GN-Nitroarginine Methyl Ester (L-NAME) significantly reduced the effects of G-CSF on rCBF, NO surge, infarct volume, and neurological deficits. We conclude that G-CSF increases rCBF through a NO surge mediated by Akt/eNOS, which partially contributes to the beneficial effect of G-CSF on brain I/R injury.Granulocyte-colony stimulating factor (G-CSF) protects brain from ischemic/reperfusion (I/R) injury, and inhibition of nitric oxide (NO) synthases partially reduces G-CSF protection. We thus further investigated the effects of G-CSF on ischemia-induced NO production and its consequence on regional cerebral blood flow (rCBF) and neurological deficit. Endothelin-1 (ET-1) microinfused above middle cerebral artery caused a rapid reduction of rCBF (ischemia) which lasted for 30 minutes and was followed by a gradual recovery of blood flow (reperfusion) within the striatal region. Regional NO concentration increased rapidly (NO surge) during ischemia and recovered soon to the baseline. G-CSF increased rCBF resulting in shorter ischemic duration and an earlier onset of reperfusion. The enhancement of the ischemia-induced NO by G-CSF accompanied by elevation of phospho-Akt and phospho-eNOS was noted, suggesting an activation of Akt/eNOS. I/R-induced infarct volume and neurological deficits were also reduced by G-CSF treatment. Inhibition of NO synthesis by L- Cerebral stroke is a medical emergency which has been one of the leading causes of death and disability worldwide. Cerebral ischemia/reperfusion (I/R) injury is most frequently observed in the vascular territory of the middle cerebral artery (MCA). Therapeutic intervention by reestablishing blood flow is an essential strategy. Therapeutic benefit of thrombolytics such as recombinant tissue-plasminogen activator (rt-PA) remains limited by hemorrhagic side effects and narrow therapeutic time window . A neuroGranulocyte-colony stimulating factor (G-CSF) is a cytokine that can penetrate the blood brain barrier , 3 to exNitric oxide (NO) is a key regulator of vascular tone . It is kAll experimental protocols were approved by the Animal Care and Use Committee of the Tzu Chi University, Taiwan, in accordance with guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed under a 12-hour light/dark cycle with free access to food and water. Utmost efforts were made to minimize the suffering and the number of animals used.Male Sprague-Dawley rats (250 g to 350 g) were anesthetized by intraperitoneal pentobarbital  or urethane . Under anesthesia, a femoral cannula (PE-50 polyethylene tube) was inserted for the monitoring of arterial pressure. The arterial pressure was recorded with MP35 amplifier . Body temperature was maintained automatically with a heating pad  at 37.5 ± 0.5°C. Animal was placed in a Stoelting stereotaxic apparatus  during the induction of ischemia and real-time measurement of NO level or rCBF, respectively. A midline incision of the scalp was made to expose the skull, and two burr holes were drilled for insertions of microinjection tube for ischemic induction and insertions of laser Doppler Flowmeter probe or NO-selective electrode, respectively. For evaluation of the neurological deficit, animals went through the same procedures but without the femoral cannulation.μL of saline was microinfused (1 μL/min for 10 minutes) via a 30-gauge needle positioned in proximity to the root of MCA (stereotaxic coordinates: anterioposterior (AP) 0.0 mm, mediolateral (ML) 5.2 mm, and dorsoventral (DV) 8.0 mm relative to bregma). A >75% reduction of rCBF resulting from the ET-1 infusion served as the criterion of successful induction of ischemia.Endothelin-1  was used to induce transient vasoconstriction of the MCA to achieve I/R as described previously –16 with n = 7), in which 10 μL of saline was microinfused (1 μL/min) proximate to the root of MCA at 0 min; Group 2: G-CSF (n = 11), in which 200 μg/kg of G-CSF , dissolved in 0.5 mL normal saline, was subcutaneously injected at 0 min without I/R induction; Group 3: I/R (n = 22), in which ET-1, 400 pmol in 10 μL saline, was microinjected (1 μL/min) proximate to the root of MCA; Group 4: I/R + G-CSF, in which G-CSF 50 μg/kg (n = 4 for measurement of blood flow) or 200 μg/kg , dissolved in 0.5 mL, was subcutaneously injected immediately followed by the ET-1 infusion; Group 5: NOS inhibitor + I/R + G-CSF (n = 17), in which NOS inhibitor L-NG-Nitroarginine Methyl Ester  30 mg/kg, dissolved in 0.5 mL saline, was intraperitoneally administered 30 minutes prior to I/R + G-CSF administration. Rats had free access to food and water before the experiments.Rats were randomly assigned as groups 1 to 5: Group 1: sham control  inserted during surgery and connected to a Laser Doppler Blood Flow Perfusion Monitor 403A . The probe was placed in the striatum at AP 0.0 mm, LM 3.0 mm, and DV 5.0 mm. The rCBF data of the baseline control period were normalized by averaging rCBF values over a 10-minute time period before drug administration by using the following formula: percentage change = [(df)/dF]−3, 10−4, 10−5, and 10−6 M) of the stable NO donor standard, S-nitroso-N-acetyl-DL-penicillamine  [Regional NO levels were monitored with a NO-selective sensor probe that consisted of porphyrin-electroplated, Nafion-coated, and carbon fiber electrodes  connected to a NO detector  as described previously . The ele, Japan) . The caln = 4 for each group) for Western blot were performed as described previously with some modifications [μg) were loaded onto 8%-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed, followed by blotting of the protein onto a polyvinylidene difluoride (PVDF) membrane , which were then blocked with 5% nonfat milk in 0.05% Tween-Tris-buffered saline. The membrane was then probed with primary antibodies overnight at 4°C with gentle rotation. The primary antibodies and concentrations used were as follows: phospho-eNOS (Ser 1177) , eNOS , phospho-nNOS (Ser 1416) , nNOS , phospho-Akt (Ser 472/473) , Akt , and β-actin . Following washing and incubation with the respective secondary antibodies  for 1 hour at room temperature, the membranes then reacted with chemiluminescent ECL Plus Western blotting detection system . The bands were visualized by exposure to X-ray films  and developed later. Intensities of bands were quantified with a densitometric analysis system  and calculated as the optical density × area of band.Rats were decapitated under deep anesthesia at 0, 15, and 30 minutes and 1 hour after various treatments. The brains were rapidly removed and the ipsilateral striatal was separated and rapidly frozen in liquid nitrogen and stored at −80°C until further analysis. Protein samples  for 20 minutes at 37°C in the dark. The sections were photographed and the unstained area  was quantitated using Image J . These sections were also used for verification of correct positioning of the ET-1 infusion tube, laser Doppler probe, or NO selective sensor probe.Modified Neurological Severity Score (mNSS) was evaluated before (0) and on 1, 2, 3, 7, and 14 days after I/R in I/R, I/R + G-CSF and L-NAME + I/R + G-CSF, respectively. Neurological function is graded on a scale of 0–18 . The mNSS is a composite of motor, sensory, reflex, and balance tests .t-test. The statistical comparisons among multiple groups were made using one way ANOVA followed by Bonferroni correction. In all instances, n refers to the number of animals in a particular group. A P value of less than 0.05 was considered statistically significant.All values  are presented as means S.E.M. and analyzed by Prism software for Student's n = 8, 50) was 39.9 ± 1.2 minutes  subcutaneously given at the onset of ischemia shortened the TFR50 resulting in an earlier reperfusion , it was adopted for subsequent experiments  and induced an early reperfusion as evidenced by the shorter TFR50 (P < 0.001). The basal systemic blood pressure was 101.6 ± 3.0 mmHg (n = 34), and no significant differences existed between the I/R and I/R + G-CSF rats (data not shown). These results indicated an accelerated recovery of blood flow by G-CSF.Intracerebral infusion of ET-1 to the proximity of MCA reduced rCBF immediately by 75% of the basal within 5 minutes (resulting ischemia) and maximally by 86.7% within 8.1 ± 0.6 minutes  and reversed the TFR50 from 24.0 ± 2.2 minutes (I/R + G-CSF 200 μg/kg) to 44.8 ± 6.1 minutes (L-NAME + I/R + G-CSF) (P < 0.01). Control animals with saline, G-CSF, or L-NAME alone had no significant effect on rCBF (data not shown). These results indicated that NO produced by NOS might participate in the effect of G-CSF on rCBF during I/R.Ample evidence suggests that NO production from eNOS is neuroprotective –22, espe in vivo voltammetry [P < 0.05). G-CSF also significantly increased cumulative regional NO concentration during the 0–15- and 16–30-minute periods (P < 0.05), but not 31–45- and 46–60-minute periods (P < 0.05).We further measured the changes of NO by real-time NO detection in the striatal tissue via a NO-selective sensor probe coupled with antammetry . Regiona periods . As compThese findings might indicate a new mechanism by which G-CSF reduces ischemic damage by promoting early recovery of rCBF through a fast production of NO, the NO surge. NO in turn will dilate cerebral blood vessels and improve collateral blood flow .P < 0.05). As compared with I/R group, G-CSF treatment (I/R + G-CSF) significantly increased the levels of phosphorylated eNOS to 15.66 ± 4.82-fold at 15 minutes (P < 0.05), and the elevation diminished at the 30- and 60-minute period in both I/R and I/R + G-CSF groups.To elucidate the mechanism of G-CSF-induced NO surge in ischemic brain tissue, we delineated the Akt/eNOS signalling pathway by western blotting Figures  and 4. TP < 0.01). As compared with I/R group, G-CSF treatment (I/R + G-CSF) had no significant effects on phosphorylated nNOS expression at 15 minutes but reduced the phosphorylated nNOS expression at 30 (P < 0.01) and 60 minutes (P < 0.05)  .P < 0.01). As compared with I/R group, G-CSF treatment (I/R + G-CSF) significantly increased the expression of phosphorylated Akt to 9.46 ± 2.34-fold at 15 minutes, but there were no differences at 30 and 60 minutes, respectively (P < 0.01). The G-CSF treatment (I/R + G-CSF) group had no significant effects on total eNOS, nNOS, and Akt expression as compared with I/R group (data not shown).The I/R increased the levels of phosphorylated Akt to 3.97 ± 1.24-, 7.23 ± 1.99-, and 2.57 ± 0.39-fold, respectively, at 15, 30, and 60 minutes  were considered in the G-CSF-induced enhancement of NO production, because the G-CSF effects on NO production were immediate . The cat in vivo context.Previous study has indicated that eNOS can be activated by the serine/threonine protein kinase Akt (protein kinase B) on serine 1177, leading to the production of NO , which p3) as compared to I/R group (287.32 ± 50.51 mm3) (P < 0.05). Neurological deficit test also indicated that G-CSF treatment  significantly reduced the mNSS . L-NAME pretreatment  blunted the neuroprotective effects of G-CSF in reducing total infarct volume (P < 0.05 as compared to I/R + G-CSF group) and mNSS .