A). We found that time-dependent changes in the CSF and blood levels of the two biomarkers were similar, but that the correlation between the biomarkers and QA was lower for NSE (ρ = 0.444) than for S100B (ρ = 0.668). Because data were longitudinal, we also conducted cross correlation analyses, which indicated a directional flow and lag-time of biomarkers from CSF to blood. For S100B, this lag-time could be ascribed to BBB integrity, whereas for NSE it could not. Upon inferential modelling, using generalized least square estimation (S100B) or linear mixed models (NSE), QA (p = 0.045), time from trauma (p < 0.001), time from trauma2 (p = 0.023), and CSF biomarker levels (p = 0.008) were independent predictors of S100B in blood. In contrast, for NSE, only time from trauma was significant (p < 0.001). These findings are novel and important, but must be carefully interpreted because of different characteristics between the two proteins. Nonetheless, we present the first data that indicate that S100B and NSE are cleared differently from the central nervous system, and that both the disrupted BBB and additional alternative pathways, such as the recently described glymphatic system, may play a role. This is of importance both for clinicians aiming to utilize these biomarkers and for the pathophysiological understanding of brain protein clearance, but warrants further examination.Brain protein biomarker clearance to blood in traumatic brain injury (TBI) is not fully understood. The aim of this study was to analyze the effect that a disrupted blood–brain barrier (BBB) had on biomarker clearance. Seventeen severe TBI patients admitted to Karolinska University Hospital were prospectively included. Cerebrospinal fluid (CSF) and blood concentrations of S100 calcium binding protein B (S100B) and neuron-specific enolase (NSE) were analyzed every 6–12 h for ∼1 week. Blood and CSF albumin were analyzed every 12–24 h, and BBB integrity was assessed using the CSF:blood albumin quotient (Q Even though neither is brain specific, both are highly brain enriched and have been associated with a worse intracranial condition and long-term functional outcome following TBI.8–10 In fact, some centers event utilize one or both in clinical routine work,12 hence making these two “biomarkers” of particular interest to the neurotrauma translational research field.Traumatic brain injury (TBI) is one of the most common causes of death and disability,13 For simplicity, and to align with the clinical research field, we refer to all of these barriers as “BBB.” In addition, a transport route denoted “the glymphatic system” has recently been described in experimental models.14 Although it has been suggested in pre-clinical models that brain-enriched proteins in CSF passively leave the brain through this perivascular “glymphatic” system,15 others claim that the BBB integrity plays a more decisive role.16–18 In TBI studies, in which the patient likely has concomitant injuries to multiple CNS barriers, the gold-standard strategy for assessing BBB disruption (BBBD) is the CSF to blood albumin quotient (QA).19–25 Previous studies of TBI and subarachnoid hemorrhage assessing how serum levels of S100B are associated with QA have not shown significant correlations.26 However, these studies have been limited by a lack of longitudinal analyses and high frequency sampling during constant CSF drainage. The trajectory of secondary pathophysiological mechanisms following TBI likely require a high temporal resolution,3 multi-compartment monitoring, and modeling to elucidate biomarker and QA dynamics. In aggregate, how brain-enriched proteins leave the injured brain warrants further research, as it may increase understanding of brain injury pathophysiology in several conditions and improve the utility of biomarkers in clinical decision making.The clearance mechanism of protein biomarkers from brain to blood is not fully understood, but is of importance for clinical interpretation and pathophysiological understanding. The adult central nervous system (CNS) comprises three anatomical barriers, namely: (1) the blood–cerebrospinal fluid (CSF) barrier, (2) the blood–brain barrier (BBB), and (3) the arachnoid barrier.A, over time utilizing a high-sampling frequency from multiple compartments in severe TBI patients.We aimed to assess if, and how, clearance of brain-enriched proteins (S100B and NSE) from brain to blood is affected by BBBD, measured as Q27 undertaken at the NCCU at Karolinska University Hospital  between January 1, 2010 and March 1, 2013. Written, informed consent was acquired from next-of-kin. The study was conducted in accordance with the Declaration of Helsinki and Swedish law. Ethical approval was provided by the Stockholm County branch of the Central Ethical Review Board (#2009/1112-31/3), now called the Swedish Ethical Review Authority.The included patients were part of a prospective observational studya priori using power analysis was unfeasible because to our knowledge, no assumptions on effect size have been described in the literature using the same approach as in our study. However, using data from our group,26 we could estimate our sample sizes for a cross-sectional rather than a longitudinal data set. Aiming for a correlation coefficient r = 0.60 between S100BCSF and S100Bblood, we found the estimated sample size needed to reach 80% power on the 0.05 significance level (one-sided test) using the R28 package pwr29 to be 15 patients. The effect size estimate was larger than the one previously reported from our group (r = 0.45),26 but smaller than what has more recently been reported30 (r = 0.79), leading us to believe that this was a valid effect size assumption. We therefore set out to include >15 study subjects.Sample size estimation Because of the sporadic availability of research personnel, recruitment was made periodically. Patients were not randomized, because no group-specific interventions were conducted. Inclusion criteria for the patients were as follows: (1) age 18–75 years, (2) Glasgow Coma Scale (GCS) score 3–8 (unconscious at admission), and (3) computerized tomography (CT) verified structural intraparenchymal intracranial injury. Exclusion criteria comprised: (1) GCS 3 and bilaterally non-responsive pupils, (2) slit ventricles  insertion impossible), (3) unsurvivable injury, (4) unconsciousness of etiology other than TBI, (5) absence of CT-verified intraparenchymal intracranial injury, (6) other concurrent systemic terminal disorder, and (7) impossibility of follow-up .27 Briefly, upon admission, patients received an EVD . The drain was connected to a four-way stopcock , connected to a LiquoGuard® CSF-pump  set to a constant drainage velocity of 2 mL CSF per hour. CSF was collected in, and samples were obtained from, the drainage bag in the LiquoGuard system in order to minimize the risk for infections, and freeze blocks were used to keep the CSF cold in order to limit protein degradation. The collected pool of CSF and readily acquired arterial blood were analyzed at 6-and 12-h intervals, for both S100B and NSE. Albumin (plasma and CSF) was analyzed once to twice daily. S100Bblood was analyzed by electrochemiluminescence immunoassay , albuminplasma was analyzed by colorometric bromocresol purple (BCP)-binding assay and albuminCSF was analyzed by immunoturbitity on a Roche Cobas/Modular platform . NSEblood, NSECSF, and S100BCSF were analyzed by immunoluminometric assay on a LIAISON XL system . S100Bblood and S100BCSF were analyzed on different platforms because of local procurements and inability to run CSF samples on the more modern Cobas/Elecsys platforms. Each individual assay has shown robust within- and between-run similarity,31 thus enabling longitudinal sampling as employed in this study. Some claim that the different platforms are not entirely interchangeable,32 whereas others have found excellent associations between the methods (r = 0.932).31 It is noteworthy that, to our knowledge, no studies have examined bicompartmental similarities . This is of importance, because absolute between-assay agreement is of greater importance when identical samples from the same compartment are examined on multiple platforms. However, for the scope of this study, the relative relationship was of greater interest. All laboratory assays were performed at the Karolinska University Laboratory in accordance with local guidelines. Aside from this, patients were treated in accordance with local routine at the NCCU, as previously described.8The detailed study setup has been described previously.33 (head and non-head) Abbreviated Injury Scale34 (AIS), Injury Severity Score35 (ISS), and significant multitrauma,36 all of which were acquired upon hospital admission. It is of note that an AIS of 6 at admission is impossible, as it denotes an unsurvivable injury. CT variables upon admission were graded using the Marshall CT Classification,38 in which classifications V and VI were collapsed into one category (“mass lesion”), as we used only the admission CT. For comparison, we also assessed the Rotterdam39 and Stockholm CT scores.40 Radiological brain injury progression upon subsequent examination was evaluated employing a similar strategy as in previous work.41 Glasgow Outcome Scale42 (GOS) was assessed as described previously.27 In short, a neurorehabilitation board-certified physician (P.H.G.) examined patients 6 months following the TBI. GOS comprises 5 categories: (1) dead, (2) persistent vegetative state, (3) severe disability, (4) moderate disability, and (5) low disability.Baseline clinical data were defined and acquired as follows. Clinical variables comprised GCS,43 BBBD was defined as the quotient between CSF and blood albumin (Equation 1), because that constitutes the literature gold standard.43 The reference intervals used for all proteins in the current study were defined using reference intervals stipulated by the Karolinska University Laboratory during the time of patient inclusion or at present  if normally distributed, or else median (interquartile range [IQR]). Categorical data were presented as count (%). In graphical depictions and calculations, biomarker values were converted to log10-transformed values, unless otherwise stated. A p value ≤0.05 or a 95% confidence interval (CI), where the range of the CI did not contain the value 0, was considered significant.Statistical analyses were conducted using R,48  of 0.5 days (12 h). Multiple measurements occurring within one lag were averaged. Last, cross-correlation was conducted per patient, whereupon results were pooled for all patients and lags. For all cross correlations, original data  were used.Correlations (regular correlations are here referred to as “momentary” correlations) between biomarkers in different compartments and Q52 Independent of analysis type, we modelled a within-patient variance-covariance matrix, because we used repeated-measures data. The variance-covariance matrix was constructed as a time series model, for which we determined stationarity, autocorrelation, and partial autocorrelation. We deemed that the data were stationary using the Kwiatkowski–Phillips–Schmidt–Shin test, and therefore abstained from autoregressive integrated moving average modeling.53 The variance-covariance