Introduction. Although quarry operations have high economic significance, the effects they cause to the workers in terms of excessive noise production cannot be overlooked. This cross-sectional study assessed the extent of noise exposure and its influence on hearing capabilities among quarry workers in Ashanti region. Methods. The study involved 400 workers randomly selected from five quarries in Ashanti region from April to June 2012. Data was collected using structured questionnaires, physical examination, and audiological assessments. A logistic regression model was fitted to assess independent predictors of hearing loss. Results. All the machines used at the various quarries produced noise that exceeded the minimum threshold with levels ranging from 85.5 dBA to 102.7 dBA. 176 (44%) of study respondents had hearing threshold higher than 25 dBA. 18% and 2% of these were moderately (41–55 dBA) and severely (71–90 dBA) impaired, respectively. Age, duration of work, and use of earplugs independently predicted the development of hearing loss. Use of earplugs showed a protective effect on the development of hearing loss . Conclusion. This study provides empirical evidence on the extent of damage caused to quarry workers as a result of excessive noise exposure. This will support the institution of appropriate protective measures to minimize this threat. Occupational health is an important public health concern for the working population . VariousThe growing attention for NIHL is due to the fact that, unlike many injuries or illnesses, hearing loss may be permanent and irreversible. Exposure to excess noise is first observed as an increase in the threshold of hearing (threshold shift), as assessed by audiometry . This isAlthough noise is associated with almost every work activity, some activities are associated with particularly high levels of noise. In general, sounds above 85 dB are considered harmful, depending on how long and how often one is exposed to them and whether you wear hearing. Previous literature shows that workers in mines, quarries, sawmills, textile factors, printing presses, and many others work with machines that produces noise much higher than the tolerated levels and therefore expose workers to potential hearing loss . In the Although the situation could be improving in developed countries as a result of more widespread appreciation of the hazard and the institution of protective measures, evidence from developing countries suggests that average noise levels are well above the occupational level recommended in many developed nations , 10. IncIn Ghana, there is very little data on the effect of noise exposure on quarry industrial workers. More so, the increasing number of patients seeking medical attention for their ears at Hearing Assessment Centre at the Komfo Anokye Teaching Hospital (KATH) in Kumasi between 1995 to 1998  has callThis was a descriptive cross-sectional study conducted in Kwabre East District of Ashanti region of Ghana. Kwabre District, carved out of the former Kwabre Sekyere District in 1988, is located almost in the central portion of Ashanti region. It is within latitude 6°44′′ north and longitude 1°33′′ to 1°44′′ west. The district shares common boundaries with Afigya-Sekyere District to the north, Kumasi metropolitan area to the south, Ejisu-Juaben District to the southeast, Atwima District to the west, and Offinso District to the northwest. The District has a total land area of 246.8 square kilometers constituting about 1.01% of the total land area of Ashanti region. Kwabre District is part of the Greater Kumasi City region, which is made up of Kumasi Metropolitan Area and the surrounding Districts. According to 2000's population and housing census, the district has a total population of about 205,372 people. The physiography of the area consists of scarps formed by the extensive quarry activities going on in the area. The main land usage in the area can be categorized into stone quarrying and agriculture. The quarries are mainly located near the mountains, where granite deposits have been detected. The quarry developers lease the land from the owners at a fee until the time the quarry work is finished. The main commercial purposes of quarrying are building stones and quarry dust. Agricultural activities in the area are mostly subsistence and cash crop farming.The study population was drawn from workers of the five (5) quarry companies, namely, Northern mine quarry, A. Kannin mine, Taysec quarry company, KAS quarry company, and Siemens quarry company. Workers who have been working at the respective quarry for more than six months were included in the study. Workers who had previous history of hearing loss and those not directly involved in the quarrying process were excluded. The study comprised 400 workers from the various quarry companies. The composition of respondents from the quarry companies was as follows: A. Kannin , KAS quarry , Siemens quarry , Taysec , and Northern mines and quarry .k, was developed for each quarry. Based on this interval, the kth worker was interviewed on each interview day until the required sample size was reached for each quarry. The administration of the study was focused mainly on the industrial noise pollution and its impact on the hearing capabilities of workers. The desired sample size was calculated based on Kirkwood and Sterne [The surveys were conducted from April to June 2012. Based on the average number of clients per day for a specific facility and the desired sample size, a recruitment interval, Data were collected mainly through interviewing. Participants were given consent form to sign and had all their concerns and questions answered before data collection began. All tools employed in the research were developed using standard procedures, pretested, and revised to ensure their validity and reliability and research assistants were trained to ensure uniformity in the administration of questionnaires. The various instruments used for measuring sound levels were the Casella sound level meter (in the recording of the noise levels in the various companies) and pure-tone audiometer .The weighted networks in a meter are electronic circuits whose response to low frequencies and to very high frequencies is reduced in a specified manner. In general, four different weightings have been standardized internationally as A, B, C, and D. Network A is used in industrial setting and this was used throughout the study. To check for vibration, shock, and excessive heat, a calibrator was attached to the microphone of the meter and the reading was then compared with the calibrator's value. The meter was adjusted when required to bring it into calibration.A biologic calibration of the pure-tone audiometer was performed daily using supposedly normal people with normal hearing prior to any audiological evaluation of the subjects. An otoscopic examination was also performed on each subject to exclude wax or any discharge in the ear canal or perforation of the eardrum to rule out possibility of conductive hearing loss. To overcome bias on the hearing acuity by ambient noise influence, five (5) of the subjects each from the various companies were randomly selected to undergo audiometric retesting at Hearing Assessment Centre at Komfo Anokye Teaching Hospital (KATH). The hearing thresholds obtained for these groups were found not to be different from those obtained within the premises of the factories. Audiometric tests were performed on employees working in the area with noise level exceeding the threshold limit value of 85 dBA using screening Audiometer, (AS608) obtained from the Hearing Assessment Centre of the Department of Ear, Nose and Throat (ENT) of KATH. Hearing acuity was measured at 5 dBA interval over a range of octave band frequencies from 500 to 8000 Hz. Hearing was considered normal if the threshold level was less than or equal to 25 dBA at the frequencies 250, 500, 1000, 2000, 4000, and 8000 Hz. However, the intensity of the stimuli was increased beyond 25 dBA at any frequency until a response was obtained. Individuals with a characteristic notch of four (4) KHz depicting the classical sign of NIHL were analysed. The degree and type of hearing loss were also determined using Goodman's  and Carhp value of <0.05.The outcome variable was hearing threshold (dBA). Hearing threshold beyond 25 dBA is classified as having hearing loss and hearing loss was further classified based on its severity . The explanatory variables were age, duration of exposure, gender, and use of earplugs. The response of each subject and the data obtained from the administration of the physical instruments were scrutinized carefully and categorized in tables and graphs. Data was analyzed with R 3.1.1 . The ana∗(loglikelihood) + 2∗(number  of  parameters  in  the  model), that is, −2L + 2p. Lower AIC is an indication of a better likelihood.A correlation between age and duration of exposure was assumed and tested using Pearson's correlation. This was because the variable appeared to be approximately normally distributed. The explanatory variables included in the model were also tested for collinearity using the variance inflation factor (VIF). The choice of best-fitted model was based on Akaike's Information Criterion (AIC). This was proposed by Akaike  as a meaA written permission was sought from the management of the various quarry companies and ethical clearance was granted by the Committee for Human Research and Population Ethics (CHRPE) at the School of Medical Science (SMS) of the Kwame Nkrumah University of Science and Technology (KNUST) and Komfo Anokye Teaching Hospital, Kumasi.Majority of the respondents were males (81.4%). The mean age (SD) of the respondents was 41.7 (9.20). Most of the respondents were Christians (59%) and about 29% were Muslims. With respect to their level of education, 47.2% had junior secondary or middle school education. About 17% had tertiary education and 14 (2.3%) had no formal education. Majority had worked in the quarry for up to 10 years and 24.7% had worked in the quarry for less than 5 years. 132 (33%) of the respondents used earplugs and 61% of respondents who wore no earplugs had hearing thresholds above 25 dB as against 36% among those who wore earplugs.All the companies had different production units with more or less the same type of machinery. p < 0.001), indicating the influence of the working environment on the hearing threshold of a respondent. As shown in The mean hearing threshold among all workers was 27.32 dBA and 176 respondents (44%) had hearing thresholds higher than 25 dBA. Comparatively, a higher mean threshold was observed among workers at Taysec company (29.98 dBA) followed by Northern mines and quarry (26.43 dBA) . More thr = 0.465) (= 0.465) . The VIF= 0.465)  were verp value of 0.05.Age, duration of exposure, and use of earplugs significantly predicted hearing impairment among quarry workers . In the Occupational health is an important concern of the working person. Various elements concerning a person's working environment can predispose one to developing a disease process. Quarries are such organizations with high noise production levels as a result of their activities. The aim of this study was to look at NIHL among quarry workers in Ashanti region of Ghana. This study found a high prevalence of hearing impairment among the quarry workers and all machines used at the various sites produced noise levels that exceed the limiting threshold.Results from this study indicate that all companies studied have different production units with similar type of machinery and the noise levels ranged from 85.5 dBA to 102.7 dBA. This reveals that all the machines used at the various companies produced sound that exceeds the minimum threshold of 85 dBA as recommended by World Health Organization , therebyThese, however, might not be limited to quarries alone. Studies on noise levels from other work settings including the study in Ghana by Boateng and Amedofu  on the iThis study revealed that 44% of the quarry workers had hearing thresholds of more than 25 dB. This was, however, higher than the other prevalence reported from Sudan , 30.6%, Generally, there is an established association between the age of workers and hearing loss –25. In tThe study also revealed a significantly positive association between duration of work at the quarry and hearing impairment. According to the United States National Institute of Deafness and Other Communicable Disorders , long orWearing earplugs or other protective devices has been recommended for individuals involved in a loud activity. The proportion of respondents with hearing impairment was higher among those who did not wear earplugs. The multivariable analysis showed that use of ear protection decreased the odds of having hearing impairment among quarry workers. This is consistent with the outcome of the study by Hong , which fAlthough the study accessed the exposure to noise, there might still be other underlying factors, which might have contributed to hearing loss. These sources of noise produce what is called sociocusis and their effect on hearing loss might not differ from occupational hearing loss . The autThis study found that most of the machines used in the quarry exceed the tolerable threshold of sound thereby having the potential of damaging the hearing status of workers. Noise levels measured among the quarries workers studied also exceed the limit set by the Ghana Environmental Protection Agency (EPA) under the EPA Act of 1994 (Act 490) which permits light industrial areas. It is recommended that EPA embark on regular monitoring to access noise levels and ensure that companies do not emit noise greater than the tolerable limits. Use of earplugs showed a protective effect on development of hearing loss. Efforts to ensure access and use of earplugs by quarry workers and quarry companies could be beneficial in reducing the absolute prevalence of hearing impairment especially among the elderly and long serving workers who have been shown to be at an increased risk of developing hearing loss.


