The tests were applied in a double-blind, randomized, repeated-measures, cross-over design. When compared to PLA, CAF ingestion increased mean power output  and reduced the total time . Furthermore, anaerobic contribution during the 2200-, 2400-, and 2600-m intervals was significantly greater in CAF than in PLA (p<0.05). However, the mean anaerobic [64.9±20.1 vs. 57.3±17.5 W] and aerobic [167.9±4.3 vs. 161.8±11.2 W] contributions were similar between conditions (p>0.05). Similarly, there were no significant differences between CAF and PLA for anaerobic work (26363±7361 vs. 23888±6795 J), aerobic work (68709±2118 vs. 67739±3912 J), or total work (95245±8593 vs. 91789±7709 J), respectively. There was no difference for integrated electromyography, blood lactate concentration, heart rate, and ratings of perceived exertion between the conditions. These results suggest that caffeine increases the anaerobic contribution in the middle of the time trial, resulting in enhanced overall performance.The purpose of the present study was to investigate the effects of caffeine ingestion on pacing strategy and energy expenditure during a 4000-m cycling time-trial (TT). Eight recreationally-trained male cyclists volunteered and performed a maximal incremental test and a familiarization test on their first and second visits, respectively. On the third and fourth visits, the participants performed a 4000-m cycling TT after ingesting capsules containing either caffeine (5 mg.kg Pacing strategy can be defined as the changes in power output/velocity that occur throughout a time trial (TT) in order to reach the end point in the fastest possible time It has been suggested that athletes subconsciously monitor some aspects derived from their anaerobic energy expenditure so that near-zero values of the anaerobic reserve are never reached during a TT −1 body mass) has been reported to increase both the total anaerobic energy contribution and performance during time-to-exhaustion tests 2max) after caffeine ingestion (5 mg.kg−1). It is believed that caffeine may increase glycolytic turnover due to an increase in phosphofructokinase (PFK) activity, resulting in an increased rate of ATP resynthesis 2a receptor While research suggests the choice of pacing strategy does not affect the total anaerobic contribution −1 body mass) compared to placebo. However, anaerobic contribution, pacing strategy and muscle recruitment have not been measured in these studies. Therefore, to date, it still is not known if the improved short-distance cycling TT performance caused by caffeine ingestion is due to an increased anaerobic contribution, an increase in muscle recruitment, or both. Furthermore, any changes in anaerobic metabolism would also be expected to affect the work-rate distribution (pacing strategy) during a TT Similar to time-to-exhaustion tests, improvement in short-distance cycling TT performance has been also found following caffeine ingestion Therefore, the aim of this study was to investigate the effects of caffeine on pacing strategy, distribution of the anaerobic contribution, and muscle recruitment during a 4000-m cycling TT. We hypothesized that, if caffeine increases the anaerobic energy contribution at any particular point of the trial, it would also modify the pattern of power output distribution during the TT, increasing the total anaerobic contribution and overall performance.−1, from 5 to 6 times per week, ∼2.5 h per session and had been training for the last ten years without any long interruption (>2 months). Participants were informed about the experimental procedures and signed an informed consent form before the investigation. This study was approved by the Ethics and Research Committee of the Federal University of Alagoas.Eight trained male cyclists volunteered to participate in this study. The sample size required was estimated from the equation max) and VO2max. On the second visit, the participants performed a 4000-m cycling TT as a familiarization session. On the third and fourth visits, participants performed a 4000-m cycling TT using a double-blind, randomized and repeated-measures crossover design. One hour before starting the experimental session, the participants ingested one capsule containing either 5 mg.kg−1 body mass of caffeine (CAF) or cellulose (PLA). Capsules were ingested one hour before the test as this has been described as an adequate time for caffeine digestion and absorption 2), electromyography activity (EMG), heart rate (HR), rating of perceived exertion (RPE) and blood lactate concentration [La] were measured during the tests. Participants were informed to refrain from consuming caffeine-containing substances  or performing heavy training for 24 h before each experiment. A list of caffeine-containing substances was given to participants before every trial. Participants were also asked to complete a 24-h food record before the first experimental trial and to replicate it in the following experimental trial. Similarity between diets before the trials was checked later by analysis of the writing 24-h food records.Each participant visited the laboratory on four occasions. On the first visit, participants’ anthropometric measurements were recorded including body mass, height and percentage body fat 2, VCO2 and VE were obtained using an automatic gas-exchange analyzer  and subsequently converted to thirty-second averages. The gas analyzer was calibrated before each test using room air and known gas concentrations (16% O2 and 5% CO2), and the ventilometer with a three-liter syringe . VO2max was assumed when at least one of the following criteria was attainted: HR greater than 90% of the maximal age-predicted value; RER greater than 1.10; VO2 rate of increase smaller than 150 mL.min−1max was determined as the highest PO maintained during the last completed stage. When the last stage was not completed, the POmax was calculated from the following equation Each participant performed a maximal incremental test on a cycle simulator  that consisted of a 3-min warm-up at a PO corresponding to 100 W, followed by increments of 30 W every 3 min until voluntary exhaustion or when the participants were not able to maintain the pedal frequency between 80–90 revolutions per minute (rpm). During the entire test, breath-by-breath measurements of VOLCS is the power output in the last complete stage performed, t is the time in seconds sustained in the last incomplete stage, 180 is the duration of each stage, and 30 is the increment of PO between the stages.Where: POAt least 48 h after the incremental test, the participants performed a familiarization session for the maximal voluntary isometric contractions (MVC) and a 4000-m cycling TT. For the TT, the seat was adjusted vertically and horizontally to each cyclist before the trial, and cycling shoes were used to secure the feet to the pedals. The seat position was recorded and replicated during all subsequent experimental sessions. The participants underwent a warm-up at 100 W for 5min at 90 rpm. All participants started in a fixed gear ratio , but participants were allowed to change gear ratio as desired immediately after the trial had started. Participants were asked to complete the 4000m cycling TT as quickly as possible. During the trial, feedback about the distance covered was provided verbally every 200-m.In the morning of the experimental test, the participants arrived at the laboratory at 0800 h after consuming breakfast between 0700 h and 0715 h. The breakfast was standardized and consisted of 60% carbohydrate (CHO), 25% lipids and 15% protein, without caffeine. One hour before the test, the participants ingested one capsule containing caffeine or placebo, with 150 ml of water. Then, the participants rested for 45min and performed the MVCs. Thereafter, the participants started a 5-min warm-up at 100 W (90 rpm) followed by a 5-min rest and the 4000-m cycling TT. The same instructions and procedures given in the familiarization session were adopted during the experimental sessions.2 and VCO2. The PO was recorded every second . During the MVC, and every 200-m during the time trial, the EMG of the right vastus lateralis (VL) was measured . The HR was measured with a heart rate transmitter coupled to the gas analyzer. The RPE was recorded every 1000-m using the Borg 15-point scale, ranging from “6” (no effort) to “20” (maximum effort) During the warm-up and the time trial, the respiratory gas exchange was measured breath-by-breath to determine VOPrior to the MVC, the participants performed a standard warm-up consisting of four 5-s isometric contractions of the quadriceps muscles at an intensity corresponding to 50, 60, 70 and 80% of their subjective maximum; there was 30s of passive rest between repetitions Prior to the collection of electromyography signals, hair was removed by shaving, the skin lightly abraded to remove the outer layer of epidermal cells, and oil and dirt were removed from the skin with an alcohol swab to reduce skin impedance. A bipolar surface electrode Ag/AgCl  was positioned over the VL muscle and the reference electrode in a neutral location (bone structure: tibia). Adhesive tape  was used to fix the electrodes on to the skin. The placement and localization of the electrodes were in accordance with the recommendations of SENIAM met) during the warm-up was calculated using the following equation The aerobic and anaerobic contributions were calculated following the model adopted by Foster et al. met is the metabolic power; VO2 corresponds to oxygen uptake and RER the respiratory exchange ratio.Where: Pmet. During the 4000-m TT, Pmet was calculated every 200 m, assuming that the RER was equivalent to 1.00 aer) was calculated every 200-m by multiplying the gross mechanical efficiency by Pmet. The anaerobic power output (Pan) was obtained by subtracting the overall power output from Paer.The gross mechanical efficiency was determined by dividing the warm-up power by Pan and Paer responses during the trials were compared using a two-way analysis of variance with repeated measures, with condition (CAF vs. PLA) and distance  as factors. When necessary, subsequent post-hoc comparisons were made using Bonferroni correction. The paired Student’s t-test was used to compare the mean values of dependent variables  between the CAF and PLA conditions. The effect size (ES) and the 95% of confidence interval (95% CI) were calculated to verify caffeine effects on performance, as suggested by Conger et al. Data distribution was analyzed using the Shapiro-Wilk test. The RPE, HR, [La], iEMG, PO, PSince all of the data were normally distributed in both conditions (p>0.05), parametric tests were used to identify statistically significant differences between CAF and PLA for all dependent variables.The mean PO during the 4000-m cycling TT was significantly greater in the CAF than in the PLA condition  . AlthougParticipants adopted a fast-start strategy in both the CAF and PLA conditions (p<0.05), but the PO remained elevated longer in CAF . The PO an and Paer was not significantly different  between CAF and PLA ingestion  in CAF than in PLA . On the other hand, Paer was not significantly different between the conditions (p>0.05) at any distance interval .The mean Pngestion . Howevern in PLA . There winterval . No signinterval . Time toThere was no significant difference between CAF and PLA conditions for the average iEMG of the vastus lateralis during the trial . In acco−1), pre-TT (CAF: 1.5±0.7 vs. PLA: 1.3±0.6 mmol.L−1), and post-TT (CAF: 9.7±1.6 vs. PLA: 9.0±2.5 mmol.L−1). Finally, the mean VO2 during the TT was similar between CAF and PLA conditions  in both conditions and thereafter remained constant throughout the test; there was no significant difference between the conditions. Similarly, the RPE increased progressively from 1000 m (PLA: 11.0±1.7 and CAF: 11.1±2.0 units) to 4000 m (PLA: 16.3±2.5 and CAF: 16.4±2.2 units) in both conditions, but there was no significant difference between them . The , suggesting that CAF was not able to increase anaerobic contribution at the end of the trial. However, a similar end spurt after having produced greater power throughout the middle portion of the TT is an interesting and meaningful outcome. It would be expected that a greater power output in the middle would result in greater failure or attenuated ability to produce an end spurt. Instead, it is plausible to suggest that the influence of caffeine also appears in the end spurt enabling participants to produce a similar sprint finish even after producing more power in the middle portion.Concerning the pacing strategy adopted, the athletes adopted a fast-start in both conditions, but were able to maintain a greater PO during the middle of the trial  in CAF versus PLA. The changes in the PO were mirrored by similar changes in the P−1 body mass of caffeine one hour before the main trial, as adopted in the present study, is sufficient to significantly raise caffeine plasma concentration A potential limitation of the present study is that circulating caffeine concentrations were not measured. However, the participants were asked not to consume caffeine in the 24 h prior to each experimental test. Adherence to this advice was controlled via diet records, which indicated that participants followed the recommendations. In addition, it has been demonstrated that the ingestion of 5 mg.kg−1 of caffeine, compared with a placebo. The improvement in the performance with caffeine intake resulted from a greater anaerobic energy contribution in the middle of the trial, whereas the aerobic energy contribution and total anaerobic energy expenditure were not significantly different.In conclusion, the results of the present study suggest that athletes were able to complete a 4000-m cycling TT more quickly when ingesting 5 mg.kg


