The reduction rate of ECD at 3 months was greater in the TR group (42.3% ± 17.2%) than in the NTR group , though reduction rate of ECD at 6 months was not significantly less in the TR group. We found that preoperative temperature change in donor corneas may adversely affect ECD following DSAEK.To examine the influence on postoperative endothelial cell density (ECD) caused by preoperative temperature change from 4 °C to room temperature in donor corneas for Descemet stripping automated endothelial keratoplasty (DSAEK). This retrospective, case-controlled comparison of 100 eyes that underwent DSAEK using imported donor corneas transferred from an overseas eye bank . Fifty donor corneas experienced temperature reversal for ECD measurement (TR group), and postoperative outcomes were compared with 50 disease-matched cases that did not experience temperature changes before DSAEK (NTR group). The main outcome measure was endothelial cell loss and reduction rate of ECD at 1, 3, and 6 months following DSAEK. ECD at 3 months following DSAEK was significantly less in the TR group (1458 ± 494/mm To assess the tissue for endothelial damage and to examine the endothelial cell density, imported donor corneas shipped to Japan were rewarmed to room temperature from 4 °C preservation for ECD measurements, then preserved at 4 °C prior to surgery. We have experienced cases with relatively high ECD loss after DSAEK when using imported postprecut donor corneas. While seeking the reason for ECD loss, we speculated that preoperative temperature change may be responsible in corneas that required temperature reversal during tissue re-evaluation prior to surgery. Because of this concern, donor endothelial examination was discontinued as of January 2015. To study the effect of preoperative temperature changes on postoperative ECD, we conducted this case-control study.Recently, Descemet stripping automated endothelial keratoplasty (DSAEK) has become the leading procedure for the treatment of bullous keratopathy; however, postoperative decreases in endothelial cell density (ECD) remain a major concern. A number of potential causes for ECD loss have been reported, including longer death-to-preservation timeDonor-related factors, including age, sex, preoperative ECD, death-to-preservation time, death-to-operation time, central graft thickness, and graft size.Recipient-related factors, including age, sex, central corneal thickness, original diseases, and best spectacle-corrected visual acuity (BSCVA).We conducted a single-center, retrospective, case-control study, involving 100 eyes that underwent DSAEK using imported postprecut donor corneas transferred from an overseas eye bank  in Tokyo Dental College between June 2010 and December 2016. No organs were procured from prisoners. The use of imported donor corneas was approved by the Ethical Committee of Tokyo Dental College and was performed in accordance with the tenets set forth in the Declaration of Helsinki (I 18–19). Our institutional review board waived the requirement for informed consent for this retrospective study. Patient data was anonymized before access and analysis. The original diseases for DSAEK included pseudophakic bullous keratopathy (PBK), post-laser iridotomy bullous keratopathy, Fuchs endothelial corneal dystrophy (FECD), endothelium decompensation following corneal transplantation, or pseudoexfoliation syndrome . A minimum of 100 cells were counted at the central area. Postoperative measurements of ECD were performed using a noncontact specular microscope  at 1, 3, and 6 months postoperatively.Visual acuity was measured using the Snellen visual acuity chart; we analyzed the results using logarithm of the minimum angle of resolution (logMAR) equivalent units, including manifest refraction, spherical equivalent, and cylindrical error.Six experienced surgeons were involved in the DSAEK procedures. Descemet’s membrane and diseased endothelium were stripped from the planned graft area using the reverse Sinskey hook . Venting incisions were made to improve graft adherence. A 4.5–5-mm clear corneal incision was created using a slit knife. The donor cornea was cut with a diameter of 7.0–8.75 mm, depending on the recipient corneal diameter. The trephinated donor lenticule was inserted into the recipient anterior chamber using a Busin glide . Once the donor graft was properly unfolded and well centered, air was injected into the anterior chamber. After the anterior chamber was filled completely with air for 10–15 min, the air was reduced to prevent pupillary block. No peripheral iridectomy was performed. Following DSAEK, each patient received a topical application of antibiotics  and 0.1% dexamethasone  eye drops, five times daily; the number of daily administrations was then tapered over the following several months.P-values less than 0.05 were considered statistically significant.The ECD and reduction rate of ECD were expressed as either mean ± standard deviation (SD) or range, and were compared using an unpaired Student’s t-test. Comparisons between categorical variables were analyzed via the chi-squared test. P = 0.46), sex (P = 0.28), or central corneal thickness (P = 0.52).The demographics of all patients are summarized in Table 2 and 2652 ± 278 cells/mm2; and the respective mean death-to-operation times were 6.1 ± 1.1 and 6.9 ± 0.8 days . Reasons for graft failure in the TR group included endothelial rejection (n = 1), endothelium decompensation (n = 1), and herpetic keratitis (n = 1).Graft survival rates at 6 months were 94.0% (47 eyes) and 100% (50 eyes) in the TR and NTR groups, respectively (P = 0.084). In the TR group, 42 eyes (91.3%) exhibited improved vision at 6 months postoperatively. One eye experienced a decline in vision due to the recurrence of herpetic keratitis; an eye with FECD showed slow visual recovery due to an unknown cause. In the NTR group, 38 eyes (92.7%) exhibited improved vision at 6 months postoperatively. Two eyes (5.3%) experienced a decline in vision due to subepithelial opacification or slow visual recovery from an unknown cause. Postoperative BSCVA did not significantly differ between the two groups .Although the mean preoperative BCVA in the NTR group tended to be better than in the TR group, there were no significant differences between the two groups  (Table P = 0.014), though ECD at 6 months was not significantly less in the TR group. The reduction rate of ECD at 3 months revealed a significant difference (P = 0.044), though reduction rate of ECD at 6 months was not significantly less in the TR group. The mean preoperative ECD was similar between the two groups without a history of trabeculectomy or postoperative re-bubbling to exclude the factor of preoperative iris damage or postoperative toxicity of air (P = 0.93). The ECD at 3 months showed a significant difference (P = 0.017). The reduction rate of ECD at 3 months revealed a significant difference (P = 0.012) (Table There were no significant differences in either surgeons or graft diameters between the TR + and TR- groups. The mean preoperative ECD was similar between the two groups (1) Table . Postopeet al. reported that rates of ECD loss were comparable between eyes that underwent penetrating or endothelial keratoplasty (EK)6. Particularly, ECD reductions during early postoperative periods are relatively high, regardless of improvements in surgical techniques. There are numerous factors that may potentially affect ECD loss during early postoperative periods following EK. Kitazawa and associates recently reported that donor corneas contained a significant number of dead endothelial cells, and that the presence of these cells may be responsible for early declines in ECD following DSAEK7. Their report indicated that proper management of donor corneas is key to successful long-term preservation of the corneal endothelium following DSAEK.There is an existing concern regarding postoperative ECD loss following endothelial keratoplasty. Recently, Dickman 8. Although these changes were statistically significant, the impacts on clinical outcomes seemed to be relatively low. Through discussion with eye bank members and other researchers, we realized that additional manipulation of donor corneas, particularly temperature changes, might adversely affect donor corneal endothelium. In the present case-control study, we observed that the use of donor corneas that experienced temperature reversal resulted in statistically significant decreases in ECD following DSAEK. The mean ECD loss at 3 months postoperatively was 44.5% for the TR group and 34.3% for the NTR group. The difference of the mean endothelial cell loss in corneas with temperature changes was approximately 10%; this impact seemed greater than those caused by post lamellar dissection procedure or overseas transportation. There were no significant differences between the two groups in other conditions such as patient background, donor background, graft condition, or surgical procedures. Further, there were no differences in ECD between eyes with and without either a history of trabeculectomy or postoperative re-bubbling. It seems likely that temperature changes in donor corneas affected endothelial survival following DSAEK. Although we did not find significant differences in graft survival rate or BSCVA, the differences in ECD seemed to be at 3 months and at 6 months after DSAEK. It is assumed that the factors except for donor corneas had a great influence on ECD at 6 months. Long-term observation of these cases may find worse graft survival in eyes that underwent DSAEK using donor corneas with TR. Although the exact mechanism underlying the association between temperature changes and ECD loss remains unclear, we speculate that the reactive oxidative stress (ROS) may play a role. Recent studies have reported that ROS induces apoptosis in corneal endothelial cells9. Rauen10et al. reported that during exposure to 4 °C, and to a lesser extent during rewarming, cultured corneal endothelial cells incurred injury mediated by ROS9. This injury was accompanied by pronounced morphologic alterations that occurred predominantly during rewarming, representing apoptotic features.As a leading hospital for corneal transplantation in Japan, we have performed many DSAEK procedures using imported donor corneas. We were particularly concerned about the long distance transportation and following lamellar dissection procedures. We and others reported that the average rate of ECD loss ranged from 1.75% to 3.7% after precut procedure, and 2.3% to 3.79% for overseas transportationIn conclusion, the limitations of this study include the small patient population, and the short duration of follow-up time. As the reason for small population, ethically unacceptable to increase the number of cases for the possibility of affecting ECD, retrospective study though further clinical and basic investigations are needed to elucidate the long-term impact on graft survival and improvement in endothelial preservation. The findings of this present study showed that preoperative temperature change in donor corneas may be related to increased ECD loss after DSAEK.


