The capacity to entrain motor action to rhythmic auditory stimulation is highly developed in humans and extremely limited in our closest relatives. An important aspect of auditory-motor entrainment is that not all forms of rhythmic stimulation motivate movement to the same degree. This variation is captured by the concept of musical groove: high-groove music stimulates a strong desire for movement, whereas low-groove music does not. Here, we utilize this difference to investigate the neurophysiological basis of our capacity for auditory-motor entrainment. In a series of three experiments we examine pupillary responses to musical stimuli varying in groove. Our results show stronger pupil dilation in response to (1) high- vs. low-groove music, (2) high vs. low spectral content, and (3) syncopated vs. straight drum patterns. We additionally report evidence for consistent sex differences in music-induced pupillary responses, with males exhibiting larger differences between responses, but females exhibiting stronger responses overall. These results imply that the biological link between movement and auditory rhythms in our species is supported by the capacity of high-groove music to stimulate arousal in the central and peripheral nervous system, presumably via highly conserved noradrenergic mechanisms. The capacity to entrain motor action to rhythmic auditory stimulation is a fundamental characteristic of our species. Widely expressed and flexibly realized in the contexts of music and dance, it is observed across cultures, develops early in life and responds rapidly to training . It is aRecent work has emphasized the importance of key acoustic and metrical properties in determining the amount of groove associated with a particular piece of music . Among aAmong metrical properties, a key role for syncopation in determining groove has long been implicated by its association with high-groove genres such as funk, soul and hip-hop . SyncopaBuilding on this research, we sought to determine whether musical groove is associated with characteristic effects on the nervous system, as well as the extent to which such effects may be dependent on spectral content and syncopation. Neural effects were assessed using pupillometry to measure time-varying pupil diameter, an accessible indicator of arousal in the peripheral and central nervous system . In addiFinally, equal numbers of male and female participants were included in each experiment, allowing us to test for sex differences in physiological reactions to groove. A clear divide in the debate over the evolutionary origins of music is between theories based on social cohesion and theories based on courtship. Social cohesion theories rationalize our capacity to entrain motor action to rhythmic auditory stimulation by referencing the fact that musically inspired motor coordination between individuals tends to increase prosociality (reviewed in SD = 4.9), and amount of musical training was not significantly different between males and females . All subjects were naïve to the research questions. The experiments were approved by the University of Vienna Ethics Committee (Protocol #00063) and all subjects gave written informed consent in accordance with the Declaration of Helsinki.Thirty-two adults  participated in this study in exchange for monetary compensation. Individuals had taken weekly music and/or dance lessons for an average of 5.4 years  connected to an Apple Mac Mini computer (model 4.1) running OSX 10.11.6. A computer monitor was used to display a fixation cross during trials and a countdown during rest periods. Stimulus presentation and the pupillometric recording were controlled by custom Matlab code . The three pupillometry experiments were run in two blocks, with an opportunity for a short break in between. Block one consisted of Experiment 3 (drum patterns) and block two consisted of Experiment 1 (music) followed by Experiment 2 (filtered music). The decision to start with the relatively simple drum patterns and end with more dynamic musical stimuli was made to mitigate subject boredom and fatigue in later stages of the experimental session. Each block began with a calibration of the eye-tracking system followed by two practice trials using example stimuli. Trials lasted 50 s: 10 s of silence followed by 40 s of stimulus playback. During trials, subjects were instructed to minimize blinking and focus on a gray fixation cross presented on a black background at the center of the computer monitor. Between trials, a 40-s countdown was presented on the monitor and subjects were encouraged to rest their eyes in preparation for the next trial. After completing the pupillometry experiments, subjects took a brief computerized survey in which they heard each stimulus a second time and provided ratings of groove, arousal, familiarity and preference  were played so that they could adjust the volume to a comfortable level, which was then maintained for the rest of the session  from the “Portland Kit” instrument in Logic Pro X. These samples were arranged to create two-bar drum patterns in 4/4 time played at 100 BPM . The number of hits on each drum type was the same across patterns. Frequency content was manipulated by altering the pitch of the snare and kick drums . In the The diameter of the left pupil and the x-y position of the subject’s gaze on the computer monitor were collected continuously throughout each trial (sampling rate = 1000 Hz). This data was processed offline in Matlab to remove artifacts introduced by blinks and visual fixations away from the cross. During blinks, the eye-tracker loses the pupil and records an arbitrary value. These samples were identified and removed including a 50 ms buffer immediately before and after . FixatioU tests in Experiment 2) to compare distributions of individual subject means calculated over time periods in which pupil diameter was maximally differentiated between experimental conditions  was used to plot interactions  vs. MLowG = 102.7% (SD = 3.8%), W = 149, p = 0.0315. Comparison of individual subject means calculated over the remaining 25 s showed greater similarity between responses, MHighG = 100.4% (SD = 4.9%) vs. MLowG = 100.0% (SD = 5.7%), W = 304, p = 0.4545.The average pupillary response to the high- and low-groove music stimuli are shown in Figure χ2(1) = 456.6, p < 0.0001, with the response to high-groove music estimated to be 0.56% greater than the response to low-groove music on average over the entire 40 s of stimulation . The interaction between groove and listener sex was also a significant predictor, χ2(1) = 131.0, p < 0.0001 , the difference was ∼1.6x larger in males, βMale = 0.018 vs. βFemale = 0.011 (SEs = 0.00080). Finally, pupil size was predicted to decrease with time post stimulus onset at a rate of ∼0.2% per second, β = -0.00019, SE = 2.4 ×10-6, χ2(1) = 5599.1, p < 0.0001.The LME model analysis showed similar results. Groove was a significant predictor of pupil dilation, 1 Figure . Post hoMHighP= 102.8% (SD = 3.2%) vs. MLowP= 99.7% (SD = 3.5%), U = 205, p = 0.0275. As in Experiment 1, comparison of individual subject means calculated over the remaining 25 s showed greater similarity between responses, MHighP= 97.6% (SD = 7.2%) vs. MLowP= 95.2% (SD = 6.1%), U = 230, p = 0.2067.The average pupillary response to the high- and low-pass filtered versions of the high-groove music stimuli from Experiment 1 are shown in Figure β = 0.016 (SE = 0.023). However, because of the reduction in statistical power associated with the between-subjects design, smaller number of subjects per condition, and increased response variance in this experiment, the main effect of spectral content was only marginally significant in this model, χ2(1) = 2.89, p = 0.0890. To address this we recalculated the LME model on the first 15 s post stimulus onset. This resulted in a much higher level of significance for spectral content, β = 0.034 (SE = 0.016), χ2(1) = 7.37, p = 0.0066, suggesting that pupil dilation was truly differentiated during this early time window. Both versions of the LME model additionally included a significant effect of listener sex, with females exhibiting greater pupil dilation than males overall, β = 0.034 (SE = 0.023), χ2(1) = 7.6, p = 0.0058 , χ2(1) = 5.4, p = 0.0207 (15-s version). Despite the relationship between spectral content and listener sex resembling that observed between groove and sex in Experiment 1 , χ2(1) = 4158.3, p < 0.0001.The LME model analysis showed similar results. Pupil dilation was estimated to be 1.6% stronger in response to high-pass stimuli than in response to low-pass stimuli over the entire 40 s of stimulation, The average pupillary responses to the drum pattern stimuli are shown in Figure χ2(1) = 1279.4, p < 0.0001. A detailed exploration of this three-way interaction is presented in Supplementary Text β = -0.003 (SE = 0.0009), χ2(1) = 61.6, p < 0.0001. The interaction between metrical structure and sex was also a significant predictor, χ2(1) = 11.8, p < 0.001 , the difference was ∼2.5x larger in males than females, βMale= 0.0075 vs. βFemale= 0.0029 (SEs = 0.0009). Finally, in keeping with the results of Experiments 1 and 2, pupil diameter was predicted to decrease with time post stimulus onset at a rate of -0.23% per second, β = -0.00023 (SE = 3.0 × 10-6), χ2(1) = 5491.2, p < 0.0001.The results of the LME model analysis informed this absence of simple differences by uncovering a significant three-way interaction between metrical structure, spectral content and listener sex, χ2 = 1279.41 Figure . Post hoβ = 49 (SE = 8.2), χ2(1) = 76.4, p < 0.0001, as well as more arousing, β = 52.4 (SE = 8.1), χ2(1) = 100.7, p < 0.0001, and more familiar β = 22.0 (SE = 7.5), χ2(1) = 26.9, p < 0.0001. Parallel to the pupillary results, a significant interaction was found between groove and listener sex for arousal ratings, χ2(1) = 4.5, p = 0.0339, with post-hoc comparisons showing that males tended to perceive high-groove stimuli as more arousing than females on average, β = 25.4 (SE = 9.5), t(71.3) = 2.67, p = 0.0563. Thus, physiological and perceptual measures suggest that responses to high- and low-groove music are more differentiated in males compared to females.The results of the survey are shown in Figure β = 28.3 (SE = 21.2), χ2(1) = 3.2, p = 0.0730, and the low-pass stimuli tended to be rated as more familiar than the high-pass stimuli, β = 22.4 (SE = 18.5), χ2(1) = 3.5, p = 0.0619. Listener sex had a significant effect on arousal ratings, β = 15.1 (SE = 15.7), χ2(1) = 4.2, p = 0.0402, but the direction was opposite to that expected based on the pupillary results: males rated the stimuli as more arousing than females despite female exhibiting stronger physiological reactions.For the stimuli in Experiment 2 , arousal β = 37.3 (SE = 9.1), familiarity, β = 54.0 (SE = 12.9), and preference, β = 59.3 (SE = 9.6), χ2s(1) = 41.9, 21.1, 27.1, and 38.7 respectively, ps < 0.0001. Models including spectral content and its interactions showed an additional significant effect, with the low-drum patterns being preferred over high-drum patterns, β = 12.0 (SE = 11.5), χ2(1) = 4.5, p = 0.0329  do not account for these effects . Intriguingly, ratings of arousal were determined to depend on listener sex in parallel with pupil dilation. The difference in average ratings between the high- and low-groove stimuli was ∼1.5x larger for males than females , this experiment was designed to probe the relative effects of low- and high-frequency spectral content on physiological arousal. Contrary to our expectations, we found greater pupil dilation in response to high-pass stimuli than the low-pass stimuli over the first 15 s of stimulation, suggesting that high-frequency content plays a critical role in the stimulating effects of high-groove music. As in Experiment 