Overall, 187 LN metastases (n = 43 patients) detected by 68Ga-PSMA-PET-CT were characterized by calculating maximum standardized uptake value (SUVmax), area, diameter and anatomical location including iliac, obturator, presacral and inguinal region. MRI achieved an overall sensitivity, specificity, positive and negative predictive value of 81.6% (CI 71.1–88.9%), 98.6% (CI 97.6–99.2%), 73.5% (CI 52.1–87.6%) and 99.5% (CI 98.8–99.8%), respectively. On a region-based analysis, detection rates differed non-significantly (ps > 0.12) in the anatomical regions. On a size-dependent analysis, detection of LN > 10 mm did not differ significantly (ps > 0.09) from LN ≤ 10 mm. In comparison to single T1 sequence evaluation, additional use of the T2 weighted sequences did not improve the overall performance significantly (p > 0.05). 3T prostate MRI represented an accurate tool for the detection of LN compared to 68Ga-PSMA-PET-CT. Especially for LN metastases smaller than 10 mm, MRI was less accurate compared to 68Ga-PSMA-PET-CT.The aim was to assess the performance of prostate 3T MRI for pelvic lymph node (LN) staging in prostate cancer (PCa), in comparison to  Therapeutic strategies are highly dependent on the assessment of the lymph node (LN) status, since it affects the therapy regimen and overall prognosis in newly diagnosed PCa2. Prior to radical prostatectomy, current guidelines recommend CT or MRI for local and LN staging as well as bone scintigraphy for osseous metastasis for patients with intermediate to high risk of recurrence according to D’Amico’s classification3. Even though choline PET is associated with certain limitations regarding the sensitivity and specificity for LN detection, it has been established as part of the clinical routine in certain centers4. Regarding MRI, especially T1 and T2 sequences are known to provide excellent anatomical information to demarcate structures and are commonly used for evaluation of local tumor infiltrations for instance in the seminal vesicles as well as local LN evaluation12. With the introduction of 3T scanners into the clinical setting, higher signal to noise ratios could be realized and a more reliable detection of LN metastases could be achieved15.Prostate cancer (PCa) is the second leading cause of death in men and the most common tumor in men in the western hemisphere18. The prostate membrane antigen (PSMA) is a highly expressed protein located at the cell surface of PCa cells and the use of 68Gallium radiolabeled PSMA inhibitor Glu-urea-Lys(Ahx)-HBED-CC showed promising results in detection of primary and recurrent disease20.Recently, PET was put back into focus for PCa staging, when novel probes targeting cell-surface receptors of PCa cells were introduced68Gallium-prostate specific membrane antigen PET-CT (68Ga-PSMA PET-CT) as reference standard for LN detection.The aim of this study was to assess the performance of clinical routine prostate 3T MRI for pelvic LN staging in PCa, compared to 68Ga-PSMA PET-CT with a mean area, size ratio and SUVmax of 0.9 ± 1.1 cm2 (range 0.1–9.2 cm2), 0.7 ± 0.2 (range 0.3–1.5) and 10.2 ± 11.5 (range 0.7–75.5). MRI detected 146 LN with a mean area of 1.0 ± 1.2 cm2 (range 0.1–9.1 cm2) and a size-ratio of 0.8 ± 0.5 (range 0.3–3.5). MRI achieved an overall sensitivity, specificity, PPV and NPV of 81.6% (CI 71.1–88.9%) 98.6% (CI 97.6–99.2%), 73.5% (CI 52.1–87.6%) and 99.5% (CI 98.8–99.8%). 41 false negative LN had a mean area, size ratio and SUVmax of 0.4 ± 0.3 cm2 (range 0.1–1.8 cm2), 0.7 ± 0.1 (range 0.4–1.0) and 6.1 ± 3.8 (range 0.7–18.9) in 68Ga-PSMA-PET-CT. 1130 true negative LN were measured in 68Ga-PSMA-PET-CT with a mean area of 0.5 ± 0.3 cm2 (range 0.1–3.5 cm2) and a size-ratio of 0.7 ± 0.2 (range 0.2–4.5). An overall of 35 false positive LN were detected in MRI with a mean area of 1.1 ± 0.7 cm2 (range 0.2–3.0 cm2) and a size-ratio of 0.7 ± 0.2 (range 0.4–1.0). Characteristics for all 1317 LN in 68Ga-PSMA-PET-CT are presented in Table Of 130 patients, 43 patients harbored 187 LN metastases in We thus were interested in possible impacts, that may have altered the results. The time between the two measures PET-CT and MRI varied between 0 to 180 days but the diagnostical accuracy did not suffer significantly from longer time intervals between the scans (χ²(2) = 1.2, p = 0.55). The impact of patients with multiple lesions to diagnostic accuracy was not significant (χ²(2) = 2.1,p = 0.36) and was deducted in all results given.In the analysis dependent on anatomical regions the sensitivities differed between 66.8% and 100% while specificities ranged between 97.1% and 100%. The highest detection rate was presented in the inguinal region with a 100% (CI 0–100%) followed by the obturator right and left region with 88.8% (CI 75.0–95.5%) and 84.9% (CI 62.8–95.0%). Presacral LN were detected with a sensitivity was 82.0% (CI 50.0–95.4%). The lowest sensitivity was presented in the iliac region with 80.7% (CI 60.0–92.1%) for the left and 66.8% (CI 46.6–82.2%) for the right region. Next, we evaluated possible differences in MRI diagnostical accuracy in the different anatomical regions. The six regions had a significant additional impact on diagnostical accuracy beyond the MRI judgments (χ²(10) = 18.7, p < 0.05). However, the source of this impact could not be pinpointed, as all single interaction effects were non-significant in logistic regression (ps > 0.12). Differences in the diagnostical accuracy were calculated relative to the iliac left region, which was arbitrary and was non-significant as can be seen at the overlapping CI of the sensitivities and specificities throughout the regions. Please refer to Table To assess a possible impact of histopathologic data to the readings, an interaction test was performed in this study. The diagnostical accuracy of MRI was significantly influenced by the results of the biopsy (χ²(1) = 4.8, p < 0.05). Sensitivity increases with greater Gleason scores from biopsy, i.e., p = 0.21 for a Gleason score of 6 and p = 0.97 for a score of 10. Gleason scores at the time of biopsy ranged from 6 to 10 with a mean of 7.8. Due to missing final Gleason scores after prostatectomy in most patients, no reliable statements could be made apart from biopsy.2 (range 0.1–9.1 cm2) and a mean size ratio of 0.8 ± 0.2 (range 0.3–1.) in MRI. False negative LN had a mean area, size ratio and SUVmax of 0.5 ± 0.3 cm2 (range 0.1–1.8 cm2), 0.7 ± 0.2 (range 0.3–1.5) and 6.4 ± 4.0 (range 0.7–18.9) in 68Ga-PSMA-PET-CT. False positive LN had a mean area of 1.2 ± 0.7 cm2 (range 0.3–3.0 cm2) and a size ratio of 0.7 ± 0.2 (range 0.4–1.0) in MRI. The sensitivities in single T1 sequence evaluation differed between 65.0% and 100% while specificities ranged between 97.7% and 100%. The highest detection rate was presented in the inguinal region with a 100% (CI 0–100%) followed by the presacral region with 82.3% (48.8–95.8%). LN in the obturator left region were detected with a sensitivity of 75.9% (CI 50.0–91.0%) followed by iliac left with 73.1% (CI 48.9–88.5%) and the obturator right region with 67.5% (CI 47.2–83.0%). The lowest sensitivity was seen in the iliac right region with 65.0% (CI 43.2–81.9%). Although the sensitivity from T1 alone was lower than combined with T2 + DWI, this difference was not significant, as can be seen from the overlapping CIs between single T1 versus T1 combined with T2 and DWI sequence evaluation. Please refer to Table Since LN detection in MRI is commonly related to T1 sequence evaluation, we assessed the performance of T1 plus T2 and DWI compared to single T1 sequence evaluation. 130 LN were detected in the single T1 evaluation presenting an overall sensitivity, specificity, PPV and NPV of 71.8% (CI 58.2–82.3%), 99.0% (CI 98.3–99.5%), 72.2% CI 47.9–88.0%) and 99.5% (CI 98.5–99.8%). Like T1 + T2, single T1 sequence evaluation displayed substantial diagnostical accuracy. Mean area was 1.0 ± 1.2 cm7.9–88.0%68Ga-PSMA-PET-CT. In the region-based analysis, the performance of MRI was not significantly (p > 0.05) different throughout the anatomical regions. The highest detection rate for MRI was achieved for the inguinal, obturator and presacral region, while the lowest sensitivities were achieved in the iliac regions. This may be owed to the challenging anatomical conditions and flow artifacts resulting from these vessels. Detection rates were higher in areas where LN can be better delineated from surrounding structures. Figures This study demonstrated that high resolution 3 T prostate MRI represents an accurate tool for the detection of LN metastases. Especially for LN metastases smaller than 10 mm, MRI was less accurate compared to In comparison to single T1 sequence evaluation, T1 combined with T2 and DWI sequence evaluation showed no statistically significant (p > 0.05) higher overall sensitivity and in the region-based analysis, underlining the relevance of sole T1 sequence evaluation. An example of additional T2 sequence evaluation is presented in Fig. On a size-based analysis, LN ≤ 10 mm in the iliac, obturator and presacral region were frequently missed, whereas LN ≤ 10 mm were detected more reliable in all anatomical regions but no statistically significant (p > 0.05) lower detection rate was seen for LN ≤ 10 mm compared to LN > 10 mm. Similar non-significant (p > 0.05) effects were found for single T1 sequence evaluation showing no significant additional impact of T2 evaluation based on LN size. For additional details please refer to the supplementary dataset.68Ga-PSMA-HBEDD-CC is an inhibitor of the glutamate carboxypeptidase II, labelled with 68Gallium21. PSMA is a cell-surface transmembrane protein found in the prostate, brain, lacrimal and salivary glands, tumor neovasculature, tubules of the kidney and intestine22. It is a 110 kDa highly glycolysated peptidase and belongs to a family of zinc-dependent exopeptidases with glutamate carboxypeptidase activity23. It is highly active in prostatic intraepithelial neoplasia and metastatic PCa22. As of today, only few numbers of studies assessed the performance of 68Ga-PSMA PET-CT for LN staging in PCa patients and the diagnostic accuracy was tested regularly against histopathology as standard reference. Recently, Hamed et al. presented a prospective study with 106 out of 165 patients presenting with local recurrence and extraprostatic metastases, in which sensitivity, specificity and accuracy of 68Ga-PSMA was 99.0%, 100% and 98.8%, respectively, compared to histopathology24. In 2016, a study was published by Herlemann et al. on 34 patients undergoing a 68Ga-PSMA PET-CT prior to pelvic lymph node dissection (PLND) reporting an overall sensitivity, specificity, NPV and PPV of 84.0%, 82.0%, 84.0% and 82.0%, respectively25. Subsequent studies also demonstrated favorable detection rates for 68Ga-PSMA PET-CT in comparison to histopathology27. These results underline the value of this imaging technique, while PLND is an invasive procedure associated with perioperative risks such as lymphedema and venous thromboembolism28. Another major limitation of histopathological assessment is the sampling error8. Skip metastases near the common and internal iliac vessels can be missed due to the limited exploration in the surgery8.29. Another input is set through the extranodal extension of LN metastases which is defined as a perforation of the LN capsule resulting in an expansion into extranodal tissues30. In 1998 Cheng et al. published a study including 269 patients, with LN metastases, presenting a significant correlation between nodal cancer volume and Gleason score recommending, that the diameter of the largest LN should be evaluated as prognostic factor of progression to distant metastasis rather than the number of LN30.In a follow-up study in 2000, Cheng et al. investigated the distant metastasis free