However, during certain neurological diseases, this balance can be disrupted. A significant disruption to the normal CSF circulation can be life threatening, leading to increased intracranial pressure (ICP), and is implicated in hydrocephalus, idiopathic intracranial hypertension, brain trauma, brain tumours and stroke. Yet, the exact cellular, molecular and physiological mechanisms that contribute to altered hydrodynamic pathways in these diseases are poorly defined or hotly debated. The traditional views and concepts of CSF secretion, flow and drainage have been challenged, also due to recent findings suggesting more complex mechanisms of brain fluid dynamics than previously proposed. This review evaluates and summarises current hypotheses of CSF dynamics and presents evidence for the role of impaired CSF dynamics in elevated ICP, alongside discussion of the proteins that are potentially involved in altered CSF physiology during neurological disease. Undoubtedly CSF secretion, absorption and drainage are important aspects of brain fluid homeostasis in maintaining a stable ICP. Traditionally, pharmacological interventions or CSF drainage have been used to reduce ICP elevation due to over production of CSF. However, these drugs are used only as a temporary solution due to their undesirable side effects. Emerging evidence suggests that pharmacological targeting of aquaporins, transient receptor potential vanilloid type 4 (TRPV4), and the Na Elevation of intracranial pressure (ICP) after a neurological injury has been reported in numerous conditions including hydrocephalus, idiopathic intracranial hypertension (IIH), oedema, traumatic brain injury (TBI), and stroke . UncontrCSF serves as a protective fluid to the brain and spinal cord, cushioning them from mechanical injury, and acts to reduce the brain’s effective weight—its actual mass is ~ 1500 g while the buoyancy provided by CSF reduces its net weight to 25–50 g . It servThe exact mechanisms of CSF secretion, flow and reabsorption/drainage are debated; however, it is understood that alterations to normal physiology can contribute to elevated ICP. The traditional view of CSF drainage is that fluid flows from the subarachnoid space through the arachnoid villi and drains into the blood of the superior sagittal sinus Fig.  13]. St. St13]. This review aims to examine existing literature—acknowledging our current understanding of CSF physiology and controversies in the field, while exploring how altered secretion and drainage may link to ICP in pathology. This will allow us to identify gaps in knowledge and to identify new targets for research within the field of CNS injury.It is estimated that around 80–90% of CSF is secreted by the choroid plexus, a highly-vascularised structure of epithelial cells located in the ventricles of the brain. The epithelial cells surrounding the capillaries compose a blood-CSF barrier (BCSFB), which selectively controls the movement of solutes and water to regulate the composition of CSF . Under nSome of the first supporting evidence for the role of the choroid plexus in CSF secretion came from Dandy , which iWhen looking to anatomical clues for the involvement of the choroid plexus in CSF secretion, ultrastructure of rat choroid plexus epithelial cells, as revealed by electron microscopy, highly resembles that of typical secretory cells with the presence of: a brush border at the apical membrane facing the ventricle, infoldings at the basolateral membrane, a high density of mitochondria, and a well-developed endoplasmic reticulum . This evThe blood–brain barrier (BBB), formed by endothelial cells that line cerebral capillaries connected by tight junctions, is the site of regulated exchange of ions, molecules and cells between the blood and the brain –26. FluiSome argue that the generation of CSF is driven by the hydrostatic pressure gradient between the blood, choroid plexus epithelial cells, and the ventricles—according to Starling’s law of filtration . This meAdditionally, alterations in CSF osmolarity have been shown to influence water flux across the choroid plexus and the BBB . This se+ concentration that is sufficient to depolarize neurons, trigger action potentials and drive repolarization further elevating cerebral K+ levels. Thus K+ homeostasis overlaps with control of water content in the brain. However, when the BBB is disrupted, the capillary endothelium acts similar to fenestrated capillaries. Therefore, both osmotic and hydrostatic pressure gradients contribute to oedema formation. When this happens, ICP and systemic blood pressure, which are determinants of hydrostatic pressure, assume an important role . Because neuronal RMP depends significantly, but not exclusively on [Kout], the maintenance of homeostatic control for extracellular potassium plays a crucial role in the regulation of neuronal firing. Several mechanisms can explain the rapid clearance of K+ from the ISF: passive diffusion through the ISF, active removal by blood flow, and neuronal reuptake. However, these mechanisms alone are not fast enough to account for the rapid K+ removal from the ISF seen under experimental conditions. Several lines of evidence suggest that brain glial cells and more specifically astrocytes, support the homeostatic regulation of the neuronal microenvironment. This phenomenon is referred to as spatial buffering of extracellular potassium . A. A104]. Increased intracranial pressure is reported as a symptom or complication in several CNS pathologies like hydrocephalus , 107, IIHydrocephalus occurs as a result of several congenital and idiopathic conditions characterised by increased fluid accumulation in the brain and swollen ventricles. Hydrocephalus is caused by disruptions to CSF secretion, flow, or drainage, which lead to increased ICP . Around A similar condition to hydrocephalus, IIH, describes elevated ICP but without concurrent lesions or CSF disruptions. The incidence of IIH varies worldwide with an estimated incidence of 0.03–2.2 per 100,000, and recent evidence has identified obesity as a major risk factor . The priPatients suffering TBI often develop oedema, which increases ICP and impairs tissue perfusion. Studies have implicated the role of aquaporins in the oedema development in cases of TBI, and upregulation of AQP4 and AQP9 is observed across the whole brain in experimental animal models . Interes+, water and Cl− promotes ion and water movement across the BBB into the ECS causes an elevation in ICP -inulin entry into the CSF. At 6-h of reperfusion, they found that 10 min of occlusion doubled the BCSFB permeability, while 30 min of occlusion trebled permeability [Reduced blood supply to the lateral ventricle choroid plexus (LVCP) was observed in two rat models of cortical ischaemic stroke: two-vessel occlusion + hypotension model; and, the commonly used, middle cerebral artery occlusion (MCAo) model. Ennis and Keep (2006) found a reduction in LVCP blood flow to 13% of control blood flow with the former model and 62% with the latter. They also investigated the influence of two-vessel occlusion + hypotension on BCSFB integrity by observing the permeability of the barrier with [eability . The infeability .Ennis and Keep  also not2+ entry into the choroid plexus epithelial cells and results in activation of Ca2+ dependent ion channels, specifically the Ca+-activated intermediate conductance K+ channel, KCNN4c. The authors of this study postulate that the channel plays a role in the regulation of CSF secretion.Recently, a nonselective cation channel has piqued interest within research of the choroid plexus. Preston et al.  have ideThis suggestion that TRPV4 is involved in the regulation of CSF is interesting considering other groups have detected an upregulation of TRPV4 in the ipsilateral hemisphere following ischaemia which contributes to greater neuronal injury . Some in−/− mice. These data implicate AQP4 in hypoxia-induced hydrocephalus and subsequent cognitive decline, likely because of increased CSF secretion. Experiments using AQP knockout mice indicate that osmotically driven water transport following ischaemia or acute water intoxication (leading to cytotoxic oedema) is mediated by AQP4 in the presence of an intact BBB [The role of aquaporins in CSF secretion was described above, and AQP expression can be altered by hypoxia, ischaemia or CNS injury . Interestact BBB . Followitact BBB . The auttact BBB . Recentltact BBB  have shoThis review summarises our current understanding of CSF dynamics with a focus on effects on ICP during neurological diseases, and highlights some of the discrepancies within the field. The implications of such findings are of high clinical relevance for understanding and treating neurological diseases where brain fluid homeostasis is impaired. Our efforts to elucidate the regulators and mechanisms involved in CSF secretion are still ongoing. Some evidence suggests the involvement of AQP1 at the choroid plexus , and mor