1, this effect is unlikely to be explained by differences in the tempo or loudness of the stimuli, which were controlled between conditions. Also in parallel with Experiment 1, the amount of pupil dilation observed in Experiment 2 depended on the sex of the listener. The relationship between spectral content and sex closely resembled that between groove and sex in Experiment 1, with females exhibiting greater pupil dilation overall, but males exhibiting greater differential sensitivity  and thus sounded more generic. Intriguingly, ratings of arousal were again determined to depend on listener sex. However, unlike in Experiment 1, the direction was opposite to that observed in pupil dilation . In sum, the results of Experiment 2 demonstrate a critical role for high-frequency spectral content in the stimulating effects of high-groove music. Together with previous work this suggests a dual account of spectral content in groove, with low-frequency content inducing auditory-motor entrainment, and high-frequency content stimulating and maintaining physiological arousal. At the level of musical phenomenology, this accords with the fact that drum-kits and rhythm sections typically pair low frequency drums with higher frequency idiophones , the later often played at more rapid sub-beat intervals.In Experiment 3, we examined the effects of systematically manipulating metrical structure and spectral content in synthetic drum patterns. Modeling the results of this experiment resulted in a significant three-way interaction between these factors and listener sex , a pontine nucleus that constitutes the largest group of noradrenergic neurons in the central nervous system . Using eThe sex differences found in this study are of particular interest with respect to ongoing debates over the evolution of human musical behavior. Although female pupillary reactions were stronger overall, male pupillary reactions differed more as a function of groove, spectral content and metrical structure. Although physiological differences between males and female reactions to music have been documented in a handful of previous studies , to our Finally, in addition to the empirical findings reported here, this work establishes a physiological correlate of the link between music and movement in our species. Because pupil dilation is relatively easy to measure, this methodology can be extended across cultures, life stages and species in a straightforward manner to assess the extent to which the physiological correlates of groove may be a function of culture, development and phylogeny, providing further insight into the biological foundations of human musicality.The experiments reported here provide the first empirical demonstration that music strongly associated with movement (“high-groove” music) stimulates stronger pupil dilation than music weakly associated with movement (“low-groove” music). This result is consistent with the hypothesis that hearing groovy music makes us want to move in part by up-regulating noradrenergic arousal in the central and peripheral nervous system. At a more detailed level, our results show that high-frequency spectra are relatively more effective at stimulating pupil dilation than low-frequency spectra. This contrasts with the wide-spread notion that low-frequencies (“bass”) hold the key to groove, instead suggesting a dual account of spectra in musically inspired movement with complementary roles for low- and high-frequency content in inducing entrainment and stimulating arousal. We also showed that syncopated rhythms stimulate stronger pupil dilation than straight rhythms, providing physiological evidence for the stimulating effects of metrical manipulation in groove. Last but not least, we found consistent sex differences across all three experiments that, if confirmed and extended by further studies, may have important implications for debates concerning the evolution of music and the human capacity for auditory-motor entrainment.DB designed the research, programmed the task, analyzed the data, and wrote the manuscript. PGA collected and analyzed the data and wrote the manuscript. MH designed the research and edited the manuscript. WTF provided resources and supervision, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To the best of our knowledge, this is the first report of leiomyoma in supranumerary testis (SNT).•Most cases of polyorchidism are found incidentally during surgery for inguinal hernia, undescended testes, torsion or testicular tumor.•There is no consensus regarding the management of SNT. If the SNT is scrotal, most authors recommend conservative management.•If nonscrotal SNT is found incidentally during surgery, orchiectomy could be performed because of increased risk of malignancy.•Treatment of intratubular germ cell neoplasia includes surveillance, orchiectomy, or low-dose external radiation. Polyorchidism, defined as the presence of more than two testicles, is a rare congenital abnormality of the male genital tract. There is no consensus regarding the management of supranumerary testis (SNT) due to its rareness. To the best of our knowledge, this is the first report of leiomyoma in SNT.We report the case of a 41-year-old man with two right testes sharing a common vas deferens and separate epididymides. Orchiectomy of right testes was performed. Pathology examination found that the superior testis was the site of a leiomyoma and the lower tesits was the site of an intratubular germ cell neoplasia (IGCN). Orchiopexy and biopsy were later performed for the left testis. Histology was compatible with IGCN. We opted for follow-up to avoid risks of hypogonadism.Polyorchidism is usually asymptomatic and found incidentally during surgery for inguinal hernia, undescended testes as in our case, torsion, hydrocele or testicular tumor. If the SNT is scrotal, and there is no other indication for surgery, most authors recommend conservative management with regular ultrasound follow-up. If nonscrotal SNT is found incidentally during surgery, orchiectomy could be performed because of increased risk of malignancy. Treatment of IGCN includes surveillance, orchiectomy, or low-dose external radiation.Different factors come into account for polyorchidsm management: the drainage system, the fertile potential of the supernumerary gonad, and its localization. In cases of uncomplicated polyorchidism, a conservative treatment, with US or MRI follow-up seems to be a rational choice without surgical complications. Triorchidism is the most common variant. Two important classifications of polyorchidism are the embryological one of Leung and the vascular one of Bergholz. Usually asymptomatic, it can be revealed by an abdominal mass, a testicular torsion or an inguinal hernia. Its management is controversial depending on different factors. To the best of our knowledge, we report the first case of leiomyoma in supranumerary testis. We summarize also the classifications and management of polyorchidism. Our work has been reported in line with the SCARE criteria .2A 41-year-old man presented to our institute for bilateral inguinal swelling and infertility for 15 years. He stated that his scrotum was always empty since childhood. There was no other medical history.Physical examination found a palpable right testis in the right lower abdomen along with an empty scrotum. The left testis was not palpable. Ultrasonography (US) showed both undescended right and left testes respectively in the right and left inguinal canals. Both testes had heterogeneous echogenicity. Right testis measured 45 × 30 × 13 mm while left testis measured 24 × 20 × 10 mm.Due to malignancy risk we decided to do an orchiopexy with testicular biopsy one side at a time. Pre-operative serum tumor markers  were normal.He was operated upon through right inguinal incision. There was no hernia sac. Two structures, identified as two right testes, were sharing a common vas deferens and a double separate epididymides. The superior testis measured 80 × 50 × 35 mm and was highly dysmorphic and suspicious, while the lower testis measured 40 × 30 × 25 mm and seemed macroscopically normal. The superior testis drains into the epididymis of the lower testis. Orchiectomy for the two right testes was performed , Fig. 2.We planned for surgical exploration directed to the left-sided cryptorchidism by an inguinal approach. An eutrophic and macroscopically normal left testis was found. A complete separation between the epididymis and the testis with a mesentery between them was noted . Testicu3Polyorchidism is rare with around 200 cases reported in the literature to date [type I: supernumerary testis (SNT) without epididymis and vas deference.--type II: SNT shares common epididymis and vas deference with ipsilateral testis.-type III: SNT has its own epididymis but shares a common vas deferens with the ipsilateral testis.-type IV: complete duplication of testis, epididymis and vas.According to Leung, types II and III together represent more than 90% of the cases .type A. Undrained testes without connection to a draining vas deferens were coded as type B. Type A testes were further divided into the subgroups A1 (own epididymis and vas deferens), A2 (own epididymis but common vas deferens), A3 (common epididymis and vas deferens), A4 (own vas deferens but common epididymis) and AX (no further description of appendixes). Type B testes were divided into the subgroups B1 (own epididymis), B2  and BX (no further description of appendixes) [Bergholz and Wenke proposed an innovative classification. A testis being drained by an outflow path was coded as Most cases of polyorchidism are asymptomatic and are found incidentally during surgery for inguinal hernia (30%), undescended testes (15–30%) as in our case, torsion (13%), hydrocele (9%) or testicular tumor . In a seThere is no consensus regarding the management of SNT due to its rareness. If the SNT is scrotal, confirmed by ultrasound or magnetic resonance imaging (MRI), and there is no other indication for surgery, most authors recommend conservative management with regular ultrasound follow-up ,4,5. If Treatment of IGCN includes surveillance, orchiectomy, or low-dose external radiation (LDRT). For patients with unilateral IGCN and a normal contralateral testis, orchiectomy is the preferred treatment option. IGCN in solitary testis is more controversial, with some advocating surveillance with testicular self-examination and others recommending early LDRT .For our patient, we opted for conservative management with regular US and follow-up to avoid risks of hypogonadism.4The diagnosis of polyorchidism is often occasional.Different factors come into account when choosing their management: the drainage system, the fertile potential of the supernumerary gonad, and its localization. Surgery should only be considered appropriate if there is an associated pathology. Indeed, in cases of uncomplicated polyorchidism, a conservative treatment, with US or MRI follow-up seems to be a rational choice without surgical complications.NO financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work.No source of funding.Ibn El Jazzar Teaching Hospital ethic comitee, Kairouan, Tunisia.Written informed consent was obtained from the patient for publication of this case report and accompanying images.Boussaffa H; concept or design, data collection, data analysis or interpretation, writing the paper.Naouar S; concept or design, data collection, data analysis or interpretation, writing the paper.Ati N; data collection, data analysis or interpretation.Amri M; data collection.Ben Khelifa B; data collection.Salem B; data collection, data analysis or interpretation, writing the paper.El Kamel R; writing the paper.This is no research study.Boussaffa Hamza & Ati Nidhal.Not commissioned, externally peer reviewed.


