Migraine is a common disorder in general population. Presence of white matter hyperintensities (WMHs) in brain MRI of migraine patients was not studied clearly. Detection of the prevalence of white matter hyperintensities in migraine patients determines its correlation with migraine severity, type and duration.Cross sectional analytic study was conducted on migraine patients attending neurology clinic Suez Canal University Hospital. Sixty-five patients with migraine aged from 18 to 50 years were included. We excluded smokers and patients with hypertension, cardiac disease, diabetes mellitus, endocrine dysfunction, oncological and hematological diseases, infectious diseases, demyelinating disorders, and Alzheimer disease. Brain MRI and laboratory investigation was done for all patients.White matter hyperintensities were significant more frequent in migraine with aura than those without aura. According to MIGSEV scale, white matter hyperintensities were highly significantly more frequent in grade III severity than grades II and I. The number of white matter hyperintensities increases significantly with increase intensity of pain during attack. The number of white matter hyperintensities increases significantly with increase intensity of nausea, disability, tolerability during attack and age. Resistance to treatment also shows statistically significant difference in increase number of WMHs.White matter hyperintensities are present in 43.1% of migraine patients. Age, presence of aura, nausea, disability during attack, resistance to treatment, and severity of headache and duration of migraine are considered a risk factor for development of white matter hyperintensities. Headache disorders are among the most common disorders of the nervous system  are referred to lesions that appeared in MRI that has infarction features but did not cause any clinical symptoms or other stroke-related signs. These lesions have common distribution in subcortical and deep white matter. In migraine with aura patients, WMH were found in deep white matter in 47% and in the periventricular region in 19% in contrast to small vessel disease where WMH found mainly at periventricular region  criteria of migraine  mild, (2) moderate, (3) intense, (4) very intenseB.Duration of attack, h: (1) < 4, (2) 4–12, (3) 12–24, (4) > 24C.Nausea: (1) non, (2) mild, (3) intense, (4) vomitingD.Disability: (1) no, (2) mild, (3) marked, (4) confined to bedE.Tolerability: (1) tolerable, (2) barely tolerable, (3) intolerableF.Resistance to treatment: (1) no, (2) yesG.Frequency of attacks: (1) < 4/year, (2) 5–10/year, (3) 1–2/month, (4) 1/week, (5) > 1/weekComplete medical history done for all selected subjects. Assessment of migraine severity had done by using Severity of migraine defined according to the main four component factors of the MIGSEV scale  as: low (grade1) if at least 1 of the 4 items with a minimum score, and no item with a maximum score; high (grade 3) if at least 1 of the 4 items with a maximum score, and no item with a minimum score OR at least 2 items with a maximum score; and intermediate (grade 2) is all other cases.Full clinical examination including neurological evaluation was done for detection of patients with focal neurological deficit. The following laboratory investigations were done for all the patients: lipid profile, fasting blood glucose, and thyroid-stimulating hormone levels.2. The matrix was 256 × 256, and the slice thickness was 5 mm, with a gap of 1 mm for all pulse sequences. For standard and accurate axial slice positioning, the anterior and posterior commissural line (AC-PC line) was used as a reference for T2-weighted and FLAIR images.Brain MRI was done for all patients. All MRI studies were carried out at Radiology Department, Suez Canal University Hospital, using 1.5 Tesla MR Imager . Circularly polarized clinical head coils were used in all patients. Sequences done are T1-weighted spin-echo sequence (Repetition Time “TR”/Echo Time “TE”: 450/15), T2-weighted turbo spin-echo sequence , FLAIR (Fluid Attenuation Inversion Recovery) sequence  and diffusion weighted (DW) images with the b value = 1000 s/mmAll original DICOM images were transferred to Philips workstation for image analysis by the same radiology consultant  who was blinded to the clinical data of patients. White matter hyperintensities, either unilateral or bilateral, are detected as high-signal-intensity punctate foci on T2WI and FLAIR images most commonly in the white matter of the centrum semiovale, contrary to small high-signal-intensity lesions seen at deep white matter of ischemic brain changes. Patients were divided into two groups: those with white matter hyperintense punctate foci and those without any lesions. WMH were considered if visible as hyperintense on T2-weighted and FLAIR images, without hypointensity on T1-weighted scans, and were larger than 3 mm.p value is considered as significant when p < 0.05 and highly significant when p < 0.01.Data were fed to the computer and analyzed using IBM SPSS