matrix hence consisted of an autoregressive-moving average (ARMA) model. We tested 25 different ARMA combinations of varying complexity, and then chose the optimal ARMA structure based on Akaike Information Criterion. For both S100B and NSE we used a first order autoregressive model with a moving average of 1; that is, ARMA. The resulting models were validated graphically.54 For general linear as well as linear mixed-model selection we employed both a step-up55 and a top-down strategy.56 Random effect structures were evaluated using restricted maximum likelihood-based estimations and fixed effects structure using maximum-likelihood estimations. Model selection was conducted by likelihood ratio tests between nested models and type I F tests. P values of the final models were generated using Satterthwaite approximations in the R package lmerTest.57 In all analyses, the dependent variable was the biomarkerblood value (log10 transformed). For S100B, the first 12 h were excluded from analysis, because these time points have been associated with an “extracranial peak.”9For inferential analysis, we compared a general linear model with correlated errors , and a linear mixed model. For both, the nlme package in R was used.56 For NSE, we had no such issues, which is why we present results from the linear mixed model. It is noteworthy that results were highly similar independent of linear mixed or marginal model design for each biomarker. Model assumptions were examined graphically with regard to homogeneity of variances, normality of variances, correlation between fitted and observed values, and individual assessment of residual autocorrelation and partial autocorrelation. For NSE, one patient (pat #17) followed a markedly different trajectory than the other patients and also constituted an outlier using the model diagnostics previously described. For validation, the results for NSE were run both including and excluding pat #17, and the results were to a large extent similar, leading us to conclude that the outlier pat #17 did not imply any major alterations with regard to overall conclusions.In general, Akaike Information Criterion was lower for the mixed-model design in the top-down strategy, whereas they were equal in the step-up strategy. For S100B, the variance-covariance matrix generated in the linear mixed model was not positive-definite, which is why we chose to use the equivalent marginal model for S100B.In total, 17 patients were recruited, of which one patient was excluded because no samples of CSF albumin had been obtained. Among included patients, there was no loss-to-follow up. Demographics are depicted both individually  and for A > 0.006–0.009  correlation and cross-correlation analyses. Cross-correlation analyses were conducted because clearance from CNS to peripheral blood might occur with a time delay. For cross-correlations, we examined three temporal relationships: (1) correlation between biomarkerCSF values and BBBD  corand BBBD ; and (3)d values .CSF and S100Bblood  as well as QA and S100Bblood . Cross-correlations for S100B showed that there was an immediate correlation between BBBD and S100BCSF (CSF and S100Bblood (blood (There was a strong positive (momentary) correlation between S100BS100BCSF , whereas00Bblood . The lagd  as well as QA and NSEblood . NSE cross-correlations demonstrated a negative lag between BBBD and NSECSF (CSF levels increased more swiftly than the BBBD occurred following an injury. NSECSF and NSEblood (blood (CSF and NSEblood lags (+2), not accounted for by the other cross correlations (-1 and 0 lags respectively). Further, this means that the delayed NSE clearance did not seem to be a consequence of a delayed intracranial NSE de novo release; that is, higher CSF levels  correlation between NSEd NSECSF  indicatiNSEblood  were posd   , where tA for blood biomarker levels, we conducted a generalized least square linear model  for S100B  patients.23 In contrast, Bellander and coworkers found a steep normalization of QA within the 1st week after TBI,26 congruent with data from Blyth and colleagues.21 This indicates that the BBB is likely more affected by the primary injury than secondary insults, and that BBBD may persist for at least 7 days, or as some have suggested, even longer.61 Consistently, some patients did not exhibit any signs of BBBD in our study. The underlying reason for this cannot be causally determined by this work because of the small sample size, but we hypothesize that differences in the intracranial injury panorama is a probable explanation. This finding further highlights the necessity of individualizing treatment following TBI. Finally, we noticed that QA and albuminCSF mimicked each other closely. Therefore, albumin levels in CSF are presumably enough to assess the BBBD, although this still necessitates CSF sampling, which is not always feasible in the clinical setting.We assessed how a trauma-induced BBBD affects brain biomarker concentrations in blood. Even though some radiological techniques have been proposed to quantify BBBD,blood was momentarily correlated to S100BCSF and QA, in accordance with previous studies.59 Others have acquired contrasting results,26 possibly because of different analysis strategies. We also noted that momentary NSEblood levels were correlated with NSECSF and QA, something that has been shown in non-TBI studies,62 whereas a previous TBI study did not show this association.26 For both biomarkers, we noted similar correlation coefficients between CSF:blood and blood:QA. This implies that a third covariate, most likely time from trauma, inferred these values. We therefore performed cross-correlations, to elucidate the underlying share time trend of the biomarker correlations.We found that S100Bserum and S100BCSF correlation have found a delay varying between 0–24 h30 and 63 Our S100B CSF:blood cross-correlation exhibited a lag of ∼12 h, corresponding to the delay seen between QA and S100Bblood. Hence, the CSF:blood and the QA:blood pattern mimicked each other, presumably indicating that there is a brief initial accumulation of S100B in CSF before clearance through a disrupted BBB. This constitutes a tentative pathway for S100B clearance to peripheral blood. In contrast, NSE exhibited a more delayed peak in cross-correlations than S100B (∼ 24 h). This is important, because a brain biomarker should confer accurate and timely information if it is to aid in clinical decision making. Moreover, in the three cross- correlations performed on NSE, de novo release of NSECSF seemed to occur more swiftly than BBBD, and BBBD occurred simultaneously to NSEblood detection. Hence, there was a delayed clearance of NSE from CSF to blood, that was not mimicked by either CSF:QA or QA:blood cross-correlations, which was the case for S100B. Data were scarce for all of these observations, but if true, this means that neither delayed BBB clearance nor delayed de novo release of NSE could explain the delayed clearance of NSECSF to blood. Hence, there is a weak but interesting signal suggesting that intracranial NSE accumulates and thereafter is cleared through a route other than the BBB. One tentative clearance pathway is the experimentally described glymphatic system,14 suggested to be a clearance route for both NSE and S100B.64 Another possible route includes the recently discovered lymphatic vascular network surrounding dural sinuses, which drains in deep cervical lymph nodes and subsequently blood, with clearance of proteins from the brain interstitium.66 The strong momentary correlations we see between CSF and blood suggests a relatively direct pathway in these glymphatic/brain lymphatic routes, which in the case of NSE is seemingly independent of BBBD. However, it should be noted that both the glymphatic and brain lymphatic vasculature have currently only been sufficiently studied in animal models. Some promising work using magnetic resonance imaging favors the existence and importance of the glymphatic system in humans,68 but larger human studies and functional characterizations are still warranted.In our cross-correlations, we noted that both S100B and NSE exhibited peak CSF:blood cross correlations at lags >0, suggesting that both biomarkers have a partially delayed clearance from CSF to blood. Previous studies examining S100Bblood, we found that QA, S100BCSF, and time from trauma were significant independent predictors. This indicates not only that S100B is dependent on the BBB for clearance to peripheral blood, but also that S100Bblood cannot be regarded solely as a marker of BBB integrity as has been suggested.69 Whereas some claim that cerebral S100B originates from the astrocytic foot processes that enfolds the BBB,30 our data rather indicate that S100Bblood is a reflection of several factors of which merely one is the extent of BBBD. In a situation in which the BBB is intact,69 it is therefore theoretically possible that the S100Bblood value is falsely low, as this biomarker will then to some extent be “trapped” in the CSF. Applying a similar approach to NSE, we found that time from trauma was retained as the only significant independent variable in the model. This means that neither NSECSF nor QA were predictive of NSEblood. Our findings cohere with one previous study that did not find any increase of NSE following chemical/osmolytic BBBD.70 In summary, there was a clear relationship between QA and S100B, whereas for NSE there was not.For inferential analysis, we employed marginal and linear mixed modelling. For S100B72 than S100B (9–14 kDa),73 implications of which could be a swifter movement across body compartments for small proteins and a longer serum half-life for larger proteins. Accordingly, NSE has been shown to exhibit an “effective half-life” of up to 48–72 h in serum compared with 24 h for S100B,74 following severe TBI. This would give a slower decline of NSEblood values than S100B values. We compensated for this by employing an ARMA variance-covariance structure to our model; however without finding any predictors of NSEblood other than time from trauma. Next, there are different sources for S100B and NSE throughout the body. S100B is present primarily in astrocytes,75 but extracranially also in muscle, bone, cartilage, adipose tissue, and melanocytes.76 The extracranial component is primarily of importance in multi-trauma patients during the first 12 h following the trauma.9 We excluded the first 12 h from analysis for S100B and monitored patients for a week, which would result in a very limited contribution of extracranial S100B to our model. In contrast, whereas NSE intracranially locates primarily to neurons,9 extracerebral NSE emanates from, among other sources, erythrocytes.77 Hence, extracerebral NSE is a confounding factor following both multi-trauma and hemolysis.9 Importantly, there is no clear period after trauma when this is important, but rather this potential confounder spans the entire data material and contributes a latent data noise. In summary, although S100B and NSE seem to have different clearance routes from the injured brain, there are differences between the proteins, which may have influenced our results.Both our cross-correlation and marginal/linear mixed model analyses hence indicate that S100B and NSE might be