The analysis of molecular haematopoietic chimerisms (HC) has become a well-established method to monitor the transplant evolution and to assess the risk of relapse after allogeneic stem cells transplantation . Different techniques and molecular markers are being used for chimerism surveillance after transplantation, including quantitative real-time PCR (qPCR) and the recently developed digital PCR (dPCR). This study aims to compare the sensitivity and accuracy of both methods to quantify HC and predict early relapse.HC was evaluated using custom PCR systems for the specific detection of the Y-chromosome, null alleles and insertion-deletion polymorphisms. A total of 281 samples from 28 adult patients who underwent an allo-SCT were studied. Increasing mixed chimerism was detected prior to relapse in 100% of patients (18 relapses).Compared with conventional qPCR amplification, dPCR predicted relapse with a median anticipation period of 63 days versus 45.5 days by qPCR. Overall, 56% of the relapses were predicted earlier with dPCR whereas 38% of the relapses where detected simultaneously using both techniques and only in 1 case, relapse was predicted earlier with qPCR.In conclusion, chimerism determination by dPCR constitutes a suitable technique for the follow-up of patients with haematological pathologies after allo-STC, showing greater sensitivity to predict an early relapse. Allogeneic Stem Cell Transplantation  is considered to be the strongest immunotherapy for a variety of malignant and non-malignant hematologic disorders. Despite its strong curative potential and improved safety, allo-SCT remains a high-risk procedure . Haematoin situ hybridisation (FISH) of blood and bone marrow cells  x 100 Number of reactions containing each target was calculated by Poisson-Plus Model distribution estimates, using the number of non-target containing reactions relative to the total number of reactions .X,Y) was computed to measure the grade of relationship between the two quantitative variables (qPCR and dPCR). In addition, the square of the Pearson correlation coefficient (R2) was computed to determine the replicability of the model, and the quality and the variability of the results that can be explained by the model. Finally, a Bland-Altmann plot was used to visualise the grade of concordance between the results obtained by both, qPCR and dPCR methods.To confirm the validity of the study and compare between both quantification techniques, the correlation between both quantifying methods was calculated. For that, Pearson correlation coefficient  was calculated to confirm the validity of the study and comparability between both techniques, indicating that both quantitative variables were positively correlated (R = 0.959). Similarly, the square of the Pearson correlation indicated a suitable repeatability of the results, accounting for R2 = 0.91, with only a few diverging values  prior to the haematologic relapse, characterised by blasts levels greater than 5% . Overall, iMC was detected earlier by dPCR in 55.6% of the total allo-SCT patients, whereas iMC was detected simultaneously by qPCR and dPCR in 38.9%, and only in one patient, iMC was identified earlier by qPCR (5.5%).Overall, the median of time between the dPCR-detected iMC and the relapse was 63 days (range: Day 14 –Day 301) versus 45.5 days (range: Day 2 –Day 100) by qPCR. This indicates that the results obtained by dPCR elucidated a gradual increment of host blood cells, iMC, earlier than when qPCR was used. Thus, concluding that more than 50% of patients, who have relapsed, would have been previously detected if the HC analysis had been performed using dPCR.Despite the continuous improvements in allo-SCT, relapse remains one of the main causes of failure of the procedure, especially in patients with acute leukaemia. HC analysis constitutes a standard procedure in the follow-up of patients undergoing allo-SCT capable to predict disease recurrence, graft-versus-host disease and graft failure. In a significant proportion of patients there is no minimal residual disease marker that allows us to monitor the risk of relapse, whereas the analysis of chimerism is possible in almost every patient. STR-PCR analysis is the gold standard for quantitative chimerism analysis recommended by the EuroChimerism consortium in the prediction of relapses ,23; howeThe advantages of qPCR-based methods over the conventional STR-PCR analysis have been demonstrated by multiple studies of HC chimerism surveillance ,24. CompClinically, it is considered that a patient has a greater risk of relapse when a gradual increase in recipient cells is detected by molecular biology techniques, which does not disappear even when exposing the patient to different treatments like immunosuppression tapering or donor leukocyte infusion. This increase, known as iMC can occur weeks, months and even years after the allo-SCT took place, and it is therefore considered a symptom of potential relapse . In this−4 to 10−3, expressed on a logarithmic scale. This is in accordance with a comparative chimerism study [In the present study, we studied 28 patients of allo-SCT using dPCR and qPCR to determine the feasibility to standardise the use of dPCR as a model method for mixed chimerism monitoring. In line with previous studies, our findings suggest that dPCR might be a more suitable method to use for the detection of haematopoietic chimerisms in the follow-up period ,20,21. Osm study , which c−3 and to distinguish the host cell increase weeks earlier that using qPCR. Clinicians, on the basis of the results of chimerism analysis, make major therapeutic decisions [The analytical accuracy and precision of dPCR play a crucial role in the study of molecular chimerism enabling to reliably differentiate between a HC increase and an oscillation of the target values at a level of 10ecisions . In concS1 TableFollow-up analysis by qPCR and dPCR was carried out using the same host-specific marker.(XLSX)Click here for additional data file.


We propose indicators to measure different aspects of independence: two assessing whether a researcher has developed an own collaboration network and two others assessing the level of thematic independence. Taken together they form an independence indicator. We illustrate how these indicators distinguish between researchers that are equally productive and have a considerable impact. The independence indicator is a step forward in evaluating individual scholarly quality.Bibliometric indicators are increasingly used to evaluate individual scientists–as is exemplified by the popularity of the many other publication and citation-based indicators used in evaluation. These indicators, however, cover at best some of the quality dimensions relevant for assessing a researcher: productivity and impact. At the same time, research quality has more dimensions than productivity and impact alone. As current bibliometric indicators are not covering various important quality dimensions, we here contribute to developing better indicators for those quality dimensions not yet addressed. One of the quality dimensions lacking valid indicators is an individual researcher’s  The use of (bibliometric) indicators for evaluating research performance has become rather common, at the level of research organizations and research teams, but after the development of the H-index also at the individual level. The number of indicators has been growing, and a paper published a few years ago already counted 108 bibliometric indicators at the individual level , 3, 4, 5In parallel, a discussion has emerged amongst evaluators and bibliometricians about the reliability and validity of the indicators, and increasingly criticism is formulated on the indicators and their use , 9, 10. The criticism on bibliometric indicators neglects the many problems with peer review, which we do not discuss here , and it In this paper, we focus on the other main point, the validity of the indicators: Do bibliometric indicators measure what we want to measure? Most bibliometric indicators focus on productivity, impact, and quality of the collaboration network. Over the years, many studies have indicated that bibliometric indicators do well as predictors of peer review in grant decisions , 16. HowWhat constitutes a high-quality researcher ? The metIn order to uncover what counts as quality in this context, one may interview those involved in setting the standards: active researchers . A previOne of the criteria frequently mentioned for jobs is independence. As science develops through original and new ideas, there is a need for independent researchers that challenge the existing common views and open up new lines of research. Independence, in this sense, means breaking with existing lines of work and trying to come with something new. To achieve higher academic positions, or to get prestigious grants, one should have shown this type of independence.earlier grants are often mentioned as an indicator of scholarly quality. However, it is not so clear how to add up grants to an indicator value, and how to weight the various aspects, such as number of grants, total amount of funding, the prestigious grants, and the same holds for operationalizing ‘stays abroad’ in order to measure mobility [The question of independence gets an additional relevance through the growth of research collaboration and team science , 22, 23.mobility .What is meant with independence? We briefly review the policy discussion about independence and then continue with a more systematic approach. Not only do peers—in interviews mentioned above–point at independence, also science organizations like the US National Academies of Science (NAS) emphasize the importance of independence, and warned that the conditions for becoming independent were deteriorating   indicators have limitations in measuring independence has been pointed out by many , 30. It “The assessment of independence is a frequently discussed issue, especially when it comes to researchers in large collaborative projects and/or young researchers. (…) Given that research today often includes networks and large collaborative research teams, it may be difficult to discern the independence of individual researchers and especially of the young. A young researcher in a network often shows many publications, but without a prominent place in the author list, which complicates the assessment of the degree of independence.”  “Independence can be documented, for example, when the applicant has been obviously senior representative of recent contributions to research, the latter not seldom exemplified by the recent scientific publications, and has conducted his (sic!) own consistent research line after the dissertation. Independence can also be shown through research responsibilities as a supervisor for a PhD student or a guest researcher.”  