This study aimed to evaluate the long-term visual outcomes of hemorrhagic retinal arterial macroaneurysm (RAM), particularly focusing on the influence of bevacizumab therapy and intraretinal hemorrhage (IRH) on the outcomes. This retrospective study included 49 patients diagnosed with hemorrhagic RAM. Patients were divided into the bevacizumab group and observation group depending on the whether they were administered bevacizumab treatment and the IRH group and the non-IRH group based on the presence of IRH at the fovea. Best-corrected visual acuity (BCVA) at diagnosis was compared with that at the final visit. Further, the BCVA at the final visit was compared between the study groups. Multivariate analysis was also performed to identify factors associated with poor BCVA at the final visit. The mean follow-up period was 24.8 ± 15.3 months. The mean logarithm of minimal angle of resolution BCVA was significantly improved from 1.37 ± 0.70 at diagnosis to 0.72 ± 0.62 at the final visit (P < 0.001). There was no significant difference in the BCVA at the final visit between the bevacizumab group and observation group (P = 0.576). However, the BCVA at the final visit was significantly worse in the IRH group than in the non-IRH group (P = 0.002). In multivariate analysis, the presence of IRH was significantly associated with poor BCVA (P = 0.007). Significant long-term visual improvement was noted in hemorrhagic RAM. However, the presence of IRH at the fovea was associated with poor visual prognosis. Bevacizumab therapy did not significantly influence the outcomes. RAM usually affects elderly females. Systemic hypertension is a well-known risk factor for RAM2. According to previous studies, 1 in 1500–4500 adults have RAM4.Retinal arterial macroaneurysm (RAM) is a disorder characterized by saccular or fusiform dilatation of the retinal artery7. Nevertheless, various approaches, including laser photocoagulation, vitrectomy with the use of an intravitreal tissue plasminogen activator, and anti-vascular endothelial growth factor (VEGF) therapy, have been attempted to improve outcomes of this condition2. To date, there is no gold-standard method to treat RAM.The RAM often regresses spontaneously10. In RAM, hemorrhage can develop at various locations—preretinal, intraretinal, or subretinal locations5. The visual outcomes are generally comparable between RAM with and without hemorrhage10. In most patients, visual acuity improves as the hemorrhage resolves5. However, the clinical course and visual outcomes substantially vary among patients. In some patients, visual acuity fails to recover with scarring, resulting in very poor visual outcomes9. In addition, the development of macular holes secondary to intraretinal hemorrhage (IRH) may impede visual recovery11. Moreover, dense vitreous hemorrhage requiring surgical intervention can develop due to massive bleeding from RAM12.Hemorrhage is a frequent presentation of RAM which can lead to a sudden loss of vision14. However, the sample size in these studies was relatively small. In addition, there is a paucity of data regarding long-term outcomes of anti-VEGF therapy15. Therefore, we aimed to evaluate the long-term clinical outcomes of 49 hemorrhagic RAM cases. We particularly focused on the difference in outcomes between patients who did and did not receive intravitreal bevacizumab treatment. In addition, we investigated the influence of IRH on the outcomes.To date, numerous studies have reported the clinical course and visual outcomes of hemorrhagic RAMThis retrospective study was conducted at Kim’s Eye Hospital. The study was approved by the Institutional Review Board (IRB) of Kim’s Eye Hospital and was conducted following the tenets of the Declaration of Helsinki. Due to the retrospective nature of this study, the need for an informed consent was waived off .Patients who had been diagnosed with symptomatic RAM at Kim’s Eye Hospital between January 2012 and January 2019 were included. The inclusion criteria were as follows: (1) patients who exhibited one-disc area or greater extent of hemorrhage and (2) patients who had hemorrhage or exudation at the fovea. The exclusion criteria were as follows: (1) follow-up duration < 12 months, (2) treatment for RAM other than intravitreal bevacizumab, (3) history of vitreoretinal surgery, (4) severe media opacity that may preclude accurate imaging of the retina, and (5) > 2 months of symptom duration.At diagnosis, ophthalmological examinations, including best-corrected visual acuity (BCVA) measurement, 90-D lens slit-lamp biomicroscopy, and fundus photography, were performed. Optical coherence tomography (OCT) and fluorescein angiography were also performed. After diagnosis, patients were treated with intravitreal bevacizumab  or closely observed without treatment at the discretion of the treating physician.The BCVA values were measured using a decimal visual acuity chart and converted to logarithm of minimal angle of resolution (logMAR) values for the analysis. Counting finger and hand motion visual acuities were converted to logMAR values 2 and 3, respectively. The central foveal thickness (CFT) was defined as the vertical distance between the internal limiting membrane and Bruch’s membrane at the fovea. This was manually measured on the OCT images. When Bruch’s membrane at the fovea was not accurately identified owing to thick retinal hemorrhage, an imaginary line was drawn between the visible Bruch’s membrane line. In this case, the distance between the internal limiting membrane and this imaginary line at the fovea was defined as the CFT. Further, ellipsoid zone disruption at the fovea was identified using OCT images taken at the final follow-up Fig. .Figure 1For all patients, the BCVA and CFT at diagnosis were compared to those at the final visit. In addition, the incidence of lesion reactivation and ellipsoid zone disruption was estimated. Lesion reactivation was defined as aggravation or new onset of hemorrhage or exudation and confirmed using fundus photography and OCT.There were no strict guidelines on the use of bevacizumab in the present study. Bevacizumab use was based on the preference of the treating physician. Patients treated with intravitreal bevacizumab within 1 month of diagnosis were included in the bevacizumab group. Patients who were closely observed without any treatment and patients who received bevacizumab injection after 1 month of diagnosis were included in the observation group. The following characteristics were compared between the two groups: age, sex, presence of diabetes mellitus, presence of hypertension, use of anticoagulants, systolic and diastolic blood pressure, extent of hemorrhage, presence of foveal IRH, presence of submacular hemorrhage, and follow-up period. In addition, the BCVA and CFT at diagnosis and the final follow-up, the incidence of lesion reactivation, and the incidence of ellipsoid zone disruption were compared between the two groups.16 was also identified.Patients were also divided into the IRH group and the non-IRH group according to the presence of IRH at the fovea. The following characteristics were compared between the two groups: age, sex, presence of diabetes mellitus, presence of hypertension, use of anticoagulants, systolic and diastolic blood pressure, extent of hemorrhage, presence of submacular hemorrhage , type of treatment , and follow-up period. In addition, the BCVA and CFT at diagnosis and the final follow-up, incidence of lesion reactivation, and the incidence of ellipsoid zone disruption were compared between the two groups. In the IRH group, the incidence of fluffy hemorrhageTo identify factors associated with BCVA at the final visit, multivariate analysis was performed. BCVA at the final visit was divided into two categories: logMAR ≥ 0.4 and logMAR < 0.4. The cutoff value logMAR 0.4 was arbitrarily determined based on the author’s clinical experience. The following factors were included in the analysis: age, sex, presence of diabetes mellitus, presence of hypertension, use of anticoagulants, systolic and diastolic blood pressure, extent of hemorrhage, presence of IRH or submacular hemorrhage, follow-up period, and type of treatment . Due to a close association between the presence of IRH and submacular hemorrhage, the association of these factors with the visual outcome was separately analyzed: IRH was included as a factor in one multivariate analysis and submacular hemorrhage in another one.The BCVA at diagnosis and that at the final follow-up were compared between the two groups. Comparisons of the BCVA at diagnosis and the final follow-up between (1) the bevacizumab group and the observation group and (2) the IRH group and the non-IRH group were additionally performed using the alternative logMAR values for counting finger and hand motion visual acuity: counting finger = logMAR values 2, hand motion = logMAR values 2.3. Patients were analyzed further according to their follow-up period: a long follow-up group with a follow-up period ≥ 20 months, and a short follow-up group with a follow-up period < 20 months.U test with or without Bonferroni correction, Fisher’s exact test, and the chi-square test. The association of factors with the final visual outcome was analyzed using binary logistic regression. P-values < 0.05 were considered statistically significant.Statistical analyses were performed using SPSS . The values were compared at different time points using a paired t-test. Comparisons between study groups were performed using the Mann–Whitney Forty-nine patients (49 eyes) were included in the study Table .Table 1CAmong the patients, 12 were males and 37 were females. The mean patient age was 78.0 ± 8.2 years, and the mean follow-up period was 24.8 ± 15.3 months. During the follow-up period, cataract surgery was performed in four patients. Thirty-three patients (67.3%) were initially treated with intravitreal bevacizumab. In these patients, mean 1.9 ± 0.9 injections were administered during the follow-up period. Among the remaining 16 patients (32.7%) who did not receive any treatment initially, intravitreal bevacizumab was administered to one patient during the follow-up because of lesion reactivation.In all 49 patients, the mean logMAR BCVA was 1.37 ± 0.70 at diagnosis, 0.82 ± 0.67 at 6 months, and 0.72 ± 0.62 at the final follow-up. BCVA at the final follow-up was significantly better than that at baseline (P < 0.001). During the follow-up period, three lines or greater improvement in BCVA was noted in 39 (79.6%) patients, whereas three lines or greater deterioration in BCVA was noted in one (2.0%) patient. The BCVA remained stable in the other nine patients (18.4%). The mean CFT was 673.5 ± 263.9 µm at diagnosis, 232.5 ± 75.7 µm at 6 months, and 228.4 ± 99.5 µm at the final follow-up. The CFT at the final follow-up was significantly lower than that at baseline (P < 0.001). During the follow-up period, lesion reactivation was noted in six patients (12.2%), of which five patients developed reactivation within 6 months of diagnosis.Thirty-three patients (67.3%) were included in the bevacizumab group, and 16 patients 32.7%) were included in the observation group. Table .7% were Both groups had similar characteristics, except for a greater extent of hemorrhage in the observation group than in the bevacizumab group (P = 0.039).Changes in the BCVA and CFT in the two groups are shown in Fig. In the bevacizumab group, the mean logMAR BCVA was 1.32 ± 0.76 at diagnosis, 0.75 ± 0.70 at 6 months, and 0.68 ± 0.65 at the final follow-up. The mean CFT was 631.6 ± 253.9 µm at diagnosis, 235.0 ± 85.2 µm at 6 months, and 213.9 ± 96.3 µm at the final follow-up. Lesion reactivation was noted in three (9.1%) patients, and ellipsoid zone disruption was noted in 13 (39.4%) patients at the final follow-up. In the observation group, the mean logMAR BCVA was 1.48 ± 0.53 at diagnosis, 0.95 ± 0.59 at 6 months, and 0.79 ± 0.56 at