μg/kg body weight G-CSF over a period of 72 hr, for the treatment of acute ischemic stroke which occurred within 9 hours of symptom onset. However, G-CSF failed to meet the primary  and secondary  endpoints in AXIS 2. As discussed by the authors, the reasons for the failure of G-CSF remain obscure. As opposed to 5 consecutive subcutaneous injections of ~10 μg/kg body weight/day in treating neutropenia, the extraordinary high dose of G-CSF infusion in AXIS 2 was proved to be safe in a previous IIa trial, and thus the dose might not be the cause of failure.Although ample animal studies and several human trials have demonstrated the potential of G-CSF as a candidate for early stroke drug, the failure of AXIS 2 is a huge setback . AXIS 2 The decrease in mean arterial blood pressure (MAP) and increase in heart rate were unexpected in G-CSF-treated patients. The authors speculated that the decrease in MAP was caused by the direct effect of G-CSF on the vasculature with a lowering of peripheral resistance. Since NO can cause vessel dilation, it will be interesting to learn if G-CSF, when given as early as possible after stoke onset, can help in the recovery of rCBF. Our current study also shows that a NO surge that occurs immediately after stroke is beneficial. It will also be of great interest to learn if G-CSF can exert its therapeutic effect when given simultaneously in the rt-PA therapy. Thus, it is still too premature to deny the application of G-CSF in early stroke therapy.G-CSF is beneficial in terms of promoting early recovery of rCBF, reduction of total infarct volume, and neurological deficit. We have provided evidence supporting enhanced NO production via the activation of Akt/eNOS pathway as the probably mediator of such beneficial effects. Taken together, our results suggest that G-CSF may have the potential as a novel therapeutic agent or part of a therapeutic regimen for acute stroke.


Ascertainment of the pattern and temporal change of biomarkers in preclinical (asymptomatic) sporadic Alzheimer's disease (AD) will increase knowledge about early pathogenesis and facilitate interventional therapeutic trials.In this prospective longitudinal study, repeated cerebrospinal fluid (CSF) collections and cognitive evaluations were performed in cognitively healthy elderly individuals during a 9-year period.42 levels predicted subsequent development of clinical AD 9 years later. Noteworthy, one-third of individuals with pathologically low baseline Aβ42 levels remained cognitively intact during follow-up. No further decrease in Aβ42 was seen in those with low levels already at baseline.Low CSF β-amyloid (Aβ)42 predicts sporadic AD at least 9 years before dementia onset and has plateaued already at this time. However, many individuals can harbor brain amyloid accumulation over a decade without signs of cognitive deterioration, which could implicate how CSF biomarkers are used to identify preclinical AD in future interventional therapeutic trials.CSF Aβ Increasing evidence suggests that cerebral accumulation of β-amyloid (Aβ) can be detected 5–20 years before dementia onset in AD, when using cerebrospinal fluid (CSF) AβSeveral studies imply that CSF can identify cognitively healthy elderly individuals that are at increased risk of subsequent development of cognitive decline In this prospective and longitudinal study, we investigated CSF biomarkers repeatedly over 9–10 years in individuals, who all were cognitively healthy at baseline. The cognitive performance and development of dementia were determined during the clinical follow-up. Great care was taken to minimize drop-out during the study.22.1The objective of this study was to model within-person neurodegenerative biomarker trajectories in preclinical AD using repeated assessments of CSF biomarkers and cognitive performance as well as to investigate the predictive ability of CSF biomarkers to identify future development of clinical dementia. It is a prospective, longitudinal, observational study on initially cognitively healthy elderly volunteers recruited through advertisement in year 2002 in the city of Malmö, Sweden 2.2In total, 62 individuals could be recruited of which 54 performed baseline lumbar puncture and CSF collection. All participants also underwent comprehensive examination including physical, neurologic, and psychiatric evaluation, computed tomography (CT) of the brain, and cognitive testing at baseline. Cognitive follow-up was offered after 3, 5, and 9–10 years with renewed lumbar puncture after 5 and 9–10 years. Individuals with baseline CSF values and clinical cognitive follow-up after 9 years (n = 44) were included in the main analyses of the present study . Only a In the subgroup of participants who were not available for the 9-year follow-up visit n = 10, , medical2.3Cognitive testing included MMSE  Cognitive diagnosis was based on clinical evaluation by a physician experienced in dementia disorders and was later confirmed by a consensus group of experienced physicians. The consensus group was blinded to biomarker values and had only access to medical history, cognitive test results, and CT scan results. Diagnosis criteria used in regular clinical settings were applied, i.e. MCI 2.4g at 4°C for 10 minutes to eliminate cells and other insoluble material. Pending biochemical analyses samples were immediately frozen and stored at −80°C without being thawed or refrozen.Lumbar puncture was performed in a sitting position with CSF obtained from the L3/L4 or L4/L5 interspaces. All CSF were collected in plastic (polypropylene) tubes, gently mixed to avoid gradient effect. Samples were then centrifuged at 2000 × 42, total tau (T-tau), and phosphorylated tau (P-tau) using xMAP technology  was performed using the same batch of reagents for each CSF acquisition. A large random sample of baseline and 5-year follow-up CSF was analyzed together with 9-year follow-up CSF to assure concordant assay values between all three analysis occasions. CSF values are given in nanograms per liter.Analysis of Aβ2.5The study was approved by the regional ethics committee at Lund University. All participants gave their written informed consent at baseline and at each follow-up.2.6APOE) ε4 allele. Finally, a model including CSF levels, delayed word recall, and all covariates were created. Results are presented as hazard ratio (HR) with the 95% confidence interval (CI). For baseline CSF levels, standardized z-scores were used so that HR reflects change per one standard deviation (SD). Cox regression models were performed on all participants with baseline CSF measurements and at least one cognitive follow-up (n = 53), thus including the individuals that died during the follow-up period.IBM SPSS statistics version 22 was used for statistical analysis. Nonparametric tests were used because of the low number of cases and the nonnormal (Gaussian) distribution of CSF biomarker levels. Differences between diagnosis groups were calculated with Kruskal-Wallis test followed by Mann-Whitney U test when applicable. Mann-Whitney U test was used for dichotomized variables. Paired samples were analyzed using Wilcoxon signed-rank test. Cox regression models were set for prediction of AD and DLB as well as cognitive impairment. Baseline CSF levels and baseline delayed word recall score were added separately to the model with the following covariates: age, gender, and presence of apolipoprotein E  were included in the main analyses of the present study, as described in 3.242 levels at baseline compared with the cognitive stable individuals and OD individuals, with MCI at intermediate levels  had CSF Aβ42 levels compared with normal levels for prediction of subsequent development of AD or DLB. Cox regression model revealed that cognitively healthy individuals with low baseline Aβ42 levels had an increased risk of subsequent AD or DLB with an HR of 11.9  for each SD decrease of CSF Aβ42 levels when adjusted for age, gender, presence of APOE ε4 allele, and baseline delayed word recall. In contrast, CSF tau and P-tau did not predict development of AD/DLB over 9 years and the ratios of CSF Aβ42/tau or Aβ42/P-tau did not improve the predictive ability compared with CSF Aβ42 alone.3.342 at baseline  and four had normal baseline levels . Thus, CSF Aβ42 predicted cognitive impairment in general (MCI or dementia) with a sensitivity of 67%, specificity of 88%, positive predictive value of 67%, and negative predictive value of 88%. Cox regression model revealed that cognitively healthy individuals with low baseline Aβ42 levels had an increased risk of subsequent cognitive impairment with an adjusted HR of 3.9  for each SD decrease of CSF Aβ42  developed some form of cognitive impairment during the 9-year follow-up . Of thesCSF Aβ42 B.3.4P > .05). However, the group of individuals that developed AD/DLB had significantly lower delayed memory scores already at the 5-year follow-up compared with those in the other groups . When added to the Cox regression model, baseline delayed word recall score (ADAS-cog item 3) contributed to predict development of AD and DLB with HR of 2.8  for each not correctly given word. However, this predictive ability disappeared if CSF Aβ42 levels were removed from the model. This is in contrast to CSF Aβ42 that remained an independent predictive factor irrespective of delayed word recall. Hence, CSF Aβ42 levels is the driving predictive factor 9 years before diagnosis in this cohort, and episodic memory only contributes if amyloid status is taken into account.No difference in baseline cognitive test results was seen between the individuals that developed AD/DLB at follow-up compared with those who remained cognitively stable  and at follow-up after 9 years  compared with the cognitively normal individuals with normal baseline CSF Aβ42 levels.Individual demographics and cognitive follow-up data, for each of the participants with low baseline CSF Aβ3.642 levels  and an increase