Not remaining too close to the research interests of the PhD supervisor(s); (ii) Having an own collaboration network. Independence (for early career researchers) should be distinguished from other—related–quality dimensions  he/she want to address and to choose the best strategies and approaches to address that problem(s). Independence in this definition is something with a time dimension: independence emerges over the years. It implies in the first place that a young researcher becomes independent from the environment where he/she was educated. As PhD student, and as Postdoc, a researcher works in projects generally designed by others, often the PI who also functions as a supervisor. After having done that for a while, the researcher should have become skilled and experienced to formulate his/her own research questions and projects at challenging research fronts, unguided by the former supervisors. This, of course, does not mean that independent researchers avoid collaboration, as almost all research nowadays is collaborative. In fact, building up relevant collaboration networks is one of the resources needed for excellent research. The challenge is to formulate indicators that can tease out the level of independence from a network of dependencies.ego-network of the researcher: (i) the eigenvector centrality of the former supervisor in the co-author ego-network of the researcher, and (ii) the clustering coefficient of the former supervisor in the ego-network of the researcher.If independence means that a researcher does not depend anymore on the environment where he/she was trained as a researcher, we would expect to see that in the collaboration network. An independent researcher is expected to collaborate with a variety of others, but less so with the former supervisor(s). Therefore, independence can be defined as the quality of the co-author network of the researcher. The size of the network indicates how the environment of a researcher perceives his or her contribution. The more someone has to contribute, the more other researchers want to collaborate, so the more co-authors someone has. A young researcher is often introduced in the academic world through the supervisor. At the beginning of the career, the co-author network of a young researcher is therefore expected to be embedded in the network of the supervisor; something that may be helpful in the first career steps. But, after a while, the co-author network of an independent researcher will significantly differ from the supervisors’ co-author network. This can be measured using two network properties of the co-author eigenvector centrality expresses influence in the network: in contrast to the degree centrality, the eigenvector centrality takes into account the connectedness of other nodes. A node is important  if it is connected to other important nodes. So even if the supervisor is not connected to all nodes in the researcher’s network, if he/she is connected to the important (highly connected) nodes, the supervisor’s eigenvector centrality is still high. The clustering coefficient measures the extent to which a node is part of a clique in the larger network. The more a supervisor is part of a specific clique within the researcher’s network, the less the researcher’s network coincides with the network of the supervisor, suggesting higher independence.The The researcher is the center of his/her own ego-network, and will, therefore, have an eigenvector centrality of 1. The more independent a researcher is, the more distinct co-author cliques he/she will be part of, and that is expressed in a low (down to 0) clustering coefficient. If the two network scores of the supervisor are similar to those of the researcher, independence is low; the more the researcher’s network is his/her own, the lower the eigenvector centrality (approaching 0) and the higher the clustering coefficient (approaching 1) of the former supervisor will be.The proposed indicator may not work in all research fields. If there is only one co-author clique (clustering coefficient = 1), it would be impossible to assess the individual independent contribution of the researcher under evaluation. This may be the case in research fronts where ‘everyone’ is on all papers (hyper-authors) as in some physics subfields. Also in fields where single-authored papers are the dominant mode, the indicator would not work, as co-author networks do not exist. However, in all fields, the share of co-authored papers grows fast .Being independent also means that the researcher starts to explore new topics and to develop own research lines, by following his or her own ideas. The researcher should move to new research questions not belonging to the research agenda of the former supervisor. This explains why the number of citations and publications may not be decisive in assessing the performance of researchers. The real issue is whether one publishes and is cited because of one’s own good research, and not because of the good performance history of the supervisor. This implies that after a while, the publications of the independent early career researcher should be outside the research front(s) where the former supervisors are or have been active.To measure whether the researcher developed own research lines, independent from the former supervisor, we downloaded from the Web of Science (Online version) the papers of the researcher and the former supervisor, including their co-authored publications. We selected all papers published until two years after the main career decision: appointment as tenured (associate) professor versus leave academia. The additional two years are included to account for papers that were written and possibly even accepted, but not yet published before promotion/leaving dates. For the supervisor we also include papers in the decade before the PhD project of the researcher in order to have the earlier research lines of the supervisor included in the analysis. In order to account for research lines, we use only the following document types: Articles, Letters, Proceeding Papers and Notes. Reviews are, in our understanding, not representations of an individual researcher’s research line as there are many references to research that might be remote to the researcher in question.Fij is the number of common references of paper i and paper j, and Si and Sj is the number of cited references in paper i and j, respectively.We created the joint paper network of researcher and supervisor using bibliographic coupling. This results in a network of several components and clusters representing different strands of research, which can be used to answer the question if an own research line of the researcher is visible in the network. Or are the own papers of the researcher included in one of the clusters of the supervisors’ papers? If the latter is the case, the researcher has remained within the research program of the supervisor, and no own program was developed. For the joint network, we calculate the similarity between papers as bibliographic coupling. Bibliographic coupling is a measure of similarity between papers in terms of shared cited references, and the number of shared cited references is normalized in relation to the total number of cited references in the two papers. We use Salton’s Cosine Index which varies between 0 and 1 . The bibUsing the similarity between papers, one can break down the set of papers into topical clusters. Many algorithms are available, and here we use the SLM algorithm for this . The topSome of the resulting clusters contain only papers of the supervisor, others only papers of the researcher and again other clusters contain papers of both, as well as co-authored papers. If a researcher has developed her own research line, which would imply that the researcher explores other/new questions, and refers to different literatures, then the similarity will be lower; if he/she continues within the research line of the supervisor, the similarity measure will be higher.We calculate this independence indicator in the following way: we divide the number of all topical clusters where the researcher is active but the supervisor(s) is not by the sum of these own clusters and the joint clusters of the researcher and the supervisor(s). The indicator value is between 0  and 1 .Often there is more than one PhD supervisor, and then all of them should be considered in the calculation of the indicators.One would assume that early career researchers are dependent on the supervisor and that later on the researcher starts to do things independently of the former supervisor. However, occasionally the supervisor may follow the former PhD student in these new topics, and in those cases having a shared research line would indicate that the supervisor has become dependent on the former PhD student. The latter pattern would imply that the researcher starts in a new topic and the supervisor moving in later on. In order to distinguish these two different dependence situations, the analysis includes also the publications of the supervisor over the 10 years before the PhD project of the early career researcher. Then we can test whether topics were already occupied by the supervisor or not.marginally active long before the researcher entered these topics. Then we do not consider this as a shared topic because of the time difference.It may be the case that the early career researcher has an own topic, in which the former supervisor has been The STEM fields are mainly producing co-authored papers. In the social sciences and humanities this is increasing, but here single-authored papers are still rather frequent. For those non-collaborative research fields, we may have to develop a different type of independence indicator, which is future work.Topic clustering is based on referencing behavior of researchers. This type of analysis gives an indication of independence based on the use of references for building clusters, potential users of the methodology are to be aware of that in cases the data set is small the methods do not apply, and other complementary methods have to be developed.Above, we have defined four 4 measures related to independence:1: The eigenvector centrality of the supervisor in the researcher’s co-author network;I2 The clustering coefficient of the supervisor in the researcher’s co-author network;I3: The share of own papers of the researcher Pns/Pall where Pns is the number of publications of the researcher, not coauthored with the former supervisor(s) and Pall is the total number of publications of the researcher; andI4: The share of own research topics of the researcher Tns/Tall where Tns is the number of research topics of the researcher in which the former supervisor(s) is not active, and Tall is the total number of topics of the researcher.IAfter calculating these four indicators, they can be aggregated into a single score. The researcher’s independence indicator RII is calculated as follows:1 = eigenvector centrality, I2 = clustering coefficient, I4 = share own papers, and I4 = share own topics. We use (1 –I1) as the lower values for eigenvector centrality represent higher independence, whereas for the other indicators it is the opposite. I4 is weighted double, as the range of the ‘own topics’ scores is between 0 and 0.5 whereas the other indicators I1, I2, and I3 are in a range between 0 and 1.Where Ia proof of concept, and we apply the indicators on four researchers and their supervisors to show how it works. Measuring independence using these indicators can be done for any researcher given that the person in question has publications covered in citation indexes, e.g. Web of Science. However, we are interested in the question whether