E. coli and showed that the isolatedprotein had unacceptable biological activity in mice. In the present paper, we aimed to characterize the purified rh-G-CSF byanalytical tests and developed an in vivo model by computational modelling of G-CSF. Granulocyte colony-stimulating factor (G-CSF) has a wide variety of functions including stimulation of hematopoiesisand proliferation of granulocyte progenitor cells. Recombinant human G-CSF (rh-G-CSF) is used for treatment of neutropeniain patients receiving chemotherapy. The mature bloodstream neutrophils express G-CSF receptor (G-CSFR), presentinga significant and specific mechanism for circulating G-CSF clearance. Computational studies are essential bioinformaticsmethods used for characterization of proteins with regard to their physicochemical properties and 3D configuration, as wellas protein–ligand interactions for recombinant drugs. We formerly produced rh-G-CSF in  In this experimental study, we analyzed the purified G-CSF using the analytical experiments.Then, the crystalline structure was extracted from Protein Data Bank (PDB) and molecular dynamics (MD) simulation wasperformed using Gromacs 5.1 package under an Amber force field. The importance of amino acid contents of G-CSF, to bindthe respective receptor was also detected; moreover, the effect of dithiothreitol (DTT) used in G-CSF purification was studied. The results revealed that characteristics of the produced recombinant G-CSF were comparable with those ofthe standard G-CSF and the recombinant G-CSF with the residual amino acid was stable. Also, purification conditions(DTT and existence of extra cysteine) had a significant effect on the stability and functionality of the produced G-CSF.Experimental and in silico analyses provided good information regarding the function and characteristics of ourrecombinant G-CSF which could be useful for industrial researches. E. coli and affirmed in chemotherapy-induced neutropenia by the U.S Food and Drug Administration (FDA) in 1991 for clinical usage (The granulocyte colony stimulating factor (G-CSF) serves a directing role in the growth, differentiation, and motivation of neutrophils and their precursors in the immune system . Likewisal usage . G-CSF gene with 2 different intein sequences and attained target protein in two forms: G-CSF and G-CSF plus cysteine  to check the presence of these tags and examine their effect on G-CSF binding to G-CSFR. The receptor of hematopoietic cytokine G-CSF is a member of a family of proteins referred to as the "family of cytokine receptors" and characterized by the existence of a 200-residue ligand-binding module. Alternatively, it is required to assess the effect of purification process on theprotein stability and its receptor binding. To answer thisquestion, computational studies were used in the presentresearch. It should be mentioned that such study has notbeen conducted on G-CSF protein so far. The use ofcomputational tools for determination of a new protein structure represents the most actual alternative to theexperimental procedures. As relates to characterization of physicochemical and structural properties of a protein,there is no doubt that in silico methods can resolvecomplications made by purification techniques -15. Considering very different properties of commercially available rh-G-CSF proteins, we constructed a model of the isolated G-CSF and its G-CSFR. Previously, we produced G-CSF with intein tags and purified it using DTT and pH exchange. So, the present work was designed to identify functional and structural changes, especially with respect to the extra amino acid residue of G-CSF, and intein tags as well as the presence of DTT in protein solution, and finally compare the results obtained for natural and purified G-CSF. We studied G-CSF characteristics,created our models with respect to the improved dynamicmodelling of rh-G-CSF and studied the effect of DTT and the residual cysteine on G-CSF stability and function. 5'MscI and 3' XhoI (Two tandems of G-CSF sequences linked to inteins (CBD-Ssp DnaB and Mxe GryA-CBD) were described in a previous study. This fragment purchased from GENESCRIPT Company (USA) was acquired in the pUC57 vector located at  3' XhoI . In the  3' XhoI  9). . 5'MscI The cloning, expression and intein-mediated purification were performed in pET-22b (+) vector , 10, 16E. coli host cell proteins (HCP) were studied by an immunoenzymetric assay kit  and an ELISA reader . According to the manufacturer’s protocol, 25 µL of each sample was loaded into each well. Next, 100 µL of E. coli antibody conjugated to horseradish peroxidase  was pipetted into each well. Then, these wells were covered and incubated on a rotator at 400-600 rpm for 90 minutes at room temperature (24 ± 4°C). The content of each well was dumped into waste. The wells were blotted softly but firmly tapped over an absorbent paper to remove most of the residual liquid. The wells were filled generously to overflow with the diluted wash solution using a squirt bottle or pipetting in 350 µL. Washing was repeated. From the bottom outside of the microtiter wells, any fluid residue that could interfere in the reading step, was wiped off. Then, 100 µL of the 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was pipetted and incubation was performed at room temperature for 30 minutes. Finally, 100 µL of the stop solution was pipetted. Absorbance was read at 450/650 nm. The microplate reader drew one standard curve and automatically calculated the amount of HCP in the purified protein  and an ELISA reader . Carefully, 50 µL of the purified protein was dispensed into suitable tubes that were endotoxin-free. These tubes were kept in a 37°C water bath. Blank tubes plus four endotoxin standards were run in duplicate. The blank tubes contained 50 µL of the LAL reagent water instead of the sample. At the same time, to the reaction vessel, 50 µL of LAL was added. Then, 100 µL of the substrate solution was added to the tubes (prewarmed to 37 ± 1°C) after 10 minutes. After 16 minutes, 100 µL of the stop reagent was added and mixed well. Absorbance of reaction in each tube was read at 405/410 nm by the ELISA reader using distilled water to adjust the photometer to the zero absorbance. The ELISA reader drew one standard curve and automatically calculated the amount of endotoxin in the purified protein .Isoelectric focusing (IEF) was performed using a pH 3-10 IEF gel  , 18.ThePeptide map analysis is an important analytical techniquewidely used to verify protein primary structures. Themethod is capable of specifically detecting and quantifying structural alterations in the recombinant proteins, such as those derived from N-terminal blockage, oxidation, proteolysis, or amino acid substitutions. To identify thestructure and fundamental variations, RP-HPLC was used , 18. In MD simulation was performed by Gromacs 4.6.4 package using GROMOS96 (53a6) . In thisE. coli HCP were detected by the immunoenzymetric assay kit . The results indicated that HCP was less than 100 ppm/dose and thus, they were in the permissible range (data not shown). E. coli endotoxins was less than 2 IU/mg protein. The number of bacterial endotoxins was determined by the LAL kit . The results showed that the amount of IEF was performed using a pH=3-10 IEF gel. Filgrastim main bond in the standard and purified sample was stopped at pH=6.1; also, no band was more intense than the chief one in the electropherogram attained with the reference solution (PDgrastim) . To confirm the protein structure and identify modifications, peptide mapping and RP-HPLC were done . The chrThe crystal structure of proteins was attained from PDB. Visualization of the proteins model was done by Chimera (version 1.8). To determine the structural andfunctional differences between G-CSF and cysteine-G-CSF and to find their differences in binding GCSF-R, MD simulation studies were performed. Thesimulations were carried out for all of the mentionedstructures, as mentioned in the Materials and Methods section, under explicit solvent conditions. The trajectory results obtained from MD simulation were evaluated for RMSD and RMSF. The Figure 4A depicts the RMSD variations during the simulation of G-CSF protein in the presence and absence of DTT. As shown in Figure 4A, protein in the presence of DTT was more unstable and its RMSD was greater than that in the non-DTT state. Likewise, the RMSF graph showed that in the presence of DTT, the protein’s flexibility was increased in certain regions .As shown in the RMSF diagram, in the residues 1 to 40, as well as 65 to 80, the presence of DTT led to a dramatic increase. As shown by the RMSD and RMSF diagrams, the presence of DTT increased protein flexibility. Additionally, the stability of the G-CSF protein, when bound to GCSF-R, was monitored in the presence of DTT. For this reason, in this part, the RMSF diagram of the GCSF protein was analyzed in the complex mode with GCSFR in the presence of DTT .As shown in this diagram, DTT increased the protein’s flexibility in all its amino acids. This increasein flexibility could probably cause the GCSF- GCSFR complex to become unstable and reduce the bindingaffinity between the two proteins. After G-CSF purification, it was possible to keep the extra cysteine in the N-terminal of G-CSF. Hence, to study the effect of this amino acid on the protein stability and receptor binding, we performed the computational analysis. The Figure 4D shows the variations of the RMSD associated with the natural GCSF protein and the cysteine residue. As shown, the presence of cysteine residue in the protein significantly reduced the RMSD level and stabilized the protein over time. The graph shows the flexibility of the residues for anatural and atypical protein (containing cysteine). Asshown in the RMSF diagram, the total flexibility of the structure in the atypical state was reduced, as compared with the normal one, and this reduction was significant inthe early amino acid sequence of the protein. Analysis of the two above-mentioned graphs shows that the presenceof cysteine residues reduced the flexibility and stability of the G-CSF structure . E. coli with laboratoryand in silico methods. Formerly, we produced G-CSF linked to intein  and extra cysteine on stability and function of G-CSF. The results displayed that the purified G-CSF had characteristics similar to those of the standard, and DTT reduced G-CSF stability and increased its flexibility. Also,our studies showed that the residual cysteine increasedthe stability and reduced flexibility; therefor e , it mightnot affect receptor binding. The results of computational studies of this protein were shown to be consistent with those of laboratory studi e s and the biological activityof this protein. In this way, the presence or absence ofcysteine could not have any important effect on thebiological activity of protein. Comparison of GCS-F and cysteine-GCSF results revealed that both of them werestable and probably had similar functionality. It was evenimaginable that the modified protein had higher biological activity; w e previously o bserved that cystein e -GCSFbiological activity was higher than that of the standardG-CSF (rh-G-CSF is widely used as a useful hematopoietic growth factor. Therefore, production of rh-G-CSF seems to be required. Hence, it was decided to analyze rh-G-CSF that had already been produced in o intein . The into intein  cloned o intein . This wardG-CSF . The inform ation displayed here may be important to biophar maceutical companies to produce new recombinan t proteins using new techniques. Despite various ap plications of MD simulation in protein engineering, no similar research was done on this protein. Therefore, studying the structural dynamic properties of this prote in under different conditions provided a new opportunity to study the stability and function of G-CSF.