cleared differently from CSF to peripheral blood. There are, however, important differences in biochemical, kinetic, and/or pathophysiological properties between the two proteins, potentially confounding our results. Biologically, NSE is larger (39 kDa)15 Although no patients received acetazolamide, and CSF drainage speed was constant in our study, all patients exhibited different levels of induced sedation, which in theory could affect glymphatic clearance, but how this may affect biomarker levels in humans warrants further research. In spite of being a gold standard metric for BBBD, QA is age dependent,19 which is a source of error across all studies using it. Moreover, for TBI patients, intraventricular hemorrhage26 and administration of large amount of intravenous fluids could both affect QA. For the latter, we propose that CSF albumin alone might be an adequate surrogate for QA (and therefore BBBD). Finally, in the injured brain, multiple factors are in play, and it is possible that CNS half-life is discrepant from blood half-life of certain proteins. We do not account for this in our models, and in order to do so, one needs to employ more extensive pharmacokinetic modeling, which will be the aim for future studies that are warranted. Another amenable avenue is radiological techniques68 using biomarker-specific contrast-enhancement combined with sampling techniques similar to ours in order to elucidate discrepancies between BBB and other plausible clearance pathways.The major limitation in this study relates to the inclusion rate (inclusion was dependent on senior author presence) and the consequently small study sample size. Moreover, there are other factors that may be associated with brain biomarker clearance, such as brain injury severity or type of injury sustained, but these correlations necessitate a larger sample size to study. Conversely, the data was prospectively collected with a uniquely high temporal resolution with constant CSF drainage speed; therefore making this study highly relevant in term of brain protein clearance. Another limitation is that the CSF at time point 0 in our models constituted a pool of CSF collected during the preceding 6 h; therefore, the lag times could be slightly longer than indicated in the cross-correlations, although we believe the current protocol using the CSF pump is the most delicate way to extract CSF for serial sampling using an enclosed system. Further, in animals, it has been shown that acetazolamide treatment, CSF drainage, and sleep deprivation inhibit the glymphatic system.A. However, longitudinally, this relationship was stronger for S100B than for NSE. Upon inferential modeling, blood levels of S100B were dependent on QA, which we could not observe for NSE. Although this could be the result of underlying differences between the two proteins such as hemolysis of NSE, it indicates that S100B and NSE might be cleared differentially from the injured CNS, which is novel and important for brain biomarker interpretation and clinical utility, although future studies are needed to confirm our findings.Blood biomarker levels of S100B and NSE correlate momentarily with BBB integrity, measured as Q


Global ischemia is the resulting effect of a cardiopulmonary arrest (CPA). Presently there is no effective treatment to address neurological deficits in patients who survived a CPA. Granulocyte-colony stimulating factor is a growth factor (G-CSF) with a plethora of beneficial effects, including neuroprotection. Clinical application of human G-CSF (hG-CSF) is limited due to its plasma half-life of 4 h. Therefore, novel approaches need to be investigated that would (1) enable prolonged manifestation of hG-CSF and (2) demonstrate G-CSF efficacy from studying the underlying protective mechanisms of hG-CSF. In our previous work, we used the self-complementary adeno-associated virus (stereotype2: scAAV2) as a vector to transfect the hG-CSF gene into the global ischemic brain of a mouse. As an extension of that work, we now seek to elucidate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia.A single drop of either AAV-CMV-hG-CSF or AAV-CMV-GFP was dropped into the conjunctival sac of the Swiss Webster mouse’s left eye, 30–60 min after bilateral common artery occlusion (BCAO). The efficacy of the expressed hG-CSF gene product was analyzed by monitoring the expression levels of endoplasmic reticulum stress (ER), mitochondrial dynamics and autophagic proteins over 4- and 7-days post-BCAO in vulnerable brain regions including the striatum, overlying cortex  and the hippocampus (middle brain regions). Statistical analysis was performed using mostly One-Way Analysis of variance (ANOVA), except for behavioral analysis, which used Repeated Measures Two-Way ANOVA, post hoc analysis was performed using the Tukey test.Several biomarkers that facilitated cellular death, including CHOP and GRP78 (ER stress) DRP1  and Beclin 1, p62 and LC3-ll (autophagy) were significantly downregulated by hG-CSF gene transfer. hG-CSF gene therapy also significantly upregulated antiapoptotic Bcl2 while downregulating pro-apoptotic Bax. The beneficial effects of hG-CSF gene therapy resulted in an overall improvement in functional behavior.Taken together, this study has substantiated the approach of sustaining the protein expression of hG-CSF by eye drop administration of the hG-CSF gene. In addition, the study has validated the efficacy of using hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia. Cardiopulmonary arrest is one of the major causes of death and disability in the world. One significant consequence of cardiopulmonary arrest is global ischemia. Although there have been improved emergency treatments (such as therapeutic hypothermia), the survival rate for out-of-hospital cardiac arrest patients remains approximately 8% for the past three decades . ReducedThe balance between mitochondrial fission and fusion plays an integral role in mitochondrial physiology. Several essential proteins are involved in maintaining mitochondrial dynamics, including dynamin-related protein 1  and optic atrophy 1 . Ischemia disrupts mitochondrial dynamics by downregulating fusion proteins, such as OPA1; which promotes the mitochondrial fission phenotype –15. DistG-CSF is a hematopoietic growth factor that is FDA approved for the treatment of neutropenia. G-CSF stimulates growth and differentiation of hematopoietic stem cells , and botAdult male Swiss Webster mice  were kept cages in a room with controlled light cycles (12 h/12 h light/dark) at a constant temperature (21 ± 1 °C). Food and water were provided ad libitum except during the behavioral assessment. All animal procedures were carried out following the guidelines for care and animal’s use and were approved by the institutional animal care and use committee (IACUC) of Florida Atlantic University, Boca Raton.2/air mixture using a vaporizer. During surgery, in maintaining anesthesia, isoflurane concentration was reduced to 0.5% and an intra-peritoneal (IP) injection of ketamine hydrochloride  and xylazine hydrochloride  cocktail was administered. Ventrally, a sagittal incision was made in the mouse neck (~ 1 cm length). Both salivary glands were separated to visualize the underlying common carotid arteries (CCAs). Dissections were made between the sternocleidomastoid and the sternohyoid muscles parallel to the trachea, to expose the CCAs. The CCAs were carefully separated from the respective vagal nerves and accompanying veins. A lose 5-0 silk suture loop was made around each CCA, which was used to gently lift the CCAs while the CCAs were clamped with microaneurysm clips. The CCAs remained occluded for 30 min after which micro clips were removed with subsequent reperfusion. During the surgery, core body temperature was maintained at 37.0 ± 0.5 °C by controlled water circulating heating pad. Local cerebral blood flow (LCBF) was monitored in the cerebral cortex of either the left or right hemisphere by positioning a Doppler laser probe  over the area of the middle cerebral artery (MCA) on the left or right of the skull. Only mice with a LDF signal reduced to a minimum of 50% of the baseline signal were included in the study. Mice with incomplete reperfusion  were also excluded from the study.The BCAO method used to induced global ischemia was the same as previously published . BrieflyThe green fluorescent protein (GFP) -encoding plasmid used in this investigation , but no significant difference in LCBF between the BCAO + AAV-CMV-GFP group and the BCAO + AAV-CMV-hG-CSF group . The control animals underwent surgical procedures identical to BCAO, except that the CCAs were not occluded. The BCAO animals were given a single administration of either scAAV2-CMV-hG-CSF or scAAV2-CMV-GFP , to the eye sac of the left eye 30–60 min post BCAO/reperfusion, while sham-operated mice were given 1.5 µl saline to the left eye sac. There was a significant difference in LCBF between sham and the BCAO group (https://blast.ncbi.nlm.nih.gov/)  on day 4 with the RNeasy Mini Kit , according to the manufacturer’s directions. Samples were treated to remove DNA by using the Turbo DNase (Thermo Fisher Scientific). RNA was reverse transcribed utilizing a random pd(N)6 primer and Thermoscript RT-PCR system (Life Technologies). Quantitative reverse transcription real-time PCR analysis (qRT-PCR) of the viral transcripts was obtained as previously described . qRT-PCR/) Table . We geneAnimals were deeply anesthetized by isoflurane (Phoenix) and decapitated, and then brains were rapidly removed. Using an adult mouse brain matrix , brains were sectioned coronally into six 2-mm coronal slices  and incubated for 5 min in a 2% (w/v) solution of 2,3,5-triphenyltetrazolium chloride  at 37 °C for staining followed by collecting samples for western blot .v/v) mammalian protease inhibitor cocktail and 2% (v/v) phosphatase inhibitor cocktail . Homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected while the pellet discarded. Bradford protein assay determined-protein concentrations were then resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose membrane. Nitrocellulose membrane was then incubated overnight (4 °C) with the following primary antibodies: Abcam: GRP78, ATF4, p-IRE1, OPA1 , GAD65 (1:500) and XBP-1 (1:500); Cell Signaling: GAPDH (1:3000), Bcl2 (1:1000), AKT, p-AKT and Beclin 1 ; Santa Cruz: CHOP/GADD153 (1:100), Bax (1:500) and hG-CSF ; Millipore: DRP1 (1:1000) and Imgenex: ATF6 (1:500); followed by three washes with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and then incubated with fluorescence conjugated secondary antibodies, goat IRDye 800-conjugated anti-rabbit  or anti-mouse   for 2 h at RT. GAPDH was used as internal loading controls for whole cell lysate samples. The hG-CSF antibody  was specific to human G-CSF protein produced by the human G-CSF gene. All antibodies with the respective company and catalog number are listed Additioanl file Mice brains were removed and quickly dissected on ice using a brain matrix. Sections of 2 mm coronal brain slices were divided into frontal , middle  and hind . The frontal and middle brain sections were used since the common carotid arteries supplied these areas. Western blot was done as described previously . Briefly GAPDH 1:000, Bcl2The