Purpose: Epilepsy is a highly disabling neurological disorder. Brain insult is the most critical cause of epilepsy in adults. This study aimed to find reliable and efficient biomarkers for predicting secondary epilepsy.Materials and methods: The LiCl-pilocarpine (LiCl-Pilo) chronic epilepsy rat model was used, and rat cerebrospinal fluid (CSF) was collected 5 days after status epilepticus (SE). The CSF was analyzed using the label-free LC-ESI-Q-TOF-MS/MS. Differential expression of proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The corresponding protein level in the CSF of patients with encephalitis in the postacute phase was determined using ELISA and compared between patients with and without symptomatic epilepsy after encephalitis during a 2-year follow-up.Results: The proteomics and ELISA results showed that the protein level of kininogen (KNG) was obviously elevated in both CSF and hippocampus, but not in serum, 5 days after the onset of SE in LiCl-Pilo chronic epilepsy model rats. In patients with encephalitis, the protein level of KNG in the CSF in the postacute phase was significantly elevated in patients with a recurrent epileptic seizure during a 2-year follow-up than in patients without a recurrent seizure.Conclusion: KNG in the CSF may serve as a potential biomarker for predicting epileptogenesis in patients with encephalitis. Epilepsy is a highly disabling neurological disorder characterized by recurrent epileptic attacks . RepeateThe expression levels of certain miRNAs  and a seCompared with peripheral blood, the cerebrospinal fluid (CSF) better reflects pathological changes in the brain. Lumber puncture has long been proved to be a safe and essential examination in many neurological disorders, especially in inflammation and infectious brain diseases, which are also common causes for symptomatic epilepsy. In patients with traumatic brain injury, the levels of C-tau in the CSF were used to predict the long-term mortality, motility, and cognitive function after traumatic brain injury –14. Yet,In the present study, the proteomic technique was used to analyze changes in protein expression levels in the CSF 5 days after the onset of status epilepticus (SE) in the LiCl-pilocarpine (LiCl-Pilo) chronic rat epilepsy model. After evaluating the occurrence of spontaneous recurrent seizures (SRS) by video monitoring during 6 weeks after the onset of SE, the differently expressed proteins in the CSF between naïve control and SRS-detected LiCl-Pilo rats were screened. Validation by enzyme-linked immunosorbent assay (ELISA) and Western blotting indicated that the expression level of kininogen (KNG) was significantly upregulated in the CSF of rats with SRS. Further, the CSF was collected from a group of patients with encephalitis in the postacute phase, and a 2-year follow-up was conducted to verify whether the patients developed SRS. This study demonstrated that patients with secondary epilepsy showed a significantly higher KNG level in the postacute phase compared with the levels in patients with encephalitis who did not develop SRS and in control patients. Taken together, the results suggested that the KNG level in the CSF might serve as a potential biomarker of early epileptogenesis.ad libitum and maintained under a reversed 12-h light/dark cycle (light on at noon). Particular efforts were made to minimize the number of animals used and the potential for animal suffering.A total of 84 male adult Sprague–Dawley (SD) rats were bought from Shanghai Laboratory Animal Center. They were housed in a controlled laboratory environment with regular animal chow and water The rats were housed for 1 week prior to the study to minimize the potential stress of human interaction. All groups of rats were of similar weight (200 ± 10 g) and age. The rats in the LiCl-Pilo group were injected intraperitoneally with LiCl  18 h before seizure induction. Atropine sulfate  was injected intraperitoneally 30 min before seizure induction. Finally, a single dose of pilocarpine  was administered to induce seizures. Rat behavior was scored according to the Racine scale  and dried by Savant Speedvac. Finally, sample was reconstituted to 40 μl (2% acetonitrile + 0.1% formic acid + H2O) and 20 μl was analyzed by Nano LC-MS/MS.Each sample of 15 μl CSF was diluted using 55 μl 100 mM NH4HCO3 and 10 μl 100 mM DTT at 60°C for 1 h. After incubation, 10 μl of 450 mM (83.23 mg/ul) was added for carboxymethylation, and the sample was allowed to incubated for 30 min in dark. Protein digestions were conducted over with trypsin (100 ng/μl) in a 1:20 trypsin-to-protein mass-ratio. Digestion was performed overnight at 37°C and further incubated at 56°C for another 20 min in the next day morning. The tryptic peptides were dried using Savant SpeedVac (Thermo Scientific) and resolved in 20 μl 0.1% formic acid. Sample was then desalted and purified individually by 10 μl ZipTip pipette tip system (Millipore). Protein attached to the resin in Ziptip was dissolved in 20 μl eluting buffer  coupled with an ESI-Q-TOF mass spectrometer . Each peptide sample was separated using a solvent system with solvent A consisting of 99.9% water and 0.1% formic acid, and solvent B consisting of 99.9% acetonitrile and 0.1% formic acid. The peptides were eluted with gradients 2–20% B in 75 min, 20–80% B in 15 min, 80% hold for 15 min, 2% hold for 15 min with a constant flow rate of 350 nl/min. The LC setup was coupled online to a Q-TOF using a nano-ESI source  in data dependent acquisition mode . The Source Capillary was set at 2400 v. the flow and temperature of dry gas was 2.0 1/min and 120°C respectively. The mass spectrometer was set as one full MS scan followed by 10 MS/MS scans on the 10 most intense ions from the MS spectrum.Tandem mass spectra were processed using PEAKS Studio version 7.5 (Bioinfor Inc. CA). PEAKS DB was set up to search the uniprot_2016_02 Rat database (29982 entries) assuming the digestion enzyme Trypsin. PEAKS DB was searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 15.0 PPM. Carbamidomethyl of cysteine was specified as a fixed modification. Deamidated of asparagine and glutamine and oxidation of methionine were specified in Mascot as variable modifications. Peptides were filter by FDR 1%. PEAKS Q was used for peptide and protein abundance calculation. Normalization was performed on total ion chromatogram. Medians were used for averaging. Different expressed proteins were filtered if their fold change were over 1.5 fold and PEAKS $$significance−10logP-value over 15.The bilateral hippocampus and serum were collected from another parallel group of rats to compare the protein levels in hippocampus, and serum. Five days after model establishment, the rats were quickly decapitated under deep isoflurane anesthesia. The peripheral blood was collected from neck vessels and centrifuged . The serum was finally aliquoted and frozen (–) for subsequent analysis. The intact brain was promptly removed and placed in ice. The bilateral hippocampus was separated and stored at −80°C for further experiments.The KNG protein level in the CSF and serum was tested using commercial ELISA kits, Rat Kininogen ELISA Kit (ab157742) and Human Kininogen ELISA Kit (ab108876) . Sample analysis was conducted according to the instruction provided. Samples were diluted using a 1 × diluent solution. Dilution ratio in the rat sample was 1:20 (CSF) and 1:10000 (plasma). Dilution ratio in the human sample was 1:100 (CSF) and 1:20000 (plasma).The hippocampal tissues were homogenized on ice in RIPA buffer  using the auto TissueLyser-24  (2 × 30 s). Complete Protease Inhibitor Cocktail  was added into the RIPA buffer. Tissue homogenates were centrifuged , and the supernatants were finally aliquoted and frozen (–) for subsequent analysis.Using the bicinchoninic acid protein assay kit , the concentration of all protein extracts was measured. Samples, each containing 30 μg of total protein, were loaded and separated by electrophoresis with 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then electrotransferred to polyvinylidene difluoride membranes . The membranes were then blocked in 5% bovine serum albumin (BSA) or 5% non-fat milk. BSA or non-fat milk was dissolved in 1 × TBST . After blocking, the membranes were incubated with primary antibody overnight at 4°C. Antibody dilutions were 1:1000 for KNG  and 1:5000 for β-actin . After incubation, the membranes were washed in 1 × TBST (3 × 10 min) with primary antibodies and then incubated with horseradish peroxidase–conjugated goat antirabbit immunoglobulin G  for 2 h at room temperature. The secondary antibody was diluted in 5% non-fat milk. The membranes were then rewashed with 1 × TBST (3 × 10 min). Finally, the protein bands were detected using an enhanced chemiluminescent reagent, and ImageJ software  was used to quantify the intensity of each band. Relative protein levels were determined using the ratio of the band intensity of the target protein to that of its respective β-actin loading control.Twelve patients with encephalitis in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine were enrolled. Five patients with functional disorders served as controls. Written consents for participation in the study were obtained from patients. The following inclusion criteria were applied according to the previous studies , 20: (1)As CSF tests are essential in encephalitis diagnosis, the collection of CSF is easy and harmless to patients. The CSF of last lumbar puncture before discharge of patients was collected. At that time, encephalitis was well-controlled after proper therapy. Routine tests of CSF, including cell number, protein level, and glucose, recovered to normal, and the patient was ready for discharge. The acute phase of encephalitis was over, yet spontaneous recurrent symptomatic epilepsy did not appear in most patients. This timing, “the postacute phase,” was approximate to what was defined as epileptogenesis period in a chronic epilepsy animal model . The CSFPatients were followed up for 2 years. Symptomatic epilepsy secondary to encephalitis was diagnosed if unprovoked epileptic seizure appeared during follow-up, regardless of seizure occurrence during acute phase.t-test or one-way analysis of variance (ANOVA) with a significance level of P < 0.05.Data were expressed as means ± standard error of the mean (SEM). Statistical analysis was conducted using the Among the 53 rats in the LiCl-Pilo group, 28 met the standards defined by SE. Five rats died and 23 survived 5 days after SE. After CSF collection, nine (9/23) rats died. One rat was excluded because of blood contamination of CSF. The numbers of rats in all three groups enrolled in CSF collection are shown in The 13 rats in LiCl-Pilo group were video monitored 6 weeks after SE for 3–6 days. A total of 66 attacks of Racine 2 or more serious seizures were observed. The average seizure frequency was 1.25 ± 1.14 per day. The numbers of epileptic attacks (Racine 2 or severer) during video monitoring in each rat are displayed in n = 12), 1.02 ± 0.07 mg/mL , and 0.88 ± 0.06 mg/mL  . Subsequent proteomic analysis detected 146 proteins; 6 of them were differently expressed  , KNG is reported –26, thisn = 10, 2.61 ± 0.41 μg/ml) than in the LiCl   and naïve groups    . Similarn = 4, 4.42 ± 0.84 fold) than in the LiCl   and naïve groups (n = 4)  . The increased CSF level of KNG was concomitant with enhanced expression in the hippocampus but not in peripheral blood, indicating that this upregulation was due to an increased synthesis in brain tissue rather than peripheral synthesis.The level of KNG in the hippocampus was examined using Western blotting and in peripheral blood using ELISA technique to explore the source of KNG in CSF in another group of rats. In the hippocampus, the level of KNG was significantly higher in the LiCl-Pilo group (y ANOVA) . In contA total of 12 patients with encephalitis were further enrolled from December 2013 to April 2014 to verify whether the increase in the KNG level in the CSF was related to epileptogenesis in humans. The clinical manifestations and laboratory examinations of encephalitis patients are shown in n = 12) was significantly higher than that in controls  . However, the level of KNG in serum of encephalitis patients  was not significantly different than that in controls   (As shown in  t-test) .p = 0.057) than in controls , but both failed to showed significance , though higher than in controls, indicating that the increased CSF KNG level in early epileptogensis period did not depend on the occurrence of seizures during acute phase. On the contrary, the serum KNG level in all three groups was not significantly different .Of the 12 patients with encephalitis, seven had symptomatic seizures during acute phase. Thus, the next question was whether the occurrence of acute phase symptomatic seizure had an impact on the increased CSF KNG level in the postacute phase. As shown in n = 5, 2.39 ± 0.29 μg/ml) than in patients without secondary epilepsy   or controls  . On the contrary, the KNG level in serum was not significantly different among the three groups , influencing the choice of proteomics methods. Various methods of proteomic analysis are used to analyze rodent CSF, including isobaric tags for relative and absolute quantitation (iTRAQ), a method widely used owing to its high sensitivity and relatively satisfactory reproducibility. However, an iTRAQ test usually requires at least 50 μL of sample with 5 mg/mL protein concentration, and can only run 8–12 samples in one test. On the contrary, the label-free proteomic analysis uses spectral counting, which is a frequency measurement that uses tandem mass spectrometry (MS/MS) counts of identified peptides as the metric to enable protein quantitation, hence requiring much less sample volume, lower protein concentration, and no additional sample preparation. Using label-free proteomics method, changes in protein expression were analyzed in the CSF of each rat individually in this study.The timing of CSF collection is crucial. In a LiCl-Pilo chronic epilepsy rat model, the pilocarpine-induced SE works as a severe brain insult, causing recurrent seizures 7 days on average after the onset of SE. The earliest, first unprovoked seizure reported is 5 or 6 days after the SE –31. TherNo strictly defined epileptogenesis period exists for patients with encephalitis with concomitant secondary epilepsy. The period between inflammation process and first unprovoked seizure can be regarded as epileptogenesis period. In this study, we collected the CSF right after inflammation of encephalitis had been controlled. In the acute phase, seizures can be displayed as one of the symptoms in some patients with encephalitis. However, whether it should be regarded as secondary epilepsy is difficult to ascertain. International League Against Epilepsy (ILAE) once defined unprovoked seizure in epidemiologic studies as one occurred >7 days after acute CNS infection . In thisThe CSF contains more than 60% of albumin and several other high-abundance proteins , 34. TheIn order to screen potential biomarkers for early epileptogenesis, the ideal design would include control rats that experienced SE but failed to develop SRS. However, in our study almost every rat displayed SRS 6 weeks after the onset of SE except only one rat. Besides, the fact that the rat (rat #4) failed to show seizure in 4 days monitoring is not sufficient to decide that the rat had no SRS after SE, because 4 days is too short to establish a “no SRS after SE” group. In this study we video monitored rats' activity continuously and watched the video very carefully and completely. During the chronic period, we monitored each rat for 3–6 days at ~6 weeks after SE. We reviewed over 1,000 h of videos to detect spontaneous seizures. Due to the limitations of the technology and workload, it was not practical to monitor the rats continuously for the 6-week period between the initial induction of SE and the chronic phase of SRS.To fill this gap, we enrolled encephalitis patients with and without unprovoked seizure during 2 years follow-up. Though the sample size was small, it further confirmed the results from our animal study. We will enroll more patients in future study to re-confirm the results.The relationship of kininogen to epileptogenesis has not been explored previously. Although we did not examine whether early stage elevation of kininogen in CSF contributes to the development of spontaneous recurrent seizures, the involvement of bradykinin receptors, including B1 and B2 receptors, are well-established , 35–39. Another important question was about the source of CSF KNG elevation. The upregulation of KNG levels in the CSF but not in the peripheral blood during epileptogenesis, both in LiCl-Pilo model rats and in patients with encephalitis with concomitant secondary epilepsy, together with the increase in the hippocampal KNG level in LiCl-Pilo rats, suggested that the upregulation of KNG level in the CSF may resulted from increased intra-cerebral synthesis.A previous study found that during the process of inflammation induced by lipopolysaccharide (LPS), the expression of KNG increased in multiple parts of the brain, including cerebellum, brainstem, hypothalamus, and, most prominently, in choroid plexus . AnotherWe showed in this study that injection of LiCl alone also results in upregulation of KNG when compared to naïve control animals. Lithium has been reported to cause pronounced peripheral pro-inflammatory changes, characterized by an increased percentage of CD3–/CD11+ cells, decreased CD4:CD8 ratio and a sudden increase of IL-1β serum levels . These cThere is also concern that KNG may leak from serum to CSF in LiCl-Plicarpine rats, because BBB disruption is a common pathological change both in encephalitis and in epilepsy rat models . Thus, fKinins have been reported to be involved in many brain disorders related to secondary epilepsy, including cerebral vascular diseases and neurodegenerative diseases , implyinJZ was involved in study design, data acquisition, data analysis and interpretation, and drafting of the manuscript. XW and LH were responsible for patient enrollment and follow-up. JL and YK helped JZ with collecting rats' CSF and hippocampus samples. SL and QL were involved in study design, data analysis and interpretation, drafting of the manuscript, study supervision, and critical revision of the manuscript for important intellectual content.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.


Background: Disruptions in cerebrospinal fluid (CSF) flow during aging could compromise protein clearance from the brain and contribute to the etiology of Alzheimer’s Disease (AD).Objective: To determine whether CSF flow is associated with cognitive deficit in elderly patients (>70 years).Methods: We studied 92 patients admitted to our geriatric unit for non-acute reasons using phase-contrast magnetic resonance imaging (PC-MRI) to calculate their ventricular and spinal CSF flow, and assessed their global cognitive status, memory, executive functions, and praxis. Multivariable regressions with backward selection (criterion p < 0.15) were performed to determine associations between cognitive tests and ventricular and spinal CSF flow, adjusting for depression, anxiety, and cardiovascular risk factors.Results: The cohort comprised 71 women (77%) and 21 (33%) men, aged 84.1 ± 5.2 years . Net ventricular CSF flow was 52 ± 40 μL/cc , and net spinal CSF flow was 500 ± 295 μL/cc . Ventricular CSF flow was associated with the number of BEC96 figures recognized . Spinal CSF flow was associated with the WAIS Digit Span Backward test , and categoric verbal fluency  and semantic verbal fluency .Conclusion: Patients with lower CSF flow had significantly worse memory, visuo-constructive capacities, and verbal fluency. Alterations in CSF flow could contribute to some of the cognitive deficit observed in patients with AD. Diagnosis and treatment of CSF flow alterations in geriatric patients with neurocognitive disorders could contribute to the prevention of their cognitive decline. The cerebrospinal fluid (CSF) is an important part of the central nervous system, as it allows exchange of water, small molecules and proteins between the brain parenchyma and arterial and venous blood , by eithThe increase of intracranial pressure during the cardiac cycle causes a flow from the blood and brain interstitial fluid to the CSF, and a net CSF flow toward its extracerebral compartment and venous blood . Since tTo the authors’ knowledge, there are no published studies that investigated the relationship between CSF flow alterations and cognitive deficit in the elderly, adjusting for cardiovascular risk factors for the development of neurocognitive disorders. The purpose of this study was therefore to evaluate the association of CSF flow in the brain ventricles and cervical spine with cognitive deficit  in a cohort of elderly patients (>70 years) admitted to our geriatric unit for non-acute reasons. The hypothesis was that reduced CSF flow would be associated with cognitive deficit. Improved knowledge of such associations could guide the development of medical or surgical treatments to limit or prevent cognitive decline in the elderly .We enrolled 115 consecutive patients admitted to our geriatric unit for non-acute reasons between October 2015 and March 2018. The inclusion criterion was patients aged over 70 years. Twenty-one patients were excluded because of contraindications to phase-contrast magnetic resonance imaging (PC-MRI) for analysis of brain fluid motion, and two patients died before completing neurocognitive assessments. This left a study cohort of 92 patients  who undeThe PC-MRI was performed using a 1.5-T machine. Conventional morphologic image sequences were first acquired in the sagittal and axial planes. The CSF flow acquisition planes were then selected perpendicular to the presumed direction of flow through the Sylvius aqueduct [representing the ventricular flow ] and thePhase-contrast magnetic resonance imaging data were transferred to a Sparc 10 workstation (SUN Microsystems) and analyzed using an in-house image processing software with Interactive Data Language . This soAll patients underwent a battery of neurocognitive tests that assessed global cognitive status, memory, executive functions, praxis, as well as depression and anxiety . Global Mild cognitive impairment was diagnosed based on the criteria of Petersen et al. , while AVascular risk factors were diagnosed based on a comprehensive geriatric assessment performed at our geriatric unit. Blood samples were obtained after a minimum of 10 h of fasting. The diagnosis of diabetes was attributed with blood glucose levels above 7 mmol/L, anemia with hemoglobin levels below 12 (women) or 13 (men) g/dL and inflammation with CRP levels above 10 mg/L. Likewise, the reference range was 4.1–6.5 mmol/L for total cholesterol, 0.6–1.8 mmol/L for triglycerides and 35–50 g/L for Albumin. Malnutrition was defined by Albumin levels <35 g/L.r = 0.231, p = 0.037), while the spinal CSF flow was correlated with the backward WAIS digit span task  and the GB recall test . Multivariable regressions with backward selection (criterion p < 0.15) were performed to determine associations between the aforementioned cognitive tests and thirteen independent variables, to adjust for depression, anxiety, and cardiovascular risk factors  and all cognitive test results. The ventricular CSF flow was correlated with the BEC96 visuo-constructive subscale   and 21 (33%) men, aged 84.1 ± 5.2 years , with BMI 25.2 ± 4.8 kg/m.6–40.0) . The MAD.6–40.0) .p = 0.034) and were worse for patients with anemia ; (ii) the GB recall results increased with spinal CSF flow  and were worse in patients with malnutrition   the WAIS Digit Span Backward results improved with increasing spinal CSF flow  .p = 0.025) and decreased in patients with malnutrition  (p = 0.024) and in men , and was decreased in patients with diabetes ; (iii) semantic verbal fluency increased with spinal CSF flow (×100)  and was decreased in patients with diabetes .Multivariable analysis also revealed that (i) the number of BEC96 figures recognized increased with ventricular CSF flow  ; (ii) caThe main finding of this study is that CSF flow was associated with cognitive test results. Ventricular CSF flow was associated with the number of