Some studies have indicated the potential for midazolam as a novel therapeutic cancer drug; however, the mechanism by which midazolam affects cancer cells needs to be clarified. This systematic review aims to summarize the progress in assessing the molecular mechanism of midazolam as an anticancer agent.With its high worldwide mortality and morbidity, cancer has gained increasing attention and novel anticancer drugs have become the focus for cancer research. Recently, studies have shown that most anesthetic agents can influence the activity of tumor cells. Midazolam is a  Cancer has become the most common disease worldwide and is the leading cause of death . CurrentThe chemical structure of midazolam (dormicum) is 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazobenzodiazepine .A receptor in the central nervous system benzodiazepine. The results were limited to English or Chinese language.Any study that clearly stated a link between midazolam and cancer, both in vitro and in vivo, was included. Studies were excluded based on the following criteria: (1) conference abstracts, reviews, conference papers, case reports, editorials, comments, news, congresses, and letters; (2) non-English or Chinese.The quality of all eligible studies was assessed by two independent reviewers using the EBLIP Critical Appraisal Checklist . The extA total of 822 studies were identified in PubMed, Embase, Web of Science, Ovid evidence-based medicine, Chinese science and technology periodicals , and Chinese Biomedical Literature (CBM) using our search strategy. After detailed screening, 12 studies were considered eligible for inclusion in this review  had no effect. Although pancaspase inhibition had a strong protective effect, flumazenil could not inhibit midazolam-induced apoptosis. Midazolam induced apoptosis via activation of the mitochondrial pathway in a concentration-dependent manner. The induction of apoptosis by midazolam is presumably unrelated to GABAignaling .α subunit of eukaryotic initiation factor 2 (p-eIF2α) are typical ER stress markers [α/ATF4/CHOP is an essential signal pathway regulating ER stress [α, ATF4, ATF3, and CHOP was observed, suggesting that midazolam may induce apoptosis via the ER stress pathway [The endoplasmic reticulum stress (ER stress) pathway, also known as the Unfolded Protein Response, is the response of the cell to a dangerous buildup of unfolded or misfolded proteins in the ER –41. C/EB markers . eIF2α/AR stress . In MA-1 pathway . Midazol pathway , but Bid pathway , 46. Mid pathway . Thus, m pathway .In decreasing order, midazolam showed the greatest toxicity for HL-60 cells, epidermal keratinocytes, oral squamous cell carcinoma (OSCC), and glioblastoma cells. Midazolam did not induce the generation of apoptosis markers in OSCC cells  but did induce many vacuoles, mitochondrial swelling, and cell membrane rupture . MidazolAutophagy is a newly recognized innate defense mechanism that has been observed in cancer and is a physiological program that enables the body to deal with the destruction of cells . AutophaHowever, in MA-10 cells, the staining and expression of LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes, was observed following midazolam treatment, suggesting that midazolam induced autophagy in MA-10 cells .The rate of cell proliferation is always determined by cell cycle distribution. There are four phases in the cell cycle including G1, G2, S, and M; G1 and G2 phases are gap phases; S phase is the synthesis phase during which the genetic material is duplicated; and the M phase is where mitosis partitions the genetic material and the cell divides , 50. As 1 phase and a reduction of cells in the G2/M phase in a time- and dose-dependent manner [Another study that looked at the effect of midazolam in mouse Leydig tumor cells showed an accumulation of MA-10 cells in the sub-Gt manner . It was t manner .0/G1 cell cycle arrest in the human head and neck squamous carcinoma FaDu cell line by regulating cell cycle regulators [On the other hand, a study by Dou and coworkers showed that midazolam triggered Ggulators .The human E1A binding protein, p300, also known as EP300 or p300, is encoded by the EP300 gene and regulates the activity of many genes in tissues throughout the body . It playMidazolam was shown to inhibit the proliferation of FaDu cells, a cell line from a squamous cell carcinoma of the hypopharynx, and attenuated the mRNA and protein levels of p300. The knockdown of p300 resulted in an upregulation of p21 and p27 proteins and downregulation of p-Rb protein. Thus, it appeared that midazolam inhibited the proliferation of FaDu cells via downregulation of p300 expression .Similarly, another study on FaDu cells showed that midazolam was able to inhibit the growth and proliferation of FaDu cells . HoweverA further study demonstrated that midazolam inhibited the growth and proliferation of SW480 colonic adenocarcinoma cells in a time- and dose-dependent manner and downregulated ubiquitin-specific protease 22 (USP22) expression. With the use of USP22 small interfering RNA (Si-RNA), they were able to silence USP22 expression and found that SW480 cell proliferation was inhibited, while P21 and P27 expression was upregulated, and pRB downregulated. Thus, a feasible mechanism by which midazolam inhibits proliferation may be via the mediation of cyclin-dependent kinase inhibitor/retinoblastoma protein (CDKI/RB) pathways through the downregulation of USP22 .Midazolam has been shown to inhibit the in vitro growth and differentiation of two murine myeloid leukemia cell lines (WEHI 3B (JCS) and M1 cells) in a dose-dependent manner . MidazolThere has only been one animal model study. This was carried out in BALB/c-nu mice bearing K562 and HT29 cell human tumor xenografts. The results showed that midazolam inhibited growth of the cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in the xenograft mice. The mechanism of inhibition of carcinogenesis by midazolam may be a suppression of reactive oxygen species (ROS) production leading to modulation of apoptosis and growth regulatory proteins .It is interesting to consider the possibility that, besides its use as an anesthetic agent, midazolam may have the ability to prevent or inhibit tumor development. New insights are rapidly being gained into the role of the midazolam in cancer treatment. In this review, the protective role of midazolam in cancer and the potential mechanisms underlying this have been described. Studies suggest a critical role for midazolam in influencing many signaling pathways on which cancer cells death is induced including necrosis and apoptosis. Midazolam can also inhibit the proliferation of cancer by inhibiting cell cycle progression. However, the impact of midazolam on other behavior of tumor cells, such as invasion and metastasis, remains to be further studied.μM, even achieving 200 μM or 1000 μM. The effect of different concentrations of midazolam on tumor cells was shown in In relating observations in vitro to molecular events in vivo, a main focus is the different concentrations of midazolam used. In vitro studies, the concentrations of midazolam were usually from 0 to 100 The above discussions on the anticancer activities of midazolam are based mostly on studies with lymphoma cells and Leydig tumor cells. These theories will serve as a basis for researchers to explore the effect of midazolam on other tumors. For example, we are investigating the effect of midazolam on lung cancer cells (A549), and we found that midazolam could induce the apoptosis of A549 cells through regulating signal transducer and activator of transcription 3 (Stat3) signaling pathway. Although the detailed mechanism by which midazolam acts on cancer cells remains elusive, the results described above suggest that midazolam could present a potential therapeutic in various cancers. Further study into the role of midazolam in the prevention of cancer is crucial if translation from the laboratory to the clinical setting can occur.


In the frame of assistive robotics, multi-finger prosthetic hand/wrists have recently appeared, offering an increasing level of dexterity; however, in practice their control is limited to a few hand grips and still unreliable, with the effect that pattern recognition has not yet appeared in the clinical environment. According to the scientific community, one of the keys to improve the situation is multi-modal sensing, i.e., using diverse sensor modalities to interpret the subject's intent and improve the reliability and safety of the control system in daily life activities. In this work, we first describe and test a novel wireless, wearable force- and electromyography device; through an experiment conducted on ten intact subjects, we then compare the obtained signals both qualitatively and quantitatively, highlighting their advantages and disadvantages. Our results indicate that force-myography yields signals which are more stable across time during whenever a pattern is held, than those obtained by electromyography. We speculate that fusion of the two modalities might be advantageous to improve the reliability of myocontrol in the near future. A prosthetic wrist adds at least one DOF to the device and further improves its potential dexterity, but empowering amputees to control such artifacts (myocontrol) is still an open issue.The human hand is a prodigious natural tool, comprising 27 bones and 33 muscles, resulting in a total of 22 degrees of freedom (DOFs) , mechanomyography (MMG), ultrasound imaging also known as sonomyography (SMG), force myography (FMG), functional magnetic resonance imaging and more are considerable options , but despite the specified remarkably large sensitivity range (0.2N–40N), they have non-negligible hysteresis at high forces, no guarantee of repeatability and a non-linear transfer function. Nonetheless, Castellini and Ravindra , linking it to the housing. Even if the elastic and damping properties of the membrane have not been subject to further investigation so far, it is assumed that the membrane may increase the signal stability, i.e., it could hypothetically add a mechanical filtering to the bio-signal. This solution offers the capability to create any combination of FMG and sEMG electrodes. In order to gather signals from the complete circumference of the forearm, the sensors are placed evenly spaced around the forearm/stump. This arrangement, also called low-density surface electrode layout or uniform electrode positioning  of the signals of both the sEMG and FSR sensors, and their wireless transmission. As the microcontroller natively supports only 15 AD-channels, AD-conversion of up to 32 sensors is realized via analog multiplexing, providing a maximum sampling rate of 192.5 Hz for each channel. Since the sEMG sensors already provide rectified and filtered signals with an evaluable bandwidth limited to 10 Hz, the provided sampling frequency is an overshoot  and the prosthesis . Hence, the board can also relay control commands to a hand prosthesis. The final cost of the system is estimated to be below 150€.Honor 6 with a commercial value of about 300€), on which the data processing is performed. This device is equipped with a quad-core Cortex-A15 processor running at 1.7 GHz, 2 GB of RAM and a 3100 mAh Li-Po battery, claimed to keep the smartphone running for 2 full days at moderate usage. Its operating system is Android 4.4 KitKat. The smartphone weighs 132 g and easily fits in a pocket (7 × 0.8 × 14 