Management of cerebrospinal fluid (CSF) leak during minimally invasive lumbar tubular microdiscectomy poses challenges unique to the surgical approach. Primary repair can be limited via tubular retractor systems, and onlay graft and dural sealant are often the treatment of choice intraoperatively. Postoperative persistent CSF leak may lead to intracranial hypotension (IH) and positional headaches. Early epidural blood patch (EBP) efficacy in the treatment of spinal CSF leaks of both spontaneous and iatrogenic origin is well-established in numerous studies. However, there is no consensus on treatment of persistent IH symptoms for patients undergoing lumbar tubular microdiscectomy. We describe the clinical courses of two patients who were treated with early EBP for IH symptoms following CSF leak during tubular microdiscectomy. Both patients underwent intraoperative repair with onlay autologous tissue graft followed by dural sealant after discectomy was completed without evidence of pseudomeningocele, but they developed postoperative positional headaches and presumed IH. Both patients received an early EBP with an immediate and complete resolution of positional headaches sparing them reoperation and/or lumbar drainage. EBP should be considered as a first-line treatment to treat postoperative IH symptoms without pseudomeningocele after iatrogenic CSF leak during tubular microdiscectomy. Tubular microdiscectomy has become a widespread method of treating lumbar disc herniation due to its minimally invasive approach and favorable benefit and risk profile . A rare Case 1A 34-year-old female presented with intractable left leg radiculopathy. She reported moderate pain in her lower back with pain and numbness radiating down the left S1 distribution. She had no motor weakness or altered bowel or bladder function. Magnetic resonance imaging (MRI) demonstrated L5-S1 disc herniation compressing the S1 nerve root (Figure Case 2A 49-year-old male presented with intractable and progressive pain in the lower back and left leg. Additionally, he had weakness of left plantar flexion. MRI revealed a large disc herniation at L5-S1 with compression of the left S1 nerve root Figure . The patThe use of transmuscular tubular retractor systems represents a safe and efficacious surgical approach for the treatment of lumbar disc herniation ,2,7. CSFIn our cases, the CSF leaks were intraoperatively sealed with autologous tissue and a dural sealant. However, both patients reported significant spinal headaches postoperatively indicative of IH. Although the precise diagnosis of IH requires a brain or spinal MRI, orthostatic headache is a classical symptom of IH . If imagThere are several complications associated with an EBP. For instance, paresthesia, neck ache, facial nerve palsy, and lumbovertebral syndrome have been reported after the procedure ,12. AddiWe present our cases to outline a paradigm of using EBP as management for postoperative IH symptoms in patients with iatrogenic CSF leak associated with tubular lumbar microdiscectomy and without pseudomeningocele. EBP is a well-documented treatment for CSF leaks of both spontaneous and iatrogenic etiologies. In a study of 30 patients with spontaneous CSF leak, a complete cure was obtained in 77% of patients after application of EBP . In anotWe have demonstrated that the efficacy of EBP extends to the treatment of the postoperative sequelae of CSF leak associated with tubular microdiscectomy. In the absence of other complications such as pseudomeningocele, the application of EBP complements well the minimally invasive methodology of tubular spinal surgery, which may obviate the need for more aggressive treatments for CSF leak such as lumbar drain and exploratory reoperation. Patients may benefit from future studies examining the success rate of intraoperative EBP, which may enable patients to avoid re-exploration, and consequently an extended hospital stay and increased risk of infection.


LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) homologous genes have been reported to play key roles in promoting the initiation of floral meristems in raceme- and cyme-type plants. Asteraceae, a large family of plants with more than 23,000 species, has a unique head-like inflorescence termed capitulum. Here, we report a floral defective plant of the garden cosmos named green head (gh), which shows homogeneous inflorescence, indistinguishable inflorescence periphery and center, and the replacement of flower meristems by indeterminate inflorescence meristems, coupled with iterative production of bract-like organs and higher order of inflorescences. A comparison of the LFY- and UFO-like genes (CbFLY and CbUFO) isolated from both the wild-type and gh cosmos revealed that CbUFO may play an important role in inflorescence differentiation into different structures and promotion of flower initiation, and the reduced expression of CbUFO in the gh cosmos could be associated with the phenotypes of the flower-defective plants. Further expression analysis indicated that CbUFO may promote the conversion of inflorescence meristem into floral meristem in early ray flower formation, but does not play a role in its later growth period. Arabidopsis thaliana and Antirrhinum majus develop the raceme-type inflorescences, with main inflorescence axes growing indefinitely and bearing flowers in lateral positions or lateral axes that reiterate this pattern. Petunia and tomato develop the cyme-type inflorescences, in which the main inflorescence meristems terminate in a flower after producing a new inflorescence meristem that reiterates this pattern. Unlike the raceme- or cyme-type inflorescences, the panicle-type inflorescence is extensively branched and is largely characteristic of grasses, such as oat and rice [Flower initiation and development, two pivotal events during the life cycle of flowering plants, play a key role in the reproductive fitness of plants, as well as the successful production of crops. The branch bearing flowers of a plant are defined as the inflorescence, and the arrangement of flowers within the inflorescences is known as the inflorescence architecture ,2. Durinand rice ,7. Arabidopsis, an antagonistic interaction between TFL1 (TERMINAL FLOWER 1) gene and flower meristem genes, such as LFY (LEAFY) and AP1 (APETALA1), has been suggested to regulate the inflorescence architecture [TFL1 is specifically expressed in the center of inflorescence meristems to suppress the expression of LFY and AP1 and prevent the meristems from gaining the floral identity, while LFY and AP1 are strongly expressed in floral meristems to repress TFL1 expression in this area and promote the floral identity [LFY encodes a plant-specific transcription factor, with its orthologs present in most plant species as single-copy genes. Loss of function of LFY results in the conversion of flowers into inflorescence shoots, and the ap1 mutation enhances the lfy mutant phenotype, indicating that LFY and AP1 act synergistically to determine the floral meristem identity (FMI) [LFY and AP1 homologs, suggesting that the gene-regulatory networks controlling inflorescence and flower development are largely conserved among flowering plants. However, pfo  mutant of Lotus japonicas [an (anantha) mutant of tomato [UFO (UNUSUAL FLORAL ORGANS), aside from LFY or AP1 orthologs [Arabidopsis [ufo mutants display defects in petal and stamen development [pfo and an mutants, loss of function of the UFO ortholog in petunia DOT (DOUBLE TOP) leads to complete conversion of floral meristems into inflorescence meristems, indicating that DOT, as well as PFO and AN, is fully required to specify FMI [LFY/FLO and UFO/FIM homologue genes play a key role in promoting the initiation of floral meristems in raceme- and cyme-type plants, respectively.Extensive studies have been performed on the molecular regulation of inflorescence architecture and flower development for the three major inflorescence types. In the model plant itecture ,9. It haidentity . LFY encty (FMI) . In receaponicas  and an  and chrysanthemum (Chrysanthemum sp.) [Asteraceae, a large family of plants with more than 23,000 species, has a unique head-like inflorescence, termed as capitulum, to distinguish itself clearly from other plants. The capitulum is a pseudanthium or false flower that resembles a solitary flower, but is actually comprised of numerous flowers ,18,19. Tmum sp.) ,23. MostCosmos bipinnatus) is a popular ornamental plant in Asteraceae. The capitulum of cosmos is comprised of only one whorl of ray flowers at the periphery and about seven whorls of spiral-arrangement disc flowers. Recently, we have found that a floral defective plant of the garden cosmos displays a defect in flower meristem identity, resulting in reiterative initiation of inflorescences with bract-like organs rather than flowers in the capitulum. We named this plant green head (gh). Here, we report the morphological and ontogenetic characterization of the capitulum in the gh cosmos and compare the development process of inflorescence and flower development between the wild-type and flower-defective plants. Meanwhile, we tried to explore the potential cause for this mutation by cloning the LFY and UFO homologs and examining possible variations of their coding sequences and expression patterns in the gh cosmos. Our research confirms the role of LFY and UFO homologous genes in regulation of inflorescence and flower development previously reported in Asteraceae and enriches the information about the genetic regulation of flower development in Asteraceae. Cosmos  and consists of two morphologically and functionally different types of flowers: a whorl of large and showy ray florets formed at the capitulum periphery, usually numbering eight for the single-flowered type of varieties, and about seven whorls of smaller disc florets at the center A,B,D. Thgh cosmos cannot form normal flowers, including the ray florets and disc florets. In the outer ring of the inflorescence, two whorls of leathery bracts and one whorl of membranous bracts can be seen to develop. Next to the last series of involucral bracts, we can see the whole inflorescence is homogeneous, the periphery and center of the inflorescence becomes indistinguishable, and no ray florets can be seen. In other words, the whole inflorescence structure becomes monotonous with a tightly folded structure emerging at the place where the normal ray or disc florets should grow  and histological section analysis, the early inflorescence development of the wild-type cosmos can be divided into two different stages: the inflorescence primordia development stage and flower primordia differentiation stage. During