recognized BEC 96 figures, while spinal CSF flow was associated with the WAIS digit-span backward test, categoric and semantic verbal fluencies, as well as the Grober-Buschke recall test (borderline). Taken together, these results suggest that CSF flow influences memory, visuo-constructive capacities, and verbal fluency. Interestingly, our findings are coherent with CSF flow values reported in recent literature. Our measures of ventricular CSF flow were comparable to values reported for healthy adults, but higher than those reported for healthy elderly volunteers, while our measures of spinal CSF flow were slightly higher than those reported for all elderly patients, except those with amnesic mild cognitive impairment .It is unclear how CSF flow alterations could affect cognition. While CSF flow could be influenced by blood perfusion , the cogNormal pressure hydrocephalus (NPH) is a rare form of neurocognitive disorder, in the elderly – characterized by the triad of dementia, gait apraxia and incontinence – thought to be caused by impaired CSF circulation . Its effIn this study, multivariable analysis revealed negative associations between diabetes and executive functions , between malnutrition and episodic memory and executive function , and between anemia and working memory (indicated by the WAIS digit span backward test). In addition, men had greater categoric fluency, which could be explained by the fact that they were on average younger than the women . Our findings are consistent with the literature: Diabetes is an important predictor of cognitive decline in the elderly and is associated with deficits in attention and executive functions . MalnutrThis study is the first to report direct associations between CSF flow and cognition, and could improve our understanding of the complex etiologies and symptoms of neurocognitive disorders in the elderly. It shows that altered CSF flow is associated with cognitive deficits in elderly patients, and thus suggests that treatments aiming at restoring normal CSF flow could reduce cognitive deficits. This is in accordance with a clinical observation in our practice, of a patient who recovered from debilitating neurocognitive disorders after sleep apnea treatment and ventriculoperitoneal shunting . HoweverIn conclusion, this study revealed direct associations of ventricular and spinal CSF flows with cognitive scores in a geriatric population with cognitive impairment. CSF flow was associated with memory, visuo-constructive ability and verbal fluency. It is therefore possible that CSF flow alterations are responsible for at least a part of the cognitive deficit observed in our patients. Better diagnosis and treatment of CSF flow alterations in geriatric patients suffering from neurocognitive disorders is therefore recommended.clinicaltrials.gov (NCT02578303). All procedures were performed according to the Declaration of Helsinki.Written informed consent was obtained from all patients for their participation and confirmed by their next-of-kin if necessary. The study protocol was approved by an independent Ethical Review Board (CPP Amiens: 2015/6) and the National Data Protection Authority (CNIL:150075B-31). The study was registered in JA-Z and OB designed the study, collected the data, and edited the manuscript. J-MS designed the study and edited the manuscript. JV wrote and edited the manuscript and performed the statistics. MS wrote and edited the manuscript. A-SM, SD, and YD collected the data. MD and KM validated the statistics.MS and JV are employed by ReSurg SA . The remaining 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.


Testis tissue xenografting complemented with cryopreservation is a feasible technique for fertility preservation in children with malignancy receiving gonadotoxic therapy and for endangered species with high neonatal mortality rate. However, xenografted testis of human and most endangered species are known to undergo spermatogenic arrest. In this study, we xenografted immature rat testis onto immunodeficient male mice to investigate the plausible underlying causes of spermatogenic arrest. Histological analysis of xenografted testes collected 8-wk post-grafting showed incomplete spermatogenesis with pachytene-stage spermatocytes as the most advanced germ cells. Although the levels of serum luteinizing hormone and testosterone were normal in recipient mice, those of follicle stimulating hormone (FSH) were significantly high, and specific receptors of FSH were absent in the xenografts. The xenografts demonstrated dysregulated expression of Sertoli cell-transcriptional regulators (WT1 and SOX9) and secretory proteins (SCF and GDNF). In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis. Further stereological analysis of xenografts can demonstrate precise cellular composition of xenografts to decipher interactions between germ and somatic cells to better understand spermatogenic arrest in xenografted testis. Xenografting complemented with cryopreservation of testis tissue can find application in fertility preservation in children with malignancy receiving gonadotoxic treatment and in the conservation of endangered animals with high neonatal mortality rate. Production of live offspring from cryopreserved-xenografted testis of rabbit2, and more recently from pig3, showed the applicability of these techniques. Of the 23 species of mammals used as donors for testicular tissue xenografting to date, 15 showed complete spermatogenesis1. In the remaining 7 species, including endangered ungulates , Iberian lynx5 (an endangered feline), common marmoset6, laboratory rat9 and humans13, spermatogenic arrest occurred at the spermatogonia, spermatocyte, or round spermatid stages. We recently showed that spermatogenesis was incomplete and arrested at the spermatocyte stage in xenografted testis from an endangered ungulate (Indian spotted mouse deer)14. It is known that genetics, hormonal, thermal, and toxic factors are implicated in spermatogenic arrest in humans15. Although exogenous gonadotropin treatment of the recipient mice was demonstrated to aid completion of spermatogenesis in xenografted testis of certain species, these results were inconsistent17. Therefore, the key factors that lead to spermatogenic arrest in xenografted testis still remain unclear, and the underlying mechanism needs to be investigated to increase the efficiency of xenografting. Further insights into spermatogenic arrest in xenografts would help in developing methods to overcome it.Ectopic testis tissue xenografting offers a practical method for understanding the mechanism of spermatogenesis and testicular maturation. This technique has been used for the production of mature gametes by grafting small pieces of testis tissue under the dorsal skin of immunodeficient mice recipients18. Development and differentiation of germ cells require the interaction of germ cells with the supporting Sertoli cells in the epithelium of the seminiferous tubules. Failure of germ cell–somatic cell interactions could be one of the causes of spermatogenic arrest. Because rat testis xenografts show spermatogenic arrest9, they can therefore serve as a suitable model for studying spermatogenic arrest. The purpose of the present study is to identify the factors that play a role in spermatogenic arrest using the rat-to-mouse testis xenograft model by evaluating endocrine changes in the recipients, and changes in protein expression in xenografts.Normal spermatogenesis and fertility are dependent upon paracrine interactions between the somatic cells and the germ cells, and endocrine support from the pituitary glandAt 2-, 4-, and 8-wk post-grafting, xenografts and seminal vesicles were recovered from recipients and weighed Table . FolliclSerum gonadotropins FSH and LH levels were raised in the recipients at 2- and 4-wk post-grafting , a spermatogonia-specific protein, was low in xenografts at 2- and 4-wk post-grafting when compared with that in donor and in situ control testis .Xenografts collected at 2-, 4-, and 8-wk post-grafting, donor, and in situ control testis (P < 0.05).Expression of 3 beta-hydroxysteroid dehydrogenase (HSD3B) protein progressively increased in xenografts, by 2 folds at 2 wk and 4 folds at 4 wk, compared with that of the donor testis  was comparable with that in the donor, in situ control testis  expression in xenografts at 2- and 4-wk post-grafting was not different from that in the donor or the in situ control testis (P > 0.05). Inhibin beta b subunit (INHBB) level was lower in xenografts collected at 2- and 4-wk post-grafting when compared with that in donor and in situ control testis  was low in xenografts at 2-wk post-grafting  decreased significantly in xenografts at 2-wk . Since completion of spermatogenesis and appearance of mature sperm in rat testis in situ was reported 6-wk post-partum25, an inadequate grafting period could not be a likely cause of spermatogenic arrest in xenografts in our study.Histological evaluation of the xenografts collected at 8-wk post-grafting showed the presence of pachytene-stage spermatocytes as the most advanced germ cells, which suggests establishment of a synchronized hormonal interaction between the recipient’s pituitary and the xenografted immature rat testis that induced spermatogenesis. However, the absence of elongated spermatids in the xenografted testis indicated that the spermatogenesis remained incomplete. These results are consistent with previous reports in which incomplete spermatogenesis was reported in xenografted rat testis at 8-wk post-grafting with a spermatogenic arrest at pachytene-stage spermatocytes21 and pig26. The elevated gonadotropin levels likely stimulated Sertoli cell and Leydig cell proliferation, and maturation in xenografted testis. The restoration of serum LH levels to a level similar to that in the intact control mice suggests establishment of feedback loop between xenografts and recipient pituitary. However, failure to restore FSH level in recipients could be due to absence of a concomitant rise in inhibin level, because inhibin reportedly suppresses FSH secretion from the pituitary by a negative feedback loop26. Protein expression analysis of xenografts revealed that expression of INHBB level (a subunit of inhibin) was indeed low in xenografts collected at 2- and 4-wk post-grafting when compared with that in donor and in situ control testis. However, in xenografts collected at 8-wk post-grafting, INHBB level was significantly elevated. Although a constantly elevated FSH level may have caused an elevation in INHBB expression in xenografts at 8 wk, it was likely insufficient to lower the serum FSH level in the recipient. In earlier studies, an intermediate to elevated FSH level was reported in recipients of mice testis21; however, in recipients of pig testis, restoration of FSH level to a level similar to that in the intact control was reported to be due to elevated inhibin level26. However, spermatogenesis was completed in xenografts of mice and pig testis in both studies, unlike in the present study, where spermatogenesis remained incomplete in rat testis xenografts. This discrepancy could be because of the difference in the donor species.In castrated recipients, gonadotropin levels were raised, which could be a result of a loss of inhibition by the testicular hormones, as observed in earlier studies using donor testis from neonatal mouse27, indicating that spermatogenesis was initiated but not completed in the absence of FSHR. Because AR expression was unchanged in xenografts when compared to that in in situ control testis, it is likely that the AR-specific signaling may not have a significant role in spermatogenic arrest in the xenografts.Loss of FSHR from the xenografts could be attributed to sustained high serum FSH concentration in the recipients or to a higher sensitivity of rat Sertoli cells to elevated FSH level in castrated recipients. Interestingly, loss of FSHR from the xenografts did not seem to impair induction of spermatogenesis but likely prevented its completion. This finding is consistent with that in a previous study in which it was found that FSHR-deficient males were fertile but displayed small testes and partial