ASPM and MCPH1 genes have been implicated in the adaptive evolution of the human brain . Curiously, experimental attempts have failed to connect the implicated SNPs in these genes with higher-level brain functions. These results stand in contrast with a population-level study linking the population frequency of their alleles with the tendency to use lexical tones in a language . In the present study, we found a significant correlation between the load of the derived alleles of ASPM and tone perception in a group of European Americans who did not speak a tone language. Moreover, preliminary results showed a significant correlation between ASPM load and hemodynamic responses to lexical tones in the auditory cortex, and such correlation remained after phonemic awareness, auditory working memory, and non-verbal IQ were controlled. As in previous studies, no significant correlation between ASPM and cognitive measures were found. MCPH1 did not correlate with any measures. These results suggest that the association between the recently derived allele of ASPM is likely to be specific and is tied to higher level brain functions in the temporal cortex related to human communication.The  ASPM and MCPH1 genes are expressed in cerebral cortical ventricular and proliferative zones ASPM A44871G  and MCPH1 G37995C  with broad measures of human cognition phenotypes, including working memory ASPM and MCPH1The The evolutionary increase in brain size and the emergence of language capacity in humans have been subjects of inquiry ASPM/MCPH1 and speech sound perception is a population study by Dediu and Ladd ASPM and MCPH1 are associated with the processing of simultaneous acoustic cues within a short temporal window, as lexical tone speakers are required to process both the syllable and pitch as an integral unit. An examination of the association between these genes and lexical tone processing, a specific linguistic function, would mitigate the potential difficulties faced by earlier studies of broad phenotypic cognitive measures.A clue to a possible connection between ASPM/MCPH1 genes and lexical tone perception. In the present study, we tested native speakers of American English of European descent (Caucasians) who had not been exposed to a tone language. Non-tone language speakers were chosen because they allowed for the examination of a genotype-phenotype association without the influence of significant environment (linguistic) factors. Furthermore, non-tone language speakers show highly variable responses in tone perception, which has been attributed to neurophysiological and neuroanatomical factors ASPM, and/or MPCH1 gene load, the number of derived alleles. Such results would indicate that individuals who have a higher gene load would demonstrate more efficient  neural encoding of tones.The Dediu and Ladd population findings provide the basis of our experimental study: there is an association between This study is approved by the Northwestern University Institutional Review Board. All participants provided informed written consent in accordance with the Institutional Review Board, Northwestern University. This study examines a gene that has been previously identified and no new genetic information has been generated.ASPM, a subgroup consisting of 13 subjects  between 21 to 34 years old (mean  =  25.54) also participated in an fMRI adaptation (fMRI-A) experiment in which they processed lexical tones [Non-fMRI subjects consisted of 19 subjects  between 18 to 27 years old (mean  =  20.5)]. Subjects who participated in the fMRI-A experiment were also tested on the Test of Nonverbal Intelligence (Third Edition) for nonverbal IQ Thirty-two younger adults (less than 35 years old) who reported to be Caucasians participated in this study. All subjects were native speakers of American English with no previous experience with any tone languages. All subjects passed a hearing screening at 30 decibel Hearing Level for the frequencies of 500, 1000, 2000, and 4000 Hz. All participants were tested on the Tone Perception test, as well as Sound Blending (SB) and Auditory Working Memory (AWM) tests of the Woodcock Johnson-III Tests of Cognitive Abilities The Tone Perception test is identical to the one used in previous studies Participants performed an orthogonal loudness judgment task while listening to the four Mandarin tone categories (Tone 1 (T1), Tone 2 (T2), Tone 3 (T3), and Tone 4 (T4) that are phonetically described in terms of pitch as “high-level”, low-rising, low-dipping, and high-falling respectively) in the MRI scanner. This orthogonal task was used to ensure that participants were attentive throughout the experiment. Loudness judgment does not interfere with pitch processing, as seen in previous studies ASPM allele load for the tone-change condition . Since participants performed at ceiling during the loudness judgment task for both conditions, task performance was not considered as a factor during analyses.In the fMRI experiment, lexical tones were presented in two conditions. In the repeat condition, the speech sound containing a tone was repeated four times , within a TR (repetition time) of 12 seconds. Therefore, in the repeat condition, the same pitch pattern  was repeated. All four tone categories were used with equal probability. In the tone-change condition, the tone varied within the TR of 12 s . Therefore, in this condition, the pitch pattern varied within a TR . The order of the tones was randomized within each TR. Irrespective of condition, the task was to indicate if the second stimulus was louder or softer than the first stimulus. Regression analysis was conducted separately for the two conditions. As a control, we expected no relationship between BOLD activity and 3. Square waves modeling the events of interest were created as extrinsic model waveforms of the task-related hemodynamic response. The waveforms of the modeled events were then used as regressors in a multiple linear regression of the voxel-based time series. Normalized beta values signifying the fit of the regressors to the functional scanning series, voxel-by-voxel for each condition, were used for group analyses. Anatomical and functional images from each subject were normalized to a standard stereotaxic template (ICBM 152). To examine the effect of allele load on BOLD responses to tones, at the second (group) level, we conducted multiple regression analysis performed using the program 3DRegAna (http://afni.nimh.nih.gov/pub/dist/doc/program_help/3dRegAna.html) implemented within AFNI ASPM load, MCPH1 load, and MCPH1/ASPM load as regressors. For these various analyses, individual voxel thresholds were set at a p < 0.05 (corrected for multiple comparisons). Corrected threshold was determined based on a Monte Carlo simulation (using AFNI function AlphaSim) of significant voxels within the contrast “all listening conditions” minus “silent conditions”. The deactivation network was excluded to avoid artifacts generated from significant deactivation during the listening task, consistent with the previous studies that examined repetition suppression of auditory word information Magnetic Resonance Images were acquired using a Siemens 3T Trio MRI scanner. For each participant, a high resolution, anatomical T1-weighted 3D volume was acquired axially   =  3.36 ms, flip angle  =  9°, TI  =  900 ms, matrix size  =  256×256, FOV of 22 cm, slice thickness  =  1 mm). Functional T2*-weighted images were acquired axially using a susceptibility weighted EPI pulse sequence  were acquired in an interleaved measurement). A sparse sampling method was used, which allows for stimuli presentation in silence MCPH1 G37995C and ASPM A44871G genotypes were determined by polymerase chain reaction (PCR) amplification of DNA followed by direct sequencing of PCR products. Primers used for PCR amplification of MCPH1 G37995C were 5′-TTTCAAAGGAAGAAATAAACTTGC-3′ and 5′-GAGGTGAATGGGAGCCATGT-3′; ASPM A44871G was amplified using primers 5′-AGGGCTGCAGTTCTCATTCAG-3′ and 5′-GCCCACTGAAGCTTTTGGTAG-3′. PCR conditions included denaturation at 94°C for 3 min followed by 45 cycles at 94°C for 30 sec, 55°C for 45 sec, and finally 72°C for 1 min. For each subject and for each gene, the number (load) of derived alleles was calculated. The G and C allele are derived alleles for ASPM A44871G and MCPH1 G37995C, respectively. For both SNPs, we calculated the load of derived alleles for each gene in each subject. Therefore, the load of derived alleles for ASPM A44871G is 0, 1, and 2, for AA, AG, and GG alleles, respectively. The load of derived alleles for MCPH1 G37995C is 0, 1, and 2 for GG, GC, and CC, respectively. For ASPM, we found 14, 12, and 6 subjects with the AA, AG, and GG genotype, respectively . For MCPH1, there were 10 and 22 subjects with the GC and CC genotype, respectively . When both genes were examined together, there were 6, 12, 8, and 6 subjects with 1, 2, 3, and 4 combined derived alleles, respectively . Both of our samples (fMRI and non-fMRI subjects) were in Hardy-Weinberg equilibrium. All statistical analyses in this report are based on two-tailed tests.Genomic DNA was extracted from buccal swab samples using QIAamp DNA mini-kit (Qiagen). ASPM load  and MCPH1 load .We estimated the correlation between Tone Perception performance and derived allele load for each gene. We used non-parametric tests as our primary analysis because the distributions were non-normal for both ASPM A44871G , even after age, SB, and AWM were partialed out   and these two genes were found. When subjects who participated in the fMRI experiment were analyzed separately from those who did not participate in the fMRI experiment, there remained a significant correlation between the load of derived alleles for ASPM and Tone Perception . No such correlation between Tone Perception and the load of derived alleles for MCPH1 was found in either sample . For combined MCPH1/ASPM, significant correlation was found only in the Non-fMRI subjects , but not in the fMRI subjects .We found a significant positive correlation between Tone Perception and the load of derived alleles for   0.015] . No signASPM and Tone Perception correlation results, we used Fisher’s combined probability test For the ASPM and the neurophysiological basis of tone perception was found predominately in the right temporal cortex , we can conclude that this relationship is specific to the encoding of tones. Furthermore, no such relationship was found with MCPH1.Until now, the hypothesis that the derived alleles of ASPM/MCPH1 and more specific measures of language has been examined Although the association between Our study contributes to the current literature on the molecular genetics of human communication by not only considering its relationship with deficits ASPM/MCPH1 with lexical tone perception. After we found a significant correlation between ASPM and tone perception in this sample, we recruited a second group with the fMRI experiment added. While the sample size for the present study may appear small, it is consistent with human imaging genetic studies It is worth pointing out that the present study was designed to examine two populations of subjects tested during two consecutive periods of time. We first conducted a behavioral study linking MCPH1 and our phenotypes could be due to our small sample size. It could also be related to the fact that we have a somewhat skewed distribution for the derived C allele. No subjects were homozygous for the ancestral G allele. Because the ASPM/MCPH1 haplogroup depends on the distribution of the MCPH1group, a skewed MCPH1 distribution could have also affected the results.The lack of a significant association between ASPM is associated with lexical tone and this association can either favor or disfavor tone. Other research groups hypothesize more specifically that the derived allele contributes a positive effect on human brain functions ASPM and tone irrespective of the direction of the relationship. Interestingly, we found a positive relationship, whereas a negative relationship was found by Dediu and Ladd. The significant but opposite pattern of results between Dediu and Ladd and the present study could be attributed to differences in methodology. Dediu and Ladd ASPM and tone perception behaviors by examining different cultural groups.Dediu and Ladd In addition to potential contribution of gene-gene and culture-gene interaction effects, other factors related to inter- and intra-population comparisons might have contributed to differences in our results. Dediu and Ladd examined populations of subjects who acquire their native languages as children, whereas we examined individual differences in adults. Experience-driven effects started from a young age could affect genetic contribution to a phenotype that we could not observe in the present study. Furthermore, language differences at the population level are results of numerous historical and geographical factors, which could not be easily compared with effects of individual differences.ASPM and MCPH1 did not find a significant association between those SNPs and neural and cognitive phenotypes, some recent studies focusing on other SNPs of the same genes found significant results. Rimol et al. [2010] found sex-dependent associations between neuroanatomical measures and non-exonic SNPs of these two genes in an ethnically homogeneous Norwegian discovery sample and an ethnically heterogeneous North American replication sample. Similarly, Wang et al. [2008] found in Chinese males an association between cranial volume and a non-synonymous SNP of MCPH1 other than the SNP that we examined. Future studies should examine the contribution of these SNPs and sex-dependency to tone perception.It is worth noting that although earlier research focusing on the SNPs that we examined in ASPM. For example, future research can examine whether ASPM and spoken language processing is restricted to lexical tones, to all speech sounds, to all speech sounds requiring simultaneous processing within a short temporal window, or whether they are also linked to other specific linguistic functions such as syntactic processing in a larger population of participants. Future studies can also examine other linguistic and speech-specific phenotypes.An obvious limitation to the current study is the small sample size , although it is consistent with a number of previous studies connecting higher-order functions and genetics