histological analysis involved nissl staining and immunofluorescence and was carried out by a blinded observer to the study. Mice were perfused transcardially with phosphate-buffered saline  followed by 4% paraformaldehyde. Brains were removed, post-fixed in 4% paraformaldehyde overnight at 4 °C, and cryopreserved with 30% sucrose, then freeze at − 80 °C in an anti-freeze solution . Sections were further process using nissl staining or immunofluorescence.2) of the striatum and three non-overlapping visual fields (0.25 mm2) of the cortex were randomly selected, while two non-overlapping visual fields (0.25 mm2) of CA3, CA2 and CA1 were randomly selected for statistical analysis. Images were captured with an epifluorescence microscope, converted to 8-bit grayscale, threshold, size excluded by setting a minimum pixel area size was set, watershed to separate close cells and automated counted using NIH Image J. Neurons with round and palely stained nuclei were considered surviving cells, whereas shrunken cells with pyknotic nuclei were considered dying cells.For each animal, twelve serial sets of coronal brain sections (40 µm) were cut on Leica cryostat between 1.94 and -2.46 posterior to bregma. Mice brain sections were washed twice for 15 min in 0.1 M PBS and incubated in 0.5% cresyl violet staining solution for 10–15 min at room temperature. Following incubation, the sections were washed with distilled water and dehydrated gradually in ethanol , then placed in xylene as a final processing step. Sections were cover slipped with mounting media. Ten consecutive sections (four sections for striatum and cortex and six sections for hippocampus) per brain were analyzed . Five non-overlapping visual fields (0.25 mm2) of the cortex, two non-overlapping visual fields (0.32 mm2) of the hippocampus’s CA3 and CA1 were selected at random for statistical analysis. Another set of three consecutive sections per brain were analyzed for ChAT and NeuN colocalization in the forebrain . Fluorescence signals were detected by a Zeiss LSM700 confocal laser scanning microscope. Collected images were quantified for GAD and CHAT immuno-positive (GAD+ and CHAT+ respectively) neurons by converting images into 8-bit grayscale, threshold, size excluded by setting a minimum pixel area size and automated counted using NIH Image J software. Negative controls were performed by omitting the primary antibody (data not shown).Free-floating sections were blocked in blocking buffer  for 1 h. at room temperature. Sections were incubated with rabbit anti-GAD, 1:200 (laboratory of Dr. Jang Yen Wu), mouse anti-Chat, 1:500; (Santa Cruz: sc-55557) or rabbit anti-NeuN, 1:500 (Millipore Sigma: MAB377) overnight at 4 °C followed by incubation with anti-rabbit Alexa Fluor 488-conjugated, anti-mouse Alexa Fluor 488-conjugated , or anti-rabbit Rhodamine Red-conjugated, 1:500 (Jackson ImmunoReach AB2338022) for 2 h at RT. After washing with TBS-T , the sections were mounted on saline-coated microscope slides with Prolong Gold antifade reagent (Invitrogen). Ten consecutive sections per brain were analyzed for GAD, with four sections for cortex and six sections for the CA3 and CA1 (n = 4 per group). Four non-overlapping visual fields (0.32 mm3) arranged to form a 30° corner; a small opening was left along the joint between the two boards. The mouse was placed 12 cm from the corner and allowed to walk into the corner so that the vibrissae on both sides of the animal's face made contact with the two boards simultaneously. Before the BCAO procedure, we conducted behavior tests (stratification) on all mice to screen for mice with no turning asymmetry (n ≥ 16). Each mouse took part in ten trials, after which we calculated the percentage of turns to each side, recording only those turns involving full rearing along one of the boards. This stratification procedure excludes mice with a pre-test score of 80–100% asymmetric turns (n = 4).All behavioral paradigms were performed by an observer blinded to the study. The corner test, which determines an animal’s asymmetric direction of turning when encountering a corner, is used to indicate brain injury. We used an experimental corner setup composed of two boards  as described . The actp < 0.05. Data are represented as the mean ± SEM independent experiments were performed in duplicates or triplicates. In figures p < 0.05 is labeled as * or #p < 0.01 is labeled as ** or ## and p < 0.001 is labeled as *** or ###based on the respective comparison.Data were analyzed using GraphPad Prism 7.0 software . Behavioral measurements were analyzed by two-way ANOVA repeated measures, followed by Tukey’s post hoc test. All other analyses were performed with one-way ANOVA, followed by Tukey’s post hoc test. Differences in p-values were considered significant if The mortality rate (4%) was low in our 30 min BCAO model of global ischemia. Mice were randomly assigned to receive either AAV-CMV-GFP or AAV-CMV-hG-CSF pre-BCAO. All animals subjected to BCAO showed a significant reduction in LCBF. Mice designated to receive AAV-CMV-GFP showed a 62% ± 5% drop in baseline LCBF while AAV-CMV- hG-CSF designated mice showed a 69% ± 8% drop in baseline LCBF  into the left eye 30–60 min post-reperfusion. Then performed qRT-PCR (left hemisphere) for hG-CSF mRNA expression and western blot (right hemisphere) for hG-CSF protein expression in sham-operated, AAV-CMV-GFP group and the AAV-CMV-hG-CSF group. After administering the viral vectors, we observed a dramatic increase, (≥ 5-fold) 4 days post-viral transfection of both hG-CSF mRNA and protein expression in the AAV-CMV-hG-CSF treated group compared to both sham mice and the AAV-CMV-GFP treated mice .Using cresyl violet (staining DNA and RNA nucleotides) to stain nissl bodies, we observed that hG-CSF gene expression reduced cellular loss, 4 days post-BCAO, by 50% in the striatum . During mitochondrial dysfunction, the mitochondria undergo fission (fragmentation), increasing DRP1. Our data from western blotting showed that global IR injury significantly increased the expression level of the mitochondrial fission protein, DRP1  exists as two isoforms, GAD65 and GAD67. GAD is enzyme in neurons that catalyzes the decarboxylation of glutamate to gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain. Therefore, the cellular expression of GAD is indicative of GABAergic neurons. We observed that at 4 days post-BCAO, the levels of GAD immunopositive neurons were reduced in the cortex, and in the CA1 and CA3 hippocampal cornus of the AAV-CMV-GFP mice compared to sham  , 43. TheLocomotor activity in an open field area such as the force actometer chamber is used as a behavioral paradigm for testing functionality after ischemic brain injury. We examined mice 4- and 7-days post-BCAO and found that mice treated with the AAV-CMV-hG-CSF gene showed relatively high locomotor activity at the first 20–30 min during the 60 min test Fig. k, l. ThiGene therapy is emerging as an exciting and effective therapeutic strategy applied to several neurological disorders and neurodegenerative diseases. The AAV serotype 2 (AAV2) viral vector used in this current study is commonly used for gene delivery in rodent models of stroke and neurodegeneration , 45. In 9 pfu; 1.5 µl) of the scAAV2 vector carrying the hG-CSF gene into the conjunctival sac of the eye is a reliable approach for delivering sustained hG-CSF into the brain. This result validates our previous study that demonstrated MRI based detection of mRNA for hG-CSF cDNA after eye drop AAV-CMV-hG-CSF administration  ER due to the decreasing ER ER . We obse2+) ER through the inositol 1,4,5-trisphosphate receptors (IP3 Rs), by direct interference of this receptor channel [Recent studies have revealed that a fraction of cytosolic Bcl2, Bax/Bak is located at the ER membrane and that pro-apoptotic Bax/Bak modulates the UPR by directly interacting with IRE1 . Further channel . These t channel . An over channel . The fusBoth ER stress and mitochondrial dysfunction inevitably proteinate autophagy (“self-digesting”) . While aThe neuroprotective effect of hG-CSF gene therapy in the frontal and middle brain sections would translate to protecting various populations of neurons in these brain regions. GABA, the CNS’s main inhibitory neurotransmitter is synthesized in GABAergic neurons from glutamate by the two isoforms of GAD; GAD65 and GAD67. GAD67 is present throughout the cytosol, whereas GAD65 preferentially localizes in axonal terminals , 77. GABPrevious studies employing rodent models of transient global ischemia and permanent global ischemia have indicated that the number of ChAT positive neurons decreases in the basal forebrain and the hippocampus. Such decreases could be reversed by treatment with neuroprotective agents, concomitant with the reversal of behavioral deficits . SimilarThe protective effect of hG-CSF gene therapy is reflected in improved functional behavior at 4 days and 7 days post-BCAO. Interestingly, locomotor activity improved for the first 20 min for the hG-CSF gene group at 4 days. Improved locomotion for the first 20 min was enhanced for a longer time period of 30 min at 7 days post BCAO This improved behavior at 7 days over 4 days indicates that the longer constitutive expression of the hG-CSF protein by the hG-CSF gene boosted protection, as reflected in improved behavior. In our previous study , we haveAlthough the global ischemic (transient BCAO) model used in this study does not provide a complete cessation of blood flow to the entire brain compared to other models, such as ventricular fibrillation/defibrillation, the transient BCAO model has however, been successfully used in several studies to simulate forebrain ischemic pathology of cardiopulmonary arrest/resuscitation (CPR) , 83. TheCurrently, no pharmacological agents have been proven to effectively improve neurological outcomes in hospital-discharged survivors of a cardiac arrest. In this study, we demonstrated the efficacy of human G-CSF gene therapy in re-establishing ER homeostasis, preserving mitochondria dynamics and reducing autophagy’s detrimental action in a mouse model of transient global ischemia. The single administration of the hG-CSF gene provides a sustained expression of the hG-CSF protein, thereby overcoming the 4-h half-life limitation of plasma hG-CSF. Furthermore, our eye drop method of administering the hG-CSF gene is noninvasive. However, translational of AAV2-CMV-hG-CSF gene therapy to stroke trial could be challenging due to limited information available for G-CSF gene therapy using the AAV-viral vector as a delivery vehicle. Recent approval from the FDA for AAV-delivered gene therapy for a rare eye disease namely, Leber congenital amaurosis  could seAdditional file 1: Fig. S1. Local cerebral blood flow (LCBF). Laser Doppler Flowmeter (LDF) recording of LCBF: (a) sham-operated mouse showing marginal change in blood flow (b) AAV-CMV-GFP mouse pre- BCAO (mean baseline flow 94.31 PU), during 30mins BCAO  and reperfusion (mean 154.77 PU). (c) AAV-CMV-hG-CSF mouse pre- BCAO (mean baseline flow 81.99 PU), during 30mins BCAO (mean 10.39 PU) and reperfusion (mean 110.61 PU). (d) Quantitative bar graph of LCBF during 30 mins BCAO. Pre-BCAO, mice were randomly assigned to be treated with either AAV-CMV-hG-CSF (n = 21) or AAV-CMV-GFP (n=27). Sham operated mice (n=16) were not subjected to BCAO. Data were presented as percentage (%) value of baseline LCBF. Values are mean ± SEM. * * * P < 0.001 vs vs sham-operated mice. One-way ANOVA, post hoc Tukey test.Additional file 2: Fig. S2. Representative example of western whole blot. Western blot of GRP78 and loading control at 4-day post-BCAO. First lane shows protein marker. Lanes 1-3 represents the front (striatum and overlying cortex) region of the brain. Lane 1- Sham, lane 2 – AAV-CMV-GFP and lane 3 – AAV-CMV- hG-CSF. Lanes 4 – 6 represents the middle (Hippocampus) region of the brain. Lane 4- Sham, lane 5– AAV-CMV-GFP and lane 6 – AAV-CMV- hG-CSF. Molecular weights of GRP78 and GAPDH 78 kDa and 37 kDa respectively. Densitometry is taken as quantitative intensity of bands and normalized against GAPDH.Additional file 3: Table S1. List of Antibodies used for Western Blotting