the indicator is valid, that is whether it measures independence as taken into account by e.g., panels that select applications for positions or jobs. In order to do that one needs a set of researchers which are similar in various dimensions  but differ in independence. With such data, we can give a tentative answer to the question whether the measured independence relates to e.g., career success.This paper intends to be For a study on research careers , researcFor the current study, we use four researchers from that sample. Researchers RA and RB are from the same STEM specialty, as are researchers RC and RD. RA and RC became full professor, and researchers RB and RD left the university and took a position in industry. For all researchers, the decisive career moment was in their late thirties, some nine years after receiving their PhD degree. To calculate the indicators, we downloaded the bibliometric data (WoS) of the publications of the researchers, as well as those of their PhD supervisors . We cleaned the data manually. The data cover the career from the start of the PhD research up to their late thirties. For the supervisor(s) we also include publication data of a decade before the start of the PhD trajectory of the researcher, in order to cover the relevant research portfolio of the supervisors.The four researchers all had a good performance level . If publWe first compare RA and RB. One of the researchers had a successful academic career (RA with supervisors SA1 and SA2) and became full professor. The other researcher (RB with supervisor SB) left the university for an industrial R&D lab–so productivity and impact were not decisive here .eigenvector centrality of 1. Within the ego-network of RB, SB has a high eigenvector centrality (0.91), indicating that SB is almost as central in the network of RB as RB herself. In contrast, the eigenvector centrality of SA1 in the ego-network of RA is very low (0.09), indicating SA1’s (and similarly SA2’s) marginal position in the network of RA. In contrast, the clustering coefficient of SB is low (0.11), as low as RB’s clustering coefficient (0.09), but the clustering coefficient of SA1 is high (0.43), very different from the clustering coefficient of RA (0.07). This indicates that SA1 (and SA2) is connected to a specific subset of nodes only. Consequently, we may conclude that RB hardly has an own network, whereas RA does have one.All researchers co-authored with their supervisor. In case of RA, about 20% of his publications are co-authored with his two supervisors, and for RB this is 95%, all in the period under. So, some researchers collaborated much more intensively with the supervisor than others did. These different collaboration levels result in rather different network scores: the researchers have in their own ego network by definition an The other two indicators measure the difference in research lines (topics) between the researcher and the former supervisor. Research lines are analyzed by creating a network of papers, based on bibliographic coupling. Papers that refer to similar literature are in this way clustered. One may calculate the similarity between two oeuvres as the average of the (cosine based) similarity between papers from the oeuvres–which is explained in the methods section. Bibliographic coupling shows that the research lines of RA and SA1/SA2 differ, whereas the research lines of RB and SB are very similar. Visualization of the paper networks illustrates these findings.Researcher RA worked intensively with supervisor 2 in topic T14 (middle of the map). Other joint clusters but with much fewer papers of the researcher are T6 and T9. But after receiving the PhD degree, RA started to work on other topics, in which neither of the supervisors has been active: T0, T5, and T12. Researcher RA has 83% of the papers and 43% of research topics without the participation of the supervisors. To investigate the further development of RA, we also created a bibliographic coupling map of the papers of RA and SA for another ten-year period (not included here). RA remains detached from his former supervisor(s), and his own research lines have clearly developed extended over that period, unconnected to the work of former supervisors.So, unlike RA, RB has hardly any work outside of the large network with SB: only 4% of the papers and 14% of the topics are without SB. RB, although productive and highly cited, did not develop new research lines but remained close to the work done with supervisor SB. Concluding, two similarly talented researchers in the same field and with about the same number of co-authors, productivity and impact, show strongly different patterns in their relation to the supervisor, and this is reflected in the scores for the proposed indicators. As we showed in own topics of the researcher may differ. This is indeed the case: A large share of RC’s papers (83%) are not co-authored with the supervisor SC, and consequently, half of the topics (50%) RC is active in are not covered by the supervisor. RD, on the other hand, has no research topics without one of the two supervisors . This confirms that we indeed need more sophisticated indicators for independence than “some papers without the former supervisor” as e.g., the ERC formulates for the starting grant.RD is also interesting from another perspective. RD has two supervisors, who not co-authored any paper. At the same time, we see RD modestly authoring and co-authoring with supervisor SD1 in topic T12, and frequent authoring and co-authoring with supervisor SD2 in topic T10 . Given tThis paper started by defining quality as a more-dimensional construct and argued that there is a need for extending the indicators portfolio reflecting this multi-dimensionality. Then we argued that independence is an important dimension, which is emphasized in a number of studies and reports. We proposed four indicators for this: two for the position of the former supervisor in the co-author network of an early career researcher, and two for the position of the former supervisor in the topic network of the early career researcher. The four indicators give a rather consistent picture: each of them places the researchers in about the same ranking, and together they may constitute a valid overall indicator of independence. The overall indicator scores clearly distinguish RA and RC from RB and RD . When usInterestingly, RA had an equal output and a lower impact than RB, and RC had a somewhat lower output and a much lower impact than RD. But RA and RC had the successful academic career whereas the others stopped, indicating that the commonly discussed publication-based and citation-based indicators may not be really informative. At this career stage, one assumes rather good numbers of publications (productivity) and citations (impact), and differences between those may not decisive, whereas other evaluation dimensions may play a role. In our cases, the career success does correspond with the independence scores. We do not suggest that independence is always the decisive variable, as job selection is a multi-criteria decision-making problem. But it is at least one of the more important quality dimensions.team science, it is not anymore possible to show independence by single authored papers, therefore there is a need for more sophisticated assessment methods, and our proposal in this paper seems to be a step in that direction. Further testing and further development are needed, and several issues need to be addressed in order to improve the indicator:In this era of The researchers we used as examples all came from STEM fields; using the WoS data then seems a reasonable approach. However, as is often argued, for other fields the WoS is not covering enough of the output. Partly this is a statistical issue: If the WoS papers are representative for the whole oeuvre, also in terms of co-authorships, then relying on the WoS data may not be problematic. Nevertheless, we would agree that the indicators should be tested on other datasets too. This was done for a social science case and a computer science case –two fielThe independence indicator is based on a model of science which assumes research fronts, where researchers build on (and cite) earlier work in order to contribute to the growth of knowledge. It is regularly argued that this model does not fit on all fields, as the concept of knowledge growth does not apply to all disciplines. Especially fields within the social sciences and even more in the humanities have different goals, such as providing meaning. The main audience in such fields may not be scholars, but other audiences–such as the general public. The differences in the intellectual and social organization of the disciplines  may leadAnother extension would be to introduce a temporal dimension–one could imagine that an early career researcher starts a new topic without the supervisor(s) being involved, with the supervisors later jumping on that bandwagon when it proves to be successful. Then a joint topic is not proof of dependence–on the contrary. This phenomenon was discussed for researcher D and the test we did should be included when using the proposed indicators.Similarly, dependency may not only occur with the previous supervisor, but also with other collaborators in the early career. To control for that, the indicators should probably be calculated for all frequent co-authors of the early career researcher.For individual evaluations, data cleaning and calculations can be easily done. However, it would be useful to test on a large sample whether the independence indicator predicts success better than other variables. In such a case data collection and cleaning may be resource and time intensive. That type of procedure indeed should be done for all indicators: their real-life value should be shown in a multivariate prediction of career or grant success. If the independence indicators as defined here have validity, such test would show whether independence does make a difference.Overall, this paper illustrates how to derive more indicators for different quality dimensions of scholarly work. We would emphasize that this is the better way to move forward: if current indicators are not adequate (enough) as claimed e.g., in the Leiden Manifesto , one shoS1 DatasetData files used to calculate ego network measures.(XLSX)Click here for additional data file.S2 DatasetData files used to calculate the share of independent research topics.(XLSX)Click here for additional data file.


Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An alr gene knockout strain  was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.The broad-spectrum amino acid racemase (Alr) of  These potential functions include metabolic , structural  plus down_homology  and up_cut  plus up_homology  were used in the initial round of PCR to provide fragments downstream and upstream of the alr gene, and also engineer an overlap sequence . Overnight culture of P. putida KT2440 supplied the template DNA (2 μl). Following the initial PCR, the SOE PCR was performed using ~20 ng of gel purified DNA (Thermo Scientific GeneJET gel extraction kit) from each of the two above reactions, also using primers down_cut and up_cut. All reactions were completed using PrimeSTAR HS DNA Polymerase (TaKaRa) according to the provided instructions . The SOE PCR provided a fragment containing 500 bp upstream and downstream of alr, including an engineered restriction enzyme cut site on each end, as well as 18 nucleotides at the beginning and end of the alr gene. Parts of the alr gene  were not deleted in order to achieve an in-frame deletion and also minimize any impact on the transcription of upstream and downstream genes. Vector pEX18Kan . After completing the washes, the cells were resuspended in 800 μl cold buffer A2X and 30 μl chloroform was added. The cell mixture was vortexed briefly and maintained on ice for 15 min. The treated cells were centrifuged at 10,600 × g (Eppendorf Centrifuge 5424) at 4°C for 10 min. Five-Hundred microliter of the supernatant was transferred to a new tube and kept on ice. This fraction represents “periplasmic” protein. The remaining, intact cell pellet was washed twice with 1 ml buffer A2X and then resuspended in 1 ml of the same buffer. The cells were lysed by sonication twice at 60 V for 10 s with a 2 min pause on ice in between. The lysate was centrifuged at 4°C at maximum speed  and 500 μl of the supernatant was transferred to a new tube and maintained on ice. This fraction represents the “cytoplasmic” protein, which includes intracellular protein as well as any remaining periplasmic protein. The identity of each protein fraction, periplasmic and cytoplasmic, was confirmed via enzyme assays using glucose-6-phosphate dehydrogenase  for the alkaline phosphatase enzyme and glucose-6-phosphate/NADP+ (Sigma) for glucose-6-phosphate dehydrogenase . The enzyme assays were quenched with 40 μl 1 M Na2CO3. P. putida wt as well as Δalr cultures at exponential and stationary growth phases were used to isolate periplasmic and cytoplasmic protein, which was subsequently used to determine Alr racemase activity according to Radkov and Moe  were harvested in 50 ml tubes and resuspended in 5 ml 20 mM KH2PO4 (pH 7.2) with 1 mM DTT. The cells were sonicated (ten cycles at 60 V for 20 s with 2 min pause on ice in between each cycle) and Triton X-100 was added to 0.5% final concentration (v/v). The suspension was vortexed for 30 s and incubated at 55°C for 30 min. The treated lysate was centrifuged at 200,000 × g at 20°C for 20 min . The recovered pellet was rinsed four times with 1 ml water without resuspension and then centrifuged once more using the same parameters. The pellet was resuspended in 1 ml 0.1 M ammonium acetate, pH 6.0. The pellet was broken using the pipette tip during resuspension, as the material becomes highly compacted after centrifugation. The resuspended pellet was incubated with 1 mg ml−1 lysozyme  at 37°C with shaking (220 rpm) for 2.5 h. After incubation, the mixture was centrifuged at 18,000 × g for 10 min and transferred to a new tube. Two volumes of cold acetone were added, the mixture was vortexed for 30 s and then centrifuged at 18,000 × g. The entire supernatant was transferred to a glass vial and evaporated overnight under air. The remaining solid was resuspended in 900 μl 6M HCl, transferred to a polypropylene 2 ml microfuge tube and incubated at 95°C in a hot plate for 12 to 16 h. After hydrolysis, the solution was cooled to room temperature and added to 900 μl 6M NaOH. The resulting solution was derivatized using Marfey's reagent (Sigma) and analyzed via HPLC  after 2, 4, 6, 8, and 10 h of incubation at 28°C with shaking. The cells in each culture were pelleted via centrifugation and the supernatant was derivatized using Marfey's reagent and analyzed via HPLC as described above [see  according to the manufacturer's instructions. cDNA libraries were prepared using an established protocol . The template-switching primer was CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGG+G (the last position is occupied by a locked nucleic acid). Following cDNA synthesis, the samples were purified using AMPure magnetic beads (Beckman Coulter). Lastly, the cDNA was enriched using Phire Taq polymerase (Thermo-Fischer) and the following Illumina-specific PCR primers 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ and 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′. Each library was assessed on a 2% agarose gel and the number of PCR cycles was varied until a smear was obtained in the 200–500 bp range according to agarose gel results. AMPure magnetic beads (Beckman Coulter) were used to purify the enriched DNA samples. DNA quantification, library pooling, and MiSeq sequencing were performed at the Advanced Genetics Technologies Center at University of Kentucky. Gene expression analysis was conducted using the CLC Genomics Workbench software (CLC bio). RNA-seq data has been submitted to the NCBI BioProject database under accession PRJNA471301.ol Hunt, . Brieflyd-AAs and their corresponding l-stereoisomers  were used to determine the growth profile for P. putida KT2440 and Δalr strains  according to the PCR band size.A collection of 19  Figures , S2. Eac Figures . The finP. putida KT2440. We used the chloroform method for obtaining the protein fraction, referred to here as “periplasmic,” that is released upon incubation of the cells with chloroform. The cellular fraction that remained was lysed to obtain the “total” fraction that comprised remaining periplasmic protein as well as the cytoplasmic protein. The goal of this experiment was to determine whether Alr activity could be detected in the periplasmic fraction, and therefore to confirm previous suppositions about its localization. Because of the nature of the fractionation, it is not possible to completely exclude cytoplasmic protein from the “periplasmic” fraction, nor is it possible to exclude periplasmic protein from the “total” fraction. Therefore, we sought to determine the relative quantities of cytoplasmic protein in the “periplasmic” fraction and the relative quantities of periplasmic protein in the “total” fraction using assays to detect the relative activity of a known cytoplasmic enzyme  in both “periplasmic” and “total” fractions and the relative activity of a known periplasmic enzyme  in the “periplasmic” and “total” fractions. It was not our goal, using this method, to exclude Alr activity in the cytoplasm, only to detect its activity in the periplasm. Based on the relative activity of AP in the “periplasmic” and “total” fractions  compared to the activity in the “total” fraction (209.7 ± 8.2 nmol min−1 mg−1). Normalizing the “total” fraction to 100% activity, this gives the “periplasmic” fraction approximately 64% activity, which is in line with the relative activity of AP in the “total” vs. “periplasmic” fraction stated above (72–77%). No Alr activity was detected in either the “periplasmic” or “total” fractions from the Δalr mutant strain.With this in mind, Alr activity was highly abundant in the “periplasmic” fraction of in vivo by measuring d- and l-Lys quantities in the growth medium when the wt and Δalr strains were grown in LB medium  was detected in the wt growth medium after 4 h of incubation .Alr activity was monitored m Figure . No signt Figure , inset. d-Lys in the supernatant and the periplasmic localization of the Alr enzyme, we next considered whether Alr activity had an impact on peptidoglycan structure. No significant differences (p > 0.05) were observed in the stationary phase composition of peptidoglycan between the wt and Δalr strains  between the two strains.Due to the presence of ns Table , precludalr strains using RNA-seq. More than half of the P. putida KT2440 genome was covered by at least one paired-end read and approximately 10% by 10 or more paired-end reads  have RPKM values (reads per kilo base per million mapped reads) larger than 200 and fold-change differences higher than 3. Several genes known to be involved in d-Lys catabolism were significantly overexpressed (p < 0.05) in the wt relative to the Δalr strain , the Δ1-piperideine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase (Pip2C/Pyr2C reductase), which converts Pip2C into l-pipecolate (PP3591), and downstream genes acting on l-pipecolate and subsequent catabolites (PP5257 and PP5258). These activities can be mapped to the Lys catabolism pathway from Figure alr strain was PP3593 (fold-change 27), which is a putative AA transporter/AA-binding protein localized to the periplasm  were catabolized as the sole source of C and N, while six l-AAs  yielded minimal growth  in V. cholerae  upregulated in the wt strain related to catabolism of AAs, suggesting that under these growth conditions the products of Alr activity do not appear to impact properties beyond metabolism of AAs.Consistent with its putative periplasmic target sequence, the Alr enzyme was found to be active in periplasmic fractions from both exponential and stationary phase cultures. As expected the Δan Table . RNA-seqP. putida KT2440. The Δalr strain showed diminished capacity for catabolism of the two l-AAs that are the best substrates for Alr (Arg and Lys), implying that in each case conversion to their corresponding d-enantiomer promotes catabolism. Previous work has shown that Alr is involved in catabolism of l-Lys  for conversion of d-Arg to l-Arg for subsequent catabolism in Pseudomonas aeruginosa (Li and Lu, P. putida KT2440 (Radkov and Moe, d-Arg catabolism, independent of l-Arg, has not been characterized. Further work will be required to determine the mechanism of d-Arg catabolism in P. putida KT2440.From our observations, elimination of Alr activity had the most significant impact on the metabolic capacity of l-Lys and l-Arg through conversion to their respective d-enantiomers. While the d-AAs resulting from Alr activity are produced, at least partly, in the periplasm, they are not incorporated into peptidoglycan under these conditions. Further, despite accumulation of d-Lys in LB growth medium, this does not appear to act in any broader signaling capacity. Rather, the excess of d-Lys promotes expression of genes that are necessary for d-Lys catabolism. At this time, however, it is not clear why Alr is localized to the periplasm in P. putida KT2440 when the d-AAs produced are further catabolized in the cytoplasm. This work adds to a growing body of data indicating that d-AAs play myriad roles in the physiology of bacteria.Data presented here support a role for Alr in the catabolism of LM and AR designed the experimental work. AR performed the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Of note, treatment of patients with high-dose aspirin led to a decrease in theconcentrations of the prothrombic mediator thromboxane A2, reduced braininfarcts, and decreased death in patients with TBM. Together, these findings identifya CSF SPM signature that is associated with disease severity and 80-d mortality inTBM.