the final follow-up. The mean CFT was 693.9 ± 231.3 µm at diagnosis, 231.3 ± 71.9 µm at 6 months, and 235.4 ± 101.8 µm at the final follow-up. Lesion reactivation was noted in three (18.8%) patients, and ellipsoid zone disruption was noted in nine patients (56.3%) at the final follow-up. There was no significant difference in the BCVA at diagnosis (P = 0.724), the BCVA at final visit (P = 0.576), the CFT at diagnosis (P = 0.924), and the CFT at the final follow-up (P = 1.000) between the two groups. In addition, there was no significant difference in the incidence of lesion reactivation (P = 0.377) and ellipsoid zone disruption at the final visit between the two groups (P = 0.266).Nineteen patients were included in the IRH group and 27 patients were included in the non-IRH group. Three patients in whom the presence of IRH was not accurately determined were excluded from this comparison. Table Both groups had similar characteristics, except for a greater extent of hemorrhage in the IRH group than in the non-IRH group (P = 0.019).Changes in the BCVA and CFT in the two groups are shown in Fig. In the IRH group, the mean logMAR BCVA was 1.71 ± 0.68 at diagnosis, 1.26 ± 0.73 at 6 months, and 1.11 ± 0.63 at the final follow-up. The mean CFT was 701.1 ± 262.2 µm at diagnosis, 221.7 ± 101.8 µm at 6 months, and 198.1 ± 101.2 µm at the final follow-up. Lesion reactivation was noted in two (10.5%) patients, and ellipsoid zone disruption was noted in 15 (78.9%) patients at the final follow-up. Fluffy hemorrhage was noted in nine patients. In the non-IRH group, the mean logMAR BCVA was 1.06 ± 0.59 at diagnosis, 0.54 ± 0.46 at 6 months, and 0.49 ± 0.49 at the final follow-up. The mean CFT was 613.3 ± 238.2 µm at diagnosis, 237.6 ± 54.1 µm at 6 months, and 250.6 ± 99.2 µm at the final follow-up. Lesion reactivation was noted in four (14.8%) patients, and ellipsoid zone disruption was noted in seven (25.9%) patients at the final follow-up. The BCVA at diagnosis (P = 0.006) and the BCVA at the final follow-up (P = 0.002) were significantly worse in the IRH group than in the non-IRH group. In addition, the CFT at the final follow-up was significantly lower in the IRH group than in the non-IRH group (P = 0.037). There was no difference in the CFT at diagnosis between the two groups (P = 0.384). There was no significant difference in the incidence of lesion reactivation (P = 1.000). The incidence of ellipsoid zone disruption at the final visit was significantly higher in the IRH group than in the non-IRH group (P < 0.001). Figure In the multivariate analysis using IRH as a factor, older age (P = 0.036) and the presence of IRH (P = 0.007) were found to be associated with worse logMAR BCVA < 0.4 at the final follow-up  and the presence of submacular hemorrhage (P = 0.041) were found to be associated with worse logMAR BCVA < 0.4 at the final follow-up Table .Table 5AWhen divided into two groups, according to the follow-up period, 23 patients were included in the long follow-up group (> 20 months) and 26 patients were included in the short follow-up group (< 20 months). In the long follow-up group, the mean logMAR BCVA was 1.39 ± 0.67 at diagnosis and 0.75 ± 0.55 at the final follow-up. In the short follow-up group, the values were 1.36 ± 0.74 at diagnosis and 0.68 ± 0.69 at the final follow-up, respectively. There was no difference in the BCVA at diagnosis (P = 0.991) and at the final follow-up (P = 0.333) between the two groups.When the counting finger and hand motion visual acuities were converted to logMAR values 2 and 2.3, respectively, the mean logMAR BCVA was 1.28 ± 0.68 at diagnosis and 0.68 ± 0.65 at the final follow-up in the bevacizumab group. The values were 1.48 ± 0.53 at diagnosis and 0.79 ± 0.56 at the final follow-up in the observation group. There was no significant difference in the BCVA at diagnosis (P = 0.362) and the BCVA at final visit (P = 0.238) between the two groups. In the IRH group, the mean logMAR BCVA was 1.64 ± 0.56 at diagnosis and 1.11 ± 0.63 at the final follow-up. In the non-IRH group, the values were 1.06 ± 0.59 at diagnosis and 0.49 ± 0.49 at the final follow-up. The BCVA at diagnosis (P = 0.003) and at the final follow-up (P = 0.001) was significantly better in the non-IRH group than in the IRH group.In our patients, the visual acuity significantly improved; three lines or greater visual improvement was noted in most patients. However, the visual improvement was limited in some patients with ellipsoid zone disruption at the fovea. The incidence of lesion reactivation was relatively low.18. Thus, management of subretinal hemorrhage using vitrectomy and tissue plasminogen activator injection, pneumatic displacement, or both have been considered useful therapeutic options for hemorrhagic RAM2. In addition, laser photocoagulation has been widely used to treat the RAM lesion2. After the advent of anti-VEGF agents, anti-VEGF therapy has also been used as the first-line treatment2 or an adjunctive to laser photocoagulation19. Although these treatment methods are effective, there is no gold-standard treatment for RAM; many doctors also recommend close observation without treatment2.Treatment for RAM primarily focuses on subretinal hemorrhage and the RAM lesion itself. The blood has a negative impact on the retina, causing iron toxicity and mechanical damage21. In a study by Chanana and Azad, a marked decrease in edema was observed after bevacizumab injection20. This favorable outcome was also reported by a study by Jonas and Schmidbauer21. After these preliminary studies, researchers have demonstrated that anti-VEGF therapy is also effective in hemorrhagic cases. Javey et al. were the first to report on outcomes of bevacizumab in hemorrhagic RAM22. In their report, marked improvement in visual acuity was noted with a hemorrhage resolution after bevacizumab injection22. Pichi et al. analyzed the efficacy of bevacizumab therapy in 37 patients, of which 19 had hemorrhagic RAM13. In their study, a significant anatomical and functional improvement was noted during the 6-week follow-up period. In addition, there was no notable difference in treatment outcomes between hemorrhagic RAM and exudative RAM. In a study by Cho et al., faster visual recovery was noted in patients treated with bevacizumab than those who were not23. A recent study by Mansour et al. also showed that anti-VEGF therapy is effective for the treatment of RAM24.In our study, approximately two-thirds of the patients were treated with intravitreal bevacizumab. Anti-VEGF therapy for RAM was first introduced to control exudation from the aneurysmAlthough the lack of prospective case–control clinical trials limits the solid conclusion regarding its efficacy, previous studies have suggested that anti-VEGF therapy has some valid effect in RAM treatment. However, the influence of initial bevacizumab therapy on the long-term outcomes of hemorrhagic RAM is unclear.In our study, we compared the long-term outcomes of hemorrhagic RAM (mean 24.8 months) between patients with and without bevacizumab therapy. The visual and anatomical outcomes were similar between both groups, suggesting that bevacizumab therapy may not significantly influence the long-term outcomes. There were no strict guidelines on the use of bevacizumab in the present study. There was no significant difference between the bevacizumab group and the observation group, except for the hypertension. However, the lack of selection guideline in the study result should be kept in mind.16. In their study, 23 patients were treated using vitrectomy, subretinal injection of tissue plasminogen activator, and air tamponade. The final visual outcome was measured at 6 months; postoperative visual acuity was significantly worse in patients with IRH than patients without IRH. Doi et al. have suggested that the presence of both subretinal hemorrhage and IRH may accelerate photoreceptor damage by exposing the photoreceptor cells to the blood from both sides of the subretinal space and the outer plexiform layer16. In our study, the incidence of submacular hemorrhage was higher in patients with IRH than those without IRH, suggesting that the presence of submacular hemorrhage may influence the poor visual outcome in patients with IRH. Furthermore, both IRH and submacular hemorrhage were found to be closely associated with poor visual outcome.In this study, the influence of IRH at the fovea on the long-term outcomes was also evaluated. Patients with IRH showed worse visual outcomes with a higher incidence of ellipsoid zone disruption than those without IRH. In addition, in multivariate analysis, the presence of IRH was significantly associated with poor visual outcomes. Doi et al. recently investigated the influence of macular IRH on short-term visual outcomes of hemorrhagic RAM16 This may highlight the negative influence of IRH on the prognosis of hemorrhagic RAM.In our study, patients were treated with intravitreal bevacizumab or did not receive any treatment. In addition, our study was based on longer follow-up data with a larger study population. Despite these differences, our study result—significantly worse anatomical and functional outcomes in patients with IRH than in patients without IRH—was consistent with that reported by Doi et al.16, was noted in approximately half of our patients in the IRH group. In the study by Doi et al., the incidence of fluffy hemorrhage was 64.7% among the patients with macular IRH16. A similar shape of IRH was previously reported in neovascular age-related macular degeneration, especially in polypoidal choroidal vasculopathy25. To date, only a few clinical studies have evaluated the nature and clinical significance of this peculiar type of IRH25. Further histopathologic studies are needed to better elucidate its origin and impact on adjacent retinal tissue.Fluffy hemorrhage, which represents IRH spreading radially26. However, the long-term incidence of lesion reactivation in hemorrhagic RAM is unclear. In our study, lesion reactivation was noted as 12.2% patients during the mean follow-up of 24.8 month. Although the incidence was not high, most events occurred within the first 6 months, suggesting the need for close observation during this period.Previous reports have shown that recurrent hemorrhage can develop after one successful RAM treatmentIn addition to its retrospective nature, this study has some limitations. First, this study was not controlled. There was no strict guideline on the use of bevacizumab. Moreover, the use of bevacizumab was not randomly assigned to patients. Second, this study included only hemorrhagic RAM cases that involved the fovea. Thus, our results may not be valid for exudative RAM or cases without foveal involvement. Third, all the patients were treated with intravitreal bevacizumab or observed without treatment. Thus, our results do not reflect outcomes in patients who underwent other treatments, such as surgical displacement of hemorrhage, laser photocoagulation, or anti-VEGF therapy using ranibizumab or aflibercept. Fourth, there was a large variation in the follow-up period among the patients. However, since the visual outcomes were comparable between the long and the short follow-up groups, we believe that the variation in the follow-up period may not significantly influence the study results. Lastly, in multivariate analysis, the cutoff value of logMAR 0.4 was arbitrarily determined.In summary, during the mean follow-up of 24.8 months, a significant visual and anatomical improvement was noted in hemorrhagic RAM. In our patients, the presence of IRH at the fovea was associated with poor long-term outcomes, whereas bevacizumab therapy did not significantly influence the outcomes. Considering the retrospective nature of the present study, however, further experimental studies are required to reveal the influence of IRH more accurately and the effect of bevacizumab therapy on vision prognosis.