in CSF T-tau levels  between baseline and follow-up after 9 years, whereas CSF P-tau remained stable. Baseline CSF Aβ42 levels correlated negatively with baseline CSF P-tau levels . Baseline CSF P-tau levels also correlated negatively with CSF Aβ42 levels at follow-up . Moreover, baseline CSF P-tau levels were significantly higher in the individuals with low baseline CSF Aβ42 when compared with those with normal baseline CSF Aβ42 , but not at follow-up 9 years later.Thirty-six of the individuals with CSF measurements at baseline had at least one repeated CSF acquisition during follow-up period (year 5 or year 9), of which 23 individuals had from all three occasions . On grou42 levels were observed in 10 of these individuals and no further decrease over time occurred  converted from normal CSF Aβ42 levels at baseline to pathologic low levels during follow-up. Compared with those with normal CSF Aβ42 levels also at follow-up, these individuals had higher CSF P-tau levels at baseline  as well as at follow-up after 9 years , whereas follow-up cognitive test results, age, sex, APOE genotype, and education did not differ.Low baseline CSF Aβoccurred . Instead42 levels (at baseline or converters during follow-up) compared with those cognitively stable with consistent normal CSF Aβ42 levels. Finally, we observe that all individuals with MCI and AD/DLB diagnoses tend to increase over time in CSF T-tau levels even though this was not statistically significant  of the individuals with low baseline CSF Aβ42 levels remained completely cognitive intact  and AD (amyloidopathy) 542 levels predict development of AD at least 9 years before dementia onset with a very high negative predictive value. Data obtained from the repeated CSF measurements imply that CSF Aβ42 levels have decreased and reached a plateau already a decade before clinical onset of dementia in sporadic AD. Surprisingly, many individuals can harbor brain amyloid accumulation over a decade without any signs of cognitive deterioration, which will clearly implicate how CSF biomarkers are used to identify preclinical AD in future interventional therapeutic trials.1.Systematic review: Traditional sources (such as PubMed) were reviewed with focus on predicting sporadic dementia in cognitively healthy individuals. A handful publications have investigated these elongated preclinical stages, although very few with follow-up exceeding 5 years. Instead, current evidence is mainly derived from evaluating autosomal dominant Alzheimer's disease (AD).2.Interpretation: Our findings improve the understanding of how cerebrospinal fluid AD-biomarkers can be interpreted in absence of cognitive symptoms. They align with current hypothesis that β-amyloid is an early marker of AD but also confirm difficulties of interpreting their predictive relevance on individual level. This could implicate their use for identifying preclinical AD in future therapeutic trials.3.Future directions: The article elucidates the need for long follow-up in preclinical AD studies. This to clarify (1) how long individuals can harbor brain amyloid accumulation without cognitive impact and (2) which biomarker combination that correctly identifies preclinical AD with impending risk of dementia conversion.Low CSF Aβ


Streptomyces coelicolor, γ-butyrolactones (GBLs) and antibiotics were recognized as signalling molecules playing fundamental roles in intra- and interspecies communications. To dissect the GBL and antibiotic signalling networks systematically, the in vivo targets of their respective receptors ScbR and ScbR2 were identified on a genome scale by ChIP-seq. These identified targets encompass many that are known to play important roles in diverse cellular processes , and they formed regulatory cascades, sub-networks and feedforward loops to elaborately control key metabolite processes, including primary and secondary metabolism, morphological differentiation and stress response. Moreover, interplay among ScbR, ScbR2 and other regulators revealed intricate cross talks between signalling pathways triggered by GBLs, antibiotics, nutrient availability and stress. Our work provides a global view on the specific responses that could be triggered by GBL and antibiotic signals in S. coelicolor, among which the main echo was the change of production profile of endogenous antibiotics and antibiotic signals manifested a role to enhance bacterial stress tolerance as well, shedding new light on GBL and antibiotic signalling networks widespread among streptomycetes.In model organism  Streptomyces are important bacteria by producing a variety of active secondary metabolites and as one of the model systems for bacterial morphological development. Small molecule signals, such as γ-butyrolactones (GBLs) and antibiotics, have been reported to play vital roles in coordinating secondary metabolism and morphological development in Streptomyces species53Microorganisms in the nature environment are overwhelmed with diverse chemicals and are engaged in extensive interactions with their community to modulate gene expression23Streptomyces coelicolor A3(2), ScbR was characterized as a receptor of GBLs synthesised by the product of the scbA genescbR, scbR2 and scbA genes are located in the cpk cluster, which determines production of the polyketide coelimycin P1scbR-scbA intergenic region to repress scbR while activating scbA in response to GBLskasO, the pathway-specific activator gene of the cpk clusterS. coelicolor by modulating the interaction of ScbR2 with adpA (encoding the master regulator of morphogenesis) and redD (the direct activator gene of the red cluster for Red production)adpA nor redD is a target of ScbR. The scbR2 mutant displayed a complete loss of production of Act, Red and the calcium-dependent antibiotics (CDA), and showed precocious development of aerial hyphae6scbR mutant, which mainly showed delayed Red productionscbR-scbA intergenic region to shut down GBL synthesis and in the promoter region of kasO to control the production of coelimycin P110S. coelicolor is largely unknown. Moreover, homologs of ScbR and ScbR2 are widespread among streptomycetes, but studies regarding to them were all focussed on the regulation of genes for antibiotic biosynthesis12S. coelicolor.In the model organism Streptomyces phenotypes. The interplay among ScbR, ScbR2 and other regulators revealed intricate cross talks between signalling pathways triggered by GBLs, antibiotics, nutrient availability and stress.In this work, targets of ScbR and ScbR2 were deciphered using chromatin immunoprecipitation followed by sequencing (ChIP-seq) and combined with transcriptomic expression analysis. These targets were engaged in diverse physiological processes, but a major role of them was to control secondary metabolism and elicit stress responses. Furthermore, ScbR and ScbR2 mediated regulatory cascades, feedforward loops (FFLs) and sub-networks were extracted to control scbR mutant showed delayed Red and reduced Act productionscbR in S. coelicolor M145. In ΔscbR, a delay of aerial development was noted after growing for 36 h on SMMS plates . Peaks locating in the coding regions was first excluded, and attention was focused on regulation occurred in promoter regions , especially those associated with genes that were well studied or vitally annotated were chosen for further validation . Thus, 2To gain further insights into the binding sites of ScbR or ScbR2, all binding sequences of ScbR or ScbR2 were submitted to the MEME algorithm9sco6268, which was shown to bind with both ScbR and ScbR2 , were then used as references for binding site extraction using the MEME algorithm, and the predicted binding sites were shown in To validate the MEME-predicted binding motifs, recombinant ScbR and ScbR2 were further used to conduct DNase I footprinting analysis as described beforend ScbR2 . Footpri′ copies , resembl′ copies . To predsco6268 (which encodes a histidine kinase) in the cpk cluster, in addition to the promoter of kasO9cpk cluster were transcriptionally activated in the scbR and scbR2 mutants .When analysing the function of their targets, a predominant role of ScbR and ScbR2 was to regulate secondary metabolism both directly and via regulatory cascades and loops. By direct targeting, they both interacted with the promoter of  mutants . Since S mutants . In thisafsK as a target of ScbR and ScbR2. In ΔscbR, consistent with the diminished production of Act and Red, expression of afsS was greatly reduced (by 0.317 fold). Remarkably, AfsK is also implicated in polar growth and hyphal branching by phosphorylating DivIVA, and high AfsK activity could cause growth impedimentafsK expression (1.405 fold) in ΔscbR.On the other hand, ScbR and ScbR2 made up the first step in some regulatory cascades . One cascprA, which encodes another close homologue of ScbR, was bound by ScbR and ScbR2, and cprB, also encoding a close homologue of ScbR, was a ScbR2 target. Both CprA and CprB were reported to be involved in the regulation of antibiotic production and sporulationscbA (unpublished results). SCO6323, encoding another ScbR homologue in S. coelicolor, was also demonstrated here to be under the control of ScbR and ScbR2. Therefore, a complex regulatory network among GBL receptor homologues in S. coelicolor intervenes to control complex phenotypes  and absA1/A2 was induced , ScbR2 bound more promoter regions within the  induced  in ΔscbRgap1and pyk2 , were found as targets of ScbR and ScbR2 (gap1 in ΔscbR and ΔscbR2 (0.650 and 0.768 fold) suggests a slowdown of primary metabolism. Although, transcription of pyk2 was marginally changed in ΔscbR, it was reduced in ΔscbR2 (0.783 fold). Another important target gene of ScbR and ScbR2, sco4921 (accA2), encodes the A subunit of acetyl-CoA carboxylase, which is the key enzyme involved in converting acetyl-CoA to malonyl-CoA, thus providing precursors for antibiotic synthesis (notably polyketides such as Act and coelimycin P1) (accA2 in both ΔscbR and ΔscbR2 (1.650 and 2.464 fold) would direct more acyl-CoA flux toward malonyl-CoA for antibiotic production. NarG3, a component of respiratory nitrate reductasenagE2, which encodes the only permease for GlcNAc, a primary source of carbon and nitrogen for streptomycetes28afsK was under direct control from ScbR and ScbR2, downstream its response regulator AfsR was also closely involved with nitrogen and phosphate metabolism by binding with the promoters of the corresponding regulatory genes glnR and phoR/phoP3031The involvement of ScbR and ScbR2 in primary