and cancer-specific 5 year survival showing that extranodal extension was not significantly (p > 0.05) associated with distant metastasis free and cancer-specific 5 year survival in contrast to nodal cancer volume which was significantly (p > 0.05) associated with poorer 5 year survival31. A meta-analysis published in 2017 by Luchini et al. investigated the risk of recurrence through extranodal extension resulting in an elevated risk for patients with extranodal extension in PLND to develop a biochemical recurrence or distant metastasis32.Histopathological evaluation of LN is mainly dependent on morphologic criteria such as enlarged diameter or rounded LN or an increased LN volume12. The performance for LN staging is still considered to be challenging12. A meta-analysis of Hövels et al. included 24 studies with a mean sensitivity and specificity of 39.0% and 82.0% with ranges of sensitivities and specificities of 6.0–83.0% and 65.0–99.0%, respectively for LN detection by MRI prior to PLND while magnetic field strength however remained unknown for all investigated studies5. Sensitivity, specificity, PPV and NPV of 71.4%, 94.7%, 62.5% and 96.4%, respectively, were presented in a study by Kim et al. in 2010 for non-contrast enhanced T1 and T2 sequences of 1.5T MRI using surface coils for LN staging in comparison to histopathology6. In 2017, Gupta et al. reported sensitivity, specificity, PPV and NPV of 25.9%, 98.6%, 70.0% and 91.4%, respectively for LN detection using non contrast enhanced 1.5T MRI9.Today, MRI of the prostate is a clinical routine and highly sensitive imaging procedure for staging of patients and detection of extracapsular and seminal vesicle infiltration due to its excellent anatomical resolution5. In 2016, Barchetti et al. compared the performance of 1.5T MRI with non-contrast enhanced T1 plus T2 sequences in comparison to 18F-Choline PET-CT exams in 152 patients with biochemical recurrence reporting a sensitivity, specificity, PPV and NPV of 98.0%, 99.0%, 97.0% and 98.0%, respectively10. This good performance of 1.5T MRI has to be seen in the context of the chosen reference standard. Tulsyan et al. examined the usefulness of 68Ga-PSMA PET-CT in 36 patients with a biopsy proven PCa with a minimum Gleason score of 8 and PSA blood levels >20 ng/ml for LN staging in comparison to non-contrast enhanced 1.5T MRI11. 29 out of 36 patients presented LN in 68Ga-PSMA PET-CT in contrast to 20 MRI positive patients, which resulted in a concordance of 72% between both modalities without sensitivities or specificities given in the manuscript11.Since area, LAdm, SAdm and size ratio are major signs for malignancy in MRI, high resolution 3T MRI showed an improved detection rate of small LN ≤ 10 mm in our study. Interestingly, a generally lower threshold for malignancy (LAdm of <10 mm) presented a better performance in comparison to histopathology than a higher threshold in a previous study, which is comparable to our findingset al. published a study on recurrent PCa after failure of primary radiation therapy using histopathology as reference standard for LN detection by contrast enhanced 3T MRI applying an endorectal coil. They reported a sensitivity and specificity of 60.0% and 85.7%, respectively, for an unknown number of LN15. A further study from Zhang et al. compared contrast enhanced multiparametric MRI at 3T and 68Ga-PSMA PET-CT against histopathology for a cohort of 42 patients prior undergoing radical prostatectomy with PLND and found to be equal regarding the diagnostic accuracy14. A total of 51 LN out of 621 resected LN were defined malignant in histopathology and sensitivities, specificities, PPV and NPV of 96.1%, 99.5%, 94.2% and 99.7% for MRI and 96.1%, 99.7%, 96.1% and 99.7% for 68Ga-PSMA PET-CT were reported14. In our study 68Ga-PSMA-PET-CT was set as reference standard with an overall sensitivity, specificity, PPV and NPV of 81.6%, 98.6%, 73.5% and 99.5% for MRI.Regarding the use of 3T MRI, Zattoni et al. did not present LN detection rates according to anatomical regions while showing a higher overall detection rates with comparable results in the size-based analysis compared to our study. This may be due to the rather small cohort of 42 patients and the elevated mean PSA blood level with 52.3 ng/ml in Zhang et al. compared to 15.8 ng/ml in our study. Furthermore, the delay between PET-CT, MRI and surgery remained unknown. Moreover, the used malignancy criteria for MRI in Zhang et al. were SAdm > 10 mm, a rounded LN with a SAdm > 8 mm, increased contrast enhancement or a diffusion restriction in DWI and ADC map14. Our study chose a stricter malignancy criteria with a LAdm excess of 10 mm or a rounded LN defined through the quotient of SAdm divided through LAdm was present. In contrast to Zhang et al., the investigated MRI sequences in this study did not include the examination of DCE sequences and no use of contrast agent in our study, since gadolinium is associated with certain risks including nephrogenic systemic fibrosis especially in patients with chronic kidney disease33.In contrast to our study, Zhang et al. compared to our study. The comparable results of our study underline the validity of 68Ga-PSMA-PET-CT as reference standard compared to histopathology.In summary, higher PSA blood levels may indicate more advanced tumor stages, the unknown delay of the procedures, the additional use of DCE sequences and contrast agent may have resulted in the slightly higher detection rates in Zhang 68Ga-PSMA PET-CT was performed. A delay of up to 180 days between both imaging techniques might have influenced the size of the LN, but because PCa is a slow growing cancer entity, this time delay was considered of minor relevance. The performance of MRI detection was shortened through challenging localization near big vessels, and therefore interobserver variability cannot be excluded. Pitfalls of physiological tracer uptake were limited through parallel evaluation in CT. Size variations or necrosis of LN may have biased the examination as well. Given that size is a determining factor of malignancy in MRI, the size-based analysis could be biased leading to better sensitivities for LN > 10 mm compared to LN ≤ 10 mm. To limit this effect, the examination was done by two experienced readers and a size ratio was included to detect small but malignant LN based on a previous study in T1 sequences29.Limitations are the retrospective character of this study and the relatively small number of patients involved. No histopathological confirmation of the metastases seen in 68Ga-PSMA PET-CT. Especially for LN metastases smaller than 10 mm, MRI was less accurate compared to 68Ga-PSMA-PET-CT. The authors suggest that 68Ga-PSMA-PET-CT should be used for primary lymph node staging and for patients with biochemical recurrence.In conclusion, high resolution 3T prostate MRI represents an accurate tool for the detection of LN metastases. The detection rates of MRI were lower for metastases in complex anatomical regions, compared to 68Ga-PSMA PET-CT and a 3 Tesla prostate MRI within 180 days. 1170 patients received a 68Ga-PSMA PET-CT between October 2013 and May 2018. No MRI or external MRI was available in the local database in 855 cases. 185 cases exceeded the maximum time interval of 180 days. The remaining cohort consisted of 130 patients. All Patients had histological proven prostate cancer with mean age of 72.1 ± 7.5 years (range 51–89 years) and received a 68Ga-PSMA PET-CT and a 3T MRI within 72.7 ± 48.4 days (range 0–180 days). The mean delay between diagnosis and the first examined scan (PET-CT or MRI) in this study was 1.7 ± 3.4 years (range 0–17 years) with a median of 0 years. When multiple studies were present, the scans with the shortest delay were used in this investigation. All other examinations were not used as part of the reading. Patients in this study did not receive any surgery, radiotherapy, change in the regime of chemotherapy or change of hormonal treatment within the delay between both modalities. There was no artificial delay of treatment in the patients investigated in this study. Prostate specific antigen (PSA) blood levels were 15.8 ± 23.6 ng/ml (range 0.2–196 ng/ml) collected within 26.5 ± 43.0 days (range 0–193 days) to the 68Ga-PSMA PET-CT and a median core needle biopsy Gleason score of 8 (range 6–10) was reported. Patients characteristics are presented in Table The institutional ethics review board of the Department of Radiology at the Charité Universitätsmedizin Berlin, Germany approved this retrospective study and it was performed in accordance to current guidelines. The local database was screened for patients, who received a 68Ge/68Ga generator (Eckert and Ziegler) was used for elution of 68Ga prior to labelling with PSMA-HBED-CC 34. After injection of 129.0 ± 26.2MBq of 68Ga-PSMA-HBED-CC, a low dose CT for attenuation correction  and anatomical mapping was acquired within 89.0 ± 42.5 min immediately before the PET scan, using a Gemini TF 16 Astonish PET-CT scanner 35. All 130 patients underwent a non-contrast-enhanced prostate multiparametric MRI at our institution at 3T . Standard prostate MRI acquisition protocol included high resolution T2 weighted high resolution turbo spin echo sequences , including DWI sequences  with generation of an ADC map from b 0, b 160 and b 1200 images of the DWI. Native T1 high resolution isotropic volume sequences . No endorectal coils were used throughout all exams.A standard 68Ga-PSMA PET sequences were automatically fused for the evaluation process. Since the acquisition of a MRI of the pelvis is limited to a localized area while 68Ga-PSMA PET-CT covered the whole body, the aortic bifurcation was set as the upper border for positive LN in 68Ga-PSMA PET-CT. Consensus reading was performed by two readers. All images were analysed independently in a blinded and random order.Visage 7.1 (Visage Imaging) was used as the standard software package. Low dose whole body CT sequences and 29. Signal intensity was no criteria for definition of a LN as metastatic. The size ratio is reported throughout the manuscript. All LN were characterized in six levels according to the adjacent anatomical structures dividing them into presacral, inguinal LN and alongside the large arterial vessels. LN alongside the common and external iliac arteries were characterized as the iliac right and iliac left region as superordinate levels and LN near the internal iliac and obturator artery were characterized as obturator right and obturator left region. Please refer to Fig. For LN examination in 3T MRI data sets, T1 TSE, T2 TSE and DWI sequences were used. T1 sequences were used in the first step, followed by high resolution T2 TSE and DWI to assess their additional value. The diameter of suspicious LN were measured in axial planes through manual delineation of a ROI, resulting in size, long-axis diameter (LAdm) and short axis diameter (SAdm). SAdm was defined as the rectangular line of the LAdm. A LN was defined as positive in MRI when the SAdm divided through LAdm exceeded the size ratio of 0.8 or if the LAdm was ≥10 mm, based on a previous study on LN detection in T1 sequences68Ga-PSMA avid LN were measured in the axial plane at maximum diameter using the CT sequence for delineation of the ROI after evaluation of tracer uptake in 68Ga-PSMA PET overlay at isocontour of 50%. LN were defined positive, when an abnormal focal tracer signal with a higher signal intensity than the surrounding background was detected in 68Ga-PSMA PET and a LN in CT could be allocated to the signal14. LN size evaluation did not affect the definition of positivity in 68Ga-PSMA PET-CT, which is comparable to a previously published study14. In addition, maximum standardized uptake values (SUVmax) were assessed. All visible LN on CT without