cm) with its 5-inch display. A C# application similar to the one used in Strazzulla et al. , joined the experiment . Each subject received a thorough description of the experiment, both in oral and written form. Informed written consent was obtained from all participants. Experiments with sEMG and FMG were approved by the Ethical Committee of the DLR.https://www.youtube.com/watch?v=KCUwoxuAdYQ, and the prototype by Ottobock which appears for instance in Amsuess et al. (The experiment consisted of performing ten times the following sequence of wrist and hand movements: (1) wrist flexion, (2) wrist extension, (3) wrist pronation, (4) wrist supination and (5) power grasp. To enforce the opening of the hand the relaxed stance was used, in order to mimic a more natural form of myocontrol. Each movement was visually stimulated on the screen of the smartphone (the name of the required motion would appear on the screen), while the experimenter was visually checking that the movement was actually being enforced, to ensure a correct execution. Each stimulation was administered as follows: the visual stimulus would appear for 2 s to allow the subject reach the full movement, then for 6 s data were captured representing the maximal activation for that particular movement , then the stimulus would disappear for 2 s to allow the subject return to the resting position. The sequence was administered in the same order to all subjects. The choice of this set of movements was motivated by the well-known importance of controlling at least the wrist pronation/supination ; they were advised to perform each movement bilaterally Figure . The recstellini .The signals recorded during the experiment were stored to the smartphone's internal memory, then analyzed off-line. Low-pass filtering was applied to both signals  in order to remove high-frequency disturbances. This is a standard procedure in the field  we evaluated Fisher's Separateness Index , where SB is the between-clusters scatter matrix, while SW is the within-clusters scatter matrix. SB is given by i, μj are the means of clusters Ci, Cj, while x are the samples in each cluster. Each pairwise Fisher's index was averaged across all subjects and collected in a matrix S = {sij}. Fisher, , definedsEMG/FMG regression for myocontrol: A comparative regression accuracy analysis was performed, in order to assess whether sEMG, FMG or their juxtaposition would be significantly better in the framework of wrist/hand prostheses control. The learning algorithm of choice was Incremental Ridge Regression with Random Fourier Features, already successfully used multiple times, e.g., in Gijsberts et al. (f(x) = wTx, where x denotes the sensor values, w is a weighting vector and f(x) is the predicted output; Random Fourier Features further employ a non-linear mapping from the input space to a higher-, finite-dimensional feature space, where the linear regression is more likely to succeed. . Ten-fostrongly disagree to 5 - strongly agree). The scoring in this survey is such that the answers to the strongly agree positive questions and to the strongly disagree negative questions generate a higher impact over the final score. The NASA Task Load Index provides an overall workload score on six subscales: Mental, Physical and Temporal Demands; Own Performance, Effort and Frustration. For each subscale, the answer could be in a range of 21 points, reaching from very low to very high  Brooke, , the NASLastly, Travis's survey consists in a series of “reaction cards” with adjectives that could be applied to the system to be tested; the user is asked to select the five cards that most closely match their personal reactions to the system. For the experiment, we used a list of 75 adjectives instead of the cards (most of them based on Travis's questionnaire), then the subject was asked to choose all adjectives he or she felt more related with the device. After that, in a more precise selection, the user had to choose only the 5 most important words and try to give a simple reason about his or her decision.x-axis).Figure while the subjects are holding the position than sEMG signals: this is apparent by looking at the “plateaus” created by the FSRs while each movement was enforced; as opposed to that, sEMG signals exhibit the typical oscillating down-ramp pattern due to muscular motor-unit recruitment .Visual inspection seems to indicate that FMG signals are more stable over time Figure t-test p < 0.001).Figure p > 0.05).Table For the SUS, the total result of each subject and the mean score are presented in Table For the NASA TLX, the total result for each subscale of each subject and overall workload are shown in Table The first two surveys applied comprised suitable results with a usability score of almost 85% and an overall workload of 25%.Finally, for the desirability survey proposed by Travis, two kinds of results were obtained, the first one using all the words chosen by the user in the first selection, and the second one considering only the 5 final selections. In order to have a different visualization, two word clouds have been created Figure , where tSimple, Intuitive, Easy to use, Familiar, Reliable and Stable. An important thing to mention about this last survey is that even though the instructions and the questions were oriented toward the device, some answers referred to the experiment performance. For instance, the adjective familiar was chosen in some cases because the subject had already performed other experiments with EMG sensors before taking part in this specific experiment.Considering only the most common adjectives in the final selection, the device can be considered To highlight the results of the user satisfaction surveys, a radial chart Figure  was builThis work had two main aims: to assess whether a wearable combined sEMG/FMG device would be accepted by human subjects using it, and to determine whether significant qualitative and quantitative differences could be observed between sEMG and FMG signals obtained during a simple experiment aimed at myocontrol.Consider Section 3.4, in particular Figure The keywords obtained in Travis's survey, the workloads obtained in the NASA TLX survey, and the successful performance reflected in the SUS test, already give hints about the usage risk of a possible future medical device. Of course, this cannot replace a thorough risk analysis and a widespread usability study. Furthermore, it must be remarked that the positive results obtained in the surveys could have been influenced by the subjects' behaviors themselves, as explained by Travis. All in all, it is worthwhile to stress that no online experiment was performed in this study, therefore, from the user survey results, we can provide no conclusive results about either the performance of this device in general, or the usefulness of a combined sEMG/FMG approach in myocontrol.overall stability over time than sEMG, meaning that, e.g., the variance of its signals is lower than that of sEMG, while human subjects are engaged in repetitive, fatiguing tasks. This is probably due to the lower influence that muscle fatigue has on FMG signals, due to its nature. Now, from the qualitative/quantitative comparison of sEMG and FMG carried out in Sections 3.1 and 3.2, two statistically significant differences between the two approaches emerge, namely (a) that FMG signals are more stable over time during single movements too, and (b) that they generate better separated patterns in the input space. Most likely, (b) is a consequence of (a); we speculate that this difference might arise from the very nature of the sEMG signals, which exhibit noise due to the recruitment of motor units while keeping an isometric hand/wrist posture. Of course, FMG would not be affected by this problem. All things considered, it seems reasonable to claim that FMG signals are more stable than sEMG ones.In previous literature , presents the drawback of being affected by hysteresis. Although the return to the resting state is apparent, this induces the FMG signals even to rise during the resting states, but not to come back exactly to zero or the previously measured resting states after a movement is performed. As opposed to this, sEMG signals remain almost at zero even when the user is not in the same initial position; this seems reasonable, since the resting phase involves no muscle activation. This problem can be countered by employing a smarter technique to gather FMG signals, for instance the capacitive approach, or (high-density) tactile sensing. Also, a fully-fledged, online FMG approach will need to take into account the inevitable artifacts generated by the arm/forearm movement  and those induced by touching the socket, bumping into objects, laying the stump on a table, etc.What we can conclude, we believe beyond any reasonable doubt, is that sEMG and FMG can be miniaturized and employed in such a framework, and that they carry different kinds of information, leading to different behaviors and signal features. We speculate that a structured sensor-fusion approach  might lead to a better exploitation of each modality's characteristics.medically certified FMG approach and device, which might dramatically raise its costs. Again, we believe that an integrated approach is the way ahead to improve myocontrol using this still novel technique alongside sEMG. To this aim, the assessment of the wearable sEMG/FMG device we carried out is promising, as it shows that at least the required electronic machinery can be embedded in an effective, light and acceptable device.One last remark is in order about the cost of each approach. Apparently, FMG is up to two orders of magnitude cheaper than sEMG, but this is mainly due at this stage to (a) it being enforced through Force-Sensing Resistors, which might not live up to the expectations as previously remarked; and (b) the necessity, in the very end, to produce a In this article, we have described a wearable, integrated sEMG/FMG system, targeting myocontrol and human intent detection. The experiment we conducted endorses the system's high degree of usability, indicating that it has the potential to become an integrated medical product. Still, the autonomous mobility of about 11 h is restricted by the cell phone battery, thus recharging strategies during the patient's daily use or the deployment of a secondary device could be a solution for now. In near future, the development of a highly integrated, low energy system, where the phone serves only for teaching and displaying information, seems to be advisable. Additionally, we explored the application of FMG as a potential complement to sEMG and provided evidence that the two techniques can be integrated, but that a smart sensor fusion approach might be required to obtain the best results. Results from user satisfaction surveys presented in the paper give strong indication for the setup to be on the right track. In the near future, more experiments are planned to check the feasibility of mixed sEMG/FMG for online myocontrol, possibly down to the level of individual finger movements, and immersed in daily-life activities.System Design BV, MC, CC; Software Design MC, BV; Test setup and conduction of user Surveys ER, MC, CC; Analysis and Interpretation of data ER, CC; Drafting of manuscript MC, ER, BV; Internal Revision BV, CC; Critical Revision BV, CC, MC, ER; Final Approval MC, ER, BV, CC; Agreement on accountability by MC, ER, BC, CC.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.