The appropriate treatment strategy for T1N0M0 lung large cell neuroendocrine carcinoma (LCNEC) was not well illustrated. We evaluated the efficacy of different surgery types and adjuvant therapy on patients with T1N0M0 LCNEC.Patients diagnosed T1N0M0 LCNEC from 2004 to 2016 were identified in the surveillance, epidemiology, and end results (SEER) database. Clinical characteristics, treatment and survival data were collected. The efficacy of surgery type and adjuvant therapy stratified by tumor size was assessed. Overall survival(OS) was evaluated by the Kaplan-Meier method, and relevant survival variables were identified by the Cox proportional hazard model.P=0.000). No matter tumor size less than 2 cm or 2 to 3 cm, lobectomy was significantly prolonged survival. Compared with no lymph nodes removed, lymph nodes dissection was associated with more remarkable OS(P<0.000). 4 or more regional lymph nodes dissection predicted better OS compared with 1 to 3 regional lymph nodes dissection(P=0.014). After surgery, adjuvant chemotherapy did not contribute to extended survival in patients with tumor less than 2 cm(P=0.658), and possibly for tumor 2 to 3 cm(P=0.082). Multivariate analysis showed that age and lobectomy were independent prognostic factors(P=0.000).From 2004 to 2016, 425 patients were included in this study, 253 (59.5%) patients received lobectomy, and 236 (55.5%) patients had 4 or more lymph nodes removed. Patients received lobectomy had better survival than those received sublobar resection was more effective in prolonging survival than 1 to 3 lymph nodes dissection. Adjuvant chemotherapy was not associated with extended survival for tumor less than 2 cm, and possibly for tumor 2 to 3 cm. Large cell neuroendocrine carcinoma (LCNEC) was traditionally classified as a subtype of non-small cell lung cancer (NSCLC), accounting for approximately 3% of all lung cancer , 2. HoweLCNEC has a relatively high recurrence rate after surgery, the 5-year survival rate is only 40% even in early stage . For staLCNEC was considered as a specific solid tumor with poor prognosis. The appropriate choice of surgery type and adjuvant therapy was uncertain, particularly in T1N0M0 disease. Thus developing a consensus on optimal treatment of patients with T1N0M0 is necessary. In this study, we used data from the surveillance, epidemiology, and end results (SEER) database, to investigate the efficacy of different surgery type and adjuvant therapy on T1N0N0 LCNEC.The SEER database was supported by the National Cancer Institute (NCI), and providing information including cancer incidence, staging, and patient survival from 18 population-based cancer registries. Patients diagnosed with LCNEC according to the ICD-0-3/WHO 2008 from 2004 to 2016 were recruited. All patients were screened according to criteria as follows: 1) tumor located in “lung and bronchus”, pathological types was LCNEC; 2) tumor diagnosed from 2004 to 2016; 3) tumor diameter less than 3 cm, without lymph nodes and distant metastases; 4) patients who with the second primary tumor were excluded; and 5) patients without detailed information about surgery type were excluded. The primary endpoint of this study was OS. Demographic data included race, gender, age at diagnosis, and tumor differentiation.P less than 0.10 on univariate regression analysis were subsequently examined in a multivariable model. Cumulative survival and differences were calculated using the Kaplan-Meier method and the log rank-test, respectively. P value <0.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS (version 22.0).Inter-relationships between variables were analyzed using the chi-square test or Fisher’s exact as appropriate. Cox proportional hazards regression was performed to assess risk factors. Variables with a A total of 4135 patients were diagnosed with LCNEC between 2004 and 2016, of which 425 patients were included in this study. The clinical characteristics of all patients were shown in P=0.002, Among the 425 patients, most patients had surgery , while 44 cases (10.4%) did not receive operation. 236(55.5%) patients performed regional lymph nodes dissection, with 4 or more lymph nodes removed. 73(17.2%) patients just removed 1 to 3 lymph nodes. 90(21.2%)patients had no lymph nodes removed. Chemotherapy was performed in 74 patients, and 22 patients had radiotherapy. Compared with chemotherapy group, patients treated with chemotherapy were younger P=0.002, Table 2.P=0.000, P=0.000, P=0.02, P=0.000, We examined the efficacy of different surgery types on OS. Comparing with non-surgery group, patients with lobectomy, sublobar resection, or extended resection had better OS(P=0.142). When stratified by tumor size, adjuvant chemotherapy did not contribute to prolonged survival for tumor size less than 2 cm, this study has some limitations. Firstly, this is a retrospective study with a relatively large sample of T1N0M0 patients, some clinical features were not detailed enough, so the conclusions may have a bias. For example, the absence of pathological differentiation data in 120 patients limited our ability to analysis its predictive efficacy. Unfortunately, this is a database-based analysis, and we can’t get the tumor tissue, a pathological review is difficult. According to the traditional understanding, T1N0M0 patients are treated with surgery first and do not need adjuvant therapy, so the subgroups submitted to adjuvant and non-surgical treatment is relatively low. Secondly, we also lack information about chemotherapy regimens, making it difficult to evaluate the potential advances of postoperative adjuvant chemotherapy. Finally, the disease-free survival time is an important criterion for evaluating surgical methods and was not available from SEER database.Since LCNEC was rare, and the treatment of LCNEC was simulated as the regimen of NSCLC and SCLC. We performed a population-based analysis, and the results showed that lobectomy was an independent protective factor. Besides, we observed that the number of regional lymph nodes removed significantly affected survival. Postoperative adjuvant therapy was not able to prolong survival. This is the largest study discussing treatment and outcome of T1N0M0 LCNEC, providing clues to understand the treatment and conduct prospective studies of T1N0N0 LCNEC.https://seer.cancer.gov/).Publicly available datasets were analyzed in this study. This data can be found here: Surveillance, Epidemiology, and End Results (SEER) database  and Guangdong Medical Science and Technology Research Fund (C2018063).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.


Objective. This review aims at exploring and compiling research evidence on medicinal plants used in the management of bacterial infections in Kenya, with a focus on their efficacy and safety. Methodology. A comprehensive web-based systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines was executed to highlight the Kenyan medicinal plants used for the management of bacterial infections in Kenya. This review includes studies published until January 2021 from the PubMed, Science Direct, AJOL, and Google Scholar databases. Results. A total of 105 Kenyan medicinal plants belonging to 43 families have their in vitro activity against various human pathogenic bacteria evaluated. Plants from the Lamiaceae, Rutaceae, and Fabaceae families were the most commonly studied. Aloe secundiflora, Toddalia asiatica, Senna didymobotrya, Warbugia ugandensis, Tithonia diversifolia, Fuerstia africana, Olea africana, and Harrisonia abyssinica were the plants frequently evaluated within Kenya. The plants with the strongest antimicrobial activities were Toddalia asiatica, Hagenia abyssinica, Ocimum gratissimum, Harrisonia abyssinica, Senna didymobotrya, Olea Africana, Camellia sinensis, and Tarmarindus indica. Conclusion. Based on a published work, it is evident that traditional medicine is seemingly an acceptable and efficient system among Kenyan communities in the management of bacterial infections. Kenya's rich biodiversity with diverse secondary metabolites presents a promising source of new therapeutic alternatives with possibly different mechanisms of action against bacteria.Kenya's vision 2030 partly aims at ensuring adequate health care for all, and the integration of traditional healthcare practices into the national healthcare system would present a more rapid alternative towards the realization of universal health coverage in Kenya. Currently, research on Kenyan medicinal plants with potential antibacterial activity remains vastly fragmented across numerous literature studies and databases; thus, it is imperative to collate and appraise these data for the ease of future research and possible clinical application.  Vibrio cholera outbreaks, and methicillin-resistant Staphylococcus aureus (MRSA) from hospitalized patients have been reported [Despite the remarkable investment in health care witnessed over the past decade, microbial infections remain a major threat to human and animal health and are a cause of morbidity and mortality especially in low- and middle-income countries (LMICs). The rising cases of antibiotic resistance present a major health problem globally, and there is an immediate need for strategies to manage it as it relentlessly compromises the effectiveness of antimicrobial therapy and increases the threat of therapeutic failure –3. Due treported .Herbalism is the most preferred form of traditional medicine and is highly lucrative in the international market with annual sales ranging from US dollar 5 billion in Western Europe to US dollar 14 billion in China . Irrespein vivo and in vitro; however, there has been little systematic appraisal of their benefits [As a developing nation with numerous healthcare challenges such as the high costs of medications, Kenya needs to grow its scientific base and create logical and effective solutions to manage them. Laboratory investigations and various clinical trials have often suggested the positive effects of phytomedicines both benefits . Due to benefits . CriticaA comprehensive web-based systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on identification, screening, eligibility, and inclusion was executed to highlight the medicinal plants used for the management of bacterial infections in Kenya. This review covers published literature from 1994 up to January 2021 obtained from the PubMed, Science Direct, African Journals Online (AJOL), and Google Scholar databases. Grey literature  from theThe literature search was performed using search terms identified from previous similar reviews. The Boolean search operators (AND and OR) were used to effectively combine the search terms . The folScreening of search outputs was performed in two stages. First, the title and abstract of identified journal articles/theses were overviewed based on PICO (Participants Intervention Comparison and Outcomes) and the studies classified as “yes” or “no” based on the information provided by the title and abstract. Thereafter, suitable articles/theses were downloaded and critically assessed for inclusion in the review .The studies eligible for inclusion were limited to the English language. The assessment of eligibility of studies was performed by