TCs) and their telopodes (Tps) have been found in various organs of many mammals, including in lower animals. However, knowledge of TCs in lower animals is still very limited. This study identified TCs and their Tps in the ileum of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata), by transmission electron microscopy. The TCs/Tps were found near epithelial cells, glandular cells and unmyelinated nerve fibres. Moreover, exosomes were also found to be present in between TCs/Tps and these cells.Telocytes ( Andrias davidianus (Amphibia: Caudata), using transmission electron microscopy (TEM) to improve our understanding of amphibian tissue regeneration Many investigations have documented that TCs and their Tps are resident in various organs of many mammals, including human, pig, gerbil, mouse, rat and degus After euthanizing on the ice, four farmed , 2.5‐year‐old Chinese giant salamanders (weight: 0.99–1.12 kg) were killed, and the ileums were excised. Small pieces of ileum were fixed in 2.5% glutaraldehyde/PBS. The specimens were sectioned with a LKB‐V ultramicrotome . The ultrathin sections were observed and photographed using a JEM‐1200EX TEM .In the TEM images, TCs and their Tps segments were located in the lamina propria of the ileums from the Chinese giant salamander Figs , 2, 3, 4In the previous studies, TCs were identified in gastrointestinal system of mammals, for example human, mice and rats The authors declare that there are no conflicts of interest.


A substantial number of prospective epidemiological studies have been conducted to investigate the association between biomarkers of inflammation and immune function and risk of colorectal cancer. Although pre-diagnostic concentrations of these biomarkers, especially C-reactive protein, have been associated with a higher risk of colorectal cancer in some studies, this association does not seem to have a robust support without hints of bias. Future prospective studies should evaluate multiple inflammatory biomarkers with longitudinal measures over the follow-up taking advantage of new multiplex cytokine quantification arrays and use more sophisticated joint or biomarker pattern statistical approaches to capture the complex and dynamic interplay between biomarkers and risk of colorectal cancer. Large collaborative consortia and Mendelian randomization studies should be encouraged to diminish the threat of biases and improve the reliability of this literature. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide that accounted for 1.36 million new cases and 694,000 deaths in 2012 .Systemic inflammation is a pathway through which several of the aforementioned risk factors may lead to colorectal neoplasia. For example, obesity is associated with a state of chronic inflammation induced by excessive production of storage lipids and high circulating concentrations of glucose, both of which create a proinflammatory oxidative environment that has been associated with colorectal carcinogenesis , 7. AdipHowever, the link between inflammation and colorectal neoplasia is not only substantiated at a systemic level, and it has been further hypothesized that sporadic colorectal neoplasia arises from colonic areas with chronic subclinical inflammation. Several lines of evidence implicate chronic inflammation as a key predisposing factor to colorectal cancer in IBD . First, The aim of this review is to examine and critically appraise the associations in the literature between biomarkers of inflammation and immune function and risk of CRC and through which infer about the strength of the evidence linking systemic and/or chronic low-grade colorectal inflammation to colorectal carcinogenesis.C-reactive protein (CRP) is a sensitive but non-specific systemic marker of inflammation that is produced mainly in the liver in response to cytokines released by phagocytes during infection, trauma, surgery, burns, tissue infarction, advanced cancer, and chronic inflammatory conditions . The cirP heterogeneity, 0.006; I2, 52 %) [A total of 18 prospective epidemiological studies have been conducted to date to investigate the association of pre-diagnostic high-sensitivity CRP concentrations with risk of CRC  •. The su2, 52 %) .Table 1CThe first prospective study was published in 2004 from the CLUE II cohort of Washington County, MD, with 172 CRC cases and 342 matched controls and found a twofold statistically significant increase in the risk of colorectal cancer comparing persons with the highest versus lowest fourth of C-reactive protein . The twoThe evidence linking CRP concentrations with colorectal adenoma development, a precursor in the natural history of colorectal neoplasia, is weaker. Pre-diagnostic CRP concentrations were not associated with colorectal adenoma in our study in the CLUE II cohort . A subseGiven that high-sensitivity CRP is a circulating marker for inflammation, the lack of a consistent association with colorectal neoplasia raises questions on its use as a marker of the inflammatory process associated with colorectal cancer. When we investigated whether increased CRP is a marker of colonic inflammation, we found no such association in a colonoscopy-based study •, which In 2012, our group critically appraised the literature of 98 biomarkers and cancer risk evaluating 847 individual studies to shed light on the existence of potential biases and identify associations with robust evidence for their presence ••, 45. WCRP gene and colorectal cancer risk [CRP variants to be associated with CRC risk [CRP and/or genetic scores thereof as instruments of the circulating CRP concentrations in order to correct for potential biases of the observational association and have generally reported null results [Further evidence suggesting a lack of a causal link between CRP and colorectal neoplasia comes from candidate gene association studies that have observed mixed results for the association of genetic variants in the cer risk , 48–52 aCRC risk . Mendeli results , 53, 54, results . However results . Future  results –58, to eInterleukin-6 (IL-6) is a multifunctional cytokine produced by a variety of hematopoietic and non-hematopoietic cells  and exerP heterogeneity, 0.18; I2, 35 %) [II’yasova and colleagues found that serum IL-6 concentrations were not statistically significantly associated with the risk of colorectal cancer in the Health Aging and Body Composition Cohort . Similar2, 35 %) •.The epidemiological studies have not in general reported results by tumor location, except for the Health Professionals Follow-up Study that observed a statistically significant increased risk of colon cancer with higher IL-6 concentrations, but the estimate was attenuated and not statistically significant anymore after excluding cases that occurred within 2 years of the baseline blood draw . No assoIL6 genetic polymorphisms and risk of CRC has been evaluated in several candidate gene studies with largely null results [The association between  results , 62–64.  results .TNFa, especially rs1800629, and risk of colorectal cancer [TNF-α is another pleiotropic cytokine that is elevated in response to infection and tissue damage and has been shown to be involved in the pathogenesis of IBD . Howeverl cancer , 62–64.The literature evidence regarding the association of circulating concentrations of inflammatory biomarkers, other than CRP, IL-6, and TNF-α, and the risk of colorectal cancer is very sparse. Serum amyloid A (SAA) is a non-specific inflammatory marker produced in the liver in response to infection, trauma, and other inflammatory states and has been hypothesized to be a more sensitive marker of inflammation over CRP for certain diseases , 71. HowThe adipose tissue is an active endocrine organ that secretes several cytokines and hormones, collectively termed adipokines , which have been demonstrated to have pro- or anti-inflammatory functions. The association between adipokines and colorectal cancer risk has been investigated in several epidemiological studies, the results of which have been mixed . The desCalprotectin is a calcium- and zinc-binding protein released by activated innate immunity cells. It is found in cells, tissues, and fluids throughout the body and has been studied in a variety of inflammatory conditions . Fecal cThe overall literature evidence for an association between inflammatory biomarkers and risk of colorectal cancer is inconclusive. Although concentrations of inflammatory biomarkers, especially CRP, have been associated with colorectal cancer risk in some studies, this association does not seem to have a robust support without hints of bias . This does not necessarily mean that these biomarkers are not truly associated with colorectal cancer, but there is still substantial uncertainty about them. If any potential causal association is identified in the future between an inflammatory biomarker and risk of colorectal cancer, then this might lead to the potential development or repurposing of drugs targeting receptors of inflammatory markers.There are several ways to improve this evidence in the future. First, the measurement and estimation of a single biomarker and colorectal cancer association in most of the existing studies do not consider the complex synergisms among the many inflammatory biomarkers. Recent developments in serum cytokine quantification technology include multiplex arrays that can measure multiple biomarkers in each sample and better evaluate the complex and dynamic nature of inflammatory responses with substantial cost and sample savings . When maThird, large collaborative consortia should be encouraged in this field. The use of standardized definitions and protocols for exposures, outcomes, confounders, and statistical analyses may diminish the threat of biases and improve the reliability of this literature. Many of the biases may be further lessened if studies were more completely and transparently reported according to published guidelines, such as the Strengthening the Reporting of Observational Studies in Epidemiology statement and its extension for Molecular Epidemiology –86. Stat