tracer uptake were measured in LAdm and SAdm to be defined as true negatives. All LN depicted in PET-CT were divided into the 2 subgroups LN > 10 mm and LN ≤ 10 mm. MRI sensitivity was calculated for both size groups and for each anatomical region. Please refer to Fig. All 68Ga-PSMA PET-CT was set as reference standard in this study.http://www.medcalc.org; 2017) and R software  was used for multi-level logistic regression. We used logistic regression in order to assess the diagnostic quality of MRI in contrast to our gold standard. The overall fit of a logistic regression model corresponds to the overall predictive accuracy and is as such related to comparable analysis techniques like ROC analysis. We utilized likelihood-ratio chi-square tests to calculate p-values within logistic regression. More specifically, we applied multi-level logistic regression to satisfy our hierarchical data structure (lymph nodes within patients) Multi-level regression adjusts for clusterings in data, i.e., for effects that some patients a conspicuous lymph node might be more likely to have more conspicuous lymph nodes36. Multi-level models include such effects and can even capture person-wise differences in diagnostic accuracy . In our analyses below, we tested for each predictive model whether such person-wise differences were statistically significant. The common properties of a diagnostical test  were calculated from logistic regression and confidence intervals (CI 95%) were given through logistic regression using the method proposed by Coughlin et al.37. When no confidence intervals could be calculated due to perfect agreement of MRI and PET-CT, it is highlighted in the tables. A p-value p < 0.05 was considered statistically significant.Descriptive statistics were done using MedCalc Statistical Software version 17.6 (MedCalc Software bvba; Dataset 1


Haplophyllum villosum (Rutaceae), a perennial herb from Iran, a new 4,8-diaryl-3,7-dioxobicyclo--octane type lignan, eudesmin A (1), together with four known compounds–eudesmin (2), haplamine (3), umbelliferone (4) and scopoletin (5)–were isolated from aerial parts of the plant. The structures of the compounds were elucidated using NMR spectral analysis  as well as UV, IR and MS spectra and comparison with previously reported data.During our phytochemical investigation of  Haplophyllum of the family Rutaceae is made up of about 70 species distributed worldwide, with about 30 of them being found in Iran. They are mainly found around the Mediterranean region of Europe and through western Asia up to Siberia ..Haplophypara-position of the aromatic ring is normally oxygenated with hydroxyl, methoxyl or methylenedioxy groups, but it is not the case with the current compound, as can be seen in the multiplicity of the signals and their couplings. This argument is further substantiated by a recent publication of a new 9-norlignan glycoside 6  and 6.87 (1H) as two broad singlets octane] and named eudesmin A (1).In lignans the coside 6  from Cessinglets . The con2) was also isolated from all three extracts as colorless prisms with m.p. 103–105 °C  was also isolated from all three extracts as colorless needles with m.p. 200–201 °C (+ . : see 13C-NMR : see Eudesmine (2). Colorless prisms with m.p. 103–105 °C . UV λmax nm (log ε), CHCl3: 281.1 (1.44) and 243.3 (1.94). IR νmax cm−1 (KBr disc): 2935, 2837, 1592, 1512, 1023, 748. EIMS m/z (% rel. int): 386 [M]+ (85), 355 (12), 219(22), 205 (10), 189 (28), 177 (68), 165 (100), 151 (72), 138 (35). 1H-NMR (CDCl3) and 13C-NMR (CDCl3): see –105 °C . The compound was recrystallized from CHCl3 to afford colorless needle-shaped crystals with a Rf value of 0.24 (98% CHCl3:2% MeOH) and m.p. 200–201 °C . Obtained as colorless needle-shaped crystals with m.p. 223–225 °C . Showed a blue fluorescent spot on TLC plate when visualized under long wave UV light developed with EtOAc:hexane (1:4) with a Rf value of 0.12. m.p and m.p. 200–203 °C -3,7-dioxabicyclooctane] (1) to which the trivial name eudesmin A has been assigned was isolated and identified from the aerial extracts of Haplophyllum villosum along with another lignan, a quinoline alkaloid and two coumarins. The crude extracts were found to be good free radical scavenger but none of the compounds exhibited such behaviour.A new lignan identified as [4,8-


Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 °C and pH 6.5 and maintained stability at a broad range of pH (5–7.5) and temperature (below 35 °C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides.Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium  Both Gldic bond . In addidic bond . Recent dic bond . Howeverdic bond . Recent dic bond ,9.Low molecular weight chondroitin sulfate (LMWCS), produced by the depolymerization of CS, has been shown to promote the absorption and ultimately increase the uptake of iron through LMWCS-mediated inorganic iron complexes in Caco-2 cells . In compd-GlcA and N-acetyl-β-d-GalNAc with the concomitant formation of an unsaturated C4–C5 bond within the GlcA that is located at the non-reducing end [Flavobacterium heparinum and shows a random endolytic mode of action by cleaving polysaccharide substrates. On the other hand, the second enzyme, ChSase AC II shows an exolytic mode of action by cleaving disaccharides from the non-reducing end of the polysaccharide chains one at a time [Enzymatic digestion has been an effective method for the preparation of bioactive CS oligosaccharide. In contrast to vertebrates, chondroitinase (ChSase) from microorganisms utilizes a β-eliminative mechanism to breakdown CS, splitting β-1,4-glycosidic bonds between β-cing end . Chondrocing end . ChSase t a time . Since tt a time .Arthrobacter sp. CS01. The characterization and analysis of degradation products were also performed. These products were suggested to have the potential for medical and industrial applications.In this study, a new ChSase AC II, was expressed and purified from a newly isolated marine bacterium Arthrobacter sp. LHYM225. According to the phylogenetic position of 16S rDNA . Analysis of the alignment of 16S rDNA gene sequences revealed that the identity of strain CS01 exhibited a 99% similarity to the 16S rDNA , CS01 waArthrobacter sp. CS01, we found an open reading frame  consisting of 2370 bp called ChSase AC II. The recombinant plasmids, pPIC9K-ChSase AC II and pPICZαA-ChSase AC II, were successfully constructed and confirmed by DNA sequencing and transformed into various P. pastoris for protein expression. The results showed that the enzyme activity of the recombinant ChSase AC II was highest in the P. pastoris X33 compared to the other strains  column, with specific activity of 18.7 U/mg and 53.4% recovery  [Bacteroides thetaiotaomicron (37 °C) [Bacteroides stercoris (37 °C) [Sphingomonas paucimobilis (40 °C) [B. thetaiotaomicron (7.1) [F. heparinum (6.8) [B. thetaiotaomicron (7.6) [S. paucimobilis (6.5) [The effects of temperature, pH, metal ions and surfactants on the enzyme activity of ChSase AC II were further determined. The ChSase AC II enzyme activity gradually improved with a rise in temperature from 25 °C to 37 °C and was optimum at 37 °C A. This w 40 °C) , Bactero (37 °C) , Bactero (37 °C) , and Sph0 °C , Baon (7.1) , F. hepaum (6.8) , B. theton (7.6) , and S. 2+, but strongly inhibited by Hg2+ which reduced it to 43.2%. However, Mn2+ markedly increased the activity to 115.78%  of 458.1, 458.2 and 779.4, which corresponded to the unsaturated disaccharide of CS-A (△Di-4S − H)—, CS-C (△Di-6S − H)—, and unsaturated tetrasaccharide of HA (o-HA4 − 2H + Na)—, respectively. To further confirm the changes in product formation and substrate consumption during degradation, the hydrolyzed samples were analyzed by high performance liquid chromatography (HPLC). As shown in As shown in Escherichia coli DH5α was used in all the cloning experiments. The plasmid pPIC9K, pPICZαA, wild-type P. pastoris strain GS115, KM71, and X33 were used for the eukaryotic expression of ChSase AC II. The different media used for strains cultivation are listed in The CS-degrading bacterial strains used in this study were isolated from the gut of sea cucumbers. The samples from the Weihai Sea Cucumber Farm  were immersed, homogenated, and diluted with sterilized seawater and spread on selective agar plates. The plates were incubated for 3 days at 28 °C to form detectable colonies. At least 100 strains were inoculated into an agar-free selective medium, and the ChSase activity in the culture supernatant was assayed. Among the isolates, the most active strain CS01 was selected for further studies. Genomic DNA from strain CS01 was extracted using a total DNA extraction kit , and then sequenced in Lingen Biotechnology Co., Ltd. . The 16S rDNA sequence was blasted and aligned with closely related sequences retrieved from GenBank using the BLASTn  and CLUSTAL X  program, respectively, and the phylogenetic tree was constructed with the MEGA 4.0 software .Ecor I site) and downstream primer: 5’-ATTTGCGGCCGCCTAGCGGTGCAGCGTGACCTC (the underlined sequence is the position of a Not I site). The purified ChSase AC II fragment was digested with Ecor I/Not I and ligated into the downstream primer of alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and pPICZαA. These were termed pPIC9K-ChSase AC II and pPICZαA-ChSase AC II, respectively. After digestion by the restriction enzyme Pme I, the linearized form of the plasmid pPIC9K-ChSase AC II was transformed into P. pastoris GS115 and KM71 by electroporation at 1.5 kV in a 0.2 cm gap electroporation cuvette, while the plasmid pPICZαA-ChSase AC II was transformed into P. pastoris X33. The recombinant plasmid pPIC9K-ChSase AC II was selected on MD plates containing 2 mg/mL Geneticin selective antibiotic (G418), and the recombinant plasmid pPICZαA-ChSase AC II was selected on YPDS plates containing 100 μg/mL zeocin. To express the ChSase AC II in P. pastoris, recombinants were grown in 50 mL YPD medium at 28 °C and 200 rpm. When OD600 reached approximately 4.0, the cells were collected by centrifugation at 4 °C and 10,000 rpm for 10 min, re-suspended in 200 mL BMGY medium and then incubated at 28 °C and 200 rpm for 72 h. Methanol was added to a final concentration of 0.5% at every 24 h to maintain the induction of ChSase AC II.To obtain the eukaryotic expression vector, the ChSase AC II gene was amplified by PCR using the following primers—upstream primer: 5’-CCGGAATTCATGACGCACGAAGTATCCCGACG-3’ . The purity of the fractions was assessed using SDS-PAGE, and the protein concentration was determined using Coomassie Brilliant Blue staining. ChSase AC II enzyme activity was measured by increasing the absorbance to 232 nm until double bonds were formed in the reaction. CS degradation was assessed using 20 mM Tris-HCl, pH 7.5, at 37 °C. A millimolar absorption coefficient of 5.1 L/(mol·cm) was used in the calculations. One international unit was defined as the amount of protein needed to form 1 µmol of 4,5-unsaturated uronic acid/min .max and Km of ChSase AC II were determined using a 0.03125–2 mg/mL substrate of CS-A, CS-C, and HA. The assay of enzyme activity was defined as described previously. The Km and Vmax values were calculated by using the linear regression plots of Lineweaver and Burk.The kinetic parameters, V2HPO4-citric acid buffer and from 7.0 to 9.0 in the 20 mM Tris-HCl buffer. To determine the thermostability of ChSase AC II, the residual activity was measured after incubating it at various temperatures (25 °C–50 °C) for 180 min. Meanwhile, the pH stability depended on the residual activity after the enzyme was incubated in buffers with different pH (4.5–9.0) for 6 h. The effect of metal ions and surfactants on the enzyme activity were analysed by incubating the purified enzyme at 4 °C for 6 h in the presence of