at least two people, independently, using the Critical Appraisal Skills Programme (CASP) appraisal checklist as a guide . This stn = 15000), 89857 studies were excluded based on the title and abstract. Three hundred (300) full-text articles were assessed for eligibility, from which 211 were excluded based on scope, methodological approach, and very little/no bioactivity reported. A total of seventy-nine (79) studies regarding the in vitro antibacterial activity of Kenyan medicinal plants were ultimately included in the review. No in vivo studies within Kenya on Kenyan medicinal plants with antibacterial activity were found.The study included research data from the pharmacological assays/ethno-medicinal studies reporting on Kenyan medicinal plants used for the treatment of bacterial infections. The initial database search identified a total of 105, 157 articles. After removing the duplicates , Toddalia asiatica (5), Senna didymobotrya (5), Warbugia ugandensis (5), Tithonia diversifolia (4), Fuerstia africana (4), Olea africana (4), and Harrisonia abyssinica (4) were the plants frequently evaluated within Kenya. The plants with the strongest antimicrobial activities were Toddalia asiatica, Hagenia abyssinica, Ocimum gratissimum, Harrisonia abyssinica, Conyza sumatrensis, Senna didymobotrya, Aloe secundiflora, Olea Africana, Vernonia glabra, Camellia sinensis, Tetradenia riparia, and Tarmarindus indica as they exhibited high mean inhibition zone values or low minimum inhibitory concentration (MIC) values. The zones of inhibition (ZOIs) were interpreted as low activity (1 mm–6 mm), moderate activity (7 mm–10 mm), high activity (11 mm–15 mm), and very high activity (>16 mm) . The frequently analyzed plant parts were leaves (37%), bark/stem bark (47%), fruits/seeds (5), pods (1%), and roots (24%). Water and methanol were the most used solvents for plant extract preparation, whereas ethanol and dichloromethane were the least utilized solvents used , bark/stem bark (47%), and roots (24%) were the most utilized plant parts against bacterial infections. The variances in their antimicrobial activities could be due to the synergistic or antagonistic actions of various secondary metabolites present .Camellia sinensis) that is often famed for its antioxidant activity, exhibited good antibacterial activity zone of inhibition (ZOI) of 21.3 Ł} 0.33mm against E.coli and a ZOI of 22.3 ± 0.50 mm against S. aureus compared to gentamicin 22.3 Ł} 0.50 mm (against E. coli) and ZOI 23.7 Ł} 0.33 mm (against S. aureus) at a concentration of 0.1 mg/ml [S. aureus. The extract also displayed a ZOI of 18 ± 0.0 mm against E. coli compared to ZOI of 10 ± 0.0 mm of streptomycin against E. coli 20 ± 0.0 mm, ZOI of 18 ± 0.0 mm, and MIC 200 mg/ml against E. coli compared to streptomycin 10 ± 0.0 mm [Vigna subterranea) had an MIC value of 7.72 ± 0.35 μg/ml for E. coli, 12.5 ± 0.32 μg/ml for S. aureus and 7.95 ± 0.10 μg/ml for P. aeruginosa at 100 μg/ml, and showed zone of inhibition of 27 ± 0.74 mm, 25.3 ± 0.40 mm, and 25.1 ± 0.24 mm E. coli, S. aureus, and P. aeruginosa, respectively [From the review, several common foods/spices are reported to have potential antibacterial benefits. For example, green tea  stem bark exhibited pronounced antimicrobial activity as indicated by zone of inhibition diameters of 24 mm (MRSA) and 22 mm (M. gypseum) compared to 22 mm and 14 mm, respectively, by the commercial hand wash (50 mg/ml). In the model hand washes efficacy experiment, the formulated herbal detergent attained a 78.8% reduction of pathogenic load as compared to 67.9% reduction with the commercial hand wash [The ethanolic extract of ectively . A herbafluoride . Also, tand wash .Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis, Candida albicans, Escherichia coli, Streptococcus mutans, Enterococcus faecalis, and Lactobacillus acidophilus). The evaluation of herbal suspensions  by Ngari et al. [Unfortunately, despite the surge in the consumption herbal products and the limited number of standardized herbal products in the Kenyan market, the pharmacovigilance for herbal medicines is nonexistent in Kenya . The inci et al.  reportedi et al. , 156.W. ugandensis, M. piperita, and S. aromaticum had ZOI of 33.1 ± 0.85 mm, 20.3 ± 1.71 mm, and 19.3 ± 1.65 mm against E. coli, S. aureus, and P. aeruginosa, respectively, compared to that of co-trimoxazole of 27.5 ± 0.7 mm, 8.5 ± 0.5 mm, and 10 ± 0.0 mm, respectively. Product HS5 composed of Aloe vera gel, W. ugandensis, and W. sominifera had a ZOI of 25.3 ± 0.25 mm, 20.25 ± 1.26 mm, and 21.5 ± 1.71 mm against E. coli, S. aureus, and P. aeruginosa, respectively, compared toco-trimoxazole 27.5 ± 0.7 mm, 8.5 ± 0.5 mm, and 10 ± 0.0 mm, respectively. This biological activity is attributed to the presence of various secondary metabolites in plants [The use of herbs as combinations is common practice with many herbal practitioners and aimed at giving better results as compared to single herbs and also treating more than one ailment . Ngari en plants .Mbuthia et al. evaluated the synergistic properties of water-soluble green and black tea extracts with penicillin G. The antimicrobial results showed a marked increase in the inhibition zone diameters on the combination of green tea extracts with penicillin G. The catechins, theaflavins, and thearubigins are the antimicrobial agents present in tea .4 Synergμg/ml was considered very high; 100–500 μg/ml, high; 500–1000 μg/ml, moderate; 1000–4000 μg/ml, low; and anything above this, inactive. The plants with the strongest antimicrobial activities were Toddalia asiatica, Hagenia abyssinica, Ocimum gratissimum, Harrisonia abyssinica, Conyza sumatrensis, Senna didymobotrya, Aloe secundiflora, Olea Africana, Vernonia glabra, Camellia sinensis, Tetradenia riparia, and Tarmarindus indica as they exhibited high mean inhibition zone values or low minimum inhibitory concentration (MIC) values.Disc diffusion method was the preferred method to assay for antibacterial activity. A clearing zone of 9 mm or greater for Gram-positive and Gram-negative bacteria was used as the criterion for designating significant antibacterial activity . The in Harrisonia abyssinica had a ZOI of 20 ± 1.6 mm compared to gentamycin (ZOI of 18 ± 1.2 mm) against S. aureus and a ZOI of 30. ± 1.7 mm against E. coli compared to gentamycin ZOI of 15.1.3 mm against E. coli [Croton macrostachyus exhibited ZOI (23.66 mm) compared to (21.33 mm) amoxicillin against S. aureus and ZOI (18.0 mm) compared to (17.58 mm) amoxicillin against P. aeruginosa. The methanolic extract had an MIC of 37.50 mg/ml compared to 18.75 mg/ml cefpodoxime against S. aureus and 18.75 mg/ml compared to 9.372 mg/ml cefpodoxime against P. aeruginosa [Several plants exhibited a high activity superior or comparable to the standard antibiotic drugs ; the met E. coli . The metruginosa .Toddalia asiatica, Hagenia abyssinica, Senna didymobotrya, Aloe secundiflora, and Camellia sinensis displayed good activities; thus, they may be considered for the assessment of in vivo activity and possibly formulated into different consumable forms. Korir et al. recommended that for plants with very low or no activity, bioactivity on all parts of the plants, for example, root, stem bark, and leaves combined ought to be done against a wide variety of pathogenic bacteria in order to conclusively report that a certain plant is inactive [Plants such as inactive .Despite herbal remedies being affordable, their lack of efficacy and safety evaluation is a great impediment to their acceptance into mainstream medicine. The safety assessment of herbal remedies remains a challenge as most of the studies of herbal medicines are directed at the toxicological properties of single plant formulations, yet most herbal preparations, especially those used in traditional medicine, contain multiple herbs .From this review, 45% of the plants were relatively safe, 44% of the plants have not been assessed for their safety, and 11% of the plants were reported to have high toxicity . The plaW. ugandensis and Prunus africana, most of the plants identified in this review are largely available and are not under any serious threat to become extinct. Since most of them are obtained from wild habitats, sustainable use of the reported medicinal plants against bacterial infections is advised as a conservation measure. The cultivation of wild medicinal plants is an important approach to safeguard the herbal industry. Biotechnological techniques such as plant cell or tissue culture, biochemical conversions, and clonal propagation of indigenous medicinal plants are another potential strategy in improving herbal medicine [Other than medicine .This review demonstrates the potential of medicinal plants to treat bacterial infections alongside justifying the use of these plant traditional medicine. It may serve as a starting point of research geared towards the clinical application of these plants. There is a need for standardization to improve the acceptance of herbalism by mainstream health practitioners.in vivo activity of plants displayed remarkable in vitro activity. Plants exhibiting strong antibacterial activity can be evaluated for their interactions with conventional antibiotics, and those displaying synergistic activity may provide useful leads in antibiotic therapy.Further research into the


This model system was established with single or multiple precursor amino acids, including lysine–glucose (Lys/Glu), asparagine–glucose (Asn/Glu), tryptophan–glucose (Trp/Glu), and a combination of these amino acids (Com/Glu). Kinetic parameters were calculated by multiresponse non-linear regression models. We found that the k values of the AGEs and of acrylamide decreased, while those of harmane increased in the Com/Glu model when heated to 170 and 200°C. Our results indicated that the precursor amino acid of acrylamide and AGEs compete for α-dicarbonyl compounds, leading to a decrease in the formation of AGEs and acrylamide. Moreover, compared with asparagine, the precursor amino acid of β-carbolines was more likely to react with acetaldehyde by Pictet–Spengler condensation, which increased the formation of harmane and decreased the formation of acrylamide via the acrolein pathway.This study investigated the effect of multiple precursor amino acids on the simultaneous formation of acrylamide, β-carbolines , and advanced glycation end products (AGEs) [i.e., N The Maillard reaction (MR) refers to the interaction between protein's free amine groups and carbohydrate's carbonyl groups , 2 and iAcrylamide is major harmful products of MR, which was first identified in high-temperature–processed, starch-rich food including french fries, potato chips, coffee, and bakery products . Currentε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) are typical AGEs found in food including tea, milk, meat, and bread (Advanced glycation end products (AGEs) refer to a group of structurally complex and chemically stable Maillard reaction harmful products (MRHPs) generated in the last stage of the MR . Nε- including harmane and norharmane are a series of complex food carcinogens and mutagens, which can form at lower temperatures without the presence of creatine and creatinine , 15. ToxUntil now, although