spermatogenic failure29. This is because the transplanted testis initially lacked complete blood supply8, thereby hampering germ cell survival. Following the establishment of vascular supply from the host mice, proliferation and differentiation of the surviving germ cells was restored. However, there was subsequent increase in UCHL1 expression from 2- and 4-wk to 8-wk post-grafting, but it was still lower than that in the donor or the in situ control testis, which suggests incomplete recovery and persistence of initial deficit.A low expression of spermatogonia-specific protein (UCHL1) in xenografts could be a result of loss of spermatogonia, which is reportedly common in xenografted testis30, which could not be confirmed because of the absence of stereological analysis in our study. However, these findings corroborate the recovery of seminal vesicle weight with increase in post-grafting duration in the recipient mice; in turn, this indicates restoration of serum testosterone level23. We found in an earlier study that Leydig cells could regenerate despite extensive degeneration of seminiferous tubules in ectopically autografted mice testis, leading to restoration of the weight of seminal vesicle31. Therefore, despite a low expression of LHR, the Leydig cells in xenografts were likely able to produce sufficient testosterone because of their increased number or enhanced steroidogenic activity.Elevated HSD3B and LHRH expressions at 8-wk post-grafting could be associated with a progressive increase in Leydig cell number (hyperplasia)32, in xenografts at all collection time points. However, precise change in cell number could not be confirmed because of the lack of stereological analysis in our study. Sertoli cells in immature rat testis were previously reported to proliferate efficiently following xenografting8. However, we observed dysregulation of Sertoli cell-specific proteins in our study. There was a significant decline in the expression of WT1 and SOX9. WT1 mutation has been reported to be associated with spermatogenic defects in humans33. Similarly, a recent study reported that ablation of SOX9 in Sertoli cells leads to complete degeneration of the seminiferous tubules34. We also observed elevated levels of SCF and GDNF in xenografts at an early grafting period in our study, which likely stimulated Sertoli cell proliferation, the onset of differentiation of spermatogonia, germ cell differentiation, and progression into meiosis. Previous studies have shown that SCF supports the survival of adult germ cells, onset and maintenance of spermatogenesis and germ cell differentiation and progression into meiosis35, and elevated levels of GDNF favors self-renewal of spermatogonia36. The expression of CLDN11, a transmembrane protein component of the tight junctions in the Sertoli cell BTB37, was found to be elevated in spermatogenic defective testis and during hypospermatogenesis, and spermatocytic and maturation arrest39. The raised CLDN11 expression in our study corroborates spermatogenic arrest in xenografts at 8-wk post-grafting.Sertoli cells are the only somatic cells present in the seminiferous tubules that secrete growth factors (such as SCF and GDNF), express transcription regulators (such as WT1 and SOX9), and establish blood-testis barrier (BTB) in the seminiferous epithelium that are essential for germ cell development and differentiation. Presence of a steady population of Sertoli cells in xenografts was suggested by consistent expression of VIM, a structural protein and Sertoli cell-specific marker in rat testisIn the current study, we assessed the endocrine status of recipients, and histological and protein expression changes in the xenografts to decipher the mechanism of spermatogenic arrest but could not perform stereological analysis. Therefore, any changes in germ and somatic cell numbers need to be further confirmed by stereological analysis, and further investigations are warranted to analyse precise cellular composition of xenografts and decipher interactions between germ and somatic cells to further understand spermatogenic arrest in xenografted testis. In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis.Rattus norvegicus) pups at age 7 days post-partum were used as testis donors (n = 30). Testis from adult male rats (9-wk-old) was used as in situ control (n = 4), because it was demonstrated that testis xenografts have a gene expression profile similar to that of testis tissue in situ40. Immunodeficient male nude mice  were used as recipients (n = 15). Nude mice were kept under specific pathogen-free conditions, and food, water, and bedding were autoclaved before use. All animals used in this study were housed in 12 hours light: 12 hours darkness cycle at constant temperature, and provided food and water ad libitum.All animal procedures were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (IACUC) of the Kyoto University, Japan . Wistar rat  HEPES  until grafting. Four rat testes were ectopically grafted onto the back of castrated nude mice recipients. Briefly, mice were anesthetized with Avertin . For castration of recipient mice, a ventral medial incision was made in the abdomen and the testes were removed, following which the peritoneum and skin were closed using absorbable suture . During the same surgery, each mouse received two incisions (~5 mm) on each side of the back . One piece of testis was inserted through each incision. The incisions were sutured and the mice were allowed to recover and returned to their cages.Testes were collected from 7-day-old rat pups following euthanasia by CO41. The average weight of seminal vesicle from non-grafted intact adult nude male mice  was taken as control.The recipient mice were anesthetized as described above, and blood was collected at 2-, 4-, and 8-wk post-grafting by cardiac puncture with a heparinized syringe for hormonal assay. Serum was immediately separated by centrifugation  and stored at −20 °C for further analysis. For the collection of xenografts and seminal vesicles, the same recipient mice were euthanized by cervical dislocation. Xenografts were collected from recipient mice at 2-, 4-, and 8-wk post-grafting, and the percentage of xenografts recovered was calculated Table . The weiin situ control testis (n = 4) tissues were fixed in Bouin’s solution followed by tissue dehydration with increased concentrations of alcohol in a series. After processing, the tissues were embedded in paraffin, sectioned (7-µm thick), stained with hematoxylin and eosin (H&E), dehydrated, mounted in Vectamount , and observed under a light microscope . Establishment of spermatogenesis was assessed by morphological evaluation, as previously described14. A xenograft without distinct cell types and tubules was classified as either degenerated or non-degenerated if it contained even a single seminiferous tubule. More than 40 seminiferous tubule cross-sections were examined randomly in a xenograft to investigate the status of spermatogenesis. The most advanced germ cell type and differentiated germ cells were identified by their morphology and location in the seminiferous tubule. The xenografts with evidence of spermatogenesis (17 out of 19 recovered xenografts) were further used for data analysis. The percentage of tubules with the most advanced germ cell type was presented. The percentage of seminiferous tubules and degenerated tubules within a graft were also presented. The percentage of seminiferous tubules and degenerated tubules within a graft was also calculated.For histological analysis, donor testes (n = 3), xenografts collected at 8-wk post-grafting (19 xenografts recovered from 5 donors), and adult Serum levels of FSH and LH were determined using ELISA kits  according to the manufacturer’s instructions. All samples were tested in triplicate. The sensitivities of the mouse FSH and LH assays were 0.313 ng/ml and 0.31 mIU/ml, respectively.in situ control (n = 4). Protein concentration was determined by the Bradford method using a Bio-Rad protein assay kit . The lysed samples (40 µg) were subjected to electrophoresis on 8% , 10% , or 12%  SDS–polyacrylamide gel. The gels were transferred onto PVDF membranes . The membranes were blocked with Starting Block TBS blocking buffer  for 1 h at room temperature. The blocked membranes were incubated with one of the following primary antibodies overnight at 4 °C: UCHL1 , HSD3B , SOX9 , CLDN11 , SCF , ACTB , INHBB , FSHR  , WT1 , GDNF , LHR , AR  , and VIM . The membranes were then washed with TBS-T and incubated with goat anti-rabbit, goat anti-mouse, or rabbit anti-goat HRP-conjugated secondary antibody  in TBS-T for 1 h at room temperature. After washing with TBS-T, immunoreactivity was detected by chemoluminescence using Hyperfilm ECL  against ECL™ Prime chemiluminescent substrate . To control protein loading on the gels, the membranes were stripped, blocked and further probed with ACTB antibody.The xenografts were analyzed for the expression of testis-specific proteins by western blot analysis. Total protein from all xenografts recovered at 2- and 4-wk post-grafting and xenografts with meiotic germ cells collected at 8-wk post-grafting was extracted upon homogenization by sonication in a dissolving buffer . Single-strength ProteCEASE-50, EDTA-free protease inhibitor  was added to the dissolving buffer before protein extraction. All xenografts collected at 2-, 4-, and 8-wk post-grafting from a recipient were pooled for protein isolation (n = 5 for each collection time point). Protein sample from pooled xenografts, at a given collection time point  from at least 4 recipients was used for western blot analysis. Pooled proteins from the testis of 7-day-old rat donors (n = 5) represented the starting material and 9-wk-old rats were used as Immunoreactivity of blots was detected by chemoluminescence using Hyperfilm ECL  against ECL™ Prime chemiluminescent substrate . Hyperfilms were scanned in high resolution, and digital images were exported to Image Lab™ Software Version 6.0.1 (Bio-Rad Laboratories) for image densitometric analysis. The densitometric value for a given antibody was normalized with densitometric value of ACTB (loading control) for each lane in every blot. To generate the graphs and statistical analysis, normalized densitometry data from at least 4 blots were considered.The results were presented as mean ± S.E.M. The statistical analyses were performed by ANOVA. Normality and homogeneity of variances were checked and confirmed to match with parametric assumptions prior to running ANOVA. Significant differences between the means were determined by analyzing the data using the Tukey honest significance difference (Tukey HSD) test. The level of significance was set at P < 0.05.Supplementary file 1