—Colas, R. A., Nhat, L. T. H., Thuong, N. T. T., Gómez, E. A., Ly,L., Thanh, H. H., Mai, N. T. H., Phu, N. H., Thwaites, G. E., Dalli, J. Proresolvingmediator profiles in cerebrospinal fluid are linked with disease severity and outcomein adults with tuberculous meningitis.Tuberculous meningitis (TBM) is the most lethal form of tuberculosis infection,characterized by a dysregulated immune response that frequently leads to neurologicinjury and death despite the best available treatment. The mechanisms driving theinflammatory response in TBM are not well understood. To gain insights into thesemechanisms, we used a lipid mediator–profiling approach to investigate theregulation of a novel group of host protective mediators, termed specializedproresolving mediators (SPMs), in the cerebrospinal fluid (CSF) of adults with TBM.Herein, using CSF from patients enrolled into a randomized placebo-controlled trialof adjunctive aspirin treatment, we found distinct lipid mediator profiles withincreasing disease severity. These changes were linked with an up-regulation ofinflammatory eicosanoids in patients with severe TBM and a decrease in the productionof a number of SPMs. CSF proresolving mediator concentrations were also associatedwith 80-d survival. In survivors, we found a significant increase in proresolvingmediator concentrations, including the lipoxygenase 5–derived 13-seriesresolvin (RvT)2, RvT4, and 15-epi-lipoxin B Mycobacterium tuberculosis is responsible for more deaths globally thanany other infectious disease. When it infects the brain and meninges to cause tuberculousmeningitis (TBM), which represents 1–5% of all forms of tuberculosis, it eitherkills or severely disables around a half of all sufferers despite the best availabletreatment . These mediators include the arachidonic acid (AA)-derived lipoxins (LXs),the eicosapentaenoic acid (EPA)-derived E-series resolvins (RvEs), the docosahexaenoic acid(DHA)-derived resolvins (RvDs), protectins (PDs), and maresins (MaRs), as well as therecently described n-3 docosapentaenoic acid (DPA)-derived resolvins (RvDn-3DPA), protectins (PDn-3 DPA), and maresins (MaRn-3 DPA)  . Recent henotype–11. Theshenotype, 12, 13.2, preventing brain infarcts (a common life-threateningcomplication of TBM) and enhancing the resolution of intracerebral inflammation through theincreased expression of SPMs (We recently tested the hypothesis that the addition of aspirin to standard TBM treatment(antituberculosis drugs and corticosteroids) may further improve outcomes by inhibitingthromboxane (Tx)A of SPMs . The trivs. those that died during the study. Finally, we also foundthat in aspirin-treated patients there was a dose-dependent alteration of the CSF lipidmediator profiles.Little is known about the regulation of lipid mediators, and in particular SPM, in thecerebrospinal fluid (CSF) during TBM. Thus, in the present studies, we investigated whetherbaseline CSF lipid mediator concentrations were altered with increasing disease severity.We found that concentrations of a number of SPM families were reduced with increasingdisease severity. This was linked with an up-regulation of inflammation-initiatingeicosanoids, including prostaglandins (PGs) and cysteinyl leukotrienes (LTs). PretreatmentCSF lipid mediator concentrations were also found to be predictive of outcome, withdistinct lipid mediator profiles obtained in those patients that were alive at the end ofthe study The patients in the current study were enrolled in a clinical trial of adjunctiveaspirin, the design and conduct of which have been previously described . BrieflyLumbar puncture was performed before the start of treatment and 30 d after treatmentinitiation, per normal clinical care with CSF archived at −80°C untillater testing. Clinical progress and neurologic and drug-related adverse events wereassessed daily until discharge from hospital and monthly thereafter until 8 mo, whena final clinical assessment was made.All samples were extracted using solid-phase extraction columns as previouslydescribed . Prior tIn the analysis of peptide-conjugated lipid mediators eluting in methanol fraction,an Agilent Poroshell 120 EC-C18 column (100 mm × 4.6 mm × 2.7μm) was kept at 50°C, and mediators were eluted using a mobile phaseconsisting of methanol:water:acetic acid at 55:45:0.1 (v/v/v) over 5 min that wasramped to 80:20:0.1 (v/v/v) for 2 min, maintained at 80:20:0.1 (v/v/v) for the next 3min, and ramped to 98:2:0.1 (v/v/v) over 3 min. This was kept at 98:2:0.1 (v/v/v) for3 min. A flow rate of 0.65 ml/min was used throughout the experiment. QTrap 5500 wasoperated in positive ionization mode using scheduled multiple reaction monitoringcoupled with information-dependent acquisition and enhanced product ion scan .r2values of 0.98–0.99.Each lipid mediator was identified using established criteria, including matchingretention time to synthetic and authentic materials and at least 6 diagnostic ions. Calibrahttp://www.R-project.org/) (n (%) for categorical data are provided in thesupplemental document.We performed all statistical analyses and data derivation in R (ct.org/) , Prism 8R-RvD1, and17R-RvD3), PDs , protectin conjugate in tissue regeneration (PCTRs) , and MaRs , maresins conjugated in tissueregeneration (MCTRs) , the n-3 DPA-derived 13-seriesresolvins (RvTs) , RvDn-3 DPA , PDn-3DPA , andMaRn-3 DPA , the RvE , and the AA-derivedLXs . Separately, we combined theconcentrations of proinflammatory eicosanoids: AA-derived LTs ,cysteinyl LTs (cysLTs) , PGs, and Txs(TxB2).To assess the overall regulation of proinflammatory and proresolving mediatorbiosynthetic pathways in the CSF, we combined the concentrations of bioactivemediators from each of the lipid mediator families together with their biosyntheticpathway markers. For this purpose, we combined the concentrations of the RvDs 1 or survivors] and test groups.Investigations into the flux down each of the mediator families was conducted bydetermining the fold change in the concentration of the different mediator familiesindicated previously, between the control groups  was found to besignificantly associated with the mortality after adjustment for multiple testing. We next tested whetherlipid mediators found to be differentially regulated between the 2 groups werecorrelated with outcome. Using LASSO regression analysis, we found that CSFconcentrations of 15-epi-LXB4 together with those of the immunosuppressivemediator PGD2 were the stronger predictors of mortality.OPLS-DA analysis of CSF lipid mediator profiles provided further support for thedifferences in lipid mediator concentrations between those who died and those whosurvived, with CSF lipid mediator concentrations from each of these patient groupsgiving 2 distinct clusters . We next queried whetheractivity of the SPM biosynthetic enzymes was reduced in nonsurvivors, thuspotentially accounting for the reduced SPM levels in the CSF of these patients.Assessment of monohydroxylated fatty acid products of these enzymes indicated thatthe activity of all 4 enzymes was significantly up-regulated in nonsurvivors whencompared with survivors . Thus, these findingsdemonstrate that the reduction in SPM concentrations was not due to either limitedsubstrate availability of reduced activity of the biosynthetic enzymes.In order to gain insights into the mechanisms contributing to the differences in CSFlipid mediator concentrations in TBM survivors and nonsurvivors, we next investigatedwhether the concentrations of essential fatty acids that are substrates in thebiosynthesis of these molecules were different between these 2 patient groups. Ofnote, CSF concentrations of all fatty acids were significantly higher in nonsurvivorswhen compared with survivors . We compared lipid mediator profiles obtained after 30 d ofaspirin (1000 or 81 mg/d) or placebo  cotreatment. OPLS-DAanalysis demonstrated that, whereas d-30 CSF lipid mediator profiles between the81-mg and placebo groups were not markedly different, lipid mediator profiles frompatients given 1000 mg aspirin gave distinct clusters compared with patients givenplacebo . Assessmentof CSF lipid mediator concentrations demonstrated a reduction in TxB2concentrations , the inactive further metabolite of theimmunosuppressive, and prothrombotic mediator TxA2.. The reduction in TxB2concentrations was found to reach statistical significance at d 30 posttreatmentinitiation in patients given 1000 mg aspirin after adjustment for multiple testing.We next conducted statistical analysis to assess whether cotreatment with aspirinchanges lipid mediator profiles between baseline and d 30 when compared with placebo.For this analysis, we started with 93 patients for whom we had matched samples forlipid mediator profiles at d 0 and 30. We excluded 13 patients who received aspirinfor <30 d and 1 patient who was found to be a statistical outlier for lipidmediator concentrations. This left a total of 79 patients that were included in theanalysis , the biosynthetic pathways leading to the formation ofthese molecules were down-regulated. Future studies powered to interrogate this questionwill need to establish whether daily administration of high-dose aspirin down-regulatesthese pathways in the CSF.Studies conducted by Vane et al.  demonstr (et al. , 37 founIn summary, the present findings uncover novel mechanisms in the pathophysiology of TBMthat may have relevance to all forms of tuberculosis. Disease severity is associatedwith a differential expression of lipid mediators that includes a reduction in theconcentrations of several SPM, an observation that is also linked with a poorprognosis.http://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