a large number of studies have focused on formation pathways, the influencing factors and inhibition methods for the individual harmful compounds or homologs with similar structure. The formation mechanisms of the above-mentioned MRHPs have only been investigated using MR chemical model systems with a single substrate amino acid and a single reducing sugar , 19. TheBased on an aqueous MR chemical model system, the effect of multiple precursor amino acids including Lys, Asn, and Trp on the simultaneous formation of acrylamide, β-carboline HAs, and AGEs was investigated. The present study offers insight into the reaction mechanism and provides mathematical evidence for predicting the simultaneous formation of acrylamide, β-carboline HAs (harmane and norharmane), and AGEs (CML and CEL) in high-temperature-processed cereals and potato-based foods.Nε-(carboxymethyl)lysine (CML), d4-CML, Nε-(carboxyethyl)lysine (CEL), and d4-CEL were obtained by Santa Cruz Biotechnology Co. . Glucose and 13C6-glucose, lysine and d4-lysine, acrylamide and 13C3-acrylamide were purchased from Sigma-Aldrich . Other amalytical grade chemicals were purchased from Sinopharm Chemical Reagent Co, Ltd. .The standards of tryptophan, and asparagine, acetaldehyde, acrolein, glyoxal and methylglyoxal, and nonafluoropentanoic acid were purchased from J&K Scientific Co, Ltd. . harmane, norharman, According to previous studies with some modifications , 24, aquNext, 20 ml reaction solutions were added to glass reaction vials with Teflon caps. The reaction vials were fully sealed as much as possible to avoid water evaporation and oil absorption. The reaction vials were heated at 170 and 200°C for 0–21 min using a Jintan thermostat-controlled oil bath . Each glass reaction vial was preheated for 5 min before sealing and inserting it into the oil bath. The reaction vials were taken out of the oil bath at predetermined heating times , cooled in an ice bath to stop any further reaction, and stored at −80°C for further analysis.4-lysine, and 200 μg/ml 13C6-glucose.After centrifugation and dilution, the 300 μl reaction sample were added to 300 μl of an internal standard containing 100 μg/ml dm/z 133 → 74 (asparagine); m/z 147 → 84 (lysine); m/z 151 → 88 (d4-lysine); m/z 151 → 88 (d4-lysine); m/z 383 → 203 (glucose); and m/z 395 → 209 (13C6-glucose).The identification and quantification of lysine, asparagine, tryptophan, and glucose were conducted on an Acquity UHPLC system equipped with a triple quadrupole MS . The separation of analytes was performed on a Waters Atlantics dC18 column  using a gradient elution with 0.1% formic acid (pH 6.8) (solvent A) and acetonitrile (solvent B). The solvent composition was 0–0.1 min, 5% A; 0.1–10 min, 5–100% A; 10–10.5 min, 100–5% A; and 10.5%−15 min, 5% A. The flow rate was 0.3 mL/min, the column temperature was 30°C and the injection volume was 10 μL. The capillary voltage was 3.5 kV. The temperature of the ion source and that of the desolvation gas were 130 and 350°C, respectively. The fragment used for quantification were o-phenylenediamine. In addition, 100 μl of 2,3-hexanedione (4 μM) was added as an internal standard. Then the mixture was kept at 4°C in the dark for 12 h.Analysis of α-dicarbonyl compounds including glyoxal (GO) and methylglyoxal (MGO) was carried out as described previously . Reactiom/z 131 → 77 (GO), 145 → 77 (MGO), and 187 → 77 .The derivatives of α-dicarbonyl compounds were determined by using a Waters 2695 LC system with a Micromass Quattro Micro triple quadrupole MS and Waters X-Bridge C18 column  at a flow rate of 0.3 mL/min. The gradient mixture was started from 30% A and increased to 90% and 100% A in 5 and 3 min, respectively. Then it decreased to 30% A in 2 min and the 30% A remained for 5 min, mobile phase A was 1% formic acid and B was methanol. The α-dicarbonyl compounds were identified with the same MS parameters used in the analysis for CML and CEL. The fragments used of the α-dicarbonyl derivatives were 2HPO4, and 1.0 ml 0.2% 2,4-dinitrophenylhydrazine, added sequentially. Then the mixed solution was heated in a water bath at 65°C for 20 min. After cooling to room temperature, 5 ml hexane was added twice for extraction of acrolein. Then 2 ml hexane was filtered through a 0.22-μm organic microporous membrane for HPLC analysis. Lichrospher C18  column was selected for HPLC separation. For each sample, 10 μL was injected onto the column and allowed to elute isocratically at 30°C with 62% acetonitrile in water at a flow rate of 1.0 mL/min. The detection walvelength was 365 nm. A run time of 20 min was enough for complete elution of sample components.According to the method reported by Uchiyama et al.  with somThe concentrations of melanoidins were determined at 470 nm on a spectrophotometer . The concentrations of melanoidins were calculated from the Lambert–Beer equation with an extinction coefficient of 282 L/mol·cm, a value derived for melanoidins formed from glucose and asparagine , 29.4-CML and 66.0 ng of d4-CEL. Chromatographic separation was performed on a Waters 2695 liuid chromatograph (LC) module coupled with a Waters X-Bridge C18 column . The mobile phase A was acetonitrile and mobile phase B was NFPA (5 mM). The flow rate was 0.3 mL/min, the injection volume was 5 μL and the column temperature was set to 40°C. The gradient elution was performed as follows: 0–0.1 min, 5% A; 0.1–5 min, 5–60% A; 5–8 min, 60%−100% A; 8–10 min, 100% A; 10–12 min, 100–5% A; 12–20 min, 5% A.According to Jiao et al.  and Yu e4-CM, CEL, and d4-CEL, the fragments m/z 205 → 84, m/z 209 → 88, m/z 219 → 84, and m/z 223 → 88, respectively, were used for quantification.The LC was interfaced with a Micromass Quattro Micro triple quadrupole mass spectrometer . The mass spectrometer (MS) was operated in an electrospray ionization (ESI) positive mode with multiple reaction monitoring (MRM). The capillary voltage was 3.6 kV. Temperatures of the ionization source and the desolvation gas were 110 and 400°C, respectively. The flow rates of the cone gas and desolvation were 50 and 600 L/h, respectively. Argon gas was used as the collision gas in the collision cell. For CML, d13C3-acrylamide. The identification and quantification of acrylamide, harman, and norharman were also conducted on an Acquity UHPLC system equipped with a triple quadrupole MS . The parameters of UHPLC-MS were same as section 2.3. The fragment used for quantification were m/z 72 → 55 (acrylamide); m/z 75 → 58 (13C3-acrylamide); m/z 169 → 115 (norharman).After centrifugation and dilution, the 300 μl reaction sample were added to 300 μl of an internal standard containing 100 μg/ml k) were estimated based on the non-linear curve fitting function of the Origin 8.5 software  and SAS 9.0 software . Moreover, the R-squared (R2) values were calculated to determine the fitting performance of the non-linear fitted models  between means were identified by the least significance difference procedure.Analysis of all components was conducted in triplicate, with values reported as the mean ± standard deviation of three independent experiments. Statistical analysis was carried out using the general linear model procedure of Statistix software 9.0 . Significant differences  in the different model systems. The Asn/Glu model had a higher reactivity, which generated more ethanal than in the other models. The content of ethanal in the Asn/Glu model was 97.25 ± 8.91 μg/ml at 170°C and 62.97 ± 7.97 μg/ml at 200°C, which was 51.92–73.98% and 24.01–34.20% higher, respectively, than in the other model systems.Aldehydes are also important MR intermediates and are degradation products of glucose at high temperatures . As showp < 0.05) in each of the model systems. At the two heating temperatures  than the results seen in the Asn/Glu model.The kinetics of acrylamide in the different models are shown in p < 0.05) than that in the Trp/Glu model.In general, the contents of harmane and norharmane increased with time when heated to 170°C . The higp < 0.05) than that in the Lys/Glu model. In addition, the content of CEL produced in the Com/Glu model was also significantly lower than the content of CEL in the Lys/Glu model. The content range of CEL was from 22.40 ± 6.36 to 33.48 ± 4.02 μg/ml (for the Com/Glu model) and from 39.25 ± 2.17 to 49.67 ± 1.70 μg/ml (for Lys/Glu) at 170°C, while the ranges were from 16.31 ± 1.54 to 30.66 ± 0.79 μg/ml (for the Com/Glu model) and from 35.01 ± 1.66 to 46.99 ± 0.30 μg/ml (for the Lys/Glu model) at 200°C.The trends for the two AGEs, CML and CEL, are shown in In addition to the already discussed MRHPs, melanoidins are generated via aldol condensations and the polymerization of carbonyl compounds and are considered as the main indicator of the browning state . The kin1-k18) were calculated by the Marquardt non-linear least squares regression.According to available experimental data, and the reaction scheme proposed in 3, k4, and k5) in the Com/Glu model system was significantly reduced when compared with the kinetics in the Lys/Glu and Asn/Glu model systems (p < 0.05). Previous studies also found that the addition of Lys to the Asn/Glu model can significantly reduce the production of acrylamide, but those studies did not consider the formation of CML and CEL , as Amrein et al.  in the Com/Glu model was significantly reduced compared with their formation in the Asn/Glu model. As a result, acrylamide (k8) produced by the acrolein oxidation pathway was also significantly reduced. In contrast, a significant increase in the content of harmane generated in the Com/Glu model was observed. As shown in 6 and k7) in the Com/Glu model increased significantly compared with its formation in the Trp/Glu model. According to Herraiz et al. , the Key Research and Development Plan of Guangdong Province, China (Grant No. 2019B020212011), and the National First-class Discipline Program of Food Science and Technology (No. JUFSTR20180201).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