Aim. To investigate whether dyspepsia symptoms differ depending on the presence or absence of cold hypersensitivity in the hands and feet (CHHF). Methods. In all, 6044 patients were recruited and provided with a questionnaire about CHHF and dyspepsia. Based on their responses, subjects were divided into a CHHF group  and a non-CHHF group . The groups were compared in terms of their usual digestion status, using chi-square tests and logistic regression analyses to calculate the propensity score and odds ratios (ORs). We analyzed the participants' responses to questions on dyspepsia symptoms. Results. After matching, chi-square tests indicated that the CHHF group had higher frequencies of the following symptoms: bad digestion, poor appetite, discomfort in the upper abdomen, motion sickness, epigastric burning, postprandial fullness, nausea, and bloating. Additionally, CHHF was associated with an increased OR for dyspepsia  compared with the non-CHHF group. Conclusion. This study confirmed that CHHF patients have elevated frequencies of most dyspepsia symptoms. In traditional Korean medicine, pattern identification is important to both understanding the patient's status and writing a prescription. There are several methods of pattern identification, including 8-principle pattern identification, constitutional pattern identification, and visceral pattern identification , 2. The Because cold and heat pattern identification is diagnosed based on both the patient's symptoms and 4 examinations by a doctor of Korean medicine, objective evidence is necessarily insufficient for diagnosis. Further, evidence on the effects of cold and heat status on the body also remains uncertain. To resolve these issues, recent studies have attempted to provide objective standards for cold and heat, including the development of a cold pattern questionnaire by Ryu et al.  and a st Huangdi Neijing [Therefore, we decided to conduct the present study using a narrower range of inclusion criteria and a more specific scope of research. First, rather than investigating the dual states of cold and heat, we limited our investigation to differences between cold and noncold individuals. Second, rather than recruiting subjects based on various symptoms of coldness, we specifically analyzed cold hypersensitivity in the hands and feet (CHHF), which is a representative cold symptom. CHHF is relatively common symptom in Korea and is more frequently observed in women than in men . Patient Neijing , which sPrevious studies have also reported that cold hypersensitivity and dyspepsia are correlated , 9. HoweThis cross-sectional study was conducted between November 2006 and August 2014. All of the questionnaire data, including CHHF and dyspepsia status, were compiled from the Korean Medicine Data Center (KDC) of the Korea Institute of Oriental Medicine (KIOM) . Using tThose who responded “cold” to the question “are your hands cold or warm?” and those who responded “cold” to the question “are your feet cold or warm?” were classified as the CHHF group. Those who responded “warm” or “normal” to the both of these questions were classified as the non-CHHF group. We excluded those who stated that they were cold in response to only 1 of these 2 questions because the presence of CHHF symptoms appeared to be unclear. For similar reasons, we excluded those who stated that they were unsure in response to either question.The questionnaire included 9 items that refer to common complaints in Korea and were derived from descriptions in the Rome II classification . In addihttp://dx.doi.org/10.1155/2016/8948690).The details of each question were as follows. To the question “how is your digestion?” the subjects chose either “1. good” or “2. bad.” To the question “how is your appetite?” and an item related to anorexia, the subjects chose “1. very good,” “2. good,” “3. average,” or “4. not good.” The criteria for these responses were as follows: “1. very good” refers to the desire to eat more foods despite satiety after meal; “2. good” refers to the case in which one feels hungry at mealtimes and wants to eat food; “3. average” refers to the case in which one eats meals at mealtimes but does not have a good appetite; and “4. not good” refers to the case in which one has no appetite at mealtimes and does not have a good sense of taste, even when eating. For the items on dyspepsia symptoms , respondents were asked to choose 1 of the following answers: “1. often,” “2. sometimes,” and “3. rarely.” “1. often” refers to greater than or equal to 2 times per week, “2. sometimes” refers to greater than or equal to 3 times per month, and “3. rarely” refers to less than or equal to 2 times per month . The chi-square test was used to analyze the frequencies and percentages of responses to digestion-related questions in the CHHF and non-CHHF groups. In addition, logistic regression was performed to calculate the odds ratios (ORs) for dyspepsia in the propensity-matched group as well as in the original groups. The OR was determined for each dyspepsia-related item in the CHHF group compared to the non-CHHF group. The statistical significance level was set at P < 0.05.The statistical program SPSS 21.0 for Windows  was used for statistical analysis. The general characteristics of the subjects were matched using a propensity score consisting of sex, age, and BMI, with the matching process involving a minimum distance scoring method. n = 1958; 66.3%) than men . The female-to-male ratio was much higher in the CHHF group  than in the total study sample. The mean ages in the CHHF and non-CHHF groups were 44.6 and 47.4 years, respectively. The mean height and weight in the non-CHHF group were 1.7 cm taller and 6.8 kg heavier, respectively, than the corresponding values in the CHHF group. The mean BMIs in the CHHF and non-CHHF groups were 22.0 and 24.1, respectively. The general characteristics were significantly different between CHHF and non-CHHF groups before matching . After propensity score matching, the total number of patients in each group was 640, with no statistically significant differences in general characteristics between the groups  in every dyspepsia item. A higher proportion of the CHHF group reported bad digestion and not good appetite compared to the non-CHHF group, and the frequency of all dyspepsia symptoms  was also higher.Before matching, the CHHF group and non-CHHF group significantly differed (P < 0.001), discomfort in the upper abdomen, motion sickness, epigastric burning, postprandial fullness, nausea, and appetite (P < 0.05). The frequency of dyspepsia symptoms was higher in the CHHF group compared to the non-CHHF group, while there was no statistically significant difference between the two groups in items of vomiting, exhaustion when hungry, belching, and epigastric pain (related to digestion)  .As can be seen in P < 0.001), vomiting, epigastric burning, nausea, and epigastric pain (P < 0.05) significantly differed between the groups, and there was no significant difference for not good appetite, discomfort in the upper abdomen, exhaustion when hungry, and belching. The OR was highest for bad digestion (2.423) before matching and for bloating after matching (1.883)  .For the OR of each response (“often” and “sometimes”) for dyspepsia symptoms after matching, both responses (“often” and “sometimes”) for bloating significantly differed between groups. There was a significant difference in either “often” or “sometimes” responses for discomfort in the upper abdomen, vomiting, motion sickness, exhaustion when hungry, epigastric burning, postprandial fullness, nausea, and epigastric pain, and there was no difference in belching .We chose to investigate the relationship between CHHF (including various cold symptoms) and indigestion because of the “spleen and 4 extremities” theory that was described in Huangdi's classic text , which iCHHF refers to a condition in which one experiences discomfort in daily living because of cold symptoms in the limbs. It is more inclusive than Raynaud's phenomenon and includes decreased temperature in the hands and feet, as well as the subjective sensation of cold. CHHF is suspected to induce spastic peripheral vasoconstriction, but no specific, certain cause has yet been identified. In Korea, CHHF is a relatively common symptom and occurs more often in women than in men . The diaDyspepsia is a digestive function disorder that refers to the collective symptoms of the upper gastrointestinal tract. In general, the term “dyspepsia” denotes functional dyspepsia; in this study, we therefore excluded persons who had been diagnosed with chronic gastritis, gastroduodenal ulcers, esophagitis, fatty liver, hepatitis, and digestive tract tumors. Through these exclusions, we sought to remove as many cases of organic dyspepsia as was feasible. In Rome III , functioIn the present study, the data were compiled from questionnaire responses that had been collected by KDC. Participants who noted having cold hands and feet were assigned to the CHHF group, while participants who had neither cold hands nor cold feet were assigned to the non-CHHF group. Between-group differences in indigestion were analyzed using the KDC digestion questionnaire. Several recent studies have used the KDC data, including those by Do et al.  study onThe aim of this study was to verify whether there was a difference in functional dyspepsia frequency according to the presence of CHHF and, if so, which of the various digestion-related symptoms differed. In summary, patients with CHHF showed a high frequency of dyspepsia, with this tendency generally maintained after matching. Bloating in particular significantly differed between groups both before and after matching: the highest OR after matching was 1.883 and the responses “often” and “sometimes” were significantly more common in the CHHF group.2 versus 44.6 years and 22 kg/m2, resp.). We concluded that these differences could harbor considerable bias in examining the relation between CHHF and dyspepsia. Therefore, the patients were matched in the CHHF group and non-CHHF group using the propensity score matching method (640 patients per matched group). There was no difference in sex, BMI, and age after matching. These differences in general characteristics confirmed that CHHF was influenced by sex and BMI, as in previous studies [The general characteristics of subjects differed markedly depending on the presence of CHHF. In the CHHF group, the female-to-male ratio (81%) was much higher compared to the non-CHHF group 56%), and the mean age and BMI were higher in the non-CHHF group  functional dyspepsia symptoms, especially bloating.Supplementary Table1: This table shows the entire questionnaires on cold hypersensitivity in the hands and feet (CHHF) and dyspepsia. The subjects were asked to answer the questionnaire based on their usual status within the past 6 months. The criteria for appetite responses were as follows: “1. Very good” refers to the desire to eat more foods despite satiety after meal; “2. Good” refers to the case in which one feels hungry at mealtimes and wants to eat food; “3. Average” refers to the case in which one eats meals at mealtimes, but does not have a good appetite; and “4. Not good” refers to the case in which one has no appetite at mealtimes and does not have a good sense of taste, even when eating. Furthermore, the criteria for responses regarding digestion symptoms were as follows: “1. Often” refers to greater than or equal to 2 times per week, “2. Sometimes” refers to greater than or equal to 3 times per month, and “3. Rarely” refers to less than or equal to 2 times per month. Supplementary Figure 1: This figure shows odds ratios (ORs) for dyspepsia before and after propensity score matching, according to CHHF status. The ORs of responses (“Often” and “Sometimes”) were analyzed for each symptom, except for digestion status and appetite.


