Cosmetic makeup significantly influences facial perception. Because faces consist of similar physical structures, cosmetic makeup is typically used to highlight individual features, particularly those of the eyes  and mouth . Though event-related potentials have been utilized to study various aspects of facial processing, the influence of cosmetics on specific ERP components remains unclear. The present study aimed to investigate the relationship between the application of cosmetic makeup and the amplitudes of the P1 and N170 event-related potential components during facial perception tasks. Moreover, the influence of visual perception on N170 amplitude, was evaluated under three makeup conditions: Eye Shadow, Lipstick, and No Makeup. Electroencephalography was used to monitor 17 participants who were exposed to visual stimuli under each these three makeup conditions. The results of the present study subsequently demonstrated that the Lipstick condition elicited a significantly greater N170 amplitude than the No Makeup condition, while P1 amplitude was unaffected by any of the conditions. Such findings indicate that the application of cosmetic makeup alters general facial perception but exerts no influence on the perception of low-level visual features. Collectively, these results support the notion that the application of makeup induces subtle alterations in the processing of facial stimuli, with a particular effect on the processing of specific facial components , as reflected by changes in N170 amplitude. Women in a number of societies throughout the world have traditionally used cosmetic makeup to modify the visual perception of their facial beauty , a proceWhile faces consist of similar physical structures, cosmetic makeup is typically used to highlight or emphasize individual features . Indeed, research has indicated that such enhancements are particularly relevant for the eyes  and the mouth  . ApplicaChanges in facial perception can be detected via the recording of event-related brain potentials (ERPs), from which studies have identified face-sensitive P1 and N170 components. As the P1 and N170 ERP components are typically regarded as markers for processing the perceived faces, these components are useful in examining the effect of cosmetics on face perception.P1, an early positive component of the ERP, typically peaks approximately 100 ms after the presentation of facial stimuli. Reported to reflect the processing of low-level visual features such as contrast , P1 amplComparatively, N170 is a negative component evoked at the onset of facial perception that is characterized by a posterior-temporal scalp distribution   and peakWhile the P1 component of the ERP appears to reflect a response to lower-level visual features, the N170 component appears to be driven by wide-scale facial perception . In addiThe current literature indicates that the eyes play a central role in facial perception and representation. In particular, a recent MEG study demonstrated that participants require significantly longer to perceive eyes presented in isolation than when presented as a facial component . AccordiIn addition, Eye shadow, Lipstick, and No Makeup.Therefore, it remains unclear whether the eyes or mouth exert a greater influence on N170 amplitude. As previously mentioned, cosmetic makeup is most frequently applied to the eyes  and the mouth  . MulhernEye shadow and Lipstick/No Makeup conditions. Alternatively, if N170 amplitude is more reflective of the processing of the mouth than the eyes following the application of cosmetics, a significant difference would be expected between the Lipstick and Eye shadow/No Makeup conditions. Moreover, because the application of cosmetics to female faces has been observed to increase facial contrast  were selected for the present study. All participants exhibited normal or corrected-to-normal vision and had no history of psychiatric or neurological disorders.All participants provided written informed consent prior to participation in the study, in accordance with the Declaration of Helsinki. The ethics committee of Otemon Gakuin University formally approved this experiment and the recruitment of participants from the Otemon Gakuin University student population.1,2 and included front-facing views of almost identical luminance. All images depicted neutral expressions. In total, 30 visual stimuli were used for the present study , Eye Shadow (wearing blue eye shadow only), and No Makeup (no makeup applied). Each image was digitally edited to include cosmetics of the same color  and reconstructed from the original using an application software (YouCan Makeup) of iPad3 . The No Makeup condition was also used as the adapting image for the experiment and was presented prior to each stimulus for comparison. In addition, each image was edited to feature the same hairstyle and color (black), as reconstructed from the original using an iPad. All stimuli were airbrushed using Adobe Photoshop 12 to remove any outstanding features or blemishes and were subsequently processed using Photoshop software to ensure background consistency. All stimuli were presented in the same orientation on a white background. All faces were presented in a front-facing view and were equated for mean luminance  and size using Adobe Photoshop 12 software . Faces occupied a visual angle of  3.4° × 4.0°. All faces were presented in the center of a 22-inch cathode ray tube monitor  that was placed 100 cm in front of the participants. The screen resolution was 1280 × 1024, with a refresh rate of 100 Hz.The images selected as visual stimuli included color pictures of the faces of 10 young, adult Japanese women. The stimulus faces were unfamiliar to all participants in the study. The pictures were obtained from various websitesA previous study  reportedLipstick, Eye Shadow, or No Makeup) was presented for 500 ms, followed by an inter-stimulus interval of 500 ms; and (IV) a judgment screen was presented for 1000 ms . The inter-trial interval varied randomly between 500 and 1500 ms.Participants were seated comfortably 100 cm in front of a 22-inch cathode ray tube monitor on which stimuli were presented using a Multi Trigger System . Each trial was completed as follows: (I) a fixation mark (+) was presented for 500 ms, followed by an inter-stimulus interval of 1000 ms; (II) an adapting facial stimulus (No Makeup) was presented for 500 ms, followed by an inter-stimulus interval of 1000 ms; (III) a target face stimulus  with a target face and to identify the type of target face as quickly and accurately as possible. Participants were required to respond by pressing one of three buttons that corresponded to 1, 2, and 3 with their right index finger to indicate whether the facial stimulus belonged to the Lipstick, Eye Shadow, or No Makeup condition, respectively. Reaction time was measured using a digital timer accurate to 1 ms, beginning with the onset of stimulus presentation and finishing once participants had responded to the stimulus. Participants performed 10 practice trials, followed by three blocks of 80 trials . The order of conditions was randomized within each block. In total, 30 stimuli were presented in random order, with equal probability. In each trial, the adapting face and target face were obtained from the same person.On the judgment screen, the three target faces were assigned a number: 1 = Electroencephalography (EEG) and Electrooculography (EOG) data were acquired using a 128-channel Sensor Net  and recorded via the standard EGI Net Station 5.2.01 package. EEG and EOG results were recorded using Ag/AgCl electrodes from the 10-5 system  and eachEye shadow, Lipstick, and No Makeup) from 200 ms before to 1000 ms after stimulus presentation, and were baseline corrected using the 200 ms pre-stimulus window. Based on previous studies . P1 amplitude was analyzed using a three-way (3 × 2 × 2) repeated-measures ANOVA with regard to condition , hemisphere  and electrode placement , while post hoc comparisons were performed using the Bonferroni test. Similarly, N170 amplitude was analyzed using a three-way (3 × 2 × 6) repeated-measures ANOVA with regard to condition , hemisphere  and electrode placement , while post hoc comparisons were performed using the Bonferroni test. ERP was analyzed using Greenhouse–Geisser corrections applied to p values associated with multiple degrees of freedom repeated-measures comparisons.Reaction time was analyzed using a one-way repeated-measures analysis of variance (ANOVA) for each condition ; Lipstick, 412 ± 98 ms; No Makeup, 391 ± 73 ms .No main effect was detected for Figure 2 displays the grand-averaged EEG waveforms for all conditions  at two electrode sites (O1/O2). For each condition, an enhanced positive ERP was identified 50–110 ms after exposure to the target face. This positive ERP was identified as P1. Table 1 displays the mean P1 amplitude for all conditions at four electrode sites (O1/O2 and PO3/PO4). No significant main effect was detected with regard to condition [F = 0.49, p = 0.77, hemisphere on P1 amplitude [F = 0.42, p = 0.53, electrode placement produced a significant effect on P1 amplitude [F = 11.95, p = 0.003, p < 0.05). No significant interactions were observed between the variables .Figure 3 displays the grand-averaged EEG waveforms for all conditions  at four electrode sites (P7/P8 and PO7/PO8). For each condition, an enhanced negative ERP was identified 120–180 ms after exposure to the target face. This negative ERP was identified as N170. Table 2 displays the mean N170 amplitude for all conditions at 12 electrode sites . A significant main effect was detected for condition [F = 3.39, p = 0.05, electrode placement on N170 amplitude [F = 7.07, p = 0.002, Lipstick condition was significantly greater than for the No Makeup condition (p < 0.05). No significant main effect was detected with regard to hemisphere on N170 amplitude [F = 0.29, p = 0.60, condition × hemisphere, condition × electrode, and hemisphere × electrode) , however, a significant interaction was detected for all three (condition × hemisphere × electrode) [F = 2.88, p = 0.04, Lipstick condition was significantly greater than for the No Makeup condition in the left hemisphere (PO7) and right hemisphere (PO10) (p < 0.05). Furthermore, simple effect analyses demonstrated that N170 amplitude was significantly greater at the P8 and PPO10h placement sites than the PO8 site in the right hemisphere for all conditions  (p < 0.05). Moreover, in the left hemisphere during the No Makeup condition, N170 amplitude was significantly greater at P5 than P7 (p < 0.05).Eye Shadow/Lipstick) to a model face without makeup (No Makeup). Subsequently, P1 and N170 amplitudes were analyzed using EEG and EOG during a facial perception task with three target faces; , wherein the adapting