Recognizing patients with ventriculomegaly who are at risk of developing acute hydrocephalus presents a challenge for the clinician. The association between disturbed cerebrospinal fluid flow (CSF) and impaired brain compliance may play a role in the pathogenesis of hydrocephalus. Phase contrast MRI is a noninvasive technique which can be used to assess CSF parameters. The aim of the work is to evaluate the effectiveness of phase contrast MRI in recognizing patients at risk of acute hydrocephalus, based on measuring the pulsatile CSF flow parameters in the Sylvian aqueduct and prepontine cistern in children with ventriculomegaly.The aim of the work is to characterize the parameters of cerebrospinal fluid (CSF) flow in the Sylvian aqueduct and prepontine cistern in children with ventriculomegaly with regard to patient age and symptoms. We hypothesize that the relationship between CSF flow parameters in these two regions will vary according to analyzed factors and it will allow to recognize children at risk of hydrocephalus.A group of 26 children with ventriculomegaly (five girls and 21 boys) underwent phase contrast MRI examinations . Amplitudes of average and peak velocities of the CSF flow through the Sylvian aqueduct and prepontine cistern were used to calculate ratios of oscillation and peak velocities, respectively. The relationship between the oscillation coefficient, the peak velocity coefficient, and stroke volume was then assessed in accordance with age and clinical symptoms.p = 0.0094). Moreover, these patients tended to develop symptoms more often (p = 0.0612). No significant age-related changes were observed in CSF flow parameters.The peak velocity coefficient was significantly higher in patients with hyper-oscillating flow through the Sylvian aqueduct (3.04 ± 3.37 vs. 0.54 ± 0.28; Phase contrast MRI is a useful tool for noninvasive assessment of CSF flow parameters. The application of coefficients instead of direct values seems to better represent hemodynamic conditions in the ventricular system. However, further studies are required to evaluate their clinical significance and normal limits. The key point to understand the nature of hydrocephalus lies in understanding the nature of CSF circulation. This issue has been broadly studied since 1940 . NeverthThe phase contrast MRI technique, invented in the 1980s, allows for the noninvasive evaluation of the CSF flow. It does not require a contrast administration because this technique assumes that atomic spins moving in the same direction as a magnetic field gradient produce a phase shift that is proportional to the velocity of these spins. Previous applications of the phase contrast MRI have demonstrated that the oscillatory character of CSF flow is consistent with the cardiac cycle and respiratory rate , 11, 23.The Linninger mathematical model of CSF hydrodynamics is more congruent with data obtained from the phase contrast MRI than traditional concepts. According to hydrodynamic theory, the main cause of hydrocephalus is the loss of brain compliance and elasticity, resulting in development of an increased transmantle pulsatile gradient between the intraventricular lumen and subarachnoid spaces. Studies have supported this theory only in small groups of adults , 28, 30.The study group comprised 26 children (five girls and 21 boys) with ventriculomegaly or at risk of hydrocephalus because of completed intraventricular hemorrhage. Patient age ranged from 7.2 months to 12 years, on average 3.5 years (± 3.05 years). The most common causes of ventriculomegaly were idiopathic, the presence of a cyst interfering with physiological CSF flow, or intraventricular bleeding. The exclusion criteria comprised any brain malformations not associated with ventriculomegaly, aqueduct occlusion, or cerebral shunt implantation. The study protocol was approved by the ethics committee (protocol number-95/2015).The patients underwent the phase contrast MRI examinations (Philips 3T Achieva) according to the following protocol. ECG electrodes were placed in standard positions and ECG trace was obtained. First, the structural brain images were acquired. CSF BFFE sequence sagittal scans were used to determine the plane for the CSF QF sequence . The CSF QF sequence plane was positioned perpendicular to the Sylvian aqueduct and prepontine cistern at the level of the inferior colliculi of the quadrigeminal plate , headaches (n = 4), paresis (n = 17), mental retardation (n = 13), the increased head circumference growth rate (n = 15), signs of chronic increased intracranial pressure (n = 2). Eight children did not show any clinical symptoms . Although a p = 0.0612) .No significant differences were observed in any of the analyzed CSF flow parameters with regard to the presence of clinical symptoms, age, or decreased brain compliance (stroke volume ≥ 42 μl) (Table p = 0.938). The theoretical age of the change in CSF hemodynamic pattern (2 years of age) did not affect the frequency of the hyper-oscillating flow occurrence: for children aged over 2 years, eight had an oscillation coefficient < 1 while six had an oscillation coefficient ≥ 1; for those aged 2 or less, six had an oscillation coefficient ≥ 1 while four had an oscillation coefficient < 1 (p = 0.4640).As far as age was concerned, no significant difference was found between the mean age of children with hyper-oscillating flow  and of children with oscillation coefficient < 1  (No significant relationship was found between decreased brain compliance (stroke volume ≥ 42 μl) and age or with CSF hemodynamics and age Tables .Table 3CNeuroendoscopic ventriculo-cystocysternostomy was performed in one patient due to the presence of a suprasellar arachnoid cyst that impaired outflow of the CSF from the third ventricle. Due to increasing head circumference, MRI examination was performed. Both the oscillation coefficient (14.38) and the peak velocity coefficient (7.7) were relatively high in comparison with the rest of the group. The measured stroke volume was 54 μl indicating decreased brain compliance. Based on clinical presentation and MRI results, the patient was qualified for shunt implantation. The procedure was performed with no complication and resulted in clinical improvement.A key novel aspect of this study is that in contrast with the majority of similar studies, which deal with adults, it concerns children with ventriculomegaly. Secondly, instead of assessing the direct parameters of CSF flow in the Sylvian aqueduct, we analyzed coefficients, including the flow through the subarachnoid space, thus giving a better insight into the equilibrium between the ventricular system and the subarachnoid space.The study has three central findings. In children with ventriculomegaly, hyper-oscillation of the CSF flow through the Sylvian aqueduct is associated with significantly greater peak flow velocity. Secondly, there is a trend in symptomatic children to develop a hyper-oscillating type of flow. Finally, age-related changes of the pattern of CSF hemodynamics may be disturbed in patients with ventriculomegaly.microvessels theory, hypothesizing that CSF flow not only manifests as a bulk flow from the choroid plexus to the arachnoid villi, but also as a free admixing between the subarachnoid space and extracellular space . This exchange is controlled by the glymphatic system [Understanding the dynamics of the movement of CSF appears a key for proper characterization of the pathomechanism of hydrocephalus. CSF flow is influenced by the blood pressure wave over the cardiac cycle and by the respiratory rate to a lesser extent . Accordic system , 8, 26 ac system .The creation of a precise CSF hydrodynamics model remains a challenge. A transmantle pressure gradient exists both in healthy volunteers and patients affected by hydrocephalus, and although the pressure is slightly higher in the hydrocephalus patients, it is still too low to be the main force responsible for driving the CSF movement , 29, 30.Phase contrast MRI was first performed in 1980, when Holland observed the disappearance of an MRI signal associated with the oscillating movements of CSF molecules , 4, 6. TThe most popular and reliable parameter for assessing CSF is a stroke volume, which represents the volume of CSF flowing back and forth during the cardiac cycle . DespiteN = 6) than the Sylvian aqueduct (on average 3.82 ± 2.12 mm/s.), the opposite is true for patients with NPH (10.82 ± 1.16 mm/s in the prepontine region and 13.14 ± 2.64 mm/s in the Sylvian aqueduct). Hydrodynamic theory assumes that decreased compliance between the arterial system and subarachnoid space results in greater pulsation of the brain parenchyma and the formation of a water hammer effect; this causes a transmantle oscillatory gradient resulting in the enlargement of the cerebral ventricles. Therefore, the coefficient of CSF flow through the subarachnoid space and the Sylvian aqueduct may be a better indicator of decreased brain elasticity than the absolute peak velocity flow value [p = 0.0612). Both these findings may be signs of increased CSF absorption resistance. This hypothesis is best represented in the illustrative case of our patient with ventriculo-cystocysternostomy, who presented a high oscillation coefficient (14.38) and peak velocity coefficient (7.7), together with a stroke volume of 54 μl, indicating decreased brain compliance.A great degree of variation has been found in CSF flow parameters depending on the study: the upper limit of the flow rate ranges from 18 to 24 ml/min , 11, andow value , 18. PubOther explanation for observed differences in CSF flow parameters between children and adults may result from distinct characteristics of the CSF hydrodynamics. Arachnoid villi are fully developed in the fetus before birth, but they only begin functioning at late infancy. Before then, in neonates, infants, and young children, the main role in CSF absorption plays the interstitial and perivascular spaces as well as perineural lymphatic channels . It has also been observed that in heathy children younger than 2 years, CSF mainly returns to the cerebral ventricular system, which indicates that CSF absorption takes place through the minor CSF pathways. The opposite situation takes place in older children and adults . AlthougOur most interesting finding was lack of any significant relationship between the marker of brain elasticity and hyper-oscillating flow in the aqueduct. This could be the result of an inappropriate limits assumed for brain elasticity. Perhaps, the stroke volume of ≥ 42 μl assessed by Bradley as significant for the adult patients who had lost brain compliance does not apply to children. Alternatively, more samples could be needed for a more accurate analysis: the group with the stroke volume ≥ 42 μl only comprised five children. However, a significant relationship was found between the oscillating coefficient and the flow velocity coefficient. The two measurements should not be treated as associated data because the oscillating coefficient describes the character of CSF flow rather than its absolute speed.There are two major limitations of this study. The first is that the study sample was too small to provide sufficient power. However, our aim was to present a new perspective for assessing CSF flow parameters as coefficients for the assessment of dynamic equilibrium rather than as direct values in a particular location. The second limitation was the lack of a control group. However, due to the fact that most children in the study were under 6 years old, the lower limit for performing MRI examination without sedation/anesthesia, it was not justifiable to expose healthy children to potentially harmful side effects. In future, we plan to gather a larger sample of patients and analyze the differences in CSF flow parameters between patients who require intervention, as in our illustrative case, and those who remain in a stable condition. It is also worth to mention that application of the phase contrast MRI may be associated with technical limitation, concerning placement of the ROI in the prepontine cistern. Particularly, when this space is narrow or scarred . In these cases, adjustment of the ROI size and its placement in some distance from the basilar artery are vital and, according to our experience, possible in almost all cases.The peak velocity coefficient was significantly higher in children with hyper-oscillating CSF flow in the Sylvian aqueduct. Moreover, this type of flow occurred more often in symptomatic children. Finally, probably, due to ventriculomegaly, the physiological maturation of CSF flow pattern was disturbed. However, diagnostic limits of CSF flow parameter values in patients affected by neurological symptoms require further research based on larger groups with a control group composed of healthy children with normal-size ventricular system.


Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established.Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi.Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an “M1-like” to a more “M2-like” activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ).Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic “M1-like” macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.The online version of this article (10.1186/s12931-017-0708-5) contains supplementary material, which is available to authorized users. Using a transgenic mouse model where we can induce GM-CSF in the airways (BAL levels approximately 300 pg/mL) of WT mice with doxycycline administration during active influenza infection, we achieve levels of GM-CSF that prevent mortality from lethal influenza A virus infection. The GM-CSF excess rescued mice from an acute respiratory distress syndrome (ARDS)-like illness and stabilized lung mechanical parameters, improved clearance of exudate protein, and disconnected M1 activation by type II IFNγ.Each year, influenza A virus (IAV) affects a significant proportion of the population  and causcsf2−/−) mice [The effect of local elevation of GM-CSF on IAV infection in the lung has been investigated in transgenic models with expression of GM-CSF under the control of constitutive or doxycycline-inducible promoters in lungs of alveolar or small airway epithelial cells of GM-CSF knockout (/−) mice , 4. Diff/−) mice , 5 and A/−) mice .The question of whether therapeutic administration of GM-CSF to the airways after establishment of the infection would confer protection has never been addressed. In this study we use a more clinically relevant model to examine whether supra-physiologic levels of GM-CSF in the airways, induced after IAV infection at the peak of virus replication, provided therapeutic benefit. Using GM-CSF-inducible mice on the WT C57BL/6 genetic background we show that airway GM-CSF over-expression starting at 3 days post infection (dpi) provides protection from mortality and prevents the degeneration of multiple lung mechanical properties. To examine the mechanism of protection conferred by therapeutic GM-CSF levels, we measured respiratory and biochemical parameters of lower airway disease, and analyzed the transcriptome of FACS-sorted AMs and exudative macrophages (EM) from IAV-infected mice. Our findings demonstrate that GM-CSF restores proteostasis of exudate proteins and redirects responsiveness of AMs and EMs from an M1-like to an M2-like activation state, and prevents mortality from influenza-induced ARDS.The double transgenic GM-CSF (DTGM) mice were bred as previously described , but thi2O, tidal volumes (Vt) 10 mL/kg, respiratory rate (RR) 150 breaths per minute (bpm) and an fraction of inspired oxygen (FiO2) of 0.21. Oxygen saturations were measured using the MouseOx Plus pulse oximeter  via the thigh sensor. Lung mechanic parameters were generated from the flexiVent rodent ventilator using the forced oscillation technique as previously described [Mice were anesthetized with ketamine/xylazine . The trachea was cannulated via tracheostomy with a 19G blunt needle and the cannula was secured in place with a suture. Mice were kept sedated using isoflurane inhalation  through the flexiVent and were paralyzed with 1 mg/kg vecuronium i.p. Mice were ventilated using baseline settings of positive end expiratory pressure (PEEP) 3 cm HBAL samples were collected as previously described, and after centrifugation at 150 g for 10 min, BAL supernatants were removed and immediately frozen at −80°C until batch analysis. Proteins were measured with kits as detailed in Additional file After IAV infection, the entire lung was removed from each mouse and placed in 2 mL of TRIzol , weighed, homogenized on ice using a polytron homogenizer for 15-30s intervals, and frozen in aliquots at -80 °C until RNA extraction. DNA was then extracted using chloroform and RNA was precipitated using isopropanol. Quantitative RT-PCR for IAV M1 copies per lung was performed as previously described  using thSingle cell suspensions were prepared from BAL and lung as described in Supplemental Methods. Single cell suspensions from lung digests were placed at 4°C and then surface stained in Hank’s Buffered Saline Solution (HBSS) with 3% FBS with fluorochrome-conjugated monoclonal antibodies  to keep only reads that have at least 80% of bases with a quality score of 20 or more  and reads left with 10 bases or longer after being end-trimmed with reads with a base quality score of b20 .RNA was phase separated using chloroform and the aqueous phase containing RNA was removed following centrifugation and precipitated overnight at −20°C using ice cold isopropanol. RNA was washed with 75% ethanol then solubilized in RNase-free water. Optical density values of extracted RNA were measured using NanoDrop (Thermo Fisher Scientific) to confirm an A260:A280 ratio above 1.9. RNA integrity number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Pico Kit to confirm RIN above 7. The cDNA libraries were prepared using the SMARTer® Ultra® Low Input RNA Kit for Sequencing - v3 (Clontech) followed by Nextera XT DNA Library Prep Kit (Illumina) as per the manufacturer’s instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies) and Kapa Library Quantification Kit (Kapa Biosystems). The libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 (Illumina) and run for 50 cycles using a single-read recipe  according to the manufacturer’s instructions. Illumina CASAVA pipeline  was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter. Additional quality filtering used FASTX-Toolkit (A bowtie2 index was built for the mouse reference genome (GRCm38) using bowtie version 2.1.0. The RNA-seq reads of each of the 38 samples were mapped using TopHat version 2.0.9  suppliedwww.qiagen.com/ingenuity) to identify upstream signaling pathways. Significance was measured by Fisher’s exact test with a q < 0.2 cut-off.We arbitrarily chose to further analyze the 5% most highly expressed gene transcripts in AM or EM cell populations from IAV-infected DTGM or LM mice using Ingenuity Pathway Analysis . Normally distributed data were analyzed using student’s t-test, and non-normally distributed data using Wilcoxon signed-rank test. Survival analysis was calculated by using the log-rank test. All data points are means ± standard error of the mean (SEM) unless otherwise stated. Graphs were created using Prism 6 for Mac OS X .50) of our PR8 IAV preparation. Female wild-type mice were much more susceptible than males with an LD50 approximately 5-fold lower than males  were purchased and we determined the lethal dose 50%  promoter after oral administration of doxycycline (1 mg/mL in water ad libitum)  of PR8 IAV and were administered doxycycline in drinking water. GM-CSF overexpression (DTGM) conferred a significant survival advantage as compared to wild-type levels . Weight loss and recovery were similar in the two groups, however, because of survivor bias likely artificially elevating the average weights of surviving LM mice . However, GM-CSF did not significantly increase median oxygen saturations (% SpO2) levels as compared to LM mice at either 7 or 10 dpi (data not shown).Lower respiratory tract IAV infection can lead to impaired oxygenation due to V:Q mismatch and decrease lung compliance due to the infiltration of inflammatory cells and an increase in lung water weight . Given t2O/mL, Fig. 2O*s/mL, Fig. 2O, Fig. To gain insight into whether GM-CSF conferred any lung mechanical benefits, lung mechanics scans were performed by the forced oscillation technique and PV loop curves were generated Fig. . As expeGiven that two of the lung mechanical parameters that are maintained by GM-CSF, K and G, are correlated with dynamic processes at the small airway or alveolar level, namely alveolar size  and chan8 M1-copies total lung copies of IAV PR8 matrix 1 (M1) via RT-PCR and the viral copies decreased to 0.8-2 × 107 by 10 dpi, though there was no statistically significant difference with GM-CSF overexpression  macrophages. To investigate this immune cells were characterized and enumerated in single cell suspensions of BAL and lung enzymatic digests by multi-parameter flow cytometry using a 12-color panel of macrophage and granulocyte surface markers  and F4/80+, CD11b+, SiglecFneg/dim cells that have been termed exudative macrophages (EMs) [Alveolar macrophages have been shown to be necessary for protection from IAV –27. GM-Cers Fig. . We speces (EMs) . Our typCcl17, Cxcl3, and CCL6, and the down-regulation of Cxcl9, and Arg1, the prototypic marker of M2 macrophage polarization . The concentration of MMP12 was approximately 6-fold higher in GM-CSF overexpressing mice at 10 dpi as compared to LMs . We also examined the ratio of the two chemokines (CXCL9: CCL17) as an intrinsic property of the BALF to probe macrophage polarization by chemokine expression and supra-physiologic GM-CSF levels significantly decreased this ratio more than ten-fold in IAV-infected mice .To validate these macrophage transcript differences we measured the chemokines CCL17 and CXCL9, and the M2-associated metalloprotease, MMP12, in BAL fluid by ELISA. CCL17 was significantly induced by GM-CSF . The IPA software allows the construction of an upstream analysis that calculates the likelihood that an upstream regulator is involved given the gene set provided , as well as a composite score of activation depending on the state of downstream genes being increased or decreased in quantity (activation z-score). For both the upstream regulator analysis, and the subsequent canonical pathway analysis, we used a stringent p-value of overlap cutoff of 1E-10.Lastly, we attempted to determine which signaling pathways were affected by GM-CSF overexpression during IAV infection by analyzing our transcriptomes with Ingenuity Pathway Analysis (IPA) software (Qiagen). We analyzed the effect of GM-CSF overexpression on the mean logIPA predicted that GM-CSF activates Table  several In terms of canonical pathway analysis one pathway, "eukaryotic initiation factor 2 (eIF2) signaling", was activated in both AMs and EMs, whereas "Fc-γ receptor-mediated phagocytosis in macrophages and monocytes" was inhibited in both populations Table . “InterfIn this study we examined the effect of elevated GM-CSF levels during IAV infection on clinical, lung physiologic and biochemical markers in a mouse model, and then used RNA-sequencing to ascertain the differential effects of elevated GM-CSF levels on the transcriptomes of the two predominant airway macrophages present during the peak of IAV infection. Our finding that elevation of airway GM-CSF during active IAV infection confers protection from mortality from IAV is novel. Multiple preclinical mouse studies have described the observations that the absence of GM-CSF increases susceptibility to IAV , 4, 25, GM-CSF over-expression led to an increase in macrophage expression and BAL fluid levels of CCL17 and MMP12, whereas a decrease in CXCL9 or monokine induced by gamma interferon (MIG). These protein data, in addition to our macrophage transcriptome data, suggest that high levels of GM-CSF push the typically classically activated M1-like monocytes/macrophages in the lung during IAV towards an M2-like phenotype. Interestingly, a recent investigation showed that the presence of M1-like monocytes are a major determinant of IAV pathogenicity in patients and strengthened this notion with a mouse model demonstrating that adoptive transfer of M2 as opposed