Hand hygiene is one of the most effective ways to remove germs, prevent the spread of infectious pathogens, and avoid getting sick. Since the COVID-19 pandemic began, health authorities have been advocating good hand hygiene practices.The primary aim of this study is to field test a prototype smart handwashing station deployed in a school setting during the COVID-19 pandemic.We deployed a smart handwashing station and examined key technological considerations including connectivity, security, and data management systems, as well as the health and safety of users.The smart handwashing station was deployed for 10 days in a school setting in Australia during the COVID-19 pandemic. The smart handwashing station’s electrical components remained operational during field testing and underwent robust cleaning protocols each day. The handwashing station was used 1138 times during the field test and there was no COVID-19 transmission at the school during the testing.This study demonstrates that a personalized feedback approach using technology can successfully be implemented at a school and can provide a platform to improve hand hygiene among school-aged children. COVID-19, a contagious infectious disease caused by SARS-CoV-2, has emerged as a global health crisis and pandemic ; howeverResearch has shown that COVID-19 can remain on environmental dry surfaces ; when a Previous studies have shown that health education on hand hygiene alone is often ineffective at changing hygiene behaviors. Current hand hygiene education, such as visual signage, is generic and may not be personalized or unique to the individual. Increasingly, posters are made available at sinks to support the public in effective hand hygiene technique. Previously, hand hygiene posters showed multiple steps and therefore may have been difficult to follow. Recently, a simpler 3-step hand hygiene technique was found to improve technique and compliance compared to a 6-step technique . HoweverThere are limitations to the current methods of measuring hand hygiene compliance and performance. The World Health Organization (WHO) guidelines recommend that hand hygiene compliance should be assessed by direct observation by trained observers as the gold standard. Direct observation is currently recommended because it is the only approach that can detect all hand hygiene opportunities and actions to assess the number of times hand hygiene was required as well as the appropriate timing of handwashing . Hand hyIoT-enabled smart devices were used in a hospital to measure how often hospital workers washed their hands; sensors monitored the flow of people and tracked disinfectant usage . A limitDuring the COVID-19 pandemic, there has been a call for novel, digital behavioral interventions to facilitate adoption and maintenance of individual-level preventive behaviors . In thisIn this study, we developed a smart handwashing station, incorporating a 365 nm UV light emitting diode (LED) light source, digital camera, and processing electronics within housing constructed from plastic and mounted on a stand. A commercially available pressure sensor mat (Radio Parts Pty Ltd) was connected via cable to a custom data acquisition system that reported pressure information in real time over a USB connection. Both the pressure sensor mat and the handwashing station were connected to a tablet (Acer Incorporated) via a USB cable. A software application written in Python triggered the camera to take a photo each time a person stood on the pressure sensor mat, and then every 5 seconds that they remained standing on the mat. All images were captured with timestamp information. Data was only stored locally on the tablet, and was periodically collected and sent to an off-site location. Although the device was supervised in this installation, it could easily be fixed to a specific location using a laptop-style antitheft lock.To check the smart handwashing station was connected and recording usage data correctly, observational testing was performed in Brisbane, Australia. Three volunteers  stood on the pressure sensor mat and used the smart handwashing station 10 times. The time-stamped data collected by the station was then compared with observational data.The temperature of the smart handwashing station following 2 hours of continuous operation was recorded using an infrared handheld thermometer (ThermaTwin TN410LCE Infrared Thermometer). The UV radiation emitted by the UV light source was measured using a UV intensity meter  fitted with a digital sensor . The detector head of the sensor was positioned with consideration of where a person’s hands would be placed during use.The field study was conducted during the COVID-19 pandemic in April and May 2020, when handwashing was required every day. One smart handwashing station was deployed to a school located in Queensland, Australia. Children and adolescents aged 5-18 years attended the school, with over 1500 students enrolled. The smart handwashing station was placed near the school entry and outside handwashing facilities in a high-traffic area accessed by all students at the start of the day and during breaks. A moisturizer that contains a UV-fluorescent compound (BREVIS Corporation) was dispensed using a touchless automatic system (Décor House).Feedback was obtained from end users, including teachers and teaching assistants, to design and refine the device. Questions regarding logistics were asked . In addition, email and phone contact details of the researchers were displayed and provided to the teachers for complaints, technical issues, or further information during the deployment. The deployment of the smart handwashing station was to assess device functionality and not human subjects research; therefore, we obtained an institutional ethics review board exemption from the Human Research Ethics Committee of the Queensland University of Technology.Observational testing of the handwashing station dichotomized usage variables to categorical data: handwashing usage data was coded “yes” if timestamped data was recorded to the tablet and coded “no” if the handwashing station was used but no data was recorded to the tablet. The Cohen κ score was calculated to determine if there was agreement between categorical variables for handwashing usage. Values of 0.4-0.6 were considered moderate agreement, values of 0.6-0.8 were considered substantial agreement, and values of 0.8-1.0 were considered almost perfect agreement. GraphPad Prism  and SPSS  were used for analyses.A prototype smart handwashing station was developed, which uses a UV light source and digital camera to provide personalized feedback on hand hygiene . First, The smart handwashing station integrated IoT approaches to track usage and collect images of users’ hands. Observational testing of the smart handwashing station demonstrated there was perfect agreement with observed handwashing and station-recorded usage  as a medical device in order to be lawfully sold in Australia. The definition of what constitutes a medical device has recently been updated in Australia by the TGA, with amendments aligning more closely with the equivalent definitions in the European Union’s Regulation (EU) 2017/745. The Federal Drug Administration (FDA) ensures that the claims made by a medical product accurately reflect its risks and benefits in the US market. The regulatory framework determining the medical device approval processes are based on relative risk. Medical devices include a diverse range of devices, from toothbrushes to software. Accelerated approval may be allowed by many regulators if a device can provide evidence that it is substantially similar to an existing device in terms of materials and safety, or if the risk class is sufficiently low as to need relatively little safety data.While observation is accepted as the gold standard when evaluating handwashing effectiveness, it can be costly and time-consuming, as well as difficult to implement during a pandemic when social distancing and shelter-in-place restrictions are imposed by many governments . MicrobiA limitation of this study is that we did not analyze the usage data or the captured image data to investigate behavior change. In addition, the duration of field testing was only 10 days. Detailed qualitative data was not collected from users. Future research could expand on the enablers and barriers to hand hygiene in schools. In this proof-of-concept study, we did not monitor students’ hand hygiene compliance or handwashing quality. The placement of the station needs to be within close proximity to handwashing facilities and the station was not optimized for hand sanitizer. The Centers for Disease Control and Prevention in the United States recommends using alcohol-based sanitizers only when soap and water are inaccessible. Handwashing using soap and water is more effective for removing most types of microorganisms than using hand sanitizers . WearingFuture research should explore image analysis approaches to objectively measure hand hygiene levels in the community. Testing satisfaction with the smart handwashing station could be expanded to other settings, including public spaces such as airports, shopping centers, recreational venues, and workplaces. The smart handwashing station shows significant promise for generating relevant data for public health authorities on hand hygiene practices in the community. The smart handwashing station data could be used to inform targeted training as part of health programs in areas with poor handwashing, as well as assist inventory management systems to ensure appropriate levels of soap are always available.Handwashing is an essential process to reduce the spread of infectious pathogens. This study developed a handwashing station, which used a simple and cheap method that was easily understood by school-aged children. To assist schools to protect their students from outbreaks, the smart handwashing station could help build awareness of the importance of hand hygiene and provide personalized feedback. The usage data generated by this device could also benefit health programs by informing public health authorities of areas with low handwashing where targeted training may increase hand hygiene. This study provides evidence for the technical feasibility of smart handwashing stations in a school setting.