Saccharomyces cerevisiae. ALR comprises 198 to 205 amino acids (approximately 22 kDa), but is post-translationally modified to three high molecular weight species (approximately 38 to 42 kDa) found in hepatocytes. ALR is present in mitochondria, cytosol, endoplasmic reticulum, and nucleus. Mitochondrial ALR may be involved in oxidative phosphorylation, but also functions as sulfhydryl oxidase and cytochrome c reductase, and causes Fe/S maturation of proteins. ALR, secreted by hepatocytes, stimulates synthesis of TNF-α, IL-6, and nitric oxide in Kupffer cells via a G-protein coupled receptor. While the 22 kDa rat recombinant ALR does not stimulate DNA synthesis in hepatocytes, the short form (15 kDa) of human recombinant ALR was reported to be equipotent as or even stronger than TGF-α or HGF as a mitogen for hepatocytes. Altered serum ALR levels in certain pathological conditions suggest that it may be a diagnostic marker for liver injury/disease. Although ALR appears to have multiple functions, the knowledge of its role in various organs, including the liver, is extremely inadequate, and it is not known whether different ALR species have distinct functions. Future research should provide better understanding of the expression and functions of this enigmatic molecule.‘Augmenter of liver regeneration’ (ALR)  was originally found to promote growth of hepatocytes in the regenerating or injured liver. ALR is expressed ubiquitously in all organs, and exclusively in hepatocytes in the liver. ALR, a survival factor for hepatocytes, exhibits significant homology with ERV1  protein that is essential for the survival of the yeast,  Saccharomyces cerevisiae). Thus it is not a surprise that ALR is expressed ubiquitously in all organs, and may have tissue-specific functions. Furthermore, post-translational modification of the 22 kDa native ALR protein to three high molecular weight species (38 to 42 kDa), and presence of ALR in mitochondria, cytosol, endoplasmic reticulum, and nucleus indicate that ALR may play an important role in various physiological functions in a cell. Current evidence indicates that ALR may be involved in mitochondrial oxidative phosphorylation, reduction of cytochrome c, and in regulation of the activities of certain proteins by its sulfhydryl oxidase activity as well as by inducing Fe/S maturation of proteins. Thus, although ALR appears to have multiple functions, the knowledge of its roles in various organs, even in the liver, is very inadequate. In this article, the history and the current status of ALR research is reviewed. The intent of this summary is to stimulate further research to unravel the many facets of the important functions of this enigmatic protein in physiology and pathology.Following the discovery that the liver possesses a remarkable ability to regenerate, pursuit to identify factors that are involved in this phenomenon led to the discovery of many mitogens, co-mitogens, and inhibitors. Among them was a protein initially named hepatic stimulatory substance or hepatopoietin and now commonly known as ‘Augmenter of liver regeneration’ (ALR). ALR was subsequently purified, and cloned in rat, mouse, and human. Both native and cloned ALR augment liver regeneration following partial hepatectomy and prevent pathology of portacaval shunt surgery in animal models. ALR is produced and secreted exclusively by hepatocytes in the liver and stimulates synthesis of TNF-α, IL-6, and nitric oxide in Kupffer cells via a G-protein coupled receptor. ALR may also exert actions on hepatocytes in an autocrine manner. Interestingly, intracellular ALR was found to be a survival factor as its depletion causes rapid mitochondrial dysfunction and apoptotic/necrotic death of hepatocytes. In this regard, ALR exhibits significant homology with ERV1 protein  named Essential for Respiration and Viability 1 (ERV1) . Interestingly, both ERV1 [The nucleotide and amino acid sequences of the rat and mouse ALR (198 amino acids) are 96% and 90% homologous, respectively . The humoth ERV1  and ALR oth ERV1 ,25. The oth ERV1 ,27, and ERV2 in the yeast, with 30% homology to ERV1 at the carboxy terminal [Subsequent work identified another gene terminal ,29. Thisterminal . While EWestern blot analysis showed that a 22 kDa- and some higher molecular weight (38 to 40 kDa) proteins react with the antiserum raised against amino terminal peptide segment of the rat ALR . SubsequALR mRNA is expressed in heart, brain, spleen, lung, skeletal muscle, kidney, liver, and testis of rat and mouse, with maximal expression in the testes followed by the liver ,20. ALR ALR is also found in all regions of the brain, co-localized with GFAP in neurons, and is specifically present in the nucleus and external envelope of mitochondria. Western blot analysis demonstrated two dimers, which under reducing condition migrated as two bands with MW of 21 and 23 kDa . Low levin vivo, [Early experiments showed that the crude extract of regenerating or hyperplastic liver, but not the unmodified adult liver, contains ALR-like activity that augmented partial hepatectomy-induced liver regeneration and prevented portacaval shunt-induced hepatic atrophy ,11. Basein vivo,  argued fS. cerevisiae[ERV1 gene or null mutation in the haploid strain led to temperature-conditional mutation resulting in alterations of the mitochondrial inner membrane, complete loss of mitochondrial genome, arrest in cell-division cycle, and death of the yeast after a few replication cycles [in vivo stability of the ERV1 mRNA and protein could be a reason for delayed death of the yeast with disrupted ERV1 gene, despite the original low level of ERV1 expression [The indication that ALR might be a protein of critical physiological importance was provided by the finding that ALR’s yeast ortholog ERV1 is essential for the biogenesis of mitochondria, normal mitochondrial morphology, and stable maintenance of mitochondria in erevisiae,25,40. Terevisiae. Disruptn cycles ,40,42. Ipression .S. cerevisiae with mutated ERV1 gene, inhibition of ALR synthesis with ALR-mRNA antisense oligonucleotide (ALR-AS) caused rapid (within hours) apoptotic/necrotic death of hepatocytes [In contrast to a lag period of a few days before the death of atocytes . The accatocytes . These rS. cerevisiae, respectively, led to the question whether these homologous proteins are functionally interchangeable. Both the 15 kDa (short) and 22 kDa (long) cloned ALR proteins were found to prevent portacaval shunt-induced liver atrophy in dogs [S. cerevisiae with mutated ERV1 gene from death. In addition to the absolute necessity of their carboxy terminal sequences in preventing cellular atrophy or loss of viability, these observations provided the proof that ALR and ERV1 proteins have structural and functional similarity. Indeed, growth arrest and death of S. cerevisiae due to deletion of carboxy terminal sequence from ERV1 could be restored by substitution with ALR’s carboxy terminal sequence [The mitochondrial presence of ALR and ERV1, necessary for the survival of hepatocytes and  in dogs ,20 sugge in dogs  as well  in dogs  that lacsequence ,44. In csequence ,40. Howeet al.[As mentioned above, the 22 kDa rrALR does not stimulate DNA synthesis in rat hepatocytes ,22. Furtet al. reportedet al.. The effet al.. Why theet al.. Since Eet al.,48, it wet al.. Howeveret al..In vitro, polyamines were found to play an important role in growth factor induced DNA synthesis in cultured rat hepatocytes [In vivo, polyamines are required for hepatic regeneration [in vivo and DNA synthesis in vitro[in vitro actions of ALR on hepatocytes to its role in their replication in vivo is ambiguous. Since ALR is released from the liver soon after partial hepatectomy in rats (21) and exogenously administered recombinant rat ALR augments liver regeneration in this model (22), it will be critical to investigate whether the proliferative response occurs by ALR’s direct action on hepatocytes and/or via mediators released from the non-parenchymal cells. Obviously, the attendant gene and protein expression pattern related to liver regeneration in the context of changes in hepatic and circulating ALR, and their relationship to other mitogens/co-mitogens and growth inhibitors will be the areas that should be addressed rigorously.The short rhALR stimulates NFκB activation  and polyatocytes ,52. In vneration ,54, and neration . HGF and in vitro. Thus, A in vitro. Several in vitro-67. Upon in vitro. TogetheAnother important mechanism of the role of ALR in unabated liver regeneration might be its ability to prevent hepatocyte injury by affecting cells of the immune system, infiltrating inflammatory cells and resident macrophages. Earlier work demonstrated that ALR inhibits the lytic activity of hepatic natural killer (NK) cells . While h. In vivo, it is likely that ALR promotes liver regeneration after partial hepatectomy [Immediate release of ALR from the rat liver following partial hepatectomy  indicateatectomy ,22 by statectomy  and cirratectomy  via mechatectomy . These eatectomy . In contatectomy .et al. [S. cerevisiae does not express QSOX. These observations suggested that ALR/ERV1 may also function as sulfhydryl oxidase. Indeed, ERV1 and ERV2 [et al.[Coppock et al.  identifiet al. ). Severaet al. -79. Howeand ERV2 ,39,40,80and ERV2  were fouand ERV2 ,40,82. Tand ERV2 . Howeverand ERV2 ,40,81,82and ERV2 -85, TIM1and ERV2 , and supand ERV2 . A funct2 [et al. showed tet al.[in vivo[Allen et al. reportedet al. and ALR et al. can alsoet al.,91. Func.[in vivo. Such ox.[in vivo.Another function associated with the ALR molecule is its ability to induce Fe/S maturation of cellular proteins. The entire ERV1/ALR sequence, including the poorly conserved amino terminus, is required for ALR/ERV1 to catalyze Fe/S assembly of cytosolic but not mitochondrial proteins . Replaceet al. [Considering the abundance of ALR in the liver and its secretory nature, researchers have examined changes in circulating and tissue levels of ALR in certain pathological conditions. Tanigawa et al.  found siet al.  suggest Hepatic ALR increases rapidly but transiently following portacaval shunt in rats, accompanied by increases in HGF, TGF-α, and TGF-β . DespiteIncreased mRNA and protein expression of ALR was observed in liver from patients with cirrhosis, and hepatocellular and cholangiocellular carcinoma . PredomiAn area of investigation that needs to be explored is the pathological developments in the liver due to mitochondrial ALR deficiency. Given its role in oxidative phosphorylation, mitochondrial ALR deficiency might induce oxidative stress causing abnormal hepatocyte functions. In this regard, we have found that mitochondrial ALR deficiency causes hepatocyte injury  and accuALR is an enigmatic protein whose functional characteristics and their underlying mechanisms are not well understood as yet despite the fact that the protein was discovered more than 35 years ago and its gene cloned 18 years ago. Originally identified as an augmenter of liver regeneration, and assumed to be present only in the hyperplastic liver, we now know that ALR is present ubiquitously in multiple forms among eukaryotes, and its presence in various subcellular organelles and extracellular compartments suggests that it may have distinct and important physiological functions. Although physiologically present as a 22 kDa protein, a large body of work has been done using the short form of ALR (15 kDa) or its dimer. Curiously, the short ALR has been shown to be equipotent in stimulating DNA synthesis in hepatocytes as HGF and TGF-α, while the 22 kDa protein is unable to stimulate this reaction. This critical observation begs a question whether the mitogenic property of short human ALR could be exploited as a therapy necessitating liver regeneration in pathological conditions. Certainly, ALR plays a critical role in maintaining viability of hepatocytes, and whether it performs similar function in cells of other organs remains to be determined. Although enzymatic activities  are associated with ALR, they are relatively weak as compared to that of other known enzymes. It is possible that ALR is highly selective in performing its enzymatic function for specific proteins. Clearly, significant further work has to be done to understand the multiple roles of this protein. Perhaps genetically engineered mouse models (lacking or over-expressing ALR in specific organs) will be required to reveal its functions.ALR: Augmenter of liver regeneration; EGF: Epidermal growth factor; ERV: Essential for respiration and viability; GFER: Growth factor ERV-like; HGF: Hepatocyte growth factor; IGF: Insulin-like growth factor; IL: Interleukin; NO: Nitric oxide; rrALR: Recombinant rat ALR; rhALR: Recombinant human ALR.The author does not have any financial competing interests to disclose. The content of this article does not represent the views of the Department of Veterans Affairs or the United States Government.