APOE) genetic polymorphism has been associated with a wide range of disorders. Knowing that some APOE alleles are associated with obesity and endocrine disorders that are common in obesity, the present study aimed at exploring associations between APOE polymorphisms and endocrine functions in subjects with obesity undergoing bariatric surgery. Methods: Analyses of hormones in blood collected before and one year after bariatric surgery were examined. The APOE alleles were grouped as follows: E2 = ε2ε2 + ε2ε3; E3 = ε3ε3 + ε2ε4; E4 = ε3ε4 + ε4ε4. The APOE groups were analysed as nominal and ordered groups (E2-E3-E4) with a linear mixed model to predict the hormonal effects of the groups. Results: Forty-nine women (79%) and thirteen (21%) men with a mean age of 47.7 (SD 8.5) years were included in the study. The adiponectin level was significantly lower (p < 0.05) in the E2 group compared with the E4 group. Adiponectin and cortisol were positively and negatively associated, respectively, with the ordered APOE groups. Conclusions: The ordered APOE groups E2-E3-E4 were significantly associated with high and low levels of adiponectin and cortisol, respectively. The findings indicate APOE-mediated effects on body weight and metabolic functions in subjects with morbid obesity.Background: Obesity is an interplay between genes and the environment, including lifestyle. The genetics of obesity is insufficiently understood. Apolipoprotein E ( The cause of obesity and overweight is an energy imbalance between calories consumed and expended . LifestyGhrelin and the adipokines leptin and adiponectin have appetite-regulating effects . AdiponeEndocrine disorders are common in subjects with obesity. Cushing’s syndrome, growth hormone deficiency, hypothyroidism and pseudohypoparathyroidism are associated with obesity and are secondary causes of obesity . Sex horAPOE) genetic polymorphism has been associated with a wide range of disorders. A recently published study reported associations with 37 outcomes representing 18 distinct disorders [APOE ε4ε4 was associated with a reduced prevalence of obesity (OR = 0.78), and ε2ε2 with an increased prevalence of obesity (OR = 1.22) compared with ε3ε3. Apolipoprotein E could be the link between APOE genetic polymorphism and obesity. The conventional conception has been that apolipoprotein E promotes obesity by the increase in lipid to white adipose tissue. More recently, apolipoprotein E3 in the brain has been associated with fat accumulation and obesity, and hepatically expressed apolipoprotein E3 has the opposite effect [Obesity is an interplay between genes, epigenetics and the environment, including lifestyle . Genes’ isorders . APOE ε4e effect . In a pre effect .APOE alleles are associated with obesity and that endocrine disorders are common in subjects with obesity, the present study aimed to explore associations between APOE polymorphism and endocrine functions in subjects with obesity undergoing bariatric surgery.Knowing that some This retrospective cohort study used data from the prospective cohort study MO-BiPS . The data are subsets of data used for other purposes in previously published papers ,13.2 or BMI > 35 kg/m2 with obesity-related complications and referred to the obesity unit at Innlandet Hospital Trust, Gjøvik, Norway for evaluation of bariatric surgery, were available for inclusion. Subjects with present or previous somatic or psychiatric disorders or drug or alcohol abuse were excluded at the doctors’ discretion.Subjects with morbid obesity, defined as BMI > 40 kg/mIn the first six months after inclusion, the participants went through a behavioural weight loss intervention with three separate one-hour-long individual consultations and seven weekly group meetings with information about the operation, dietary recommendations and physical activity advice. During the last three weeks prior to bariatric surgery, they followed a strict “crispbread diet” or an alternative powder diet, both with a maximum of 4200 kJ . BariatrData from the visits immediately before surgery and the follow-up visit one year later were used in this study. Participants with complete datasets from these visits were included in the analyses.2);Demographic and anthropometric data: age (years), gender , height (m), body weight (kg) and body mass index ;Thyroid Stimulating Hormone (TSH); reference values women/men: 0.5–3.6/0.27–4.2 mIE/L;Free Thyroxin (fT4); reference values women/men: 8.0–21/12–22 pmol/L;Free Triiodothyronine (fT3); reference values women/men: 2.8–7.0/2.7–6.3 pmol/L;Reverse Triiodothyronine (rT3); reference values women/men: 0.23–0.54/0.14–0.54 pmol/L;Parathyroid hormone (PTH); reference values 1.5–7.0 pmol/L;Cortisol; reference morning values: 138–690 nmol/L;Sex Hormone Binding Globulin (SHBG); reference values women/men: 23–100/8–60 nmol/L;Testosterone; reference values: women < 50 years of age: ≤1.9; ≥50 years of age: ≤1.1 nmol/L, men 20–40 years: 7.2–24; ≥40 years: 4.6–24 nmol/L;Free Testosterone Index (FTI) (testosterone (nmol/L) × 10/SHBG (nmol/L)): women 0.1–0.3; men age 20–39: 2.4–11.8; age 40–64: 1.5–7.3;HbA1c; reference values 4.7–6.0%.This study used the following data from the prospective MO-BiPS cohort study:Jansen et al. described the hormone analyses in detail .Leptin; reference values for BMI < 25: women/men: 80–2500/< 950 pmol/L;Adiponectin; reference values women/men 4–22/2–20 mg/L;Ghrelin (acylated and non-acylated); reference values: 686–1731 pg/mL.The adipokines were analysed at the Hormone Laboratory, Oslo University Hospital, Oslo, Norway  with competitive radioimmunoassay (RIA). Ghrelin, leptin and adiponectin were not included in the accreditation. The results were used in another context in a recently submitted paper by Jansen A. et. al.APOE alleles were determined. There is no consensus regarding the grouping and order of the APOE variants. In particular, grouping of the ε2ε4 is difficult. The following order of the APOE variants was chosen: ε2ε2, ε2ε3, ε3ε3, ε2ε4, ε3ε4, e4ε4, which is in accordance with other papers, e.g., the comprehensive review by Lumsden et al. [APOE variants and because some groups were small , we combined the genetic variants into three groups: E2 (ε2ε2 + ε2ε3), E3 (ε3ε3 + ε2ε4) and E4 (ε3ε4 + ε4ε4) according to Han et al. [Genotyping. The n et al. . Based on et al. . This isn et al. ,12,17, tGenotyping was performed at the Department of Medical Biochemistry, Oslo University Hospital—Rikshospitalet, Oslo, Norway using real-time polymerase chain reactions, described by Hestad et al. .t-test. A linear mixed regression model for repeated measurements was used to analyse predictors of the hormones, and the results were reported as the estimate  with 95% confidence interval and p-values. The APOE groups E2, E3 and E4 were analysed as nominal groups with comparisons between the groups one-to-one and as an ordered variable with rank order: E2-E3-E4. p-values < 0.05 were judged as statistically significant. The normality of the residuals of the mixed model linear regression was confirmed by the visual inspection of QQ-plots. The analyses were performed with IBM SPSS Statistics for Windows, version 27.0 . The p-values were adjusted for multiple testing ad modum Benjamini–Hochberg with R-Studio version 1.4.1106 and reported as q-values. Because the results showed associations between adiponectin and the APOE genetic variants, HbA1c was added post hoc as a dependent variable.Descriptive data are reported as mean (SD) and number with proportion (%). Values before and after surgery were compared using a paired One hundred and forty-two subjects were included in the main study, twenty-one withdrew during the behavioural intervention and one hundred and twenty-one underwent bariatric surgery. Sixty-two participants who had complete data before and 12 months after surgery were included in this study. APOE groups.There was a significant difference in adiponectin between the groups E2 and E4, differences in HbA1c  between E2 and E3 and differences between E3 and E4 adjusted for age, gender, BMI and point of time. No significant associations were detected after adjusting for multiple testing. APOE groups and the hormones presented as the regression lines adjusted for the mean of the other variables.APOE (E2-E3-E4) and adiponectin and cortisol, respectively. No significant associations were observed between the APOE alleles and the other hormones.In our current study, the main findings include the positive and negative associations between the ordered Adiponectin is a hormone that passes into the brain and has an appetite-regulating effect, increases energy expenditure and thermogenesis, potentiates the effect of leptin and reduces body weight ,19. AdipIn addition to the appetite-regulating effects, adiponectin is an indicator of insulin sensitivity. The positive association between E4 and adiponectin indicates a protective effect of ε4 on diabetes, as reported in the meta-analysis by Lumsden et al. . Our finAdiponectin has also been associated with unfavourable effects, such as chronic heart failure, chronic kidney disease, cognitive impairment and Alzheimer’s disease . The disAPOE polymorphism. High cortisol values are associated with overweight [APOE polymorphism and disorders of the adrenal glands [Screening for endocrine disorders such as hypogonadism, polycystic ovary syndrome, Cushing’s syndrome, thyroid dysfunction, diabetes, growth hormone deficiency and pseudohypoparathyroidism is mandatory in subjects with severe obesity ,24. In terweight . The negl glands .Glucocorticosteroids inhibit adiponectin secretion in animals, healthy persons and non-obese subjects with Cushing’s syndrome . The inhAPOE polymorphism with thyroid and parathyroid disorders, sex hormones, and the adipokines ghrelin and leptin were observed [APOE polymorphism and body weight and weight loss after surgery [APOE genotypes and endocrine functions could indicate that change in weight is a metabolic effect and not a direct APOE genotype effect.In accordance with other studies, no statistically significant associations of observed . In a pr surgery . In the The study population was an unselected and representative group of subjects referred for evaluation of bariatric surgery. The treatment followed national and international guidelines with a lifestyle intervention before surgery, which was performed with standard techniques, and regular follow-ups after surgery. A broad spectrum of hormones known to be related to obesity, or possibly related to obesity, was analysed. Information about the lipid profiles would have been desirable.The unbalanced gender distribution in favour of females limits the external validity. All the analyses were adjusted for gender, and the limited number of participants made gender-specific analyses inappropriate. The results might represent female-specific effects.APOE variants, and this depends on the study population. Instead of having each individual genotype ε2ε2, ε2ε3, ε3ε3, ε2ε4, ε3ε4 and e4ε4 as separate groups, we decided to combine genotypes pairwise into three groups: E2 (ε2ε2 + ε2ε3), E3 (ε3ε3 + ε2ε4) and E4 (ε3ε4 + ε4ε4) to obtain larger groups whilst ensuring clear E2 and E4 profiles. A different grouping or leaving out the participant with ε2ε4 could have given other results but has not been performed.There is no consensus about the grouping of the p-values were adjusted for multiple testing. This strengthening of the methods, which removed all significant associations, demonstrates the uncertainty of the results. Similarly, because of the relatively small sample size, particularly in the APOE subgroups, negative results do not exclude an effect (type II error). Discrepancies between this and other studies could also be due to the study population , different ethnic groups and the study design. The results could be specific for subjects with obesity. The lack of a reference group with healthy subjects is a limitation. Nevertheless, the results indicate endocrine effects of clinical importance of APOE polymorphism in subjects with morbid obesity. Confirmations from larger studies are desirable.The explorative design with multiple testing increases the possibilities of type I errors. To reduce type I errors, APOE polymorphism seems to have endocrine effects of clinical significance in subjects with morbid obesity. The ε4 allele was positively and negatively associated with adiponectin and cortisol, respectively, indicating effects on metabolic functions and thus body weight.