the inflorescence primordia development stage, the whole inflorescence is approximately 0.2–1 mm in diameter, and little difference can be found between the wild-type cosmos and the uberance A,E. At auberance B,C. The uberance B,C. At tuberance B. The inuberance B,C, folluberance D,E. Whenuberance F. The rauberance . Compareapitulum H,I. Thesapitulum , followeapitulum G,H. Withild type . In the nt stage H. Then, nt stage E,F. ThisLFY and UFO were cloned and identified from a wild-type cosmos plant, which were named as CbLFY (FLORICAULA/LEAFY like gene in Cosmos bipinnatus) and CbUFO (UNUSUAL FLORAL ORGANS like gene in Cosmos bipinnatus), respectively. CbLFY had an open reading frame (ORF) sequence of 1254 bp and encoded a putative protein of 417 amino acids (Chrysanthemum x morifolium). It also contains some specific regions, such as a putative short Leu zipper, a basic region formed by a core of Arg and Lys residues, and an acidic region, which can also be found in other angiosperm FLO/LFY proteins (CbLFY represents the LFY ortholog in cosmos. CbUFO had an ORF sequence of 1308 bp and encoded a putative protein of 435 amino acids containing F-box (UNUSUAL FLORAL ORGANS like protein in Gerbera hybrid cultivar). Phylogenetic tree analysis indicated that the CbUFO protein is clustered together with HaUFO (Helianthus annuus) and GhUFO, the orthologs of UFO from species in Asteraceae  of CbLFY and CbUFO were further amplified from 15 lines of wild-type cosmos and 3 individuals of the gh plant. Three different putative amino-acid sequences of CbLFY  were obtained from the wild-type plants and one unique putative amino-acid sequence (named CbLFY-m) from the gh plants. A multiple alignment of these sequences revealed that the gene function might not be affected by the amino acid variation at the six sites in the wild-type CbLFY sequences -PCR . The results showed that the CbLFY expression was absent from roots, leaves, and stems, but was detectable in vegetative buds and strongly detectable in flower buds , forming inflorescence meristem; Period 2 (P2), forming flower meristem; and Period 3 (P3), forming flower organs. CbLFY was most highly expressed at the early stage (P1), followed by a gradual decrease at the later stages (P2 and P3); CbUFO, however, showed the highest expression level at the middle stage (P2) (The transcription levels of wer buds G. Statiswer buds H. Statisage (P2) C,D.CbLFY and CbUFO were further investigated by in situ hybridization during the three inflorescence development stages. At the P1 stage, the CbLFY transcripts were uniformly and highly accumulated in the whole dome-shaped inflorescence meristem, and the expression was also detected in the basal part of the involucral bracts , and the CbLFY expression was slightly lower in the gh cosmos than in the wild type, with a significant difference (p < 0.05) in two lines of gh and no significant difference in one line  from the wild type at P3 stage (CbUFO remained at a low level during the three periods (gh showed a significant difference (p < 0.01) from the wild type, especially at the P2 stage when flower meristem was being formed in the wild-type plants, but no significant difference was observed at P3 stage . Similar to the phenotypes of over-expressed LFY and UFO in Arabidopsis [CbLFY transgenic lines have different degrees of early flowering phenotypes and the lateral shoots and inflorescences all turned into flowers in the transgenic lines with a severe phenotype , implying CbUFO can also play a role in the early development of inflorescence. This is different from the gerbera research reporting that GhUFO was not expressed in the naked IM [CbUFO reached the highest level at the P2 stage when the flower meristem was forming, confirming that it may play a more important role in controlling the floral meristem identity as the master regulator of FMI in cosmos, with the same function in cymose species and gerbera [CbUFO expression was only detected in disc flowers, but not in developed ray flower, indicating that CbUFO gene may play a different role in regulating disc and ray flowers. A further look at the process for determining the fate of ray flowers showed that CbUFO is strongly expressed in the early stage of determining ray flower primordia, followed by a weak expression and finally the absence of CbUFO when the ray flower primordia are totally determined  might be caused by the decreased expression of UFO-like gene.Currently, we still cannot confirm whether the capitulum inflorescence in Asteraceae plants belongs to raceme or cyme inflorescence based only on the expression pattern of  markers , and thegh) plants of Cosmos bipinnatus used in this experiment were naturally collected from the field at the experimental station of the College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, China. The plants were grown under natural conditions using standard agricultural practices. The gh cosmos was obtained from the natural mutation of the WT cosmos. They did not grow closely together, but in the same area and the same environment. We collected the seeds of wild-type plants nearby, hoping to obtain gh plants from these seeds. We also preserved the mutant plants by asexual propagation, such as cutting and tissue culture.The wild-type (WT) and flower-defective . The outer bracts of the capitulum were removed to expose the central inflorescence meristem and the developing flower primordia when needed.Flower buds (0.5-4 mm) of the wild-type and Samples of young capitulum at different developmental stages (0.5-4 mm) for histological sections were collected and fixed in FAA  at room temperature for 2 days, followed by embedding in paraffin after treatment with different concentrations of alcohol and xylene. The paraffin-embedded samples were cut in 8 µm , then examined and photographed using the BX53 Microscope (OLYMPUS) equipped with a U-HSCBM (OLYMPUS) digital camera.gh cosmos were collected at early developmental stages and hand dissected under a stereomicroscope. When necessary, excessive involucral bracts were removed to expose the central inflorescence meristem and the developing flower primordia. The samples were first stored in 2.5% glutaraldehyde for 24 hours at 4 °C, followed by rinsing in 0.1 mol/L phosphate buffer (pH7.5) and dehydration by alcohol  in turn. Finally, the samples were dried at the critical point of Autosampler 810 and plated with gold in a Hummer V sputter coater. The treated samples were examined by SEM . The obtained SEM images were further edited using Adobe Photoshop CS5.For SEM analysis, a time series of the capitula (0.5-4 mm) of the wild type and Total RNA was extracted from the young capitula at different developmental stages using Total RNA Kit . First-strand cDNA was synthesized using cDNA synthesis . Genomic DNA was extracted from different developmental stages of the apical bud by the modified CTAB-based method .LFY and UFO homologs. The conserved fragments of CbLFY and CbUFO were amplified with cDNA synthesized from the young capitula mRNA, and the full-length cDNA sequences of CbLFY and CbUFO genes were obtained using 3′-RACE and TAIL-PCR [Gene-special primers were designed based on the conserved regions of TAIL-PCR . The prihttps://blast.ncbi.nlm.nih.gov/) and aligned using ClustalW program in MEGA 5.0 . The phylogenetic trees of the LFY-like and UFO-like proteins were constructed using MEGA 5.0 with the Neighbor-Joining (NJ) method with 1000 bootstrap replicates. The sequences of LFY-like and UFO-like proteins were retrieved from GenBank , capitula with a diameter of 0-1 mm that were forming inflorescence meristems; period 2 (P2), capitula with a diameter of 1-2 mm that were forming flower meristems and ectopic bracts for the wild type and gh plants, respectively; period 3 (P3), capitula with a diameter of 2-4 mm that were forming flower organs and secondary inflorescence meristems in wild-type and gh plants, respectively. RNA samples for the qPCR analysis were collected from three independent plants of the wild-type and gh , respectively. CbEF1α, an EF1α homolog gene in cosmos, which was cloned by RT-PCR and confirmed by sequence-blast with homologous genes in NCBI, was selected for data normalization and calculation of the relative mRNA levels. The real-time quantitative RT-PCR was performed with the SYBR Premix Ex Taq  and the ABI7500 Real-Time System . The reaction was carried out in a 10 μl RT-reaction mixture containing 5 μL 2× SYBR Green Master Mix (TAKARA), 0.2 μL forward and reverse primers (10 μmol/μL), and water to a final volume of 10 μL, under the following cycling conditions: an initial denaturing step of 10 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, and 34 s at 60 °C. All qPCR experiments were carried out in three biological replicates. All the data were analyzed using the 2CT−ΔΔ method [http://www.ncbi. nlm.nih.gov/tools/primer-blast/) and shown in Real-time quantitative RT-PCR was performed to measure the T method . All thegh cosmos were collected at early developmental stages for in situ expression analysis. Tissues were fixed by dehydration in an increasing gradient series of ethanol, clearing with xylene, and embedding in Paraffin [A time series of capitula of both the wild-type and Paraffin ,37. The CbLFY (307 bp) and CbUFO(300 bp) according to Braissant and Wahli, 1998 [Gene specific probes were synthesized for li, 1998 , using ali, 1998 . SectionCbLFY and CbUFO genes were amplified and cloned into pMD-18T vector, respectively . For genetic transformation, we used the modified pmv vector to create the overexpression constructs 35S:CbLFY and 35S:CbUFO. Agrobacterium tumefaciens GV3101 containing the respective expression vectors was used for Arabidopsis (Col-0) transformation via the floral dip method [v/v) sodium hypochlorite solution followed by 95% (v/v) ethanol. Murashige and Skoog (1/2MS) medium supplemented with 50 mg/L kanamycin sulfate  and 50 mg/L cefotaxime sodium salt  was used to screen transformants. Then, the plants that survived were transplanted into soil and grown under long-day conditions (16 h light/8 h dark) at 22 °C to obtain self-pollinated seeds. The transgenic plants of the T2 generation lines, which fitted a segregation ratio of 3:1, were chosen to record flowering time and floral phenotype. Seedlings were grown in a growth incubator at 22 °C under long day (LD) conditions . Flowering time and the number of rosette leaves were measured when the plants bore a 1 cm long inflorescence. Three transgenic lines of CbLFY and two transgenic lines of CbUFO were chosen for analysis of the expression by real-time quantitative RT-PCR. Arabidopsis EF1α gene was used as the reference