metabolism was mainly observed at three critical nodes in carbon flow, nitrate respiration and acetylglucosamine (GlcNAc) transport. Two genes encoding enzymes in glycolysis, nd ScbR2 . Reducedycin P1) . OverexprpsD and rpsL, and were also present inside and upstream of ribosomal RNA genes (rpsL and rpsD in ΔscbR2 (1.418 and1.871 fold) could contribute to increased antibiotic production. In streptomycetes, mutants of σ factors implicated in stress-response are also perturbed in antibiotic productionscbR-scbA intergenic regionkasO promoter is recognised by the HrdB sigma factorsigR in ΔscbR2 (1.620 fold) was speculated to increase cysteine synthesis to meet the high demand of N-acetylcysteine in coelimycin P1 biosynthesiscysM by SigRStreptomycesAlso ScbR2 controlled specific targets other than that shared with ScbR. The ribosome is a key node in cell associating with antibiotic-inducing responses and bacterial drug resistanceNA genes , showingnagE2 is also under multi-level regulation including repression by DasR (a master GlcNAc-responsive regulator)2344actII-orf4, a known target of DasR, AtrA and ROK7B72528redZ promoteractII-orf4 and atrA are shown here to be under the direct control of ScbR2 , kanamycin (50 μg/mL), apramycin (50 μg/mL), hygromycin (50 μg/mL) or chloramphenicol (50 μg/mL) when necessary.Bacterial strains used in this study are listed in the ScbR and ScbR2 monoclonal antibodies were prepared with recombinant proteins as antigens by CoWin Biotech Co. Ltd as described beforescbR was verified by PCR, showing that a 502 bp fragment internal to scbR gene was deleted.To construct ΔscbR, a 1903 bp an a 1548 bp homologous arm were amplified from M145 genome with primers scbRLarmF/scbRLarmR and scbRRarmF/scbRRarmR, and digested with HindIII and BamHI, and BamHI and EcoRI, respectively. Digested fragments were then ligated with pKC1139 digested with HindIII and EcoRI to obtain the plasmid pKC1139-∆scbR, which was then conjugated into M145 to obtain the ΔscbR strain. Disruption of S. coelicolor M145 were grown in liquid SMM and harvested at 30 h and 42 h for ScbR and ScbR2 ChIP experiments, respectively. DNA obtained from ChIP experiments was then sonicated into shorter fragments and TruSeqTM DNA Sample Prep Kit-Set A was used to create a pair-end DNA library, which was subsequently amplified with TruSeq PE Cluster Kit and sequenced using Illumina Hiseq2500. MACShttp://www.ncbi.nlm.nih.gov/geo/, number GSE64903).ChIP experiments were carried out as described previouslyS. coelicolor for hybridization according to the manufacturer’s published protocol (Agilent). Hybridization signals were extracted with Feature Extraction software to obtain raw data, which was introduced into GeneSpring GX software to set parameters and data obtained were then normalised. Six probes were designed for each gene and three biological replicates were analysed. Offset data of each gene were removed firstly based on criterion: Spores of M145, ΔscbR and ΔscbR2 were inoculated in liquid SMM medium and grown at 30 °C. M145 and ΔscbR were harvested for RNA extraction at 30 h, M145 and ΔscbR2 were harvested at 42 h for RNA extraction. RNAs were then subjected to customized 8*60k Agilent microarray of absA1, actII-orf4 and cdaR were amplified from genomic DNA with primers absA1pGR/absA1pGF, actII-orf4pGF/actII-orf4pGR, and cdaRpGF/cdaRpGR, respectively. Plasmid backbone was amplified from plasmid pLC-gusPromoters of How to cite this article: Li, X. et al. ScbR- and ScbR2-mediated signal transduction networks coordinate complex physiological responses in Streptomyces coelicolor. Sci. Rep.5, 14831; doi: 10.1038/srep14831 (2015).


Neisseria meningitidis and the normal CSF, suggesting a potential role for neopterin in meningococcal meningitis detection and screening applications. To estimate the best performance of neopterin as a marker of bacterial infection, principal component analysis (PCA) was performed in a selected region (640–720 cm−1) where the most prominent SERS peak at 695 cm−1 arising from neopterin was observed. The calculated specificity of 95 % and sensitivity of 98 % clearly indicate the effective diagnostic efficiency for differentiation between infected and control samples. Additionally, the limit of detection (LOD) of neopterin in CSF clinical samples was estimated. The level of neopterin was significantly higher in CSF samples infected by N. meningitidis (48 nmol/L), compared to the normal (control) group (4.3 nmol/L). Additionally, this work presents a new type of SERS-active nanostructure, based on polymer mats, that allows simultaneous filtration, immobilization, and enhancement of the Raman signal, enabling detection of spectra from single bacterial cells of N. meningitidis present in CSF samples. This provides a new possibility for fast and easy detection of bacteria in CSF and other clinical body fluids on a time scale of seconds. This method of detection produces consistent results faster and cheaper than traditional laboratory techniques, demonstrates the powerful potential of SERS for detection of disease, and shows the viability of future development in healthcare applications.A highly efficient recognition unit based on surface-enhanced Raman spectroscopy (SERS) was developed as a promising, fast, and sensitive tool for detection of meningococcal meningitis, which is an extremely serious and often fatal disease of the nervous system . The results of this study confirmed that there were specific differences in SERS spectra between cerebrospinal fluid (CSF) samples infected by The online version of this article (doi:10.1007/s00216-016-9535-7) contains supplementary material, which is available to authorized users. It may also be useful to distinguish active forms of disease from latent ones. Within the group of bacterial infections it was shown that patients with symptoms lasting for at least 5 days had significantly higher neopterin concentrations than patients with acute illness. Investigations on critically ill patients in intensive care units evaluated neopterin levels as a tool to discriminate patients with systemic inflammatory response syndrome with and without infectious etiology. Neopterin levels were found to have a specificity of 78 % for discriminating infectious and noninfectious etiology of critical illness .The measurement of the neopterin levels can provide reliable information regarding the disease diagnosis, stage, prognosis, and is also important for monitoring the response to therapy. Screening of neopterin concentrations in blood donations allows one to detect acute infections in a nonspecific way and improves safety of blood transfusions. Up to now several analytical procedures have been applied for evaluation of the neopterin level in blood using mainly high pressure liquid chromatography (HPLC)  and enzyHowever, both of these methods are effortful and expensive, and moreover they require technical equipment and highly qualified personnel. Additionally, the difficulties and inaccuracy of existing neopterin assays have been presented, and a number of factors have been shown to affect the validity and quality of such measurements –23. To t11-fold. A chemical mechanism is also believed to take place in SERS enhancement owing to the charge transfer between adsorbed molecules and the metal conduction band of metallic nanostructures. This chemical mechanism may provide an enhancement factor (EF) of less than 4 orders of magnitude [As was mentioned, the main method for determination of the selected immune markers relies heavily on various ELISA kits. Alternative approaches during recent decades have usually used fluorescent antibody assays, evanescent wave interference, and electrochemical methods. Current successes in nanotechnology and instrumentation development have led to recognition of biomolecular systems based on surface-enhanced Raman spectroscopy (SERS) with a higher sensitivity and a more clear visualization of bioanalytes. In brief, surface-enhanced Raman scattering is a vibrational spectroscopy that relies on enhancement of the electromagnetic field due to resonance between the excitation light and surface plasmons of the SERS nanostructures , 25. Thiagnitude , 27. Botagnitude . Anotheragnitude . The SERagnitude , therapeagnitude , drugs aagnitude , microoragnitude , and celagnitude .Neisseria meningitidis using surface-enhanced Raman spectroscopy. Anton Weichselbaum first isolated N. meningitidis from the CSF of a patient [Neisseriaceae family [. N. meningitidis is one of the three main bacteria that cause acute bacterial meningitis, along with Streptococcus pneumoniae and Haemophilus influenzae [In this study we present our research efforts aimed at detection of specific bacterial infection caused by  patient . This bae family . N. menifluenzae .N. meningitidis only infects humans and the average incubation period is 4 days, but it can range between 2 and 10 days. Meningitis caused by this bacteria is usually very serious  and requires rapid detection and urgent medical attention with appropriate antibiotic therapy. Taking into account the high mortality rates, rapid detection of bacteria in CSF and subsequent effective treatment are essential.N. meningitidis. Moreover, besides the Si/ZnO/Au platform used for neopterin level calculation, a new SERS substrate based on a polymer mat was applied for simultaneous filtration, immobilization, and enhancement of the Raman signal for the detection of single bacterial cells.In this work we present also, for the first time, the possibility of using neopterin levels for diagnosis of meningococcal meningitis disease in CSF clinical samples already diagnosed by microbiological techniques. Additionally, the obtained results were compared with ELISA as the reference method. We use SERS combined with a multivariate statistical method  to differentiate between CSF control clinical samples  and CSF clinical samples infected by N. meningitidis strain were obtained from the National Reference Centre for Bacterial Meningitis (NRCBM) in the National Medicines Institute (NMI) in Warsaw. The neopterin levels in CSF were estimated by a commercial ELISA test .Neopterin was purchased from Tocris Bioscience . Water (resistivity over 18 MΩ cm) was purified using a Milli-Q plus 185 system and used in all experiments. The CSF and −1 at resolution of ca. 2 cm−1. The typical acquisition time was 10 s for a single SERS measurement. The obtained spectra were processed with OPUS software provided by Bruker. All spectra were smoothed, baseline