To investigate the prevalence and risk factors of asymptomatic colorectal diverticulosis in Taiwanese general population.From January 2009 to December 2011, consecutive asymptomatic subjects undergoing a health check-up were evaluated by colonoscopy. The colorectal diverticulosis was assessed, and a medical history and demographic data were obtained from each subject. Logistic regression analysis was conducted to search the risk factors of colorectal diverticulosis.Of the 1899 asymptomatic subjects, the prevalence of colorectal diverticulosis was 13.5%. On univariate logistic regression analysis, age over 60 years old, male, adenomatous polyp, current smoking and heavy alcohol consumption were significantly associated with diverticulosis. Multivariate logistic regression analysis revealed that age over 60 years old , adenomatous polyps  and heavy alcohol consumption  were independent predictors for colorectal diverticulosis.The prevalence of asymptomatic colorectal diverticulosis was 13.5% in Taiwan. Age over 60 years old, adenomatous polyp and heavy alcohol consumption may affect the risk of development of the disease. A diverticulum is a herniation through a weak site of the bowel wall that produces a small outpouching . AlthougColorectal diverticulosis shows both geographic and ethnic variability. It is rare in Africa and Asia but common in the United States, Europe and Australia ,6. In WeCurrently, the risk factors for colorectal diverticulosis remain unclear and the results of risk factors for colorectal diverticulosis in Asian population are quite conflicting. Some people in Taiwan underwent health check-up including colonoscopy at their own expense because of various reasons. We therefore conducted this cross-sectional study to investigate the risk factors for colorectal diverticulosis in Taiwan.From January 2009 to December 2011, consecutive asymptomatic subjects aged ≥ 20 years old undergoing a colonoscopy during a health check-up were included into this study. The subjects were excluded if they reported symptoms of lower gastrointestinal tract disease including rectal bleeding, a marked change in bowel habits, or lower abdominal pain that would normally require medical evaluation. Other exclusion criteria were a history of colitis, colorectal polyps or colorectal cancer, prior colonic surgery, undergoing a sigmoidscopy, a colonoscopy, or a barium enema within the previous 10 years. The study protocol was approved by Institutional Review Board at Kaohsiung Veterans General Hospital and all participants provided written informed consent.A complete history and physical examination were performed for each subject undergoing the health check-up. All subjects were carefully queried regarding the presence of abdominal symptoms in the previous 1 month. Subjects who responded negatively were classified as asymptomatic subjects and were enrolled into this study. All the participants received anthropometric and blood biochemical tests including fasting plasma glucose, serum triglyceride and high-density lipoprotein (HDL)–cholesterol level, and underwent total colonoscopy. Colonoscopies were performed by three experienced endoscopists  using the Olympus PCF-Q240AL and PCF-Q260AL endoscopy  after the subjects had fasted overnight. Bowel preparation was performed with oral saline lexative following the protocol of diagnostic colonoscopy. The patients were carefully examined for colorectal mucosal lesion. If colorectal diverticula were observed, their location and type was recorded carefully. Distribution type was defined as the following: right-side colon, involving the cecum, ascending colon, or transverse colon; left- side colon, involving the splenic flexure, descending colon, sigmoid colon, or rectum; or bilateral, involving the entire colon. Colorectal polyp was defined as a protuberance into the lumen from the normally flat colonic mucosa. All visible polyps were removed and examined histologically by the pathologist. The pathology types of colorectal polyps were subsequently categorized into hyperplastic polyps and adenomatous polyps.To assess the relationship between clinical characteristics and asymptomatic colorectal diverticulosis, the following data were recorded for each subject: age; gender; educational status; consumption of tobacco, alcohol, coffee, tea, spicy foods or betel nut, exercise habit, whether vegetarian or not and long-term use of non-steroidal anti-inflammatory drug (NSAID). All variables were categorized for data analyses.p value less than 0.05 was considered significant. Significant variables revealed by univariate analysis were subsequently assessed by a stepwise logistic regression method to identify independent clinical factors predicting the presence of colorectal diverticulosis. All statistical analyses were performed using SPSS version 17.0 .The chi-square test or Fisher’s exact test was employed to investigate the relationship between the rate of colorectal diverticulosis and clinical characteristics. These variables included the following: gender; age ; education status ; BMI ; regular NSAID use at least 1 year (yes or no); colorectal polyps (hyperplastic polyps or adenomatous polyps); smoking status ; consumption of alcohol ,coffee, tea or spicy foods and exercise habit ; betel nut habit and vegetarian (yes or no). Metabolic syndrome was defined according to the modified National Cholesterol Education Program Adult Treatment Panel III definition for South Asians and Chinese. A From January 2009 to December 2011, 1899 asymptomatic subjects  was recruited into this study. Among them, 256 subjects (13.5%) had colorectal diverticulosis. Diverticula were located predominately in the right-side of the colon in 52.3% (n = 134), left-side in 25% (n = 64), and bilaterally in 22.7% (n = 58) of the subjects (Table Table The current study demonstrated that the prevalence of colorectal diverticulosis were 13.5% in asymptomatic Taiwanese. Old age, heavy alcohol consumption and adenomatous polyps are risk factors of colorectal diverticulosis. To our knowledge, this is the first work investigating the prevalence and risk factors of colorectal diverticulosis in an asymptomatic Taiwanese population based on colonoscopic findings.The location of colorectal diverticulosis in subjects from this study was also different from that of Western countries . In WestThis study revealed a strong positive association between colorectal adenomatous polyps and diverticulosis by univariate and multivariate analysis. The association of colorectal diverticulosis and the risk of colorectal adenomas is controversial. Morini et al. found significantly more adenoma/advanced adenoma in the sigmoid colon of patients with diverticula than in controls in a study of Italian patients undergoing colonoscopy . In a reOur study also showed that consumption of alcoholic drinks three times or more per week was an independent predictor for colorectal diverticulosis in asymptomatic subjects. A Danish study found a relative risk of 2 for alcoholics to have admissions due to colonic diverticular disease . Song etDespite of the findings, this study had certain limitations. First, self-selection bias of the population in this trial was possible because all enrolled subjects underwent self-paid health examination, which usually indicated better economic status than the general population in Taiwan Table . Second,The prevalence of colorectal diverticulosis is 13.5% in this cross-section study of asymptomatic Taiwanese subjects, with 52.3% of the cases on the right side of the colon. Advanced age and alcohol consumption are identifiable risk factors for the development of colorectal diverticulosis. These findings suggested that lifestyle modification including abstaining from alcohol might potentially decrease the risk of developing colorectal diverticulosis. Colorectal adenomatous polyps was also associated with the presence of colorectal diverticular disease but the causative relationship is vague. Further studies are warranted to investigate the pathophysiological mechanisms underlying the association of alcohol consumption with the risk of colorectal diverticulosis.


Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α− inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15′) on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the βN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between βN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1β2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the β-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1β2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive βN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1β2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in β+/α− interfaces, we found that βN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. βN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics β+/α− anesthetic site occupancy. Our results show that βN265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in β+/α− sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for βN265 in anesthetic binding within the β+/α− transmembrane sites. A receptors A receptors consist of α, β, and γ subunits, arranged β-α-β-α-γ counterclockwise viewed from the extracellular space A receptors, based on crystallography of distantly related pLGICs Etomidate and propofol are potent general anesthetics that act as positive allosteric modulators and agonists at GABACaenorhabditis elegans glutamate-sensitive chloride channels (GluCl) Allosteric co-agonist sites for etomidate and propofol are located between the extracellular ends of α-M1 and β-M3 transmembrane helices; the transmembrane β+/α− interfaces . CombineA receptor sensitivity to alcohols and anesthetics A receptors. Interchanging the β-M2-15′ asparagine of β2 or β3 and the homologous serine of β1 accounts for the remarkable specificity of etomidate for receptors containing β2/3 subunits Xenopus oocytes indicated that drug efficacy was reduced and that affinity for receptors might be weakened. Similar studies of α1βN265Mγ2L receptors were inconclusive due to the absence of anesthetic effects A receptors locate βN265 either near or within the β+/α− site  mutants A βN265 has been detected. One photoreactive propofol analog incorporates at β3H267 A receptors, consistent with a role in binding.In addition to the α-M1 and β-M3 residues identified above, M2 domain residues may contribute to the β+/α− transmembrane sites for propofol and etomidate. The focus of this study, β-M2 position 265 (M2-15′), is of particular and longstanding interest as one of the first sites where mutations were found to affect GABA/α− site , and in versus efficacy, we applied both direct and indirect structure-function approaches. We first performed pharmacological sensitivity and thiol modification-protection studies of α1β2N265Cγ2L GABAA receptors expressed in Xenopus oocytes and monitored with voltage-clamp electrophysiology, using etomidate at concentrations up to 300 µM (intending to achieve high site occupancy). These experiments revealed that etomidate neither affects α1β2N265Cγ2L receptor function nor inhibits reaction with a water-soluble thiol modifier. We also explored βN265M effects in GABAA receptors containing previously characterized second point mutations: a modifiable and anesthetic-protectable cysteine (α1M236C), and a mutation that mimics bound anesthetic (α1M236W). Our results reveal that βN265M reduces both anesthetic binding affinity at β+/α− sites and transduction of gating effects associated with anesthetic binding.To discriminate between the effects of βN265 mutations on anesthetic binding Xenopus laevis frogs were used as a source of oocytes for voltage-clamp electrophysiological experiments. Frogs were housed in a veterinarian-supervised facility and used in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All