In the present study, a codon optimized gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) was cloned and over-expressed in Escherichia coli BL21 (DE3) using both conventional inducer-based and auto-induction approaches in a shake flask scale and the yield of GM-CSF expression and biomass production was identified. Results showed higher biomass production and expression yield for GM-CSF in case of auto-induction comparing with IPTG-induction. The auto-induction approach was also performed in a fed batch fermentation process in a 2-L bioreactor scale. The feeding strategy yielded an amount of 300 mg/L GM-CSF within 20 h of induction. However, most of the over-expressed GM-CSF was produced as inclusion bodies and following purification and refolding, a final yield of 90 mg/L was achieved. These results suggest that auto-induction approach can be effectively applied in fed-batch fermentation for the large scale production of GM-CSF; however, further optimization of purification process is obligatory to increase the purification yield.A novel strategy to increase protein expression yield is unintended induction of expression in complex media, called auto-induction. This method can be used to express proteins under control of the  Saccharomyces cerevisiae (Sargramostim™), while the other one is a non-glycosylated protein derived from Escherichia coli expression system (Molgramostim™)  shows more in-vitro biological activity and less half-life compared with glycosylated protein (Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of colony-stimulating factors which regulates proliferation, differentiation, and survival of hematopoietic cells including mature neutrophils, macrophages, and dendritic cells . This cymostim™) , 5. Ther protein .E. coli is one of the most popular expression platforms for production of heterologous proteins , a protease with potential toxicity for host bacteria  using both conventional IPTG induction-based and auto-induction approach in a shake flask scale. In addition, the auto-induction approach was performed in fed batch fermentation in a 2-L bioreactor scale. Furthermore, here different aspects regarding purification and refolding procedures following auto induction of the recombinant GM-CSF are reported and discussed.In the current study, a codon optimized gene encoding GM-CSF was cloned and highly expressed in Bacterial strain and plasmidrGM-CSF) for its expression in E. coli. The sequence also included NcoI (5′) and XhoI (3′) restriction sites, an amino-terminal 6xHis-tag and a stop codon. The fragment was synthesized by Biomatik Company (Canada) and obtained in pET28a expression plasmid . Finally, E. coli BL21 (DE3) was transformed with the recombinant pET28-GM-CSF and used for subsequent expression experiments.The amino acid sequence of human GM-CSF was used to design a codon optimized DNA sequence (Auto-induction medium -1): 0.5 glucose, 2.0 lactose, 5.0 glycerol, 10.0 tryptone, 5.0 yeast extract, 7.1 Na2HPO4, 6.8 KH2PO4, 3.3 (NH4)2SO4, 0.12 MgSO4, and 1X metal solution. The metal stock solution  included 50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2 mM each of CoCl2, CuCl2, NiCl2, Na2MoO4, and H3BO3  compared with other media; high phosphate decrease the sensitivity to kanamycin . Then, expression of the protein was induced by addition of 1 mM IPTG at 37 °C for 3 h.A single recombinant colony was transferred to 5 mL of Luria–Bertani (LB) broth containing 25 µg/mL kanamycin, and the culture was incubated at 37 °C and 180 rpm for 16 h. On the following day, this culture was added to 50 mL fresh LB medium in 150 mL shaker flask and the cells were incubated at 37 °C until they reached the exponential phase 2SO4 as a nitrogen source, and Na2HPO4 ,KH2PO4 as buffering agents, was added after 7 h as a pulse.Bioreactor scale cultivation and production was performed in a 2-L stirred-tank bioreactor  at 37 °C for 25 h. The pre-inoculum culture was transferred to a 250 mL shaker flask containing 50 mL LB broth supplemented with 1% glucose and shaken at 37 °C and 180 rpm overnight. This culture was inoculated (1:1000) into the bioreactor containing 1250 mL of ZYP-5052 medium under sterile conditions. During the entire process, the dissolved oxygen level (DO) was maintained at approximately 30% air saturation by automatically adjusting the agitation speed and air flow rate. Antifoam (Propylene glycol) was added manually when necessary. The supplementary medium (112.5 mL), containing glycerol and lactose as carbon sources and inducer, , and subsequently disrupted through sonication  in an ice-bath. This lysate was incubated in the presence of 10 U/mL benzonase  for 15 min at 25 °C to eliminate DNA and RNA. The soluble and insoluble fractions of the cell lysate were separated by centrifugation at 7,500×g for 10 min. The insoluble fraction was solubilized with guanidinium lysis buffer and centrifuged at 7,500×g for 10 min to remove the insoluble debris. The obtained supernatant was loaded onto a nickel affinity column  and the recombinant GM-CSF was purified as described by the manufacturer  was dialyzed against 500 mL of refolding buffer 1  at 4 °C overnight to slowly remove urea. The next day, the protein solution was dialyzed overnight at 4 °C against 500 mL of refolding buffer 2 . Finally, the dialysis buffer was replaced with refolding buffer 3  and dialyzed overnight at 4 °C. Analytical methods During the bioreactor cultivations, the samples were taken every hour for cellular growth and protein analysis. The cell growth was monitored using measurements of the optical density (OD) of culture medium at 600 nm. In this regard, the samples were diluted between 1:1 and 1:20 in 0.9% NaCl solution. For determination of dry cell weight (DCW), 10 mL of the samples were harvested and centrifuged at 4 °C and 4,700 rpm. Afterwards, the cell pellets were washed with 10 mL of 0.9% NaCl solution and centrifuged again. The supernatants were removed and the cell pellets were dried overnight at 80 °C before being weighed. Protein expression was evaluated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the protein production rate was estimated by densitometry analysis of acrylamide gels using TL120 software  . Bovine Protein expression in shake flaskrGM-CSF coding sequence was synthesized on the basis of codon optimized nucleotide sequences corresponding to the amino acid sequence of MolgramostimTM and the synthesized gene was obtained as an expression frame in the pET28a plasmid (pET28-GM-CSF). E. coli BL21 (DE3) harboring the pET28-GM-CSF plasmid was used for over-expression of the rGM-CSF using both conventional IPTG induction and auto-induction approaches in shake flask scale, and the expression was analyzed by SDS–PAGE. As it is shown by The The identity of the over-expressed protein was verified by Western blotting using anti-6X His-tag antibody. Western blot analysis indicated that the expressed rGM-CSF was a His-tagged fusion protein with a molecular weight of 16 kDa .Auto-induction of rGM-CSF in a 2-L bioreactorE. coli BL21 (DE3) harboring pET28a (rGM-CSF) in a 2-L bioreactor was presented in 600. OD600 of 1 corresponded to approximately 0.395 g of DCW per liter culture of E. coli BL21 (DE3). The cellular growth was illustrated with a diauxic (two phase) growth curve. First, an exponential cell growth was observed, as the bacterium consuming glucose has the higher maximum specific growth rate. The initial carbon source was exhausted after 5 h of cultivation and following a lag phase, cellular growth continued with a lower growth rate because of the application of lactose as the main carbon source. In the auto-induction approach, the cellular concentration reached an OD600 of 8 within 25 h of cultivation.The profiles of auto-induced culture of 4)2SO4 as a nitrogen source, and Na2HPO4, KH2PO4 as buffering agents were added, in order to avoid reduction of protein synthesis rate as a result of insufficient carbon source or inducer. The use of this feeding strategy yielded a yield of 300 mg/L rGM-CSF within 20 h of induction, corresponding to a protein-specific production rate of 95 mgprotein/g DCW.As shown in Purification and refoding of GM-CSFPurification of rGM-CSF was performed using Ni-NTA affinity chromatography under denaturing condition which led to about 90% purity , and oveE. coli by an auto-induction strategy for the first time. To the best of our knowledge, this is the first study that described the scale-up of GM-CSF auto-induction from shake flask to fed-batch bioreactor scale. Considering the wide range of GM-CSF therapeutic applications, various strategies for production of proper amounts of this protein with reasonable costs have been considered. Here, we reported high-yield production of rGM-CSF in E. coli has been reported to be very difficult because bacterial cells stopped growing and eventually lysed upon induction due to toxicity of the recombinant protein , and E. coli GJ1158, and the estimated production yield following intein-mediated purification reported to be 7 mg/L and 20 mg/L, respectively  and reported higher cell density and lower expression yield for high oxygenation conditions (Maximum oxygen transfer rate (OTRmax)∼120 mmol L−1 h−1) compared to moderately aerated conditions (OTRmax∼30 mmol L−1 h−1) . However, Mayer and coworkers (600 = 25) and product (1.3 g/L) yields without oxygen limitation  . Under hL−1 h−1) . Therefooworkers  performemitation . They comitation .E. coli. The over-expressed protein mainly aggregated as inclusion bodies which can be purified using one-step affinity chromatography under denaturing conditions and subsequently refolded in-vitro by slow dialysis against buffers containing arginine and a combination of reduced and oxidized thiols. Moreover, we successfully scaled up auto-induction of GM-CSF from shake flask to fed-batch bioreactor. A pulse feeding of lactose and glycerol was used to provide adequate inducer and carbon source during 25 h of cultivation. Considering different therapeutic and research applications of GM-CSF, the auto-induction approach has the potential to be used for economical production of this protein in large amounts. Furthermore, the efficiency of the purification and refolding procedures are going to be evaluated by undergoing studies on the structure and biological activity of the purified rGM-CSF.In the present study, we reported an effective strategy for over-expression of rGM-CSF in cytoplasm of


F = 3.864, p = 0.032; partial η2 = 0.205] and cadence . Group contrasts showed significantly higher values for RAS-TT. Stride length results did not vary between the groups. LOC, 3MWT, and IEB did not indicate group differences. One patient was withdrawn from TT because of pain in one arm. The study provides first evidence for a higher efficacy of RAS-TT in comparison to the standard approaches TT and NDT in restoring functional gait in SP. The results support the implementation of functional music in neurological gait rehabilitation and its use in combination with treadmill training.The use of functional music in gait training termed rhythmic auditory stimulation (RAS) and treadmill training (TT) have both been shown to be effective in stroke patients (SP). The combination of RAS and treadmill training (RAS-TT) has not been clinically evaluated to date. The aim of the study was to evaluate the efficacy of RAS-TT on functional gait in SP. The protocol followed the design of an explorative study with a rater-blinded three arm prospective randomized controlled parallel group design. Forty-five independently walking SP with a hemiparesis of the lower limb or an unsafe and asymmetrical walking pattern were recruited. RAS-TT was carried out over 4 weeks with TT and neurodevelopmental treatment based on Bobath approach (NDT) serving as control interventions. For RAS-TT functional music was adjusted individually while walking on the treadmill. Pre and post-assessments consisted of the fast gait speed test (FGS), a gait analysis with the locometre (LOC), 3 min walking time test (3MWT), and an instrumental evaluation of balance (IEB). Raters were blinded to group assignments. An analysis of covariance (ANCOVA) was performed with affiliated measures from pre-assessment and time between stroke and start of study as covariates. Thirty-five participants  completed the study . Significant group differences occurred in the FGS for adjusted post-measures in gait velocity [Clinical Trial Registration: https://www.drks.de/drks_web/, identifier DRKS00014603 About 60% of all stroke patients (SP) have difficulties with walking . These an = 2,658 patients) revealed clear therapeutic effects on gait velocity and walking endurance, the latter only for TT with body weight support (Treadmill training (TT) with and without body weight support has been shown to improve functional gait in stroke patients effectively. A meta-analysis comparing 44 trials ( support . However support . Lee's wn = 197) that showed improvements in functional gait performance  (Rhythmic-auditory stimulation (RAS) is defined as a therapeutic application of pulsed rhythmic or musical stimulation in order to improve gait or gait related aspects of movement . It has  length) . The app length) . The RAS length) .While RAS and TT have proven to be effective for gait training in SP, the efficacy of its combination (RAS-TT) in the early course of rehabilitation in SP has not been investigated to date. Therefore, we hypothesized that RAS-TT in the early course of rehabilitation would improve the clinical efficacy of TT for SP. The purpose of the present study was to investigate the functional improvements of