promotion of meiotic crossover between homologous chromosomes -20. Meiotic homologous recombination begins when the double-strand breaks (DSBs) are formed in DNA by Spo11 protein. Holliday junction (formed during homologous recombination) resolvase is activated by MSH4-MSH5 and MLH1-MLH3 in the same pathway . MammaliMLH3 is associated with recombination nodules at early pachytene. This study demonstrated that there was an association between a functional polymorphism and female infertility. This finding can be an indication of this fact that the MLH1–MLH3 pathway plays an important role in making crossovers during mammalian meiosis and female fertility. Our findings in the present study are biologically plausible, based on the previously reported function of MMR components. Cytological studies in mice have shown that MLH3 gene polymorphism may be a genetic risk factor for unexplained infertility in women. The further functional study will continue to provide new evidence of the role of the MLH3 in pathogenesis of female infertility and will help to further elucidate the mechanisms of meiotic recombination in mammal. Therefore, the


The hope for ‘normalization’ of the next generation of children with hearing impairments receives a new boost whenever new technology such as cochlear implants or new concepts in identification, intervention, and education are introduced into the field of deafness. These boosts run the risk of producing unrealistic expectations for the future, and neglecting the needs of the present generation of people with hearing impairments.By showing that behaviour problems of children with hearing impairments did not improve with early confirmation of hearing loss, Stevenson et al.In addition, their findings of the relatively small improvement in the receptive language ability of those confirmed before 9 months of age highlight the fact that congenital hearing loss continues to have devastating consequences.In line with existing research, the authors showed in a previous paperBut given that children with PCHI achieve almost normalized receptive language scores, and even including all the biological and social factors which influence mental health in general, it still leaves us with the question whether a reduced ability to hear itself contributes significantly to mental health problems observed in those with impaired hearing.This idea is supported by findings that the level of language skills was highly correlated with the degree of hearing loss but mental health problems, especially with peer relationships, were not.3The above leads us to ask the crucial question: what kind of evidence-based interventions need to follow UNHS in order to support families to actively foster the development of a strong identity and positive mental health of their child with PCHI, beyond the drive for ‘normalization’.