animal procedures for this study were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee . To ameliorate suffering, frogs were anesthetized by immersion in 0.2% tricaine prior to harvesting oocytes . Laparotomy wounds were minimized (<0.5 cm), and infiltrated with 0.25% bupivicaine to provide post-procedure analgesia. To reduce the number of frogs used, each frog was subjected to a maximum of six oocyte harvests with at least 8 weeks recovery between procedures.Adult female A receptors p-Chloromercuribenzenesulfonic acid sodium salt (pCMBS) was purchased from Toronto Research Chemicals . Salts and buffers were purchased from Sigma-Aldrich.R(+)-Etomidate (2 mg/ml in 35% propylene glycol:water) was from Bedford Laboratories . Propofol was purchased from Sigma-Aldrich  and stored as stock solution in DMSO. Alphaxalone was purchased from MP Biomedical  and prepared as a stock solution in DMSO. Drugs were diluted into electrophysiology solutions on the day of use. Maximal experimental concentrations of propylene glycol (<4%) and DMSO (≤0.1%) produced no functional effects on GABAA receptor α1, β2, and γ2L subunits were cloned into pCDNA3.1 vectors . Plasmids encoding concatenated dimer (β2-α1 and β3-α1) and trimer (γ2-β2-α1 and β3-α1-δ) subunit proteins were a generous gift from Professor Erwin Sigel  5A3PAQ2A3PA2Q5) between the C-terminus of β2 and the N-terminus of the mature α1 (without its leader sequence). The γ2L-β2-α1 trimer also contains a 23 residue linker (Q5A3PAQ3AQA3PA2Q5) between γ2 and β2. In β3-α1 and β3-α1-γ2L constructs , linkers between β3 and α1 were composed of 23 amino acids (Q5A3PTGQA3PA2Q5), and that between α1 and γ2L subunits is 10 amino acids (Q4TGQ4). Mutations in cDNAs were created with oligonucleotide-directed mutagenesis using QuikChange kits . Clones from each mutagenesis reaction were subjected to DNA sequencing through the entire cDNA region to confirm the presence of the mutation and absence of stray mutations.cDNAs for human GABAXenopus oocyte expression were performed as previously described 2, 1.8 CaCl2, 5 HEPES, pH 7.5). Peak current responses to varying GABA concentrations (range 0.1 µM to 10 mM) alone or co-applied with anesthetics, were measured in Xenopus oocytes using two microelectrode voltage clamp electrophysiology, as previously described nd or 3rd sweep for normalization. Picrotoxin-sensitive spontaneous channel activity was measured by applying 2 mM PTX, followed by >5 minute washout and a maximal GABA response test. Etomidate (10 µM) or alphaxalone (2 µM) were used as gating enhancers with high GABA concentrations to estimate maximal GABA efficacy. Oocyte currents were low-pass filtered at 1 kHz  digitized at 1–2 kHz  and recorded digitally .Messenger RNA synthesis and −1s−1. A subset of oocytes were modified first in the absence of etomidate, then in the presence of etomidate. Modification rates in both conditions were independently fitted with linear least squares.The pCMBS concentrations used for modification experiments were chosen so that initial modification rate conditions  were maintained at up to 40 s exposure. Oocytes were repetitively stimulated with GABA pulses every five minutes until at least three sequential current responses were constant (±5%). For modification and protection experiments, oocytes were exposed to pCMBS  for 5 or 10 s, followed by 5–10 min wash in ND96. Responses to both low GABA (EC10) and high GABA (1 to 3 mM) were tested after each cycle of pCMBS exposure and wash. After up to ten cycles of modification and wash, maximal modification effect was checked using 10× higher pCMBS or 100 s pCMBS exposure. Modification rate analysis was performed on data from individual oocytes. For β2N265C receptor modification, spontaneous current after modification was plotted against [pCMBS] × time and fitted with single exponential functions. For α1M236C modification, the ratio of low GABA to high GABA responses were calculated, normalized to the pre-modification control, and plotted against [pCMBS] × time (mM×s). Linear least squares fits to the first three to five points were used to determine the initial modification rate in M50 is the half-maximal activating concentration and nH is Hill slope. Etomidate-dependent direct activation of receptors was analyzed similarly.Analyses for agonist concentration-responses, etomidate-induced left shift, and allosteric co-agonist model fitting followed our approach described elsewhere 50 shift ratios were calculated from the difference in log(GABA EC50) values (Δlog(EC50)) measured in the presence of 3.2 µM etomidate versus control.ECPTX) normalized to PTX-sensitive leak currents  co-agonist mechanism with two equivalent sites each for GABA and etomidate ; Eq. 3 w0 is a dimensionless basal closed:open gating equilibrium variable, KG and KE are dissociation constants for respectively, GABA and etomidate binding to inactive receptors, and c and d are the respective (dimensionless) ratios of dissociation constants in activated versus inactive receptors. The maximal agonist efficacies of GABA and etomidate are inversely related to, respectively, L0c2 and L0d2.In Eq. 3, L50) is assumed to be the anesthetic dissociation constant (KE).Because α1M236C is fully protectable by etomidate A receptor was constructed using the Prime module in Schrödinger Maestro 9.7 . The structure was based on the GluCl structure template PDB 3RHW A subunit to GluCl were performed using the ClustalW algorithm. Alignments were gap-free in all M1 through M3 domains. The intracellular ends of GABAA subunit M3 and M4 were established by the strong alignment of homologous sequences in the middle of these domains and the length of the helices in the GluCl crystal structure. Intervening M3–M4 loops of GABAA subunit sequences were then truncated and replaced with that of the modified GluCl used for crystallography. Alignment introduced a modest number of gaps into extracellular domains and M4 sequences. Some of these were edited out or moved to preserve local GluCl secondary structure. The final sequence alignments  A homology model for α1β3γ2L GABAA receptor transmembrane domains A receptors for spontaneous activity, GABA EC50 and efficacy, and sensitivity to both etomidate modulation and direct activation , produced currents that did not fully reverse with pCMBS washout (−1s−1 (n = 6). Addition of GABA increased the apparent rate of modification to 3800±600 M−1s−1, (n = 3). In contrast, control experiments in wild-type α1β2γ2L receptors revealed no functional effects of 2 mM pCMBS exposures up to 60 s .Exposing α1β2N265Cγ2L GABA washout . These cAbsent any data to guide estimation of etomidate affinity in α1β2N265Cγ2L receptors, we tested protection using a range of concentrations up to 300 µM, which fully protected β2M286C from modification in both the absence and presence of GABA There are several hypotheses for this lack of etomidate protection at βN265C: 1) etomidate may bind normally, but not near the βN265 residue, implying that the βN265C mutation eliminates drug transduction/efficacy; 2) the βN265C mutation may reduce affinity for etomidate so that site occupancy remains low at concentrations up to 300 µM; and 3) etomidate may bind near βN265, but without obstructing pCMBS access. To address some of these possibilities, we assessed the effects of combining βN265M with previously characterized second mutations in the etomidate site. In order to reduce potential receptor assembly variations caused by subunit-subunit interfacial mutations, we also used concatenated dimer and trimer subunit assemblies for these double-mutant experiments.A receptors with cysteine substitutions at α1M236 are modified by pCMBS and that bound etomidate blocks α1M236C modification A receptors assembled from concatenated subunit dimers and trimers, α1M236C is both pCMBS modifiable and protected by both etomidate and propofol .Voltage-clamped oocytes expressing β3-α1M236C dimers and β3-α1M236C-γ2L trimers produced GABA-activatable receptor-channels that were both modulated and directly activated by etomidate ; Table 1A receptors produced GABA-activated currents that were insensitive to both etomidate modulation and direct etomidate activation (50 ≈ 300 µM (95% CI = 215 to 423 µM) and is similar in the presence of GABA . Assuming that the PC50 estimates reflect etomidate occupancy, we infer that etomidate binds very weakly (dissociation constant ≈ 300 µM) to both inactive and GABA-activated β3N265M-α1M236C/β3N265M-α1M236C-γ2L receptors, in comparison to activated β3-α1M236C/β3-α1M236C-γ2L receptors (dissociation constant ≈ 1 µM).Double-mutant β3N265M-α1M236C/β3N265M-α1M236C-γ2L GABAtivation . Currenttivation . Modifictivation . Etomidativation  or presetivation . Using 3tivation . LogistiWe also demonstrated that propofol at 30 µM, like etomidate, reduced the rate of pCMBS modification at α1M236C by about 90% in activated free subunit α1M236Cβ3γ2L receptors (not shown) and in concatenated β3-α1M236C/β3-α1M236C-γ2L receptors . In compTo study the effect of βN265M mutations on β+/α− site transduction, without the need to establish drug site occupancy, we used another mutation. The azi-etomidate photolabeled α1M236 sidechain abuts the β+/α− cleft where etomidate binds, and α1M236W mutation mimics the positive allosteric effects of anesthetic binding while reducing etomidate modulation 5050  residue in the binding and efficacy of etomidate and propofol within their established β+/α− interfacial sites. Cysteine substitution at βN265 produced receptors that were profoundly insensitive to etomidate (We used both direct and indirect structure-function strategies to investigate the role of the GABAtomidate . Moreovetomidate . The absA receptors 0 in Eq. 3), 2) altering anesthetic binding to inactive receptors (KE), and 3) altering anesthetic efficacy (d), which is equivalent to selectively altering binding to activated receptor states (dKE).The challenge of distinguishing whether ligand-gated ion channel mutations affect agonist binding, efficacy, or both, is well-known E) and active (dKE) receptors are approximately 50 µM and 0.5 µM  is also in good agreement with that for free-subunit α1M236Cβ2γ2L receptors(dKE ≈ 1 µM) 50 ≈ 1 µM; Both current and previous d 0.5 µM , 7A. Thud 0.5 µM , 7A, comn states . This acn states . The MWCE ≈ 300 µM) and GABA-activated (dKE ≈ 300 µM) receptors (E ≈ 50 µM vs. 300 µM), while efficacy (d) is weakened about 100-fold per site. Together, reductions of both drug affinity and efficacy account for the approximately 600-fold reduction in apparent etomidate binding affinity for GABA-activated receptors (dKE ≈ 0.5 µM vs. 300 µM).In comparison, protection experiments in double-mutant concatenated β3N265M-α1M236C/β3N265M-α1M236C-γ2L receptors indicated only ∼50% occupancy at 300 µM etomidate, implying extremely low-affinity binding to both inactive  and propofol els e.g.  also shoA receptor modulating efficacy A receptors If βN265 sidechains contact anesthetics, how also to explain the absence of anesthetic photolabeling at this residue? The large effects of βN265 mutations on etomidate sensitivity are fully consistent with complementary structure-function data on etomidate derivatives, if we assume limitations on their binding orientation. Modification at the ester leaving group of etomidate, including appending large photoreactive groups, preserves anesthetic activity and GABAA receptors form other transmembrane anesthetic sites, including other inter-subunit pockets, intra-subunit helical bundles, and the ion channel Finally, GABAFigure S1A receptor subunit amino acid sequences with GluCl.Alignment of GABA Each subunit sequence was independently aligned as described in methods. Predicted secondary structure domains are indicated by blue bars for beta sheet and red bars for alpha-helix.(TIF)Click here for additional data file.