face presented prior to the target face was the No Makeup condition. The results of the present study demonstrated that N170 amplitudes were significantly greater in response to the Lipstick condition than to the No Makeup condition, while no significant effect was detected for cosmetic makeup on P1 amplitude.The present study aimed to investigate changes in P1 and N170 amplitude in response to the application of cosmetics during a facial perception task. This experiment adopted an ERP adaptation paradigm, in which participants were required to compare a model face with makeup  drew attention to the mouth during facial perception.Such findings provide support for the notion that the application of cosmetics significantly influences facial perception . In the us study , whereinHowever, because different amounts of makeup affect attractiveness , it is pIn addition, According to No Makeup and Eye Shadow conditions in the present study, the eyes nonetheless play a significant role in facial perception (While N170 amplitudes did not significantly differ between the rception . AccordiLipstick), but was unaffected by the No Makeup condition. In addition, no significant main effect was identified with regard to condition for P1 amplitude. Therefore, the present results support the notion that the application of cosmetic makeup alters facial perception. These findings subsequently indicate that cosmetic makeup produces a significant effect on facial perception, influencing the processing of specific facial features, with a particular focus on the mouth, as reflected by changes in N170 amplitude.The present study found that N170 amplitude was significantly increased in response to the application of cosmetic makeup (HT designed the experiments, performed the experiments and EEG data recording. HT performed the EEG data analysis and statistical analyses and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Moreover, there is a profound constraint imposed by the anchoring of the centromere  to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies ( A fundamentally important step in the repair of a broken chromosome by homologous recombination is the identification and use of a homologous donor sequence to repair the DSB –6. In euSaccharomyces cerevisiae [Recently we developed an ectopic donor assay to study DSB repair efficiency in haploid revisiae . A site-revisiae ,7. In sorevisiae  and by Zrevisiae  also reaHere we have extended our analysis to examine the correlation between contact frequencies and repair for intrachromosomal events. We find again a strong correlation with contact frequency but also see additional constraints imposed by the centromere and by the very high level of contacts made by nearby intrachromosomal sequences.leu2 sequences inserted on Chr5, while a homologous 2-kb LEU2 donor was placed at 4 positions on the same chromosome or at 20 locations on different chromosomes. Repair occurs predominantly by synthesis-dependent strand annealing in which by a patch of DNA newly copied from the donor to replaces the 117-bp HO cleavage site sequences [In our previous study, we mainly focused on the correlation between chromosome organization and recombination frequencies in interchromosomal noncrossover gene conversion events . A DSB wequences . We and equences .LEU2 gene inserted 625 kb from the left end on chromosome 2 (Chr2) and a donor was inserted at different sites across the chromosome (CEN2 and 188 kb from the right telomere (http://www.yeastgenome.org/). The efficiency of DSB repair of each of the 12 strains was measured by plating cells on YEP-galactose plates to continuously induce the HO endonuclease, compared to the same number of cells plated on glucose-containing medium. Virtually all of the survivors repaired the DSB by ectopic gene conversion rather than by nonhomologous end-joining, which occurs only in 0.2% of cases [To examine intrachromosomal repair in more detail, we constructed a series of 12 strains, in which a DSB was created within a 2-kb romosome . The DSBr = 0.82 (P = 2 x 10−3) with +/-10 kb window around the donor and r = 0.85 (P = 5 x 10−4) with +/-20 kb window around the donor. Thus, intrachromosomal recombination is strongly constrained by the likelihood that two sequences will come into contact, as we saw for interchromosomal events.Repair efficiencies were then plotted with respect to the total contact frequencies between the DSB region and the donor region, which is calculated by adding up all the interaction reads, measured by , betweenWe note that when donors were located within 50 kb from a telomere (locus 1 and 12), their measured viabilities were higher than expected based on their contact frequency . It has LEU2 donor from the left end of Chr2, it becomes evident that donors located close to the centromere display a low repair rate compared to donors located within a chromosome arm  among four of the MCM21 deletion strains whose donors were close to CEN2 . For strains with wild type CEN2 (cutsite-deleted derivatives of strains YWW113 and 119), both mother and daughter cells gave rise to colonies in each of 45 cases. For the modified strain YWW216 , lacking the HO cutsite, 16 of 29 daughters failed to produce colonies, and for modified strain YWW231 , 7 out of 20 daughter cells failed to give rise to colonies. Finding approximately 1/3 of pedigrees failing to properly disjoin the GAL::CEN chromosome is consistent with our previous study and confirms that when galactose was added to induce HO it also would disrupt normal CEN function [To confirm that transcription did indeed perturb centromere function in these strains we carried out pedigree analysis to measure missegregation of the chromosome by the appearance of daughter cells that failed to inherit Chr2 . To be sfunction .leu2::HOcs at 625 kb and an intrachromosomal ectopic LEU2 donor at a different location with a strain carrying a URA3-selectable marker and a leu2-K donor at 625 kb; that is, at the allelic position to the DSB  and allelic (leu2-K) donors by PCR-amplifying the repaired locus followed by KpnI digestion and determining the proportion of repair events resistant to KpnI digestion. This analysis was carried out both from colonies of individual recombinants (KpnI+) intrachromosomal donor was substantially reduced (LEU2 was placed at site 2 (122 kb), which was 36% viable in the haploid strain assay, analysis of pooled diploid cells (which were nearly 100% viable) showed only 12% of the repair events used this intrachromosomal donor. For site 8 (532 kb), which was a highly efficient donor (85%) in the haploid assay, its use was decreased to 58% in the diploid (strain YWW210), with the remainder coming from the allelic locus . Previously we have shown that increasing homology from 1 kb to 2 or 3 kb on each side of the DSB had a very significant effect on the efficiency of ectopic DSB repair ,10, has In haploid yeast genome, the sixteen chromosomes adopt a preferential 3D conformation with centromeres clustered at spindle pole body and telomeres loosely associated at the nuclear envelope, the so-called Rabl configuration . Our preHere we show that chromosome conformation capture data also provide strong predictions for intrachromosomal DSB repair frequencies. Sites within approximately 100 kb of the DSB, which show very high levels of contact frequency, reach a plateau in their ability to recombine, but beyond that distance repair roughly diminishes with distance as the donors are placed closer to the centromere. However, at increasing distance from the centromere on the opposite chromosome arm, repair frequencies increase. This pattern is consistent with the Rabl configuration of chromosomes and, further, that a site 200 kb on the left arm is approximately as able to recombine as one on the right arm, despite being much further away as viewed along the chromosome itself. These results suggest that the left arm is not tethered away from the DSB site. The high level of accessibility of sites on the left arm that are distant from the centromere is not evident in the Hi-C data, which is swamped by interactions close to the site of interest .Our results demonstrate a strong constraint on the ability of centromere-proximal sequences to recombine with distant loci, although Agmon et al.  showed tMCM21 leads to the partial dislocation of centromeres from the SPB [CEN2 function by galactose-induced transcription proved to cause a modest but statistically significant increase in the ability of centromere-proximal sequences to recombine. We note that even with GAL::CEN disruption, 2/3 of cells are able to maintain proper chromosome segregation, so the effect of disrupting GAL::CEN would not necessarily be expected to have a larger consequence.Despite reports that deletion of  the SPB , this dePreviously we had shown that there could be a strong completion between an intrachromosomal donor and a competitor at an allelic site for spontaneous mitotic recombination ,6. Here,ho hmlΔ::ADE1 mataΔ::hisG hmrΔ::ADE1 leu2::KAN ade3::GAL::HO ade1 lys5 ura3-52 trp1 Chr2.625kb::leu2::HOcs). The specific locations of the donors inserted on Chr2 and the derivatives containing either mcm21Δ or a GAL::CEN replacement of CEN2 are presented in MCM21 was accomplished by transforming cells with a PCR-amplified NAT-marked deletion, copied from the yeast genome knockout collection [URA3::GAL::CEN3 sequence was amplified from pJH870 using PCR primers cen2::GAL-CEN3 p4 and cen2::GAL-CEN3 p5 to replace the CEN2 region. The sequences of the primers used in strain construction are presented in All strains were derived from YCSL305 (llection . The URAMAT) that carried the leu2::HOcs at 625 kb and an intrachromosomal ectopic LEU2 donor at different locations with another MAT-deleted strain carrying a URA3 selectable marker and a leu2-K donor at 625 kb . Mating was accomplished by transforming the second strain with a TRP1-marked MATα plasmid, which was not retained after mating.Diploid strains were constructed by mating a-like strains . The contact frequency between the DSB and the donor is determined by adding up all individual contacts around the donor location. The position of the HO cleavage site is given by a red arrow.(TIF)Click here for additional data file.S2 FigThe centromere is tethered to the spindle pole body and the telomeres are clustered at the nuclear envelope.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigCEN2 was replaced by a URA3 marked GAL-CEN3 fragment through homologous recombination. The conditional chromosome contains a GAL1 promoter adjacent to CEN3.Wild type (TIF)Click here for additional data file.S5 FigURA3-leu2-KpnI sequence was excluded in the PCR-based analysis by using short amplification times, as indicated by smaller arrowheads. (B) An example of donor usage measurement on agarose gel . The top band (1045 bp) represents leu2-KpnI repair product. The lower two bands (732 bp and 313 bp), digested by KpnI, represent LEU2 repair product. The intrachromosomal donor relative usage (%) was calculated as the intensity of the sum of lower two bands divided by the total intensities of the three bands. (C) Plot of intrachromosomal donor relative usage versus contact frequency (±10 kb around donor and ±25 kb around DSB). The intrachromosomal donor locations and their corresponding viabilities (%) in haploid strains are shown in blue. Error bars indicate one SD from three independent experiments.(A) Scheme to assess ectopic and allelic donor usage from a population of cells. The 3kb (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