to M1 macrophages results in better outcomes . The obs−/− and IFNGR1−/− mice that interferon signaling is necessary for protection from IAV [−/− mice on both the Balb/c and C57BL/6 backgrounds demonstrated improved survival to lethal IAV infection [The role of interferons during IAV infection is also nuanced. While it has been shown using IFNARfrom IAV , it is pfrom IAV , and Sunfrom IAV . Recentlnfection . In theinfection , howevernfection .Stat1 promoter [Trim24 transcripts in our sorted airway macrophages, but GM-CSF over-expression did lead to increased expression of another TRIM family member, Trim16, that also acts as a E3 ubiquitin ligase that can heterodimerize with other TRIMs [Our data suggest that high levels of GM-CSF inhibit interferon signaling in airway macrophages, though the mechanism is not clear. Canonically, GM-CSF signaling acts through JAK2/STAT5 , though promoter , thus inpromoter  while TRpromoter . Furtherpromoter . Howeverer TRIMs . Future GM-CSF enhanced exudative macrophage expression, and 8 dpi BAL fluid levels of MMP12, or macrophage elastase, which is best known for its requirement for the development of smoke-induced emphysema in mice . HoweverCsf2−/−) genetic background [Csf2−/− mice [Csf2−/− /SPC-GM mice, in which T2AECs express high levels of GM-CSF constitutively, came to disparate conclusions as to the role of AMs, dendritic cells and epithelial cells [+/+ model results in non-physiological proliferation of both T2AEC cells and AMs [+/+ model also illustrates that prolonged lung exposure to supraphysiologic levels of GM-CSF leads to desquamative interstitial pneumonia (DIP) [Our current model of GM-CSF induction on the wild-type background differs from our previous work using the inducible model generated on the GM-CSF knockout  . We did ia (DIP) . Concentia (DIP) –71.Our data demonstrate that in vivo high airway levels of GM-CSF profoundly rescue mice from lethal influenza pneumonia. While in vitro GM-CSF is canonically described as an M1-polarizing cytokine, our data demonstrates that in vivo, during IAV infection, GM-CSF instead temporizes the type II interferon-induced M1 polarization of airway macrophages. The exact mechanism through which high levels of GM-CSF block M1-macrophage polarization is still not known, and is the focus of our ongoing research.Additional file 1: Table S1.All protein concentration measurements were made as described in the manuscript text using the reagents and kits listed. (TIFF 3075 kb)Additional file 2: Table S2.Multi-parameter flow cytometry was utilized to characterize the alveolar and exudative macrophages as shown in Fig. Additional file 3: Figures S1A-D.Lethal dose 50% (LD50) determination of influenza virus strain A/Puerto Rico/8/1934 (PR8) in female  and male  mice, demonstrating an LD50 of 728 vs. 3728 fluorescent focus units (FFU) in female and males, respectively. (ZIP 87 kb)Additional file 4: Figure S2A and B.Characterization of the Double Transgenic GM-csf (DTGM) mouse model. In the absence of influenza A virus infection, GM-CSF levels (A) in bronchoalveolar lavage (BAL) fluid was low, near the limit of detection in littermate (LM) and DTGM mice. Upon influenza A virus infection DTGM mice without doxycycline-induction (DTGM noDox) demonstrate "leakiness" that corresponds to the peak of type II interferon levels at days 7-8 post-infection. DTGM +Dox mice demonstrate supra-physiologic levels of GM-CSF in BAL fluid at all time points after induction. DTGM mice were less susceptible to IAV infection (B) even in the absence of doxycycline induction, whereas doxycycline administration to LM mice had no effect. (ZIP 55 kb)Additional file 5: Figure S3A and B.Measurement of serum proteins in BAL fluid. Elevated levels of GM-CSF neither affected the quantity of mouse albumin (A) nor IgM (B) in BAL fluid at 10 and 14 days post-infection. (ZIP 39 kb)Additional file 6: Figures S4A-D.https://www.luminexcorp.com) at the indicated time points. (ZIP 74 kb)Characterization of the kinetics of BAL cytokines. Type I interferon (A), type II interferon (B), type III interferon (C), and IL-10 (D), were measured in BAL fluid from wild-type  or DTGM +Dox (red bars) mice by multiplex analysis (Luminex,


Compartmentalization of HIV-1 has been observed in the cerebrospinal fluid (CSF) of patients at different clinical stages. Considering the low permeability of the blood-brain barrier, we wondered if a reduced selective pressure by neutralizing antibodies (NAb) in the central nervous system (CNS) could favor the evolution of NAb-sensitive viruses in this compartment. Single genome amplification (SGA) was used to sequence full-length HIV-1 envelope variants (453 sequences) from paired CSF and blood plasma samples in 9 subjects infected by HIV variants of various clades and suffering from diverse neurologic disorders. Dynamics of viral evolution were evaluated with a bayesian coalescent approach for individuals with longitudinal samples. Pseudotyped viruses expressing envelope glycoproteins variants representative of the quasi-species present in each compartment were generated, and their sensitivity to autologous neutralization, broadly neutralizing antibodies (bNAbs) and entry inhibitors was assessed. Significant compartmentalization of HIV populations between blood and CSF were detected in 5 out of 9 subjects. Some of the previously described genetic determinants for compartmentalization in the CNS were observed regardless of the HIV-1 clade. There was no difference of sensitivity to autologous neutralization between blood- and CSF-variants, even for subjects with compartmentalization, suggesting that selective pressure by autologous NAb is not the main driver of HIV evolution in the CNS. However, we observed major differences of sensitivity to sCD4 or to at least one bNAb targeting either the N160-V1V2 site, the N332-V3 site or the CD4bs, between blood- and CSF-variants in all cases. In particular, HIV-1 variants present in the CSF were more resistant to bNAbs than their blood counterpart in some cases. Considering the possible migration from CSF to blood, the CNS could be a reservoir of bNAb resistant viruses, an observation that should be considered for immunotherapeutic approaches. HIV-1 replication in the central nervous system (CNS) occurs early after infection  and is menv gene due to the role of the Envelope (Env) glycoproteins in the cell tropism . Longitudinal blood plasma samples were available for four patients, but no additional sequential CSF sample was available for any of them. Characteristics of the participants and the samples are described in env gene. A total of 453 single genome sequences were obtained with a mean of 19 sequences (9–28) per sample. Phylogenetic analysis demonstrated the absence of cross-contamination between samples from different individuals and showed the broad diversity of the strains which were related to seven different clades  [nn) [P values < 0.01) and a distinct monophyletic CSF subpopulation with boostrap value ≥ 0.70 was observed [Compartmentalization was studied for 330 sequences obtained from the contemporaneous plasma/CSF pairs of the 9 patients. It was assessed based on interpretation of neighbor-joining trees and statistical evidence using three methods ,32: the st)  and the st) [nn) . CSF virobserved ,20. CSF p < 0.001) , in agreement with the tree topologies   sequences by determining the mean average pairwise distance (APD) within each compartment. We also analyzed the mean average pairwise distance between compartments (BC-APD). For three of five individuals with compartmentalized viral populations , the mean APD was significant lower in CSF compared to blood plasma  . In contpologies . Region ctively) .In order to provide insights into viral population dynamics in these patients developing neurological disorders, we performed a Bayesian phylogenetic analysis for the four subjects with longitudinal plasma samples. In three out four patients—KP, GK and KU—the viral populations were clearly divided into two major evolutionary lineages and the CSF compartmentalized populations were detected mainly in one of them, suggesting a selective advantage of one particular lineage to establish infection in the CNS . Time toThe longer period of divergence between plasma and CSF compartmentalized populations was observed for subject RO, with an estimated duration of 8.2 years before the time of the lumbar puncture . This esOverall, the data suggest different patterns of evolution of CSF- and blood- viral populations. Compartmentalized variants of the CSF could have originated from variants seeding CNS years ago (up to 8 years) and could have evolved independently since then. Otherwise they could have emerged and diversified a shorter time before neurocognitive impairment.env gene amplified by RT-PCR from the plasma and the CSF of each patient. Infectious pseudotyped viruses were obtained for six of the nine subjects, including four individuals with longitudinal plasma samples and compartmentalized viral populations , and two subjects with equilibrated viral populations . We compared their sensitivity to autologous neutralization by measuring the 50% inhibitory concentration (IC50) by serially diluted purified IgG from autologous sera collected at different time points. As expected, the viral population sampled from blood plasma was either resistant (IC50 > 1 mg/mL) or moderately sensitive (IC50 = 0.13–1 mg/mL) to the contemporaneous autologous neutralization  and the IC50 of both PGT145 and VRC03 were increased by 16 and 15 fold respectively (i.e. increased resistance) when compared to the blood population in patient BA. The differences of sensitivity to bNAbs in the two sampled compartments were observed regardless of compartmentalization status, suggesting that the CSF populations had specific properties.bNAbs target specific epitopes on the envelope spikes, thus interfering with the functionality of the envelope glycoproteins and preventing virus entry. We compared the sensitivity of the six pairs of contemporary plasma- and CSF-derived pseudotyped viruses to bNAbs representative of the major sites of vulnerability: PGT121, which targets a glycan-dependent epitope within or near the V3 loop of gp120; PGT145 and PG16, which recognize glycan-dependent quaternary epitopes in the V1V2 region; VRC03, which targets the CD4-binding site of gp120; 10E8 which targets the membrane-proximal external region (MPER) of gp41; and 8ANC195 which targets a conformational epitope overlapping both envelope subunits. The IC50 . All of them, either issued from plasma or CSF, were CCR5-tropic as determined by both genotypic and phenotypic assays. The sensitivity to MVC was almost similar in each pair of CSF- and blood- populations for all participants. The two cases with equilibrated viral populations presented sensitivity to sCD4 in the same range. However, two of four individuals with CSF-compartmentalized populations showed highly different sCD4 IC50 . The incenv sequences from paired CSF and blood plasma samples, we tried to identify sequence signatures that would be specific of the localization to the CNS. After alignment completed by the GUIDANCE program to exclude positions for which there were uncertainties, we performed a signature pattern analysis using the Viral Epidemiology Signature Analysis (VESPA) software in order to examine the amino acid differences between CSF sequences and blood plasma sequences for each individual. A Fisher’s exact test was performed for each site to determine