corrected, and normalized.Raman and SERS spectra of analyzed samples were recorded using the Renishaw inVia Raman system with 1024 × 256 pixel Peltier-cooled RenCam CCD detector. All measurement were performed using ×20 microscope objective , focusing the 785-nm laser to a spot size of approximately 5 μm. SERS spectra were acquired from less than 5 mW of incident laser power at ambient conditions using a back-scattering geometry. The SERS spectra were recorded between 400 and 1600 cmSEM measurements were conducted using the FEI Nova NanoSEM 450 with an accelerating voltage of 10 kV under high vacuum.Atomic layer deposition (ALD) was used for zinc oxide layer deposition on Si(100) at 100 °C. Diethylzinc and deionized water were used as precursors, and nitrogen was used as purging gas . The prol-lactide) (PLA) mats were purchased from MECC Co., Ltd., Japan and cut into squares (area of 0.25 cm2).The poly(−2 mbar and current of 25 mA were applied.Si/ZnO layers and electrospun polymer mats were covered with a thin layer of sputtered gold (ca. 90 nm) using PVD equipment from Leica (model EM MED020). Au target was obtained from Mennica Metale Szlachetne, Warsaw, Poland. During this procedure a vacuum level of 10N. meningitidis of serogroup B (603/2011) used during the study serves as a reference strain in polymerase chain reaction (PCR) in the NRCBM.Clinical CSF samples were obtained as a part of routine activity of the NRCBM and were analyzed anonymously. All the data were collected in accordance with the European Parliament and Council decision for the epidemiological surveillance and control of communicable disease in the European Community , 39. ThuN. meningitidis [In the case of negative culture, the NRCBM has been receiving clinical materials, including CSF, from patients with suspected invasive meningococcal disease. The DNA isolated from these samples was used for PCR to identify ngitidis , 41.The strain was identified on the basis of typical morphology of colonies, Gram stain, oxidase test, and API NH test  according to the manufacturer’s instructions. Serogroup was determined by slide agglutination tests using commercial antisera (Remel).Neisseria meningitidis used in the experiment was obtained from the NRCBM in Warsaw, Poland. To multiply microbial organisms, we cultivated them on solid BHI (brain heart infusion) growth medium at 37 °C for 24 h. After that, some bacterial colonies were redispersed in saline solution (sterile 0.9 % NaCl solution) and centrifuged for 5 min at 4000 rpm (so as not to destroy the cell membrane). The centrifugation process in the fresh saline solution was repeated four times to obtain a solution of clean bacterial cells, at a concentration of N. meningitidis of 106 CFU/mL. The density of bacterial cells was determined by counting the amount of colonies which had grown on the Petri dish from a known amount of medium. The count was taken after 1 day of cultivation at 37 °C. Before carrying out Raman measurements, 10 μL of an aqueous bacterial solution was placed over the SERS substrate. Measurements were taken after 5 min.−1). All the data were transferred to the Unscrambler software  where PCA was performed. PCA is a multivariate technique that reduces the dimensionality of complex spectroscopic data from many wavenumber assignments to a few principal components (PCs), making it easier to identify the majority of variations within the spectra [The SERS spectra were prepared for PCA using a two-step approach. First, OPUS software (Bruker Optic GmbH 2012 version) was used to smooth the spectra with the Savitsky–Golay filter, the background was removed using baseline correction, and then the spectra were normalized using a so-called Min-Max normalization (the area of band around 963 cm spectra .X) is transformed into two smaller matrices according to the formula X = TPT + E where T is the matrix of scores, P is the matrix of loadings, and E is the error matrix. The PCA method enables one to understand the sources of variation in the obtained SERS data, e.g., the plot of loadings vs. the wavenumber indicates the most important diagnostic vibrations in the spectra.PCA reduces the complexity of high-dimensional SERS data from many wavenumber assignments to several PCs. Each PC represents a linear combination of the original variables . The first component  is the most important one and accounts for as much variation in the data as possible. In the PCA model, the big spectral set matrix (N. meningitidis infection) and for CSF infected by this pathogen. Additionally, the obtained data allow one to calculate the diagnostic sensitivity and specificity according to the formula [In our study, PCA was carried on two different data sets, consisting of spectra obtained for normal CSF  and the normal (control) CSF samples (1b) deposited onto the Si/ZnO/Au substrate. CSF of the healthy patient contains no erythrocytes and up to five leukocytes per microliter [−1 were assigned to vibrations of the nucleic bases of DNA and lipids [−1 are associated with CH2 deformation, the amide III, the symmetric ring breathing bands of phenylalanine and protein, and C–C stretching, respectively [−1. The prominent SERS peaks located at 659, 728, 963, 1006, 1096, 1134, and 1469 cm−1 can also be consistently observed in infected samples  in which the marker band of neopterin appears (695 cm−1) is devoid of other intensive vibrations and allows label-free analysis of neopterin. There are also distinctive SERS features and intensity differences for normal and infected CSF samples in the spectral ranges of 1200–1260, 1270–1380, and 1500–1650 cm−1, indicated by the gray-shaded columns in Fig. −1 due to the C–H in-plane bending of phenylalanine and/or tyrosine and amide III band of proteins (α-helix) disappear and a new band at around 1240 cm−1 is observed, indicating transition from an α-helical to β-sheet structure [−1 was found to be higher in normal CSF samples, suggesting a decrease in the relative amount of tryptophan and also larger amount of neutral amino acids like phenylalanine, histidine, and isoleucine [N. meningitidis. It should be highlighted that these changes in the three regions mentioned above appear only in real clinical samples of CSF infected by N. meningitidis bacterium. Figure N. meningitidis (b) versus that of normal CSF with added 15 nmol/L solution of neopterin (a). As can be seen, for samples of CSF with neopterin “artificially” inserted  were not observed. This is clear evidence that these spectral features are correlated only with anomalous metabolism of patients with bacterial infection.Neopterin appears in human CSF, blood , and urine, and its increased levels indicate activation of the immune system involved in the pathogenesis and/or affected by malignant diseases. An earlier literature report  reveals ectively . In thiscroliter . The intd lipids . The feaectively . Aromati samples  depositeeopterin  can be ooleucine  in CSF wESM Fig.  only theAlthough significant spectral differences between normal and infected CSF have been clearly observed in SERS spectra, this simplistic analysis of the experimental observations uses only limited number of SERS peaks or SERS regions. Therefore, multivariate statistical analysis in the form of PCA has been exploited to utilize the whole spectra and to automatically determine the diagnostic marker bands for improving the efficiency of CSF diagnosis and also to find out the statistical significance of the proposed method. This analysis method is known as an excellent approach towards reducing dimensionality of Raman data and is widely used by researchers in discriminating cancer tissues from that of a control subject , for ide−1. In the first step we found that three PCs  are the most diagnostically significant and explain 94 % of the variance in the data. The resulting PC scores plots  where the most prominent SERS peak at 695 cm−1 arising from neopterin is observed  that are important for group separation. Figure rin Fig. . That isN. meningitidis-infected CSF SERS spectra. The stepwise analysis in this area gives a more effective diagnostic efficiency for distinguishing between infected and control samples. A specificity of 98 % and sensitivity of 95 % were obtained. These results demonstrate that the marker band of neopterin (695 cm−1) can be used for meningococcal meningitis detection in CSF samples.Figure N. meningitidis and in healthy controls using the SERS technique. Figure −1) versus the concentration of neopterin in normal CSF  and the correlation coefficient of 0.998 was calculated. The linear dependence of the SERS intensity at 695 cm−1 (y) on the neopterin concentration (x) in the whole examined region was evident. Moreover, the limit of detection (LOD = 1.6 nmol/L) was assessed on the basis of the signal-to-noise method [N. meningitidis. The neopterin concentrations of the same samples were also evaluated using a commercial ELISA kit . Furthermore, the results were evaluated by comparing with control samples and estimated values are described in Table The measurement of neopterin concentration in human body fluids such as serum, urine pleural fluid, and CSF provides an insight into cell-mediated immune responses. In this study we also examined the neopterin levels in samples infected by CSF Fig. . The neoe method . This caIt should be highlighted that for normal healthy control samples of CSF the level of neopterin was below 4.3 nmol/L. A value below 5 nmol/L is a typical CSF neopterin concentration calculated for patients without a modified immune system . In contThese results clearly demonstrate that neopterin may be used as a marker in the meningococcal meningitis diagnosis and monitoring of this infection.N. meningitidis in CSF samples. The applied technique is simple, fast, and allows filtration and/or concentration of CSF composites  within a small area of the SERS nanostructures. The setup for the proposed method consists of a Büchner flask, a filter funnel, and a vacuum pump [N. meningitidis bacterium, which ranges from 0.6 to 1.0 μm [, we are able to separate these species. The largest leukocytes (the main component of CSF) remain on the polymer mat, while the smaller proteins and bacteria pass through. Figure N. meningitidis after filtration on the SERS polymer platform. Figure N. meningitidis has not been presented in the literature; therefore we attempted, for the first time, the SERS measurements and band assignments of this bacterium. Figure N. meningitidis multiplied before SERS experiment by