In typical cases, normal pressure hydrocephalus (NPH) manifests itself with thetriad of gait disturbance, which begins first, followed by mental deteriorationand urinary incontinence associated with ventriculomegaly (on CT or MRI) andnormal cerebrospinal fluid (CSF) pressure. These cases present minor diagnosticdifficulties and are the most likely to improve after shunting. Problems arisewhen NPH shows atypical or incomplete clinical manifestations (25–50% of cases)or is mimicked by other diseases. In this scenario, other complementary testshave to be used, preferentially those that can best predict surgical outcome.Radionuclide cisternography, intracranial pressure monitoring (ICP) and lumbarinfusion tests can show CSF dynamics malfunction, but none are able to confirmwhether the patient will benefit from surgery. The CSF tap test (CSF-TT) is theonly procedure that can temporarily simulate the effect of definitive shunt.Since the one tap CSF-TT has low sensitivity, it cannot be used to excludepatients from surgery. In such cases, we have to resort to a repeated CSF-TT(RTT) or continuous lumbar external drainage (LED). The most reliable predictionwould be achieved if RTT or LED proved positive, in addition to the occurrenceof B-waves during more than 50% of ICP recording time. This review was based ona PubMed literature search from 1966 to date. It focuses on clinicalpresentation, neuroimaging, complementary prognostic tests, and differentialdiagnosis of NPH, particularly on the problem of selecting appropriatecandidates for shunt. Normal pressure hydrocephalus (NPH) is characterized by the triad of gait disturbance,progressive mental deterioration and urinary incontinence associated with enlargement ofthe ventricular system and normal cerebrospinal fluid (CSF) pressure. In NPH, the CSFpressure may be normal at one spinal tap, but episodes of increased CSF pressure dooccur in this syndrome. For this reason, other terms such as “intermittent pressurehydrocephalus”, “adult hydrocephalus syndrome”, and “adult symptomatic hydrocephalus”could be deemed more appropriate.2 have estimated theannual incidence of idiopathic NPH to be between 1.8/100,000 and 5.5/100,000inhabitants, with a prevalence of 22/100,000.NPH has been regarded as a rare cause of dementia with estimates ranging from 0% to 5% ofall demented patients. This variable rate depends on whether the diagnosis is basedsolely on clinical and neuroimaging data or on improvement after a shunt(shunt-responsive NPH). Epidemiological data on NPH incidence and prevalence are scarce,but recent surveys in Germany and Norwayhydro-, water+ cephalus, head), the excessive accumulation of CSF can be due toan obstruction into the brain ventricles  or to an impairment of CSF flow distally to the fourthventricle (“communicating”), almost always at the level of the basal cisterns, asmost often happens in NPH. In about 50% of patients with communicating NPH there isa known cause , such as meningitis, subarachnoidhemorrhage, or cranial trauma, while the other 50% of cases are idiopathic(INPH).3 SNPH may presentat any age, while INPH usually presents in the 6th or 7thdecade of life.4The CSF is normally produced in the choroid plexus within the lateral and fourthventricles, from which it passes through the foramina of Luschka and Magendie andenters the cisterna magna, then bathing the superior cerebral convexities in thesubarachnoid space, and finally being absorbed by the arachnoid granulations, mainlyin the superior sagittal sinus. In hydrocephalus  and,later on, urinary urgency or incontinence. These cases present minor diagnosticdifficulties and they are the most likely to improve after shunting.13The complete triad is seen in 50–75% of cases, with gait and cognitive disturbancesoccurring in 80–95%, and urinary incontinence in 50–75% of cases.14Nevertheless, the term “gait apraxia” has been considered inappropriate in INPH,since these patients may execute walking movements without difficulty when supportedor lying down, in spite of freezing their gait as soon as they try to startwalking.15 Therefore, thisgait disturbance has also been explained by a disconnection between the frontalcortex and the basal ganglia, uninhibited antigravity reflexes, and co-contractionof agonists and antagonists during walking.16 This explanationis in line with findings of reduced blood flow and oxygen metabolism in the basalganglia as well as in the frontal lobe, thalamus, hippocampus, and periventricularwhite matter.18In fully developed INPH, the cardinal sign is a broad-based, short-step, magneticgait with start hesitation and increased instability on turning, often with falls.In mild cases the gait may be merely ataxic and wide-based. Although INPH gaitshares the features of gait in Parkinson disease, progressive supranuclear palsy,and cerebellar ataxia, the nature of INPH gait is closest to apraxia of gait, whichmay be explained by gait ignition failure, probably caused by frontaldysfunction.19Additional motor signs are commonly found at examination: postural instability withtendency to fall backwards , spastic paraparesis,hyper-reflexia, paratonic rigidity, and primitive reflexes such as snouting,palmomental, sucking, grasping, and Babinski sign.21The cognitive impairment is typically of the “subcortical” type, withinattention, memory impairment, psychomotor slowing, apathy, and difficulty withexecutive functions. Unlike Alzheimer’s disease, “cortical” signs such asapraxia, agnosia and aphasia are rare; and delayed memory recall is severelyimpaired, while delayed recognition is less affected or even normal. INPHdementia syndrome is usually mild, and when it is the preceding or predominantclinical sign, particularly if severe, then the most probable diagnosis isAlzheimer’s disease and not NPH. The cognitive deficits are often accompanied byother mental symptoms which constitute an astheno-emotional syndrome,sometimes with impaired wakefulness and mental confusion.22 Detrusor overactivity mostlyunderlies urinary urgency/frequency and incontinence in INPH.23 As the disease progresses,even fecal incontinence may occur.Usually presents thirdly after gait and cognitive disorder, but at early stagesurinary frequency and urgency may be present. These symptoms are due tostretching of periventricular nerve fibers with subsequent loss of voluntarysupraspinal control (uninhibited) of bladder contractions.The diagnosis of NPH is usually based on the following criteria:(1) a history of gait disturbance, progressive mental deterioration, andurinary urgency or incontinence;(2) hydrocephalus, defined as Evans’ ratio  above 0.30 on computerized tomography (CT) or magneticresonance (MR) image; and(3) a mean CSF pressure below 18 or 20 cm of water.24 into“probable”, “possible” or “unlikely” on the basis of history, clinical findings,brain imaging data, and CSF opening pressure. Even if we adhere to these criteria,which can yield a 65% positive predictive value and an 82% negative predictivevalue,25 we cannotdefinitively rule out other dementing conditions. Therefore, the “gold standard” fordiagnosis remains clinical improvement after CSF shunting. In the Japaneseguidelines for INPH,26 surgicalindication should be determined by the clinical symptoms, MRI findings, and CSF taptest, with the diagnosis of “probable” INPH being based on improved gait after theCSF tap test or continuous CSF drainage, whereas the diagnosis of “definite” INPH isbased on improvement of symptoms after shunt surgery.Particularly for research purposes, INPH can be classified according to the 2005consensus guidelines20 INPH maysometimes mimic Parkinson’s disease,28 but INPH can be distinguished by its more broad-based, wideroutward rotation of the feet, diminished step height, relatively preserved armswing, and more erect trunk.29 Inelderly patients, other more common conditions than these can also explain gaitdifficulties  and urinary incontinence . Differentiation from Alzheimer’sdisease (AD) is rarely difficult, since in AD the dementia precedes and predominatesover the motor and urinary symptoms, which typically manifest later on, often yearsafterwards; while in AD “cortical” symptoms prevail such as aphasia, agnosia,apraxia, as well as amnesia with impaired recognition .Differential diagnostic problems may arise in some patients with atypical orincomplete clinical manifestations, as well as in patients with “subcortical”dementia ,which can mimic the clinical picture of INPH. Subcortical arterioscleroticencephalopathy is in fact much more common than INPH, and should be included in thedifferential diagnosis as the most probable cause of the “classical”triad.13The limitations and uncertainties associated with the clinical diagnosis of NPH havestimulated the search for more accurate methods and criteria for selection ofpatients for shunt surgery, which can benefit 25–80% of these patients but hascomplication rates (35–52%) that dissuade us from shunting every case of suspectedNPH.32The testing should cover the cognitive areas most impaired in INPH: attention,speed of information processing, working memory, executive functions, and memory.33 By thesame token, the presence of enlarged basal cisterns and Sylvian fissures and offocally dilated sulci tend to support rather than exclude the diagnosis ofshunt-responsive INPH.34 Innormal aging and degenerative processes such as Alzheimer’s and Pick’s disease,on the contrary, the thinning of the gyri and dilation of the sulci are moregeneralized, occurring to a similar degree in the affected brainregions.35In typical cases of INPH, computerized tomography (CT) and magnetic resonanceimaging (MRI) show ventricular enlargement out of proportion to cerebralatrophy, with associated ballooning of frontal horns, periventricularhyperintensities, thinning and elevation of the corpus callosum, and widening oftemporal horns without evidence of hippocampal atrophy. The presence ofperiventricular hyperintensity and associated subcortical lacunar infarctions inNPH does not predict poor surgical outcome and should not