B-cell dysregulation has been implicated but not fully characterized in pediatric opsoclonus-myoclonus syndrome (OMS), a neuroblastoma-associated neuroinflammatory disorder.To assess the role of B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), two critical B cell-modulating cytokines, as potential biomarkers of disease activity and treatment biomarkers in OMS.Soluble BAFF and APRIL were measured in cerebrospinal fluid (CSF) and serum by ELISA in 433 children ). BAFF-R receptors on circulating CD19+ B cells were measured by flow cytometry. A blinded scorer rated motor severity on the OMS Evaluation Scale. Immunotherapies were evaluated cross-sectionally and longitudinally.P = 0.006), and reduction by adrenocorticotropic hormone or corticotropin (ACTH) (−61%) or corticosteroids (−38%) was seen at each level of severity. In contrast, CSF APRIL was normal in OMS and OIND and unaffected by immunotherapy. When the entire OMS dataset was dichotomized into ‘high’ versus ‘normal’ CSF BAFF concentration, the phenotype of the high group included greater motor severity and number of CSF oligoclonal bands, and a higher concentration of inflammatory chemokines CXCL13 and CXCL10 in CSF and CXCL9 and CCL21 in serum. Serum APRIL was 6.7-fold higher in the intravenous immunoglobulins (IVIg) group, whereas serum BAFF was 2.6-fold higher in the rituximab group. The frequency of B cell BAFF-R expression was similar in untreated and treated OMS. Longitudinal studies of CSF BAFF revealed a significant decline in ACTH-treated patients (with or without rituximab) (P < 0.0001). Longitudinal studies of serum APRIL showed a 2.9-fold increase after 1 to 2 g/kg IVIg monotherapy (P = 0.0003).The mean CSF BAFF concentration, which was elevated in untreated OMS and OIND, correlated with OMS severity category (Striking distinctions in BAFF/APRIL signaling were found. OMS displayed heterogeneity in CSF BAFF expression, which met many but not all criteria as a potential biomarker of disease activity. We speculate that CSF BAFF may have more utility in a biomarker panel than as a stand-alone biomarker, and that the selective upregulation of both serum APRIL by IVIg and BAFF by rituximab, as well as downregulation of CSF BAFF by ACTH/steroids, may have utility as treatment biomarkers. Opsoclonus-myoclonus syndrome (OMS) is a neuroinflammatory disorder of children and adults, which is demonstrably paraneoplastic in about 50% of the cases . Our resTwo critical cytokines for B-cell activation, proliferation, homeostasis, and survival are a proliferating-inducing ligand (APRIL) and B-cell activating factor (BAFF) . They arOur preliminary work on BAFF showed an increase in OMS and decrease by corticotropin (ACTH) or corticosteroids . The preThis prospective, case–control, translational research study is part of the ‘Study of Cytokines in Children with Opsoclonus-Myoclonus Syndrome’  . Approxin = 120; girls n = 156), who were enrolled after consent was signed by parents and assent by older children. OMS groups included untreated, currently treated, and previously treated patients. Videotapes were made using a standardized procedure and later scored blinded on the OMS Evaluation Scale to yield a total score [The cross-sectional part of the study involved 276 children with OMS, mean age (SD) 3.8 ± 3.0 years .Controls comprised children with various non-inflammatory neurological disorders (NIND), including ataxia, developmental disorders, headache, movement disorders, seizures, and miscellaneous disorders. Their mean age was 8.4 ± 5.6 years, and 96 were boys and 91 were girls. Children with OIND, who were evaluated locally and through the Center for Pediatric-Onset Demyelinating Disease in Alabama, served as a comparison group for specificity. Their diagnoses included acute disseminated encephalomyelitis, encephalitis, meningitis, multiple sclerosis and related disorders, and miscellaneous disorders. The OIND mean age was 7.7 ± 5.8 years  or without (n = 11) rituximab. There were 16 boys and 26 girls, mean age 3.7 ± 2.8 years (range 1.8 to 18 years). CSF was obtained before and at 8.0 ± 2.1 months of treatment. In the APRIL study, 20 new children with OMS were recruited for monotherapy with intravenous immunoglobulins (IVIg) under FDA BB-IND No. 5839. There were 11 boys and nine girls, mean age 4.6 ± 1.4 years (range 1.8 to 6.4 years). They received standard monthly clinical doses of 1 or 2 g/kg, and serum was collected before and at 4.1 ± 3.9 months of treatment.Two longitudinal OMS studies were performed. In the BAFF study, 42 children from the cross-sectional study were treated using ACTH-based conventional therapy with (n = 22) for CSF BAFF, 8.5% (n = 20) for serum BAFF, 7.8% (n = 10) for CSF APRIL, and 4.8% (n = 8) for serum APRIL. The corresponding intra-assay CV was 4.8% (n = 11), 6.0% (n = 10), 7.2% (n = 6), and 6.7% (n = 6). Freezer storage time and the concentration of CSF BAFF (P = 0.21) or APRIL (P = 0.16), or serum concentrations, were not correlated.CSF and serum were collected on ice, aliquotted, and stored at −80°C in biorepository freezers. Samples were thawed on the assay day and BAFF and chemokines were measured using Quantikine ELISA kits per instructions by the manufacturer . The comparison chemokine panel included CXCL9, CXCL10, CXCL12, CXCL13, CCL17, CCL21, and CCL22 kits from the same vendor. Human APRIL ELISA kits were purchased from eBioscience . Assays were performed in duplicate on undiluted samples, each 96-well plate containing both control and OMS samples. Samples with values above the highest standard were re-run at a 1:2 or higher dilution. Outliers were verified by repeat measurement. The kit user had no patient contact. BAFF sensitivity was 0.73 ± 6.7 pg/mL in CSF, and 1.5 to 11.9 pg/mL in serum; APRIL, 0.4 pg/mL. The inter-assay coefficient of variance (CV) was 9.4% , no correlation between age of controls and CSF BAFF concentration (P = 0.72) or CSF APRIL (P = 0.19) was found, and the age range of the groups was equivalent.Because of inter-individual differences and the large range of values inherent in human cytokine and chemokine data, both means and medians were analyzed. Cross-sectional groups were compared by analysis of variance (ANOVA) with Tukey post-hoc tests, and primary comparisons were between controls and untreated OMS, and between untreated and treated OMS. Medians were analyzed secondarily by the Kruskal-Wallis test with Dunn’s post-hoc test. Two group comparisons were made by two-tailed P < 0.008), nine chemokines/cytokines and oligoclonal bands (P  < 0.0055), and five lymphocyte subsets (P  <  0.01) to control for the multiple comparisons that were made.Secondary analysis was performed on the OIND group compared to the other groups. Also, OMS immunotherapy groups were bundled based on common treatment effects for graphic presentation. Lastly, the dataset was dichotomized based on cytokine levels 2 SD above the control mean to determine if two different phenotypes emerged, and family-wise Bonferroni corrections (α at 0.5/n) were performed separately on six clinical variables , significantly lower mean CSF BAFF concentrations (pg/mL) were found for ACTH , steroid , ACTH + IVIg , steroid + IVIg , ACTH + other , and steroid + other . In each of those groups, the mean BAFF concentration was below, though not significantly different than, the control mean. Of actively treated patients, 22% had CSF BAFF concentrations below the lowest controls. In previously treated patients, mean CSF BAFF concentration did not differ significantly from controls.In comparisons of individual OMS treatment groups P = 0.04) and controls (P < 0.0001), though there was no significant difference in medians. In four OIND, whose diagnoses included encephalitis, neurolupus, and developmental delay, the CSF BAFF concentration was above the highest control in the 369 to 1,398 pg/mL range. The CSF:serum BAFF ratios did not differ between these groups.The OIND group also manifested an elevated mean CSF BAFF concentration compared to untreated OMS . When all ACTH groups (n = 90) were compared to all steroid groups (n = 62), there was no significant difference in CSF BAFF concentrations: 91 ± 78 vs 123 ± 95 pg/mL, respectively. Only the combined ACTH group was significantly lower than the IVIg group (184 ± 136 pg/mL).ACTH and steroid monotherapy groups did not differ significantly in CSF BAFF concentrations (P < 0.0001). Compared to untreated OMS, there were significant ratio reductions in treated groups. The lowest ratios were for ACTH , steroids , ACTH + IVIg , steroid + IVIg , ACTH + other treatments , and steroid + other treatments .The mean CSF/serum BAFF ratio was 64% higher in untreated OMS than in controls (P < 0.0001).The mean serum BAFF concentration did not reflect CSF concentrations Figure B. It wasThe CSF concentration of APRIL did not differ significantly between the control group and OMS cross-sectional groups Figure A. NeitheCombined IVIg treatment groups displayed significantly higher serum APRIL concentrations Figure B; so didP  <  0.0001); in untreated OMS, it was 2.8-fold higher (P  <  0.0001).As a result of increased serum APRIL, the CSF:serum APRIL ratio was lower by 53% in the IVIg monotherapy group, 60% in the ACTH + IVIg group, and 81% in the steroid + IVIg group compared to untreated OMS. In controls, the CSF:serum APRIL ratio was 5.8-fold higher than the CSF:serum BAFF ratio (n = 271) ; so did the median . For OMS duration category, there was no significant relation to CSF BAFF (P = 0.11). The CSF:serum BAFF ratio correlated with OMS total score , OMS duration , and CSF B cell frequency . When all groups receiving either ACTH or steroids were analyzed separately from all remaining OMS groups, they manifested the lowest CSF BAFF concentrations regardless of OMS