TNF) α, rs1800630; TNF receptor super family (TNFRSF) 1B, rs1061624; interleukin (IL)-1A, rs1800587; IL-1B, rs16944; IL-4R, rs1801275; IL-6, rs1800796; IL-10, rs1800872; IL-1 receptor-associated kinase 1 (IRAK1), rs1059702; C reactive protein (CRP), rs1130864.Aging process is accompanied by a chronic sub-clinical systemic inflammation. This study aimed to assess the association between hearing impairment and polymorphisms of genes encoding cytokines deeply-committed to the inflammatory response and immune homeostasis in an elderly Japanese population. Data were collected in the Longitudinal Study of Aging surveyed biennially between 1997 and 2010. The participants without any missing information at baseline were 1,957 individuals, and the gross accumulated number of 8,675 subjects (40–89 years of age) was analyzed. Two hearing impairment criteria were taken as the better ear pure-tone average (PTABE) greater than 25 dB and greater than 40 dB. We analyzed cumulative data using generalized estimating equations to investigate the effect of 9 polymorphisms, namely, tumor necrosis factor , 1.211 (CI: 1.053-1.392) in model after adjustment for possible confounders. Using the criterion of PTABE >40 dB as disabling hearing impairment, the association remains significant in TNFRSF1B rs1061624, but not in TNF-α rs1800630. No other polymorphisms showed a significant association.The odds ratios for the hearing impairment (PTABE >25 dB) risk under additive genetic model were significant in TNF-α rs1800630 and TNFRSF1B rs1061624 contributed to the incremental risk of hearing impairment in the elderly. TNF-α and TNF receptor interactions play a pivotal role in the pathogenesis of the inflammatory response, and also cause programmed cell death and cell proliferation. The present observation implied the signalling cascades of TNF were involved in ear aging.The present population-based cohort study demonstrated that  Chronic low-grade inflammation is the hallmark of the aging process and is now accepted as a fundamental pathogenic mechanism in the development of several age-related degeneration including cardiovascular disease , atherosThe low-grade proinflammatory status appearing during the aging process has become termed ‘inflammaging’, coined by Franceschi et al. . PhysiolTNF-α C-863A, rs1800630; TNF receptor superfamily member (TNFRSF) 1B G593A, rs1061624; IL-1A -889C/T, rs1800587; IL-1B -511C/T, rs16944; IL-4R G1902A, rs1801275; IL-6 C-572G, also known as C-634G, rs1800796; IL-10 A-592C, rs1800872; IL-1 receptor-associated kinase 1 (IRAK1) T587C, rs1059702; CRP +1444 C > T, rs1130864. Our ultimate aim is to get closer to the pathophysiological elucidation of presbycusis as an age-related disease.The subjects in the present analyses were derived from the National Institute for Longevity Sciences - Longitudinal Study of Aging (NILS-LSA), a population-based biennial survey of a cohort of approximately 2,200 community dwellers. In the present study, we intended to clarify the relationship between hearing impairment and nine polymorphisms of genes encoding inflammatory mediators, namely, Table TNF-α and TNFRSF1B with risk increments for hearing impairment, based on the criterion of PTABE >25 dB. The odds ratios (ORs) for hearing impairment (PTABE >25 dB) risk in TNF-α rs1800630 were 1.120  in model 1, 1.192 (CI: 1.020-1.392) in model 2, and 1.172 (CI: 1.005-1.367) in model 3. The ORs in TNFRSF1B rs1061624 were 1.151 (CI: 1.021-1.297) in model 1, 1.210 (CI: 1.052-1.393) in model 2, and 1.211 (CI: 1.053-1.392) in model 3. The significance was still observed even after adjustment for possible confounders in model 3. Using the criterion of PTABE >40 dB as disabling hearing impairment, the association between polymorphic allele and hearing impairment remain significant in TNFRSF1B rs1061624, but not in TNF-α rs1800630. The ORs for hearing impairment (PTABE >40 dB) risk in TNFRSF1B rs1061624 were 1.272 (CI: 1.052-1.538) in model 1, 1.293 (CI: 1.045-1.599) in model 2, and 1.275 (CI: 1.026-1.584) in model 3.The results of multivariable analysis using logistic regression with generalized estimating equations (GEEs) were demonstrated in Table In the other 7 SNPs, no significant risks of hearing impairment in either criterion were observed in models with different combination of moderator variables.TNF-α rs1800630 and TNFRSF1B rs1061624 may be involved in risk increment of age-related hearing impairment. The significant ORs observed both in two hearing impairment criteria implied a substantial impact of TNFRSF1B rs1061624 on the susceptibility of age-related hearing impairment, since hearing impairment of the better ear means bilateral impairment and thus WHO defines disabling hearing impairment.In the present study, we assessed the contribution of polymorphic genes encoding inflammatory mediators to hearing loss in an elderly Japanese community-dwelling population. The results suggested that TNF-α is a proinflammatory cytokine that is a master regulator of vascular proatherogenic changes and the pathogenesis of inflammatory diseases . It servMany researchers have substantiated emergence of TNF-α and other mediators in the inflammatory response of inner ear. TNF-α is secreted mainly by activated macrophages, monocytes, T cells, B cells, and fibroblasts . In the TNF-α, IL-1β, and N-methyl D-aspartate receptor subunit 2B (NR2B) genes in cochlea and inferior colliculus of mice after intraperitoneal injections of salicylate, then concluded that salicylate treatment resulting in tinnitus augmented expression of the TNF-α, IL-1 β genes in cochlea and inferior colliculus [Recently inflammatory responses in inner ear under various damaging conditions including noise-overstimulation have been reported ,17. Fujilliculus .One study in experimental animals demonstrated that the use of a TNF-α-neutralizing agent could prevent the sensorineural hearing loss secondary to induced pneumococcal meningitis . MeanwhiTNF-α, TNFRSF1A and TNFRSF1B genes have been identified, however functional data pertaining to these polymorphisms in scarce. Rs1800630 is a common functional polymorphism, involving a C to A substitution at position −863. It is located in the proximal promoter of the TNF-α gene. The presence of DNA sequence variations in regulatory region might interfere with transcription of TNF-α gene, and with increase in the susceptibility to human wide range of diseases . Qidwai & Khan reviewed TNF-α gene polymorphism and disease prevalence comprehensively [TNF-α polymorphism rs1800630 A-allele was associated with lower specific anti-pneumococcal IgG levels compared with children carrying C⁄C genotype of rs1800630, in one instance [Through extensive examinations of expression and function, some genetic variations have been shown to explain inter-individual variation. SNPs in the ensively . In the instance . In the TNFRSF1B gene. One clinical cancer investigation demonstrated that the TNFRSF1B rs1061624 genotype was a predictive factor of clinical response i.e., a complete response or not, to treatment with a 5-fluorouracil (5-FU)/cisplatin (CDDP) - based chemoradiotherapy in Japanese esophageal squamous cell carcinoma patients [TNFRSF1B rs1061624 was associated with risk increment of age-related hearing impairment defined as a hearing level of PTABE >25 dB and PTABE >40 dB.There are limited data regarding the functionality of the various SNPs in the patients . Carryinpatients . In our IL-1A rs1800587 polymorphism [IL-6 rs1800796 minor-allele were potentially more susceptible to SSNHL than noncarriers [IL-4R polymorphism Q576R, also known as G1902A, rs1801275 [TNF-α rs1800630 and TNFRSF1B rs1061624, were found with the risk of age-related hearing impairment.Recently, we found a significant association of risks for both sudden sensorineural hearing loss (SSNHL) and Ménière’s disease with morphism , and alscarriers . From ons1801275 , was reps1801275 . In the TNF-α upstream region [EDN1, MTHFR, MTR (folate metabolism), UCP1, UCP2, and FABP2, oxidative stress related SNPs [Several potential limitations of the present study should be cited. The genes in the selection of the present analyses were highly polymorphic respectively. To cite an example, 32 SNPs lies in transcription factor–binding site of 20 transcription factors have been detected in the m region . But rested SNPs –30.In addition, we were not able to obtain each inflammatory cytokine level in individuals. However, recent experimental observation, which explored the cochlear cytokine expression pattern under normoxia and hypoxia, suggested presence of tissue-specific regulatory pathways even among three different cochlear tissues; the organ of Corti, stria vascularis together with spiral ligament, and modiolus . Given tFrom the aspect of methodology, there may be some other way to define a criterion for hearing impairment by different age groups, such as comparison with normal hearing levels according to the International Organization for Standardization (ISO) standard 7029:2000 . ISO-702TNF-α rs1800630 and TNFRSF1B rs1061624 between the HapMap-JPT values and those obtained for the current participants. The ancestral allele of the rs1061624 single nucleotide variation is A-allele. The reference allele in the present analyses was G-allele, and G-allele frequencies of the rs1061624 polymorphism were released in the frequency reports as 0.535 in HapMap-JPT (Japanese in Tokyo), 0.539 in 1000 Genomes Project JPT (Japanese in Tokyo), 0.390 in AoD_Japanese, 0.507 in JBIC-allele (752 anonymous unrelated Japanese volunteers), and 0.580 in JPT_GENO_PANEL.The strength of this study is found in a feature of the NILS-LSA subject group. The NILS–LSA cohort consists of inhabitants who were selected randomly from resident registrations and stratified according to both age and sex. The risk of genotypic bias in subjects was therefore likely to be minimal. There were no significant differences in genotype frequencies of TNF-α rs1800630 and TNFRSF1B rs1061624 contributed to the incremental risk of hearing impairment in the elderly Japanese population. TNF-α and TNF receptor interactions play a pivotal role in the pathogenesis of the inflammatory response, and also cause programmed cell death and cell proliferation. The present observation implied that the signaling cascades of TNF-α were involved in age-related hearing loss.The present population-based cohort study demonstrated that The NILS-LSA is a comprehensive and interdisciplinary study to observe age-related changes and consists of various gerontological and geriatric measurements, such as medical examinations, blood chemical analysis, body composition, anthropometry, physical function, nutritional analysis, psychological tests, and visual and auditory function. The participants were community-dwelling adults of Aichi Prefecture in central Japan, who were randomly selected from resident registrations, stratified by both age and sex in cooperation with the local government. The numbers of men and women recruited were similar, and age at baseline was 40 years to 79 years, with