cultivation in a liquid LB (Lysogeny broth) growth medium and then deposited on the Si/ZnO/Au substrate. As can be seen in Fig. −1 appears in the SERS spectrum of N. meningitidis. This band is observed in many types of bacterial species like Escherichia coli, Salmonella enterica, Staphylococcus epidermidis, Staphylococcus aureus, and Bacillus megaterium [N. meningitidis. The other observed bands can be attributed to amino acids , or to C–C vibrations in phosphate binding of DNA, respectively [Surface-enhanced Raman spectroscopy is a powerful technique for the rapid and accurate detection of pathogenic bacteria in biological fluids . In our uum pump . The detuum pump , albuminuum pump ), and tho 1.0 μm , we are gaterium , 55. Thigaterium , 60. Jargaterium . Typicalectively .Fig. 6Th−1) and CH2 vibrations (1450 cm−1) [N. meningitidis. The most intense bands at 736, 1002, 1330, and 1450 cm−1 were also observed in the remaining part of a filtrated solution where the presence of, inter alia, bacteria is expected  . Table Sted Fig. . As exemN. meningitidis. Additionally, a new class of SERS substrates based on a polymer mat was developed for simultaneous filtration, immobilization, and enhancement of the Raman signal, which allows the detection of single bacterial cells of N. meningitidis present in CSF samples. This opens a new route for simple identification of bacteria in CSF and other clinical body fluids on a time scale of seconds. In conclusion, neopterin emerges as an important biomarker and a strong predictor of bacterial meningitis infections. In the very near future, this study will be extended to a larger number of clinical samples to improve the diagnostic sensitivity and selectivity.This work presents a new label-free method for neopterin detection based on the SERS technique and the possibility of using this method for determination of the neopterin levels in CSF. It was also shown that CSF neopterin evaluation may be used in determining the bacterial meningitis infections caused by ESM 1(PDF 70 kb)Below is the link to the electronic supplementary material.


Tachycardia and high troponin levels prognosticate for poor outcomes in human sepsis . ReducinTo determine whether non-survival is associated with early changes in troponin release and circulating catecholamine levels.ad libitum. Fluid resuscitation  was commenced at 2h. At 6h, echocardiography was used to measure heart rate and stroke volume. Animals were observed until 72h to assess survival. In a second experiment septic animals underwent echocardiography at 6h followed by sacrifice and blood and tissue sampling. We here report plasma catecholamine and troponin T levels (measured by ELISA) in predicted survivors and non-survivors, and sham-operated controls.Male Wistar rats (325±15g) underwent insertion of tunneled carotid arterial and jugular venous lines under isoflurane anaesthesia, followed by immediate i.p. injection of 4µl/g faecal slurry. Control animals were treated identically but without i.p. injection of slurry. Once awake, attachment to a swivel-tether system allowed animals to move freely and access food and water Septic animals n=16) had a mortality rate of 56%, with death occurring between 18-36h. A heart rate cut point of 460/min measured at 6h prognosticated 3-day survival with sensitivity of 0.88 and specificity of 0.92. Clinical features of illness at this timepoint were however mild. Table 6 had a mAn association was seen between eventual non-survival and tachycardia, low stroke volume and myocardial injury (denoted by high troponin) at 6h after induction of sepsis. The impact of modulating cardiac stress on outcome merits further study.


Diffusion tensor imaging (DTI) is sensitive to white matter tract pathology. A core signature involving the corticospinal tracts (CSTs) has been identified in amyotrophic lateral sclerosis (ALS). Raised neurofilament light chain protein (NfL) in cerebrospinal fluid (CSF) is thought to reflect axonal damage in a range of neurological disorders. The relationship between these two measures was explored.n = 25 CSF, 40 serum) and healthy, age-similar controls . Within-group correlations between NfL and DTI measures of microstructural integrity in major white matter tracts  were performed using tract-based spatial statistics.CSF and serum NfL concentrations and DTI acquired at 3 Tesla on the same day were obtained from ALS patients (NfL levels were higher in patients compared to controls. CSF levels correlated with clinical upper motor neuron burden and rate of disease progression. Higher NfL levels were significantly associated with lower DTI fractional anisotropy and increased radial diffusivity in the CSTs of ALS patients, but not in controls.Elevated CSF and serum NfL is, in part, a result of CST degeneration in ALS. This highlights the wider potential for combining neurochemical and neuroimaging-based biomarkers in neurological disease. Neurofilament in cerebrospinal fluid (CSF) and serum has emerged as a much-needed candidate biomarker for the fatal adult neurodegenerative disorder amyotrophic lateral sclerosis (ALS).6In addition to neurochemical markers, measures derived from noninvasive magnetic resonance diffusion tensor imaging (DTI), such as fractional anisotropy (FA) and radial diffusivity (RD) have been found to be sensitive to microstructural white matter alterations. While the former metric represents the degree of anisotropy of the diffusion of water molecules in axons and decreased FA is a frequent correlate of brain tissue injury, RD is a measure of diffusion perpendicular to the axon, and therefore commonly linked to the microstructure of myelin.11While numerous studies concluded that NfL or MRI measures show promise as biomarkers for diagnosis or as predictors of disease progression in different neurological disorders, to date there has been little attempt to directly relate the two.Gaining insight in how and where in the brain these different measures relate to each other in health and disease may assist the development of improved, combined biomarker panels. Therefore, we investigated the relationship between CSF NfL levels and DTI measures of white matter microstructural integrity in a cohort of ALS patients and healthy controls, and furthermore attempted to correlate neurochemical data with clinical scores in the patient group. We repeated the analyses for serum NfL concentrations, for which there was a large cohort available with both measures.Prevalent and incident cases of ALS from a large tertiary referral clinic were offered participation in Phase 1 of the Oxford Study for Biomarkers in MND (“BioMOx”) between 2009 and 2013. Ethical approval for all procedures was obtained in advance , with written informed consent obtained from all participants. All patients in this study were apparently sporadic, that is, reporting no family history of ALS or frontotemporal dementia (FTD), and diagnosed by one of two experienced neurologists  according to standard criteria. The revised ALS Functional Rating Scale (ALSFRS-R) was used to assess disability on the day of study (M. R. T.). Disease duration was calculated from symptom onset to scan date in months, and the rate of progression was determined as (48 − ALSFRS-R)/disease duration. A clinical UMN clinical burden score of 15 comprised the number of pathologically brisk reflexes .CSF and serum samples were processed within 1 h of extraction and on the same day as the MRI (M. R. T.). Samples were centrifuged, aliquoted, and stored in polypropylene tubes at −80°C until analysis. An electrochemiluminescence enzyme-linked immunosorbent assay (ELISA) was used to quantify NfL as previously described.123 voxel size, TE/TR/TI = 4.7/2040/900 msec). Acquisition time for the MP-RAGE image was 6 min. Whole-brain DTI images were acquired using an echoplanar imaging sequence . In addition, four images without diffusion weighting were acquired. Furthermore, a field map was acquired using a gradient echo imaging sequence  to account for distortions caused by field inhomogeneities.Scans were performed at the Oxford Centre for Clinical Magnetic Resonance (OCMR) using 3 T Siemens Trio scanner  with a 12-channel head coil. For each subject T1-weighted images were obtained using a 3D Magnetization Prepared-Rapid Acquisition Gradient Echo (MP-RAGE) sequence .All images were analyzed using tools from the FMRIB Software Library , axial diffusivity , and L2 and L3 maps were created using DTIFIT by applying a diffusion tensor model to each voxel. RD maps were created by averaging the L2 and L3 maps (RD = (L2 + L3)/2).Each subject’s DTI scans were corrected for head motion and eddy currents and then brain-extracted to remove any non-brain voxels. To correct for Bhttp://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FMRIB58_FA), and then averaged to create a study-specific template to which each subject’s FA map was then nonlinearly registered. Next, the mean FA image was created and thinned to create a mean FA skeleton, which represents the centers of all tracts common to the group. Each subject’s aligned FA data were then projected onto this skeleton. The same operations that were used to register the individual FA images to the study-specific template and project FA values onto the mean FA skeleton were subsequently applied to the individual MD, L1, and RD images.All individual FA images of all subjects were nonlinearly registered to a standard FA template , and superior longitudinal fascicle (SLF) in Montreal Neurological Institute (MNI) standard space. The CST and CC are obvious a priori region-of-interest (ROI) choices in ALS. The SLF was chosen based on previously published studies reporting involvement of extra-motor tracts in ALS. For the TBSS analysis, where the final ROI results from the intersection of the atlas-based masks and the mean FA skeleton mask, the left and right CST and CC masks were thresholded at “45” and the left and right SLF masks thresholded at “10”  within each region of interest, using the threshold-free cluster enhancement (TFCE) approach.13Voxel-wise General Linear Model (GLM) was applied using permutation-based non-parametric testing to regress CSF and serum NfL concentrations with the pre-processed TBSS images separately in patients and controls (controlling for age by inclusion as a covariate of no interest). Results were considered significant for CSF and serum NfL concentrations were separately correlated with age in both groups, and with UMN score and progression rate in the ALS group .Twenty-five