severity . However, when the OMS dataset was split into no IVIg groups versus all IVIg groups, significant differences were found for IVIg at each level of OMS severity  and the non-tumor OMS group  did not differ significantly in mean serum BAFF concentration (P = 0.79), or in medians (P = 0.42). Mean serum APRIL concentration in the tumor group (30.3 ± 70 ng/mL) and non-tumor group (19.7 ± 30 ng/mL) also was not significantly different (P = 0.22).The tumor OMS group , though the r value was low (r = 0.24). There were no significant correlations with CSF IgG concentration or the albumin ratio.The OMS combined dataset was analyzed for various correlations. CSF BAFF, not APRIL, correlated with CSF leukocyte count (n = 23), there was a significantly higher concentration of CSF CXCL13 and CXCL10, CSF oligoclonal band number and frequency, and serum CXCL9 and CCL21 compared to the ‘normal’ BAFF group (n = 253). The clinical characteristics of this group were greater total score (motor severity) and a higher frequency of untreated patients. CSF T cell frequency was lower in the ‘high’ BAFF group (80.3 ± 8.1%) than the ‘normal’ BAFF group (85.2 ± 7.0%) (P = 0.004). There were no significant between-group differences after Bonferroni corrections for CSF or serum APRIL, plasma CXCL12, serum CCL17, serum CCL22, or for CSF lymphocyte subset frequencies  (data not shown).The whole OMS dataset was divided into two groups as described under statistical analysis Figure . In the P = 0.06), serum CXCL9 (P = 0.08). When the sample was increased by inclusion of all non-ACTH/steroid groups, there were 10 ‘high’ and 106 ‘normal’. The statistically significant results were for CSF CXCL13 (P = 0.01), CSF OCB frequency (P = 0.04) and number (P = 0.03), serum CXCL9 (P = 0.02), and total score (P = 0.02); CXCL10 and CCL21 were not significant. Thus, the ‘high’ group appeared statistically underpowered when the dataset was delimited.Because certain treatments alter CSF BAFF concentrations, the analysis was then performed only for untreated OMS, for which there were 10 ‘high’ and 34 ‘normal,’ with non-significant trends: CSF CXCL13 (n = 6), 97.1 ± 5.4% for conventionally-treated OMS (n = 9), 90.1 ± 12.2% for multimodal immunotherapy (n = 13), and 96.9 ± 2.2% in controls, with no significant differences between groups (P = 0.45). Median frequencies also did not differ significantly .BAFF-R was expressed on almost all circulating CD19+ B cells, whether from controls or OMS. Mean BAFF-R frequency was 94.5 ± 9.3% for untreated OMS (P < 0.0001). It declined significantly in children receiving ACTH-based immunotherapy . Post-treatment CSF BAFF concentration was in the normal range. Although there was an overall 71% reduction in motor severity  (P < 0.0001), the change in BAFF did not correlate with the change in total score, even when patients without a decrease in BAFF were excluded .The mean CSF BAFF concentration in pre-treatment OMS was higher than in controls , B-cell chemoattractants , and oligoclonal bands -5. Such ACTH: Adrenocorticotropic hormone or corticotropin; APRIL: A proliferating-inducing ligand; ANOVA: Analysis of variance; BAFF: B cell activating factor; BAFF-R: BAFF receptor; CD: Cluster of differentiation; ELISA: Enzyme-linked immunoassay; IVIg: Intravenous immunoglobulins; NIND: Non-inflammatory neurological disorders; OIND:Other inflammatory neurological disorders; OMS: Opsoclonus-myoclonus syndrome.Richard M. Ransohoff is a member of the Scientific Advisory Board of Chemocentryx and holds stock in the company. He is a member of the Scientific Advisory Board of Vertex. The rest of the authors have nothing to disclose.MRP was the principal investigator, and participated in the drafting/revising of the manuscript, the study concept and design, acquisition of data, analysis and interpretation of data, and statistical analysis. EDT was the co-investigator, and participated in patient recruitment and evaluation, study concept and design, acquisition of data, and revision of the manuscript. NRM was the laboratory researcher, and participated in the acquisition and analysis of data, and statistical analysis. ALT was the flow cytometrist, and participated in the acquisition and analysis of data, and revising of the manuscript. JAC was the study statistician, and participated in the review of the manuscript. JMN participated in OIND patient recruitment and evaluation, and vetting of the manuscript. RMR was the project consultant, and revised the manuscript. All authors have read and approved the final version of the manuscript.


Streptococcus pneumoniae meningitis complicated with right 6th cranial nerve palsy. He presented with fever, headache and neck stiffness for 3 days and was treated with intravenous antimicrobial agents including linezolid and dexamethasone successfully. An association between CSF HGF concentrations and CSF proteins is observed . This result showed that HGF level in CSF might act as an additional marker in patients with bacterial meningitis.Hepatocyte growth factor (HGF) is a multifunctional cytokine that has well-defined mitogenic, motogenic, and morphogenic functions on the epithelial cells. Strong increases of HGF concentrations in cerebrospinal fluid (CSF) are only present in patients with acute bacterial meningitis. We report a 15-year-old young man with drug resistant  S. pneumoniae meningitis who was followed up by serial CSF HGF levels.Hepatocyte growth factor (HGF) is a multifunctional cytokine that has well-defined mitogenic, motogenic and morphogenic functions on the epithelial cells.9/l with granulocyte 81%, lymphocyte 12% and monocyte 6%. Platelet count was 513000/mm3. Lumbar puncture revealed an opening and closing pressure of 310 mmH2O, and 195 mmH2O, respectively. His CSF was turbid with a white cell count of 36/mm3 with 63% granulocyte, lymphocyte 30% and monocyte 7%. CSF protein level was 236 mg/dl and sugar 36 mg/dl (serum glucose 131 mg/dl). A gram stain of the spinal fluid showed numerous polymorphonuclear leukocytes and gram positive cocci in pairs and in chains. Since it was a holiday, the CSF was left at room temperature for 10 hours before processing. Culture of the CSF and blood yielded Streptococcus pneumoniae . He was treated with meropenem 2 g every eight hours with simultaneous intravenous dexamethasone and glycerol. Fever subsided 2 days later but recurred after 4 days of treatment. A repeat lumbar puncture showed a white cell count of 4850/mm3, with 99% granulocyte. CSF protein was 159 mg/dl and sugar 56 mg/dl. Blood culture again yielded Streptococcus pneumoniae. Echo-cardiogram showed absence of any intracardiac vegetation. Antibiotics was switched to vancomycin 1g every 12 hours and cefotaxime 2 g every 6 hours intravenously. His fever subsided but the headache persisted. On the 15th day of admission, rifampin at a dose of 600 mg every 12 hours was added due to recurrence of fever. No skin rash or eosinophilia was found. Another repeat lumbar puncture was done 2 days later showing a white cell count of 84/mm3, with CSF protein 68 mg/dl and sugar 49 mg/dl. Due to persistent fever and headache, all antimicrobial agents were discontinued and shifted to intravenous linezolid 600 mg every 12 hours. His fever subsided 4 days after. Brain magnetic resonance imaging showed presence of leptomeningeal enhancement. A follow up lumbar puncture done after 6 days of linezolid showed a white cell count of 80/mm3 with CSF protein level of 101mg/dl and sugar 43 mg/dl. Intravenous linezolid was given for 7 days and he was discharged under stable condition. He was followed up at out patient department for 1 year without any neurologic sequelae.A 15 year-old young man presented to our hospital with fever, neck stiffness and headache for 3 days. He had a history of right distal radius fracture 4 years ago and recovered completely. At presentation, physical examination revealed an acutely ill-looking, febrile patient with evident nuchal rigidity. Neurologic examination showed right 6th cranial nerve palsy. His blood pressure was 113/74 mmHg, pulse rate 88/min, and respiratory rate 18/min. Laboratory results showed a white cell count of 18.4 × 10The CSF samples stored at −70 ºC were centrifuged at 3000 g for 15 min prior to analysis. Dynamic changes of CSF HGF concentrations was determined by ELISA, using a commercially available kit . The serial CSF parameters and levels of HGF are shown in S. pneumoniae meningitis. In a meningitis study done in 2004, Turkish researchers found that there were no significant correlations between HGF levels in CSF and other parameters of CSF, including cell count, total protein and glucose levels.S. pneumoniae meningitis, researchers conducting a study in neurosurgical patients found that the maximum and minimum measured concentrations of linezolid in the CSF were 10.8 g/ml and 6.1 g/ml.The present study showed that changes in CSF HGF concentrations are correlated with levels of CSF protein. Linezolid may act as an alternative treatment for patients with drug-resistant S. pneumonaie meningitis.In conclusion, we suggest that CSF HGF concentrations might provide additional information in patients with bacterial meningitis. Although further study is needed to verify our observation, we suggest that linezolid may act as an alternative treatment choice for patients with drug-resistant