M.tb and bacille Calmette–Guérin (BCG) from infected THP-1cells. First, M.tb and BCG infected THP-1 at MOI 10:1. Twelve hours postinfection, intracellular bacteria of M.tb and BCG were collected, whereas the two bacilli cultured in 7H9 broth media were used as the control. Then four groups of bacilli were subjected to proteomic analysis, and differential proteomic profiles between M.tb and BCG were comparatively analyzed with bioinformatics tools. As a result, we identified a total of 1,557 proteins. Further, they were divided into four groups for comparison of M.tb versus BCG under 7H9 culture (shorten as out), M.tb in (intracellular) versus M.tb out, BCG in versus BCG out and M.tb in versus BCG in. Between M.tb in versus BCG in, a total of 211 differentially expressed proteins were found. Eight proteins like ESAT-6 distributed in six RDs and some known proteins related to virulence. Besides, five uncharacterized proteins were differentially expressed. Further analysis revealed enriched pathways were associated with glyoxylate and dicarboxylate metabolism pathways. In M.tb out versus BCG out, a total of 144 differential proteins were identified and mainly involved in metabolism pathways. Then, 121 differential proteins in the group of M.tb in versus M.tb out were enriched in ribosome and oxidative phosphorylation related to adaptation to the host environment. The group of BCG in versus BCG out shared the same trend of different pathways to the M.tb in versus M.tb out. Finally, 42 proteins were identified to be up-regulated only in intracellular M.tb including eight RD proteins, whereas 22 up-regulated uniquely in intracellular BCG. Besides, only two proteins (Pks13 and Rv1405c) were commonly up-regulated in intracellular M.tb and BCG. Further, some unknown proteins were uniquely up-regulated in the intracellular M.tb and BCG. These findings provide valuable data for further exploration of molecular mechanism for M.tb virulence and BCG immune response.Tuberculosis is the second cause in infectious diseases leading to human death. Understanding the virulence mechanism is inevitable if the disease needs to be fully cured. Therefore, this study aimed to reveal this mechanism by comparing proteomic profiles of intracellular and extracellular virulent strain  Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis (TB), remains a serious global public health threat. As an extremely successful intracellular bacteria, M.tb can survive and cause disease correlated to their unique immune evasion mechanism. Despite the availability of anti-TB drugs, the normal treatment time lasts 6 to 12 months. In addition, increasing multidrug-resistant TB and extensively drug-resistant TB are bringing great difficulties to realize End-TB strategy proposed by the World Health Organization , an attenuated strain of M. bovis that developed more than a century ago and remains the only licensed TB vaccine in humans for 100 years. Although the efficacy of BCG against active TB in adults has been challenged, it saves millions of lives by preventing severe, miliary, and meningeal TB, especially for children  is a strain of bovine origin isolated by the National Animal Tuberculosis Para-Reference Laboratory (Wuhan) at Huazhong agricultural university, Wuhan, China   for 12 h, at 37°C. For infection, the two logarithmic growing cultures (M.tb and BCG) were centrifuged and resuspended with Hanks balanced salt solution (HBSS) (Gibco). The bacterial concentrations (colony-forming units/ml) were estimated by measuring the OD600 values and infected at MOI  of 10:1. After 12 h, the extracellular bacteria in the culture medium were collected. The cells were washed twice with RPMI 1640 fresh medium and incubated with a fresh RPMI 1640 medium containing 100 µg/ml gentamicin to kill extracellular bacteria.THP-1 cells were seeded at 30 × 10The bacteria were confirmed by acid-fast staining. Briefly, the smears of infected THP-1 cells were permeabilized with 0.4% triton X-100 for 10 min and washed twice with phosphate-buffered saline. Finally, the acid-fast staining was performed using acid-fast staining solution kit  according to the manufacturer’s instruction.g for 2 min at 4°C. The pellet was collected and added 1 ml 1% sodium dodecyl sulfate (SDS) for resuspension.For intracellular bacterial proteins, the infected cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer  containing protease and phosphatase inhibitors  and subjected to be centrifuged for 15,000g for 10 min. The pelleted washed by HBSS and centrifuged at 4,000g for 10 min. Then, 1 ml HBSS was resuspended, pelleted, and further lysed with sonication. The parameters were set as 1 s/1-s intervals, 3-min time, and 80-W power. After sonication, the samples were centrifuged at 15,000g for 15 min to remove insoluble particles, repeated once to further exclude precipitation. The bacterial proteins that contained membrane proteins were collected, and the concentration was determined by BCA protein assay kit (Beyotime). Finally, the sample was stored at −80°C for the following steps.Bacteria growing to logarithmic phase were centrifuged at 4,000g for 20 min at 4°C and the concentrated protein solution on the filter was added 100 µL of TEAB (100 mM) and centrifuged at 12,000g for 20 min. Then 100 µl of 100 mM TEAB and 2 µl trypsin (1 µg/µl) were added to each tube and then incubated at 37°C for 12 h. Finally, the digested peptides were centrifuged at 12,000g for 20 min, 50 µl of 100 mM TEAB were added and repeated centrifugation again. The generated peptides were dried by vacuum centrifugation and re-dissolved with 100 µl of 0.5 mM TEAB. The 40 µl of each sample were transferred into a new tube and 100 µl of the iTRAQ label reagent dissolved in isopropanol was added. Finally, 200 µl of high-performance liquid chromatography (HPLC) water were added and incubated for 30 min to terminate the reaction.For tryptic digestion and iTRAQ labeling, 50 µg of each protein sample was mixed with 120 µl reduction buffer , pH 8.0) on a 10 K ultrafiltration tube, and incubated at 60°C for 1 h, and then IAA (3-indoleacetic acid) was added at a final concentration of 50 mM and stayed at room temperature for 40 min under protection from light. The solution was then centrifuged on the filter at 12,000A 1100 HPLC system (Agilent) was used for reversed-phase (RP) chromatographic separation with an Agilent Zorbax Extend RP column . Mobile phases A (2% acetonitrile in HPLC water) and B (98% acetonitrile in HPLC water) were used for the RP gradient. Trypsin peptides were separated at a steady flow rate of 300 µl/min, monitored at 210 and 280 nm. Then elution buffer at every minute was collected, lasting for 8–50 min. The separated peptides were lyophilized for mass spectrometer (MS) detection as follows.All analyses were performed by a Q-Exactive MS  equipped with a nanospray flex source . The peptides were separated on a C18 analytical RP column (75 µm × 15 cm) at a flow rate of 300 nL/min and a linear gradient of 70 min. A full MS scan was obtained in the mass range of 300 to 1600 m/z. Ten strongest peaks in the MS were fragmented by high-energy collision dissociation. The resolution of the obtained MS/MS spectrum is 17,500 with the maximum injection time of 50 ms. Q-E dynamic exclusion was set to 15 s.M.tb database. The false-positive rate of peptide identification was controlled below 1%.All of the Q exactive MS/MS raw data were analyzed by using proteome discoverer v.2.2  software and the uniprot-M.tb and BCG from the macrophages were defined M.tb in and BCG in, whereas the extracellular bacteria cultured in 7H9 medium were defined M.tb out and BCG out. So, the four groups are BCG in versus M.tb in, BCG out versus M.tb out, M.tb in vs. M.tb out, and BCG in vs. BCG out.To compare the proteomics with 1,557 proteins, we divided them into four groups. Among them, the intracellular bacteria of Mycobacterium tuberculosis database. For the obtained search results, only unique peptides were contained for iTRAQ labeling quantification, and peptides with global false discovery rate (FDR) values from fit less than 1% were considered for further analysis. Based on the screened credible proteins, the difference multiple fold change (FC) value and difference significance p value of each comparison group were calculated. The proteins with significant differences were screened according to the criteria of FC >1.5 or FC < 2/3 and p < 0.05. Different proteins in four groups were analyzed by heatmaps and volcano plots based on a threshold FC >1.5 and p-value < 0.05. Using the OmicsBean omics data integration analysis cloud platform (http://www.omicsbean.cn/), the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed on the selected differential proteins. The KEGG analysis was used corrected p-value < 0.05 as a threshold for finding significantly enriched terms in the input list of differentially expressed proteins. To find the shared mechanism for both M.tb and BCG necessary mechanism to infection, the differential proteins with twofold difference were checked to analyze and identify commonly regulated proteins in different groups.Database searches were performed for proteins analyzed by RP liquid chromatography and MS. Experimental data were analyzed by Proteome Discoverer™ 2.2  software. The data are from the Uniprot–M.tb and BCG in red and THP-1 in blue, demonstrating the infection was successful  each in triplicate numbering 1 to 3 were divided into three groups 1, 2, and 3 by pooling the same number of the group into one group first for analysis of qualitative proteins by base search. Then, the data were filtered with a global FDR < 0.01 to obtain quantitative protein 1 to 3. Finally, we identified 2,442, 2,625, and 2,584 valid proteins for groups 1 to 3 samples  and RP chromatography . Combini samples . In the e trends . Taken tM.tb in were comparatively analyzed. The dysregulated proteins total to 211 between the intracellular M.tb and BCG. Almost equal numbers of genes were down-regulated (109) and up-regulated (102). Compared with the group of BCG out and M.tb out, their gene expression profiles in this group showed more diversity . Rv2588c, Rv1762c, and Rv0025 were the proteins uncharacterized   and RD regions EspK (RD1), EsxA (RD1), EsxO (RD7), LpqG (RD9), Rv0223c (RD4), epiA (RD6), Rv2073c (RD12), and Rv1513 (RD6). The functional studies of Rv2073c, EpiA, and Rv1513 remain not clear and deserve further study.The proteomic profiles of BCG in vs. iversity . The topcterized . On the ds down) . TB22.2 nfection . In addiM.tb, it might be used by BCG to adapt to the environment within macrophages. On the other hand, the intracellular M.tb relies more on TB pathway than BCG shown by the differential proteins including ESAT-6 (esxA), a protein in RD1, pstS1, and lprA up-regulated, whereas pstS3 down-regulated in M.tb compared with BCG.KEGG pathway of the differential expressed genes was further performed and showed that glyoxylate and dicarboxylate metabolism; valine, leucine, and isoleucine degradation; and TB are the top three pathways enriched . GlyoxylM.tb out was analyzed. The cluster heatmap showed that both BCG and M.tb samples were clustered into two groups. In M.tb, there are more genes downregulated, whereas the trend was opposite in BCG samples . Rv3699 was uncharacterized, and Rv1358 was defined as transcriptional regulator, but the detailed functions remain unknown   generates production in low-virulence strains of mycobacteria to help persist in host macrophages and a crucial chaperon protein necessary for the survival of M.tb in the dormant phase  . For Rv1ld down) . It giveb or BCG . In addint phase . Psts3 ise  . For BCG in M.tb . It suggM.tb in versus M.tb out were found (M.tb in and M.tb out showed that most (>70%) of the differential genes are repressed in M.tb in of macrophages compared with M.tb out , but the protein currently uncharacterized. RspF, 30S ribosomal protein S6, was modified by ATP-dependent glutamate ligase RimK and affected bacterium growth   . Eccd3, M.tb are encountered with constant pressure; therefore, the intracellular bacteria need to coordinate its metabolic