the following reagents—10 mM KCl, NaCl, FeCl2, FeCl3, MnCl2, ZnCl2, CoCl2, BaCl2, HgCl2, CdCl2, MgCl2, CaCl2 and CuCl2, 0.5% (v/v) SDS, Tween-80 and EDTA.The optimal temperatures of ChSase AC II were found by measuring its activity to be from 25 °C to 60 °C. The optimal pH was determined to be from 4.5 to 7.0 in the 20 mM Nam/z 100–2000 amu). To determine the changes in product formation and substrate consumption during degradation, samples taken at different reaction times  were analyzed by the HPLC using PL aquagel-OH 30 column  under the following conditions—the mobile phase used a buffer composed of NaNO3 (0.2 M) and NaH2PO4 (0.01 M), the column temperature was 25 °C, the flow velocity was 0.5 mL/min, and a refractive index detector (RID) was used.To investigate the mode of action, the reaction mixtures (1 mL) containing 1 μg purified enzyme and 2 mg substrates  were incubated at 37 °C for 0, 5, 15, 30 min, 1, 2, 3, 4, 5 h. The degradation products were first analyzed by TLC with the solvent system (N-butanol/acetic acid/ethyl acetate/water 2:1:1:1) and visualized by heating the TLC plate at 120 °C for 10 min after spraying with 10% (v/v) sulfuric acid in ethanol. To further determine the composition and degree of polymerization (DP) of the products, the degradation products were analyzed by negative-ion ESI-MS  was cloned, expressed, purified, and characterized. The enzyme showed maximal activity at 37 °C and pH 6.5. It was stable at a broad pH range (5–7.5) and retained above 80% activity after incubation at 37 °C for 180 min. It had broad substrate specificity towards CS-A, CS-C and HA, suggesting the potential for it to be used as a candidate for industrial applications to produce oligosaccharides. In addition, further works should be focused on improving the enzyme activity and determining the catalytic direction of ChSase AC II.The ChSase AC II from a newly isolated marine strain (


Nymphaea tetragona Georgi (Nymphaceae) is traditionally used in Asia for the treatment of diarrhea, dysentery and fever. The plant contains various active compounds, including methyl gallate (MG) which are reported to inhibit bacterial virulence mechanisms. This study aimed to evaluate the alterations on viability, membrane potential and integrity of Salmonella enterica Serovar Typhimurium exposed to MG in combination with Tylosin (Ty), which is relatively inactive against Gram-negative bacteria, but it is commonly used as a feed additive in livestock. Besides, the effects of sub-inhibitory concentrations of the combination (MT) on the interaction between S. Typhimurium and the host cell, as well as on the indirect host responses, were characterized. Flow cytometry, confocal and electron microscopic examinations were undertaken to determine the effects of MT on S. Typhimurium. The impacts of sub-inhibitory concentrations of MT on biofilm formation, as well as on the adhesion, invasion and intracellular survival of S. Typhimurium were assessed. The result demonstrated significant damage to the bacterial membrane, leakage of cell contents and a reduction in the membrane potential when treated with MT. Sub-inhibitory concentrations of MT significantly reduced (P < 0.05) the biofilm-forming, adhesive and invasive abilities of S. Typhimurium. Exposure to MT drastically reduced the bacterial count in macrophages. Up-regulation of interleukin (IL)-6, IL-8 and IL-10 cytokine genes were detected in intestinal epithelial cells pre-treated with MT. This report is the first to describe the effects of MT against S. Typhimurium. The result indicates a synergistic interaction between MG and Ty against S. Typhimurium. Therefore, the combination may be a promising option to combat S. Typhimurium in swine and, indirectly, safeguard the health of the public. Salmonella enterica Serovar Typhimurium is one of the causative agents of food poisoning that is transmitted to humans from food animals, such as pigs and chickens. Pigs and chicken are asymptomatic carriers that harbor S. Typhimurium mainly in the intestine. Consumption or mishandling of contaminated pork and poultry products are the primary causes of gastroenteritis in human  Pte. Ltd.). Three hundred Nano molars of both the forward and reverse primers were used in 20 μL reaction mixture. The reaction conditions included denaturation at 95°C for 3 min, 40 cycles of amplification at 94°C for 10 s, 60°C for 30 s and melting from 65 to 90°C. The housekeeping genes β-actin and GADPH were used to normalize gene expression. The fold changes of cytokines gene expression were analysed using the 2-ΔΔCT method and the statistical difference were determined using the Student’s t-test in GraphPad Prism 6 . The sequences of all primers used in this experiment are listed in Infection of intestinal epithelial cells with ne genes . The effP < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism 6 . One-way and two-way analyses of variance (ANOVA) were conducted to compare the mean values among treatment groups. S. Typhimurium strains were 512, 1024 and 128 μg/mL, respectively. The MBCs of MG and PG were twice the MIC values in both strains; however, the MBC of Ty exceeded 4 mg/mL. Similar MIC values were obtained for both of the tested agents in RPMI and MEM.The MICs of MG, Ty and PG against the two MT was effective, as confirmed by the reduced MICs of 128 and 256 μg/mL for MG and Ty, respectively, for both strains. The FICI was calculated to be 0.5, indicating synergism between Ty and MG. Furthermore, an additive effect (FICI = 0.56) was found between Ty and PG, in which the MIC values were reduced to 64 μg/mL. Therefore, MT was chosen for subsequent experiments.in vitro time–kill activities of MT were performed for both strains  were observed after 4-h exposure to 1/2× MT (5.8 ± 1.2%) and 1× MT (21.2 ± 4.8%). In contrast, those grown in the presence of MG, Ty and 1/4× MT (green fluorescent stain) indicated that the cell membrane was intact.The confocal microscopic images of SYTO9/PI-stained S. Typhimurium, treatment with 1/2× MT and 1× MT caused a remarkable modification in morphology, such as rough surface (red arrow), membrane blebbing (yellow arrow) and leakage of cellular contents (blue arrow)  . Marked S. Typhimurium (ATCC 14028) that had been incubated with different concentrations of MT for 2 h was determined by measuring the ratio of red-to-green fluorescence intensity  lower when compared with fluorescence ratios of 0.80, 0.83 and 1.10 in MG-treated, Ty-treated and non-treated S. Typhimurium, respectively. However, treatment with 1/4× MT did not produce a significant change in the fluorescence ratio (P > 0.05).The MP of ntensity . The ratP > 0.05) in planktonic bacterial growth (mean OD600) in the presence and absence of sub-inhibitory concentrations of MG, Ty and MT. Particularly, MT reduced S. Typhimurium biofilm formation in a concentration-dependent manner. Biofilm formation by S. Typhimurium (ATCC 14028) treated with 1/8× MT and 1/4× MT was decreased by 57.3 and 55.9%, respectively  could cause half of the maximum inhibition (EC50) of S. Typhimurium (LVPP-STI15) biofilms while the EC50 value for S. Typhimurium ATCC 14028 biofilms was 1/12.5× MT (a combination of 10.24 μg/mL MG and 20.5 μg/mL Ty). Conversely, the maximum biofilm inhibitions caused by MG and Ty were 6.86 and 6.48%, respectively (P > 0.05) in the biofilm inhibition ability of MT between the two strains. Besides, neither the combination nor each agent dispersed the pre-formed biofilms of both strains.The study demonstrated an insignificant difference  (P < 0.05), 1/8× MT  (P < 0.0001) and 1/4× MT  (P < 0.001) relative to the infected and non-treated cells. The counts of adhered S. Typhimurium (ATCC 14028) in cells pre-treated with 1/8× MT and 1/4× MT were significantly lower relative to those pre-treated with MG (73.6%) (P < 0.01) and Ty (86.1%) (P < 0.001) alone. Moreover, the invasion of S. Typhimurium strains LVPP-STI15 and ATCC 14028 to Caco-2 cells was reduced in a concentration-dependent manner in cells pre-treated with 1/16× MT , 1/8× MT  and 1/4× MT  when compared with the infected and non-treated cells (P < 0.0001). The counts of invaded bacteria in cells treated with 1/4× MT and 1/8× MT was significantly reduced with respect to those of MG (P < 0.05) and Ty (P < 0.001) alone. Interestingly, the total counts of adhered (P < 0.05) and invaded (P < 0.01) bacteria reduced significantly in the presence of MG alone in comparison to infected and untreated cells, but to a lesser extent compared to MT.The effects of sub-inhibitory concentrations of MG, Ty and MT on the adhesion and invasion of S. Typhimurium was observed after incubation with various concentrations of MT for 4 and 8 h in comparison to MG and Ty alone. The bacterial load (ATCC 14028 and LVPP-STI15) in cells incubated with 1/16× MT, 1/8× MT and 1/4× MT for 8 h was decreased significantly (P < 0.001) by 79.14 and 88.6%, 79.1 and 81.99%, and 82.7 and 87.75%, respectively, compared with the load in the infected and non-treated cells. Interestingly, MG significantly (P < 0.5) decreased the intracellular S. Typhimurium count from 4 h of incubation, in contrast with Ty, as well as the infected and non-treated cells.After incubation for 2 h, treatment did not produce a significant difference in the percentage of surviving bacteria . HoweverS. Typhimurium induced the expression of IL-6, IL-8, IL-1β, TNFα and IL-10 genes in CaCo-2 cells after 8-h infection (P < 0.001) in the IL-6 gene expression was detected when infected cells were incubated with 1/8× MT and 1/4× MT compared to those of MG and Ty. The expression of IL-8 gene was significantly enhanced (P < 0.01) in 1/4× MT-treated cells. Treatment by ¼ x MT up-regulated the gene expression of IL-10 to a very moderate level (1.5X) by comparison to IL-6 (2.5X) and IL-8 (3X). However, neither MG nor Ty modified the genes expressions of the tested cytokines in infected cells. In addition, the effect of MT on S. Typhimurium-induced IL-1β and TNFα gene expression was not significantly different from those of MG and Ty alone. Moreover, the results of genes expressions of cytokines normalized to GADPH housekeeping gene exhibited similar patterns as shown on supplementary file (nfection . A conceary file .μg/mL) found in this study is not surprising because the outer membrane of Enterobacteriaceae, such as Salmonella, has low permeability to macrolides, including Ty, which limits the activities of these drugs [μg/mL) was comparable to the findings of Choi et al. [μg/mL) reported by Choi et al. [Salmonella isolates from chicken and pig. The difference of bacterial strains used between these studies could contribute to their difference in susceptibility to MG.The high MIC value of Ty Click here for additional data file.S1 FigS. Typhimurium (ATCC 14028) biofilm in the presence of methyl gallate (MG) (A) and tylosin (Ty) (B). The effect was not significantly different in S. Typhimurium LVPP-STI15 biofilm. Values represent mean ± SEM of four independent experiments.Crystal violet staining of (JPG)Click here for additional data file.S2 FigS. Typhimurium.Cytokine expression of IL-8 (A), IL-10 (B), IL-6 (C), IL-1β (D) and TNFα (E) in Caco-2 monolayers co-cultured with methyl gallate (MG), tylosin (Ty) and various concentrations of their combination (MT) during 8 h infection with  Cell: non-infected and non-treated control. Salmonella: infected, but non-treated cells. Results mean ± SEM of three independent experiments. MG = 32 μg/mL; Ty = 64 μg/mL; 1/16× MT = 8 μg/mL MG and 16 μg/mL Ty; 1/8× MT = 16 μg/mL MG and 32 μg/mL Ty; and ¼× MT = 32 μg/mL MG and 64 μg/mL Ty. *P < 0.05 compared to infected, but non-treated cells; τP < 0.05 compared to Ty; πP < 0.05 compared to MG; and #P < 0.05 compared to both MG and Ty.(JPG)Click here for additional data file.