There are a number of commercial assays that are routinely employed for measuring Aβ42; however, these assays give diverse ranges for the absolute levels of CSF Aβ42. In order to employ CSF Aβ42 as a biomarker across multiple laboratories, studies need to be performed to understand the relationship between the different platforms. We have analyzed CSF samples from both diseased and nondiseased subjects with two different widely used assay platforms. The results showed that different values for the levels of CSF Aβ42 were reported, depending on the assay used. Nonetheless, both assays clearly demonstrated statistically significant differences in the levels of Aβ42 in CSF from AD relative to age-matched controls (AMC). This paper provides essential data for establishing the relationship between these assays and provides an important step towards the validation of Aβ42 as a biomarker for AD.Alzheimer's disease (AD), which is characterized by a degeneration of neurons and their synapses, is one of the most common forms of dementia. CSF levels of amyloid  These assays give diverse values for the levels of CSF Aβ42 [β42 values from these different assays are unclear, but researchers agree in the importance of standardizing assays for CSF Aβ42 [Alzheimer's disease (AD) is the most common neurodegenerative disorder. Because the disease is often difficult to detect and diagnose at an early stage, a tremendous need exists for the identification and characterization of biomarkers that can be used to diagnose early-stage AD, or for monitoring new therapies for AD in clinical trials. Much interest has been generated regarding the use of CSF Agression , 2. SeveSF Aβ42 , 4. The β42 CSF measurements from two widely used Aβ42 ELISA assays using the same CSF sample. Our data extends their findings by directly comparing human CSF samples from both diseased and non diseased subjects with two different widely-used assay platforms, one of which uses colorimetric detection, while the other employs electrochemiluminescence (ECL) detection. Comparisons were made to determine if the assays gave similar values and were able to distinguish CSF from Alzheimer's subjects or age-matched controls based.Schoonenboom et al. compared the AμL aliquots of each CSF sample were placed in randomly assigned wells in a lo-bind polypropylene tray . CSF was stored at −80°C until analysis. All samples were assayed in duplicate in both Aβ42 assays, conducted by the same experienced scientist.Human cerebrospinal fluid (CSF) samples were purchased from Precision Med, Inc. , which is in compliance with all applicable rules and regulations for human sample collection and dissemination. CSF samples from 23 individuals with AD  and 22 age-matched controls  were tested. To avoid any effects from multiple freeze-thaw cycles, 60 β-Amyloid1–42  is a solid-phase ELISA for measuring the levels in human CSF utilizing colorimetric detection with peroxidase-labeled streptavidin. The ELISA was performed as outlined by the manufacturer's instructions. Absorbance at 450 nm was measured on the SpectraMax M2 , and analysis was preformed using Softmax 5.2 software. The limit of detection (LOD) of 50 pg/mL was calculated by the manufacture as the mean of 8 determinations of the sample diluent.Innotest βx-42 assay  was preformed as outlined by the manufacturer's instructions. Analysis was performed using MSD workbench version 3.0.17.3 . An LOD of approximately 20 pg/mL was calculated by the manufacture based on data obtained from 4 different product lots (MSD 96-well MULTI-ARRAY Human (6E10) Auct lots . All calP value < 0.05 was taken to indicate statistical significance. Statistical testing for differences in mean Aβ42 level among the two diagnosis groups (AD and AMC) was made using a two-sided, two-sample t-test.All statistical analyses and graphics were performed using either SAS JMP version 8 or R version 2.9.0. A β1–42 ELISA standard curve showed a dynamic range of 125 to 2000 pg/mL, with an LOD of 50 pg/mL, and the average CV based on sample duplicates was 3.9%. The Aβx-42 ECL assay standard curve showed a dynamic range of 12 to 3000 pg/mL, with an LOD of approximately 20 pg/mL, and the average CV based on sample duplicates was 7.1% (data not shown). The Aβ42 levels in the 45 CSF samples determined by ELISA and ECL assay are shown in β42 levels of all the CSF samples were above LODs in both assays. The means of Aβ1–42 measured by ELISA were significantly lower in AD patients compared with AMC , (P < 0.0001*). The average CV based on sample duplicates was 6%. Mean Aβx-42 measured by ECL assay were also significantly lower in AD patients compared with AMC  (P < 0.0001*). The average CV based on sample duplicates was 8.4%. Although absolute concentrations varied between the ELISA and ECL, the correlation coefficient for CSF Aβ42 was r = 0.819 and highly significant (P < 0.0001) (The A 0.0001) .β42 levels in human CSF. Both the ELISA and ECL assay showed that Aβ42 was higher in the AMC than the AD group. These observations in subject differentiation are similar to other published reports [We provide here a direct comparison between two commonly used assays, ELISA and ECL assay, in measuring A reports , 4–6.β42 levels were 2.6-fold higher in the ECL assay relative to the ELISA. The observed differences may be related to a number of factors including the matrix , the purity of the calibrators, and differences in the affinity of the capture and detection antibodies [β1–42. In contrast, ECL assay uses an undisclosed antibody, which recognizes Aβx-42 as the capture antibody. There is also a lack of synchronization between the two assays due to the difference between the two calibrators. We conducted an experiment swapping the calibrators between the two kits. Both assays detected the other kits' calibrator; however, the % recovery was not at an acceptable level (data not shown).We observed that in general Atibodies , 7. The β42 concentrations are different and could be a contributing factor to the underlying difference. The ELISA signal is detected with a peroxidase-labeled streptavidin antibody and the result is colorimetric, which provides the desired sensitivity but less dynamic range. The ECL signal is detected by incorporating a SULFO-TAG labeled antibody that emits light upon electrochemical stimulation initiated at the electrode, which provides sensitivity similar to the ELISA but a broader dynamic range.Although both are plate-based methods, the detection technologies for measuring the CSF Aβ42 measurements [β42 in human CSF. The present study provides an important first step by comparing and establishing the relationship between two widely used platforms for measuring Aβ42.Currently, quality control efforts are under investigation to evaluate interlaboratory variance components and to aid in the standardization of CSF Aurements . In addi


Microglial activation and the proinflammatory response are controlled by a complex regulatory network. Among the various candidates, macrophage colony-stimulating factor (M-CSF) is considered an important cytokine. The up-regulation of M-CSF and its receptor CSF-1R has been reported in brain disease, as well as in diabetic complications; however, the mechanism is unclear. An elevated level of glycated albumin (GA) is a characteristic of diabetes; thus, it may be involved in monocyte/macrophage-associated diabetic complications.in vitro. Immunofluorescence, real-time PCR, immunoprecipitation, and Western blot analyses revealed the up-regulation of CSF-1R in GA-treated microglial cells. We also detected increased expression and release of M-CSF, suggesting that the cytokine is produced by activated microglia via autocrine signaling. Using an enzyme-linked immunosorbent assay, we found that GA affects microglial activation by stimulating the release of tumor necrosis factor-α and interleukin-1β. Furthermore, the neutralization of M-CSF or CSF-1R with antibodies suppressed the proinflammatory response. Conversely, this proinflammatory response was augmented by the administration of M-CSF.The basal level of expression of M-CSF/CSF-1R was examined in retinal microglial cells We conclude that GA induces microglial activation via the release of proinflammatory cytokines, which may contribute to the inflammatory pathogenesis of diabetic retinopathy. The increased microglial expression of M-CSF/CSF-1R not only is a response to microglial activation in diabetic retinopathy but also augments the microglial inflammation responsible for the diabetic microenvironment. Recent evidence strongly suggests that microglial activation plays a central role in the inflammation induced by experimental and human retinopathy -4. Microin vitro, suggesting that GA contributes to microglial inflammation in diabetic retinopathy. Chronic hyperglycemia in diabetes, through the nonenzymatic glycation of free amino groups in proteins by glucose, leads to the formation of labile Schiff base intermediates that undergo Amadori rearrangement, leading to the relatively stable early adducts ketoamine or fructosamine . Eventually, these Amadori products form irreversible advanced glycation end products (AGEs) . The lysates were centrifuged at 14,000 × 5 cells per well in 1 mL of serum-free medium with or without graded GA. The supernatant was collected at the indicated times after treatment with the conditioned medium from the control and experimental groups, and the amounts of TNF-α, IL-1β, and M-CSF were quantified by ELISAs , in accordance with the manufacturer's instructions. Each experiment was performed three times.Purified microglial cells were incubated overnight in 24-well plates at 2.5 × 10Statistical analyses were performed using one-way ANOVA, followed by post-hoc analysis with the Bonferroni  or Dunnett's test . All quantitative data are presented as the mean ± SD. Values of P < 0.05 were considered statistically significant.LW performed the experiments, designed the protocol, performed the statistical analysis and drafted the manuscript. XGZ and JCH designed the protocol and coordinated the study. TJ participated in the microglial culture. All authors have read and approved the final manuscript.The authors declare that they have no competing interests.