gene to normalize small differences in template amounts. The expression analysis was carried out using 14-d-old seedlings.The full-length cDNA sequences of the p method . The traAll data analyses were performed using SPSS statistical software . Values were expressed as the mean ± standard deviation (SD) of three replications. Statistical significances of the differences between various groups were analyzed using the Fisher’s least significant difference (LSD) procedure. For comparison between two groups, Student’s t-test was used. A p-value of < 0.05 and <0.01 was considered significant and highly significant, respectively.gh); (ii) the gh capitulum produces more whorls of green involucral bracts and remains indeterminate for a longer time than the wild type; (iii) the whole inflorescence becomes homogeneous, the periphery and center of the inflorescence become indistinguishable, and its flower meristem is replaced by an indeterminate inflorescence meristem, leading to iterative production of bract-like organs and higher order of inflorescences. Given that CbLFY and CbUFO have the functions of conserved proteins as their counterparts in Arabidopsis, and the gh phenotype is not derived from the CDS mutation of the CbLFY or CbUFO genes, the gh phenotype could be associated with the reduced expression of CbUFO in the gh cosmos. Expression analysis of both wild-type and gh cosmos indicated that CbLFY was more involved in promoting the early inflorescence development, while CbUFO was more involved in inflorescence differentiation into different structures and promoting flower initiation. A further analysis of the expression of the CbUFO gene in early ray flower formation revealed that the CbUFO gene may play a different role in regulating disc and ray flowers. CbUFO is strongly expressed in the early stage of determining ray flower primordia, followed by a weak expression and finally the absence of its expression when the primordia of ray flowers are totally determined. The overall results indicated that CbUFO may promote the conversion of inflorescence meristem into floral meristem in ray flowers, but does not play a role in its later growth period.In this study, a floral defective plant in cosmos was identified to show three major characteristics in contrast to the wild type: (i) its capitulum cannot form normal florets, but produces a “green flower head” (named as


Standard dosing of meropenem (2 g t.i.d.) produces CSF concentrations of only 1–2 mg/L which is inferior to the clinical breakpoint for most Gram-negative bacteria. There is therefore concern that dosing must be increased in order to achieve therapeutic CSF concentrations for bacteria with susceptibility close to clinical breakpoints. Yet, the effects of high-dose meropenem on CSF concentrations are not well described in literature. We therefore determined meropenem CSF-levels in a patient who was treated with 15 g/day of meropenem.Acinetobacter baumannii  was isolated from blood cultures and CSF. The MIC for meropenem was > 32 mg/L (R), and we opted for a combination therapy of meropenem, colistin and fosfomycin. Meropenem was given at an unusual high-dose (15 g/day) with the aim of achieving high CSF concentrations. CSF concentrations peaked at 64 mg/L. Yet, the patient succumbed to an intracranial bleed into a preexisting cerebral contusion.Our patient suffered from a brain trauma and an external ventricular drainage was implanted. Later, a carbapenemase-producing High-dose meropenem can achieve CSF levels largely superior to those achieved with commonly recommended dosing regimens. Though our patient succumbed to an intracranial bleed which could be regarded as a severe adverse event, we suggest that meropenem dosing can be increased when pathogens with increased MICs are found in the CSF. More in vivo data are however needed to determine the safety of high-dose meropenem. Meropenem is a frequently used antimicrobial for post-neurosurgical meningitis/ventriculitis . It has Acinetobacter baumannii brain abscess where relevant CSF concentrations could be attained with very high-dose meropenem. To our knowledge, this is the first report on meropenem CSF concentration with such a high-dose regimen.The usual regimen of 2 g meropenem every 8 h results in CSF concentrations of 1–2 mg/L which is lower than the clinical breakpoint for susceptibility for most Gram-negative organisms . TherefoOur patient developed an intracranial abscess after placement of an external ventricular drain because of a severe brain trauma. CSF results are given in Table A. baumannii  was isolated from blood cultures and CSF. The MIC for meropenem was determined using a gradient test  and for colistin via commercial broth microdilution . European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied. The MIC for meropenem was > 32 mg/L (R) and for colistin ≤ 1 mg/L (S). Colistin, fosfomycin and meropenem were given because clinical data point to improved outcomes even in carbapenem-resistant A. baumannii infections using combination therapy [A carbapenemase-producing  therapy . Meropen2 initially but decreased to 23–40 mL/min/1.73 m2 after day 26, most probably due to the concomitant infusion of colistin. CSF meropenem concentrations varied between 31 and 38 mg/L when 10 g/day were given over a time period of 12 days. The increased dosage of 15 g/day for 3 days resulted in a peak CSF concentration of 64 mg/L (Fig. Since creatinine clearance has been found to be an important covariate of meropenem pharmacokinetics , we moniCommonly recommended meropenem doses for meningitis/ventriculitis are 2 g every 8 h . Yet, meA prolonged 4-h meropenem infusion of 2 g every 8 h leads to a probability of a target attainment rate of > 90% for most Gram-negative organisms in the blood compartment, when the MIC of a Gram-negative organism is ≤ 8 mg/L. Monte Carlo simulations suggested that, to reach this with the same dosing regimen in CSF, the MIC has to be as low as ≤ 0.5 mg/L . In a ph


Developing novel therapeutic agents to treat amyotrophic lateral sclerosis (ALS) has been difficult due to multifactorial pathophysiologic processes at work. Intrathecal drug administration shows promise due to close proximity of cerebrospinal fluid (CSF) to affected tissues. Development of effective intrathecal pharmaceuticals will rely on accurate models of how drugs are dispersed in the CSF. Therefore, a method to quantify these dynamics and a characterization of differences across disease states is needed.Complete intrathecal 3D CSF geometry and CSF flow velocities at six axial locations in the spinal canal were collected by T2-weighted and phase-contrast MRI, respectively. Scans were completed for eight people with ALS and ten healthy controls. Manual segmentation of the spinal subarachnoid space was performed and coupled with an interpolated model of CSF flow within the spinal canal. Geometric and hydrodynamic parameters were then generated at 1 mm slice intervals along the entire spine. Temporal analysis of the waveform spectral content and feature points was also completed.Comparison of ALS and control groups revealed a reduction in CSF flow magnitude and increased flow propagation velocities in the ALS cohort. Other differences in spectral harmonic content and geometric comparisons may support an overall decrease in intrathecal compliance in the ALS group. Notably, there was a high degree of variability between cases, with one ALS patient displaying nearly zero CSF flow along the entire spinal canal.While our sample size limits statistical confidence about the differences observed in this study, it was possible to measure and quantify inter-individual and cohort variability in a non-invasive manner. Our study also shows the potential for MRI based measurements of CSF geometry and flow to provide information about  the hydrodynamic environment of the spinal subarachnoid space. These dynamics may be studied further to understand the behavior of CSF solute transport in healthy and diseased states. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a devastating neurological disorder of predominately sporadic origin  that leaA current challenge in identifying treatments for ALS is finding reliable measures of efficacy. Historically, survivability is one of the main metrics used in this determination . ThroughTo be effective, delivery of IT therapies rely on transport within CSF, movement of the drug across the meninges, transport along the perivascular spaces and finally absorption into CNS tissue. The use of in vivo measurements along with computer models of CSF solute transport could help maximize drug dispersion and help avoid toxicity. Magnetic resonance imaging (MRI) is an effective tool for non-invasively measuring CSF flow. Several studies have already used phase contrast MRI (PCMRI) to measure and reconstruct CSF flow dynamics in silico –14.The goal of the present study was to characterize CSF flow dynamics and geometry in people with ALS compared to healthy controls. A few groups have made strides towards characterizing CSF dynamics in conditions such as Chiari malformation –21, SyriInclusion criteria for people with ALS were diagnosis of clinically suspected or definite ALS and able to tolerate the MRI scan without contraindications. Exclusion criteria included: presence of connective tissue disorder, previous history of cardiovascular disease, intracranial mass/deformity, CSF leak, spinal cord tethering, spina bifida, or myelomeningocele. Ultimately, eight participants with ALS were recruited from a regional adult population. The size of this population and restriction on travel compensation further limited recruitment of people with ALS in this pilot study. Ten healthy adult controls were also recruited based on the same exclusion criteria and the inclusion criterion of tolerance for the MRI scan without contraindications. Prior to each MRI scan, subject height, weight, waist circumference, heart rate, and blood pressure were recorded. MRI data collection was performed at Inland Imaging in Spokane, WA.MRI measurements were obtained on a Siemens 3T Skyra . Identical CSF flow measurements were taken for all subjects at six vertebral locations, Foramen Magnum (FM), C2–C3, C5–C6, T4–T5, T11–T12, and L3–L4 using PCMRI with retrospective gating from pulse oximetry for 30 cardiac phases  magnetic resonance (MR) images of the complete spinal subarachnoid space (SSS) anatomy was acquired for each subject Fig. d. These The segmentation of MRI data was performed using the open-source program, ITK-SNAP . The MR image set for each spinal segment was manually reconstructed from an axial view with the semi-automatic contrast-based segmentation