similar numbers of participants in each decade of age . Most of medical check-up are examined repeatedly every two years, and venous blood samples for genetic test were collected only at the first-wave examination. Sera and DNA samples were stored in deep freezers for later examination. The study protocol was approved by the Committee of Ethics of Human Research of the National Center for Geriatrics and Gerontology . Written informed consent was obtained from all participants. The details of the NILS-LSA have been described elsewhere ,33.Data were collected from the NILS-LSA participants who partook at least in the first-wave examination. Those who did not consent to have blood samples taken, and those who lacked results for any of the nine SNP genotype analyses were excluded. The participants at baseline were 2,149 adults, and the cumulative data were 9,358 samples in accumulated total, which were compiled from the first through the sixth-wave examination surveyed between 1997 and 2010. Participants filled out a series of questionnaires in advance of the examination visit. Ear disease history and experience of occupational noise exposure were taken account as basic informations in an evaluation of age-related hearing impairment. Histories of hypertension, diabetes, dyslipidemia, stroke, and cardiac disease were also taken from the self-reported account. Lifetime smoking history was categorized as never or ever. Those who had a deficiency of any required information for the present analyses and those who did not complete the hearing test were excluded. The subjects for the present analyses without any missing information were 1,957 individuals at baseline, and the gross accumulation number of subjects, regardless of repetitive visits, was 8,675 adults 40 years to 89 years of age. The mean number of repetitive visits was 4.4.TNF-α C-863A, rs1800630; TNF receptor superfamily member (TNFRSF) 1B G593A, rs1061624; IL-1A -889C/T, rs1800587; IL-1B -511C/T, rs16944; IL-4R G1902A, rs1801275; IL-6 C-572G, also known as C-634G, rs1800796; IL-10 A-592C, rs1800872; IL-1 receptor-associated kinase 1 (IRAK1) T587C, rs1059702; CRP +1444 C > T, rs1130864 were investigated.Genomic DNA was extracted from peripheral blood lymphocytes using the standard procedure and polymerase chain reaction (PCR) amplification was performed. Nine polymorphisms of genes encoding inflammatory mediators, namely, Genotyping was carried out using a fluorescence method and melting curve method. Details including of primer sequences and PCR conditions are listed in Table IL-4R and IL-6 .Genotype distributions of 9 SNPs in participants at first-wave examinations of NILS-LSA before application of exclusion criteria are shown in Table Air-conduction pure-tone thresholds at octave intervals from 0.5 kHz to 8 kHz were measured in a sound-proof booth by trained examiners using a standardized protocol and a diagnostic audiometer . The pure-tone average threshold level of the better ear at frequencies of 0.5, 1, 2, and 4 kHz (PTABE) was used as an index of hearing status. Hearing impairment was defined as a PTABE greater than 25 dB according to WHO grades . The WHOt-test was used to assess differences of continuous variables between two groups, and comparisons of categorical variables were performed using the chi-square test. Fisher’s exact test was also used, as and when the number of cell samples was less than five. Unless otherwise noted, values are expressed as mean ± standard deviation.Statistical analyses were conducted using the Statistical Analysis System (SAS) version 9.1.3 . For univariate analysis, a Cumulative data were analyzed using GEEs, which take into account the dependency of repeated observations within subjects. GEE is a common statistical model to analyze epidemiological longitudinal data. GEE models were fitted using the GENMOD procedure of Statistical Analysis System version 9.1.3. The GENMOD procedure fits generalized linear models. The correlation structure was specified as autoregressive. We took mode of inheritance in analyses as additive genetic model, which is the prevailing analytical model in genetic epidemiology, assumes that there is a linear gradient in risk with increasing numbers of variant alleles . ORs for the additive genetic models of 9 SNPs with the risk of hearing impairment were analyzed. As a combination of moderator variables, three models were analyzed. In model 1, no moderator variables were adjusted; in model 2, age and gender were taken as moderator variables; and in model 3, any history of noise exposure, ear disease, hypertension, diabetes, dyslipidemia, stroke and cardiac disease, and lifetime smoking habit was adjusted in addition to age and gender.


The most common methods for obtaining human resting metabolic rate (RMR) use either a ventilated hood connected to a metabolic cart (VH_MC) or calculation by many prediction equations utilizing the person’s height and weight. These methods may be inherently inaccurate. The objective of this study is to compare the accuracy for the measurement of RMR by three methods: a new whole room indirect calorimeter specific for this purpose (RMR_WRIC), VH_MC and calculation by the Mifflin equation (ME).2), carbon dioxide (VCO2) and RQ (VCO2/VO2) measured after a 12-h fast with both the VH_ MC and the RMR_WRIC in a randomized fashion. The resting state was documented by heart rate. The RMR was also calculated using the ME, which was compared to both the RMR_WRIC and the VH_MC. All simulated and human metabolic data were extrapolated to 24-h and analyzed .First, the VH_MC  and RMR_WRIC  were subjected to 10, one-hour ethanol (99.8 % purity) and propane (99.5 % purity) combustion tests, respectively, for simulated metabolic measurements. Thereafter, 40 healthy adults  had one-hour RMR , ventilation (liters) rates of oxygen (VOp < 0.05), VO2 (p < 0.05), VCO2 (p < 0.05) and the RQ. Similarly the RMR_WRIC underestimated simulated RMR (p < 0.05) and VO2 while overestimating VCO2 and the RQ. There was much greater variability in the simulated metabolic data between combustion and the VH_MC as compared to that of the RMR_WRIC. With regards to the volunteers, the RMR, RQ, VO2 and VCO2 determined by the VH_MC tended to be lower in comparison to these measurements determined by the RMR_WRIC. Finally, RMR calculated utilizing the ME was significantly (p < 0.05) less than the RMR_WRIC but similar to that obtained by the VH_MC.Comparing stoichiometry to actual combustion, the VH_MC underestimated simulated RMR (The RMR_WRIC was more accurate and precise than either the VH_MC or ME, which has implications for determining energy requirements for individuals participating in weight loss or nutrition rehabilitation programs. Inaccurate short duration RMR measurements commonly done in many research and clinical health care institutions can lead to large errors in extrapolated 24-h sedentary energy requirements. This is a report of a new indirect calorimetric method that is both accurate and comfortable for human subjects thereby eliminating many of the problems that are associated with short-duration metabolic measurement techniques.Indirect calorimetry has made significant contributions to the knowledge of energy needs of humans. For example, the effects of aging , changesThe first practical clinical measurements of RMR were obtained in 1918 by the respiratory-valve and spirometer (Tissot) technique . This deA major disadvantage of all the past and present metabolic carts is the necessity for the use of ventilated hoods, face masks or nose clips that may cause discomfort or anxiety in subjects . This caTo eliminate these problems, a new WRIC was designed and built specifically for the measurement of RMR in humans. The present report evaluates the accuracy of RMR measurements with the new RMR_WRIC and compares it to the VH_MC and to that calculated with the ME  using th2 and CO2 concentrations (%). This research grade instrumentation is accurate to 0.001 % for mass air flow (liters), O2 and CO2 concentrations. Moreover, WVP of the sample gas stream is measured directly to 0.001 Kpa and utilized to continuously correct the VO2 and VCO2, along with mass air flow (L). This eliminates the need for any type of desiccants to dry the sample gas stream during metabolic measurements. The lack of desiccants eliminates any potential errors due to incomplete removal of moisture prior to analysis of sample gases and mass air flow [2 and CO2 gas concentrations in order to account for the corrected room volume of 4,500 L in their response time to metabolic changes of the subject [2 and VCO2, along with the RQ (VCO2/VO2) are then calculated (20) every minute during the RMR measurement. A schematic of the entire experimental protocol is shown in Fig. The RMR_WRIC at the New York Obesity Nutrition Research Center in Manhattan, NY is a 4,500 liter climate controlled room equipped with a standard size hospital bed (91 × 203 × 20 cm) and a small stereo for music entertainment. A large window and an intercom system are available so family members and investigators can observe and talk to the subject during RMR measurements. This new RMR_WRIC eliminates the need for any kind of head apparatus. The respiratory exchange is measured by the Promethion (model GA-06/ FG-01) integrated whole room indirect calorimeter system . This is accomplished by flowing a fixed 100 L/min of fresh air through the RMR_WRIC chamber and obtaining a sample on the exhaust side of the system for measurement of Oair flow . Moreove subject . Energy 2, VCO2 and the RQ of 0.67 to that obtained through actual combustion. Similarly, 10 one-hour propane  combustion tests were performed to determine the accuracy and precision of the RMR_WRIC. This was accomplished by comparing the stoichiometry of propane for EE , VO2, VCO2 and the RQ of 0.60 to that obtained through actual combustion. Ethanol [Ten one-hour ethanol  combustion tests were performed utilizing a burn kit  to determine the accuracy and precision of the VH_MC . This was accomplished by comparing the stoichiometry of ethanol for EE , VO Ethanol  and prop2, VCO2 and RQ by the Vmax program manager (Ver. 21-1A) for the VH_MC and Expedata (Ver.1.4.15) for the RMR_WRIC. The results from each combustion test were then compared to that calculated from the respective stoichiometry.To simulate human subject protocols for RMR measurements, each one-hour ethanol or propane combustion test was comprised of both a 15 and 45-min acclimation and measurement periods, respectively. Mean EE across the 45-min measurement period was then multiplied by 1440 (minutes in 24-h) to obtain extrapolated 24-h simulated RMR  for both methods. The burn rate (g/min) for both ethanol and propane were determined by obtaining the weight prior to and after completion of each combustion test using a calibrated analytical balance . The accuracy of the analytical balance was confirmed prior to the initiation of each ethanol or propane combustion test by placing calibration weights of the expected weight ranges on the balance. Prior to