Nova Scotia has the highest reported incidence in Canada, but risk is localized to identified disease-endemic regions. Ixodes scapularis ticks, which transmit Borreliaburgdorferi, the causative agent of Lyme disease (LD), are endemic to at least 6 regions of Nova Scotia, Canada. To assess the epidemiology and prevalence of LD in Nova Scotia, we analyzed data from 329 persons with LD reported in Nova Scotia during 2002–2013. Most patients reported symptoms of early localized infection with rash (89.7%), influenza-like illness (69.6%), or both; clinician-diagnosed erythema migrans was documented for 53.2%. In a separate serosurvey, of 1,855 serum samples screened for antibodies to B.burgdorferi, 2 were borderline positive (both with an indeterminate IgG on Western blot), resulting in an estimated seroprevalence of 0.14% (95% CI 0.02%–0.51%). Although LD incidence in Nova Scotia has risen sharply since 2002 and is the highest in Canada , the estimated number of residents with evidence of infection is low, and risk is localized to currently identified LD-endemic regions. Borrelia burgdorferi, which is transmitted to humans by infected ticks. In Nova Scotia, Canada, the vector is the blacklegged tick, Ixodes scapularis. Approximately 300,000 cases of LD occur in the United States each year (Ixodes ticks are now endemic to parts of British Columbia (I. pacificus), Manitoba, Ontario, Quebec, New Brunswick, and Nova Scotia (I. scapularis)  is an emerging vector-borne infection caused by B. burgdorferi to characterize and estimate the risk for features of LD in Nova Scotia.Since the first case of locally acquired LD was reported in 2002, the number of human infections in Nova Scotia has risen sharply; 154 cases were reported in 2013 alone. LD can manifest with localized disease, most commonly erythema migrans (EM), or disseminated illness with neurologic, cardiac, and/or joint involvement. In the United States, up to 7% of cases are asymptomatic , and sampling was proportionate to the Nova Scotia population in five 10-year age groups for ages 10–59 years and 1 age group for ages 60–64 years. The Research Ethics Board of each DHA approved the serosurvey. One Research Ethics Board required an opt-out option for patients undergoing blood collection during the study period, achieved by publicizing the study using posters and asking patients who did not want their serum used to identify themselves at the time of collection. No patients opted out.B. burgdorferi using a commercially available enzyme immunoassay (EIA) that used a whole-cell sonicate of B. burgdorferi . Samples were tested at the Capital DHA microbiology laboratory. In accordance with Canadian and US guidelines (We detected antibodies to http://qgis.org/en/site/). Population estimates were based on 2011 census data from Statistics Canada  years of age, and most (76.9%) were male . Only 26A total of 307 (93%) case-patients reported living in or traveling to an endemic region in Nova Scotia . Of the A total of 263 (80%) cases had serologic evidence of infection. Of the 66 cases classified as “probable: EM + endemic exposure” that were reported to Nova Scotia public health professionals, 10 did not have any information about laboratory testing, 42 did not have serologic testing, and 14 had serologic test results that were negative by the 2-tier algorithm. Of these 14 patients, 3 were negative on the whole-cell EIA, and 11 were positive by the C6 ELISA but negative by WB.The annual number of serologic tests for LD in Nova Scotia increased from 1,659 in 2010 to 2,421 in 2013. Testing rates varied by DHA , rangingB. burgdorferi, 215 (11.6%) screened positive by the whole-cell EIA  and were sent to the NML for confirmatory testing. Of these, 17  were positive or equivocal by the C6 ELISA. None were positive by IgG WB using Centers for Disease Control and Prevention (CDC) criteria, but 2 samples were borderline positive  and 0.98% (95% CI 0.56%−1.60%), respectively   were positive on the C6 ELISA. Although some data suggest that classifying LD on the basis of C6 alone is more sensitive than the 2-tier algorithms  and not recommended. Thus, surveillance data might underestimate incidence if not all clinical cases are captured. We recognize that estimating the extent of underreporting is difficult and that underreporting could vary geographically. However, in the cohort reported here, 42 of 66 cases classified as “probable: EM + endemic exposure” followed the Infectious Diseases Society of America guidelines (>65-year age groups.Another limitation is that the serosurvey samples might not be representative of the population at risk. We used samples of residual serum from diagnostic testing that might be biased toward a population with more medical co-morbidities or different risk and health-seeking behaviors (B. burgdorferi. However, as tick populations continue to expand, we expect LD rates to continue to increase. Residents of, and travelers, to Nova Scotia need to be vigilant and take precautions to reduce their risk for LD when they venture into regions where ticks are present. Because only a minority of patients will report a tick bite, physicians should be aware of the manifestations of LD and consider it when patients have compatible symptoms and exposure to an endemic region, through residence or travel, in Nova Scotia.Although LD incidence is increasing in Nova Scotia, infections appear to be restricted to regions within the province where populations of infected blacklegged ticks are known to be endemic. These findings support a targeted approach to public health risk messaging. Our seroprevalence study suggests that <1% of Nova Scotia residents have been exposed to


Macaca sylvanus). We tested whether grooming is related to kinship, rank and friendship. Furthermore, we tested whether grooming is reciprocated or exchanged for rank related benefits (i.e. lower aggression and increased tolerance whilst feeding). We found that in general grooming was reciprocally exchanged, directed up the hierarchy and at the same time affected by friendship and kinship. Grooming was more frequent among individuals with higher friendship values as well as amongst related individuals. We also divided our data set on the basis of rank difference and tested if different power asymmetries between individuals affected the tendency to exchange grooming for rank related benefits and grooming reciprocation. In support of our initial hypothesis our results show that the reciprocation of grooming was a significant predictor of grooming interactions between individuals of similar rank, but not between those individuals more distantly separated in the social hierarchy. However, we did not find any evidence for grooming being exchanged for rank related benefits in either data set. Our results, together with previously published studies, illustrate the behavioral flexibility of macaques. It is clear that multiple studies of the same species are necessary to gather the data required for the solid comparative studies needed to shed light on patterns of grooming behavior in primates.Grooming is one of the most conspicuous social interactions among nonhuman primates. The selection of grooming partners can provide important clues about factors relevant for the distribution of grooming within a social group. We analyzed grooming behavior among 17 semi-free ranging female Barbary macaques ( Grooming behavior involves the careful inspection and subsequent cleaning of other individuals’ fur and may occupy up to 20% of the daily time budget in non-human primates . MoreoveTraditionally grooming has been considered to be an altruistic behavior that is costly to the groomer (e.g. lost time and energy) and beneficial to the recipient  . The timStrong positive social relationships  have Macaca assamensis [M. arctoides [Silk et al.  suggestesamensis , M. arctrctoides ). This trctoides . BarbaryPatterns of grooming interactions and their driving factors are not expected to be the same across different species of primates. Several characteristics of social hierarchy, particularly dominance steepness, vary among species and may have important consequences for grooming interactions . DominanMacaca fuscata) data from different groups and populations are available. This well-studied species is highly variable with regards to social behavior, and serves as a reminder that data from several groups per species is important for detailed comparative studies  (no permission IDs were given).The study was conducted in the Apes’ Den troop of Barbary macaques living in the Apes’ Den in the Upper Rock Natural Reserve, Gibraltar. This group is semi-free ranging, provisioned daily by the Gibraltar Ornithological and Natural History Society (GONHS), and visited by tourists. The study included all 17 adult females (age ≥ 3 years) present in the troop at the beginning of the study . The troop also included six adult males and up to 15 juveniles and infants. Three new immigrant males  joined the troop in the second part of the study. All adult subjects were individually recognized and well-habituated to the presence of human observers  and focal instantaneous sampling   using the lme4 package [i) by saying that the expected response variable value increases exp(bi)-times  when the predictor value increases by one unit. In the case of categorical predictors , exp(bi) shows how many times larger the mean response value for the particular predictor state is than the mean of observations for the other (reference) state. This interpretation using the exponential function can also be applied to the end points of the estimated confidence intervals.Linear mixed effect models (LMM) were used to test the effect of kinship, rank and friendship on grooming interactions. Grooming was represented by two measurements: grooming rate  and grooming