Hand gesture and grip formations are produced by the muscle synergies arising between extrinsic and intrinsic hand muscles and many functional hand movements involve repositioning of the thumb relative to other digits. In this study we explored whether changes in thumb posture in able-body volunteers can be identified and classified from the modulation of forearm muscle surface-electromyography (sEMG) alone without reference to activity from the intrinsic musculature. In this proof-of-concept study, our goal was to determine if there is scope to develop prosthetic hand control systems that may incorporate myoelectric thumb-position control. Healthy volunteers performed a controlled-isometric grip task with their thumb held in four different opposing-postures. Grip force during task performance was maintained at 30% maximal-voluntary-force and sEMG signals from the forearm were recorded using 2D high-density sEMG (HD-sEMG arrays). Correlations between sEMG amplitude and root-mean squared estimates with variation in thumb-position were investigated using principal-component analysis and self-organizing feature maps. Results demonstrate that forearm muscle sEMG patterns possess classifiable parameters that correlate with variations in static thumb position . Of importance, this suggests that in transradial amputees, despite the loss of access to the intrinsic muscles that control thumb action, an acceptable level of control over a thumb component within myoelectric devices may be achievable. Accordingly, further work exploring the potential to provide myoelectric control over the thumb within a prosthetic hand is warranted. In forming hand gestures and grip patterns, activation of both extrinsic and intrinsic hand muscles is necessary  is a small amplitude, time-varying signal detected from the surface of the skin and reflects the summed activity of superficial and deep motor-units from near-by co-contracting muscles. Activity in motor-units is driven by neural commands acting through the motor nerves innervating those muscles. Therefore, sEMG signals are directly linked to the neural-command associated with movement. This makes the use of sEMG a compelling non-invasive method for user control and interfacing in prostheses, and has resulted in a range of commercial myoelectric prosthetic devices entering the market over the last decades. However, the control over grip-transitions during activities of daily living remains a significant challenge for the majority of myoelectric prosthetic users to master. Intention detection based on user-modulation of sEMG, through implementation of real time classification algorithms, adaptive learning methods, binary classifications or pattern recognition . All subjects provided informed written consent prior to the tests and reported to have no prior history of nerve damage, hand surgery, existing neuromuscular pain, tremor, epilepsy, and/or movement disorder.Subjects were seated and the position of their upper arm, forearm and wrist was standardized by use of a specially designed apparatus incorporating grip force measurement . ExperimOnce subjects could demonstrate that they could perform a grip that targeted each finger pad, an estimate of the maximal voluntary contraction (MVC) force between the thumb and hand was determined. Graphical user interfaces (GUIs) were then used to provide the participant with visual cues indicating which finger pad to oppose, the start and end times for a contraction effort and a 30% MVC grip force target level.Simultaneous HD-sEMG signal acquisition occurred whilst the subject followed the GUI instructed grip tasks. The GUI randomized the opposition order of grip tasks to provide a total of 30 grip trials at each of the four grip postures , and each 30% MVC task was sustained for 5 s. A variable resting time interval of 5–7 s between efforts was provided. No subject reported fatigue nor was fatigue evident as a failure to sustain the required force throughout a session.Monopolar HD-sEMG signals were acquired using 128 channels of a multichannel bioelectrical signal amplifier . Data were acquired from participants performing the different thumb opposition grip tasks as described above.Two regions of interest (ROIs) were recorded simultaneously via two separate 13-by-5 electrode grids with an 8 mm inter-electrode distance . The two ROIs corresponded to sites overlying the posterior and the anterior forearm musculature . To stanThe HD-sEMG signals were acquired at a frequency of 2,048 samples per second, with a fixed gain of 1,000 V/V, a bandwidth of 3–900 Hz and a zero-phase notch filter to remove the 50 Hz mains interference and its harmonics. The sampled HD-sEMG data was then used to create iso-potential maps computed from averaged RMS values for each channel. The averaged RMS values were calculated, as detailed in Sebelius et al.  and BettThe RMS is a convenient measure reflecting the change of electrical activity generated by a contracting muscle and is widely used for feature recognition/distribution  was calculated to highlight the local differences specific to each of the four thumb positions in both ROIs.As differences in the global RMS values are seen to vary within each thumb posture during different grip tasks, feature extraction was applied to the averaged 300 ms overlapped windows of RMS vectors separated by thumb opposition and by grid, in order to understand the amplitude variation for each task.Finally, the thumb variations were classified using self-organizing feature maps (SOFM). The SOFM is an unsupervised artificial neural-network algorithm which aims to discover underlying structure by clustering the input data quantified by their Euclidean distance  and the Neural-Network Pattern Recognition (NNPR) Matlab built-in toolboxes were applied as supervised methods. These methods use backpropagation and are designed to minimize the sum of square error , thereby providing to be a suitable benchmark. The networks were presented with 30% of the labeled data, 35% used for testing and the remaining 35% was used for validation. The function used to assess the maps performance was the mean squared error value (MSE) between the outputs and the targets, where lower values meant greater accuracy and zero meant no error.In each experiment, the participant opposed their thumb to each of the four different target postures, as previously described, totaling 120 repetitions (30 per opposition).RMS values for each isometric effort sustained were estimated using 300 ms window size with a 50 ms overlap for each electrode site in both ROI. The resulting windows were further averaged generating a highlighted spatial distribution map of each of the four opposition tasks. In A total of 1,920 vectors  for each opposition and for each were then processed using the Classification Learner toolbox. The resulting clusters highlighting the amplitude variation across positions further separated by electrode grid position is shown in The resulting 60% of the FVs containing the isometric effort of HD-sEMG data acquired from each participant was used to train the SOFMs. A resulting hit histogram, separated by thumb position, on the trained network during a test is shown in The performance of the trained SOFMs was assessed through quantization, topographical and combined error as quality measures. The outcomes of these measures are shown in The remaining 40% of the data were used to test the SOFMs to compare the results of the un-presented datasets to the already trained network. The precision and success is shown on The different thumb positions were further inspected reducing the dimensions through PCA . This anThis accentuates that important, classifiable differences are found in the HD-sEMG patterns extracted from the extrinsic hand muscles while the hand performs different grips.Supervised neural networks (NN), such as LM and the NNPR were applied using the MATLAB Pattern Recognition toolbox for further data validation. The HD-sEMG data inputs were provided with the appropriate labels so that the network could recognize which cluster each of the presented FV belonged to. The NN performance was assessed through the values of cross-entropy and percentage of error , and conIt can be observed from the It remains a technical and economic challenge to provide adequate and consistent user control of powered hand prosthesis from the small numbers of sEMG electrodes implemented in current devices with limited processing power and running simple signal processing protocols. As the complexity and functional capability of next generation powered anthropomorphic hands increases, so does the need for better and more intuitive user-control. Presently, no myoelectric prosthesis provides sEMG based control over thumb components. This is surprising from an anatomical, physiological and functional perspective as the thumb is the digit which gives the human hand its greatest adaptability in grip and gesture formation, and it is the digit most often coupled to the motions of the other digits in grip and object manipulation. The thumb is estimated to be contribute to at least 50% of normal overall hand function . All subjects provided informed written consent prior to the tests.AA-G and BC jointly planned the research protocol. AA-G carried out the testing, signal acquisition and analysis. AA-G and BC jointly analyzed and discussed the findings. Both contributed to the production of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.