This review discusses recent evidence that shows PO acts via the multiple inositol polyphosphate phosphatase (MIPP) to regulate gene expression. As a consequence, PO inhibition causes both a transient, rapid increase in IP3 and a long-term elevation of IP signalling. This pathway is evolutionary conserved, being present in both the social amoeba Dictyostelium and human cell systems, and has potential implications for mental health.Inhibition of prolyl oligopeptidase (PO) elevates inositol phosphate (IP) signalling and reduces cell sensitivity to lithium (Li Bipolar mood disorder, also know as manic-depression, is a prevalent mental illness with a significant socio-economic impact. The biological causes of this major mental disorder remain unknown and, despite a strong genetic evidence for predisposition, there are currently no universally agreed consensus candidate genes that confer risk. Li+ is the most commonly used mood stabilizer used in the treatment of bipolar mood disorder and is particularly useful as a prophylactic, suppressing the re-occurrence of the illness. However, Li+ is taken in high doses, has side-effects and is not effective in all cases. Although a number of enzyme targets have been well characterised, how Li+ treatment mediates its action also remains unknown. This review describes recent results investigating how PO modulates cell sensitivity to Li+, and how this may relate to the treatment and molecular origins of bipolar mood disorder.An intriguing aspect of prolyl oligopeptidase (PO) biology is its relationship with bipolar mood disorder and lithium (Li+ identified was inositol monophosphatase (IMPase) [1A). The two most studied are PIP2 and PIP3 (PIP3) [2 are important in membrane trafficking in the cell [3 (IP3) by specific IP3 5’phosphatases to IP2 and then further dephosphorylation by inositol 1-polyphosphate phosphatase (IPP), a second lithium sensitive enzyme with a protein structure closely related to IMPase [1A). Alternatively myo-inositol is synthesised de novo by isomerisation of glucose-6-phosphate through the action of inositol synthase (Ino1) [+. This has the potential to deplete the cellular concentration of inositol, however in many cases inositol can be taken up from the environment via transporter proteins in the membrane [+ and other mood stabilizers may also lower inositol up-take potentially enhancing their effects [The first enzyme target of Li(IMPase) . This de3 (PIP3)  althoughed are PI,5P2 and  effects .2 is a substrate for the enzyme phospholipase C (PLC), which, following cell stimulation, releases the second messenger, IP3. This binds the IP3 receptor on the endoplasmic reticulum to release Ca2+ from its intracellular store [2+ elicits a range of cellular processes, including cell motility, cell survival and neurotransmitter release. Li+ is well documented in lowering IP3 and cellular Ca2+ levels in cultured cells, however its effects on the brain tissue can vary on cell type or species [PIPar store . This tr species , and may species .3. This is synthesised from PIP2 following stimulation of class 1 PI3 kinases (PI3Ks). PIP3 is also an important cell regulator being involved in many processes including cell growth, survival and chemotaxis [3 acts as a binding site for PH domain proteins, bringing them in contact with activator and effector proteins. For example, PIP3 promotes contact between the protein kinases  and PKB  allowing  phosphorylation and activation of PKB [However, there are other inositol containing signal molecules in the cell, and a particular interesting molecule is the phospholipid, PIPemotaxis . Once gen of PKB , 13.3 could be sensitive to Li+ had not been tested. However, a direct effect of Li+ on PIP3 has recently been shown in the social amoeba Dictyostelium [Dictyostelium is a single celled eukaryote with a close phylogenetic relationship to the animals. It grows as unicellular amoeba, but when depleted of nutrients enters a developmental program to form a multicellular fruiting body [2A). This is a highly differentiated structure comprising a small number of cell types. The majority of cells differentiate into spore cells, which are resistant to environmental stress, but germinate to release amoeba when conditions favour cell growth. The remaining cell types form the fruiting body structure that supports the spore head. Early Dictyostelium development is dependent on chemotaxis towards extracellular pulses of cAMP, which brings cells together into the multicellular aggregation. PIP3 based signalling is an important part of this process and is sensitive to Li+ .Surprisingly, until recently the notion that PIPostelium . Dictyosing body  . Exactly the same effects are seen in mutants that lack all five Dictyostelium PI3Ks, or cells treated with the PI3K inhibitor LY294002 [3 concentrations, phosphorylation of PKB or translocation of a GFP-fused PH domain showed that Li+ indeed suppresses PIP3 synthesis following cAMP stimulation [3 signalling is also suppressed in the human HL60 neutrophil-like cell line following stimulation with the ligand fMLP. Consistent with inositol depletion, over-expression of IMPase in Dictyostelium cells reverses the effects of Li+.LiLY294002 , 17. Meamulation . Similar+, IMPase over-expressing cells show higher levels of stimulated PIP3 than wild type cells, indicating that inositol synthesis is limiting for cell signalling. This comes as a surprise as the steady-state concentration of PIP2 is 200 times that of PIP3 and is unaltered by Li+ treatment. However, when newly synthesised PIP2 is measured, Li+ causes a 60% decrease that mirrors that of PIP3. Over-expression of IMPase reverses the effect of Li+, whereas PIP2 concentrations are higher than wild type cells in the absence of Li+ [2 that is sensitive to Li+ treatment.Interestingly, in the absence of Lie of Li+ . These r+ target is GSK-3, a highly conserved protein kinase which is involved in a plethora of cellular processes, including differentiation and development, growth and survival, chemotaxis and metabolism, as well as controlling glycogen synthesis [+ inhibits GSK-3 with a Ki of 2.0 mM for the GSK-3β isoform and an IC50 of 3.5 mM for the GSK-3α isoform [2+, and that this Mg2+ dependency is competed by Li+. As Li+ is a non-competitive inhibitor with regard to its substrate and ATP, it would appear that the Li+ sensitivity of GSK-3 is via a Mg2+-binding site that is distinct from the Mg-ATP binding site present in all other kinases [+ sensitive protein kinase.The second major Liynthesis . As a co isoform , 20. Bio kinases . Consist+ is phosphoglucomutase (PGM) [+ as IMPase and GSK-3. Interestingly, GSK-3 is a negative regulator of glycogen synthase, reducing glucose conversion to glycogen, and hence Li+ inhibition of GSK-3 would lead to decreased glucose. As glucose-6-phosphate is the substrate for Ino1, inositol synthesis would be expected to be sensitive to altered activity of both PGM and GSK-3. This suggests that Li+ treatment could potentially be able to reduce inositol biosynthesis via simultaneously inhibiting all three enzyme targets .A third enzyme target of Lise (PGM) . This en+ has also been suggested to have a non-enzymatic action by disruption of a protein complex containing β-arrestin2, the phosphatase PP2A and PKB [+ treatment releases PKB from the complex increasing its activity, and in turn phosphorylating and inactivating GSK-3 . Exactly how this works is unclear, and in fact it has frequently been reported that inhibition of GSK-3 feeds back to elevate its own phosphorylation via PKB [In contrast, Li and PKB . The com via PKB -26, so i+ has a variety of effects on Dictyostelium development, which can be distinguished by dose and timing. In addition to its effect on chemotaxis and PIP3 signalling, lower concentrations of Li+ cause altered multicellular development, where the one population of stalk cells enlarges at the expense of the spore head [Dictyostelium GSK-3 gene is mutated [Dictyostelium development and GSK-3 mutants are incapable of both cAMP signalling and chemotaxis [2A). However loss of GSK-3 activity has very different effects from the chemotaxis deficit that arises due to suppression of PIP3 signalling [ Dictyostelium offer an easy means of distinguishing between different lithium targets on the basis of altered cell behaviour, something that is often difficult with other cell systems.Liore head . This is mutated . GSK-3 iemotaxis  . The first mutant to be fully characterised was lisA. This mutant arose from an insertion into the Dictyostelium PO gene, DpoA, and lacked all PO activity. Importantly, no suppression of the GSK-3 mutant phenotype was seen when GSK-3 and DpoA mutations were combined. However, loss of dpoA or treatment with PO inhibitors elevates IP3, suggesting that PO is a modulator of IP signalling [Using resistance to Li mutants   is a histidine acid phosphatase that dephosphorylates the higher order inositol phosphate IPmediates -35. It amediates . Withoutmediates , 38, and3 [3 following PO inhibition [3 concentrations. Consistent with these observations, MIPP mutants are Li+ hypersensitive, showing a significantly stronger effect of Li+ on chemotaxis . Mutants that combine both DpoA and MIPP mutations are Li+ hypersensitive, not resistant, placing MIPP genetically downstream of DpoA [Mutants lacking DpoA have decreased MIPP activity, leading to increased IP3 , whereashibition . This su of DpoA .3 concentrations rise by 30 minutes and then drop to basal values within an hour. Both in vitro and in vivo assays therefore indicate a rapid change of MIPP activity following loss of PO activity.The DpoA-MIPP interaction can be reconstituted in a cell free extract using recombinant PO and a semi-purified MIPP enzyme made from cells over-expressing MIPP. Addition of PO inhibits MIPP activity, and can be reversed by inclusion of a PO inhibitor. How this is actually achieved is unclear. Western blotting shows no increase in mobility of the MIPP enzyme, indicating that it is not directly cleaved. As MIPP is prepared as a membrane fraction, it would seem likely that direct target of PO is a co-purified peptide, whose current identity is unknown. In whole cells, IP3 following PO inhibition directly leads to Li+ resistance, there are a number of observations that argue that this is not the case [+ is an uncompetitive inhibitor of IMPase, elevating the concentration of its inositol phosphate substrates should enhance rather than alleviate Li+ inhibition [+ resistance, as seen for Li+ inhibition of PIP3. Second, in contrast to DpoA, genetic manipulation of MIPP, either by gene disruption or over-expression, has no long-term direct effect on IP3 concentration, however both have Li+ hypersensitive phenotypes. This argues that whilst DpoA mediated changes in MIPP activity lead to transient changes in IP3 levels, it is not the end point of the signal pathway, but in fact lies en route to more long-term changes in cell behaviour.Although tempting to assume that the rise in IPthe case . First, hibition . Only in3 5’phosphatase genes) [+ resistant, as consistent with an uncompetitive mode of inhibition by increasing enzyme rather than substrate concentration. DpoA mediated gene expression is dependent on MIPP, as PO inhibitors no longer elevate gene expression in a MIPP mutant strain.Further investigation showed that PO inhibition leads to decreased expression of the genes encoding inositol synthesis (IMPase and Ino1) and inositol recycling (IPP and IPe genes) . Elevatie genes) , and inc+ hypersensitivity and lowers concentrations of IP5 and IP6. IP5 has previously been shown to regulate ino1 and pho gene expression in yeast [via the extracellular protein ligand Wnt [5 or a related higher order inositol phosphate, over-expression of the IP3 kinases, inositol polyphosphate multikinase and inositol 1,3,4-triphosphate 5/6 kinase, which produce IP5, elevate Ino1 and IMPase gene expression and confers Li+ resistance [5 and IP6 regulate ino1 gene expression in yeast by regulation of chromatin re-modelling via the ATP-dependent re-modellers Swi/SNF2 and Ino80 [5 and IP6 signalling and chromatin remodelling.The exact mechanism leading to gene expression changes is not fully worked out, however there are some strong pointers to other elements of the signalling pathway . Over-exin yeast , and hasgand Wnt . Consistsistance . Furthernd Ino80 , 42. TheDictyostelium observations reveal the existence of a novel signalling pathway that is mediated via changes in PO and MIPP activity, however, is this a conserved pathway? A number of observations link PO activity to changes in IP signalling and Li+ sensitivity in mammalian systems.These 3 receptor type 1 protein expression, suggesting a regulatory relationship between PO and IP3 signalling [In rats and humans, PO activity and expression has been found throughout the tissues of the body, but it is enriched in the brain. Looking at the brain in more detail, it is clear that there is differential enrichment within the brain, so that higher PO levels are seen in hippocampal CA1 pyramidal cells, cortical interneurons and pyramidal cells, striatal spiny neurons, cells in the thalamus and cerebllar Purkinje cells . Interesgnalling , 45.3 signalling in response to Substance P [+ mediated growth cone spreading is reversed by either addition of myo-inositol or PO inhibition [+ through an inositol-depletion mechanism [+ action on these processes is suppressed by PO inhibition.In astroglioma cells, treatment with PO inhibitors or PO-specific siRNA elevates IPstance P . In primhibition . Finallyechanism . Again L5, IP6 or chromatin re-modelling is also required for this signalling