As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 μL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 μg/mL and rat plasma dilutions of 0.5–2% provide the most accurate concentration estimates when compared with rat CSF. 1000 μg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.The online version of this article (10.1007/s00216-020-02403-3) contains supplementary material, which is available to authorized users. Within pharmaceutical research, there is considerable interest in therapeutic antibodies, with 9 out of the 20 best-selling drugs in 2018 being antibody-based  and pushExamples of potential targets in neurodegenerative diseases are amyloid β and tau proteins. In Alzheimer’s and Parkinson’s disease, they form neurotic plaques containing the aggregative amyloid β 1–42 amino acid form  and neurHistorically, quantitation of mAbs in CSF and plasma samples has been performed using ligand binding assay platforms (LBA) –14. LC-MA standardized artificial CSF (aCSF) is needed as a surrogate matrix to develop and perform sensitive LC-MS methods for CSF without the need of sacrificing several animals to retrieve blank CSF. An aCSF must behave identically to CSF with regard to digestion efficiency when exposed to the bottom-up workflow. In addition, it must also mimic prevention of any non-specific binding (NSB) of analytes, as NSB to surfaces is an issue related to every process involving pipetting and storage of peptides and proteins , 33. FinVarious aCSF compositions have already been applied in other labs and are summarized in Table CSF from all species was purchased from Seralab (UK) and plasma was purchased from Cytotech (UK). SILu™Lite SigmaMAb human immunoglobulin G1 (hIgG) standards, SILu™Mab SIL hIgG, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich .ProteinWorks eXpress Digest kit  was used for the bottom-up workflow. The kit includes all reagents used in the digestion workflow Table  except fThe surrogate matrices were produced from either diluted rat plasma or BSA. In total, 11 surrogate matrices were prepared in 50 mM ammonium bicarbonate (AB50) for the following concentrations: 20, 300, 600, 1000, 1400, 2000, and 10,000 μg/mL BSA and 0.5, 1, 2, and 5% rat plasma .Eight hIgG standards were prepared in the concentration range 20–20,000 ng/mL in AB50 with 20 μg/mL BSA and diluted 1:19 in surrogate matrix by liquid handling robot  for a final calibration standard range of 1–1000 ng/mL.Manually prepared control and CSF samples were made by spiking hIgG (prepared in AB50 with 20 μg/mL BSA) in surrogate matrix or CSF in the ratio 1:9. Final samples were stored at − 80 °C until use.All analytical work was carried out using Acquity UPLC system  coupled with Xevo TQ-XS or Xevo TQ-S triple quadrupole . Separation was performed using an Acquity CSH C18 Column, 130 Å, 1.7 μm, 2.1 × 100 mm . Sample handling was performed in protein LoBind plates and tubes  with low-retention pipette tips .Quantitation by measuring selected tryptic peptides from hIgG was done following the bottom-up approach as outlined in Table The peptides were separated using 12-min gradient elution on a UPLC system with a 20 μL sample loop. Mobile phase A was MQ water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid Table . AnalysiThe setup was designed around three experiments in order to investigate which surrogate matrices could be recommended for use in quantitative analysis of CSF from preclinical species. All the experiments were performed using the bottom-up approach described in the “First, the ion suppression and digestion efficiency were investigated using the ISTD response from SIL peptides added to the digestion tubes as intact protein in a relatively large amount. From the procedure in Table The second experiment was designed to evaluate the accuracy and precision of QC samples in different surrogate matrices to determine if factors other than ion suppression and digestion efficiency could affect the estimates. Nine different surrogate matrices were spiked with hIgG at 3 levels  in 4 replicates and estimated using one calibration curve in 1000 μg/mL BSA. Spiked rat CSF was used as a reference.The last experiment evaluated species differences using a surrogate matrix of 1000 μg/mL to estimate concentrations of QC samples spiked into CSF from dog, rat, and monkey. This experiment would determine if an aCSF could be used for multiple species and provide an accurate estimate. QC samples of 500 and 1000 ng/mL in 4 replicates were estimated using GPSVFPLAPSSK 418–506 and TTPPVLDSDGSFFLYSK 937–836 signature peptides to reduce interference from endogenous peptides.Human IgG is considered a suitable analyte for evaluation of aCSF as it produces stable signature peptides of different sizes and hydrophobicity (chromatogram shown in ESM) that can be affected differently by the tested matrices. The hIgG concentration was estimated using calibration curves made in 1000 μg/mL BSA. The statistical data from the linear regression on each fragment is shown in Table p < 0.05) than observed in CSF . The two matrices, 10,000 μg/mL BSA and 5% plasma , had the highest protein content of the tested matrices. It is likely that the reduced response was caused either by ion suppression or by decreased digestion efficiency; hence, the high protein concentration matrices cannot be recommended to use as a surrogate for CSF. However, the 10,000 μg/mL BSA matrix was analyzed further to determine if ISTD is capable of correcting the reduced signal observed. The remaining matrices produced similar responses so any variation observed for the hIgG analyte in the following experiments was likely caused by factors other than ion suppression and digestion efficiency. The 1000 μg/mL BSA matrix showed the closest resemblance to rat CSF so this was chosen as the default calibration curve.Six surrogate matrices covering a broad range of protein content were investigated for their matrix effect on the ISTD signal. Each bar displayed in Fig. Nine different surrogate matrices were evaluated by spiking with three levels of hIgG  and comparing the estimates with similar samples in rat CSF. The results, including coefficient of variation and accuracy, are listed in Table The range of total protein content in the remaining matrices  is likely to cover biological variations of real CSF samples. Even the 10,000 μg/mL BSA matrix provided similar accuracy as the other matrices and could work as an aCSF, but it should be noted that the overall decreased signal caused by the large amount of protein can decrease sensitivity and increase column wear. Also, it was only possible to achieve this accuracy because a SIL signature peptide was available. A novel biotherapeutic with a unique sequence cannot rely on available SIL signature peptides. These results demonstrate the importance of the total protein concentration in the matrix since this will significantly affect the estimated concentration of a hIgG analyte. The protein content needs to be above a level where NSB is avoided but below a level where it does not affect digestion efficiency or cause ion suppression in the MS analysis as seen in Fig. Although 300–10,000 μg/mL BSA and 0.5–2% plasma all performed well as a surrogate matrices for CSF, there are some considerations to take into account. BSA from a standardized production is expected to give less batch variation due to the relatively homogenous content. There is less chance of the matrix containing any proteins or proteases that can affect the analyte of interest which assures a similar effect across different analytes. Diluted plasma is under influence from the species of origin, diet, and other factors  and has Two levels of hIgG in CSF from three species were estimated using a single surrogate matrix. Results shown in Fig. Our findings reveal that in a bottom-up LC-MS analysis of a model mAb (hIgG), it is recommended to use a BSA solution in the range of 300 to 2000 μg/mL to replicate the analytical response in rat CSF. Plasma dilutions of 0.5 to 2% can also be used as an adequate aCSF matrix; however, the BSA matrix likely offers less variation and less chance of interference from endogenous proteins. The highest protein concentration of 10,000 μg/mL BSA also provided similar accuracy to other matrices, but the signal intensities were noticeably lower which can be expected to decrease sensitivity. Matrices with particularly low protein concentration underestimated hIgG levels when compared with real CSF and must be avoided.The bottom-up LC-MS method used here for quantification of mAbs provides the ability to accurately measure 10 ng/mL hIgG in sample volumes of 10 μL rat CSF or aCSF with no additional sample cleanup after digestion. The investigated aCSF could be applied to species other than rat, as shown with standards prepared in 1000 μg/mL BSA that was able to quantify hIgG in CSF from rat, dog, and monkey within 20% of the true concentration. It is highly plausible that other species can be included as the investigated matrices covered a large range of protein contents  to account for any biological variation within or between species. We hypothesize that the identified aCSF compositions that yielded reasonable results for mAb analysis in the current study may also be useful for quantitative analysis of other biotherapeutics or protein biomarkers in CSF where digestion efficiency and NSB are important factors.ESM 1(PDF 893 kb)