time . These two variables were positively correlated in our study , however some previous studies have shown different results for grooming rate and time, thus we computed models for both. The grooming data were log transformed to increase the homogeneity of their variances. The dataset for each model comprised two lines per dyad  and in total included 272 lines for each season. All the LMMs analyses were run in  package . When pr package . Given tRank—represented the dominance relationship of the groomer relative to the groomee coded as two states: dominant and subordinate. Rank distance—represented by the absolute value of the rank distance between the groomer and groomee (based on the David`s score). Kinship—represented the maternal kin relationship between the groomer and groomee coded as two states: kin and non-kin. Friendship—measured as the time spent in contact by a given pair of individuals relative to average time spent in contact with other group members, entered as a continuous variable. Co-provisioning—measured as the percentage of time spent by individuals in a given dyad using one food source. Grooming received—grooming rate or time received by the groomer from a given groomee. Aggression received—rate of aggression received by a groomer from a given groomee. Season—coded as two states: season 1 and season 2, corresponding to the two study periods. Age difference—the absolute value of age difference between the groomer and the groomee. This last variable was added to control for dyad similarity based on age. The identities of the groomer and groomee were used as crossed random factors in the models.The models tested the effects of the following variables on initiated grooming rate or grooming time. In order to assess the differences in grooming patterns of individuals closer in rank compared to those of individuals that were distant in rank, we divided our data into two subsets based on rank distance: a) a dataset with grooming interactions among females who have higher rank distance than the total sample average rank distance and b) a dataset with grooming interaction among females who have smaller than average rank distance. Again we tested the effect of the same variables on grooming rate and grooming time. This yielded four additional models. For each model we checked homoscedasticity and distribution of residuals using regression diagnostic plots. The significance of all our models was assessed by comparison with corresponding null models.We collected a mean of 15.5 (±0.27 SD) hours of focal observation data per female in season 1 and 13.6 (±0.65 SD) hours per female in season 2. We recorded 1,362 grooming interactions among females (842 in season 1 and 520 in season 2). Females were involved in an average of 0.17 (±0.48 SD) grooming interactions per hour and for an average of 0.79 (±1.74 SD) minutes per hour. The number of grooming interactions per female ranged from 69 to 298 with an average of 160.2 interactions. Data provided in The resulting dominance hierarchies were based on 495 (with 21 unknown dyads) and 395 (with 35 unknown dyads) interactions in season 1 and season 2 respectively. The dominance hierarchies were significantly linear  and the direction of interactions was highly consistent with the resulting rank order . The model predicted that when a groomer is subordinate to a groomee the rate of grooming is 16% to 184% higher than when the groomer is dominant to the groomee. Friendship was also a significant predictor of grooming and females groomed their higher quality partners more often than low quality social partners. The model predicted that an increase of 1 in the friendship value will lead to an increase in grooming rate of 37% to 64%.The results also show that females exchange grooming for grooming with their partners as they more often groom females who groomed them back. The model predicted that an increase of 0.1 in grooming rate received is related to an increase of 5% to 22% in grooming rate initiated. Aggression received was also related to grooming rate although, contradictory to our hypotheses, this was in a positive direction, in other words those groomers who initiated more grooming received more aggression from that given partner. The model predicted that an increase of 0.1 in aggression is related to an increase from 13% up to 37% in grooming rate.The season had also a significant effect on grooming, with females grooming less often during the second season. Neither relative rank distance among grooming partners nor time spent co-provisioning were related to grooming distribution in this or any of the following models.The results were slightly different for the model based on grooming time . KinshipComparison of grooming distribution among females close in rank and distant in rank. Results of the LMMs based on the two separate data sets showed that grooming received was a significant predictor in grooming among females close in rank but not in grooming among females distant in rank. Females close in rank thus exchanged grooming reciprocally and females distant in rank did not . In addition to these general patterns, we detected differences between pairs of individuals that were particularly close or distant in rank.M. sylvanus, [M. thibetana, [Of the factors studied here the effect of kinship and rank on grooming behavior has already been demonstrated  and/or ibetana, ) is neveibetana, .The third factor, friendship, was previously demonstrated to influence positive social interactions e.g. reconciliation  and thisMacaca assamensis), Macdonald and colleagues [As mentioned above the significant relationship between grooming and rank was not surprising. The tendency of females to prefer a dominant grooming partner is usually explained by social market theory, where low ranking females have more to gain from high ranking individuals than vice versa ,6. As halleagues  also suglleagues  showed tlleagues ,48. We tEnvironmental conditions together with the more tolerant social system of Barbary macaques might explain the absence of exchange for potential rank related benefits. If this was the case we would also expect infrequent grooming up the hierarchy, but this remains present among females in our focal group. Moreover, exchange for rank related benefits has recently been documented in another study of Barbary macaques , althougWe suggest that another relevant rank related benefit may be access to mates. Although competition among females for mates has been usually overlooked  it can rAn interesting and contradictory result of our study is the positive relationship between aggression received and grooming given. While lower aggression has been considered as one of the potential benefits to be gained via grooming interactions , in our In our data we found several differences in models based on grooming rate compared to those based on grooming time. These differences suggest that individuals reciprocate grooming more on the basis of rate rather than time. In other words, individuals in a given dyad groom each other at the same rate but not necessarily for the same duration of time. This finding is unfortunately difficult to compare to other studies, as they usually use either time or rate as an exclusive parameter in their analyses (but see ) or do nFinally it should be noted that statistical analysis of grooming data involves several limitations and potential issues of the reliability of the fitted models. First, the grooming interactions are not evenly distributed across all group members and thus the resulting dataset includes many zeroes. This is a natural consequence of the fact that individuals groom others selectively and thus some dyads groom each other more often than others and some do not engage in grooming at all. Second, some combinations of predictors included in our model are rare or even non-existent e.g., females distant in rank are more often unrelated than related. This again is an inevitable consequence of the nepotistic hierarchies and matriline system of macaques. These data characteristics may limit the power and possibly even the reliability of the tests but are also impossible to avoid.Our study tested the hypothesis that grooming is exchanged for different commodities in relation to rank difference among tolerant Barbary macaque females. We found that individuals in general, and specifically those close in rank, mainly exchange grooming reciprocally. Our results thus support the hypothesis that in tolerant species reciprocal grooming will prevail over exchange of grooming for rank related benefits. However, females still directed grooming up the hierarchy and thus further studies are needed to reveal if other rank-related benefits can explain this pattern. We also suggest that the relationship between aggression and grooming is more complex than previously thought. The prediction that grooming serves to reduce the aggression received from ones grooming partner has not been supported in Barbary macaques.We thank Vaclav Hypsa for the encouragement and helpful comments made during writing of the manuscript, Stanislav Lhota for useful comments on the previous version of the manuscript and Simon T. Segar for language corrections. We also thank three reviewers for their detailed comments that significantly improved the manuscript.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.


In the Author Contributions section, Nathalie J. M. van Hees (NH), Erik J. Giltay (EG), Thomas Puvill (TP), Nadine Janssen (NJ), and Willem van der Does (WD) all should be listed as one of the persons who wrote the paper. The correct contributions are: Conceived and designed the experiments: NH EG WD. Performed the experiments: NH TP NJ. Analyzed the data: NH EG TP NJ WD ST. Contributed reagents/materials/analysis tools: ST JG. Wrote the paper: NH EG WD TP NJ ST JG.