Fixed amounts of CS with different metal matrix w/v ratios of 0.5%, 1.0%, 1.5%, and 2.0% for Mn and Zn precursors were used to synthesize ZnMn2O4–CS hybrid composites. X-ray diffraction analysis indicated the formation of polycrystalline tetragonal-structured ZnMn2O4 with a CS matrix in the hybrids. Fourier-transform infrared spectroscopic analysis confirmed the formation of ZnMn2O4–CS hybrids. Detailed investigations of the surface modifications were conducted using scanning electron microscopy; micrographs at different magnifications revealed that the composites’ surface changed depending on the ratio of the source materials used to synthesize the ZnMn2O4–CS hybrids. The antibacterial activity of the Mn3O4–CS and ZnMn2O4–CS composites was tested against various bacterial species, including Bacillus subtilis, Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa. The zone of inhibition and minimum inhibitory concentration values were deduced to demonstrate the efficacy of the ZnMn2O4–CS nanocomposites as antibacterial agents.The development of productive antibacterial agents from nontoxic materials via a simple methodology has been an immense research contribution in the medicinal chemistry field. Herein, a sol–gel one-pot reaction was used to synthesize hybrid composites of hausmannite–chitosan (Mn Since antiquity, even after many revolutionary clinical discoveries, human societies have faced tremendous obstacles posed by infection, which costs billions of dollars per year because of clinical difficulties and the lengthening of patient recovery times ,2. In caantibiotics was first used by Selman Waksman in 1941 to describe the antimicrobial agents created by certain microorganisms [The term rganisms ,7. Antibrganisms .The rise of pathogenic and putrefaction bacteria resistant to antimicrobial agents has become a major health concern. Biomedical researchers are therefore exploring pathways to improve existing antimicrobial agents. Antibacterial agents are bactericidal; they kill microorganisms by penetrating the wall or membrane of a cell of the pathogen and rendering it bacteriostatic  or by preventing its growth. The inhibition phenomenon of bacteriostatic agents involves preventing protein production or impeding bacterial vitality pathways ,10. BactAntibacterial agents are used in various applications such as water disinfection, medicine, the textile industry, and food packaging ,13. The Nanomedicines such as nanomaterials provide durable, targeted, and comprehensive antimicrobial activity at lower doses than conventional antibiotics . The nand-glucosamine polymer [Chitin, a naturally abundant biopolymer, can be obtained from crustacean shells and fishing-industry wastes . Chitosa polymer . Numerou polymer ,28,29.3O4) have been intensively investigated as potential antibacterial systems [3O4 is one of the most stable oxides and is used effectively as a catalytic material for the oxidative destruction of volatile organic compounds [3O4 is an excellent antioxidant and antibacterial system [The particle size and material type used to prepare nanoparticles are two important factors in tuning their antibacterial effectiveness ,21. Howe systems ,33. Inde systems . The gre systems . Mn3O4 iompounds . In addil system ,36,37. Vl system ,38,39.2O4–CS) with various concentrations of metal oxides. A comprehensive examination of antibacterial properties was carried out by zone-of-inhibition and minimum-inhibitory-concentration (MIC) studies against Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Salmonella typhi (S. typhi).In the present work, we focus on the sol–gel synthesis of hybrid systems of zinc manganese oxide–CS composites  with 0.5% (w/v) and purified chitin (0.25 g) liquefied 2% acetic acid (AA) were mixed in a beaker with constant magnetic stirring; the beaker solution was then transferred to hotplate at 70 °C for 2 h. Freshly prepared NaOH solution (45% w/v) was mixed dropwise till a brown precipitate formed, and the precipitate was allowed to settle as a residue at room temperature for 24 h. The excess fluid was removed, and the residue was washed several times and permitted to deposit for 30 min. The remnant was collected by vacuum filtration and dehydrated for 2 h at 110 °C. The resultant Mn3O4–CS composite is hereafter denoted as S1.Manganese chloride (MnCl2 and zinc chloride (ZnCl2) with equal w/v  and purified chitin (0.25 g) liquefied 2% AA were mixed in a beaker with constant magnetic stirring; the beaker was then transferred to a hotplate at 70 °C for 2 h. Freshly prepared NaOH solution (45% w/v) was added dropwise until the mixture formed a brown/black precipitate, which was allowed to settle at room temperature for 24 h. The excess fluid was removed, and the excess fluid was washed several times and allowed to deposit for 30 min. The remnant was collected by vacuum filtration and dehydrated for 2 h at 110 °C. The equal w/v concentrations of 0.5%, 1.0%, 1.5%, and 2.0% for MnCl2 and ZnCl2 were used to prepare the ZnMn2O4–CS composites as given in 2O4–CS composite products were stored for further characterization.MnClλ = 0.15406 nm); Fourier transform infrared (FTIR) spectroscopy ; photoluminescence (PL) spectroscopy, ; UV–vis spectroscopy ; and scanning electron microscopy .The synthesized nanocomposites properties were characterized by X-ray diffraction  with Cu Kα radiation  were rubbed onto the surface of MHA broth and cultured. Nanocomposites S1–S5 were added to various regions of the plates. Thereafter, the MHA broth plates were nurtured for 24 h at 37 °C and the zone of inhibition was estimated in millimeters. The micro dilution process experiments were carried out to assess the minimum inhibitory concentrations (MICs) for the hybrid composites. 96-well disposable plates were used for the micro dilution process. The serial dilutions of nanocomposites antibacterial agents in the liquid media were added to the microdilution trays bacterial suspension (~105 colony-forming unit/mL) and the plates were nurtured at 37 °C for 48 h. MICs were assessed as the lowest concentration of nanocomposites to inhibit the growth of the organisms. The antibacterial experiments were carried out in triplicate; their mean results are described with standard deviation (SD). The statistical one-way analysis of variance (ANOVA) was calibrated by Student’s t-test with significance of p ≤ 0.05. The statistical values were appraised by SPSS software, IBM, NY, USA (version 13.0).The antibacterial properties of the hybrid composites were investigated using the diffusion technique in sterilized Mueller–Hinton agar (MHA) medium. Different organisms  nanocomposite shows reflections indexed to the (103), (211), (220), (321), (215), and (400) planes of tetragonal-structured hausmannite, Mn3O4 (JCPDS: 80-0382). The observed pattern confirms the (211) preferred orientation of the nanosized crystallites. The XRD patterns of the ZnMn2O4–CS nanocomposites (S2–S5) show reflections indexed to the (101), (112), (200), (103), (211), (004), (220), (301), (105), (312), (321), (224), (116), and (420) lattice orientations. In addition, the peak associated with the (211) lattice orientation is particularly intense in the patterns of all of the ZnMn2O4–CS nanocomposites, indicating their preferred orientation. The observed XRD peaks are well indexed to the body-centered tetragonal structure of ZnMn2O4 (JCPDS: 77-0470). The XRD reflection intensities changed slightly as the w/v ratio of the metal sources was varied; however, the structure of the ZnMn2O4 compound was not altered. The XRD results clearly indicate the arrangements of metal atoms in the nanocomposites.A simple sol–gel chemical reaction was used to synthesize the Mn3O4–CS and ZnMn2O4–CS nanocomposites. −1 [−1, respectively, the spectrum of the S1 composite [−1 is attributed to the characteristic behavior of Mn (from Mn3O4) interacting with C=O groups [−1 [S1 nanocomposites, the peaks at 1483 and 1415 cm−1 are attributed to the symmetric stretching of CH3 groups and C–H bending vibrations, respectively [−1 are attributed to the –CH3 out-of-plane, OH in-plane, and C–N in-plane bending vibrations, respectively [−1 [−1, respectively [−1 [3O4, consistent with the XRD analysis, in S1 is confirmed by the peak at 525 cm−1 [2O4–CS nanocomposites (S2–S5), the C–C stretching and C–H bending vibrations are shifted to ~1575 and 1411 cm−1, respectively, and their intensities are increased. The Mn3O4 tetragonal lattice vibrations are shifted to 521 cm−1, whereas the ZnO vibrations are located at ~480 and 423 cm−1 in the spectra of the S2–S5 nanocomposites [2O4–CS nanocomposites.FTIR analysis was used to characterize the Mnomposite ,42. The O groups . A charaoups [−1 . For theectively ,44. Peakectively . The C–Hvely [−1 . The peaectively ,47. In t525 cm−1 . For themposites . The FTI3O4–CS (S1) and ZnMn2O4–CS (S2–S5) nanocomposites. Spherically structured patch-shaped agglomerated grains are observed in the micrographs of the S1 nanocomposite  Zn and Mn sources, sheet-like agglomerated grains with highly textured morphology are observed (w/v) to synthesize the S4 hybrid composite  were measured against B. subtilis, S. typhi, E. coli, and P. aeruginosa pathogens. For the comparison, the standard antibiotic of streptomycin (20 µg), further noted as SMN, and different CS concentrations were used as the antibacterial agent and their results are incorporated. B. subtilis Gram-positive pathogen by zone of inhibition assay. Agar plate represents the SMN and CS antibacterial activity against B. subtilis shown in the B. subtilis for CS, SMN, and nanocomposites S1–S5 are given in B. subtilis bacteria. The antibacterial activity is effectively enhanced with increasing source concentrations used to prepare the ZnMn2O4–CS nanocomposites, as validated in B. subtilis was indicated by a 19 mm of zone of inhibition for the S5 nanocomposite. B. subtilis. A low 0.5 µg/mL MIC is estimated for the S5 nanocomposite versus B. subtilis. The results reveal that small amounts of the S5 nanocomposite can effectively inhibit the growth of B. subtilis. The CS exposed the 5 µg/mL MIC versus B. subtilis.Detailed antibacterial activities of nanocomposites S1–S5 (MnE. coli Gram-negative bacteria are shown in E. coli pathogen proficiently, as validated in E. coli is provided in E. coli pathogen (E. coli bacteria. Nanocomposites S1–S5 prevent the development of E. coli, with MICs of 1.25, 0.75, 0.50, 0.25, and 0.25 µg/mL, respectively. The maximum MIC is 5.0 µg/mL against E. coli for CS. A 0.25 µg/mL MIC value versus E. coli is estimated for hybrids S4 and S5, which demonstrates the importance of the increased metal matrix ratio in synthesizing the ZnMn2O4–CS composites.The antibacterial behaviors