positions where statistically significant differences between the two compartments were observed and, for each protein alignment the number of variable sites was used to calculate the corrected Bonferroni p-value threshold. Using this approach, we identified several positions that either were shared by at least two individuals of our study or were found for one of our cases and reported in previous studies. There were thirteen positions in gp120 and six positions in gp41 where dominant amino acid in CSF sequences differed significantly from blood sequences  had evidence of viral compartmentalization in the CSF, in agreement with the previous studies.The compartmentalization of HIV-1 in the CNS has been reported in association with severe neurocognitive stages as well as in milder forms of impairment ,15–21. UThe phenotypic correlates implicated in the replication in the CNS environment and the dynamics of this phenomenon in the course of the disease are far from understood. Some genetic characteristics of neuroadaptation across HIV envelope have been reported and shown to be related to an enhanced capacity to infect macrophages –20,22–24Highly potent human monoclonal bNAbs that have been isolated these recent years target epitopes known as sites of vulnerability on the Env spikes ,39,40. TPrevious functional studies have shown that CNS/CSF-derived viruses may have adapted for efficient entry into brain macrophages which are known to express low levels of CD4 ,24,45,46env diversity was statistically lower in CSF- than in plasma-derived variants in some patients with compartmentalized virus [Despite this diversity and complexity, we tried to identify some molecular features that would be associated with the neurotropism, particularly when compartmentalization was observed. As reported by others, we found that mean HIV-1 ed virus . Focusined virus ,21. The ed virus ,48 suggeed virus . Interesed virus . Severaled virus . We idenenv clones isolated from brain or CSF. Instead of working with clones, which allow to identify the individual phenotypes of the envelope variants that are present in each compartment, we have chosen to perform our study with pseudotyped viruses expressing envelope glycoproteins representative of the viral quasi-species present in each compartment. Although we cannot exclude that bulk PCR can amplify certain predominant variants selectively and generate recombination artefacts, we believe that this approach allows to be more representative of the physiological situation occurring in each patient.In this study, we analyzed both the genotypic properties and the phenotypic properties of paired CSF and blood plasma samples from patients with HIV-1 associated neurologic syndromes. Most of the published previous studies analyzed the phenotypic properties of the Env glycoproteins of CNS/CSF variants using In conclusion, our study provides additional proofs of the complexity of the compartmentalization of HIV-1 and suggests that different independent factors are involved in HIV-1 infection of the CNS. Among them, the viral migration from or to CNS over time, eventually modified by ART treatment or its interruption, and the interaction of the HIV-1 envelope glycoproteins with CD4 would deserve to be analyzed in depth. We also showed that selective pressure by autologous NAb does not appear as a major driver of the HIV evolution in the CNS, and that CNS could be a reservoir of bNAb resistant viruses, an observation that should be kept in mind when considering passive immunization as a potential strategy to the therapy and cure of HIV-1 infection.3; range 23–319). They were not on antiretroviral treatment (ART) or, if treated, had a poor adherence to treatment.HIV-1 infected patients were selected based on the availability of contemporary CSF and plasma samples with viral load > 1 000 copies/mL. They were hospitalized for various neurologic disorders for which a lumbar puncture was required for diagnostic purpose. Diagnoses and CSF characteristics for the nine individuals are detailed in Patients were assigned identification codes (2 letters) that were unique to this analysis and could only be linked back to the original data by a single authorized personnel. The database was approved by the French watchdog committee (#2017–056). Written informed consent was collected from patients who were still in care in our hospital. The Institutional Board of the University Hospital of Tours (Espace de Réflexion Ethique Région Centre) reviewed the study for approval (#2017–021).5’- CTTRTAAGTCATTGGTCTTAAAGGYAG-3’) and the Superscript III First strand synthesis system (Invitrogen) according to manufacturer’s instructions. Single genome amplification (SGA) of the full-length HIV-1 env was conducted as described elsewhere [5’-ATGGCAGGAAGAAGCGG-ARRC-3’) and ExtM3 and the inner primers pair IntM5XE (5’-AATTCTCGAGAATTCAGAAAGAGC-AGAAGACAGTGGCAATG-3’) and IntM3MX (5’-GGCCACGCGTCTAGACTACTTTTTGACCACT-TGCCMCCCAT-3’) with the following conditions: 2 min at 94°C, then 35 cycles of 15 s at 94°C, 30 s at 55°C and 3 min at 68°C, and a final extension step of 10 min at 68°C. HIV-1 env gene products were directly sequenced according to the Dye Terminator cycle sequencing protocol  Each chromatogram was carefully inspected. Sequences with double peaks, sequences with frameshift mutations or premature stop codons were excluded from analysis. All sequences have been submitted to GenBank and assigned accession numbers KY825261 to KY825713.HIV-1 RNA was extracted from plasma and CSF 150 μL) using the Nucleospin RNA Virus kit (Macherey-Nagel). Samples with viral loads <10,000 copies/ml were first pelleted by ultracentrifugation to increase the viral RNA recovery. Reverse transcription (RT) was carried out using 16 μL of viral RNA, outer primer ExtM3  [P < 0.01) [For each individual, compartmentalization between blood and CSF viral populations was evaluated using a panel of methods available on HyPhy software package : the Wrist) , the neast) , the Slast) . Compart < 0.01) ,32.A Bayesian Markov chain Monte Carlo (BMCMC) approach was used to reconstruct phylogenies when longitudinal plasma samples were available. Sampling dates were used to calibrate the time scale and the site of sampling was implemented. Briefly, BMCMC chains of 50 million generations were performed for each analysis with a GTR + Γ4 substitution model under an extended Bayesian skyline plot coalescent model, logging every 1000 and with a 10% burn-in. All analyses were performed using an uncorrelated lognormal relaxed molecular clock implemented in the BEAST software package . Tracer http://guidance.tau.ac.il/overview.html) [https://www.hiv.lanl.gov/).The mean average pairwise distance (APD) within each compartment and the mean average pairwise distance between compartments (BC-APD) were determined using the distance matrix in MEGA6 from each multiple amino acid sequence alignment, with a JTT plus Gamma model. Signature pattern analysis was performed using the Viral Epidemiology Signature Analysis (VESPA) programme on the Los Alamos HIV database site . For eacew.html)  For eachew.html) . The Varew.html) . Both prenv genes were amplified by nested RT-PCR as previously reported, using the same primers as described above for SGA. Reverse transcription (RT) was carried out using outer primer ExtM3 and the Superscript III First strand synthesis system (Invitrogen). It was followed by the first round of PCR, using the Platinum PCR SuperMix High Fidelity (Invitrogen) with the following conditions: 2 min at 94°C, then 35 cycles of 15 s at 94°C, 30 s at 55°C and 3 min at 68°C, and a final extension step of 10 min at 68°C. A 5 μL aliquot of the products from the first round of PCR was then used as template for the second round of amplification under the same cycling conditions. Each fragment was approximately 2.6 kb in length, spanning the entire open reading frame of the HIV-1 gp160 polyprotein. The inner primers IntM5XE containing XhoI and EcoRI sites and IntM3MX containing MluI and XbaI sites [Escherichia coli (Invitrogen). Library of pCI-env plasmids were purified from transformed cultures using silica column chromatography (Macherey-Nagel). Env-pseudotyped viruses were produced as previously described [6 293T cells with 4 μg of each patient-derived pCI-env library and 8 μg of pNL4.3.LUC.R_E_ [4 TZM-bl cells, with serial five-fold dilutions of viral supernatants in quadruplicate, in the presence of 30 mg/mL DEAE-dextran. Infection levels were determined after 48 h by measuring the luciferase activity of cell lysates using the Bright-Glo luciferase assay (Promega) and a Centro LB 960 luminometer (Berthold Technologies) and the TCID50 was calculated as described previously [Full length gp160) aI sites , the ampescribed  by cotra aI siteseviously .via multiple round centrifugations with Amicon Ultra centrifugal filter devices (Millipore) in normal saline solution. IgG concentration was measured with BN ProSpec analyzer (Siemens).IgGs were purified from 1 to 2 mL of plasma samples by gravity-flow protein G affinity chromatography . The elution fraction was then desalted and concentrated Sensitivity to autologous IgGs, to bNAbs and to soluble CD4  of the pseudotyped viruses was assessed as previously described . After t4 cells per well one day prior to infection with viruses diluted to 400 TCID50/mL. Successful infection led to production of luciferase activity in target cells, measured 48 hours post inoculation. Pseudotyped viruses with AD8 (pAD8) or NL4.3 (pNL4.3) envelope were used respectively as R5 tropism and X4 tropism positive controls. A pseudotyped virus obtained by co-transfection of empty-pCI expression vector was used as negative control. Technical cutoff for R5 and X4 coreceptor usage capacity was calculated as the means plus two standard deviations (SD) of RLU detected after U373.R5 infection by pNL4.3 and U373.X4 infection by pAD8 respectively.Prediction of the coreceptor usage was performed through Geno2pheno algorithm for each sequence . A false3 TZM-bl cells per well were prepared the day prior infection. Cells were first treated for 1h at 37°C with 150 μL of three-fold serial dilutions of MVC (6 μM to 0.3 nM) before adding 50 μL of pseudotyped viruses normalized to 400 TCID50/mL. 100 μl of DMEM medium supplemented with 30 μg/mL DEAE-dextran were then added to cells. IC50 values were defined as the reciprocal of the MVC concentration required to reduce RLUs by 50%.For MVC inhibition, 8.10S1 FigSequences from both compartments (plasma and CSF) and all time points were aligned with reference sequences from HIV-1 major subtypes. Each subject formed a distinct cluster, showing the absence of contamination. HIV-1 quasispecies infecting each patient belonged to a variety of clades. Bootstrap analysis (500 replications) was used to test the reliability of the branching order. Bootstrap values ≥ 0.70 are represented by an asterisk. CPZ.CM.CAM5 was used as an outgroup.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableFor each paired CSF/blood plasma single genome sequences dataset, mean length, number of potential N-glycosylation sites and charge of V1V2, V3 and V4 variable regions are indicated, as well as the mean difference between compartments.(TIF)Click here for additional data file.S3 TableFor each paired CSF/blood plasma single genome sequences dataset, mean number of potential N-glycosylation sites on HIV-1 Env is indicated, as well as the mean difference between compartments.(TIF)Click here for additional data file.