exclude patients fromshunting.36 Thepredictive value of radionuclide cisternography has been repeatedly questioned,such that Vanneste et al.37suggested it should no longer be performed as it will not reduce the diagnosticuncertainty remaining after clinical and CT evaluation. Even MRI (with CSFvoiding sign in the aqueduct) has been criticized for its questionableadditional predictive value, besides its limited availability and high costs.Some studies39 have shown correlation between the increase ofCSF flow in the aqueduct (“void sign”) and shunt results, while in otherstudies40 thecorrelation was low , with the same frequency ofvoid sign occurring in the groups of NPH patients and healthy controls. Morerecent studies41 have shownthat the measurement of peak CSF flow velocity at the level of the aqueduct,before and after lumbar CSF drainage, by using cine phase-contrast MRI, is asensitive method to support the diagnosis of NPH and to select patients who arelikely to benefit (or not) from shunt surgery.The results of CT, MRI or radionuclide cisternography can be inconclusive andinsufficient to establish a correct diagnosis and particularly to predict whichpatients will improve after shunt surgery. A “positive” radionuclidecisternography (with ventricular reflux and convexity block) is not specific forNPH and can be seen in other dementia disorders and even in healthysubjects.Single photon emission computerized tomography (SPECT) and positron emissiontomography (PET) can show reduction of cerebral blood flow and metabolism,mainly in frontobasal and anterior periventricular regions, but their diagnosticand prognostic value is not well established and they are not part of theroutine selection procedures for shunt surgery.10The continuous long-term monitoring of ICP commonly shows elevations of CSFpressure , which have long been regarded as highly predictiveof good postsurgical outcome, particularly when they occur during more than 50%of ICP recording time.43 More recent studies2 have found that, among theinfusion-derived parameters, CSF pressure pulsatility rather than resistance toCSF outflow, is linked to shunt response.The infusion of saline or artificial CSF into the ventricle or lumbarsubarachnoid space raises the resistance to CSF outflow with subsequent increaseof CSF pressure, which in NPH patients reaches higher levels than the plateauobserved in normal individuals. Most investigators agree that a resistance toCSF flow of 18 mm Hg/ml per minute or higher, predicts good surgical outcome inthese patients.44 In spite of the positivefindings pertaining to both methods, other authors have questioned theirpredictive value.48The problem with CSF infusion tests is that their reliability depends on hightechnical expertise, which may not be available in many neurosurgical services.Furthermore, as regards ICP monitoring, the interpretation of the recordedpressure oscillations has not followed standardized criteria, and B-wavefrequency, amplitude and morphology vary according to the different sleep stagesand have been recorded even in non-hydrocephalic persons.49 andFisher50 originallydescribed the beneficial (though transient) effect of CSF removal in patientswith hydrocephalic dementia, as well as the improvement of these patients aftershunt surgery. This method was later improved by Wikkelsö etal.52 and others54 byintroducing the quantitative testing of gait and cognitive functions before andafter the drainage of 40–50 ml lumbar CSF (CSF tap test or CSF-TT). Theseauthors found that CSF-TT can predict not only the outcome of surgery but alsothe degree of improvement.Adams et al.55 or the repeated lumbar CSF taptest (RTT).56 In thesestudies, RTT taps were performed on three consecutive days and at each tap aminimum of 30 to 40 ml of CSF was removed, while the LED was performedcontinuously for 3 to 5 days, with a minimum of 150 ml of CSF drained daily. Infact, with the introduction of these two procedures the (one tap) CSF-TTsensitivity (26–61%) and positive predictive value (73–100%) was improved upon,with LED showing a sensitivity of 50–100%, specificity of 60–100%, and positivepredictive value of 80–100%.43Although these procedures could improve the accuracy of CSF-TT, they can havehigher complication rates , besides requiring hospitalization, with higher costs and greatersuffering for the patient.Lately, however, this test has been criticized for its high rate of falsenegatives, which has led some authors to introduce the continuous lumbarexternal drainage (LED)58 The selection of subtests takes into account that theyshould (1) be sensitive to NPH motor manifestations and measure cognitivefunctions that usually improve after LP and shunting; (2) be suited even forilliterate subjects, on account of the high illiteracy rate of our population ofpatients (about 15%); and (3) be low cost and easy and rapid to administer byneurologists in ambulatory outpatients. In order to rule out other diseases, wecarried out a comprehensive investigation including CT or MRI, radionuclidecisternography, CSF analysis, laboratory tests, the Mini-Mental StateExamination,60 CAMDEX,61 and use of Hachinski IschemicScores for vascular dementia,62 ICD-1063and NINCDS-ADRDA criteria for Alzheimer’s disease,64 besides gait and memory tests.At the UNICAMP hospital, in Campinas, Brazil, we have attempted to improve thepredictive value of the one tap version of CSF-TT since 1988 by increasing theamount of CSF removed and the drainage duration.19, 50 ml of CSF was removed, andthere was good correlation between shunting results  and the gait and memory tests [thetests for visuo-motor speed (cylinders test) and visuo-construction showed nocorrelation].With the first version of our CSF-TT (used from 1988 to 1995)58 50 ml to 100 ml of CSF were removed, the testsfor visuo-motor speed and visuo-constructive skills were excluded, while thegait and memory tests were maintained and tests for postural reaction wereadded. Twenty-eight (78%) of the 36 NPH operated patients improved. Eleven (30%)had postsurgical complications . The results of thissecond version were correlated to those of shunt surgery(p<0.01), particularly as regards gait test, and its additionalpredictive value was 24% compared to the predictive value of clinical andtomographic data alone.In the second CSF-TT version (in use since 1996),thmonth follow-up. If the patient had not improved, we examined the shunt functionby percutaneously testing the proximal and distal patency of the shunt , as well as by analyzing changes in ventricular size on CTscan.The second version of our CSF-TT was performed in two consecutive days and at thesame time each day. In the morning of the first day (about 11 o’clock) thepatients underwent testing of memory and gait. On the second day, early in themorning (about eight o’clock), CSF was drained by lumbar puncture (LP) for threehours (until eleven o’clock) or until a maximum of 100 ml CSF was removed. Thebest score obtained by the patient on each subtest out of these two post-LPevaluations was considered their post-LP score. Postsurgical follow-upexamination  was performed at 3 and 6months, at 1 year, and yearly thereafter. CT was repeated at the 6gait test, the examiner recorded (with a chronometer) thetime taken by the patient to walk 18 meters as quickly as possible. The meanvalue of four attempts was the patient’s gait score. In the memory test, the subject was given ten trials tolearn a list of 10 unrelated words presented orally by the examiner . The patient’s score(maximum of 10) was the mean of summed total recall across the ten acquisitiontrials. At the post-LP memory testing, the words presented before LP werereplaced by another 10 words  in order to avoid any learning effect. Improvement in thegait test was defined as an increase of at least 5%, and in the memory test asan increase of at least 20%. CSF-TT was considered positive if the patient’sperformance improved in the two tests or only in the gait test.In the 65 The surgical procedure is usually aventriculoperitoneal shunt (VPS), placing the proximal catheter within theventricles through the right hemisphere posterior parietal region, and thedistal catheter into the peritoneal cavity. In cases with previous peritonitisor abdominal surgeries, a ventriculoatrial shunt is the alternative. The VPS mayuse a flow-limiting or a differential pressure valve (DPV), which can be of thelow, medium or high pressure types. The problem with DPV is that it can causeCSF overdrainage, depending on changes of body position from supine to upright.With the recent introduction of adjustable or programmable valves (see http://www.lifenph.com/), overdrainage and underdrainage can benoninvasively managed by use of a magnetic device.As yet the only efficacious treatment for NPH has been surgical diversion of CSF.Other alternatives such as acetazolamide (250 mg to 500 mg daily) and repeatedlumbar punctures can yield mild and transient relief of symptoms, and arejustified in patients with high surgical risks.66 but the authorscaution that their data must be confirmed by further studies.Another surgical procedure is the endoscopic third ventriculostomy (ETV), whosemain indication is in NPH associated with aqueductal stenosis. In an Italianmulticenter study of 110 patients operated using ETV for INPH, 76 subjects(69.1%) remained improved after 2 years follow-up, and the complication rate waslow,57Nevertheless, the positive predictive value of the clinical-tomographic data aloneis no greater than 65%,25 andtherefore must be improved upon by using complementary prognostic tests. As regardsradionuclide cisternography, ICP monitoring and lumbar infusion tests, we agree withother authors56 that none of these tests can confirm whether thepatient will benefit from surgery, although they are able to show malfunctioning ofCSF dynamics. CSF-TT is the only test that can temporarily simulate the effect of adefinitive shunt. The one tap CSF-TT has high positive predictive value , but has low sensitivity (26–61%). Therefore, when the testreturns a negative result it cannot be used to exclude patients from surgery. Insuch cases, we have to resort to repeated (RTT) or continuous (LED) CSF removal. Themost reliable prediction would be achieved if RTT or LED were to prove positive, inaddition to the occurrence of B-waves during more than 50% of ICP recordingtime.In NPH cases, the most important question is not whether the patient has NPH or not,but whether they will benefit from shunt surgery. To tackle this question we have tofirst base our decision on clinical and tomographic data. To this end, the bestclinical predictors of good surgical outcome are short disease duration, highcognitive scores (mild or no dementia), gait disturbance preceding mentaldeterioration, dilation of temporal horns, and small sulci.