gait using a rehabilitation therapy combining RAS and TT in order to assess its clinical efficacy for patients suffering the aftermaths of a stroke.The study protocol was approved by the state authorization association for medical issues in Brandenburg, that determined on the 21st of January 2010 that no formal ethics approval was required. Patients gave their informed consent according to the Helsinki declaration.The study was designed as a prospective, single center three arm clinical study with parallel groups. We enrolled patients who performed either RAS on the treadmill (RAS-TT) or treadmill training alone (TT). A third group that received neurodevelopmental treatment following the Bobath approach (NDT) served as a control group. The patients were randomly assigned to the three training interventions by a person not involved in the study using a block randomization (software randlist). Allocations were placed in sealed sequentially numbered envelopes and were not opened until the actual study inclusion. Thus, the patients, the responsible doctor, the assessing physiotherapist, and study manager were not informed beforehand regarding the group assignment.We included stroke patients with a hemiparesis of the lower limb  or with an unsafe and asymmetrical walking pattern (by assessment of a physiotherapist). The patients had to be able to walk independently with assistive devices if necessary for at least 3 min.Criteria for exclusion were the following: significantly disturbed language perception (marked by either the Aachener Aphasietest or Token Test), cognitive impairment , major depression or productive psychosis, adjustment disorder with a need for medical treatment, peripheral arterial occlusive disease with walking distance <100 m, and coronary heart disease (instable angina pectoris).After having passed the diagnostics patients underwent a screening session on the treadmill. There they had to demonstrate a stable and sufficiently ergonomic gait. Candidates with insufficient quality of gait on the treadmill  were postponed and re-screened every week Figure .Patients were recruited in a clinical center for neurological rehabilitation in Beelitz-Heilstaetten/Germany. We included 45 stroke patients, of which ten were excluded during the intervention phase due to the following reasons: five for administrative reasons (funding of the in-patient rehabilitation was stopped) distributed over all three groups ; four patients developed acute medical complications that were unrelated to the movement therapies (occurring in group RAS-TT: 2 and group NDT: 2); one patient reported pain in the paretic arm following treadmill training (TT), which seemed to be associated with increasing spasticity. The data of 35 patients were used for data analysis  based on their actual walking disability for 30 min. During this treatment movements were directed toward an enhanced activation of the paretic side while inhibiting an immoderate co-activation of the non-affected side. NDT was applied by physiotherapists who had received an advanced training in the NDT for neurologically impaired adults.No body weight support or inclination was used for the RAS-TT and the TT interventions. Patients walked on the treadmill  holding themselves with at least one hand at the side bar. Therapists were instructed to keep the treadmill pace at a maximum level while striving for a normalized physiological gait pattern . The training time was increased continually . Therapists were allowed to set breaks for the patient when needed.For the RAS-TT intervention, patients listened to functional training music via ear plugs through an ordinary MP3 player while walking on the treadmill. The music was designed (software cubase 3 SE) according to the criteria described by Thaut et al. : clearlyAll patients were given extra conventional physiotherapy treatment either 30 or 60 min per week according to the level of impairment, which was constantly updated. All patients performed further training therapies according to their medical needs .Our primary outcome parameters were gait velocity, cadence and stride length. Secondary outcome parameters were gait symmetry, endurance and postural stability. Assessments were carried out by physiotherapists blinded for the group assignment. Pre-assessments were carried out at the beginning of the training period. Post-testing was performed on the day after completing the 4 weeks training.We included a fast gait speed test (FGS), a gait analysis with the locometre (LOC), a 3-min walking test (3MWT), and an instrumental evaluation of balance (IEB).In the FGS, patients were asked to walk safely as fast as possible toward a goal, while starting 2 meters ahead of the start mark. The time was measured with a stop watch, and the heel strikes within a marked 10 meter walk way were counted. Patients were allowed walking aids when they needed them and were asked to use them again for the post-test. The test was performed twice. For the statistical analysis the data from the second trial were used. Stride length, gait velocity and cadence were calculated using the formula presented by Flansbjer et al. . The LOCIn the 3MWT patients were asked to walk for 3 min as far as they could. The distance covered was measured in meters. The IEB was performed using a force platform . In this test, postural balance was evaluated in a standing position on a firm surface with eyes open. The duration of recording was 51 s (sampling rate: 40 Hz). The length of sway, sway area and mean lateral displacement of the center of pressure were calculated .For the calculation of the sample size an ANOVA with repeated measures for the time effect was conducted (software G * power 3.0.10) with an error probability of α = 0.05, a given power of 0.8. The required sample size was 36 for the intended effect size f = 0.25.t-tests with a level of significance of p = 0.0167. The ANCOVA was calculated on the basis of the following comparisons of covariates and post-intervention measurements for each intervention type: the linear relationship between covariates and post-intervention measures, which was assessed by visual inspection of a scatterplot, the homogeneity of regression by determining the interaction term, the normal distribution of residuals using Shapiro-Wilk's test and the homoscedasticity and homogeneity of variances using visual inspection of a scatterplot and Levene's test of homogeneity of variance. Statistical outliers were detected by identifying cases with standardized residuals >±3. The assumptions for ANCOVA were met for all assessment parameters of the LOC, the FGS, and the 3MWT. One outlier in the standardized residuals (as defined by being greater than the standard deviation ±3) was found in the LOC parameter stride length. This outlier was kept for the analysis.Due to the applied power analysis we decided to enroll a total sample of 45. As some of the data were not normally distributed and there was no fitting non-parametric statistical method for the study design, an analysis of covariance (ANCOVA) was run (SPSS 11.5). Thus, post-intervention assessment measures were adjusted for the two covariates: (1) respective pre-intervention measure and (2) the time between the stroke and the start of training intervention. Significant pre-post-effects were followed up group-wise using Bonferoni corrected There were no significant differences between the groups with respect to baseline characteristics , except for the use of assistive devices as these were notably less present in the NDT group. The groups differed also in time between stroke and study inclusion.p < 0.001; cadence: p = 0.001; stride length: p < 0.001). For TT, there were significant changes in velocity (p < 0.007) and stride length (p < 0.001) while cadence did not improve significantly (p = 0.283). In the NDT group, no significant changes were observed  = 3.864, p = 0.032, partial η2 = 0.205] and for cadence . The adjusted post-values compared by group contrasts were significantly higher in favor of RAS-TT   with moderate effect sizes. The results of NDT showed a significant improvement in stride length only (p = 0.006) while velocity (p = 0.017) and cadence changes (p = 0.102) did not reach the level of significance  = 1.861, p = 0.173, partial η2 = 0.11; for stride length: F = 1.108, p = 0.343, partial η2 = 0.069]. The analysis for cadence fell short of the level of significance with F = 3.242, p = 0.053, partial η2 = 0.178. Group contrasts here revealed a significant difference between RAS-TT and NDT (p = 0.023) while the contrast RAS-TT to TT failed to show significance (p = 0.06)  while patients in the TT group did not improve significantly (p = 0.046) despite a small effect size of d = 0.413. The group comparison with the regression analysis showed no significance  but revealed a significant group contrast between RAS-TT and TT (p = 0.033) . No significant pre-post-improvements were present when looking separately at the three groups.In the IEB, only the parameter length of sway showed a significant time effect (n = 12) at comfortable walking speed (ICC 0.93)  , but notOur balance measures showed a significant time effect on the length of sway of COP. The fact, that this improvement was distributed equally across groups while gait improvement was not could indicate that balance functions improve unrelated to special training interventions due to general recovery from sensorimotor deficits. In order to deepen the understanding of the role of movement training in the improvement of balance functions in this clientele, further investigation using balance measures as an inclusion criteria should be carried out.In conventional TT, the therapist mainly intervenes by adjusting the pace of the treadmill. Thus, the speed of gait can be regulated. The additional use of music enables the inherent correction of step frequency and gait pattern, by using auditory motor coupling. RAS has been used so far to increase cadence in gait training on the ground. Typically, cadence in TT with stroke patients is almost as high as in walking on the ground while stride length is short . This isIn our study, there was one drop-out that was attributed to TT. In general, patients coped well with this form of apparatus based therapy while performing comparable to or better than when NDT was used. Even though we used computerized music, none of the patients would consider omitting the music. A few patients reported that they became bored with the long stereotype arrangement, but still felt the advantage for their gait training and wanted to carry on with it. According to our clinical observations, the severe motoric problems stroke patients experience call for the use of clear-cut and foreseeable music. This is especially important when training on the treadmill. Nonetheless, these considerations can be realized in a more refined and artistically ambitious manner. Ideally, patients will be able to choose their preferred musical genre in the future. This could lead to a still better compliance and to additional motivational effects.Our study has some limitations. The power of the measured effects is limited by the small sample size. We were not able to follow up on patients. Therefore, it remains uncertain whether the effects would persist over a longer period of time. A weak point of our assessment battery can be seen in the absence of participation oriented assessments like the Barthel Index or functional ambulatory category.Our data suggest, that RAS-TT can be considered a form of training that optimizes gait rehabilitation in stroke patients. The need for a larger study, preferably comparing TT, and RAS-TT, remains. Furthermore, it would be interesting to investigate, whether patients with either predominantly paretic or more sensory symptoms benefit most from the music intervention.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.SM conceptualized and directed this study, performed data analysis, wrote the first draft of the manuscript. JW contributed to the conception and design of the study. All authors contributed to manuscript revision, read and approved the submitted 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.