software package version 20.0  ; Kruskal Wallis test ; and Spearman coefficient .The study protocol was approved by the ethics committee of the Faculty of Medicine of Suez Canal University 20 July, 2016, registration number is 2877.The purpose of the study was explained, and an informed written consent was taken before taking any data or doing any investigations. The participants were informed that their participation was voluntary and that they could withdraw from the study at any time without consequences. The participants were assured that all data will be considered confidential and will not be used outside this research. At the end of this study, records will be destroyed appropriately.This study included 65 patients with migraine. Their age ranged from 18 to 50 years with mean age 28.98 ± 10.16 years. Forty-eight patients (73.8%) were females, and 17 patients (26.2%) were males.We found that 55 patients (84.6%) of the patients had migraine without aura and 10 patients (15.4%) had migraine with aura; 8 of them has visual aura and 2 has sensory aura. According to severity of migraine, 32 patients (49.2%) were grade I, 20 patients (30.8%) were grade II, and 13 patients (20.0%) were grade III. Thirty-three patients (50.8%) of the studied patients had migraine for less than 5 years, and 18 (27.7%) patients had migraine for 5–10 years, while 10 patients (15.4%) had migraine for 10–15 years, and 4 patients (6.2%) had migraine for more than 15 years.According to MIGSEV scale: intensity of pain showed that 22 patients (33.8%) had mild pain, 27 patients 41.5%) had moderate pain, 6 patients (9.2%) had intense pain, and 10 patients (15.4%) had very intense pain. According to duration of headache attack, 17 patients (26.2%) had attacks less than 4 h, 27 patients (41.5%) had attacks last for 4–12 h, 11 patients (16.9%) had attacks last for 12–24 h, and 10 patients (15.4%) had attacks last for more than 24 h  had no nausea, 21 patients (32.3%) had mild nausea, 7 patients (10.8%) had intense nausea, and 4 patients (6.2%) had vomiting. According to disability during attack, 31 patients (47.7%) had no disability, 22 patients (33.8%) had mild disability, 8 patients (12.3%) had marked disability, and 4 patients (6.2%) became confined to bed. According to tolerability of headache during attack, 44 patients (67.7%) had tolerable headache, 16 patients (24.6%) had barely tolerable headache, and 5 patients (7.7%) had intolerable headache. According to resistance to treatment, 54 patients (83.1%) did not have resistance, while 11 patients (16.9%) had resistance , six of them had one lesion (9.2%)  with maximum number of hyperintensities 9 lesions, while in migraine without aura the median was (0.91 ± 1.49) with maximum number of 6 lesions. There was no statistical significant difference detected between type of aura and white matter hyperintensities. According to grade of migraine, white matter hyperintensities were highly statistically significant more frequent in grade III severity with median (2.62 ± 2.69) with maximum number of hyperintensities 9 lesions, while grade II severity has median (1.55 ± 1.73) and maximum number of hyperintensities 6 lesions, and finally grade I severity has median (0.44 ± 0.88) and maximum number of hyperintensities 3 lesions. There was no statistical significant difference detected between duration of migraine and white matter hyperintensities , intense pain (2.0 ± 1.26), moderate pain (0.93 ± 1.57), and mild pain (0.55 ± 0.96). While duration of attack and frequency of attacks show no statistical significant difference in number of white matter hyperintensities Table .Table 4NThe number of white matter hyperintensities was found to increase significantly with increase intensity of nausea during attack as mean number of hyperintensities in patients who had vomiting 2.75 ± 2.50), intense nausea (2.29 ± 1.89), mild nausea (1.38 ± 1.66), and those did not have nausea (0.70 ± 1.70). Also disability and tolerability during attack show statistically significant difference in increase number of white matter hyperintensities. Mean number of hyperintensities to those patients confined to bed (2.0 ± 2.83), marked disability (2.38 ± 1.77), mild disability (1.82 ± 2.28), and those had no disability was 0.39 ± 0.76. While mean number of hyperintensities who had intolerable headache was 3.0 ± 2.24, barely tolerable 2.25 ± 2.54, and those has tolerable headache 0.64 ± 1.08. At the last, resistance to treatment also shows statistically significant difference in increase number of white matter hyperintensities as mean number of hyperintensities in patients who had resistance to treatment was 2.27 ± 1.74 and those who did not have resistance to treatment was 1.0 ± 1.79 . Age, presence of aura, nausea, and disability during attack, resistance to treatment, and severity of headache and duration of migraine are considered a risk factor for development of WMHs, although WMHs were detected more frequent in grade III severity of migraine that is considered an association.