Hearing loss—ranging from mild to severe—afflicts large numbers of individuals of all ages. It is estimated that 40–50% of adults over the age of 65 years have some degree of significant hearing loss, with this figure rising to 83% of those over the age of 70  model in which they stress the importance of working memory for online spoken language processing, especially under poor listening conditions  to assess white matter integrity (Rachakonda et al., Together, these collected articles reflect a valuable sample of current approaches to our understanding of the effects of hearing loss on neural and perceptual processing. A theme that emerges from both the human and animal studies in this collection is that of an adaptive plasticity in the sensory, perceptual and cognitive systems that regulates performance in the face of often seriously degraded input. Challenges for future research include better understanding the link between neural consequences of hearing loss and other modifications of acoustic input (Van Engen and Peelle, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Life-space mobility reflects individuals’ actual mobility and engagement with society. Difficulty in hearing is common among older adults and can complicate participation in everyday activities, thus restricting life-space mobility. The aim of this study was to examine whether self-reported hearing predicts changes in life-space mobility among older adults.n = 848). At-home face-to-face interviews at baseline and telephone follow-up were used. Participants responded to standardized questions on perceived hearing at baseline. Life-space mobility  was assessed at baseline and one and two years thereafter. Generalized estimating equations were used to analyze the effect of hearing difficulties on changes in LSA scores.We conducted a prospective cohort study of community-dwelling older adults aged 75–90 years (p = .040). Over the 2-year follow-up, the life-space mobility score declined in all hearing categories in a similar rate . Participants with mild or major hearing difficulties at baseline had significantly higher odds for restricted life-space (LSA score < 60) at two years  compared to those without hearing difficulties. The analyses were adjusted for chronic conditions, age, sex and cognitive functioning.At baseline, participants with major hearing difficulties had a significantly lower life-space mobility score than those without hearing difficulties (mean 54, 95 % CI 50–58 vs. 57, 95 % CI 53–61, People with major hearing difficulties had lower life-space mobility scores at baseline but did not exhibit accelerated decline over the follow-up compared to those without hearing difficulties. Life-space mobility describes older people’s possibilities for participating in out-of-home activities and access to community amenities, which are important building blocks of quality of life in old age. Early recognition of hearing difficulties may help prevent life-space restriction. Difficulty in hearing is common among older adults . The prePrevious studies have suggested that hearing difficulties can complicate engagement in everyday life situations; especially those requiring communication with other people , 9. Diffactually do, as it describes total mobility, thereby providing us with a broad picture of a person’s engagement with the community [Life-space mobility reflects the size of the spatial area a person moves through in daily life, the frequency of moving and the need for assistance , 17. Whiommunity , 22, 23.ommunity , 19.We are aware of only one earlier study that has investigated the association between hearing problems and life-space mobility. After adjustment for potential confounders, Allman et al. found that hearing difficulty did not predict decline in life-space mobility in a 18-month follow-up .The aim of our study was to examine whether self-reported hearing is associated cross-sectionally with life-space mobility among 75 to 90-year-old community-dwelling older adults and, whether self-reported hearing difficulties at baseline predict changes in life-space mobility at follow-up one and two years later.n = 6), poor health (n = 5), not willing to continue (n = 6), and not reached (n = 2). The LISPE project was approved by the Ethical Committee of the University of Jyväskylä. Participants were informed about the project and signed a written informed consent.This study is part of the “Life-space mobility in old age” (LISPE) project, which is a prospective cohort study of community-dwelling older adults. A more detailed description of the study design has been published earlier . BrieflyHearing at baseline was assessed by asking the following three questions:“ Do you have difficulties hearing when having a conversation with several people simultaneously?”, “Do you have difficulty hearing when conversing with another person in the presence of noise?”, and “Do you have difficulties hearing where a particular sound  is coming from?”. The participants were asked to estimate their level of difficulty when using a hearing aid if they had one. The response categories were 1) No difficulty (0 points), 2) Sometimes, some difficulty (1 point), and 3) Yes, major difficulty (2 points). Scores were summed and the resulting scale was divided into three categories: no hearing difficulties (score 0), mild hearing difficulties (score 1–2), and major hearing difficulties (3 or higher). The reason for this categorization was that having major hearing difficulties should involve some difficulty in all three situations or major difficulty in at least one situation plus some difficulty in another situation .Life-space mobility was measured using the University of Alabama at Birmingham Life-Space Assessment (LSA) questionnaire  at baselBasic demographic and socioeconomic indicators of the study subjects included age, sex and number of years of education. Self-reported physician diagnosed chronic conditions were obtained from a list of 22 chronic conditions and with an open-ended question. Chronic conditions that could theoretically be linked to hearing difficulties and life-space mobility, namely diabetes, rheumatoid arthritis, cardiac, circulatory and neurological diseases were chosen as potential covariates. Cognitive functioning was assessed using the Mini- Mental State Examination (MMSE) [Factors previously found to be potential risk factors for life-space mobility decline and hearing difficulties were considered potential confounders , 28–31. n (MMSE) .p < .05) on the parameters and were used as covariates in the adjusted models. Education did not have a significant effect in the model and was therefore excluded from the final model.The Kruskal-Wallis test was used for continuous variables and chi-square tests for categorical variables to compare the baseline characteristics between the hearing groups. Characteristics of the participants are described using medians and interquartile ranges (IQR) or percentages. In the two-year follow-up, generalized estimating equations (GEE) models were used to test the significance of the association of self-reported hearing difficulties on changes in life-space mobility over time. In addition, logistic regression models were used to investigate whether self-reported hearing difficulties at baseline were associated with higher odds for life-space restriction at baseline and at the second follow-up. In the logistic regression analyses, the life-space mobility score was dichotomized by using a cut-off score of 60. Odds ratios for life-space restriction at the second follow-up were calculated only for those participants who had unrestricted life-space at baseline. Age and sex were included in all models. In addition, cognitive functioning, diabetes and cardiac, circulatory, and neurological diseases had also a significant effect  was 80.0 years at baseline  and 62 % of the participants were women. At baseline, the median life-space mobility score in the total sample was 64.0 , ranging from 8 to 120. In the total sample, 33 % reported no hearing difficulties, 45 % reported mild hearing difficulties and 22 % reported major hearing difficulties, while having a hearing aid was reported by 13.5 %. Baseline characteristics of the participants categorized according to self-reported difficulty in hearing are presented in Table The median age of all the participants (p < .001) or with mild hearing difficulties . Participants with mild hearing difficulties did not statistically significantly differ from those without hearing difficulties in their life-space mobility score (p = .141). After further adjustment for cognitive functioning (MMSE), diabetes, cardiac, circulatory and neurological diseases and rheumatoid arthritis, the statistically significant difference in life-space mobility score between persons with mild hearing difficulty and those with major hearing difficulty became non-significant , while between participants without hearing difficulties  and those with major hearing difficulty the difference remained significant (p = .040).Participants with major hearing difficulties had a significantly lower life-space mobility score at baseline  compared to participants without hearing difficulties , and those with major hearing difficulty 2.1(95 % CI 1.4–3.2) times higher odds for restricted life-space compared to those without hearing difficulties. After further adjustment for the covariates, the participants with mild hearing difficulty no longer differed significantly from those without hearing difficulties .p < .001) and at a similar rate (group x time p = .164) . During the two-year follow-up, the difference in life-space mobility score remained significant (p = .001) between the participants without hearing difficulties and those with major hearing difficulties. At the end of the follow-up, participants without hearing difficulties had a higher life-space mobility score  than those with either mild  or major hearing difficulties . Participants with mild hearing difficulties and those with major hearing difficulties did not differ in their life-space mobility score at two-years (p = .544). Further adjustments for cognitive functioning, rheumatoid arthritis, diabetes, cardiac, circulatory or neurological diseases did not essentially change the differences between hearing categories, as life-space mobility score remained significantly higher among participants without hearing difficulties  compared to those with mild  or major  hearing difficulties.Over the 2-year follow-up, the life-space mobility score declined in all the hearing categories (main effect of time n = 465), those with mild or major hearing difficulties at baseline had significantly higher odds for life-space restriction at the second follow-up compared to those without hearing difficulties. While further adjustments did not materially change these results, the odds ratio became borderline significant (p = .057) for those with major hearing difficulty (Table Among the participants with unrestricted life-space at baseline (LSA-score > 60, ty Table .Our results showed that hearing difficulties were associated with poorer life-space mobility in community-dwelling older adults. Life-space mobility declined steadily in all the hearing categories over the two-year follow-up period. However, persons who did not have hearing difficulties at baseline had significantly higher life-space mobility score both at baseline and at the two-year follow up compared to those who reported hearing difficulties, even when controlling for the presence of chronic medical conditions and the influence of cognitive functioning, age and sex. It is likely that this trend existed before the cohort was followed up. Thus, although the changes observed in the life-space mobility score between the hearing categories were modest, the results indicate that hearing problems may contribute to restrictions in life-space mobility over time among older adults.According to previous studies , 23, a sTo our knowledge, the 18-month follow-up study of Allman and colleagues  is the oThere may be several explanations for our findings. According to earlier studies, communication problems are the most prevalent participation restrictions mentioned by older adults with hearing difficulties . For oldHearing difficulties not only impede communication with other people, but may also impair the ability to observe environmental hazards. Acoustic information supports observation of the environment while moving , and henThis study included a large population-based sample of community-dwelling older adults and there were very few missing data in the sample. Although the participants were rather well-functioning, the sample also included people with health problems . It is tA potential limitation of our study is that hearing was self-reported. However, self-reports of hearing are commonly used in epidemiological studies, and previous studies support the validity of self-reported measures of hearing impairment , 35, 40.Further studies with longer follow-ups starting from middle-age are needed to confirm the associations reported in this study. Also, some persons were excluded from the study because they were not able to communicate due to hearing problems during the initial telephone screening or home interview. Therefore, it is likely that the number of persons with severe hearing impairment in this study was under-represented. The associations observed between hearing and life-space mobility might have been stronger had these persons participated.To conclude, the present study provides new information on longitudinal changes in life-space mobility among older people with and without hearing difficulties. Given the increasing proportion of older adults in the population, it is particularly important to understand the role of hearing difficulties as a risk factor for restricted life-space mobility and consequent decreased participation in society. Our findings emphasize the need for early assessment and recognition of hearing difficulties in order to diminish the likelihood of subsequent losses in functional capacity. In future, additional attention should also be given to the balance between individual resources and environmental demands in older adults who have hearing difficulties. More specifically, it would be important to know more precisely how the acoustic characteristics of the environment and environmental noise affect older persons’ possibilities to maintain social relationships and active participation in daily life in real world situations.