patients and 17 healthy, age-matched controls with CSF NfL levels and MRI data obtained on the same day were available for analysis . Paired serum samples and MRI data were available for a larger cohort of 40 patients and 25 controls. Participant features are summarized in Tableρ = 0.642, P = 0.001; controls: ρ = 0.677, P = 0.004).In both groups, CSF and serum NfL concentrations were significantly correlated . CSF NfL concentration was positively correlated with age  in controls, but not in patients. In patients, CSF NfL levels correlated positively with UMN score , and progression rate   versus controls  and gender distribution, we also compared CSF NfL levels between a closely age-matched subset of 15 patients  and 15 controls . The results of the group comparison remained qualitatively unchanged for this smaller cohort; with significantly higher mean CSF NfL level in patients (7446 ± 5392 pg/mL) versus controls .As in the overall sample patients and controls differed significantly with respect to age . Serum NfL concentration was also positively correlated with age  in controls, and correlated positively with UMN score , and progression rate  in patients.Mean serum NfL level was also significantly higher in patients (97 ± 90 pg/mL) versus controls  negative correlation between NfL measures and FA  in both CSTs in ALS patients  mutations compared to cases with wild-type SOD1.SOD1 “D90A” mutation showed a curious relative sparing (in DTI terms) of CST involvement compared with sporadic ALS patients matched for disability and clinical UMN involvement.Lower CSF NfL levels have been noted in patients with ALS linked to superoxide dismutase  found in ALS appear to be related to white matter damage within the CSTs as assessed by metrics derived from DTI. This provides further support for NfL as a meaningful biomarker in ALS. Prospective studies in those suspected to have ALS, in established ALS mimic disorders, and in those presymptomatic individuals carrying genetic mutations placing them at high risk of ALS, are now underway to fully explore the biomarker potential of combined neurochemical and MRI measures.


Hypoxemia may occur in young infants with severe acute illnesses or congenital cardiac anomalies, but is not reliably detected on physical exam. Pulse oximetry (PO) can be used to detect hypoxemia, but its application in low-income countries has been limited, and its feasibility in the routine assessment of young infants (aged 0–59 days) has not been previously studied. The aim of this study was to characterize the operational feasibility and parent/guardian acceptability of incorporating PO into the routine clinical assessment of young infants in a primary care setting in a low-income country.2 + 1 % for at least 10 s, heart rate displayed, and adequate signal indicators). PT included the time for one repeat attempt at a different anatomical site if the first attempt did not yield an acceptable reading within 1 min. Parent/guardian acceptability of PO was based on a questionnaire and unprompted comments about the procedure. All infants underwent physician assessment.This was a cross-sectional study of 862 visits by 529 infants at two primary care clinics in Karachi, Pakistan . After clinical assessment, oxygen saturation (Sp02) was measured by a handheld PO device  according to a standardized protocol. Performance time (PT) was the time between sensor placement and attainment of an acceptable PO reading . Median PT was 42 s . Parents/guardians overwhelmingly accepted PO . Of 10 infants with at least one visit with Sp02 <92 % on a first PO attempt, 3 did not have a significant acute illness on physician assessment. There were no PO-related adverse events.Using a commercially available handheld pulse oximeter, acceptable Sp02 measurements were obtained in nearly all infants in under 1 minute. The procedure was readily integrated into existing assessment pathways and parents/guardians had positive views of the technology.When incorporated into routine clinical assessment of young infants at primary care clinics in a low-income country, PO was feasible and acceptable to parents/guardians. Future research is needed to determine if the introduction of routine PO screening of young infants will improve outcomes in low-resource settings. Timely management of severe neonatal infections  is a crucial component of public health strategies to reduce infant mortality in low-income regions . AlthougPulse oximetry (PO) is an non-invasive method of measuring peripheral oxygen saturation (SpO2) based on the differential absorption of red versus infrared light by oxygenated hemoglobin in a narrow tissue segment (e.g. infant’s hand or foot) . The useIn primary care clinics where acute illnesses are triaged and managed, PO may be particularly useful in young infants (<2 months of age), among whom clinical features of serious illness are often subtle and non-specific. However, PO has infrequently been implemented in primary care clinics in low-income countries due to a range of both real and perceived barriers, including the availability and field robustness of low-cost PO devices, the time required to perform PO, and the challenges of accurate interpretation of PO readings by minimally-trained personnel  . Althougth and June 1st, 2013 at two primary care clinics in low-income communities of Karachi, Pakistan. The Bilal Colony (Site 1) primary care clinic serves a population of 70 000, with approximately 40 young infant visits per week. The Bhains Colony (Site 2) primary care clinic serves a population of approximately 40 000, with 30 young infant visits per week. Both clinics provide well-child care  and outpatient care for common childhood illnesses. A team of community health workers add outreach capacity through regular visits to households to detect early symptoms of illness such as newborn sepsis, and refer ill infants to these clinics for physician assessment. Seriously ill infants and children are provided transport to hospital or in case of refusal, centre-based parenteral antibiotic therapy (if indicated). Both sites are approximately located at sea level.We conducted a cross-sectional facility-based study between March 5Study procedures were performed by a pair of study personnel – a study worker and a research assistant. Each clinic site was served by a core team of two study workers and one research assistant. Less than 2 % of the visits involved other trained personnel substituting for a core team member. “Study workers” refer to the four personnel (two at each clinic) who directly assessed infants according to the IMCI algorithm, performed pulse oximetry and conducted the initial infant parents/guardian interview. These individuals had secondary school education and prior experience in health research projects involving infants and children, but they did not have professional research or health care credentials. We considered their level of training/experience to be similar to that of a CHW. ‘Research assistants’ (one at each clinic) coordinated study activities, observed study workers to record data related to timing of pulse oximetry, and conducted an exit interview with parents/guardians. Research assistants had post-secondary education and had long-standing professional involvement in research as employees of Aga Khan University. All personnel were trained in formal sessions as well as a pilot implementation phase. None of the team members had used a pulse oximeter prior to this training/pilot period.All visits by infants 0 to 59 days of age were eligible for inclusion, unless the infant was attending the clinic solely for a scheduled injectable antibiotic administration based on a previous diagnosis of suspected bacterial infection, or was too sick to undergo study procedures . Infants were initially assessed by a Lady Health Worker  according to routine clinic intake procedures, including measurement of weight and axillary temperature. Eligible infants were then referred to the PO study team who obtained informed consent from the parent or guardian  prior to proceeding with study procedures. Critically ill infants were referred directly to a clinic physician. Infants with previous study visits were eligible for an unlimited number of ‘revisits’ .The protocol was conducted in compliance with the Declaration of Helsinki and the protocol was approved research ethics committees at the Aga Khan University (Protocol No. 2006-Ped-ERC-11) and the Hospital for Sick Children (Protocol No. 1000028096). Written informed consent was obtained from all parents/guardians.A structured parent/guardian interview was conducted by the study worker to elicit sociodemographic information, reason for clinic visit, and the parent/guardian perception of the infant’s illness severity. Enrolled infants underwent clinical assessment by the study worker according to the IMCI protocol, including enquiry about a history of convulsions or poor feeding, measurement of axillary temperature  to determine presence of fever >37.5 °C), counting of respiratory rate (RR) over 1 min (fast breathing defined as RR ≥ 60), observation for the presence of lower chest wall in-drawing, and determination of the level of consciousness/movement 7.5 °C, c.2 was stable (±1 %) for at least 10 s, the heart rate was displayed during the period of SpO2 measurement and the device’s functional indicators (‘blip bars’) suggested that the signal strength was adequate . If 1 min passed without obtaining an acceptable reading, the sensor was moved to the alternate site and the PO measurement was re-attempted. If five minutes passed without obtaining an acceptable reading, the measurement was considered attempted but unsuccessful. If an acceptable Sp02 was <90 %, the measurement was repeated to confirm the low value. Infants with Sp02 <90 % were offered referral to a tertiary health facility, regardless of the presence of other clinical signs. While the study worker performed the PO procedure, a research assistant observed and recorded the procedure time, Sp02, heart rate, and signal strength indicators displayed at the time the reading was deemed to be acceptable by the study worker. The study worker also recorded any spontaneous verbal comments by parents/guardians regarding the procedure .Following the IMCI assessment, conducted by the study worker, SpO2 was measured using a commercially-available handheld PO device widely used in pediatric practice , applying the low noise cabled sensor (LNCS) YI sensor  to a left foot (first attempt) or