The segregation ratio of single- and moderately-branched types to the highly-branched type was significantly different from 3:1 but not different from 15:1 at p < 0.05, suggesting that more than one gene controls the branch number of the inflorescences. Through genome-wide comparison of single-nucleotide polymorphism (SNP) profiles between the highly-branched type bulk and the single-branch type bulk constructed using the F2 plants, we identified a major quantitative trait locus (QTL) on chromosome 3 (58.75–61.4 Mb) and a minor QTL on chromosome 2 (32.95–37.1 Mb), which explained 15.7% and 6.1% of the BN variation, respectively. FALSIFLORA (FA) and COMPOUND INFLORESCENCE (S) genes, located in the QTL peak regions, caught our attention. Sequence comparison of the FA and S genes and their promoter regions from the two parental lines revealed that both contain missense mutations in the coding regions. Segregation analysis of FA and S alleles by high-resolution melting (HRM) method confirmed that alleles for both genes from 10AS111A significantly increased the BN and the size of inflorescence. In conclusion, we propose that SNPs in coding sequences might cause changes in the function of FA and S genes, which might be important determinants of BN.The architecture of inflorescences shows extensive diversity in both branching frequency and flower number, which eventually, determines agricultural productivity. In this study, F Solanum lycopersicum L.) is an agriculturally and economically important vegetable crop worldwide. The architecture of tomato inflorescence varies greatly from a simple type having a single inflorescence branch to highly branched type having dozens of inflorescence branches. The branch number (BN) of the inflorescence is an important trait that determines the final number of fruits produced per inflorescence, which then influences the yield. In small-fruited tomato production, moderately-branched inflorescence structure has an advantage for producing a sufficient number of fruits and preventing possible yield reduction due to the small fruit size [Tomato (uit size . When thOn the basis of the inflorescence meristem (IM) growth habits, two major architectural types of inflorescences can be distinguished in tomato: monopodial and sympodial . In monoCOMPOUND INFLORESCENCE . The segregation of simple-branched type to highly-branched type in F2  generations derived from a cross between ′Wonder of Italy′ and a simple type cultivar ′Lister’s Prolific′ was close to a 3:1 ratio, suggesting that the highly-branched inflorescence is controlled by a recessive allele [S gene allele, s-classic, found from ‘Wonder of Italy’ was also identified in 22 domesticated highly-branched varieties [Previous studies showed that the highly-branched phenotype of a tomato variety ‘Wonder of Italy’ resulted from a mutation in e allele ,5. The SANANTHA (the homolog of UFO) and FALSIFLORA  also caused frequent inflorescence branching, but fertile flowers were replaced by cauline leaves or bracts, and no visible floral structures were formed in the an and fa mutants. Further studies in these three genes suggested that S was required for the maintenance of IM activity, while AN and FA formed a complex to specify flower formation [s) or block (an and fa) the IM transition to FM, leading to branched inflorescences.Mutations in other two genes, ormation . Therefos-classic allele by sequencing analysis. This observation suggested that there should be another S allele or another mechanism than s-classic, that causes inflorescence branching.Although a precise control of inflorescence branching is required for optimizing the fruit production in small-fruited tomatoes, their molecular mechanisms remain obscure. The parental line 10AS111A was derived from a ′double truss′ cultivar ′Sicilian Rogue′, which was kindly provided by the Musashi Plant Breeding Corp. We found that although 10AS111A showed a highly-branched inflorescence phenotype, it did not carry the 2 generation to identify the major genomic regions harboring QTLs associated with BN in tomato progeny of 10AS111A. The integration of QTL-seq with allele-specific association analysis delineated two candidate genes and new allelic variants within the QTL intervals governing BN of tomato inflorescence.In this study, we used the restriction site associated DNA (RAD) sequencing-based quantitative trait loci sequencing (QTL-seq)  approach1 plants were of the simple-type, having one to a few inflorescence branches  was 11 and two in 2014 and 2015, respectively (p < 0.05 (2) of the BN in two-year experiments was 62.8% and 74%, respectively , then aligned 10AS111A genomic sequence was used as the reference sequence for detecting SNPs. A total of 15,640 SNPs were identified between SB and the reference genome. All these SNPs were mapped on the 10AS111A genome with an average depth of 12×  were of a high quality, with quality scores of at least 20 and a guanine and cytosine (GC) percentage around 36%. All the sequence date  were submitted to the Sequence Read Archive (SRA) at NCBI  for unrestricted public access. These short reads from 10AS111A were mapped to the publically-available reference genome of tomato (h of 12× . The SNPh of 12× . The Δ (h of 12× .p < 0.05)  was detected. In this region, the average SNP index of SB was higher than 0.8, but lower than 0.2 for HB. We obtained a significant value of Δ (SNP-index) for this peak, which was higher than 0.5 ( < 0.05) . In addi as well . We also as well .http://www.ncbi.nlm.nih.gov/). The FA gene caught our attention due to prior knowledge of its function [FA gene and its promoter sequence from PI124039 and 10AS111A. Seven SNPs and a small indel were revealed between the two parents. One of the SNPs in the middle of exon 2 (position chr_03:61167529 C/G) [Following the results of QTL-seq , we initfunction . We re-s529 C/G)  changed S and AN [1 and P2, and identified four SNPs and a small nucleotide transition in the S gene  [We also analyzed the other two peak regions on chromosome 2 (32.95–37.10 Mb and 39.80–42.25 Mb), which were also predicted to harbor hundreds of genes. These regions included two genes which were reported to be involved in control of the IM identity, i.e., e S gene B, but noFA and S genes were used to genotype the F2 populations. In 129 F2 individuals of 2014, where the HRM analysis was conducted  for the FA locus and 33:61:36  for the S locus. In 99 F2 individuals of 2015, the segregation ratios were 21:56:22  for the FA locus and 20:56:23  for the S locus. Thus, both data fit well with the expected ratio (1:2:1) for single locus Mendelian segregation. Combining the data obtained from FA and S markers, the F2 populations were classified into nine genotypes. FA and/or S allele from 10AS111A. As expected, the genotype ffss showed the highest value of LOG2(BN) and genotype F_S_ presented the lowest value for both F2 populations , but was not significantly associated with the actual time taken (DA)  . We also with SL . MBD andFA locus .2 population derived from a cross between a highly-branched inflorescence type and single-branch type tomatoes. The observed segregation ratio of single-branch or moderately-branched type to highly-branched type was significantly different from 3:1 but not different from 15:1 at p < 0.05  in PI124039 to glutamine (uncharged) in 10AS111A  associated with BN, FN, DW, and SL  at position 479, which gives rise to a protein with an additional valine residue at position 158. Another change was a deletion of 16 bp in exon 2 resulting in a frameshift mutation. The deletion leads to the mutant plant becoming sterile, as it never produced flowers, even during the late developmental stages [2 lines, which were homozygous for the FA allele from 10AS111A, were highly branched, lacking the determinate growth characteristic of tomato inflorescence, appearing very similar to the FA mutant plant except for the production of mature flowers. All the F2 lines and 10AS111A were fertile and produced normal flowers. This may be because the mutation of FA in 10AS111A is only slightly deleterious so that it just delayed the IM maturation but did not inhibit the IM transition to FM.Molinero-rosales et al.  comparedS  (S (outside the homeodomain region) changed a valine in PI124039 to a glutamine in 10AS111A at position 194  , which mtion 194 . Since tneration , the all changed , the newmosome 2 .S gene in FA mutant inflorescences of wild-type plants and the enhancement in FA expression level in S mutant compared with the wild-type plant [FA or S alleles from 10AS111A. Plants homozygous for alleles of both the genes from 10AS111A have the highest ratio of highly-branched phenotype  and the S. pimpinellifolium accession PI124039 . The sizes of F2 population used in 2014 and 2015 were 129 and 103, respectively. Seeds were sown in 2 L plastic pots filled with 3:1 mixture of granular clay soil and peat-based soil mix . In 2014, the F2 plants were sown on 30 May and grown in a greenhouse  from seedling stage to the end of the experiment (10 November). In 2015, the F2 plants were sown on 19 January and grown in a room controlled at 28/23 °C (day/night) with natural light for one month and then transferred to the same greenhouse until 30 August. The plants were trimmed as single stem by regularly removing lateral shoots, and fertilized weekly with liquid fertilizer Hyponex .The F2), the percentage of phenotypic variance explained (PVE) by each QTL, and the frequency distribution and association of quantitative phenotypes with FA and S alleles were analyzed following the methods described by Das et al. [The first to tenth inflorescences of each plant were harvested on maturation of the first fruit of that inflorescence. A ‘branch’ of inflorescence was defined as a stretch of inflorescence axis arising from a parent branch, having no daughter branches, and bearing more than two flowers. After the inflorescences were collected, the number of leaves preceding the first inflorescence (NLF), the days from sowing to the first flower anthesis (DA), the BN per inflorescence, flower number per inflorescence (FN), the length of the main inflorescence branch (MBL), the diameter of the main inflorescence branch at the basal position (MBD), sepal length (SL), and petal length (PL) were recorded. After taking these measurements, the inflorescence axis was dried at 80 °C for 24 h and the dry weight (DW) was determined. Owing to the skewed distribution of BN, it was log transformed before analysis. The broad-sense heritability  test when Levene’s statistic was not significant but analysis of variance (ANOVA) indicated a significant group effect. Additionally, the non-parametric Jonckheere-Terpstra test was performed using SPSS 16.0.0 .1, P2, F1, and F2 plants from the 2014 experiment, using the Plant Genomic DNA Kit  following the manufacturer’s instructions. For QTL-seq [2 plants from the 2014 experiment. DNA isolated from the two bulks and two parental lines were sent to BGI-Japan  for RAD sequencing (Genomic DNA was isolated from freshly harvested leaves (about 0.02 g) of the P QTL-seq , two DNAquencing .Solanum lycopersicum SL2.40) using BWA software [p < 0.05 level were obtained by QTL-seq analysis [Adaptor sequences, low-quality reads, and duplicate reads were trimmed and filtered prior to analysis. The short reads obtained from the two DNA-bulks were aligned against the tomato genome sequence  were designed using the ‘Pick Primers’ tool (http://www.ncbi.nlm.nih.gov) (2 generations of 2014 and 2015 were performed by high-resolution melting (HRM) method [We investigated the genomic DNA sequences of the candidate genes from the two parental lines. Primers for amplifying the nih.gov) . The DNA) method . HRM ana


We aimed to develop our previously presented mechanical device, the Testis Rigidity Tester (TRT), into an electronic system  by applying tactile imaging, which has been used successfully with other solid organs. A measuring device, located at the front end of the ETRT incorporates a tactile sensor comprising an array of microsensors. By application of a predetermined deformation of 2 mm, increased pressure alters linearly the resistance of each microsensor, producing changes of voltage. These signals were amplified, filtered, and digitized, and then processed by an electronic collector system, which presented them as a color-filled contour plot of the area of the testis coming into contact with the sensor. Testis models of different rigidity served for initial evaluation of ETRT; their evacuated central spaces contained different, increasing glue masses. An independent method of rigidity measurement, using an electric weight scale and a micrometer, showed that the more the glue injected, the greater the force needed for a 2-mm deformation. In a preliminary test, a single sensor connected to a multimeter showed similar force measurement for the same deformation in these phantoms. For each of the testis models compressed in the same manner, the ETRT system offered a map of pressures, represented by a color scale within the contour plot of the contact area with the sensor. ETRT found certain differences in rigidity between models that had escaped detection by a blind observer. ETRT is easy to use and provides a color-coded “insight“ of the testis internal structure. After experimental testing, it could be valuable in intraoperative evaluation of testes, so that the surgeon can decide about orchectomy or orcheopexy.