Borrelia burgdorferi. We developed a number of oral vaccines based in E. coli expressing recombinant OspC type K, OspB, BBK32 from B. burgdorferi, and Salp25, Salp15 from Ixodes scapularis. Of the five immunogenic candidates only OspC induced significant levels of antigen-specific IgG and IgA when administered to mice via the oral route. Antibodies to OspC did not prevent dissemination of B. burgdorferi as determined by the presence of spirochetes in ear, heart and bladder tissues four weeks after challenge. Next generation sequencing of genomic DNA from ticks identified multiple phyletic types of B. burgdorferi OspC  in nymphs that engorged on vaccinated mice. PCR amplification of OspC types A and K from flat and engorged nymphal ticks, and from heart and bladder tissues collected after challenge confirmed sequencing analysis. Quantification of spirochete growth in a borreliacidal assay shows that both types of spirochetes (A and K) survived in the presence of OspC-K specific serum whereas the spirochetes were killed by OspA specific serum. We show that oral vaccination of C3H-HeN mice with OspC-K induced significant levels of antigen-specific IgG. However, these serologic antibodies did not protect mice from infection with B. burgdorferi expressing homologous or heterologous types of OspC after tick challenge.Oral vaccination strategies are of interest to prevent transmission of Lyme disease as they can be used to deliver vaccines to humans, pets, and to natural wildlife reservoir hosts of  Borrelia burgdorferi by vaccinating reservoir hosts , . Or. OrBorrek vector  and for k vector , 12]. F. FBorrelk vector , 14], ..BorreliaOuter surface protein C (OspC) is currently considered one of the most promising Lyme disease vaccine candidates due to the protein being upregulated at 34–37°C rather than 24°C , 18]. F. F18]. FB. burgdorferi for mammal (human or reservoir) targeted vaccines based on evidence that these proteins induced significant IgG immune responses when administered via a parenteral route , , , , 16] an an16] anSix to eight week old female C3H-HeN mice  were used. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee, Animal Care Protocol Application (Permit Number: 14–007). Survival anesthesia was performed under isofluorane or avertin, and all efforts were made to minimize animal suffering. Avertin was used before tick challenge. For euthanasia we used isofluorane followed by a physical method . During immunization animals were kept in groups of 4 in standard cages under regular feeding and light cycle schedules. For tick challenge mice were individually caged in wire bottom cages, which were held in FIC-2 isolators for a week. During that week mice were closely monitored for any signs of discomfort for a minimum of three times daily, engorged ticks were collected and precise records were kept for each animal. We maintain a protocol in which animals that become severely ill prior to experimental endpoint  are to be humanely euthanized. No animals died prior to the experimental endpoint.B. burgdorferi originally isolated from heart of Peromyscus leucopus infected with field caught ticks (from New York State) was used to produce our infected flat nymph tick cohort. B. burgdorferi strain BL204 (OspC type K)  and B. burgdorferi strain B31MI  were used in the neutralization assay. B. burgdorferi were cultured in Barbour-Stoenner-Kelly (BSK-H) medium at 34°C until the cells reached the log phase. The number of spirochetes was determined using dark field microscopy and quantitative PCR.A culture containing multiple OspC types of ospC type K (ospC-K), ospB and bbk32 from B. burgdorferi sensu stricto and salp25 and salp15 from Ix. scapularis were synthesized fused to the nucleotide sequence encoding the leader peptide of outer surface protein A (OspA) from B. burgdorferi and cloned into pET9c (for vaccine production) and into pET28a (for protein purification) using codons optimized for expression in E. coli . DNA constructs were then transformed into the E. coli strain BL21 (DE3) pLysS. The parental E. coli strain transformed with the empty plasmid was used as a control (Ctrl).The genes E. coli clones were grown in Tryptone Broth Yeast (TBY) medium supplemented with 50 μg/ml Kanamycin (Kn) at 37°C, shaking at 225 rpm, until it reached an OD600 of 0.8. The expression of 6xHis—recombinant proteins was induced by adding 1 mM IPTG  to the cells followed by incubation at 37°C for 3h. The cells were harvested by centrifugation at 4000 x g for 10 min at 4°C. The proteins were purified using the Ni-NTA Purification System (Invitrogen) following the manufacturers instructions. Protein concentration was determined by the Bradford protein assay , and was stored at -80°C.Recombinant E. coli were cultivated in TBY medium supplemented with 50 μg/ml Kanamycin at 37°C, 225 rpm. At an OD600 = 0.8, cells were induced with 1mM IPTG for 3 hours and grown to an OD600 of ~1. The cells were harvested by centrifugation at 20,000g for 10 min at 4°C and were washed three times with ice-cold phosphate buffered salt solution . The pellet was resuspended in ice-cold PBS supplemented with protease inhibitor cocktail  to an OD600 ~ 1. E. coli cells were disrupted with a French press  and centrifuged at 20,000 x g for 10 minutes at 4°C to isolate the cytosol fraction (supernatant) from the cell envelope (pellet). The pellet was resuspended in 1ml of ice-cold PBS 2% Triton-X114  (v/v) and was incubated at 0°C for 1 hour with frequent gentle agitation. Phase separation was performed by warming the suspension for 30 minutes in a 37°C water-bath, followed by centrifugation at 13 000 x g for 15 minutes at 25°C. Aqueous phase and detergent phase were then collected, separated from each other and were washed three times. Briefly, the aqueous phase was washed by adding fresh 10% Triton X-114 to a final concentration of 2%. The aqueous phase was rewarmed for 30 minutes in a 37°C water-bath and centrifuged at 13 000 x g for 15 minutes at 25°C. The detergent phase was washed by diluting it to 1 ml in ice-cold PBS followed by rewarming for 30 minutes in a 37°C water-bath and a centrifugation at 13 000 x g for 15 minutes at 25°C. Total extract (TE), Supernatant (SN), Cell envelope (CE), Cell Envelope/Detergent phase (CE/DET), Cell Envelope/Aqueous phase (CE/AQ), and pure recombinant proteins were analyzed on a 10% denaturing polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane  for analysis with antigen-specific- polyclonal mouse antibody. Protein was quantified by densitometry using Alpha Imager .Recombinant E. coli was cultured in TBY supplemented with Kn, and grown at 37°C, 225 rpm, to an OD600 of 0.8. Protein expression was induced with 1 mM IPTG during 3h. The cells were harvested by centrifugation at 4000xg for 10 min at 4°C and resuspended in 20% glycerol/phosphate buffered salt solution  in 1% of the initial volume. Cell suspensions in aliquots of 2 ml were frozen quickly in a dry ice bath and stored at -80°C. Aliquots of recombinant E. coli were thawed at 4°C and 400 μl (109 cells) were placed in a ball-tipped syringe for oral gavage inoculation. Groups of female C3H/HeN mice were immunized by intragastric inoculation of induced recombinant E. coli or with the empty vector (Ec) as controls. Mice received the first immunization, twice daily, for 10 days (days 1–5 and 8–12), rested for two weeks, received the first boost for 5 days (days 29–33), rested for another week and then received a second boost for 5 days (days 43–47). On day 73, mice were terminated for collection of bronchoalveolar lavage (BAL) or challenged. Low amounts of mouse blood (up to 50 μl) were obtained via tail nick on the following days: 0, 28, 42, 73, 105, 135 and 170 after immunization. Stool , bronchoalveolar lavage (BAL) and vaginal lavage (VAL) were collected on day 73 after immunization.Recombinant B. burgdorferi sensu stricto (previously cultured from heart of P. leucopus infected with field caught ticks) was injected subcutaneously in the back of the neck of C3H-HeN mice; four weeks later, batches of clean flat larvae (purchased from Oklahoma State University or from the University of Rhode Island) were placed in the back of the head of anesthetized mice; mice were checked to ensure ticks were attached and were caged individually and monitored for one week. Engorged larval ticks  were collected as they naturally fell off and allowed to molt to the next stage for about 3 months to produce nymphal ticks.Media (50 μl) containing 10,000 B. burgdorferi infected nymphal ticks on the back of the head of anesthetized mice. Five to seven days later, ticks that engorged after taking a blood meal were retrieved after naturally falling off, immediately processed for DNA extraction and stored at -20°C for further q-PCR and direct PCR analysis.Tick challenge was performed by placing six E. coli expressing recombinant proteins were tested by indirect ELISA for the presence of IgG and IgA. IgG was further isotyped for IgG1 and IgG2a. Purified recombinant 6xHis-tagged proteins were coated at 2 μg/ml on Nunc MaxiSorp™ flat-bottom ELISA plates  and indirect ELISA was performed using serum (1:100), BAL (1:1), VAL (1:1) and GUT (1:1). Goat anti-mouse IgG , goat anti-mouse IgG1 , goat anti-mouse IgG2a  and goat anti-mouse IgA  horseradish peroxidase-conjugated antibody  was used as secondary antibody.Serum, bronchoalveolar lavage (BAL), stool  and vaginal lavage (VAL) from mice orally immunized with Borrelia B31 ViraStripe IgG  was performed according to the manufacturer’s instructions using blood from immunized mice as the first antibody. A pattern of 5 out of 10 bands was considered evidence of infection.Heart, bladder and ear biopsy tissues were individually cultured in BSK-H medium with an antibiotic mixture for Borrelia (Sigma) for up to 6 weeks at 34°C. Cultures were checked every week by dark field microscopy . Cultures were deemed positive if Borrelia cells were observed in any field and negative if no Borrelia cells were observed in 10 fields.B. burgdorferi