BackgroundSevere alcoholic hepatitis is a condition with a very high mortality rate and there is a paucity of evidence regarding efficacy and safety of most available therapeutic options. The present systematic review and meta-analysis aims to assess the survival benefit of granulocyte colony stimulating factor (G-CSF) in patients with severe alcoholic hepatitis.MethodsStudies involving adult patients receiving G-CSF for severe alcoholic hepatitis were searched in MEDLINE, Ovid journals, MEDLINE nonindexed citations, and Cochrane Central Register of Controlled Trials and Database of Systematic Reviews. Pooling was conducted by both fixed and random effects model.Resultsn = 136) which met the inclusion criteria. In the pooled analysis, the 90-day survival in the G-CSF group was 80.03% (95% CI = 69.93-88.49) compared to 40.92% (95% CI = 29.76-52.58) in the Standard Medical Therapy (SMT) group. At 28 days, the Model for End-Stage Liver Disease (MELD) score lowered by 4.89 (95% CI = 4.13-5.64) in the G-CSF group compared to 4.00 (95% CI = 3.25-4.75) in the SMT group. Child-Turcotte-Pugh score declined by 2.26 (95% CI = 1.90-2.63) in the G-CSF group after 28 days compared to 0.91 (95% CI = 0.59-1.23) in the SMT group. At 28 days, Maddrey Discriminant Function score lowered by 39.79 (95% CI = 34.22-45.36) in the G-CSF group compared to 12.39 (95% CI = 6.90-17.88) in the SMT group.The initial search identified 543 reference articles; of these 24 relevant articles were selected and reviewed. Data was extracted from four studies  after 28 days of treatment. There certainly is a need for further studies, including development of personalized therapeutic dosing schedules, for G-CSF administration. Alcoholic hepatitis (AH) has a very high mortality rate, approaching 40% within the first four weeks of clinical presentation . ClinicaMultiple therapeutic modalities have been investigated for the treatment of such patients. Corticosteroid therapy is the current first-line treatment for severe AH but has remained controversial due to modest reductions in 28-day mortality and no survival benefit beyond one month . MoreoveG-CSF is an indigenous glycoprotein produced by macrophages, fibroblasts, and endothelial cells which promotes cell growth, differentiation, and function of neutrophils . In humaThere are currently several small reports suggesting benefit of G-CSF therapy in severe AH. The aim of the present meta-analysis was to systematically review the published literature and assess the efficacy of G-CSF therapy in improving survival and liver severity indices in patients with severe AH.Search methodologyA literature search was conducted using the electronic database engines MEDLINE through PubMed, Ovid, Cochrane Library , EMBASE, Cumulative Index for Nursing & Allied Health Literature, ACP Journal Club, Database of Abstracts of Reviews of Effects (DARE), International Pharmaceutical Abstracts, OVID Healthstar, and Google Scholar from January 1974 to August 2020 to identify published articles and reports addressing the use of G-CSF in patients with AH. The combinations of keywords used were  and (“Granulocyte colony-stimulating factor” or “G-CSF”). The reference list of all eligible studies was reviewed to identify additional studies. The retrieved studies were carefully examined to exclude potential duplicates or overlapping data. Titles and abstracts selected from the initial search were first scanned, and the full papers of potential eligible studies were reviewed.Study eligibilityPublished studies were eligible for inclusion if they reported the use of G-CSF for the management of severe alcoholic hepatitis. Articles were excluded if they were not written in English, no outcomes were reported, or they represented review articles or studies published as abstracts only. In studies using multiple modalities for the management of alcoholic hepatitis, data from the cohort of patients who received G-CSF were collected and analyzed. Two reviewers  independently performed study selection according to eligibility criteria. Disagreements were resolved by discussion or a third reviewer. The agreement between reviewers for the collected data gave a Cohen κ value of 1.0.Data extraction and quality assessmentThe following data were independently abstracted onto a standardized form: study characteristics , study design, baseline characteristics of the study population , the intervention details and outcomes . Risk of bias was rated for each study by two authors independently, using the Cochrane criteria for RCTs . Outcome definitionThe primary outcome of interest was 90-day survival of severe AH patients receiving G-CSF therapy compared to Standard Medical Therapy (SMT). Effect of G-CSF therapy on severity indices  was evaluated as a secondary outcome . Statistical analysis2 statistic and Cochran Q test based upon inverse variance weights [2 of 0% to 39% was considered as nonsignificant heterogeneity, 40% to 75% as moderate heterogeneity, and 76% to 100% as considerable heterogeneity. If P-value is >0.10, it rejects the null hypothesis that the studies are heterogeneous. The effect of publication and selection bias on the summary estimates was tested by Harbord-Egger bias indicator [This meta-analysis was performed by calculating pooled proportions. First the individual study proportions were transformed into a quantity using Freeman-Tukey variant of the arcsine square root transformed proportion. The pooled proportion is calculated as the back-transform of the weighted mean of the transformed proportions, using inverse arcsine variance weights for the fixed effects model and DerSimonian-Laird weights for the random effects model . Forest  weights . I2 of 0ndicator .The initial search identified 543 reference articles, of which 24 articles were selected and reviewed. Data were extracted from four studies that evaluated the efficacy of G-CSF in severe alcoholic hepatitis, which met the inclusion criterion ,23,24,25The total number of patients included in this meta-analysis was 136. Mean age of the patients in the G-CSF group was 43.44 years compared to 45.32 years in the SMT group. Three studies used G-CSF dose of 10 mcg/kg for five consecutive days ,24,25. OPrimary outcome 2 = 20.3%). Figures In the pooled analysis, 90-day survival in the G-CSF group was 80.03% (95% CI  = 69.93-88.49) compared to 40.92% (95% CI  = 29.76-52.58) in the SMT group with low heterogeneity  compared to a reduction of 4.00 (95% CI = 3.25-4.75) in the SMT group. Moreover, at 90 days G-CSF therapy further reduced the MELD score by 11.94 (95% CI = 11.04-12.84) compared to baseline. Child-Turcotte-Pugh ScoreAt 28 days, G-CSF therapy lowered the CTP score by 2.26 (95% CI = 1.90-2.63) which was significantly better than a reduction of 0.91 (95% CI = 0.59-1.23) in the SMT group. Moreover, at 90 days G-CSF therapy further lowered the CTP score by 3.21 (95% CI = 2.85-3.57) compared to baseline. Maddrey Discriminant Function ScoreAt 28 days, G-CSF therapy lowered the mDF score by 39.79 (95% CI = 34.22-45.36) which was significantly better than a reduction of 12.39 (95% CI = 6.90-17.88) in the SMT group. Moreover, at 90 days G-CSF therapy further lowered the mDF score by 65.07 (95% CI = 60.16-69.97) compared to baseline. Severe alcoholic hepatitis remains a commonly encountered clinical entity with high degree of morbidity and mortality and no satisfactory therapeutic option. Corticosteroid therapy remains the standard of medical care but literature supports only modest reductions in 28-day mortality with no long-term survival benefit . High inRecombinant G-CSF, first approved in 1991 to reduce the incidence of neutropenic fever after myelosuppressive chemotherapy, causes proliferation and differentiation of bone marrow precursor cells into mature granulocytes . FurtherSeveral case reports and small studies have investigated the use of G-CSF in severe AH. To the best of our knowledge, this is the first systematic review and meta-analysis to assess its role in improving survival and liver severity indices of patients with severe AH. Patients treated with G-CSF were noted to have significantly improved 90-day survival compared with standard medical therapy (80.03% versus 40.92%). This significant survival benefit is likely due to the role of G-CSF therapy in liver regeneration with reduced incidence of infectious complications compared to corticosteroids . As the 3) [All included studies and their pooled analysis provided evidence of improved liver function demonstrated by significant reduction in CTP, MELD, and mDF scores at 28 days. The robust decline of mDF score by 39.79 in the G-CSF group compared to 12.39 in the SMT group is reassuring. All included studies reported continued improvement of liver function with G-CSF therapy even at 90 days. No significant adverse events were reported in any of the trials. Deaths reported among patients receiving G-CSF therapy were owing to progressive liver failure with no mortality reported directly as a complication of therapy . As expe3) . Singh e3) . Minor b3) ,25.Our meta-analysis, however, has some important limitations. The number of trials included in this review is small and restricted to South Asian (India) and European populations, limiting the external validity of the results ,23,24,25In summary, G-CSF could be an additional rescue therapeutic option in patients with severe AH. It demonstrated a significant improvement of liver function and reduction in 90-day mortality with no significant adverse events. Based on the encouraging outcomes reported in the literature and our meta-analysis along with a long safety record of use in cancer patients, further randomized controlled trials evaluating the effectiveness of G-CSF in patients with alcoholic hepatitis are required.