of nanocomposites S1–S5 versus pathogen b, whereaS. typhi. The experimental results clearly demonstrate inhibition behavior against S. typhi propagation for nanocomposites S1–S5 (S. typhi is provided in the S. typhi (S. typhi bacteria. The nanocomposites S1–S5 prevent the growth of S. typhi, with MICs of 1.25, 1.5, 1.0, 0.75, and 0.25 µg/mL, respectively. The experimental results confirm that the S5 nanocomposite exhibits efficient antibacterial activity toward S. typhi, with a small MIC parameter of 0.25 µg/mL. For CS, the 2.0 µg/mL MIC value is against S. typhi.es S1–S5 a. The agS. typhi b. FigureP. aeruginosa bacterial is shown in P. aeruginosa is shown in P. aeruginosa is given in the P. aeruginosa pathogen were 8, 25, 15, 16, 22, 18, and 15 mm, respectively (P. aeruginosa was observed for the S3 composite, with the zone of inhibition of 22 mm, possibly because of surface modification. P. aeruginosa bacteria. Nanocomposites S1–S5 exhibit MICs of 1.0, 0.75, 0.50, 0.75, and 1.50 µg/mL, respectively, to prevent the growth of P. aeruginosa. A smaller MIC of 0.5 µg/mL versus P. aeruginosa is estimated for the S3 hybrid, consistent with its zone of inhibition performance. The effectiveness of the S3 ZnMn2O4–CS composite is attributed to its surface properties. The maximum MIC of 5.0 µg/mL is perceived for CS versus P. aeruginosa.The antibacterial activity of nanocomposites S1–S5 against ectively b. EnhancThe antibacterial activity of the metal-oxide-incorporated CS nanocomposites can be explained by the following mechanism ,56,57. T3O4–CS and ZnMn2O4–CS nanocomposites. XRD and FTIR analyses confirmed the formation of Mn3O4–CS and ZnMn2O4–CS nanocomposites. Morphological alterations were observed for nanocomposites S1–S5 by SEM analyses at different magnifications. The modified morphological and structural properties effectively altered the optical and luminescence results for nanocomposites S1–S5. The antibacterial activity of Mn3O4–CS and ZnMn2O4–CS were tested against Gram-positive organism B. subtilis and Gram-negative organism E. coli, P. aeruginosa, and S. typhi. Moreover, the ZnMn2O4–CS nanocomposite demonstrated superior immunoactivity against all of the microorganisms. The S5 ZnMn2O4-CS nanocomposite exhibited enhanced activities with low MIC values against B. subtilis, E. coli, and S. typhi bacteria, whereas the S3 nanocomposite exhibited enhanced activity against P. aeruginosa. Hence, the results illustrate the significance of ZnMn2O4–CS nanocomposites and their highly inhibitory properties against various pathogens.A one-pot simple sol–gel chemical reaction was used to prepare Mn


In adult mammals, retinal ganglion cells (RGCs) fail to regenerate following damage. As a result, RGCs die after acute injury and in progressive degenerative diseases such as glaucoma; this can lead to permanent vision loss and, eventually, blindness. Lipids are crucial for the development and maintenance of cell membranes, myelin sheaths, and cellular signaling pathways, however, little is known about their role in axon injury and repair. Studies examining changes to the lipidome during optic nerve (ON) regeneration could greatly inform treatment strategies, yet these are largely lacking. Experimental animal models of ON regeneration have facilitated the exploration of the molecular determinants that affect RGC axon regeneration. Here, we analyzed lipid profiles of the ON and retina in an ON crush rat model using liquid chromatography–mass spectrometry. Furthermore, we investigated lipidome changes after ON crush followed by intravitreal treatment with Zymosan, a yeast cell wall derivative known to enhance RGC regeneration. This data is available at the NIH Common Fund's Metabolomics Data Repository and Coordinating Center  website, the Metabolomics Workbench, http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53. The experimental groups were: intact control (control), optic nerve (ON) crush + vehicle (crush) and ON crush + Zymosan + CPT-cAMP (regeneration). Zymosan is a yeast cell wall preparation traditionally used to induce sterile inflammation experimentally. The addition of a cell-permeable cAMP analog (CPT-cAMP) potentiates Zymosan's action but cannot induce ON regeneration when administered alone 22.1All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and policies of the UCLA Animal Research Committee. A rat model of inflammation-induced ON regeneration was established with intravitreal injection of a yeast cell wall preparation (Zymosan A) 2.2Optic nerves were fixed in PBS plus 4% paraformaldehyde and cryoprotected overnight at 4 °C in 30% sucrose. Cryoprotected tissue was embedded in optimal cutting temperature compound and flash frozen in liquid nitrogen. Longitudinal sections of nerve (14 μm) were cut with a cryostat, mounted on plus charged glass microscope slides, and permeabilized for 30 minutes at room temperature (RT) in TBS plus 0.25% Tween 20 (0.25%TBST). The sections were then blocked with 10% normal donkey serum in TBS for 1 hour at RT and incubated overnight at 4 °C with gentle shaking in 0.1%TBST plus 2% BSA (2%BSA-0.1%TBST) and rabbit polyclonal anti-GAP43 . The sections were rinsed with 0.1%TBST and incubated for 1 hour at RT with Hoechst 33,258 in 2%BSA-0.1%TBST plus Alexa Fluor-conjugated donkey secondary antibody. Finally, the sections were rinsed with 0.1%TBST and coverslipped with an aqueous mounting medium. Images of immunostained tissues were obtained with a Fluoview FV1000 confocal microscope .2.3Specimens were stored at −80 °C. 6 mL of methanol (LC-MS grade) and 3 mL of chloroform (LC-MS grade) were added to each sample. After 2 min of vigorous vortexing and 2 min of sonication in an ultrasonic bath, the samples were incubated at 48 °C overnight . The following day, 3 mL of water (LC-MS grade) and 1.5 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4 °C for 15 min to obtain phase separation. Lower phases were collected and dried in a centrifugal vacuum concentrator. Samples were stored at −20 °C until reconstituted in 60 μL of chloroform:methanol (1:1) prior to mass spectrometric analysis.2.4Reversed-phase chromatographic separation was achieved with the Accela Autosampler, Accela 600 pump and Acclaim C30 column: 3 μm, 2.1 × 150 mm . The column temperature was maintained at 30 °C (negative mode) or 45 °C (positive mode) and tray temperature at 20 °C. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid (LC-MS grade). Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% formic acid. The flow rate was 260 μL/min, and the injection volume was 5 μL. The gradient was 35–100% solvent B over 13.0 min, 100% solvent B over 13.0–13.8 min, 100-35% solvent B over 13.8–14.5 min, 35% solvent B over 14.5–18.0 min, 0% solvent B over 18.0–20.0 min.2.56 ions and maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS (top 10) were acquired with the following parameters: resolution 17,500; AGC 2 × 105 (negative mode) or 1 × 105 (positive mode); maximum IT 100 ms (negative mode) or 75 ms (positive mode); 1.3 m/z isolation window. Normalized collision energy (NCE) settings were 40 ± 30% for the negative mode and 30, parallel with 19 ± 5% for the positive mode. Samples list is available in The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) in positive and negative modes separately for each sample. The spray voltage was 4.4 kV, the heated capillary was held at 310 °C (negative mode) or 350 °C (positive mode) and heater at 275 °C (positive mode). The S-lens radio frequency (RF) level was 70. The sheath gas flow rate was 30 (negative mode) or 45 units (positive mode), and auxiliary gas was 14 (negative mode) or 15 units (positive mode). Full scan (m/z 150–1500) used resolution 70,000 at m/z 200 with automatic gain control (AGC) target of 1 × 102.6Lipid identification and relative quantification were performed with LipidSearch 4.1 software (Thermo). The search criteria were as follows: product search; parent m/z tolerance 5 ppm; product m/z tolerance 10 ppm; product ion intensity threshold 1%; filters: toprank, main isomer peak, FA priority; quantification: m/z tolerance 5 ppm, retention time tolerance 1 min. The following adducts were allowed in positive mode: +H, +NH4, +H—H2O, +H—2H2O, +2H, and negative mode: —H, +HCOO, +CH3COO, -2H. All classes were selected for search. LipidSearch nomenclature is used .Table 2L2.7Positive and negative mode identifications of the retina and ON samples were aligned in LipidSearch 4.1, allowing calculation of unassigned peaks. The following settings were applied: product search; alignment method max; retention time tolerance 0.1 min; filters: toprank, main isomer peak; M-score 5. Only peaks with molecular identification grade: A-B (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified) were accepted. Only peaks appearing in all biological replicates were accepted. Peaks with the same annotated lipid species were merged. Lists of species and their relative abundances were uploaded to the MetaboAnalyst 4.0 2.8http://www.metabolomicsworkbench.org, where it has been assigned Project ID: PR000661. The data can be accessed directly via it's Project DOI: doi: 10.21,228/M87D53. The submission includes 94. RAW files: Q Exactive output  website, the Metabolomics Workbench, https://doi.org/10.6084/m9.figshare.7078253.v2. Species input: ON3_MetaboAnalyst.csv, ON7_MetaboAnalyst.csv, ON14_MetaboAnalyst.csv, retina7_MetaboAnalyst.csv and retina14_MetaboAnalyst.csv. Classes input: ON3_classMetaboAnalyst.csv, ON7_classMetaboAnalyst.csv, ON14_classMetaboAnalyst.csv, retina7_classMetaboAnalyst.csv and retina14_classMetaboAnalyst.csv.In addition, 10. csv files have been uploaded to Figshare 2.9Files in. csv format can be directly input to MetaboAnalyst: Statistical Analysis. The user should select the following format: samples in columns (unpaired). For the following analysis example, we replaced missing values with a small number , applied normalization to sum and log 2 transformation.Data were obtained from 3 to 4 biological replicates for each group. In a and classes b). Next, we performed hierarchical clustering and heatmap visualization of the dysregulated species . The heatmaps of significant species in the retina and ON 14 days post-crush are presented as To identify features undergoing the significant change between experimental groups, we used one-way ANOVA analysis with Tukey's post-hoc test. We examined a number of significant features at different FDR adjusted p values (Conceptualization: D.S., S.K.B., J.C.Methodology: D.S., M.P.Investigation: A.T., D.S., J.K.Analysis: A.T., D.S.Writing – original draft: A.T., D.S.Writing – review & editing: A.T., D.S., J.K., M.P., S.K.B., J.C.Resources & Funding acquisition: S.K.B., J.C.