the commencement of the combustion tests, the instrumentation related to both the VH_MC and the RMR_WRIC were calibrated according to each respective manufacturer’s instructions. From the burn rates, the EE was calculated every minute utilizing the Weir equation , along wForty healthy non-smoking adult subjects  were recruited by word of mouth to participate in this study. Medical histories were obtained by physicians familiar with the study. Exclusion criteria included age < 18 years old, recent or prescribed medications affecting metabolism, pregnancy or lactation as verified by an on-site HCG urine test, unstable weight maintenance within the last three months and reported or diagnosed claustrophobia.After informed consent was obtained, subjects were instructed to fast for 12-h and refrain from caffeine, strenuous exercise and alcohol for one day prior to their scheduled morning appointment. Anthropometrics were obtained upon arrival to the laboratory. Prior to the first RMR measurement (VH_MC or RMR_WRIC), heart rate (beats/min) was measured by wrist palpation after a one-hour supine rest to determine each subject’s baseline (resting) state. The subjects were randomized as to which of the two methods (VH_MC or RMR_WRIC) was used first. A registered dietitian recorded 24-h food recalls for the period prior to the appointment time to ensure a fasting state. Similar to the ethanol or propane combustion tests, each metabolic measurement was comprised of acclimation and measurement periods and EE was extrapolated to 24-h RMR.2 levels below 1.0%. The RMR_WRIC uses a pre-set continuous flow rate of 100 L/min and no manual adjustments were necessary as the CO2 levels within the chamber never exceeded 0.50 %. Collected raw data for the subjects were analyzed by each respective method’s instrumentation (VH_MC and RMR_WRIC) software as described above for the ethanol and propane combustion tests. Finally, the ME [The VH_MC and RMR_WRIC are located in two different laboratories on the same floor within 20 m of each other. After the first RMR measurement was completed, subjects were escorted to the next laboratory, where they rested for at least 30 min. Thereafter, heart rate was taken again in triplicate and averaged to assure a baseline state similar to the initial one. The second RMR measurement was not started until the interim average heart rate was within five beats per minute of that obtained initially. Prior to the start of each RMR measurement, respective instrumentations were calibrated as described previously. During RMR measurements subjects were asked to remain awake, relax and minimize any movements while lying in a comfortable supine position under the ventilated hood of the metabolic cart or on a standard hospital bed in the RMR_WRIC. Subjects were monitored during each of the entire one-hour RMR measurements to ensure compliance to study procedures. This is especially necessary for proper functioning of the VH_MC as this methodology utilizes a manually adjusted flow rate. The flow rate of the VH_MC was constantly monitored and adjusted to maintain exhaled CO, the ME  [24-h RMStudy procedures were approved by the Institutional Review Boards of both St. Luke’s-Roosevelt Hospital and Columbia University, and were conducted in accordance with the Declaration of Helsinki of 1975 as revised in 1983. All participants provided written informed consent before enrollment.p < 0.05) were utilized to determine any differences in all metabolic parameters between ethanol or propane stoichiometry and that obtained from actual combustion for both the VH_MC and the RMR_WRIC. A similar analysis was performed to determine any differences between both methodologies for all metabolic parameters measured in the human subjects. Moreover, this same analysis was performed to determine any differences between RMR measured by both the RMR_WRIC and VH_MC to that calculated by the ME [Power calculations  were pery the ME . Pearson2 and VCO2 for both the VH_MC and RMR_WRIC. Furthermore, a similar analysis was applied to the subject data to determine the magnitude of agreement for these same metabolic parameters between the two methods. Moreover, proportional bias (p < 0.05) was determined by regressing mean differences for combustion (ethanol or propane) for each the VH_MC and the RMR_WRIC for RMR, VO2 and VCO2 on mean values obtained by both methods. A similar proportional bias was also performed for the human subject data utilizing mean differences in the metabolic parameters obtained with each methodology. Finally, the Bland-Altman limit analysis [The Bland-Altman  limit ananalysis  was applanalysis  to deter2/VO2), VO2 (L/min) and VCO2 (L/min) calculated from ethanol and propane stoichiometry and that obtained by combustion for each method are shown in Table p < 0.05) to that obtained from actual combustion of the VH_MC  for simulated RMR, VO2 and VCO2 between ethanol stoichiometry and that obtained from actual combustion for the VH_MC as reflected by the limits of agreement shown in Table 2 and VCO2 obtained from the RMR_WRIC were of smaller magnitude as reflected by the much tighter limits of agreement as shown in Table 2 and VCO2 for the VH_MC while none existed for these values for the RMR_WRIC. This suggests that greater disagreements will occur with larger average values when using the VH-MC for measurements of RMR, VO2 and VCO2.All descriptive statistics for simulated RMR , RQ, . In comparison to the RMR_WRIC, the VH_MC underestimated RMR, RQ, VO2 and VCO2 by 9.7 ± 7.8, 2.6 ± 5.7, 10.5 ± 9.7 and 10.4 ± 8.0 %, respectively. This is further reflected in terms of the Bland-Altman limit analysis as shown in Table All subjects were at a resting state prior to each metabolic measurement as reflected by lack of significant differences in their pre 59.1 ± 9.2) and post (58.6 ± 10.1) metabolic test heart rates (beats/min). Moreover, the sequence to which method was performed first had no effect upon any of the metabolic measurements p = 0.36). With regard to the measurements obtained by both methods, no correlation was found for the RQ. However, RMR measured by both methodologies  and 5.30 ± 10.13 %, respectively. This is further reflected in the Bland-Altman limit analysis plot  and the VH_MC , the Mifflin equation underestimated their RMR  by 14.7 ± 10.1 (lot Fig.  for the 2 and VCO2, as well as the RQ with the VH_MC versus that compared with propane stoichiometry for the RMR_WRIC. This was verified by the Bland-Altman limit analysis which showed much greater limits of agreement as well as much wider confidence intervals for the VH_MC. All of the metabolic measurements during combustion validations with the RMR_WRIC were of greater accuracy and precision than that obtained for the VH_MC when compared to the respective stoichiometries. With regard to the comparison between the two methods for the human subjects, all metabolic measurements, except the RQ, were greater for the RMR_WRIC. Even though there was a relationship between the two methods in terms of RMR, the values obtained in the human subjects by the RMR_WRIC tended to be 10 % greater than that from the VH_MC. Similar results were found for the RQ with a 5 % over estimate.When compared to ethanol stoichiometry, there was greater variation in terms of RMR, VOAs noted in the results for the human subjects, significant differences were found between the two methods for RMR measurements with lower values obtained by the VH_MC. This is in contrast to the expected where RMR measured by the VH_MC would be greater due to the possibility of subject anxiety while being placed under the ventilated hood , 16. TheThe unexpected lower values for RMR with the VH_MC might be partly explained by the technological and procedural differences between the two methods. For example, the fresh air flow rates utilized for the RMR-WRIC is fixed at 100 L/min throughout the one-hour RMR measurement. This is in contrast to the VH_MC where the fresh air flow rate is constantly adjusted to maintain carbon dioxide levels below one percent while the subject is under the ventilated hood. Adjustments of greater magnitude are necessary for extremely large or obese subjects due to the small volume of the ventilated hood thus possibly compounding the errors. These constant adjustments to the fresh air flow rate might produce consistent errors by the VH_MC systems electronics. Therefore, more adjustments to the fresh air flow may translate to a greater magnitude of errors in RMR. However, this problem is eliminated with the RMR_WRIC. This is similar to the first Deltatrac Metabolic Monitors which also used a single fresh air flow setting of 40 L/min  during R2 and CO2 concentrations can lead to underestimates in calculated RMR. This might partly explain the underestimated RMR with the VH_MC. The early Deltatrac Metabolic Monitors, as well as VH_MC’s used today, rely on Naphion tubing to partly remove moisture from the sample gas stream prior to analysis. Naphion tubing only equilibrates the sample gas moisture content to that of the ambient air within the lab and an additional calculation for sample gas moisture content is utilized by the VH_MC’s software to further correct for its effects. Poor maintenance of the Naphion tubing can contribute to inadequate equilibration thus contributing to additional errors in metabolic calculations.Not accounting for all of the sample gas moisture content prior to the measurement of O2 sensor will lead to large underestimates of the ventilation rates of O2 thereby lowering RMR. Similar errors will occur for the ventilation rates of CO2 but are of a lesser magnitude due to less of an effect of sample gas moisture on the CO2 sensor. Lack of accurate accounting for moisture in the sample gas stream possibly contributed to counteracting the suspected increase in RMR with the VH_MC. This, combined with the potential for errors in the constantly changing fresh air flow rate through the ventilated hood, probably contributed to the overall underestimates of RMR in comparison to the RMR_WRIC. Moreover, this is further verified by the greater variability in all of the metabolic parameters measured by the VH_MC compared against ethanol versus that found for the RMR_WRIC against propane stoichiometry.Both methods utilize the Weir equation  for calcAll of these potential problems with the VH_MC are eliminated when RMR is measured with the RMR_WRIC utilizing the Promethion integrated instrumentation. This instrumentation does not require the removal of the moisture content of the sample gas stream since it is continuously measured and corrected for at the point of detection. Finally, the single rate fresh air flow setting eliminates any errors by the systems electronics that might occur. These technological improvements, along with improved human subject comfort of the RMR-WRIC chamber, may combine to reduce overall errors in the measurement of RMR.The RMR_WRIC is more accurate and precise in the determination of all of the metabolic parameters while providing greater comfort for the human subjects.


Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels.Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate.To enhance EPA production in M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.An ALA-preferring FADS6 from The online version of this article (doi:10.1186/s12934-016-0516-5) contains supplementary material, which is available to authorized users. Δ5, 8, 11, 14, 17) plays crucial roles in infant growth and adult health  was added in cultures.Although low temperature can prompt the conversion of LA to ALA in M. alpina  was selected as a suitable exogenous substrate, because it contains 46 % ALA and 26 % LA  in cells was slightly decreased with PSO supplementation  was supplemented in cultures. PSM is a rich source of carbon and nitrogen nutrients for microbial growth and its ALA level account for approximately 2 % of its total mass  higher than those of the PSM-free control  at 384 h, which was 73 mg/L higher than the yield obtained in shaking flasks supplemented with 50 g/L PSM . The Ma-MpFADS6-2 transformant and WT M. alpina produced 11.8 and 2.7 mg EPA per g of PSM, respectively.The Ma-MpFADS6-2 transformant which produced the highest EPA level among all Ma-MpFADS6 transformants was selected to be inoculated in 5 L jar fermenter containing 4 L of fermentation medium; as a control, WT M. alpina and 13.5 ± 1.6 g/L in Ma-MpFADS6-2 cultures , our result revealed that the EPA level was not readily increased in substrate-free cultures, and exogenous ALA had to be supplemented as the substrate for the ALA-preferring FADS6. Linseeds, perilla seeds, sesame seeds and peony seeds are rich in ALA, and among these, peony seeds have been reported to contain the highest percentage of ALA, where it reaches more than 40 % of TFA [Mortierella alpina is capable of using a variety of substrates for its growth and EPA production, including linseed oil [M. alpina and transformants were supplemented with no substrate, the free form of the ALA fatty acid, and different concentrations of PSO or PSM for enhancing EPA production -PUFA synthesis in % of TFA . Tree pe% of TFA . Howeverseed oil , crude sseed oil , vegetabseed oil , sunflowseed oil , fish meseed oil . In our M. alpina and all transformants was very low  form as an exogenous substrate. After supplementing media with lower concentrations of PSO in Ma-MpFADS6 transformants, EPA yield was significantly improved, indicating that PSO Fig. c, d. Thiina Fig. e, suggesM. alpina. Thus, EPA production was increased Ma-MpFADS6 transformants  when transformants were cultivated in 4 mL GY medium for 16 days. Recently, Tomoyo et al. [Saprolegnia diclina into M. alpina, which was inoculated in a 20 mL flask containing 4 mL GY medium and yielded 1.8 g/L of EPA under low temperature conditions. But most of the liquid in the culture had been evaporated after 7 days cultivation in our preliminary experiments. The EPA yields of our experiments were based on the larger flasks and 5 L fermenter, and we could not obtain such results from the published data.Ando et al.  over-expo et al.  transforM. alpina via manipulation of the ω3 pathway. Although only 588.5 ± 29.6 mg/L EPA was achieved in M. alpina ATCC 32222, there is a potential to significantly lower the overall cultivation costs for EPA production by addition of inexpensive PSM. One gram PSM, as a waste by-product, was converted to a high value-added commodity containing 11.8 mg EPA by microbial fermentation. This high value-added PSM can be better used in animal feed. The AA level of all transformants was not altered when EPA was produced, and our result showed that the EPA/AA ratio ranged from 1:20 in M. alpina to 1:4 in the Ma-MpFADS6-2 transformant, which enhanced the ω-3/ω-6 ratio for supplement applications and would be reasonable for the optimal ω3/ω6 ratio at ≈1:4 [Compared to their works, our study is the first report demonstrating the accumulation of EPA in  at ≈1:4 .M. alpina uracil-auxotrophic strain, representing the first report of EPA bioproduction via the ω3 pathway of M. alpina. EPA production in Ma-MpFADS6 transformants was enhanced to various degrees by supplementing cultures with no substrate, free form of fatty acid, different concentrations of PSO or PSM  was purchased from ATCC (American type culture collection) and conserved in our lab; M. alpina uracil-auxotrophic strain (CCFM501) was modified from M. alpina by Guangfei [Agrobacterium tumefaciens C58C1 (A. tumefaciens), which was kindly provided by Yasuyuki Kubo , was used as a transfer DNA (T-DNA) donor for fungal transformation; pBIG2-ura5s-ITs plasmid was modified in our lab from pBIG2RHPH2 sent from Yasuyuki Kubo.Wild-type (WT) Guangfei  in our lM. alpina was grown on potato dextrose agar (PDA) medium at 28 °C. Mortierella alpina uracil-auxotrophic strain was maintained on GY medium, consisting of 30 g/L glucose, 5 g/L yeast extract, 2 g/L KNO3, 1 g/L NaH2PO4 and 0.3 g/L MgSO4·7H2O, supplemented with 0.05 c uracil. YEP medium, consisting of 10 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl, was used for A. tumefaciens cultivation at 28 °C. Synthetic complete (SC) medium was used for screening positive transformants. Minimal medium (MM) and induction medium (IM) were used for co-cultivation of M. alpina and A. tumefaciens. The composition of these media were described previously [M. alpina were grown for 7 days at 28 °C in Broth medium, consisting of 20 g/L glucose, 5 g/L yeast extract, 1 g/L KH2PO4, 0.25 g/L MgSO4·7H2O, 10 g/L KNO3, pH 6.0. Lipid producing medium, consisting of 50 g/L glucose, 13.3 g/L KNO3, 3 g/L KH2PO4, 1 g/L Na2SO4, 0.5 g/L MgCl2·6H2O, 0.5 g/L CaCl2·2H2O, pH 6.0, was used for oil-producing cultivation. PSO  with 0.05 % Tween 80 or PSM  were added in lipid producing medium as exogenous substrates for lipid production. 50 g/L PSM was added in lipid producing medium for jar fermentation (fermentation medium). PSM in medium mentioned above was prepared by the following procedure: PSM was crushed using a mortar/pestle, sieved through a 40 mesh, boiled in double distilled water, precipitated for 10 min and filtered through quadruple layers of gauze.WT eviously . For theM. alpina uracil-auxotrophic strain. MaFADS6-I was digested and subcloned into HindIII and XhoI sites to generate a plasmid designated pBIG2-ura5s-MaFADS6-I while MpFADS6 was subcloned into HindIII and SmaI sites to generate a plasmid designated pBIG2-ura5s-MpFADS6.MaFADS6-I and MpFADS6 genes were amplified with pBIG-FMa/pBIG-RMa primers and pBIG-FMp/pBIG-RMp primers, respectively, as shown in Table A. tumefaciens C58C1 strain by electrotransformation. The detailed procedures of the ATMT method is described in Michielse et al. [A. tumefaciens harboring pBIG2-ura5s-MaFADS6-I or pBIG2-ura5s-MpFADS6 and M. alpina uracil-auxotrophic strain, genomic DNA preparation and confirmation of the existence of T-DNA in the genome of transformants were performed following our previously published methods [The binary plasmids (pBIG2-ura5s-MaFADS6-I and pBIG2-ura5s-MpFADS6) were transferred into e et al. . Co-cult methods .M. alpina was extracted using the HYQspin™ Total RNA Kit (HG403-03) and reverse-transcribed using the QuantScript RT kit (TIANGEN) according to the manufacturer’s instructions. Primers used for RT-qPCR are shown in Table −ΔΔCt method [Total RNA of transformants and WT t method .M. alpina were cultivated in 50 mL Broth medium at a shaking speed of 200 rpm at 28 °C for 7 days (constant temperature) or at 28 °C for 3 days and then at 12 °C for 8 days (low temperature). The free form of LA or ALA was added in Broth medium as a substrate with a final concentration of 0.1 mg/mL for free fatty acid investigations. PSO  with 0.05 % Tween 80 or PSM  were added in lipid-producing medium as exogenous substrates for triglyceride investigations. All strains were grown at shaking speed of 200 rpm at 28 °C for 3 days and then at 12 °C for 8 days (low temperature). All experiments were carried out in triplicate.Each stable transformant and WT M.alpina (control) were grown in lipid-producing medium with 50 g/L PSM in a 5-L jar fermenter containing 4 L of fermentation medium  at 28 °C for 3 days and then at 12 °C for 8 days with an aeration rate of 0.5 vol/vol/min (vvm) and agitation at 500 rpm, and finally aged at 12 °C for another 5 days. Cells of the Ma-MpFADS6 transformant and WT M. alpina were collected at 0, 24, 48, 72, 96, 120, 192, 264 and 384 h for determination of the growth curve, level of total fatty acids (TFA), AA, ALA and EPA, residual glucose, ammonium and dissolved oxygen (DO).One of the three Ma-MpFADS6 transformants with the highest EPA production and WT g and lyophilized. Lipids from an equivalent weight of freeze-dried cells (50 mg) and supernatant were extracted and methyl esterified as described previously [For the extraction of fatty acids, cells were centrifuged at 10,000×eviously . Fatty a


Internal auditory canal (IAC) metastasis due to leptomeningeal carcinomatosis (LMC) from gastric cancer (GC) has rarely been reported. Early manifestation of symptoms, such as hearing loss, vertigo and facial paralysis, in cases of IAC metastasis due to LMC may facilitate the early detection of brain metastasis. To the best of our knowledge, the present study is the first to report IAC metastasis due to LMC in human epidermal growth factor receptor 2 (Her2)-positive GC. This study reports a case of an Her2-positive GC patient with LMC including IAC metastasis, who presented with acute sensorineural hearing loss, ipsilateral facial paralysis and vertigo during trastuzumab containing chemotherapy. The current study also discusses the early diagnosis and management of this complicated condition, demonstrating that clinical suspicion is key for a prompt diagnosis and proper management of LMC including IAC metastasis in Her2-positive GC. Gastric cancer (GC) is the most common malignancy and the third most common cause of cancer-related mortality in Korea . AlthougThe rate of Her2 positivity in GC is 22%, which is similar to the rate of that in breast cancer (BC) . AlthougA 56-year-old male who had been diagnosed with Her2-positive advanced GC with multiple lymph node metastases, including left supraclavicular, paraaortic and retroperitoneal lymph nodes, underwent 12 cycles of combination chemotherapy composed of trastuzumab , capecitabine and cisplatin at the Konkuk University Medical Center . Cisplatin was discontinued after the eighth cycle due to cumulative peripheral neuropathy. Only capecitabine and trastuzumab were continued due to their clinical benefits. The response was a partial remission according to Response Evaluation Criteria In Solid Tumors .After the 10th cycle of chemotherapy, the patient was referred to the Department of Otorhinolaryngology-Head and Neck Surgery at the Konkuk University Medical Center, Konkuk University School of Medicine  for an evaluation of right ear fullness, which had been persisting for 15 days. Otitis media with effusion was diagnosed by an otoendoscopic examination, and the nasopharyngeal examination revealed no specific finding. Bone conduction pure tone audiometry after the treatment of the otitis media with effusion revealed mild (35dB) sensorineural hearing loss on the right side .Two weeks later, the patient suddenly developed a total hearing loss  and faciSudden hearing loss, vertigo and ipsilateral facial palsy caused by IAC metastasis are uncommon initial presenting symptoms in patients with LMC. Recently, IAC metastasis due to direct invasion into the IAC from the metastatic lesion in the petrous apex was reported, which was a different spread pattern to that in the present case . LMC, heEarly diagnosis of LMC is important in order to prevent irreversible neurological deficits. The most common symptoms of IAC metastasis include facial nerve palsy, hearing loss and tinnitus, and these symptoms are rapidly progressive in nature . Althouget al (et al, the researchers reported LMC and confusion, cognitive impairment and ataxia without hearing loss, which are different symptoms to those identified in the present case. IT trastuzumab therapy has been investigated in Her2-positive BC, and case reports indicate that this is a promising experimental treatment for patients with symptomatic LMC (LMC in Her2-positive GC was first reported by Cavanna et al . Despiteatic LMC ,15,16. Hatic LMC . LapatinIn conclusion, a clinical history of sudden sensorineural hearing loss raises the possibility of IAC metastasis, which may be a consequence of LMC, in Her2-positive GC. Prompt suspicion and an MRI are greatly helpful in the diagnosis of metastatic carcinoma to the IAC. We would like to emphasize that if hearing impairment is suspected in Her2-positive GC, LMC should be considered as a differential diagnosis, and early diagnosis and concerted therapeutic efforts are important for the management of LMC in Her2-positive GC.