culture, flat and engorged ticks were extracted with DNeasy Blood and Tissue kit according to the manufacturer instructions . The purified DNA was stored at -20°C until use. The concentration of B. burgdorferi was quantified by a Bio-Rad iQ5 Real-time System  using the iTaq Universal probes supermix (Bio-Rad). The flaB and the ospC genes were PCR amplified. The PCR reaction contained 10 μM of each primer, 5 μM of the specific probe and 5 μl of DNA in a total volume of 25 μl. The amplification protocol consisted of 10 min at 95°C, followed by 40 cycles of 95°C for 15s and 60°C for 1 min. Results were expressed as the number of B. burgdorferi per 1mg (heart and bladder), per 1mm (ear) of tissue or per tick.DNA from heart (25mg), bladder (25mg), ear tissue (2mm), http://bowtiebio.sourceforge.net/bowtie2/index.shtml). Cufflinks was used to generate the rpkm (reads per kilobase per million) of each strain from the alignment results. Data was deposited in the NIH Sequence Read Archive (SRA) accession Nr. SRP071080.Genomic DNA purified from 10 engorged nymphal ticks that fed on immunized mice was sent for next generation sequencing at Genewiz  with the following OspC general primers, OspC-f: GGAGGCACAAATTAATG and OspC-r: GACTTTATTTTTCCAGTTAC. The library was prepared using NEBNext Ultra DNA Library Preparation Kit as per the manufacturer’s instructions. Quality control was performed by Qubit dsDNA Assay and the High Sensitivity Assay on the Agilent 2100 Bioanalyser. Library was then sequenced on Illumina MiSeq 2x300 PE cartridge. BOWTIE2 was used to generate the alignment (B. burgdorferi B31MI (OspC type A) and BL204 (OspC type K) were grown at 34°C in BSK medium and cell density adjusted to ~ 104 cells per mL. Serum was harvested from mice immunized orally with E. coli expressing OspC type K and immunized subcutaneously with purified recombinant OspA (αOspA), rOspC type K (αOspCK) and control (αCtrl). Subcutaneous inoculum was prepared by combing 100 μg of purified protein with alum (1:1). The borreliacidal effect of vaccinated serum, pre-challenge (containing active complement) was tested by adding 16 μl of cells to 8 μl of mouse serum and 16 μl of BSK medium in 0.2 mL PCR tubes and incubating the reaction at 34°C for 7 days for visualization of B. burgdorferi under a dark field microscope and for quantification of growth by qPCR. Aliquots were collected and frozen on days 0, 3, 5, 7. Two (2) μl of each sample were used to quantify the FlaB copies by quantitative PCR reaction method .The borreliacidal assay was adapted from a method previously described . B. burgData are represented as mean ± standard deviation. Statistical analyses were performed using unpaired T test with equal standard deviation.ospC-K), the outer surface protein B (ospB) and the fibronectin binding protein BBK32 (bbk32) genes from B. burgdorferi sensu stricto into an E. coli expression plasmid as well as salivary gland proteins 15 and 25  from Ix. scapularis. We evaluated the localization of the recombinant proteins within the cell envelope of E. coli by cell fractionation followed by quantification by densitometry . Mice immunized orally with E. coli expressing OspC-K produced significant levels of OspC-K specific IgG antibodies with a mean antibody level of OD450 = 1.108 (± 0.303 SD) (450 = 0.322 (± 0.171 SD) was IgG1 and OD450 = 0.375 (± 0.110 SD) was IgG2a  and Ix scapularis genes  did not induce levels of antigen-specific IgGs different from the control, p>0.05 (E. coli expressing OspC-K (450 = 0.425 (± 0.175 SD) compared to 0.233 (± 0.094 SD) in the control, p = 0.0030; but also at mucosal sites distal to the gut, such as the lung (BAL) with a mean OD450 = 0.163 (± 0.071 SD) compared to 0.062 (±0.008 SD) in the control, p = 0.0004, and in the vagina (VAL) with a mean OD450 = 0.385 (± 0.289 SD) compared to 0.093 (± 0.044) in the control, p = 0.0022. Mice immunized orally with E. coli expressing B. burgdorferi genes ospB and bbk32 and Ix scapularis genes salp25 and salp15 did not produce significant levels of antigen specific IgA  , of whicas IgG2a . Differe, p>0.05  whereas g OspC-K  producedific IgA .E. coli-based oral vaccine expressing OspC-K in preventing infection with B. burgdorferi after tick challenge. We used laboratory produced flat nymphs infected with a number of B. burgdorferi, which had an infection rate of ~83%. We placed 6 flat Ix scapularis nymphs per mouse , using a total of 144 flat nymphs. After challenge, we recovered 57 of the 72 nymphs placed on the control group and 58 out of the 72 nymphs placed on the immunized group, for an average nymphal recovery rate of 80% and 81%, respectively.We determined the efficacy of an B. burgdorferi in serum from mice after nymphal challenge by Western blot. All vaccinated and unvaccinated mice seroconverted to B. burgdorferi (>5 positive bands); 100% of mice vaccinated orally with E. coli expressing OspC-K produced antibodies that bound with high avidity to OspC type A and 8/12 (67%) of unvaccinated mice developed antibodies that bound somewhat weakly to OspC type A in the Western blot , p>0.05  and ospC type A (fragment ~256bp) as an example of dissemination of other invasive OspC phyletic types  IgG levels to OspC-K dropped to levels below OD450<0.5 and remained low until mice were terminated on day 170, p<0.0001 (p = 0.3820), but we measured a significant loss in IgG1, not IgG2a, from d73 to day 170 (p = 0.0012), 3 months after challenge.We compared the levels of OspC-K specific IgG antibodies in serum following oral immunization of mice before  and after tick challenge  by ELISA . IgG antp<0.0001 . We lookp<0.0001 . DiffereB. burgdorferi (104) were treated with serum from mice immunized orally with E. coli expressing OspC-K, and immunized subcutaneously with purified recombinant OspC-K, OspA and control; cultures were incubated at 34°C for 7 days. Samples were collected on days 0, 3, 5 and 7 post-treatment for visualization of spirochetes under a dark field microscope (DFM) and quantification by qPCR targeting the Fla gene (B. burgdorferi type A and type K survived and grew in culture (DFM) supplemented with intact serum from mice immunized with OspC-K orally or subcutaneously. We quantified ~105 Type A B. burgdorferi in cultures treated with anti-OspC-K serum generated either by oral gavage or by subcutaneous injection  revealed that all immunogens were incorporated mostly within the cell envelope and that OspC, BBK32 and Salp 15 were most prevalent within the hydrophobic portion of the extract, CE/DET,  nor the heterologous type A. Others have shown that OspC protection is type specific after challenge with homologous strains via needle challenge ,  we weB. burspC type . Furtherhallenge , 37] an anB. burhallenge , but notTo understand some of the mechanisms that lead to vaccine failure we evaluated the OspC-specific antibody response during immunization and after challenge  and we p450 <0.5, , antibody production to OspC decreased to levels below OD50 <0.5,  which is, antiboded ticks . Alterna evasion , 40], [, [450 <0 evasion . If OspC to tick  lends fuB. burgdorferi in TH1 biased C3H-HeN but not in TH2 biased Balb/c mice [B. burgdorferi dissemination in the TH1 C3H-HeN background but not in TH2 Balb/c. In our study we observed that in the absence of B. burgdorferi expressing OspC, the mouse (C3H-HeN) produced equal levels of IgG1 and IgG2a to the OspC immunogen indicative of a TH1/TH2 response before challenge. After the host was exposed to B. burgdorferi expressing OspC upon challenge, dissemination of B. burgdorferi proceeded efficiently in the presence of anti-OspC antibody and the immune response seems to have switched from the initial TH1/TH2 response raised against the OspC-K immunogen, to a TH1 biased response to the pathogen.In this study we also show that oral vaccination of mice with OspC induced significant levels of antigen-specific IgG equally distributed between IgG1 and IgG2a before we infected mice via tick challenge. After tick challenge, total IgG was reduced to about half and we observed that the difference was due to the loss of IgG1, an immunoglobulin subclass associated with TH2 responses, but not IgG2a an immunoglobulin associated with TH1 responses . Interesb/c mice  suggestiB. burgdorferi carrying an OspC distinct from (type A) or homologous to (type K) the vaccinogen (type K) may have lead to immune evasion given that in either case we were able to quantify increasing amounts of live type A and type K spirochetes after treatment with serum from OspC-K immunized mice which was independent of the route of immunization , due to factors related to the immunological response to the vaccine , due to the genetic background of the mice , and to partial neutralization of B. burgdorferi by homologous serum and inefficient production of cross-protective neutralizing borreliacidal antibodies to OspC by the mammalian host as shown here and in [Our results suggest that oral vaccines based in OspC type K are ineffective. Vaccine failure in mice could be due to factors related to the biology of the spirochete , cell envelope (CE), and cell envelope partitioned into detergent (CE/DET) and aqueous (CE/AQ) phases were analyzed by cell fractionation followed by quantification of protein by densitometry using an Alpha Imager .(EPS)Click here for additional data file.S2 FigE. coli expressing OspB, BBK32, Salp25, Salp15; control mice were orally inoculated with E. coli carrying the empty vector (C). C3H/OspB and BBK32, n = 6 per group; C3H/Salp25 and Salp15 n = 4 per group. Blood, stool (GUT), bronchoalveolar lavage (BAL) and vaginal lavage (VAL) samples were collected on the day before challenge and antigen-specific IgG, subtyped into IgG1 and IgG2a  and antigen-specific IgA  were measured by ELISA and plotted as Optical Density at 450 nm (OD 450). The average of triplicate readings per mouse/per group was determined and the error bar indicates standard deviation.C3H/HeN mice were vaccinated orally with live recombinant (EPS)Click here for additional data file.