tool Fig. c, as perAd, were used to obtain the cross-sectional area of the SSS, Pc, and dura, Pd, perimeter. A user-defined function was used to calculate each of these parameters in ANSYS FLUENT .Similar to our previous studies, computational meshing was used to calculate the following geometric parameters along the spine : First, Hydrodynamic environment was assessed at 1-mm slice intervals along the entire spine by Reynolds number based on the peak flow rate, and Womersley number based on hydraulic diameter. In calculating Reynolds number, echanics , 17, 26. the SSS . CSF pul studies , 26. In For flow rate and frequency analysis, it was necessary to perform normalization and alignment of the flow data. CSF flow rate at each of the six axial measurement locations were offset-corrected such that the net flow rate corresponded to 0 mL/s. Average flow rate was calculated for the C2–C3 location . BecauseDescriptive statistics were obtained for each parameter analyzed in terms of mean and standard deviation of values at each axial location for the ALS and control groups. Average values over the entire spine were also computed for each parameter along with the total value for parameters such as total spinal cord, dura and SSS volume. Statistical analysis was conducted in MATLAB R2016b. Feature points and the individual frequency components were statistically compared with a Mann–Whitney U test. Differences were considered significant at a p-value < 0.05.The ALS group was comprised of 7 males and 1 female with average age of 56 ± 10 years. The control group included 6 males and 4 females averaging 59 ± 12 years of age. The tabulated results for all parameters quantified in the ALS group and corresponding values in the control group may be found in Table 2, Fig. 2 and 1.78 cm2 respectively, Fig. 3) was 10% less than the control group (108.2 cm3).Visual inspection of the geometries showed a similar appearance in terms of shape between ALS patients and controls. One ALS subject 102) had a focal decrease in SSS cross-sectional area  and ALS (red) groups, excluding ALS case 102 in Fig. Average CSF PWV along the spine was 36% faster in the ALS group (473 cm/s) compared to the control group (347 cm/s) Table . The aveSpectral analysis revealed that the frequency components of the normalized flow rate for the ALS (red) and the control (blue) groups are significantly different (*) for the first, second, sixth and seventh harmonic of the T11–T12 location , and for the sixth harmonic of the L3–L4 location (p = 0.0330). The frequency components are presented from the first to the seventh harmonic in Fig. All hydrodynamic parameters of interest for both the ALS group, excluding subject 102, and the control group are shown in Fig. The present study quantified geometric parameters in the spinal canal of eight people with ALS and ten healthy controls. CSF hydrodynamics were also possible to quantify and compare in seven people with ALS and ten healthy controls. This section includes the findings we feel warrant further investigation as they may have an impact on drug delivery strategies and the monitoring of ALS progression.Timing of peak systolic CSF velocity at the FM was significantly faster for the ALS group p = 0.0136). Notably, one control subject had slower peak systolic CSF flow. However, because the flow amplitude was within the normal range, this subject was not considered an outlier as with ALS subject 102. Despite inclusion of this subject in the statistical analysis, timing at the FM was still found to be significantly faster in the ALS cohort. Overall, average CSF pulse wave velocity along the spine was elevated in the ALS group 473.19 cm/s) compared to the control group (347.41 cm/s)  to derive in vitro and computer-generated models for analyzing dispersion of compounds in the SSS and pulsatile flow is consistently indicated as one of the major contributors to CSF mixing , 61–64. Several limitations exist in our study. Findings for both groups were based on a relatively small sample. Rarity and variability of the disease combined with careful screening against the ability to undergo a protracted MRI-scan presented a distinct challenge in finding participants on a regional level. Secondly, our control group was not ideally matched against our ALS group and both groups included subjects with confounding conditions. While these factors negatively impacted the statistical power of the results in this study, it is straightforward to expand this data set in future with additional subjects. Furthermore, a longitudinal study would also allow comparison of any observations with disease progression.Lastly, while raw data was collected at a relatively high resolution, micro anatomy such as nerve roots, and denticulate ligaments were not possible to visualize. While important for specific transport dynamics, nerve roots did not have a large effect on unsteady CSF velocities as show in our previous computational study . SimilarThis study characterized CSF flow dynamics alongside geometric parameters in humans with ALS as well healthy controls. We found significant differences in peak systolic CSF flow timing at the FM, as well significant differences in the spectral content of CSF waveforms between ALS and control cohorts. More modest and non-significant differences in the CSF dynamics of our ALS group showed reduced CSF flow magnitude and increased PWV. While our study lacks sufficient power to draw definite conclusions regarding the differences we observed, we believe they deserve further investigation because of their potential importance related to intrathecal solute transport. In particular, a growing interest in IT drug delivery and the possible connection of trophic and neurotoxic factors in the CSF with disease progression warrant further study of CSF dynamics in the disease state. With the high degree of heterogeneity that exists among ALS cases it may be beneficial to conduct larger, longitudinal studies to determine how changes in CSF flow correlate with disease progression. This may contribute to the understanding of the pathologic progression of ALS, particularly if the onset of a neurotoxic CSF environment and breakdown of CSF flow were to coincide.


Background: Neonatal infection and sepsis are common for preterm infants due to their immature immune system. Early diagnosis is important for effective treatment, but few early markers of systemic and neuro-inflammatory responses in neonates are known. We hypothesised that systemic infection with Staphylococcus epidermidis (SE), a Gram-positive bacteria, induces acute changes to proteins in the plasma and cerebrospinal fluid (CSF), potentially affecting the immature brain of preterm neonates.Methods: Using preterm pigs as a model for preterm infants, plasma and CSF samples were collected up to 24 h after SE infection and investigated by untargeted mass spectrometry (MS)-based proteomics. Multiple differentially expressed proteins were further studied in vitro.Results: The clinical signs of sepsis and neuroinflammation in SE-infected piglets were associated with changes of multiple CSF and plasma proteins. Eight plasma proteins, including APOA4, haptoglobin, MBL1, vWF, LBP, and sCD14, were affected 6 h after infection. Acute phase reactants, including complement components, showed a time-dependent activation pattern after infection. Feeding bovine colostrum reduced the sepsis-related changes in clinical indices and plasma proteins. Neuroinflammation-related neuropeptide Y (NPY), IL-18, and MMP-14 showed distinct changes in the CSF and several brain regions  24 h after infection. These changes were verified in TLR2 agonist-challenged primary microglia cells, where exogenous NPY suppressed the inflammatory response.Conclusion: Systemic infection with SE induces inflammation with rapid proteome changes in the plasma and CSF in preterm newborn pigs. The observed early markers of sepsis and neuroinflammation in preterm pigs may serve as novel biomarkers for sepsis in preterm infants. Preterm infants are at a high risk of bacterial infection in the neonatal period and systemic infections, leading to sepsis, are important for the overall mortality and morbidity of these infants , 2. The Staphylococcus epidermidis (SE), a skin commensal bacteria, has been reported to be responsible for up to 50% of LOS cases , are often observed as causative pathogens in neonatal infection . In survOS cases . Unlike OS cases . In our ammation . Even wiammation . The sysammation , suggestin vitro cell culture systems.Prompt diagnosis of infection in preterm infants is difficult as the clinical manifestation is often variable, subtle, and further complicated by non-infectious conditions , resultin = 40) delivered from two sows (Danish Landrace × Large White × Duroc) via caesarean section at 91% gestation age (107 ± 1 d) were individually incubated with heating (37–38°C) and an oxygen supply. Each piglet was fitted with one vascular catheter  into the dorsal aorta via the umbilical cord and an orogastric tube . Pareteral nutrition and eternal nutrition for each piglet, if applicable, started immediately after the catheterisation. A group of piglets was intra-arterially inoculated with live Staphylococcus epidermidis (SE) re-suspended in sterile saline  over 3 min. Fourteen inoculated piglets received enteral feeding of bovine colostrum  via the orogastric tube and parenteral nutrition (3 mL/kg bw/h) via the umbilical catheter . The remaining inoculated pigs received parental nutrition  only. Piglets that were not inoculated and only received parenteral nutrition (6 mL/kg bw/h) served as controls . The parenteral nutrition was formulated, as previously described, based on Kabiven  and modified to meet the requirement of preterm pigs with added vitamins and minerals   was administered via the umbilical catheter 30 min before the scheduled euthanasia for assessment of blood–CSF barrier permeability. Under anaesthesia, piglets were euthanised with a lethal intracardial injection of sodium barbital . CSF was directly collected by sub-occipital puncture from each piglet and save at −80°C for proteomic analysis. CSF samples with possible blood contamination, examined by oxyhemoglobin absorbance (λ = 414 nm), were discarded. The number of CSF and plasma samples applied to the proteomic analysis are shown in oC for later analysis. Animals were treated in compliance with the Animal Experimentation Act of Denmark, which is in accordance with the Council of Europe Convention ETS 123. The study was approved by the Danish National Committee on Animal Experimentation (2014-15-0201-00418).The right hemisphere of the brain was fixed in ice-cold 