Pspm, which controls expression of nicotine-degrading genes that are not controlled by the Phsp promoter. We identified specific nucleotides of the Pspm promoter responsible for NicR2 binding. HSP was further demonstrated as an antagonist, which prevents the binding of NicR2 to the Pspm and Phsp promoters, by locking NicR2 in the derepression conformation. The competition between NicR2 and RNA polymerase is essential to initiate transcription of nicotine-degrading genes. This study extends our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.We report the entire process underlying the NicR2 regulatory mechanism from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. NicR2 can bind to another promoter,  Pseudomonas putida S16. However, little is known regarding whole mechanism(s) regulating transcription of the nicotine degradation pathway gene cluster. In the present study, we comprehensively elucidate an overall view of the NicR2-mediated two-step mechanism regulating 3-succinoyl-pyridine (SP) biotransformation, which involves the association of free NicR2 with two promoters and the dissociation of NicR2 from the NicR2-promoter complex. NicR2 can bind to another promoter, Pspm, and regulate expression of the nicotine-degrading genes in the middle of nic2 gene cluster, which are not controlled by the previously reported Phsp promoter. We identified the function of the inverted repeat bases on the two promoters responsible for NicR2 binding and found out that the –35/–10 motif for RNA polymerase is overlapped by the NicR2 binding site. We clarify the exact role of 6-hydroxy-3-succinoyl-pyridine (HSP), which acts as an antagonist and may prevent binding of free NicR2 to the promoters but cannot release NicR2 from the promoters. Finally, a regulatory model is proposed, which consists of three parts: the interaction between NicR2 and two promoters (Pspm and Phsp), the interaction between NicR2 and two effectors (HSP and SP), and the interaction between NicR2 and RNA polymerase.Nicotine, a toxic and addictive alkaloid from tobacco, is an environmental pollutant in areas near cigarette production facilities. Over the last decade, our group has studied, in depth, the pyrrolidine pathway of nicotine degradation in  The bases responsible for the NicR2-promoter association and the homotropic effect between the NicR2 dimers were identified, which had not been reported before. Moreover, we clarified the true role of HSP as an antagonist that may prevent the binding of free NicR2 to the Pspm and Phsp promoters but could not induce the release of NicR2 bound to the promoters. Given the remarkable function of HSP, we demonstrated the significance of the competition between RNA polymerase and NicR2. Finally, we propose a model of regulation of the expression of nicotine degradation genes.Nicotine is not only a toxic and addictive alkaloid from tobacco that harms smokers  but alson detail , 12. Funentified  and B (1entified –15. Furtf-sites) . In addinic2 gene cluster  , there was an inverted repeat on the Pspm promoter, located in the vicinity of to the start codon of spmA, (TTATACCGTCGTACATATAA)   . It is knscribed . In the his gene . The upspromoter . Considepromoter . Given tCATATAA) . We demond EMSA)  and S3.10.1128/mBio.00602-19.1FIG S1β-Galactosidase assay of S16ΔspmABC. Mutant S16ΔspmABC was employed in this assay, in which spmABC was mutated. The strain was cultured in MSM+citrate medium with different compounds (SP or HSP). The Pspm promoter is the wild-type. Download FIG S1, TIF file, 1.3 MB.The Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00602-19.2FIG S2Pspm promoter by biolayer interferometry assay. In phase 1, NicR2 was incubated with the Pspm immobilized senor surface in the dilution buffer for 300 s. In phase 2, PBS-Tween buffers were applied to elute the Pspm-NicR2 complex from the senor surface for 300 s. Download FIG S2, TIF file, 0.3 MB.Determination of the association between NicR2 and Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00602-19.3FIG S3FIG S3, TIF file, 0.3 MB.Identification of binding site by EMSA. Free, free DNA; Fa, Fa fragment (a 30-bp fragment). Download Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pspm promoter used for these DNA affinity purification assays, we speculate that these two proteins were purified because they attached to the biotin, not the promoter. Therefore, we predicted that pyruvate carboxylase and an acetyl-CoA carboxylase biotin carboxyl carrier protein subunit are very likely to be unrelated to the regulation.Regarding the other proteins identified in the DNA affinity purification experiments, pyruvate carboxylase can transform pyruvic acid to oxaloacetic acid, and the acetyl-CoA carboxylase biotin carboxyl carrier protein subunit is a subunit of acetyl-CoA carboxylase. Both of these enzymes have biotin prosthetic groups. Considering that there was a biotin group at the 5′ end of the Phsp and Pspm promoters are 20-bp inverted repeats, (CTATATGTACAGCCATATAA) and (TTATACCGTCGTACATATAA), respectively. We named the half-site bases from 5′ to 3′ as left half-site bases  and right half-site bases . The right half-sites of both Pspm and Phsp promoters were strictly conserved (CATATAA), whereas three bases in the left half-site differed between the two promoters. A 6-bp spacer fragment between the half-sites in both promoters provided adequate space for the association between the two dimers since they resided on the promoters and repressed transcription  values of R-m3 and R-m4 mutants were 1,800 ± 600 nM and 570 ± 130 nM, respectively, indicating that R3 had a stronger contribution to the interaction with NicR2 than did R4  and isothermal titration calorimetry ITC) assays to assess the influence of each base on the association between NicR2 and the inverted repeat sequence. The promoter mutants were named R-m1, R-m2, R-m3, R-m4, R-m5, R-m6, and R-m7, respectively . Two bann did R4  and C. assays t10.1128/mBio.00602-19.8TABLE S2Table S2, XLSX file, 0.01 MB.Mutants applied in EMSA and ITC assays. Download Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pspm and Phsp promoters. In addition, we still observed a binding band for R-m45, supporting our deduction that R3 contributed more to the association between NicR2 and the two promoters than R4. In addition, the wild-type homotropic band was superseded by a highly enhanced R-m12 mutant nonhomotropic band, indicating that bases R1 and R2 directly participated in the homotropic interaction between the two NicR2 dimers  addition order impacts results, which was missed in previous study. When NicR2 was initially incubated with the Pspm or Phsp promoter, none of the concentrations of HSP tested was able to cause the dissociation of NicR2-promoter complexes  (in vivo but was not as effective as HSP.One of the genes controlled by the  at 4 mM . The precitrate) . This inin vitro but was able to induce the transcription in vivo led us to study how transcription starts. By comparing the whole Pspm and Phsp sequences, we found other similarities between the two promoters in addition to the inverted repeats. The –35 and –10 motifs of Pspm and Phsp promoter sequences were identical . Both motif locations on the promoters were near the transcriptional initiation site, covering the whole inverted repeat  /optical density at 600 nm (OD600) ratio in arbitrary units (AU) of S16_pUCP18k_Phsp_GFP/pME6032-NicR2 was lower than that of S16_pUCP18k_Phsp_GFP . To inhibit the leaky expression from pETDuet_NicR2, we added 1 g/liter glucose into the first group, of which the AU value of FI/OD600 was much higher than those for the other two groups. We constructed another strain DH5α-pUCP18K_Phsp_GFP to detect the influence of glucose and IPTG to the promoter activity in the absence of NicR2. The AU values of FI/OD600 of DH5α-pUCP18K_Phsp_GFP were all around 7,800 with glucose and IPTG, which means that the fluorescence signal of this strain is unrelated to the glucose and IPTG  . This stf sites) . HoweverPhsp_GFP . We alsoand IPTG . All of 10.1128/mBio.00602-19.4FIG S4Phsp promoter activity at different NicR2 concentrations. Two reconstructed strains were employed, S16_pUCP18k_Phsp_GFP (negative control) and S16_pUCP18k_Phsp_GFP/pME6032-NicR2 (with plasmid expressing NicR2). S16_pUCP18k_Phsp_GFP/pME6032-NicR2 was used to detect Phsp promoter activity at a higher NicR2 concentration in strain S16. Download FIG S4, TIF file, 1.3 MB.Detection of Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00602-19.5FIG S5Phsp promoter activity at different NicR2 concentrations. Two reconstructed strains were used, DH5α-pUCP18K_Phsp_GFP (negative control) and DH5α-pUCP18K_Phsp_GFP/pETDuet_NicR2 (with plasmid expressing NicR2). Strain DH5α-pUCP18K_Phsp_GFP was constructed for the absence of NicR2. Each strain was detected at 1 g/liter glucose and at 0 and 0.2 mM IPTG, respectively. Download FIG S5, TIF file, 1.3 MB.Detection of Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DK = 16.2 ± 1.9 μM), but it was not as strong as that for the free NicR2+HSP group (DK = 4.95 ± 0.48 μM) . For example, in the nicotine acid degradation pathway, which overlaps in part with nicotine degradation along the pyrrolidine pathway, NiaR and BpsR can be inhibited by nicotine acid or its intermediate , 19. How0.48 μM) . This inagonists , similar10.1128/mBio.00602-19.6FIG S6FIG S6, TIF file, 0.9 MB.In a previous study, structural comparison of the HSP-bound form (green) and the ligand-free form (magenta) of NicR2 reveals little conformational change . DownloaCopyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo compared to HSP  and R2 (A) are responsible for the homotropic effect between the two NicR2 dimers.Our previous studies on the pyrrolidine pathway of nicotine degradation in ng genes , to dissng genes  8). In . In P. pPspm and the Phsp promoters are exposed to RNA polymerase, and transcription of the genes of the degradation cluster is activated. The HspB enzyme is one of the transcriptional products of the cluster and catalyzes the reaction of HSP into 2,5-dihydroxy-pyridine. This decreases the concentration of HSP and eventually allows transcription to resume inhibition by NicR2  the interaction between NicR2 and effectors HSP and SP, and (iii) the interaction between NicR2 and RNA polymerase. The model reveals an integrated process from association between free NicR2 and two promoters to dissociation of the NicR2-promoter complex. This study offers an overall view of the regulatory mechanism of nicotine degradation in Pseudomonas and enriches our understanding of molecular mechanisms in biodegradation of environmental pollutants and toxicants.In conclusion, the regulatory model was summarized and proposed , which cl-(–)-Nicotine (≥ 99% purity) was obtained from Fluka Chemie GmbH (Switzerland). 3-Succinoyl-pyridine (SP) was purchased from Toronto Research Chemicals (Canada). 