Telocytes (TCs) is an interstitial cell with extremely long and thin telopodes (Tps) with thin segments (podomers) and dilations (podoms) to interact with neighboring cells. TCs have been found in different organs, while there is still a lack of TCs-specific biomarkers to distinguish TCs from the other cells.+ T cells from bronchial lymph nodes (T-BL), and CD8+ T cells from lungs (T-LL). The chromosome 17 and 18 genes were extracted for further analysis. The TCs-specific genes and functional networks were identified and analyzed by bioinformatics tools. 16 and 10 of TCs-specific genes were up-regulated and 68 and 22 were down-regulated in chromosome 17 and 18, as compared with other cells respectively. Of them, Mapk14 and Trem2 were up-regulated to indicate the biological function of TCs in immune regulation, and up-regulated MCFD2 and down-regulated E4F1 and PDCD2 had an association with tissue homeostasis for TCs. Over-expressed Dpysl3 may promote TCs self-proliferation and cell-cell network forming.We compared gene expression profiles of murine pulmonary TCs on days 5 (TC5) and days 10 (TC10) with mesenchymal stem cells (MSCs), fibroblasts (Fbs), alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8The differential gene expression in chromosomes 17 and 18 clearly revealed that TCs were the distinctive type of interstitial cells. Our data also indicates that TCs may play a dual role in immune surveillance and immune homoeostasis to keep from immune disorder in acute and chronic pulmonary diseases. TCs also participated in proliferation, differentiation and regeneration.This article was reviewed by Qing Kay Li and Dragos Cretoiu.The online version of this article (doi:10.1186/s13062-015-0042-0) contains supplementary material, which is available to authorized users. TCs were found to form a three-dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system [Telocytes (TCs) have been proposed as a new type of interstitial cells with extremely long telopodes (Tps), which were recognized in different organs, such as trachea and lung ,2, oesope system ,13,16-18e system ,19. TCs e system . TCs pree system ,21.+ T cells from bronchial lymph nodes (T-BL), and CD8+ T cells from lungs (T-LL) by global analyses [Our previous study initially approved the existence of TCs in the trachea and lung tissues of the mouse and human ,22,23. Wanalyses . The simanalyses . HoweverThe present study aimed at investigating features and patterns of TCs-specific gene profiles and exploring potential function of TCs by focusing in chromosomes 17 and 18. The significant difference in gene expression profiles of murine pulmonary TCs on days 5 (TC5) and days 10 (TC10) with the other cells, MSCs, Fbs, ATII, ABCs, PACs, T-BL and T-LL were compared to identify TCs-specific genes and the functional networks were identified by bioinformatics tools.+ T-cells from lungs and bronchial lymph nodes (GSM677065) and CD8+ T-cells from bronchial lymph nodes, lymphocytes (GSM677065) derived from mice (CL4). Murine proximal airway duct (GSM709834-GSM709836) and basal cells  were isolated from 8–12 week old C57BL/6 mice.Gene expression profiles of murine pulmonary TCs on days 5 and 10, MSCs and Fbs were obtained from our previous study . TCs werThe gene expression profiles of murine lung TCs, MSCs and Fbs from our previous study contained 23861 probes. About 13236 probes and 11532 genes were defined after eliminating the probes without corresponding official symbols. Gene expression of ATII, ABCs, PACs, T-BL, and T-LL from GEO originally included 45,101 probes. 5684 probes without corresponding official symbols were eliminated and 39,417 probes and 21,680 genes were obtained. Data integration and genes detected in all the samples were selected to analysis, of which 11532 genes were elected finally. Totally, 582 genes of the chromosome 17 and 267 genes of the chromosome 18 were focused and furthermore analyzed in the present study.Gene expression data were normalized and imported into Agilent GeneSpring GX software version 11.5.1) for further analysis. Differentially expressed genes between two samples were identified through Fold Change filtering as our previous research . The pro1.5.1 forFifty three genes were up-regulated and 92 down-regulated in chromosome 17 of TC5 as compared with other cells, and 28 genes were up-regulated and 167 down-regulated in TC10. Of genes up or down-regulated at TC5 and TC10, 16 over-expressed genes  were obtained in chromosome 17 of TCs. Among them, Trem2 was up-regulated over five folds in TC5 and TC10, as compared with others. Sixty eight genes were down-regulated in TC5 and TC10, of which C030034I22Rik was expressed in TCs 2–5 folds lower than in other cells  in chromosomes 17 and 18 , respectively , a distinct type of stromal cells. There are some minor issues which, if addressed, could improve the manuscript.1) Introduction – There are more papers already published regarding the presence of telocytes in different organs, which might be worth mentioning. Some characteristics of the immunophenotype and ultrastructure should also be described.Authors’ response: We have now described the presence of TCs in different organs and the characteristics of the immunophenotype and ultrastructure in introduction paragraph.The aim of the study is not clearly stated and therefore a strong justification for the purpose of this study must be included. Also, some discussion about the chromosome 2 and 3 gene expression profile in TCs should be mentioned.Authors’ response: We revised and highlighted the aim of our study to investigate features and patterns of TCs-specific gene profiles and explore potential function of TCs by focusing in chromosomes 17 and 18. It was not only the first time but also a new method to mine the TCs-specific gene profiles in chromosome 17 and 18. We now also have made a discussion about the study for the chromosome 2 and 3 gene expression profiles in TCs, which had not been published before we submitted this article.2) Results – It would be useful for the readers to describe name of gene (or the encoded protein) at least for the most significant ones.Authors’ response: We completely agree with this suggestion and have listed these significant genes in Tables 3) Conclusion – I would suggest that this paragraph should be re-written pointing out the specific novelty of this study and the major findings leading to some functional hypotheses. Anyway, phrases like “Data from the present study demonstrated TC-specific genes in chromosomes 17 and 18, although the mechanism of TCs-specific genes in biological process of TCs needs to be furthermore explored” must be removed since is hampering the clarity and the significance of the study. TCs diversity according to organ location is quite intriguing, even considering the proposed roles of this cell subset in immune surveillance/tissue homeostasis. The authors should comment more on this.Authors’ response: We thank the reviewer’s advice and have re-written the conclusion paragraph to highlight the specific novelty of our study and TCs-specific genes identification with its potential function in chromosomes 17 and 18. Also, negative phrases like “Data from the present study demonstrated TC-specific genes in chromosomes 17 and 18, although the mechanism of TCs-specific genes in biological process of TCs needs to be furthermore explored” have been removed.Besides, TCs was isolated from murine lung tissue in this study. We have described the proposed roles of TCs in immune surveillance/tissue homeostasis which may protect from immune disorder in acute and chronic pulmonary diseases. We think it’s really meaning to differentiate the proposed roles of TCs subsets from different organ location in further study, as reviewer suggested.4) An English language revision is mandatory. Many sentences should be rephrased. For example “The novel feature of telocytes under TEM has extremely long TPs with podomers and podoms, to contact with neighboring or distant effectors like axinal of neuron”.Quality of written English: Needs some language corrections before being published.Authors’ response: We have revised the article by an English native specialist. I hope you are satisfied with the revised version of the manuscript and hope it suitable for the publication.Although telocytes (TCs) have been found in a variety of organs, TCs-specific biomarkers still need to be evaluated. This manuscript studied the gene expression profiles of murine pulmonary TCs and compared their profiles with a variety of cell types, including mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8+ T cells from bronchial lymph nodes, and CD8+ T cells from the lung. In the study, genes of chromosome 17 and 18 were extracted and analyzed using bioinformatics tools, in order to identify TCs-specific genes and their functional networks. They found that 16 and 10 of TCs-specific genes were up-regulated, and 68 and 22 were down-regulated in the chromosome 17 and 18, respectively. Particularly, Mapk14, Trem2, MCFD2 and Dpysl3 were up-regulated, whereas the E4F1 and PDCD2 were down-regulated in TCs. Their data demonstrated that the differential expression of subset of genes in chromosomes 17 and 18 were unique features of pulmonary TCs, and they might be used as biomarkers to distinguish TCs from the other type of cells. Their findings were also suggestive of that TCs might play critical roles in immune surveillance and other intracellular functions.Quality of written English: Acceptable.Authors’ response: We appreciated Dr. Qing Kay Li’s encouraging comment on this article.