LTA4H variant benefitting from antiinflammatory steroids administered alongside antibiotics. A prior study found poor correlation between LTA4H genotype and cerebrospinal fluid levels of cytokines, key mediators of inflammation. The study used “frequentist” statistical methods that can fail to detect true differences. Using Bayesian statistics, which can detect significant differences not found by frequentist methods, we found good correlation between LTA4H genotype and cytokine levels, and cytokine levels and outcome even independent of LTA4H genotype. These findings suggest that LTA4H and additional inflammation factors affect outcome and suggest tailoring steroid therapy to cytokine levels.Despite appropriate antibiotic treatment, tuberculous meningitis carries a high mortality ascribed to overexuberant inflammation. Genetic variations in the enzyme, LTA4H, alter inflammation, with individuals carrying the inflammation-associated  Leukotriene A4 Hydrolase (LTA4H), which regulates expression of the proinflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased expression of LTA4H, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world’s population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pretreatment levels of cerebrospinal fluid proinflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis. LTA4H TT homozygosity was associated with global cytokine increases, including tumor necrosis factor. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pretreatment cerebrospinal fluid cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.Adjunctive treatment with antiinflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant in  Leukotriene A4 Hydrolase (LTA4H) gene is associated with responsiveness to dexamethasone, a potent corticosteroid   . TNF caurophages , 14.LTA4H TT genotype mediates increases in cerebrospinal fluid (CSF) cytokines and if these increases are associated with dexamethasone responsiveness. Consistent with the findings of Tobin et al. , we find that survival in response to dexamethasone is associated with significant increases in all cytokines tested before or at the start of treatment, representing innate proinflammatory, helper T cell-associated and immunomodulatory classes. While the LTA4H TT genotype was associated with increases in these cytokines, we also found that increased cytokines are associated with survival in an LTA4H-independent manner in this dexamethasone-treated cohort.Using Bayesian methods (detailed in Dataset S1) showed that none followed a normal (Gaussian) distribution. For nearly all values, log-skew-Student  was the preferred distribution class both in the dataset as a whole and for the various subsets considered in our analyses . Comparisons were performed using restricted geometric means as is appropriate for such heavy-tailed approximately logarithmically distributed data . Furthermore, unlike the previous analysis, we made no assumption that there would be a linear trend with the number of T-alleles in a given patient. Using this method of analysis, we found that TT patients had significant increases in all measured CSF cytokines, except interferon-γ (IFN-γ) and IL-4, compared to both CC and CT patients who had similar levels to one another (Dataset S2). Similarly, a comparison of cytokine levels in TT patients to those in combined non-TT (CT and CC) patients showed that these levels in TT patients were significantly higher for all cytokines except for IFN-γ and IL-4 . Since there were increased cytokine levels for both the TT genotype and for higher disease grades, we predicted that these levels would be highest in TT patients in the higher disease grades. Whereas non-TT patients had a similar pattern of increased cytokines with increasing disease grade as the overall cohort . Together, these analyses show that the default inflammatory response to tuberculous meningitis includes global increases in CSF cytokines, and suggest they are associated with a survival benefit from dexamethasone. Overlaid on these are further increases mediated by the LTA4H TT genotype.Thuong et al.  comparedwho died A. Becauswho died , 9, we hwho died B. We couwho died B. CC andLTA4H TT patients have increased CSF cytokines over their non-TT counterparts provides an explanation for why they survive better when treated with dexamethasone than their non-TT counterparts  to median cytokine values showed that increased pretreatment levels of most (8 of the 10 tested) were significantly associated with survival with dexamethasone, indicating that these higher levels are pathogenic. All cytokines were increased with increased disease grade, but only one of these, IFN-γ, was significantly increased in those with grade 3 disease. The LTA4H TT genotype was also associated with global increases in cytokine concentrations across the board in comparison to the non-TT genotype patients, but the differences were significant in only 3 of the 10 cytokines. Given that most cytokines are induced by interrelated and often shared signal transduction networks, notably the NF-κB family of transcription factors  than in Asia  (LTA4H TT homozygote genotype frequency would have been rare (1 to 4% of population) , we find that while TT patients gain a major survival advantage from dexamethasone, non-TT patients’ survival is neither helped nor harmed by it.Our initial goal in performing these analyses was to ask whether the  pathway . LTA4H T4c232e3) , 9, 25, 4c232e3) , 27. In 4c232e3) , were doLTA4H genotype and by CSF cytokine levels, potentially indicating higher and lower doses of the drug or no drug at all. Because corticosteroids exhibit a dose-dependent degree of immunosuppression of cytokines, including TNF  in a mouse inflammatory colitis model . The tuberculous meningitis cohort used for survival analysis has also been previously described (Dataset S1). Patients were admitted to one of two tertiary care referral hospitals in Ho Chi Minh City, Vietnam: Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease  or the Hospital for Tropical Diseases . Patients were grouped on study entry according to the modified BMRC tuberculous meningitis grade: Patients with a Glasgow Coma Scale (GCS) score of 15, with no focal neurologic signs, were designated grade 1; patients with GCS score of either 11 to 14, or 15 with focal neurologic signs, were designated as grade 2; and patients with GCS scores of 10 or less were designated as grade 3 (Dataset S1). Patients with cytokine measurements were classified as having definite, probable, or possible tuberculous meningitis in accordance with published diagnostic criteria , Tobin et al. . CSF for cytokine measurements was available in a smaller proportion of patients admitted to Hospital 1 than Hospital 2 (162 of 190 [55.9%] vs. 144 of 149 [96.6%]) .The Tobin et al.  study usSI Appendix, Table S2). There was no such bias in relation to survival status or disease grade severity . To determine if the Hospital 1 collection bias in the TT vs. non-TT patients changed the analysis, all data were analyzed for the whole cohort and Hospital 2 alone (Dataset S2). Of the 350 comparisons, 349 yielded similar patterns in the two datasets, with some loss of significance when considering Hospital 2 alone due to reduced patient numbers. The only comparison where there was a change in the direction was IL-5, grade 1 versus grade 2, which was significantly greater in grade 2 than in grade 1 in Hospital 2 alone, and nonsignificantly greater in grade 1 than grade 2 in the overall cohort. The analyses for the overall cohort are presented in Further analysis showed that Hospital 1 TT patients were significantly less likely to have had cytokine measurements than non-TT patients . A number of CSF samples yielded cytokine concentrations that were outside the linear ranges of the assays and had been assigned specified high or low fixed values. To handle apparent limits to the measurable value, resulting in many observations of the maximum and minimum seen value, these maximal and minimal values were spread in a process called dithering . The geometric means of both the undithered and dithered data are in SI Appendix, Table S2. The geometric means indicated in Dataset S2) and the results from the analyses of the dithered data are presented in Dataset S2.As described in Thuong et al. , 10 cytoThe Bayesian methods used for the survival analysis in Supplementary FileSupplementary FileSupplementary FileSupplementary File


Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein–protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo–electron microscopy structure of the ribosome from  Tiny pathogenic eukaryotes called microsporidia have evolved highly compact ribosomes, smaller than bacterial ribosomes, and employ diverse hibernation factors to facilitate ribosome inactivation and recovery from the latent spore stage, including the conserved eukaryotic hibernation and recycling factor Lso2. Ribosome biogenesis and functionality are energy-intensive processes accounting for 80% of a cell’s ATP usage in nutrient-rich conditions ,2. When Plasmodium falciparum, the causative agent of malaria, thereby providing a potential mechanism to control this highly infectious human pathogen The cryo-EM study by Ehrenbolger and coworkers presents a single-particle structural model of hibernating ribosome from microsporidium pathogen family organism Paranosema locustae in complex with a recycling factor Lso2. Although microsporidian ribosomes generally have reduced rRNA, correlated with minimal known eukaryotic DNA, here the authors have chosen species with a less reduced variant, and remarkably identified an expansion segment reduced to a single nucleotide. From the high-resolution structural models, the authors assigned a function for this nucleotide and could explain why it retained in evolution. The central aspect of the work is the factor Lso2 that blocks catalytic sites. The technical work is well executed, the analysis conducted with attention to details and well presented. The comparison to other characterised and more familiar ribosomal complexes is interesting and helpful.I only have minor comments:- Figure 4 and related text: perhaps indicate the local resolution for the nucleotide density to assure readers.- Methods: clarify why three P. locustae genomes were needed.Reviewer #2:This manuscript by Ehrenbolger and colleagues replicates a previously published study by Beckmann's group in which Wells and colleagues described hibernation and reactivation of 80S ribosomes with protein Lso2. Because PLoS Biology publishes "scooped" work as a means of improving reproducibility, I request the authors to replicate the most exciting and key finding of Wells and colleagues' study: their finding that Lso2 is not simply an additional hibernation factor, but a factor that helps reactivate hibernating ribosomes by predisposing them for Dom34-mediated splitting into subunits. Otherwise, I find the study by Ehrenbolger and colleagues too incremental and superficial to warrant its publication in PLoS Biology. https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000780).Specifically, I request the authors to express microsporidian homologs of Dom34-recycling system and repeat the in vitro splitting assay shown in Figure 5 of Beckmann's group study  which could undergo phosphorylation in response to growth stimuli?As a minor comment, I disagree with the authors's statement that their study highlights "diversification of the microsporidian ribosome". In my opinion, their study highlights diversification and redundancy of ribosome hibernation mechanisms. For instance, yeast S. cerevisiae can hibernate their 80S ribosomes by using either protein Lso2 or Myb protein 1. But it does not mean that S. cerevisiae 80S ribosomes have two distinct and diversified populations for each of these hibernation factors.Overall, this study is stunningly illustrated and written in a very articulate and simple manner, and I am sure the authors will be able to easily address my comments. Reviewer #3:This manuscript reports the structure of an inactive microsporidian (P. locustae) ribosome bound by the ribosome hibernation factor Lso2. The ribosome structure presented here shows an intermediate level of rRNA reduction relative to the recently published V. necatrix ribosome. The Lso2 hibernation factor also contrasts with the MDF1 and MDF2 hibernation factors in the V. necatrix ribosome, and allows direct comparison with Lso2 / CCDC124 in other eukaryotes. It is also reported that the structure contains a bound nucleotide between uL6 and uL20. This nucleotide is interpreted to serve as a replacement for an eukaryotic expansion segment that has been reduced in P. locustae.The structure of this microsporidian ribosome contributes to our understanding of the translational machinery, based on its phylogenetic position and reduced ribosome size. The Lso2 structure is likewise valuable and is complemented by functional studies in yeast . However, it seems that the presence of the bound nucleotide is interpreted quite strongly in the absence of corroborating data. The proposed model is plausible, but the nucleotide might bind adventitiously, or only in the hibernating state. Gathering the sort of genetic or biochemical evidence that could support this claim is beyond the scope of revisions. 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