right palm  and secured in place using a reusable foam wrap, according to the manufacturer’s recommendation. First, the sensor (connected to the oximeter via an extension cable) was placed on the infant and subsequently, the oximeter was powered-on, an approach similar to that which has been previously referred to as “sensor to infant first” . The Sp02 reading before making a decision regarding the need for hospital referral. Five of the 8 unmasked readings had SpO2 <90 %; in these cases, the physician was informed of the PO results to expedite clinical management. After completion of clinical and PO procedures, parents/guardians were asked to participate in a brief structured interview regarding perceptions and acceptance of PO.Infants were subsequently referred to a clinic physician, who completed a history and physical examination, and determined whether the infant required clinic-based treatment or immediate referral to hospital. To the extent that it was possible, the physician was not informed of the Sp02 prior to the determination of hospital referral. For 8 of 862 readings (0.9 %), blinding was not achieved, such that the physician knew of the SpOFollowing the initial standardized PO procedure with the Rad-5v, PO was repeated using one of three other handheld PO devices: Lifebox , Tuffsat , or Nellcor OxiMax N-65 . The original intention was to establish feasibility on a variety of hand-held oximeters; however, we made a post-hoc decision to exclude data for these additional devices from the present analysis, for several reasons: these devices were each used less frequently than the Rad-5v such that personnel did not have comparable time to develop comfort and competence with their use; the secondary devices were not consistently used in both clinics and therefore were used by different personnel and among different patient populations Table ; measure2 value on the data form). The ‘lag time’ (LT), in seconds, was defined as the period from pressing the power button on the device until an acceptable reading was obtained. Whereas PT incorporated both personnel and device performance factors, the LT (a component of PT) primarily reflected device factors. Both PT and LT included the 10 s required to confirm that the displayed Sp02 was stable (criterion for acceptable reading). Parent/guardian perceptions of PO were measured by yes/no responses to a series of prompted items regarding overall satisfaction, openness to PO in the future, perceived usefulness, and concerns about infant discomfort. Unprompted parent/guardian concerns were also included in acceptability analyses.The primary feasibility metric was the “performance time” (PT), in seconds, defined as the total time required to obtain an acceptable reading from the initiation of the procedure (when the study worker first picked up the sensor to place it on the infant) until an acceptable reading was obtained . In the original design of this study, we had aimed to explore the clinical utility of PO through analyses of the association of hypoxemia  with inDescriptive statistics included median  for non-normally-distributed variables and proportions for dichotomous/categorical variables. We considered feasibility in terms of the median PT and LT, as well as the proportion of acceptable readings obtained within 1 min (primary target) and 5 min. We investigated whether the feasibility outcomes (PT and percent acceptable readings within 1 min) were significantly associated with any of the following factors: clinic site, day of visit relative to the start of the study, age, sex, weight, reason for visit and presence/absence of at least one IMCI criteria. Differences in PT across strata  were assessed using non-parametric testing (Wilcoxon rank-sum tests). Associations between PT and continuous covariates  were also analyzed using Spearmen’s rank correlation coefficient. Differences in proportions  were analyzed by Chi-squared tests. Because clinic site was a strong predictor of PT, analyses for other factors were performed after stratification by site. Among infants with >1 visit during the study period, there was no correlation between PT at initial and first revisits . Therefore, visits by the same infant were treated independently in analyses of feasibility metrics.Of 1166 visits screened for eligibility during the study period, 1084 (93 %) were eligible for inclusion and 862 (74 %) were enrolled. Of 222 ineligible visits, five involved critically ill infants who required immediate medical management. Of the 862 included visits, 529 (61 %) were infants’ initial visits included in the study and 333 (39 %) were revisits. Characteristics of the visits and revisits differed between the two study sites with respect to age distribution, reason for visit, and parent/guardian perception of illness severity . The median PT was 42 s  and median LT was 34 s . The proportion of visits with acceptable readings increased rapidly within the first minute; in comparison, the rate of increase in the proportion of infants with acceptable readings during second attempts was relatively slower  visits at which the first attempt was unsuccessful , all (100 %) showed poor quality signals according to the device indicator, 87 % had SpOThe distribution of PT differed significantly across sites, even after stratification by infant characteristics Table . In straPO was widely accepted by parents/guardians, even at repeated encounters Table . A very 2 measurements that ranged from 72 % to 100 %, with the majority between 90 and 100 %   at which SpO2 < 95 % .At 529 initial visits, 528 infants had acceptable SpO2 of 94 %. Further SpO2 measurements at two subsequent revisits were >92 % and the infant was alive and well at the end of follow-up at 2 months of age. Of the 3 (of 10) infants who were not considered to be significantly unwell according to IMCI criteria or physician assessment at the time of first detection of Sp02 < 92 % (Table 2 <92 % measured repeatedly starting at the third visit (age 37 days). Based on the low Sp02 in combination with the presence of a cardiac murmur, echocardiography was arranged and revealed a diagnosis of tetralogy of Fallot with pulmonary atresia. The infant was referred for specialist pediatric cardiac care and surgical correction. Infant #4 was enrolled at 7 days of age during a well baby visit, at which time SpO2 was 84 % and 91 % based on consecutive measurements. The physician assessed the infant as generally well-appearing and not meeting IMCI criteria for referral; however, ten days after participation in the study, the infant died at home of an unknown cause .Ten infants with at least one acceptable Sp02 < 92 % at any visit  contributed a total of 37 visits; SpO2 < 92 % occurred at 13 of 37 (35 %) visits Table . Sp02 waThis observational study demonstrated the operational feasibility and parent/guardian acceptability of PO ‘spot checks’ in the routine clinical assessment of young infants at two primary care clinics in low-income communities of Karachi, Pakistan. Using a commercially available handheld device and a sensor-to-infant-first technique, acceptable Sp02 measurements were obtained in nearly all infants in under 1 min, from the time of initiating sensor placement to the display of a stable Sp02 value. The procedure was readily integrated into existing assessment pathways and was well tolerated by the infants. Additionally, parents/guardians had positive views of the technology at nearly all visits. These findings provide a practical basis for further research assessing the clinical effectiveness of PO in the triage and management of young infants in low-income settings.PO is a painless and portable technology that is widely used in pediatric practice to non-invasively assess and monitor cardiorespiratory function, particularly in perioperative care, emergency departments, critical care units, and in the management of patients with acute or chronic respiratory disease . DespiteThe widespread implementation of PO in low-income countries remains limited, due in part to practical barriers including lack of access to functioning devices and insufficient training in use of the technology . Where PIn-hospital implementation of PO has been previously described in The Gambia  and PapuNot unexpectedly, we observed a much lower prevalence of hypoxemia in a primary care setting compared to rates reported in hospital-based studies . Even amThe protocol for Sp02 spot checks in this study was intended to simulate a practice that could be realistically introduced into a variety of primary care settings. Pulse oximeters were used by first-level personnel , but because staff had substantial opportunities for training and practice, and were highly motivated throughout the study, they may not be representative of health care workers who would implement routine pulse oximetry outside of a research context. The small number of study workers who were trained to use pulse oximetry limited the analysis of worker, clinic and system-level factors that may have impacted the performance and feasibility of pulse oximetry in this setting, and which likely accounted for the observed between-site differences. A limitation of the Sp02 interpretation was that the criteria for acceptable readings were based only on information displayed in real-time by the handheld oximeter . Although we used a device that employs a motion-resistant algorithm that has been validated and widely implemented in neonatology , 26, it Future research efforts should aim to establish the conditions under which the introduction of PO into primary care settings improves health outcomes, ideally in conjunction with supplemental oxygen availability. For example, PO may improve clinical outcomes of ill neonates by improving the rational allocation of supplemental oxygen, prompting appropriate referrals to hospital, and identifying infants who warrant further investigations . Studies should ideally measure PO feasibility and identify context-specific barriers to implementation and scale-up, including long-term robustness of PO devices and components, data that we did not report because of the relatively short duration of this study. In addition to the initial cost of the devices, the high cost of repair and replacement of minor components, such as sensors and cables, is likely to be an important barrier to PO implementation in developing countries .There is a growing literature suggesting that PO is feasible and improves clinical care of children in low resource settings . The pre