The number of older adults in the global population is increasing. This demographic shift leads to an increasing prevalence of age-associated disorders, such as Alzheimer’s disease and other types of dementia. With the progression of the disease, the risk for institutional care increases, which contrasts with the desire of most patients to stay in their home environment. Despite doctors’ and caregivers’ awareness of the patient’s cognitive status, they are often uncertain about its consequences on activities of daily living (ADL). To provide effective care, they need to know how patients cope with ADL, in particular, the estimation of risks associated with the cognitive decline. The occurrence, performance, and duration of different ADL are important indicators of functional ability. The patient’s ability to cope with these activities is traditionally assessed with questionnaires, which has disadvantages . Several groups have proposed sensor-based systems to recognize and quantify these activities in the patient’s home. Combined with Web technology, these systems can inform caregivers about their patients in real-time .We hypothesize that a non-intrusive system, which does not use body-mounted sensors, video-based imaging, and microphone recordings would be better suited for use in dementia patients. Since it does not require patient’s attention and compliance, such a system might be well accepted by patients. We present a passive, Web-based, non-intrusive, assistive technology system that recognizes and classifies ADL.The components of this novel assistive technology system were wireless sensors distributed in every room of the participant’s home and a central computer unit (CCU). The environmental data were acquired for 20 days (per participant) and then stored and processed on the CCU. In consultation with medical experts, eight ADL were classified.In this study, 10 healthy participants  were included. For explorative purposes, one female Alzheimer patient  was measured in parallel with the healthy subjects. In total, 1317 ADL were performed by the participants, 1211 ADL were classified correctly, and 106 ADL were missed. This led to an overall sensitivity of 91.27% and a specificity of 92.52%. Each subject performed an average of 134.8 ADL (SD 75).The non-intrusive wireless sensor system can acquire environmental data essential for the classification of activities of daily living. By analyzing retrieved data, it is possible to distinguish and assign data patterns to subjects' specific activities and to identify eight different activities in daily living. The Web-based technology allows the system to improve care and provides valuable information about the patient in real-time. The number of older adults in the global population is increasing both in absolute and relative terms due to increased life expectancy . This deThe patient’s ability to cope with ADL is traditionally assessed with questionnaires  a number of sensors that are distributed in every room and (2) a central computer unit (CCU). The collected data are stored and processed by an ADL classifier based on a rule-based forward chaining inference engine. Eight ADL, such as sleeping, grooming, toileting, getting ready for bed, cooking, eating, watching TV, and seated activity were allotted for the ADL classifier to determine. The ADL were chosen in consultation with medical experts. Healthy participants were recruited by advertisements in local newspapers. They were assessed with a standardized paper-pencil test battery, which included the Montreal Cognitive Assessment (MoCA) ,25, the In total, 10 healthy mobile adults  were included in the study. The study was carried out in accordance with the Declaration of Helsinki and was approved by the local ethics board. Written informed consent was obtained from all participants prior to inclusion. No compensation for participation was provided.3 , luminescence in lx , motion (binary) , and acceleration in m/s2 . Each PCB was assembled in polyvinyl chloride (PVC) housing  capturing ambient values: temperature in °C , humidity in g/m housing  and was l x w x h=15mm x 30mm x 60mm, weight=80g), which digitalizes the analog environmental data and sends it to the CCU. A commercial available laptop, running customized Microsoft Windows 7, builds the CCU and served as data server with integrated Web link . Therefore, a low byte order marked bidirectional protocol placed on an 868 MHz carrier transmitted by frequency modulation was implemented. Even-parity error handling and frame collision detection was implemented.The assistive technology system was installed in the home of 10 healthy subjects. Installation duration for the entire system depends on the apartment layout and varied from 15-30 minutes. Once set up and initialized, the system continuously recorded the five ambient environmental values autonomously. Activities of the 10 subjects were recorded for 20 days each. The subjects were told to ignore the system as much as possible and act naturally. They were also asked to touch the sensor boxes only if absolutely necessary. The system was uninstalled after 20 days of continuous measuring.For evaluation purposes, a wireless protocol device, built in a housing with a wearable belt clip, was provided to the subjects . The proIn addition, participants were provided with a paper-pencil log book, to record every wrongly stated, forgotten, or missed ADL.First, two different sorting methods were used sequentially to increase the sorting efficiency of the acquired data . A BuckeIn summary, the data are bijectively matched to their origin compartment by the Bucketsort, and then lined up in a chronological order by Radixsort. A mathematical description regarding the efficiency of the two sort algorithms is presented in In the second step, the data were classified using an ADL classifier . The claThe used method is to obey the rules that the medical experts defined first. The forward chaining inference engine, on the other hand, works with three basic elements: (1) ambient value matrices, (2) rules, and (3) behavioral parameters. For each potential ADL, an ambient value matrix is composed of the sorted raw data. Each ambient value matrix is then compared to a set of rules defined upfront for each of the eight ADL. The rules are built under the premise of the behavioral parameter. If the ambient value matrix fits the set of rules, the activity is considered as the corresponding ADL.For example, if one looks at “toileting”, the matrix consists of the data from the five sensors in the bathroom and from the flush handle. Hence, the ambient value matrix shows a specific pattern associated with toileting. First, the forward chaining inference engine verifies the activity’s duration for toileting. Further, the forward chaining inference engine verifies if the light conditions in the bathroom change, if the subjects sit on the toilet, if the subjects flushes, and if the humidity and the temperature do not change significantly. Having these prerequisite (rules) fulfilled, the activity is stated as “toileting”. The mathematical formulas and the source code of the forward chaining inference engine are provided in Depending on the complexity of the ADL to be classified, n-steps of iteration can be done. By processing the daily routine with the developed classifier algorithm, the system allots specific patterns to one of the 8 selected ADL. The output of the classifier (the conclusion) is the ADL a subject performed at a given time throughout the day. This information is stored in the database.The calculation and algorithms were implemented in Matlab R2007b .The subject log book was updated by superimposing the data of the wireless protocol device with the paper-pencil log book. The systems’ performance (sensitivity and specificity) was then calculated by correlating the output of the ADL classifier with the ADL protocols .In addition to the 10 healthy subjects, one dementia patient was measured for explorative purpose. This was done to understand the acceptance of the system in the target group of dementia patients and also to estimate the further clinical implications. The measurement was performed similarly in all respects to the measurement in the healthy subjects. The patient was an 84-year-old female. She was receiving home care twice a week for 1 hour, and she was on her own for the rest of the time.In total, 33,939,441 data packets  were captured correctly, and 160,269 data packets (0.47%) were lost due to transmission error. Thus, an overall reliability of 99.53% was achieved.In total, 1317 ADL were performed by the participants, 1211 ADL were classified correctly, and 106 ADL were missed. This leads to an overall sensitivity of 91.27% and a specificity of 92.52% . Each suThe activity pattern of a healthy female  is shown in The feasibility and reliability of the newly developed non-intrusive sensor system (hardware and the operating software) could be proven in the field. The results show that daily living evokes variation in ambient values that can be captured by the sensor system, leading to ADL specific data patterns. Hence, it is possible to assign sensor data patterns to specific activities, whereby the most relevant sensor in the detection of ADL is the movement sensor. By analyzing the retrieved data, it became possible to identify eight ADL.Other researchers have conducted similar studies. Bang et al  used a sThe ADL classification performance of the system relied on the protocol compliance of the subjects during the measurement. We asked the subjects to record their ADL very carefully with the electronic protocol device and provided them furthermore with paper-based log books to note any errors or wrong manipulations. Overall, it is possible that subjects forgot to record an activity or recorded a wrong activity, which negatively affects the classification performance numbers. Hence, the reported performance of the ADL classifier expressed by sensitivity and specificity numbers actually represents worst-case scenario.A limitation of our system is that the sensors need a clear view of the entire room and must not be covered. This is can be solved by proper placement of the sensors in the patient’s home.As expected, the classifier showed better performance for some ADL  than it did for others . This is due to the fact that some ADL are not exclusively room related and evoke only slightly different ambient values. Such data pattern differs only poorly among one another, which has also been found by other researchers ,16,33.The system uses wireless technology with small, discrete shaped sensor boxes. Hence, no hardware or tools are needed for the installation and the set-up can be done in less than 30 minutes. All parts were built under consequent low-power conditions, which keeps the system in service for 6 months straight. During this period, no maintenance is needed. Once the system is set up, the functioning of each sensor box can be monitored at any time. This leads to a high degree of transparency regarding the transmitted, lost, and compromised data (reliability of 99.53%). Furthermore, data can be bijectively matched to its origin to take statistical advantage of paired data, which ensures valid data throughout the measurement. Moreover, by foregoing the use of cameras and microphones, the privacy of the subjects can be guaranteed at any time of the experiment. Furthermore, due to its Web link, the system can provide discrete information about the patient’s ADL performances in real-time, but also historical and statistical information about the progression of the disease.When it comes to measurement in dementia patients, we proved feasibility in one female Alzheimer patient. The acceptance of the system was good, and no significant incident or failure occurred during the measured period. The patient was not disturbed by the system.The activity map of the patient shows significant difference to the activity map of the healthy subject. While in the healthy subject, a daily structure is observable, much less structure can be found in the Alzheimer patient. Comprehensive analysis of further datasets of dementia patients can provide valuable clinical information, especially by comparing it to datasets of healthy patients.These preliminary results obtained from one Alzheimer patient are promising in anticipation of further research and the clinical implication of the system.The non-intrusive wireless sensor system can be used to acquire environmental data essential for the classification of ADL. By analyzing the retrieved data, it is possible to distinguish and assign data patterns to subjects’ specific activities and to identify eight different ADL. Thanks to the Web-based technology, the system has a high potential to improve care and provides valuable information about the patient in real-time.Based on the results of this study, there are plans to install the system in the home of Alzheimer patients, suffering from moderate to severe dementia. The data of these patients will be compared to the data of the healthy subjects to study and analyze the different behavior patterns.