The effects of acacetin on these KCa channels were determined with a whole-cell patch voltage-clamp technique. The results showed that acacetin inhibited the three subtype SKCa channel currents in concentration-dependent manner with IC50 of 12.4 μM for SKCa1, 10.8 μM for SKCa2, and 11.6 μM for SKCa3. Site-directed mutagenesis of SKCa3 channels generated the mutants H490N, S512T, H521N, and A537V. Acacetin inhibited the mutants with IC50 of 118.5 μM for H490N, 275.2 μM for S512T, 15.3 μM for H521N, and 10.6 μM for A537V, suggesting that acacetin interacts with the P-loop helix of SKCa3 channel. However, acacetin at 3–10 μM did not decrease, but induced a slight increase of BKCa (+70 mV) by 8% at 30 μM. These results demonstrate the novel information that acacetin remarkably inhibits SKCa channels, but not IKCa or BKCa channels, which suggests that blockade of SKCa by acacetin likely contributes to its anti-AF property previously observed in experimental AF.The natural flavone acacetin inhibits several voltage-gated potassium currents in atrial myocytes, and has anti-atrial fibrillation (AF) effect in experimental AF models. The present study investigates whether acacetin inhibits the Ca They are divided into three subfamilies: big (or large) conductance , intermediate conductance , and small conductance channels   and also in some non-excitable tissues    . The thrs, etc.) . In neurrization  and regurization . In the rization , endotherization , and alsrization . Resultsion (AF) , tumor cion (AF) , and oveion (AF) . A recent hearts ; however anti-AF . The dev anti-AF .Saussurea involucrata) prolongs the atrial effective refractory period and prevents or terminates the experimentally induced AF in anesthetized dogs without increasing the QT interval (IKur (ultra-rapidly activating delayed rectifier potassium current) or Kv1.5, IK.ACh (acetylcholine-activated potassium current), and Ito (transient outward potassium current)  , 2013a. Ca2 (KCNN2), pCDNA3/hSKCa3 (KCNN3), and pCDNA3/hIKCa (KCNN4) plasmids obtained as generous gifts from Dr. Nicole Schmitt  were transfected into HEK 293 cells  using Lipofectamine 2000TM. The HEK 293 cell lines stably expressing the SKCa1, SKCa2, and SKCa3 channels were established as described previously (Ca (KCNMA1) was also maintained in the same culture conditions. Cells were seeded on glass cover slips for electrophysiological recording.The pCDNA3/rSKeviously . The celeviously  stably eCa3 mutants were synthesized by the Genome Research Center, the University of Hong Kong (Hong Kong), and the mutants were generated using a QuikChange kit . After confirmed by DNA sequencing, the mutants were transiently expressed in HEK 293 cells in a 35 mm culture dish using Lipofectamine 2000TM (10 μl) with SKCa3 mutant cDNA plasmid (4 μg).The primers of SK2, 1.8 CaCl2, 10 HEPES, and 10 glucose (pH was adjusted to 7.3 with NaOH). The pipette solution contained (in mM): 20 KCl, 110 potassium aspartate, 1.0 MgCl2, 10 HEPES, 5 EGTA, 0.1 GTP, 5 sodium phosphocreatine, and 5 Mg-ATP, pH adjusted to 7.2 with KOH  was included.Acacetin  was synthesized in the laboratory as described previously in the US patent . The stowith KOH , in whicThe HEK 293 cells on a coverslip were placed into a cell chamber mounted on the stage of an inverted microscope , and superfused with Tyrode’s solution (2 ml/min). Whole-cell current was recorded with a patch clamp amplifier  as described previously . Brieflyt-tests were used as appropriate to evaluate the differences between two group means, and ANOVA was used for multiple groups. A value of P < 0.05 was considered to indicate statistical significance.The data were expressed as means ± SEM. Unpaired Student’s Ca1 current was determined in HEK 293 cells stably expressing human KCNN1. Figure 1A displays the voltage-dependent SKCa1 current recorded with 200-ms voltage steps between -70 and +80 mV from a holding potential of -80 mV in a representative cell. The current was inhibited by 10 μM acacetin (10 min exposure), and the inhibition was partially reversed by washout. Figure 1B displays the current–voltage (I–V) relationships of SKCa1 determined in another typical experiment with a voltage ramp in the absence and presence of acacetin. The I–V relationships of SKCa1 current showed a reversal potential around -70 mV and an inward rectification property, typical SKCa current as described previously  by acacetin. The concentration–response curve was fitted to a Hill equation to obtain IC50 (the concentration of inhibiting 50% current) value. The IC50 of acacetin for inhibiting SKCa1 at +80 mV was 12.4 μM .The effect of acacetin on SKeviously . The curCa2 current was determined in HEK 293 cell line expressing rat KCNN2. Figure 2A shows the voltage-dependent SKCa2 current in a typical experiment with the voltage protocol as shown in the inset. The current was significantly decreased by 10 μM acacetin (10 min exposure) at all testing potentials, and the inhibition was partially reversed by washout. I–V relationships of SKCa2 current determined by a ramp voltage protocol also showed an inward rectification. Inward and outward components of the current were decreased by 10 μM acacetin, and the inhibition was partially recovered on drug washout . Figure 2C illustrates the concentration–response relationship of acacetin for inhibiting SKCa2 current (at +80 mV). The curve was fitted to a Hill equation. The IC50 of acacetin for inhibiting SKCa2 current was 10.8 μM .The effect of acacetin on SKCa3 was determined in HEK 293 cell line expressing human KCNN3 gene. The voltage-dependent step SKCa3 current was determined with the voltage protocol as shown in the inset in a typical experiment . SKCa3 current at all test potentials was inhibited by 10 μM acacetin with 10 min incubation, and the inhibition partially recovered on washout for 10 min. I–V relationships of SKCa3 current was determined with a ramp voltage protocol in another cell  before and after application of acacetin. The current also displays an inward rectification and was reversibly decreased by 10 μM acacetin. The concentration–response curve of acacetin for inhibiting SKCa 3 current was fitted to a Hill equation . The IC50 of acacetin for inhibiting SKCa3 was 11.6 μM (with a Hill coefficient of 0.8).The inhibitory effect of acacetin on SKCa channels was investigated using SKCa3 mutants, H490N, S512T, H521N, and A537V in P-loop helix and S6, generated by site-directed mutagenesis as described previously  SKCa current and mutant currents recorded in representative cells expressing WT SKCa3 or the mutant H490N, S512T, or H521N with a voltage ramp protocol before (control) and after 10 μM acacetin. The inhibitory effect of acacetin for the mutant H490N and S512T currents was clearly reduced, compared with WT SKCa3 current. Figure 4B illustrates the percent values of current inhibition by 10 μM acacetin for WT SKCa3, and the mutants H490N, S512T, H521N, and A537V currents at +80 mV. Acacetin at 10 μM decreased the current by 45.7 ± 4.1% for WT SKCa1 current (n = 11), 21.9 ± 4.5% for H490N current , 17.9 ± 3.9% for S512T current , 48.8 ± 3.5% for H521N current , and 40.8 ± 7.6% for A537V current , respectively.The potential molecular determinant of acacetin for inhibiting SKeviously ,b. FigurFigure 5A displays the concentration–response relationships of acacetin for inhibiting WT SKCa3 current, H490N current, S512T current, H521N current, and A537V current at +80 mV. The concentration-dependent inhibition curves were fitted to a Hill equation. The IC50 of acacetin was 11.6 μM for WT SKCa3 current, 118.5 μM for H490N current, 275.2 μM for S512T current, 15.3 μM for H521N current, and 10.6 μM for A537V current, respectively. The efficacy of acacetin for inhibiting H490N current and S512T current was dramatically reduced, which suggests that acacetin blocks SKCa3 channel by interacting with H490 and S512 in the P-loop helix of the channel .Ca was determined in HEK 293 cell line expressing human KCNN4. The voltage-dependent IKCa current  was recorded with the step voltages as shown in the inset in a typical experiment before and after application of acacetin. Acacetin (10 and 30 μM) slightly decreased the current, and the inhibition was partially recovered on washout. Similar results were observed for the I–V relationships of the current recorded with a voltage ramp in another representative cell . IKCa shows a linear I–V relationship, similar to those previously recorded in HEK 293 cell line expressing IKCa  decreased IKCa (+70 mV) to 95.0 ± 4.5%  and 89.3 ± 5.5% of control , respectively. These results suggest that acacetin has a slight inhibition of IKCa current.The potential effect of acacetin on IKing IKCa . Figure Ca current was examined in HEK 293 cell line expressing human KCNMA1 gene. Voltage-dependent BKCa current was recorded with the step voltage protocol as shown in the inset in a representative cell before and after application of acacetin . Acacetin had no effect on BKCa at 3 and 10 μM, whereas it slightly increased the current at 30 μM. I–V relationships  of BKCa current determined with a voltage ramp showed a similar response to acacetin. Acacetin did not affect the current at 10 μM, but slightly increased the outward component of BKCa current at 30 μM. The BKCa inhibitor paxilline (1 μM) almost fully suppressed the current. The percent values of BKCa current at +70 mV illustrated in Figure 7C show that no significant effect of acacetin was observed at 3 and 10 μM, whereas 30 μM acacetin increased the current to 108.1 ± 5.7% of control . These results suggest that acacetin may stimulate BKCa channel at high concentration of 30 μM.The effect of acacetin on BKCa channel subtypes: SKCa1, SKCa2, and SKCa3, stably expressed in HEK 293 cells with similar efficacy. The IC50 values of acacetin for inhibiting SKCa1, SKCa2, and SKCa3 are 12.4, 10.8, and 11.6 μM, respectively. Point mutagenesis of SKCa3 channel reveals that acacetin mainly interacts with H490 and S512 in the P-loop helix of the channel. However, acacetin at a high concentration of 30 μM induces only a small decrease in IKCa channel and a small increase in BKCa channel stably expressed in HEK 293 cells. The very limited effect of acacetin on IKCa channel is similar to that reported previously for other SKCa channel blockers , and are highly conserved among mammalian species, and are identified in many organisms from Drosophila to humans -4-(pyridin-2-yl)thiazol-2-amine (ICA) and apamin, have been used for anti-AF studies (in vivo rat model (Ca channel blockers can be considered as promising anti-AF drugs. Moreover, recent studies showed that acute myocardial infarction might activate SKCa channels, and apamin, UCL-1684 or ICA reduced ventricular burden arrhythmia by prolonging ventricular action potential duration and effective refractory period in rats with acute myocardial infarction (However, the results from other groups demonstrated that blockade of SKnt of AF . Several studies , 2015. I studies . Injectiat model . UCL1684at model . Interesat model  and horsat model , intravefarction .Ca1, SKCa2, and SKCa3 channels in HEK 293 cell line expressing the corresponding genes. The blockade of SKCa channels by acacetin likely also contributes to the anti-AF effect observed in canine models in addition to blocking IKur/Kv1.5, Ito/Kv4.3, and IK.ACh (Ca blockers, is effective in improving learning and memory (Ca blocking effect of acacetin is related to its anti-cancer effect (In this study, we demonstrated that acacetin inhibited SKd IK.ACh , 2013a. d memory  remains r effect .Ca3 channel. The pore blocking of SKCa3 by acacetin is applicable to SKCa1 and SKCa2, because SKCa1, SKCa2, and SKCa3 share the same sequence in the range of pore helix1. This differs from the molecular determinants of acacetin for blocking Kv1.5 or Kv4.3 channel. On the other hand, the acacetin blockade of SKCa3 channel is different from the organic SKCa blocker NS8593 and the archetypical peptide SKCa blocker apamin. NS8593 interacts with S507 of P-loop helix and A532 of S6 domain (Ca blockers differ, it is important to develop these potential blockers into feasible drug candidates for future clinical application. A water soluble prodrug of acacetin has been developed, which can be intravenously administered for future clinical application (In our previous reports, we demonstrated that acacetin blocked Kv1.5 channel by binding to both its resting and open states by interacting with V505, I508, and V512 within the S6 domain , and inh6 domain , while a6 domain . While tlication .Ca current at concentration of 30 μM. Although the concentration for activating BKCa channel is greater than those of blocking IKur/Kv1.5, IK.ACh, Ito/Kv4.3, and also SKCa channels; this effect may account in part for the vascular dilation reported in a previous study (Acacetin showed increased BKus study .Ca1, SKCa2, or SKCa3 channels and lack of data from native cardiomyocytes. However, this does not affect the conclusion that acacetin blocks SKCa channels. Future effort is required to obtain the data for the effect of acacetin on SKCa current in native cardiomyocytes from an animal species whose heart has no or less expression of IKur/Kv1.5 and Ito/Kv4.3, because acacetin also inhibits these currents in native human atrial myocytes (A limitation of the present study was that all the experiments were conducted only in HEK 293 line expressing SKmyocytes .Ca channel blocker and inhibits three subtypes of the SKCa channels stably expressed in HEK 293 cells. The SKCa channel blocking effect may be involved in its anti-AF property previously observed in experimentally induced AF in dogs.Collectively, the present study demonstrates for the first time that acacetin is a SKK-HC, M-WJ, G-SX, YW, and G-RL conceived and designed the project. K-HC, HL, and H-YS conducted the experiments. K-HC, HL, H-YS, and G-RL analyzed the data. K-HC and G-RL prepared the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MS) exploiting multistage fragmentation (MSn). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol  in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid  were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophages.Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography–mass spectrometry (LC–The online version of this article (doi:10.1007/s12035-016-0281-9) contains supplementary material, which is available to authorized users. The size of the final C18 column used for CSF was 50 mg while that used for plasma was 200 mg. Exact details are given in Online Resource Non-esterified sterols in plasma were assayed by LC–t tests were performed against the CP group, *P < 0.05; **P < 0.01. Concentrations given in the text are mean ± standard deviation (SD). The boxplots in Figs. P values for the significance of the correlations are listed in the Tables in Online Resources P values that are below 0.05/((21*20)/2) = 0.000238 for CSF and below 0.05/((22*21)/2) = 0.000216 for plasma are highlighted in the Online Resource Tables, these are significant after a Bonferronni correction at 5 %.An ANOVA test was run for each sterol on linear and logarithmic scales. The log scale used a transformation log2+1) to avoid issues with zero and small numbers. Uni-variant  to avoidP values) against log2 fold change between RRMS and CP shown in Figs. t test was made using the modification by Welch that accommodates for different variances in the two groups.The volcano plots of statistical significance (3 (25-D3), cholestenoic and cholenoic acids in plasma. Table P < 0.01). We also see a reduction in the concentration of (25R)26-hydroxycholesterol ((25R)26-HC, 22.63 ± 5.03 ng/mL cf. 27.97 ± 7.89 ng/mL, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid  in RRMS patients. It is important to note that 25-HC is generated by macrophages [CH25H or reduced activity of the enzyme and that RRMS patients have a reduced capacity to produce 25-HC.Shown in Fig. rophages , while trophages . The levP < 0.01) [P < 0.05), as it is in patients diagnosed with PBI , compared to CP (1.54 ± 0.42 ng/mL).In agreement with some, but not all, earlier studies, the level of 24S-hydroxycholesterol (24S-HC) is reduced in plasma from the group of patients with either AD or PD compared to CP  , 26. TheP < 0.05) compared to CP  compared to CP (17.40 ± 4.63 ng/mL), as is the level of its precursor 3β-hydroxycholest-5-enoic acid . The intermediary metabolite between these two acids, 3β,7α-dihydroxycholest-5-enoic acid , is also elevated (3.71 ± 4.07 ng/mL cf. 2.12 ± 1.65 ng/mL) but not to significance. MS is a demyelinating disease and it likely that cholesterol is released in the CNS is metabolised through (25R)26-HC and 3β-HCA to 7αH,3O-CA, although the actual concentration of non-esterified cholesterol in CSF does not differ from controls (1.24 ± 0.33 μg/mL). 3β,7β-Dihydroxycholest-5-enoic acid  which may be a metabolic product of 3β-HCA, 3β,7α-diHCA or 7-oxocholesterol (7O-C) is also elevated in CSF from RRMS patients .It is generally considered that in healthy individuals CSF levels of 25R)26-HC correlate with levels of this oxysterol in the circulation . However5R26-HC cP < 0.01). The concentration of 7α,(25R)26-dihydroxycholest-4-en-3-one 26-diHCO, 0.03 ± 0.01 ng/mL, cf. 0.02 ± 0.01 ng/mL, P < 0.01) is increased as is its downstream metabolite 7αH,3O-CA, although not quite to significance . Other neurodegenerative diseases including AD and PD are also believed to result in increased cholesterol release in brain as neurons die [P < 0.05), 7αH,3O-CA  and 3β,7β-diHCA  are elevated in this disease group.The CSF from the ALS group shows differences in cholesterol metabolite concentrations compared to controls and also in the concentration of cholesterol itself  in CSF while those diagnosed with SA/UA show increased concentrations of (25R)26-HC , 7α,(25R)26-dihydroxycholesterol 26-diHC, 0.01 ± 0.01 ng/mL cf. 0.00 ± 0.00 ng/mL, P < 0.05) and 7α,(25R)26-diHCO .Patients diagnosed with PBI show an increase in concentration of 7α,(25R)26-diHCO  did any of the disease states show any statistical differences in the level of 25-HC in CSF; this is also true for the 25-HC metabolite 7α,25-diHCO  .The limit of quantification of our analytical method in CSF for most analytes is 0.01 ng/mL ; however, for 25-HC, it is somewhat higher at 0.03 ng/mL, although detection can be made at 0.01 ng/mL. Only in the patient group of SA/UA  in plasma from patients with RRMS, falling from 1.54 ± 0.42 ng/mL in CP to 1.19 ± 0.26 ng/mL in RRMS plasma , in plasma from patients with PBI and AD or PD. Non-esterified oxysterols are present in CSF at pg/mL levels [P < 0.05). The plasma data for RRMS patients, indicating a reduced capacity of macrophages to synthesise 25-HC, is in-line with the recent study by Reboldi et al. which shows that in mouse macrophages 25-HC is a mediator of negative feedback towards interleukin 1 (IL-1) family cytokine production and inflammasome activity, through binding to INSIG (insulin-induced gene) and antagonising the sterol response element-binding protein-2 (SREBP-2) driven mevalonate pathway, thereby, reducing Il1b transcription and repressing IL-1-activating inflammasomes [Ch25h knockout mice show exacerbated experimental autoimmune encephalomyelitis (EAE), an IL-17-driven inflammatory disease model of MS, and increased susceptibility to septic shock. Ch25h-deficient macrophages produce more proinflammatory IL-17A+ T cells following lipopolysaccharide (LPS) stimulation and cytokines that cooperate with transforming growth factor-β (TGFβ) to induce Th17 cells include IL-1β. Notably, after LPS stimulation Ch25h-deficient macrophages overproduce Il1b [.In the current study, we have measured the levels of non-esterified sterols, oxysterols, 25-Dd P < 0.0 in plasmL levels , and in mmasomes . Thus, ruce Il1b .Il1b transcription is not known. Reboldi et al. suggested that this may be through reducing the cellular content of sterols generated via the mevalonate pathway [Ch25h gene significantly attenuates EAE by limiting trafficking of pathogenic CD4+ T lymphocyte to the CNS [P < 0.05) in RRMS plasma  and of its metabolites 3β-HCA (P < 0.05) and 7αH,3O-CA (P < 0.01) in CSF of RRMS patients , formed in the peroxisome as a thioester is also found to be elevated in RRMS CSF  but not its downstream metabolites. Interestingly, both 3β-HCA and 3β,7β-diHCA have been shown to be neurotoxic [In contrast to plasma, there is a significant elevation in 25R)26-HC (5R26-HC (urotoxic .P < 0.05) and 7αH,3O–CA (P < 0.01) of the “acidic pathway” are elevated in the AD and PD group, while in the ALS group 7α,(25R)26-diHCO (P < 0.01) is increased  as are (25R)26-HC (P < 0.05), 7α,(25R)26-diHC (P = 0.05) and 7α,(25R)26-diHCO (P = 0.01). When plasma was analysed from these patients, none of the metabolites showed statistical differences compared to controls except 7α,(25R)26-diHCO, which unlike the situation in CSF was reduced in concentration in plasma (P < 0.05). Little is known of the regulation of enzymes of the acidic pathway of bile acid biosynthesis [CH25H in macrophages of the inflamed CNS.Patients with inflammatory CNS disease include those diagnosed with SA/UA and those diagnosed with PBI. The patients with SA/UA show elevated levels of 25-HC in CSF  excluding and (B) including the inflammatory CNS disease group). As might be expected (25R)26-HC is highly correlated with 7α,(25R)26-diHC and 3β-HCA corresponding to the first metabolites of the two branches of the “acidic pathway” of bile acid biosynthesis . A fascinating feature of the pair-wise correlations is the tight cluster indicated by the “red box” incorporating 7αH,3O-CA, two dihydroxy-3-oxocholest-4-enoic acid isomers  and 7αH,3,24-diO-CA. We do not have an authentic standard for the latter compound whose identification is based on exact mass, retention time and multistage fragmentation (MSn) spectrum and the location of the hydroxy groups on the two dihydroxy-3-oxocholest-4-enoic acid isomers is unclear. In both