6-Hydroxy-3-succinoyl-pyridine (HSP) was purified via a previously described protocol (protocol . All othPseudomonas putida S16 (DSM 28022) was cultured overnight in citrate or nicotine medium, and total RNA was extracted using an RNAprep pure cell/bacteria kit , as previously described (P. putida S16 was used as the positive-control group template.escribed . GenomicDNA-protein binding kinetics were measured using the Octet RED96 System (ForteBio). In the preparatory stage, 5′-biotin-TEG-labeled duplex oligonucleotide probes representing the A or G alleles of rs7279549 were immobilized on streptavidin-modified senor surfaces in DNA solutions at a fixed concentration (1 μM). In phase 1, protein interacted with the DNA immobilized senor surface in the dilution buffer for 300 s. In phase 2, phosphate-buffered saline/Tween (PBS-Tween) buffers were used to elute the DNA-protein compound from the senor surface. This phase was sustained for 300 s.nic2 gene cluster were identified using a 5′-RACE system (Invitrogen). First-strand cDNA was amplified by the spm-GSP1 primer, after which terminal transferase and dCTP were applied in the tailing treatment of cDNA. The dC-tailed cDNA was amplified using the abridged anchor primer (AAP) and spm-GSP2, and a nested PCR with AAP and primer spm-GSP3 was performed using this PCR product as the template. This amplification product was then cloned into the pMD18-T vector  for confirmation by sequencing. All assay primers are shown in The transcriptional start sites of the 10.1128/mBio.00602-19.7TABLE S1Table S1, XLSX file, 0.01 MB.Primers used in this study. Download Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. putida strain S16△spmABC-pME6522-Pspm was cultured in citrate, citrate with the addition of 1 mM SP, and citrate with the addition of 1 mM HSP, respectively. O-Nitrophenyl-β-d-galactopyranoside was added during the β-galactosidase activity assay. Data were normalized to the OD600 and are expressed in Miller units . P. putida S16 was cultured overnight in citrate medium, and the harvested cells were resuspended with PBS and disrupted by sonication in an ice-water bath. The insoluble material was removed by centrifugation . The crude enzyme preparation and the treated beads were then mixed, followed by incubation at room temperature for 30 min. Magnets were used to collect the beads, and protein was released from the beads by boiling in a water bath for 5 min. SDS-PAGE was performed for the detection of the sample using a 12% gel in a MiniProtean III electrophoresis cell (Bio–Rad). The single bands were cut and characterized by matrix-assisted laser desorption ionization--time of flight mass spectrometry.The promoter P. putida S16 genomic DNA. Protein NicR2 was purified as previously described , and electrophoresed at 170 V (constant voltage) for 50 min in an ice bath. The gel was stained immediately with SYBR green I according to the manufacturer’s instructions .EMSA was performed as previously described . All DNAescribed . The staSP and HSP were tested as potential small molecule effectors by EMSA. Two different methods were applied: the first method involved incubation of NicR2 with the putative effector for 15 min at room temperature prior to DNA fragment addition and a 30-min incubation, and the second method involved mixing the DNA fragment and NicR2 prior to the addition of an effector.200 instrument . The reaction cell was filled with 64 μM NicR2 solution, and the titration was performed with an initial 0.4-μl injection of 100 μM a given DNA fragment, followed by 19 injections of 2 μl of the DNA fragment spaced at 2-min intervals. Titrating buffer was used as the control. The binding stoichiometry (N) value and the equilibrium dissociation constant (DK) were calculated using Origin 7.0 software.DNA fragments and NicR2 were prepared in PBS buffer just before the ITC assay. All DNA fragments, including the inverted repeat regulatory sequence (5′-CACTAAAGCGCCCGTCGTACATATAATAAA-3′), were 30 bp in length. All samples were degassed with vacuum aspiration for five min before analysis with an ITCPhsp_GFP is a GFP-reporter plasmid. pETDuet_NicR2 and pME6032_NicR2 are NicR2 expression plasmids which can be induced by IPTG. Two reconstructed strains were used to detect the promoter activity at different NicR2 concentration in strain S16, S16_pUCP18k_Phsp_GFP and S16_pUCP18k_Phsp_GFP/pME6032-NicR2. S16_pUCP18k_Phsp_GFP is the negative control. The strains were cultured at 30°C for 12 h with suitable antibiotics and 0.2 mM IPTG.pUCP18K_Phsp_GFP and DH5α- pUCP18K_Phsp_GFP/pETDuet_NicR2, were used to detect the promoter activity at different NicR2 concentrations in strain DH5α. Each strain was cultured with different IPTG concentrations, i.e., 0, 0.2, and 1 mM, respectively. The strains were cultured at 30°C for 12 h with suitable antibiotics. Fluorescence signal and OD600 values were detected by using an ENSPIRE 2300 multimode plate reader .Two reconstructed strains, DH5α- pUCP18K_10.1128/mBio.00602-19.9TABLE S3Table S3, XLSX file, 0.01 MB.Function identification of the bases on the IR by ITC. Download Copyright © 2019 Hu et al.2019Hu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Acinetobacter baumannii (A. baumannii) infected burns.Burn wounds are one of the most important health problems all over the world because infection after burn can delay wound healing. Treating burn wounds with granulocyte-colony stimulating factor (G-CSF) is known to improve healing of injured tissue. In addition, colistin is prescribed as an effective treatment. The aim of this study was to evaluate the effect of G-CSF and colistin alone or in combination with G-CSF on wound healing of A. baumannii. In a 7-day period, burn wounds in each group were daily treated with subcutaneous injections (0.1 ml) of saline, G-CSF, colistin, and G-CSF plus colistin. After killing the animals, the size of the wound, number of leukocytes in the skin and microbial growth were evaluated. A value of p ≤ 0.05 was considered statistically significant.This study was performed between January 2016 and April 2018. Burn wounds were experimentally induced in 36 mice. The wounds were inoculated with th day of treatment, number of positive bacterial cultures in the colistin and the G-CSF plus colistin groups was lower than other groups with a significant difference.Wound healing in the G-CSF plus colistin group was significantly higher than the control group and the G-CSF group . In G-CSF+colistin group, the number of leukocytes was higher than the control group considerably (P = 0.007). On the 7Concurrent consumption of G-CSF and antibiotics can control burn infection and enhance the immune system towards wound healing. Acinetobacter baumannii is known as an important pathogen in nosocomial and burn infections in recent years  is a cytokine, which has been used to reverse neutropenia in some cases such as catatonic chemotherapy and bone marrow and hematopoietic stem cell transplantation , 10. Stubaumanni . Therefoin vitro and in vivo and the following laboratory steps were carried out:This study was performed in two steps, A. baumannii were collected from burn wounds in patients of Motahari Hospital in Tehran which is affiliated to the Iran University of Medical Sciences. The samples were identified using standard diagnostic biochemical tests.A total of 30 isolates of A. baumannii strains was checked for 8 different antibiotic disks. These include ceftazidime (30 μg) gentamicin (10 μg), amikacin (30 μg), imipenem (10 μg), cefipime (30 μg), chloramphenicol (30 μg), ciprofloxacin (30 μg) and tetracycline (30 μg), . In this study, A. baumannii ATCC 19606 was used as the reference strain for quality control.Antimicrobial susceptibility and resistance were determined using disk diffusion method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) . Antibacin-vivo step. Antibiotic susceptibility was determined by the diameter of the inhibition of growth based on the CLSI guidelines.Results were interpreted as susceptible, intermediate, or resistant according to the guidelines . In the Thirty six adult male Balb/c mice with similar age (18–20 month) and weight (20 ± 2g) were transferred from Pasteur Institute of Iran to animal laboratory of Iran University of Medical Sciences. Animals were healthy and did not have any particular illness. They were handled and taken care of, based on standard principles . The aniMice were randomly placed in 4 groups which had 9 members each .The mice were anesthetized using 10 mg/kg of ketamine and 4 mg/kg of Xylazine intraperitoneally. Afterwards, the dorsal area of the animals was shaved and disinfected by ethanol 70% . For indA. baumannii (1.5 × 108 CFU/mL) and as it was reflected in Twenty four hours after the induction of burns, the wounds were inoculated with 1 ml suspension of To evaluate the improvement process in mice, general conditions of the animal including weight, state of the immune system and also wound condition were investigated.To assess the general conditions of the animals, all of them were weighed on the first and the last day of treatment process . The meath (last) day of post treatment and the following evaluations were conducted:All animals were killed by overdose of ether anesthetics on the 7th day (after the treatment: last day) by measuring the area of the wounds with the naked eye  and on the 7aked eye . The perth day of treatment by a cell counting device (cell Counter Refreshed Sysmex kx2).To survey of condition of the immune system, immune cells were counted . Therefoth day of treatment, the surface layer of the lesion was removed by a wet sterile swab in all mice and was cultured on blood agar medium and incubated at 37°C. The cultures were checked for A. baumannii using colony morphology, microscopic Gram-stain investigation and standard biochemical tests after 24 h  and were followed by Tukey–Kramer test. A value of p≤0.05 was considered to be statistically significant.A. bumannii are shown in Evaluation of antimicrobial susceptibility and resistance the antibiotic resistance patterns of 30 clinical samples of In order to determine the amount of general improvement each mouse had experienced, all animals were weighed daily (based on gr) in each group. The mean weight of the first and the last day were calculated in each group and were compared between the studied groups. Result showed that in G-CSF+colistin group, the mean of weight of animals on the last day was higher than the mean of weight of them on the first day but not significant .Burn wounds were evaluated starting 24 hours after the induction of burn and also on the last day after treatment. Percentage of wound recovery in all treated groups was compared to other groups . The resAs A. baumannii after 24 h. Percentage of positive and negative bacterial cultures were calculated for all groups simultaneously ).A. bumannii isolates were resistant to imipenem, cefipime, ciprofloxacin, ceftazidime and amikacin * and the G-CSF group (P = 0.033)* in The new therapeutic methods improve the immune system and remove the infection for wound healing. G-CSF can help in the wound healing by reducing translocation and colonization of the bacteria, and increasing blood neutrophils , 24. In A. baumannii . Bacterial infections in burn wounds might appear to have a higher prevalence rather than other forms of trauma, because skin is the first line of defense against infections and is the place where the first alterations of cellular and humoral immune responses happen (th day of treatment after the injury induction (sig: 0.000)* * in . In this000)* in . The resIn conclusion, G-CSF can be effective in wound closure by controling the infection and improving the immune system. In fact, combination of antibiotic and G-CSF if used appropriately can control the infection through colistin and improving the immune system through G-CSF.