4% paraformaldehyde for immunohistochemistry (IHC), and different brain parts, including the prefrontal cortex, paraventricular white matter (PVWM), and hippocampus, of the left hemisphere were dissected, snap-frozen, and saved at −80The preparation of protein samples was performed using a filter-aided protocol, as previously described . ProteinSus scrofa, UP000008227, last modified 2016-08-02) (http://www.proteomexchange.org/) with the data set identifier PXD016013.Protein annotation and quantification based on mass spectra of peptides were carried out using a MaxQuant (version 1.5.2.8)  against 6-08-02) . Raw dat6-08-02) , protein6-08-02)  and inte6-08-02)  for data2 for gene expression analysis or 96-well-plates at the density of 250,000 cells/cm2 for ELISA. After overnight rest and growing in serum-free medium for 8–12 h, microglial cells were treated, in duplicates, with either 2.5 μg/mL LTA-SA  or 20 ng/mL Pam3CSK4 (InVivoGen) alone or in the presence of 0.5 μM exogenous NPY peptide  for 24 h. The level of tumour necrosis factor-α (TNF-α) in the culture was determined in triplicates using porcine DuoSetELISA kits .The primary culture of microglial cells was prepared as previously described  with min® Permanox chamber slides (NUNC), allowed to grow for 24 h, fixed with 3.7% (v/v) formaldehyde and 1% (v/v) methanol in PBS, and stained with anti-Iba1 antibody  followed by secondary Alexa Fluor 488-conjugated antibody . Cell nuclei were counterstained by DAPI using mounting media (Molecular Probes). Images were recorded using an Axiovert 100 microscope  equipped with an AxioCam MRm camera (Zeiss) and ZEN 2012 software (Zeiss). The enriched microglia culture was 90% pure, as determined by the ratio of Iba-1-positive cells to total cells  in the brain tissue, brain sagittal sections (5 μm) were probed with primary antibody against NPY  and a biotin-conjugated secondary antibody  after standard deparaffinisation, rehydration, antigen retrieval, and blocking procedure. Staining was visualised with 0.04% 3,3′-diaminobenzidine  using an ABC Peroxidase Staining Kit (Thermo Scientific). For the quantification of NPY expression in brain, whole sections were scanned at a 10 × objective magnification on a Zeiss Axio Scan Z1 (Zeiss). The region of interest was selected in similar anatomical regions across samples, and the number of NPY-positive cells was counted in the defined frames. Representative images were acquired with a BX-51 microscope  using Integrator System software (Visiopharm).The concentration of IL-18 in CSF and plasma samples was measured with a porcine IL-18 ELISA kit . The frozen tissue of two brain regions, the prefrontal cortex and PVWM, was homogenised and lysed for protein extraction. Total protein concentration was determined by a Pierce™ BCA kit  for total protein adjustment later. The concentration of NPY in two brain regions was measured with a porcine Neuropeptide Y (NPY) ELISA kit , and the final concentration was adjusted to total protein concentration.HPRT1  on this model was adopted to compare the effect between three treatment groups, i.e., Control, SE, and SE+ENT, in a pairwise fashion  into q values within each comparison of each sample type (plasma or CSF) at each time-point using the multtest package , indicating sepsis and elevated systemic and intracranial immune response in the SE piglets. Blood–CSF barrier permeability was higher in SE piglets, as shown by the higher CSF-serum fluorescein ratio (p < 0.05) relative to the controls. The blood leukocyte number decreased after birth (p < 0.05) in Control, while SE infection maintained the leukocyte count over time (p = 0.91), although generally lower than in Controls. The blood leukocyte number in SE + ENT piglets tended to decrease over time (p = 0.08), similar to Control, but at a lower level. The platelet number increased over time in all three treatment groups, while the value in SE piglets was lower than that in Control and SE+ENT pigs . The values in SE+ENT pigs were similar to those in Control.As shown in our previous report, preterm piglets inoculated with live SE developed sepsis and blood–CSF barrier disruption, and enteral feeding ameliorated these damages . The cliP = 0.17) as tested by a linear model including factors such as treatment, sex, and litter. In CSF and plasma samples, 1,960 and 735 proteins were annotated (as shown in q ≤ 0.1 in at least one treatment comparison) were selected for functional assignment ,” “immune response,” “protein processing,” “metabolism,” and “others.” Observed acute phase reactants were categorised as a separate group. The protein groups may show overlap and are not mutually exclusive. Information on proteins with differential abundance, including UniProt ID, gene name, protein name, and abundance in each group, is shown in q < 0.1), 16 proteins were affected at 12 h, but only 5 proteins at 6 h.In the CSF, 38 differential proteins were related to immune response  and NPY 1R (p = 0.08) in the hippocampus increased after SE infection, but no effect of enteral feeding was observed. The protein level of NPY in two brain regions, the prefrontal cortex and PVWM, was significantly lower in SE piglets, which was consistent with the transcription and proteomic data (Neuropeptide Y (NPY) was only detected in the CSF samples and not in plasma samples, and it was detected at a lower level in the SE pigs relative to the Control . In the l cortex . Unlike mic data . Immunohmic data .NPY transcription  was only detected in the SE and SE + ENT groups, with the highest level being in SE pigs , while no significant difference was observed between SE+ENT and SE piglets . SimilarMultiple acute phase reactants were detected in the CSF and plasma after SE infection, including those related to opsonisation , SAA, coagulation factors , α-2-macroglobulin, microbial iron uptake-inhibiting haptoglobin, and complement components and various serpins including α-1-antitrypsin . These “p < 0.05, p < 0.001, Multiple immune response proteins, including Serpin A3-6, Serpin A3-8, MBL1, vWF, haptoglobin, LBP, and sCD14, increased in abundance as early as 6 h post-SE infection. Most proteins, including LBP, vWF, Serpin A3-6, and Serpin A3-8, maintained this change until 24 h  had their expression affected in various brain regions. Enteral feeding with bovine colostrum modified the proteome response in plasma within 24 h, while less CSF proteins were affected within this time frame. Collectively, these results suggest that plasma and CSF proteins may be a rich source of early biomarkers for sepsis and neuroinflammation in preterm neonates. Furthermore, they show that early enteral feeding may be important in dampening the systemic and brain inflammatory response to infection.Besides activating leukocytes, the systemic immune system may enlist proteins supporting immune response to enter the CNS. In the current study, 15 immune response-related proteins changed in abundance in both the CSF and plasma following SE infection, and most of them changed in the same direction. The majority of these proteins were related to immune defence, for example, KNG1 was involved in blood coagulation and platelet degranulation. Other proteins were involved in acute phase response, such as A2M, properdin, C1R, C5, CPN2, haptoglobin , serpins (inter-α-trypsin inhibitors), and SAA (immune cell recruitment). LBP and sCD14 (presepsin), involved in TLR4 activation, were also stimulated by SE infection , confirmNPY in three brain regions of SE-infected pigs  indicates that brain-derived NPY, at least in part, contributes to the altered NPY level in SE-infected CSF. On the other hand, circulating NPY may also have contributed to the NPY levels in the CSF, especially in the light of the elevated blood–CSF permeability post-SE infection. Unlike that in the prefrontal cortex and PVWM, the hippocampal expression of NPY was elevated in the SE group. Besides, elevated transcription of NPY 1R, one key receptor of NPY, was only found in the hippocampus of SE piglets, supporting other studies  . Being aAmong the early response proteins in plasma, eight showed differential abundance as early as 6 h post-infection, and these included haptoglobin, Serpin A3-6, Serpin A3-8, vWF, LBP, sCD14, MBL1, and APOA4 . HaptoglStreptococcus pneumonia  to SE and enteral feeding. Due to the extremely high technical demand of this pig model, a non-inoculated but colostrum-fed group was skipped. It remains to be investigated whether the effect of colostrum feeding observed in this study is specific to the highly bioactive bovine colostrum or if it is a general effect of enteral feeding, independent of the diet type. Cerebral and systemic responses may also vary according to gestational age at birth, postnatal age, type of pathogen, and infection intensity. Clearly, both pre-clinical and clinical studies are required to clarify the several variables that affect the pathological response to systemic bacterial infection and sepsis in preterm neonates. Combined with proteomic analyses of plasma and CSF, our animal model has shown a new path to investigate both mechanisms and clinically relevant markers of sepsis in preterm neonates.S. epidermidis infection induced rapid changes in inflammation-related CSF and the plasma proteome, and early enteral feeding dampened the SE-induced changes in the plasma proteome, but with limited effect in the CSF. The affected plasma proteins may serve as new early biomarkers of Gram-positive systemic infection in newborns, which may also affect central aspects of neuroinflammation, such as microglia activation and blood–CSF barrier disruption.Our findings in this study may reflect how systemic infection by Gram-positive bacteria soon after birth may affect brain development and neuroinflammation in preterm infants. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD016013.All experimental animal protocols were performed in accordance with the Danish Animal Welfare Act and this study was approved by the Danish Committee for Animal Research.TM conceived the study, analyzed the proteomic data, performed qPCR and ELISA experiments, and prepared the manuscript. AS performed the proteomics analysis and protein annotation. SP performed the primary cell culture. AB and DN performed the animal experiment, sample collection, and assisted in interpreting the data. PS conceived the study, interpreted the data, and prepared the manuscript. P-PJ conceived the study, analyzed and interpreted the data, and prepared the manuscript. All authors drafted the work, revised it critically for important intellectual content, and approved the final version.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.