isomers, one hydroxy group is located at the 7α position while the second hydroxy group is most likely on the side chain. The pair-wise correlations between successive members of the “acidic pathway” provide strong evidence for the activity of this pathway in the CNS. The absence of correlations with 7α-hydroxycholesterol (7α-HC) confirms that the “neutral pathway” of bile acid biosynthesis is not operative in the CNS. The rate limiting enzyme of this pathway, CYP7A1, is liver specific [4-BA, 7αH,3,24-diO-CA; 7αH,3O-CA; 7α,x-diH,3O-CA; and 7α,y-diH,3O-CA cluster together, as do 3β-HCA, (25R)26-HC, 3β,7β-diHCA, 3β,7α-diHCA and 7α,(25R)26-diHC. The clustering patterns evident in the dendrogram in Online Resource It is of interest to study pair-wise correlations between CSF concentrations and specific sterol analytes which are arranged in metabolic order in Fig. sis Fig. . (25R)26specific . The corsis Fig. -BA. A faP values for the significance of correlation). As expected from the pathway outlined in Fig. 4-BA, while (25R)26-HC correlates with 3β-HCA and 7αH,3O-CA. Unsurprisingly, 3β-HCA correlates strongly with 3β,7α-diHCA, while 7αH,3O-CA correlates strongly with 7αH,3O-Δ4-BA. As evident from the dendrogram in the Fig. in Online Resources While the metabolite correlations in CSF are dominated by the “acidic pathway” of bile acid biosynthesis, the correlations in plasma are reflective of both the “acidic pathway” and of the “neutral pathway” starting with 7α-HC followed by 7α-hydroxycholest-4-en-3-one ESM 2(PPTX 109 kb)ESM 3(DOC 109 kb)ESM 4(PPTX 597 kb)ESM 5(PPTX 550 kb)ESM 6(XLSX 21 kb)ESM 7(XLSX 23 kb)ESM 8(XLSX 31 kb)ESM 9(XLSX 32 kb)ESM 10(PDF 84 kb)ESM 11(PDF 64 kb)ESM 12(PDF 54 kb)


Enterococcus (VRE) surgical site and bloodstream infection, a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome sequencing identified that donor and recipient VRE isolates were highly similar when compared to time-matched hospital isolates. Comparison of de novo assembled isolate genomes was highly suggestive of transplant transmission rather than hospital-acquired transmission and also identified subtle internal rearrangements between donor and recipient missed by other genomic approaches. Given the improved resolution, whole-genome assembly of pathogen genomes is likely to become an essential tool for investigation of potential organ transplant transmissions.In a liver transplant recipient with vancomycin-resistant  Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) could infect the donor and recipient independently, and it may be difficult to distinguish donor acquisition from hospital acquisition. This is particularly true when infection is not immediately recognized in the recipient. Automated de novo construction of high-quality bacterial genomes using long-read whole genome sequencing (WGS) is a powerful tool that can aid in donor transmission epidemiology[Donor-derived bacterial infections are a recognized early complication of solid organ transplantation (SOT). The curdemiology–5. In oudemiology. Here, wA 51-year-old woman was hospitalized for an exacerbation of systemic lupus erythematosus. The hospital course was complicated by respiratory failure from diffuse alveolar hemorrhage, acute kidney injury requiring renal replacement therapy, and ultimately irreversible central nervous system damage. She was pronounced brain dead and her liver was recovered for transplant. All cultures were negative at this time.A 65-year-old man with hepatitis C cirrhosis and hepatocellular carcinoma was admitted from home and underwent deceased donor liver transplantation. There were no significant intra-operative complications except for expected blood product requirements. The post-operative course was complicated by the development of a complex fluid collection that gradually increased in size; ongoing need for blood transfusion; and acute kidney injury requiring continuous veno-venous hemofiltration (CVVH). Renal function improved and CVVH was discontinued on post-operative day 6. Fever developed immediately after discontinuation of CVVH. Blood cultures obtained on post-operative days 7–9 grew VRE. Linezolid was started on post-operative day 10. The patient was taken to the operating room on post-operative day 13, and one liter of hematoma was evacuated. Cultures of the hematoma grew VRE. Follow-up blood cultures were negative and fever resolved. Linezolid was discontinued thirteen days after evacuation of the hematoma.Candida glabrata, respectively. The final donor culture results were communicated to our hospital on post-transplant day 15. Caspofungin was started in the recipient at this time and continued for 21 days; however, none of the recipient cultures grew C. glabrata. The recipient was eventually discharged home after a 49-day hospitalization. He has not required any further hospitalizations and is doing well approximately five years later with excellent graft function.Donor blood cultures at the donor hospital eventually grew Gram-positive cocci in pairs and chains and yeast on post-transplant days 1 and 2, respectively. These culture results were finalized on post-transplant day 6 as VRE and The donor blood VRE isolate (VRE Donor) was obtained from the donor hospital by LiveOnNY (formerly New York Organ Donor Network) for comparison with the recipient blood isolate (VRE Recipient). Ten additional VRE blood culture isolate strains in the preceding eight weeks from the recipient hospital were retrieved for comparison.s.VRE Donor, VRE Recipient and three control  isolates were selected for pulse-field gel electrophoresis (PFGE). Agarose plugs containing genomic DNA were digested with SmaI, and pulsed-field gel electrophoresis (PFGE) was performed using a previously described method[For whole genome sequencing, single bacterial colonies were grown overnight in Luria-Bertani (LB) broth and high molecular weight DNA extraction was performed as previously described. DNA libhttps://github.com/PacificBiosciences/SMRT-Analysis/wiki/SMRT-Analysis-Software-Installation-v2.2.0). Assemblies were circularized using a custom pipeline employing Nucmer[de novo assembly on the PacBio RS II. The resulting assemblies were utilized for multilocus sequence typing (MLST) and whole-genome comparative analyses. Table A in PacBio sequences were assembled using HGAP3 . Next, amplicon sequence libraries were prepared using the end repair, A-tailing, and adaptor ligation NEBNext DNA library prep modules for Illumina from New England Biolabs according to the manufacturer’s protocol. Following final size-selection with 1x volume Ampure XP beads, and secondary PCR (8 cycles) to introduce barcoded primers, multiplexed libraries were sequenced on the Illumina HiSeq 2500 platform in a single-end 100nt run format.Sequence errors in the assemblies were corrected by mapping Illumina reads to the assembled contigs with BWA mem. VariantTo help resolve potentially missed plasmids, Illumina reads were also assembled using SPAdes version http://pubmlst.org, a public database of MLST types, which includes data for Enterococcus faecium and by using core genome MLST (cgMLST) as part of Ridom SeqSphere+ (http://www.ridom.com/seqsphere/cgmlst/). The pubMLST database returned an MLST type for each genome and types for each allele in seven genes: adk, atpA, ddl, gdh, gyd, pstS and purK; these types were confirmed by SeqSphere for all strains except VRE1, for which no MLST type was given  using default settings. InitialDe novo assemblies were also to examined for potential structural differences via Nucmer alignment (MUMmer version 3.23)[Fig 1B.Phylogenetic analyses was performed using HarvestTools v1.1.2. First Pion 3.23). Lastly,ion 3.23) was usedFig 2). The control strains were considerably different, both from each other and from VRE Donor and VRE Recipient, showing greater than 6 band changes  following Tenover et al. criteria[VRE Donor and VRE Recipient had almost identical SmaI restriction enzyme PFGE patterns, differing only in the presence of a single brighter band . The resulting phylogeny identified a Donor-Recipient specific subclade within the ST736 MLST subtype. Pairwise whole genome comparison was then performed between VRE Donor and VRE Recipient which were assembled to completion. While the two isolates were nearly identical at the SNV level, containing a single SNV within the main chromosome, they were not completely structurally identical: Four intervals exhibited inserted or deleted sequence between the isolates  within the VRE Donor. The corresponding chromosomal location was interrogated across all 12 isolates by isolating a 40kb flanking interval that encodes 29 genes including 10 mobile elements. Fig 1B and Table F in Fig 1A); however, local variation (such as insertions and deletions and SNVs) still existed within each group. Closest strain pairs on the SNV phylogeny did not necessarily correspond to the closest structural matches for this region (e.g. VRE10 and VRE11), and in some cases large-scale rearrangements had occurred between two proximal isolates (e.g. VRE11 and VRE8). Together, this suggests that this region is likely to mutate or undergo rearrangement over a short period (as in the case of VRE Donor and VRE Recipient).The E in 1251 and ISEfVRE surgical site and bloodstream infection in a hospitalized liver transplant recipient meets the definition of a hospital-acquired infection, but earde novo assembly was able to clarify the unique structural differences between the donor and recipient isolate. Interestingly, this and related insertion elements have previously been associated with increased antibiotic resistance in the context of the vanA gene cluster[E. faecium[E. faecium have been shown in simulation to render MLST phylogenetic inference inaccurate[Our combination of short and long read genomic approaches highlights the increased specificity of WGS for resolving both SNV and structural differences between similar isolates. All genomic analyses revealed a strong donor and recipient grouping; however, multiple isolates had MLST types consistent with the donor and recipient. And, while PFGE did show a tighter grouping between donor and recipient, only the full e cluster. This ty. faecium. The hignaccurate. In factnaccurate, but sucIn summary, WGS provided substantial evidence that VRE infection in our liver transplant recipient was not hospital acquired as would have typically been considered but rather transmitted from the deceased donor. We expect that WGS and assembly of pathogen genomes will be increasingly important not only for understanding pathogen biology and evolution but also become a routine and essential tool for investigation of potential organ transplant transmissions in many settings. Our case also highlights the importance of communicating deceased donor culture information to transplant recipient centers so that potential preventive or preemptive strategies can be implemented as early as possible.S1 File(DOCX)Click here for additional data file.