activity to adapt either replication or quiescence statue. The key feature is to generate ATP via oxidative phosphorylation. Many bacteria transited into hibernation of ribosome when faced with stresses to growth; the M.tb also has to rearrange ribosome pathway to adapt to the environment. In conclusion, M.tb survival strategies are linked with ATP generation and ribosome regulation to commensurate with intracellular environment.KEGG pathway showed that differentiated genes were enriched on ribosome, oxidative phosphorylation, and fatty acid biosynthesis . A highlM.tb, or even more trend was seen, most of the infected BCG’s proteins were inhibited compared with BCG control , while it also lost other capacities to coordinate like ribosome pathway.Interestingly, KEGG pathway showed that oxidative phosphorylation was unique and most relevant in the group of BCG in vs. BCG, instead of both oxidative phosphorylation and ribosome in  strains . As the M.tb in clustered together, and BCG out and M.tb out clustered closer. But in between, there are clusters that form a separate group. At last, we used principal component analysis to summarize the different conditions. From the chart, we found the BCG was the farthest from the center; interestingly, the BCG in and M.tb in clustered as a whole separate group. M.tb group was close to both BCG and intracellular group but as a separate group  in macrophages, their protein expression were synchronized to the similar level.In summary, we could clearly show that BCG in and te group . The infM.tb and BCG targeting novel mechanism underlying virulence and pathogenesis focusing on the whole proteins or only membrane proteins. However, all works used the bacteria in medium culture. In current study, we compared the proteomics of both intracellular M.tb and BCG from infected macrophages and the extracellular M.tb and BCG grown in 7H9 medium. Therefore, the differential proteins including membrane proteins and whole proteins in this study revealed data more relevant to virulence of M.tb.Previously, there are several works on the differentiated proteins between M.tb in vs. M.tb out and BCG in vs. BCG out, there were 30 differential proteins up-regulated M.tb in vs. M.tb out, whereas 22 proteins in the group of BCG in vs. BCG out. However, only two proteins (Pks13 and Rv1405c) were commonly up-regulated, indicating both are essential for mycobacteria to enter THP-1 cells . Among them, 27 differential proteins were down-regulated commonly. Especially a series of oxidative phosphorylation pathway–related genes such as QcrA, QcrB, NuoL, NuoN, and CtaC that are closely related to energy metabolism were included. QcrA and QcrB are subunits of cytochrome-c complex, which is one of the transport electrons from the respiratory electron transport chain  T-cell activation and both Th1 and Th17 immunity in mice , EsxO, LpqG, and Rv1513 were identified; among them, Rv1512, Rv1513, and Rv2073c need to be investigated in the future. There are seven other known virulence-related proteins including PPE41, PPE51, VapC10, VapB19, PhoB1, PhoH2, and Eccb1. PPE41 has been shown to induce macrophage necrosis  and two less-information proteins (Rv0634c and Rv3842c) up-regulated in the group of intracellular BCG might be considered as candidate targets for vaccine research.M.tb are also mainly related to immunity and can stimulate the host’s immune response. Like Rv0494, an FadR homolog in M.tb, directly binds to long-chain fatty acyl-CoA to inhibit fatty acid synthase II regulation  were commonly up-regulated for intracellular M.tb and BCG. Further, 10 differential unknown proteins were uniquely up-regulated in the intracellular M.tb (10) and BCG (8). These findings provide valuable data for further exploration of molecular mechanism for M.tb virulence and BCG immune response.In conclusion, more than 70% of the proteins in both


Meloidogyne mali can cause damage in trees, shrubs, and herbaceous plants, and was placed on the EPPO Alert List in 2014. In the present study, we report a population isolated from Japanese maple. The recovered population is described by detailed morphological and molecular approaches. The molecular phylogentic analysis based on 28S rRNA, ITS, and mitochondrial COI genes places the population in the clade together with other M. mali sequences available in GenBank. The cloned sequences of the 28S rRNA gene revealed a high intragenomic rRNA polymorphism where the polymorphic copies are spread across M. mali clade. Similarly, we also found high variation in the mitochondrial COI gene. Among four haplotypes in M. mali, three occur in the newly found population. Our study provides the first report of intragenomic polymorphism in M. mali, and the results suggest that intragenomic polymorphism maybe widespread in Meloidogyne.Root-knot nematode,  Meloidogyne mali  is a root-knot nematode (RKN) causing significant damage by inducing root galls on its host plant and consequently reduced host growth by interfering with the uptake of water and nutrients. M. mali has a wide host range, typically on trees, but can also parasitize on shrubs and herbaceous plants  . In 2000, a root-knot nematode parasitizing elm (Ulmus chenmoui W.C. Cheng) was described as a new species, M. ulmi . Currently, M. mali has been reported worldwide from Japan, France, Italy, the Netherlands , M. arenaria , and M. javanica , which have reticulated evolution background  imported from Japan to Ningbo, China, were collected in a plastic bag. The symptoms of the trees were not observed. Nematodes were extracted in the laboratory by a modified Baermann funnel technique for 24 hrs. When second-stage juveniles and males of Meloidogyne spp. were detected, the remaining roots were studied and observed with a stereomicroscope, and females were dissected directly from galled roots. Specimens were prepared for light microscopy (LM); males and J2s were relaxed with gentle heat, fixed in a solution of 4% formaldehyde, and processed using the glycerin-ethanol method  following the protocol used by For extracting DNA, a single juvenile was handpicked, individually examined on a temporary slide, photographed, and transferred to a small drop of worm lysis buffer  = 710 ± 32.7(670-750) µm, neck = 90.0 ± 9.4(78.0-101.0) µm, stylet = 12.0 ± 1.1(10.9-13.4) µm, dorsal pharyngeal grand orifice to stylet base = 3.5 ± 0.7(2.6-4.4) µm, vulval slit length = 20.7 ± 2.1 (18.3-23.5) µm, vulval slit to anus = 18.6 ± 2.1(15.6-20.3) µm, and interphasmidial distance = 21.4 ± 1.6(19.6-23.4) µm.Mature females were completely enclosed in the root tissue, body white or slightly darker, usually spherical or onion-shaped with a short neck. Neck tapering at the anterior end, less than one-fourth of body length. The head region was small, slightly set off from the body. The cuticle was comparatively thick. The excretory pore was generally located slightly posterior to the dorsal pharyngeal gland orifice. The stylet was slender, short, and slightly backwardly sloping to the anterior concave. The general shape of the perineal pattern which have been studied ranged from low, rounded, to oval, made up of smooth striae finely spaced. The dorsal arch was mostly low and rounded. A lateral field with weak lateral lines, but the number is hard to identify, maybe two.a = 34.1, b = 14.5 , stylet = 19.7 µm, dorsal pharyngeal gland orifice to stylet base = 8.9 µm, excretory pore to the anterior end = 156.4 µm, spicules = 34.9 µm, and gubernaculum = 9.6 µm.Body length = 1,365 µm, width = 40 µm, The body was long, slender, tapering rather at both head and tail portions, and being narrower anteriorly. The body was almost straight when killed by gentle heat showing a twist in the posterior portion. The lateral field was about one-third the body width with four incisures. The excretory pore was located 21 annules behind the median esophageal bulb. The head region was not clearly set off from the body. The stylet with rounded basal knobs was also described.The body was slender, tapering at both the head and tail portions and cuticular annulation was fine but fairly distinct. The lateral field was more than one-third of body width, with four incisures. The head was slightly set off from the body, with a thin labial cap. The stylet was delicate, with small knobs sloping backward. The excretory pore was often difficult to observe. The hemizonid measured 74.4 to 85.5 µm from the head. The tail was conoid and short tapering to a finely rounded or slightly pointed terminus, sometimes more or less rounded. Cuticular constrictions were sometimes present. Hyline tail terminus was variable in length, measuring 6.8 to 12.0 µm.Compared to the original description, the morphological characteristics, especially the female perineal patterns and the juvenile tail shape are similar.But for juvenile morphometrics, small differences exist: some individuals of our population had a much longer body length, up to 507 µm, while in reported isolates, the longest one was only 466 µm . FurtherM. mali sequences available in GenBank. The monophyletic M. mali was fully supported in mitochondrial COI gene (PP = 1), but only moderately (PP = 86) and weakly supported (PP = 46) in 28S and ITS, respectively. In 28S phylogenetic tree, M. mali was sister to an unidentified Chinese Meloidogyne population forming a fully supported clade (PP = 1), while in ITS, it was sister to M. oleae Archidona-Yuste, Cantalapiedra-Navarrete, Liébanas, Rapoport, Castillo and Palomares-Rius, 2018 in a weakly supported clade (PP = 0.58) and showed an unresolved mitochondrial COI phylogenetic tree . Similarly, ITS also shows high rRNA variation where three clones were different and intragenomic polymorphisms are higher than the variation among populations. Compared with 28S rRNA, COI sequences in M. mali are more homogeneous. The haplotype network analysis recovered a total of four haplotypes among 21 sequences originated from the Netherlands, United Kingdom, Belgium, and Japan. The haplotype H3 is most common with 15 sequences, while haplotype H4 has been found only once. Remarkably, the three newly sequenced specimens belong to three different haplotypes, suggesting that our recovered population has a high haplotype diversity.A total of 17 clones from four specimens were sequenced for 28S rRNA, and analysis of acquired sequences showed an overall p-distance of 0.032, with 87 variable sites and 39 singletons in an alignment of 798 nucleotides. The phylogeny analysis suggested that these clones were generally placed across M. mali are relatively difficult, given the long asymptomatic incubation period in host trees. The tree decline was only discriminative after several years when a high population density built up.The identification and detection of M. incognita, M. javanica, and M. arenaria). Practically, preparation of female perineal pattern is challenging. Regardless of the high intraspecific variation, our findings suggest that all these variations were placed in a monophyletic clade. Therefore, the rRNA-based molecular barcoding still remains a powerful tool for M. mali delimitation.From a taxonomic aspect, less morphology and molecular data are available compared with tropical root-knot nematode   . Moreoveylenchus .M. mali population (M. mali is population-specific. The variation may be caused by a high level of intraspecific hybridization background, or the difference in reproduction strategies, as M. mali, was found to reproduce by amphimixis in the presence of males, but it remains possible to reproduce in meiotic parthenogenesis (In an examination of our pulation  using thogenesis . UnfortuMeloidogyne species are spread in long distances mainly through plants and seedling transportation. In recent years, M. mali has been repeatedly detected in imported plants from Japan. So living plants and seedlings with roots are possible to carry dangerous nematodes, and quarantine-treatment measurements, like chemical treatment, are impossible to kill all the nematodes in the roots. Quick detection and identification methods are required for national distribution research and port quarantine and inspection.