pathway.In addition, PO inhibitor treatment of HEK293 cells leads to decreased expression of the human homologues of Ino1 (ISYNA1) and IMPase (IMPA1 and IMPA2). This gene regulatory effect is suppressed by MIPP1 specific siRNAs . These rDictyostelium, DpoA expression and PO activity is developmentally regulated, being significantly up-regulated just prior to the formation of multicellularity [3 concentration. This suggests that PO may also mediate gene regulation in alternative signalling pathways to that modulating inositol biosynthesis.In lularity . It is nlularity . Interes+, at least two other mood stabilizers, valproic acid (VPA) and carbamazepine (CBZ), alter neuronal cell behaviour in the same way as Li+, and again can be reversed by either addition of myo-inositol or PO inhibition [+, but has inositol phosphate signalling in common.What insights do these observations offer into the origins of bipolar mood disorder? In addition to Lihibition . Similarhibition . The cel+ treatment.Altered PO activity has previously been linked to mental disorders. PO activity is decreased in schizophrenia patients and decreased in depression , 51. In The cause of decreased PO activity is unknown, however high doses of the cytokine IFN-α in patients undergoing immunotherapy have been associated with lowered serum PO activity; it has been suggested that this is associated with symptoms of depression . Mood diLooking at the genes regulated by PO in HEK293 cells, it is notable that IMPA2 has previously been associated with bipolar mood disorder. Elevated IMPA2 expression has been reported in post-mortem patient brains . This co3). One scenario, for example, could arise if an environmentally induced-response, perhaps via IFN-α, lead to decreased PO activity and increased IMPA1 and ISYNA1 gene expression. This would interact with aberrant regulation of the IMPA2 promoter to cause substantial elevation of inositol biosynthesis and over-active IP signalling, which in turn could be suppressed by Li+ or other mood stabilizers. Such enhanced signalling is seen in Dictyostelium when both the IMPase and Ino1 genes are over-expressed [+.On the other hand, SNP analysis has failed to find a significant association of PO alleles and risk of bipolar disorder . One intxpressed . This ca5 and IP6, and regulates gene expression. IP3 is both decreased transiently following PO inhibition through decreased MIPP activity, and in the long term via decreased gene expression. This signalling pathway appears to be conserved in human cells, and has a number of characteristics that associate with observations on mental disorders. However, further work investigating the action of PO in model systems, as well as more genetic studies, will be required to establish the validity of these associations.PO interacts with a novel intracellular signalling pathway, which regulates gene expression. This pathway requires the enzyme MIPP, which acts to regulate IP


APOE ε4 allele genotype which has been associated with poor outcome after TBI.Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed.APOE ε4 allele had similar biomarker concentrations as non-carriers.CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL . No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele was not found to influence biomarker levels after acute TBI.Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the  Sports-related mild traumatic brain injury (TBI) is a common health issue at emergency departments We have recently shown in the same cohort, that repeated subconcussive trauma in amateur boxing (with head guard) causes a mild TBI, diagnosable by elevation of the brain injury biomarkers Neurofilament Light Protein (NFL), Glial Fibrillary Acidic Protein (GFAP), Total-tau (T-tau) and S-100B in the cerebrospinal fluid (CSF) Other biomarkers may also be useful in detecting mild TBI.Neurofilaments (NF) are exclusively found in neurons. As they are mainly involved in maintaining neuronal shape and size and conduction of nerve impulses along the axons APP are expressed in neurons and have neurotrophic functions in promoting neuronal survival after axonal damage Apolipoproteins are lipid transporters with six different subclasses . Apolipoprotein A1 (ApoA1) is the major protein component of high-density lipoprotein in plasma. In the CNS, ApoA1 may be a marker of neural degeneration and increased CSF concentrations have been seen in patients with AD, Parkinson's disease and multiple sclerosis APOE gene is located on chromosome 19 and has three common alleles  where the presence of APOE ε4 is associated with unfavorable outcome after TBI APOE ε4 is a well-known risk factor for AD APOE ε4 in combination with TBI is suggested to additionally increase the risk for developing AD The APOE ε4 genotype influences biomarker concentrations after brain trauma.Although the available evidence suggests that pNFH, APPs and apoliproteins are affected by TBI, most of these markers have only been investigated after severe TBI or in animal models. To the best of our knowledge, none of these markers have been investigated in CSF after a mild TBI in the absence of obvious symptoms. The primary aim of the present study was to investigate whether pNFH, amyloid precursor proteins, or apolipoproteins in CSF and/or blood are useful biomarkers in the diagnosis and monitoring of mild TBI, The study was performed in the same cohort of boxers as our previous biomarker studies The study was designed as a prospective follow-up study. Thirty amateur boxers competing at elite level were compared to 25 healthy, age-matched controls. All boxers had completed at least 45 bouts. This number was based on the regulation of the Swedish National Boxing Federation demanding an examination with MRI, CT or EEG every 50 bouts. The control group consisted of friends or relatives to the boxers, aiming to get control subjects with a similar social background and education level to the boxers. Exclusion criteria were athletes at senior elite level in sports where head trauma may occur, e.g., soccer, ice hockey and martial arts.The Regional Ethical Review Board in Linköping, Sweden approved the study. Written informed consent was obtained from all participants.All participants filled in a questionnaire about medical history, medication, education, present occupation, information about previous concussions and quantification of alcohol and drug intake. Previous sports career was reported, to identify those who had trained in sports with risk of TBI. The questionnaire included a 10-question survey regarding previous and current symptoms of head and neck injuries based on a previous study All participants underwent a neurological examination. The neurological examination protocol included anamnestic questions about concussion symptoms, a general somatic status  and neurological status . Magnetic resonance imaging (MRI) of the brain and neuropsychological testing  were performed in all participants without the findings of any structural injuries  or other major pathological findings observed. Detailed results of these investigations will be presented in a separate paper.The lumbar puncture was performed at daytime, between 10 a.m. and 3 p.m., with the study objects in sitting position or lying on one side. For the first 18 participants a Quincke Type Point spinal needle (22 Gauge) were used, but since a few of the study participants suffered from postspinal headache, the needle was changed to a Sprotte (24 Gauge). Thereafter no more postspinal headache occurred. For each study participant 5 to 10 ml CSF was collected in a polypropylene tube , gently mixed to avoid gradient effects, aliquoted and stored at −80°C pending analysis. LPs were performed twice in the boxers: First LP 1–6 days after a bout (test A) and the second without exposure to bouts or training with blows to head for at least 14 days (test B). The control subjects underwent one LP.Blood was collected by venipuncture into whole blood and gel-separator tubes. The samples were centrifuged within 20–60 minutes, aliquoted and stored at −80°C pending analysis. Venipuncture were performed twice in the boxers: First 1–6 days after a bout (test A) and the second without exposure to bouts or training with blows to the head for at least 14 days (test B). The control subjects underwent one venipuncture.CSF NFH was analyzed using a sandwich ELISA  by a person blinded to the experimental groups. CSF AβX-38, AβX-40 and AβX-42 levels were measured by the electrochemiluminescence technology using the MS6000 Human Abeta 3-Plex Ultra-Sensitive Kit, while β-secretase cleaved soluble APP (sAPP-β) and α-secretase cleaved soluble APP (sAPP-α) were measured using the MS6000 Human sAPPalpha/sAPPbeta Kit , as decribed previously APOE (gene map locus 19 q13.2) genotyping was performed using TaqMan® Allelic Discrimination technology . Genotypes were obtained for the two SNPs that are used to unambiguously define the ε2, ε3, and ε4 alleles (rs7412 and rs429358).Statistical analysis was carried out with SPSS 16.0. Comparisons between groups were performed using the non-parametric Mann-Whitney U-test, as some of the biomarker concentrations were skewed. Comparison between test A and test B were performed with Related Samples Wilcoxon Signed Rank Test. Correlation analyses were performed with Spearman two-tailed test.All participants were included in the CSF biomarker analysis 1 to 6 days after a boxing bout (N = 30). Two participants with complications after the lumbar puncture  and two without complications declined follow-up (N = 26 at follow-up).Baseline factors about medical and social history and the 10-question survey were similar between boxers and controls and detailed results of the questionnaire have been published previously The results of pNFH are presented in There was no correlation between pNFH and boxing exposure (r = 0.251).There was a slight increase in median ApoA1between timepoint A (1 to 6 days) and B (≥14 days) for the boxers (p = 0.006), but there was no significant difference between boxers and controls. For the biomarkers sAPPα, sAPPβ and the Aβ-isoforms Aβ N-38, Aβ N-40 and Aβ N-42, no differences were seen between the different timepoints for the boxers, or between the boxing and control groups. The results are presented in detail in No significant differences were seen between the groups at any of the two time-points .The results from the analyses in the present study were compared to previously analyzed biomarkers on the same study cohort APOE ε4 carriers vs 7 of the controls. When comparing the biomarker concentrations presented in this paper and the CSF biomarkers NFL, GFAP, P-tau and S-100β Twelve of the 30 boxers were APOE ε4 genotype influences biomarker concentrations after brain trauma. The study was performed in the same cohort of boxers as our previous biomarker studies which enabled us to correlate the findings with previously detected biomarkers.The purpose of the study was to investigate whether pNFH, amyloid precursor proteins, or apolipoproteins in CSF and/or blood are useful biomarkers in the diagnosis and monitoring of mild TBI to analyze whether the pNFH in CSF increases after a amateur boxing bout (with head guard) and does not normalize within 14 days of rest, even though none of the boxers had suffered from KO and only one of the boxers had concussion symptoms (headache) after bout. None of the CSF biomarkers APP, Aβ isoforms, ApoA1, Apo E and serum Aβ demonstrated any pathological concentrations.The CSF pNFH levels significantly correlated with earlier analyzed CSF NFL This study suggests that the CSF biomarkers APP, Aβ isoforms, Apo E and serum Aβ are not useful in the diagnosis or monitoring of mild TBI or early stages of chronic TBI. However, ApoA1 significantly increased between test A and B for the boxers, although there was no significant difference between boxers and controls. This is interesting, since ApoA1 is believed to be a marker of neural degeneration and increased CSF concentrations have been seen in patients with neurodegenerative disorders such as AD, Parkinson's disease and multiple sclerosis The lack of association between mild TBI and the amyloid-related biomarkers is also interesting given that earlier studies have established a relationship between severe TBI and abnormal amyloid metabolism APOE ε4 carriers versus non carriers APOE ε4 carrier status did not influence the CSF concentrations of pNFH. Furthermore, we did not find any relationship between APOE ε4 carrier status and any of the earlier published concentrations of NFL, GFAP, T-tau or S-100β in this study group APOE ε4-positive group.A recent study on severe TBI showed higher concentrations of S-100β in the CSF of The biomarkers identified by this study and our earlier published studies with the same cohort Ice hockey and soccer are examples of popular sports with high risk for concussions and it is known that coaches and players do not always follow the return-to-play guidelines APOE ε4-carrier status did not influence biomarker concentrations after acute TBI. Thus, sub-concussive repetitive trauma in amateur boxing causes a mild TBI, and without proper rest there might be an increased risk for long-term consequences.In conclusion, pNFH significantly increased between 1 and 6 days after an amateur boxing bout (with head guard). After a rest period of at least 14 days, the concentrations decreased but were still significantly elevated compared to controls. The results of this study, and our previous studies in the same cohort, provide insight into the pathophysiology of sports-related mild TBI. Moreover, they suggest that pNFH, NFL, GFAP, T-Tau and S-100β