The testis has been identified as the organ in which a large number of tissue-enriched genes are present. However, a large portion of transcripts related to each stage or cell type in the testis still remains unknown. In this study, databases combined with confirmatory measurements were used to investigate testis-enriched genes, localization in the testis, developmental regulation, gene expression profiles of testicular disease, and signaling pathways. Our comparative analysis of GEO DataSets showed that 24 genes are predominantly expressed in testis. Cellular locations of 15 testis-enriched proteins in human testis have been identified and most of them were located in spermatocytes and round spermatids. Real-time PCR revealed that expressions of these 15 genes are significantly increased during testis development. Also, an analysis of GEO DataSets indicated that expressions of these 15 genes were significantly decreased in teratozoospermic patients and polyubiquitin knockout mice, suggesting their involvement in normal testis development. Pathway analysis revealed that most of those 15 genes are implicated in various sperm-related cell processes and disease conditions. This approach provides effective strategies for discovering novel testis-enriched genes and their expression patterns, paving the way for future characterization of their functions regarding infertility and providing new biomarkers for specific stages of spematogenesis. The testis has been identified by RNA sequencing as the organ in which the largest number of tissue-enriched genes is expressed among various organs. It has been estimated that expressions of more than 1000 genes are enriched in the testis . Tissue-www.ncbi.nlm.nih.gov/geo) followed by protein profiling using immunohistochemical data from the Human Protein Atlas portal (www.proteinatlas.org) is a useful tool for discovering highly expressed genes in each stage of spermatogenesis in the testis. Furthermore, gene expression profiles under various developmental, disease, and knockout conditions produced in GEO microarray datasets offer a platform for functional genomic studies of spermatogenesis stage-specific gene expression.Investigation of gene expression and function during spermatogenesis has been hampered by a lack of immortalized cell lines for each stage . AlternaUsing these sources combined with confirmatory gene expression measurements and pathway analysis, in this study, protein localization and signaling pathways of 15 testis-enriched genes were analyzed. The objectives of this study were to identify novel testis-enriched genes using gene expression profiles and analyze protein localization, developmental regulation and biological implications of testis-enriched genes in humans and mice. The current approach provides an effective strategy for discovering novel testis-enriched genes and their unique stage-specific expression, paving the way for future studies of normal development of the testis and associated diseases.www.ncbi.nlm.nih.gov/geo). For analyzing tissue distribution pattern of gene expression in 12 male mouse tissues and 10 man tissues, GDS3142 for mice and GDS596 for humans were downloaded and sorted  of the GEO repository in the National Center for Biotechnology Information (NCBI) archives  at The Ohio State University. Mice were raised under ad libitum feeding conditions in a mice housing facility at The Ohio State University. Mice were euthanized by carbon dioxide inhalation followed by cervical dislocation. For isolation of total RNAs, testis, muscle, liver, brain, lung, kidney, adipose tissue, thymus, spleen, and small intestine were collected from 3-month-old FVB mice (n = 3) using Trizol reagent  . Total RTo measure the quantity of RNA, a Nanodrop spectrophotometer  was used. The RNA samples were stored at -80°C until use. Approximately 1 μg of RNA was reverse-transcribed in a 20 μL total reaction to cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). The thermal cycle of the reverse transcription was 65°C for 5 min, 37°C for 52 min, and 70°C for 15 min. Exactly 1 μL of cDNA samples was used as a template for PCR in a 25 μL total reaction with AmpliTaq Gold DNA polymerase . The conditions for this reaction were 95°C for 1 min 30 s, 33 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, with an additional extension step at 72°C for 10 min. PCR products were separated by using 1% agarose gel electrophoresis. Forward and reverse primers for both humans and mice listed in supporting information were designed on different exons for multi-exon genes to avoid genomic DNA contamination.www.proteinatlas.org). A total of 15 testis-enriched proteins were analyzed for their localization in the human testis: ten of them have been published in terms of their localization in mouse testis, but not in human testis, and the rest of them have not been published regarding their localization in both human and mouse testis.Data visualizing immunohistochemically the expression patterns of selected proteins in human testes were obtained from the Human Protein Atlas portal (CYC) was used as a housekeeping gene. Reactions were performed in duplicate 25μL volumes and conditions for the qPCR were 95°C for 10 minutes followed by 40 cycles of 94°C for 15 seconds, 60°C for 40 seconds, 72°C for 30 seconds, and 82°C for 33 seconds. Relative quantification of gene expression was determined by using the 2-ΔΔCT method [Quantitative real-time PCR (qPCR) was performed on an ABI 7300 Real-Time PCR instrument  by using AmpliTaq Gold polymerase (Applied BioSystems) with SYBR green detection dye. Cyclophilin . A list of 15 testis-enriched proteins was entered into Pathway Studio. The resulting pathways were verified through the PubMed/Medline hyperlink embedded in each node.t test was conducted to compare the difference between two means. Comparison of multiple means was conducted by one-way ANOVA followed by a Tukey’s post hoc test. The significance level was set at p < 0.05.For comparison of gene expression in testis versus other tissues, one-way ANOVA followed by a Fisher’s protected least significant difference test was performed using SAS version 9.2 . A Student’s Tnp1 and human TNP1 expressions are 276- and 186-fold greater in the mouse and human testis, respectively, than an average value of other tissues. In addition, these 24 genes are expressed at very low levels in the ovary, showing that they are male-specific genes. Our literature search revealed that some, but not all, genes were reported for testis enrichment and protein cellular location in testis. For instance, testis-specific expression of murine Ldhc gene and cellular protein location of LDHC were reported in the mouse testis [Comparative analysis of GEO DataSets (GDS3142 for mice and GDS596 for humans), a public microarray repository, revealed that expressions of 24 genes in both the mouse and human testis are more than 10-fold higher than an average expression value of other tissues  with mostly spermatogonia, 21 dpp when spermatocytes are the most abundant cell type, and 91 dpp representing an adult stage with spermatids were selected. Compared to 10 days postpartum (dpp), expression of these selected genes was significantly increased at 21 dpp after weaning and/or at 91 dpp (three months postpartum) when sexual maturation occurs . These dt 21 dpp , and thit 21 dpp , suggestIn addition, our further data analysis showed that, compared to fertile normal males, expression of these testis-enriched genes was significantly decreased in teratozoospermic patients with abnormal sperm morphology according to GDS2697 . It appeSignaling pathway analysis was conducted to identify corresponding pathways related to sperm-related biological functions and disease conditions. Schematic illustration was drawn to identify cellular and metabolic processes regulated by testis-enriched genes and showed that at least 12 out of 15 spermatocyte- or spermatozoa-enriched proteins were putatively associated with various sperm-related cell processes, clinical parameters, and disease conditions .In this study, testis-enriched genes in human and mouse were arranged based on microarray based-GEO database, and 15 genes that have not been published regarding their localization in human  were selDrosophila, and it is associated with male fertility [Proteins localized in pachytene spermatocytes (PC) and round spermatids (RS) were shown in ertility . STAG3 iertility .The acrosome reaction is required for zona pellucida penetration and fertilization with oocytes , and fouThose selected testis-enriched proteins were mostly expressed in cells in the late spermatogenesis stages. The stage-specific mRNA expression of these genes showed similar patterns as shown in the GDS2390 dataset . Their eExpression of all of these testis-enriched genes was decreased in teratozoospermic patients compared to normal individuals . SpermatPathway analysis, in this study, provided comprehensive insight into the underlying biological functions and diseases involved in spermatocyte- or spermatozoa-enriched expression. As such, most of spermatocyte- or spermatozoa-enriched proteins being analyzed were implicated in a variety of sperm functions, including motility and capacitation, and multiple disease conditions such as infertility. On the other hand, three proteins  out of those 15 spermatocyte- or spermatozoa-enriched proteins were not implicated in the biological conditions possibly due to incomplete functional annotations .In conclusion, testis-enriched genes were found based on GEO profiles, and among them, protein localization of 15 genes was identified using the Human Protein Atlas. Mostly, these testis-enriched proteins were expressed in spermatocytes and/or round spermatids, and their expression significantly increased during testis development. In testicular disease conditions, expressions of these genes were significantly decreased suggesting their relation to normal spermatogenesis and testis development. Moreover, in our pathway analysis, most of these proteins exhibited multiple biological implications related to sperm function. Future studies should ascertain the potential involvement of these testis-enriched genes in male infertility.S1 TableSamples were derived from normal and teratozoospermic individuals aged 21–57 (GDS2697) and from wild-type and polyubiquitin knockout mice at 28 dpp (GDS3906).(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.