HOX genes play a key role in neural tube closure. Although abnormal HOX gene expression can lead to NTDs, the underlying pathological mechanisms have not fully been understood.Neural tube defects (NTDs) are severe, common birth defects that result from failure of normal neural tube closure during early embryogenesis. Accumulating strong evidence indicates that genetic factors contribute to NTDs etiology, among them, Hox genes in a retinoic acid (RA) induced mouse NTDs model on E8.5, E9.5 and E10.5 using RNA-sequencing and chromatin immunoprecipitation sequencing assays. Furthermore, we quantified 10 Hox genes using NanoString nCounter in brain tissue of fetuses with 39 NTDs patients including anencephaly, spina bifida, hydrocephaly and encephalocele.We detected that H3K27me3 and expression of the Hox genes. RT-qPCR revealed that these 10 Hox genes were all upregulated in RA-induced mouse NTDs as well as RA-treated embryonic stem cells (ESCs). Using ChIP-seq assays, we demonstrate that a decrease in H3K27me3 level upregulates the expression of Hox cluster A–D in RA-induced mouse NTDs model on E10.5. Interestingly, RA treatment led to attenuation of H3K27me3 due to cooperate between UTX and Suz12, affecting Hox gene regulation. Further analysis, in human anencephaly cases, upregulation of 10 HOX genes was observed, along with aberrant levels of H3K27me3. Notably, HOXB4, HOXC4 and HOXD1 expression was negatively correlated with H3K27me3 levels.Here, our results showed differential expression in 26 genes with a > 20-fold change in the level of expression, including 10 upregulated HOX gene expression induced by aberrant H3K27me3 levels may be a risk factor for NTDs and highlight the need for further analysis of genome-wide epigenetic modification in NTDs.Our results indicate that abnormal  The neural tube is considered as the precursor of the future central nervous system (CNS) which comprises the brain and spinal cord . Neural Hox family includes four clusters, Hoxa, b, c and d, which encode transcriptional regulators that are highly conserved in vertebrates  and chilled on ice. 5% of protease inhibitors were added immediately before lysis of cells, chilled on ice for 30 min, and nuclei were isolated by centrifugation (1500g for 5 min). For the preparation of histones, nuclei were incubated with four volumes of 0.2 M sulfuric acid (H2SO4) for overnight at 4 °C. The supernatant was precipitated with 33% trichloroacetic acid  and followed by centrifugation . The obtained pellet was washed with cold acetone and subsequently dissolved in distilled water. Core histone proteins of mouse and human brain samples were extracted using EpiQuik™ Total Histone Extraction kit  according to the manufacturer’s protocols. Nucleoprotein extraction was extracted from cells or mouse and human brain samples using Nucleoprotein Extraction Kit  according to the manufacturer’s protocols.Core histone proteins of cells were extracted using acid extraction . Briefly5 μg of histone was separated on a 12% SDS-PAGE for H3K27me3 and H3 detection. The blots were incubated with the primary antibody, mouse anti- H3K27me3 , anti-H3K27me2, anti-H3K27me1, anti- Suz12 monoclonal antibody , and mouse anti-H3 monoclonal antibody  overnight at 4 °C, and then incubated with secondary anti-mouse HRP conjugated antibody  for 1 h at room temperature. The blots were developed with SuperSignal West Pico Chemiluminescence Substrate  and quantitated on densitometer  using Quantity One software.Hep-G2 cells were maintained in DMEM  supplemented with 10% fetal bovine serum , treated with 1 μM RA for 24 h. The primary antibodies used for immunofluorescence staining were the mouse monoclonal anti-H3K27me3 antibody .Embryos (E10.5) were fixed in 4% paraformaldehyde overnight and processed to generate 5-μm paraffin sections. Immunohistochemistry was performed on transverse sections according to the method previously described . The priCranial neural tissue of pooled mouse embryos on E10.5 were collected and performed by ChIP-seq. ChIP was done with Simple ChIP Enzymatic chromatinIP kit (9003s) from Cell Signaling Technology and chromatin was sheared to an average DNA fragment size of 100–300 bp. ChIP-Seq libraries were prepared according to Illumina protocols. Sequencing was done with Illumina HiSeq platform. After quality of control (QC), the clean reads were obtained and have been deposited into NCBI SRA (Accession Numbers: SRP193168). The clean data were mapped to the mouse genome (mm9) using SOAPaligner/SOAP2 (Version: 2.21t), and no more than two mismatches are allowed in the alignment. Peaks of H3K27me3 ChIP-Seq signals on genome were determined using MACS2 with false-discovery rate as 0.05. Tracks of H3K27me3 ChIP-seq were viewed by UCSC Genome Browser for mm9.(CT) 2% input sample − (CT) IP sample).ChIP assays were performed using the SimpleChIP Enzymatic chromatin IP system  following the manufacturer’s protocols. Chromatin was prepared, sonicated to DNA segments between 200 and 1000 bp and then immunoprecipitated with anti-H3K27me3, anti-UTX  and anti-H3K27me2 . The immunoprecipitated DNA was analyzed by qPCR, which were performed using QuantStudio 7 Flex with SYBR Green detection. The primers used for ChIP assays were shown in Additional file HOX mRNA expression level of degradative brain tissues from human fetus samples. The RNA was extracted using miRNeasy Mini Kit . Hybridizations were performed according to the NanoString miRGE Assay Manual. Approximately 100 ng of each RNA sample was mixed with 20 μl of nCounter Reporter probes in hybridization buffer and 5 μl of nCounter Capture probes for a total reaction volume of 30 μl. The hybridizations were incubated at 65 °C for approximately 16 h, then eluted and immobilized in the cartridge for data collection, which was performed on the nCounter Digital Analyzer. The counts were analyzed by log2 transformation using nSolver Analysis Software 2.5, and normalized by housekeeping genes such as GAPDH, CLCT, GUSB, HPRT1 and PKG1 genes.NanoString nCounter system was used to analyze the 10 UTX activity was detected according to the manufacturer’s protocols of Epigenase JMJD3/UTX Demethylase activity/inhibition assay kit  using 10 μg of nuclear extracts. The UTX activity was calculated as the following formula: UTX activity (OD/min/mg) = sample OD − Blank OD/(protein amount (μg) × min) × 1000.Sv/129 ESCs were treated with 30 nM of UTX inhibitor GSK J4 sc-391114  for 6 h according to the manufacturer’s protocol.Mouse F9 cells were maintained in DMEM  supplemented with 10% fetal bovine serum . siRNA was delivered to cells using Lipofectamine 2000 according to the manufacturer’s instruction. siRNAs specific for Suz12 5′-AAGCTGTTACCAAGCTCCGTG-3′ and a nonspecific siRNA 5′-TTCTCCGAACGTGTCACGT-3′ were designed and synthesized , and the latter was transfected as negative control. Suz12 expression plasmid was purchase from Origene. After transfection for 24 h, F9 cells were treated with 1 μM RA for 24 h, and then harvested for further analysis.t test or ANOVA analysis was performed. And Pearson’s correlation analysis was used to analyze the association between HOXs expression and H3K27me3 level. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS software, version 22.0 . Data were expressed as the mean ± SD, Student’s All animals were handled in strict accordance with the “Guide for the Care and Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate Animals”, and all animal work was approved by Institutional Animal Care and Use Committee (IACUC) at the Beijing Institute of Radiation Medicine. The study using clinical samples including 39 paired human NTDs and matched normal tissues were approved by department of Lvliang area of Shanxi Province in northern China. Informed consent was obtained from all subjects or their relatives. Human samples were collected and analyzed in accordance with Capital Institute of Pediatrics approval. The Ethics Board of Capital Institute of Pediatrics approved the study protocol. All animal experiments were conducted in compliance with the guidelines of the Institute for Laboratory Animal Research, Capital Institute of Pediatrics.Additional file 1: Figure S1. Bioinformatic analysis of RNA-seq. A. Morphology of mouse NTDs embryos induced by RA. (a). Normal mouse embryo. (b). Mouse embryo showed growth retardation, neural tube close incompletely. Arrow indicates unclosed neural tube. (c). Mouse embryo showed anencephaly, enlarged heart and ventricular chambers, and short tail. Arrow indicates hindbrain, heart and tail respectively. B. Unsupervised hierarchical clustering plot of genes detected in mouse embryo cranial neural tissue. C. GO functional classification of DEGs. Blue represents cellular component, red represents molecular function, and green represents biological process. D. KEGG pathway analysis of DEGs. E. Protein-protein interaction (PPI) network of 196 genes analyzed by STRING database.Additional file 2: Table S1. Enriched GO terms of DEGs in Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and Con-E10.5-vs-RA-E10.5 comparisons.Additional file 3: Table S2. Enriched KEGG pathways of DEGs in Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and Con-E10.5-vs-RA-E10.5 comparisons.Additional file 4: Figure S2. H3K27me3/2/1 analysis in mouse NTD embryos of E10.5. A. Relative protein expression of H3K27me3, H3K27me2 and H3K27me1 in mouse NTDs embryo. Data are shown as the mean . *P < 0.05. B. Peak statistics of mouse embryos used for ChIP-seq. C. GO analysis of differential peak related gene.Additional file 5: Table S3. Differential peaks related genes of mouse NTDs embryos of E10.5.Additional file 6: Figure S3. H3K27me3/2/1 analysis in RA-induced ESCs. A. Relative protein expression of H3K27me3, H3K27me2 and H3K27me1in RA-induced ESCs. Data are shown as the mean . *P < 0.05. B. ChIP assays of H3K27me2 were performed using F9 cells treated with 1 μM RA for 24 h. Mouse IgG was used as control. Enrichment of Hox gene promoters was measured by qPCR.Additional file 7: Figure S4. Suz12 and Ezh2 decreased in RA-induced mouse NTDs and ESCs. A. RNA-seq analysis showed Suz12 expression in cranial neural tissue of RA-induced mouse NTDs embryos from E8.5 to E10.5. B. RNA-seq analysis showed Ezh2 expression in cranial neural tissue of RA-induced mouse NTDs embryos from E8.5 to E10.5. C. Ezh2 level in cranial neural tissue of RA-induced mouse NTDs was measured by RT-qPCR and Western blotting. Actb and Gapdh were used as a loading control respectively. Data are shown as the mean . *P < 0.05. D. Ezh2 level in mouse ESCs treated with RA was measured by RT-qPCR and Western blotting. Actb and Gapdh were used as loading control respectively. Data are shown as the mean . *P < 0.05. E. Relative protein expression of H3K27me3 after overexpression of Suz12 in RA-induced F9 cells. Data are shown as the mean . Different letters represent the difference had statistic significance, P < 0.05.Additional file 8: Figure S5.HOX gene expression in human spinal bifida, hydrocephaly and encephalocele. A.HOX genes were detected with NanoString in the spinal cord from human spinal bifida and normal cases. Data are shown as the mean . P < 0.05 indicates statistical significance. B.HOX genes were detected with NanoString in the brain tissues from human hydrocephaly and normal cases. Data are shown as the mean . P < 0.05 indicates statistical significance. C.HOX genes were detected with NanoString in the brain tissues from human encephalocele and normal cases. Data are shown as the mean . P < 0.05 indicates statistical significance. D. Whole genome sequencing of 100 human NTDs samples. E, F. Variant distribution allele frequency of 10 HOX genes in 100 human NTDs samples. G. The number of variants by minor allele frequency (MAF) in 100 human NTDs samples. H, I. Variant rate of 10 HOX genes in 100 human NTDs samples.Additional file 9: Table S4. Information of human NTDs samples. The human cases with anencephaly, spinal bifida, hydrocephaly and encephalocele involved in this study. Gender, gestational age, sample type and NTDs phenotype are listed.Additional file 10: Table S5. RT-qPCR primer sequences. All oligonucleotides were synthesized by Sangon Biotech.Additional file 11: Table S6. ChIP-qPCR primer sequences. All oligonucleotides were synthesized by Sangon Biotech.