Meropenem is used to manage postneurosurgical meningitis, but its population pharmacokinetics (PPK) in plasma and cerebrospinal fluid (CSF) in this patient group are not well-known. Our aims were to (i) characterize meropenem PPK in plasma and CSF and (ii) recommend favorable dosing regimens in postneurosurgical meningitis patients. Eighty-two patients were enrolled to receive meropenem infusions of 2 g every 8 h (q8h), 1 g q8h, or 1 g q6h for at least 3 days. Serial blood and CSF samples were collected, and concentrations were determined and analyzed via population modeling. Probabilities of target attainment (PTA) were predicted via Monte Carlo simulations, using the target of unbound meropenem concentrations above the MICs for at least 40% of dosing intervals in plasma and at least of 50% or 100% of dosing intervals in CSF. A two-compartment model plus another CSF compartment best described the data. The central, intercentral/peripheral, and intercentral/CSF compartment clearances were 22.2 liters/h, 1.79 liters/h, and 0.01 liter/h, respectively. Distribution volumes of the central and peripheral compartments were 17.9 liters and 3.84 liters, respectively. The CSF compartment volume was fixed at 0.13 liter, with its clearance calculated by the observed drainage amount. The multiplier for the transfer from the central to the CSF compartment was 0.172. Simulation results show that the PTAs increase as infusion is prolonged and as the daily CSF drainage volume decreases. A 4-hour infusion of 2 g q8h with CSF drainage of less than 150 ml/day, which provides a PTA of >90% for MICs of ≤8 mg/liter in blood and of ≤0.5 mg/liter or 0.25 mg/liter in CSF, is recommended.  Acinetobacter baumannii, are emerging as important pathogens of postneurosurgical meningitis in recent years, though Staphylococcus aureus and coagulase-negative Staphylococcus are still more common  for the pathogen  as one of the empirical treatments for bacterial meningitis , 10. In pathogen , 12. Thepathogen –18; howeCSF drainage is a common operation in postneurosurgery patients to drain inflammatory mediator residual cells during the operation and to manage central nervous system (CNS) symptoms –26; howe–The aims of this study were (i) to develop a population PK model for meropenem after intravenous administration in postneurosurgical meningitis patients and (ii) to recommend clinical dosing regimen optimization for this patient group, including dose, dosing interval, infusion duration, and CSF drainage rate, via PK/PD evaluation.This was a single-center, parallel, open-label, prospective clinical study conducted in Huashan Hospital Affiliated to Fudan University. It was approved by the Hospital Institutional Review Board of Huashan Hospital before execution and has been registered at ClinicalTrials.gov under identifier NCT02506686.6/liter) or proven bacterial meningitis (if Gram-negative bacteria were identified from CSF culture); (iii) CSF samples could be taken; and (iv) at least 18 years of age. The exclusion criteria were as follows: (i) patients who did not receive at least 3 days of meropenem treatment or were hypersensitive to meropenem; (ii) patients who were receiving hemodialysis, had unstable vital signs, or had lumbar puncture contraindications and so were inappropriate for sample collection; (iii) patients with decompensated liver disease  or severe renal dysfunction (defined as creatinine clearance [CLCR] of ≤10 ml/min), status epilepticus, potential neurodegenerative diseases, and other meropenem contraindications, and (iv) pregnant (with a positive urine pregnancy test at screening) or lactating women.Patients were included if inclusion and exclusion criteria were all satisfied. The inclusion criteria were as follows: (i) signed informed consent form provided; (ii) postneurosurgery patients with clinical diagnosis of probable bacterial meningitis  and China GCP. One predose CSF sample was collected and sent to the clinical microbiology department of the hospital for pathogen culture, isolation, identification, and antibiotic susceptibility testing. Treatment regimens were adjusted according to susceptibility if necessary.The meropenem  used in this study was manufactured by Dainippon Sumitomo Pharma. Meropenem at 1 g and 2 g was dissolved in 100 ml and 250 ml 0.9% sodium chloride solution, respectively, for injection. The infusion rate was kept at 1 g/h with the actual infusion start and end times recorded. The meropenem dosing regimen was determined according to the patient's body temperature, severity of systemic infection, CSF cell count, and pathogen. The total daily dose of meropenem was 3 to 6 g divided into 3 or 4 intravenous infusions. In most cases, meropenem at 1 g every 8 h (q8h) was given to patients with a white blood cell count of <1,000 × 10Blood and CSF samples were collected simultaneously after the fourth dose of meropenem for concentration testing. To increase the sample coverage of the concentration-versus-time curve, patients were randomly assigned to two groups for sample collection at predefined time points: during intravenous infusion and 10 min, 2 h, and 4 h after the end of infusion in group 1 and immediately at the end of infusion, 1 h and 3 h after the end of infusion, and immediately before administration of the next dose in group 2. Blood samples were collected from a vein of the arm without intravenous infusion. CSF samples were collected through lumbar cistern drainage or external ventricular drainage.Plasma samples were separated by centrifugation and stored in duplicate along with CSF samples at −80 ± 10°C for future analysis. Validated high-pressure liquid chromatography (HPLC)-UV chromatographic separation methods were used to extract meropenem and detect its concentrations in plasma and CSF. Methodology details can be found in our previous publications , 28.CR), serum alanine aminotransferase (ALT), CSF white blood cell count, CSF red blood cell count, CSF absolute neutrophil count, CSF glucose, CSF protein, CSF chlorine, and concomitant medications . CLCR was estimated from the Cockcroft-Gault equation  with the G77 FORTRAN compiler. The first-order conditional estimation method with interaction (FOCE-I) was used throughout the model-building process. One specific CSF compartment was added in the models to describe the meropenem disposition process in CSF –32, whic–equation  using thThe bootstrap resampling method was used to evaluate the stability and robustness of the final PK model. Resampling with replacement generated 1,000 bootstrap data sets, and the final population PK model was fitted repeatedly to each of them. Ninety-five percent confidence intervals (CIs) for the final parameters were obtained from the bootstrap empirical posterior distribution. Visual predictive checks (VPCs) were performed by overlaying observed data points with 5th, 50th, and 95th percentile curves of 1,000 data sets simulated from the final model.The steady state of the concentration-time curve for meropenem on the basis of the model developed for the currently used dosage regimens  were obtained from 1,000 simulated virtual postneurosurgical meningitis patients with different CSF rate amounts per clinical practice. The CSF drainage rate was set at 0, 50 ml, 150 ml, and 250 ml per day.fT>MIC were calculated to approximate the probability of target attainment (PTA)  . Target A total of 82 patients  with postneurosurgery meningitis were enrolled in this study, and initially 42 of them were treated with the dosing regimen of 1 g q8h, 19 with the regimen of 1 g q8h, and 21 with the regimen of 2 g q8h, according to their baseline conditions. They had different original diseases, including brain injury, brain bleeding, brain glioma, astrocytoma, acoustic neurinoma, trigeminal neurinoma, neurocytoma, meningioma, thalamic cyst, hemangioblastoma, encephalic angioma, cholesteatoma, etc. The baseline characteristics of all enrolled patients are shown in K12 = Q1 × PC/V1, K21 = Q1/V2, and k20 = daily CSF drainage volume/24.A total of 315 plasma and 297 CSF meropenem concentration data points were included in developing the population PK model. The final model contained one central compartment, one peripheral compartment, and one CSF compartment, as shown in The estimated population pharmacokinetics (PPK) parameters are shown in T>MIC in CSF are less than 90% for MICs of >0.25 mg/liter and >0.5 mg/liter, respectively, so these regimens are not recommended. The PTAs of regimens of 2 g q8h are shown in T>MIC for MICs of ≤8 mg/liter. In CSF, it has a >90% probability of achieving of achieving 50% and 100% T>MIC for MICs of ≤0.5 mg/liter and ≤0.25 mg/liter, respectively, and has a >80% probability of achieving 50% and 100% T>MIC for MICs of ≤1 mg/liter and ≤0.5 mg/liter, respectively.The trends we saw from Monte Carlo simulation results are that the probability of target attainment (PTA) increases as the total daily dose increase, while for the same dose, the longer the infusion time, the higher the PTA. CSF drainage has no impact on PTAs in plasma, but the more that CSF drained every day, the lower the PTA was in CSF. Under all circumstances of regimens of 1 g q8h and 1 g q6h including no CSF drainage, the probabilities of achieving 50% and 100% Postneurosurgery bacterial meningitis remains a severe disease associated with high mortality rates. Although CSF culture is the “gold standard” for diagnosis of meningitis and is important to establish the susceptibility of the causative microorganism to rationalize treatment, it is time-consuming. Rapid initiation of effective empirical treatment is still crucial for patient outcomes –43. Ther–In plasma, the CSF drainage rate had no impact on probabilities of target attainment. This may be because the target was set at 40% and the amount of drug elimination from CSF drainage was limited compared to the total drug amount in plasma. In CSF, the CSF drainage rate had a significant impact on the drug amount in CSF, though the trade-off between postsurgery symptom management and maintaining drug concentrations in infection site should be carefully made.CL was not identified as a covariate. The variance of PK parameters was relatively high but still meaningful, and this might be because the patients had different baseline disease conditions, different surgical operations, and different demographic characteristics. Our approach can also be used to investigate and recommend favorable dosing regimens for other antibiotics to manage this indication.The final model is more than a classic population PK model because it introduced a CSF compartment. The fixed CSF compartment volume was a little lower than the reported physiological volume , which mfT>MIC  in two patients via brain microdialysis , this apA third debate is what the most appropriate CSF drainage rate is. Although 50 ml/day provides a higher PTA for bacterial killing, it might not be enough to manage the postsurgery syndromes in some patients. Therefore, less than 150 ml/day was recommended, and the clinicians could be reminded to drain as little CSF as possible as long as the syndromes could be well managed.Acinetobacter baumannii , Klebsiella pneumoniae , and Stenotrophomonas maltophilia . The 3 patients who were infected by meropenem-resistant bacteria  were withdrawn from the study and switched to the other treatment regimens, and the clinical outcomes were not recorded. It is worthy of mention that the individual fitting plots for the 8 subjects who had pathogens isolated from CSF imply that the final model predicted the concentrations in this patient population well also.Our study has some limitations. First, it was not designed to assess clinical outcome; therefore, no control was included. Second, only 8 out of 82 enrolled patients had a positive result from CSF bacterial culture, which indicated that the diagnosis criteria for probable meningitis need to be improved. The bacteria isolated included In summary, our current study of population PK and PK/PD in postneurosurgery meningitis patients found that 2 g of meropenem every 8 h, administered as a 4-hour infusion with a limited CSF drainage rate (less than 150 ml/day), may provide the highest possibility of target attainment. However, further large, well-defined clinical trials with this patient population are required to confirm these findings.