Escherichia coli phage endolysin Lysep3 to create five different hydrophobic-modified endolysins. Compared with endogenous Lysep3, endolysins modified with hydrophobic amino acids surprisingly could kill E. coli from outside of the cell at the appropriate pH and endolysin concentration. The lysis ability of modified endolysins were enhanced with increasing numbers of hydrophobic amino acids at the C-terminus of endolysin. Thus, these findings demonstrate that the enhancement of hydrophobicity at the C-terminus enables the endolysin to act upon E. coli from the outside, representing a novel method of lysing Gram-negative antibiotic-resistant bacteria.Drug-resistant bacteria are a serious threat to global public health. Gram-positive bacterial endolysin preparations have been successfully used to fight Gram-positive bacteria as a novel antimicrobial replacement strategy. However, Gram-negative bacterial phage endolysins cannot be applied directly to destroy Gram-negative strains due to the externally inaccessible peptidoglycan layer of the cell wall; this has seriously hampered the development of endolysin-like antibiotics against Gram-negative bacteria. In this study, 3–12 hydrophobic amino acids were successively added to the C-terminus of  The excessive use of antibiotics in clinical medicine and raising livestock have resulted in an increased prevalence of antibiotic-resistant strains   were used for cloning and expressing the fusion endolysins, respectively. E. coli strains , Acinetobacter baumannii (A. baumannii) strain, and Streptococcus suis (S. suis) strain were stored in the lab. The restriction endonucleases NdeI and SalI, T4 DNA ligase, and pET-28a vector  were used to construct the recombinant vectors. Genscript Ni–NTA affinity chromatography medium  was used for purifying endolysins, and primers were synthesized by Jilin Ku Mei Biotechnology Co., Ltd.In this study, the E. coli from outside the cell , a coliphage endolysin that was previously isolated by our lab  contained a NdeI site and the protection base; downstream primers contain a SalI site. Hydrophobic amino acids were added to the 3′ end of Lysep3, as shown in Table NdeI and SalI at 37 °C for 30 min. DNA products were re-purified and cloned into plasmid pET-28a to generate the recombinant constructs pET-3, pET-5, pET-7, pET-12a, and pET-12b.The expression and purification of Lysep3 were performed as previously described  to express recombinant proteins. The expression and purification of Lysep3, Lysep3-3, and Lysep3-5 were performed as previously described  colony was cultured in 5 mL of LB medium at 37 °C with shaking at 180 rpm until the OD600 nm reached 0.45. We then removed 1 mL of bacterial suspension and diluted it 106-fold. Then, 200 μL of bacterial suspensions were added to 21 sterile 1.5 mL tubes and centrifuged at 12,000 rpm. After discarding the supernatants, 300 μL of phosphate-buffered saline (PBS) at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 were added to seven sterile tubes as control treatments. For another seven tubes, 206 μL of PBS at pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 was individually added into sterile tubes, and then 94 μL of Lysep3-5  was added to each tube and mixed well. Lysep3 was added in the same manner to the final seven tubes . All tubes were then incubated at room temperature for 30 min. Finally, 200 μL of bacterial suspension was cultured on solid LB medium at 37 °C for 16 h, and bacterial colony numbers were determined. Each group was tested three times.These experiments were performed as previously described , respectively, so that the concentration of Lysep3-5 was 1.0, 1.25, 1.5, 1.75, or 2.00 μg μL−1, respectively, before incubation at room temperature for 30 min. The 100 μL of bacterial suspension were then cultured on solid LB medium, and bacterial colony numbers were determined. Lysep3 was added to the other five tubes for comparison, with concentrations of 1, 1.25, 1.5, 1.75, and 2 μg μL−1. Each group was tested three times, and 300 μL of PBS at pH 5.0 was used as the control treatment.The cultivated BL21 (DE3) were diluted 10E. coli BL21 (DE3) colony was cultured in 5 mL of LB medium at 37 °C with shaking at 180 rpm until the OD600 nm reached 0.45. Then, 1 mL of bacterial suspension was diluted 106-fold. Subsequently, 150 μL of diluted bacterial suspension was transferred into 1.5 mL tubes, centrifuged at 12,000 rpm, and then the supernatants were discarded. A total of 206, 199, 206, 24, 95, and 120 μL of PBS at pH 5.0 were added to the tubes, as well as 94, 101, 94, 276, 205, and 180 μL of Lysep3, Lysep3-3, Lysep3-5, Lysep3-7, Lysep3-12a, and Lysep3-12b, respectively, to a final concentration of 1.75 μg μL−1. Tubes were then incubated at room temperature for 30 min. Finally, 100 μL of bacterial suspension was cultured on solid LB medium at 37 °C for 14 h, and bacterial colony numbers were determined. Each group was tested three times.A single −1 of Lysep3, and fusion endolysins were added to 1.5-mL centrifuge tubes, and then 25 μL of ethylenediaminetetraacetic acid disodium salt (EDTA) at 25 mM  was added to each tube. All tubes were incubated at room temperature for 30 min. Finally, 100 μL of bacterial suspension was cultured on solid LB medium at 37 °C for 14 h, and bacterial colony numbers were determined. Each treatment was tested three times.Equal amounts of bacteria, final concentration 1.75 μg μLE. coli strains , A. baumannii strain, and S. suis strain each was diluted 106-fold. Then, 150 μL of each strain was picked out and added into a tube, total three tubes. The bacteria suspensions were centrifuged at 12,000 rpm for 1 min, and the supernatants were discarded. Each strain was treated with PBS, Lysep3, and Lysep3-5 , and a final volume of 300 μL was reached with PBS (pH 5.0). After the treatment, the bacterial colonies were counted using the methods described earlier.The cultivated 100 μL of For statistical analyses, one-way ANOVA was performed using SPSS software . P-values < 0.05 were considered statistically significant.To explore the effect of hydrophobic amino acids on the ability of endolysins to get into the outer membrane of Gram-negative bacteria, different hydrophobic amino acids were added to the C-terminus of Lysep3, generating the constructs: Lysep3, Lysep3-3, Lysep3-5, Lysep3-7, Lysep3-12a, and Lysep3-12b . However, compared with the PBS and Lysep3 groups, Lysep3-5 significantly lysed bacteria (P < 0.05). Moreover, Lysep3-5 had the strongest lysis activity at pH 5.0 Fig. a, which −1, and there was no significant difference in bacterial lysis activity at concentrations > 1.75 μg μL−1 . In addition, it had a certain bactericidal effect on A. baumannii (P < 0.05). However, it had no bactericidal effect on Gram-positive S. suis (P > 0.05) . Compared with PBS and Lysep3 groups, Lysep3-5 had a prominent bactericidal effect on . CompareE. coli from the outside of the cell wall at pH 5.0 and concentration of 1.75 µg μL−1. Moreover, the lysis activity could be strengthened by EDTA. The fusion endolysins could effectively lyse different E. coli strains from the outside of the cell. It might bring a novel method to fight with Gram-negative antibiotic-resistant bacterial infection.Phage endolysins have been used to treat diseases caused by Gram-positive bacteria. However, due to the special structure of the cell wall in Gram-negative bacteria, phage endolysins cannot lyse the bacteria from the outside of the cell, which is not the same with the Gram-positive bacteria. This problem limits the application of endolysins. In a previous study, the endolysin could kill the Gram-negative bacteria from the outside by altering the charge number at the C-terminal of the endolysin (Ma et al. E. coli from the outside of the cell (Wang et al. The modification of phage endolysin by gene engineering to lyse Gram-negative bacteria is a novel and effective strategy. Wang et al. fused the Lysep3 with the cell wall binding domain D8, and the fusion endolysin Lysep3-D8 could lyse the host This study found that increased numbers of hydrophobic amino acids could strengthen the bactericidal ability of endolysins. However, no differences were found between the Lysep12a and Lysep12b groups, although they had different amino acid sequences Fig. . It mighEscherichia coli is a representative Gram-negative bacterium with a similar cell wall structure as other Gram-negative bacteria. In a way, it is the optimal model for studying phage endolysins in the laboratory. Therefore, endolysins were modified with hydrophobic amino acids and found to effectively kill E. coli from the outside of the cell. This indicated that modified endolysins could also lyse other Gram-negative bacteria, which would require further study, but could be a meaningful discovery. This study has important significance for the modification of endolysins and their application in clinical practice.