Objective: To highlight potential functional variants and causal genes for ischemic stroke (IS) in genomic loci identified by genome-wide association studies (GWAS).Methods: We examined the association between m6A-SNPs and IS in large scale GWAS. Furthermore, eQTL analysis was performed to evaluate the effect of m6A-SNPs on gene expression. The top associations between m6A-SNPs and gene expressions were validated in 40 individuals from the Chinese Han population. Besides, we applied differential expression analysis and Mendelian randomization (MR) analysis to detect potential causal genes for IS.Results: We found 310 (7.39%) m6A-SNPs which were nominally associated with IS. The proportion of m6A-SNPs with P < 0.05 for IS was significantly higher than the non-m6A-SNPs . We found that the IS-associated m6A-SNP rs2013162 was associated with IRF6 expression (P = 6.30 × 10−23), meanwhile IRF6 was differentially expressed between IS cases and controls (P = 6.15 × 10−3) and showed a causal association with IS (P = 3.64 × 10−4). Similar results were found for m6A-SNP rs2273235 in the NDST1 gene which was associated with cardioembolic stroke (P = 8.47 × 10−3). The associations of rs2013162 and rs2273235 with the expression of IRF6 and NDST1 were validated in blood cells (P = 0.0247 and 0.0007), respectively.Conclusions: This study showed that m6A-SNPs may affect IS risk through altering gene expressions. The results suggested that m6A might play a role in IS etiology and gene expressions that affected by m6A may be causal factors for IS. Ischemic stroke (IS) is the second leading cause of death worldwide . As a co6-methyladenosine (m6A) is a pervasive RNA modification that plays critical roles in mRNA stability, protein expression and several other cellular processes , can influence m6A by changing the RNA sequences of the target sites  and common etiological subtypes of LAS, CES, and SVS. These datasets were available at the MEGASTROKE website (http://megastroke.org/).In this study, we first investigated the effect of the male GWAS . This GW6A-SNPs in these 8 million SNPs, we annotated them using a list of m6A-SNPs which were downloaded from the m6AVar database (http://m6avar.renlab.org/). The list contains 13,703 high, 54,222 medium and 284,089 low confidence level m6A-SNPs for human  from the GWAS datasets for IS as matched background, and then determined if the proportion of m6A-SNPs with P < 0.05 was significantly higher than the proportion of non-m6A-SNPs with P < 0.05 in the 1,000 sets for each trait. Besides, to assure that allele frequency differences between m6A and non-m6A-SNPs are not driving the conclusion that there is overrepresentation of m6A-SNPs in stroke cases, we selected from allele frequency bins of 1–5, 5–10, 11–20, 21–30, 31–40, and 41–50% for each set of non-m6A-SNPs to more precisely mirror the distribution of m6A-SNPs.Among IS-associated SNPs, we determined if m6A-SNPs may participate in gene expression regulation through exerting influence on RNA modification, thus they may be associated with gene expression level. We carried out the cis-acting eQTL analysis to obtain evidence on associations between the identified m6A-SNPs and gene expressions in HaploReg . To validate the eQTLs we also tested genotypes and mRNA expression levels in peripheral blood mononuclear cells (PBMCs) of 40 unrelated Chinese Han individuals (age range from 27 to 67) using RT-PCR method to obtain additional evidence to support the top IS-associated m6A-SNPs. PBMCs were isolated from 15 ml peripheral blood by density gradient centrifugation using Lymphoprep . Total RNA and DNA were extracted in the same lab according to the instructions recommended by the manufacturer. The study was approved by the ethical committee of Soochow University. The written informed consent was obtained from all of the participants.The m6A-SNPs showed cis-eQTL effects on were associated with IS based on the expression profile data (GSE22255) available in the GEO database (http://www.ncbi.nlm.nih.gov/geo). GSE22255 contained data of gene expression levels in PBMCs from 20 IS cases to 20 controls  analysis , 305 (6.72%), 279 (6.18%), and 205 (6.26%) were nominally (P < 0.05) associated with AIS, LAS, CES, and SVS, respectively. The proportion of m6A-SNPs which have GWAS P < 0.05 for AIS was significantly higher than the non-m6A-SNPs . But it's not for LAS , CES , or SVS , which were analyzed in smaller sample than AIS. In the original METASTROKE GWAS, SNPs with minor allele frequencies (MAF) ≥ 0.01 were examined. Therefore, the MAF of SNPs considered in our study was ≥0.01. In fact, the MAF of about 90% of the 321 IS-associated SNPs were >0.05. The distributions of MAF were not different between m6A-SNPs and non-m6A-SNPs with P < 0.05 (goodness-of-fit test P = 0.1551). While selected from allele frequency bins of 1–5% (number of SNPs sampled n = 583), 6–10% (n = 663), 11–20% , 21–30% (n = 720), 31–40% (n = 628), and 41–50% (n = 539) for each of the 1,000 sets of non-m6A-SNPs to mirror the distribution of m6A-SNPs for AIS, we also found that the proportion of m6A-SNPs with P < 0.05 was significantly higher than the non-m6A-SNPs .We found about 4,000 mP < 0.05/4000 for roughly 4000 SNPs). The association between m6A-SNPs rs11559309  and AIS reached the significance level of 1.25 × 10−5, followed by rs7398833 (P = 1.78 × 10−5) in CUX2 gene, rs5213 (P = 2.46 × 10−5) in KCNJ11 gene and rs10832778 (P = 4.25 × 10−5) in NCR3LG1 gene. The most significant m6A-SNPs for LAS, CES, and SVS were rs174535 (P = 6.05 × 10−5) in MYRF gene, rs2273235 (P = 3.99 × 10−4) in NDST1 gene and rs1887812 (P = 4.05 × 10−4) in BRDT gene  might alter regulatory motifs (CUX2) was associated with expression of ALDH2 (P = 1.47 × 10−5), ATXN2 (P = 2.74 × 10−6), FAM109A (P = 3.41 × 10−3) and SH2B3 (P = 1.27 × 10−10), and rs174535 (MYRF) was associated with expression of FADS1 (P = 2.37 × 10−31), FADS2 (P = 6.68 × 10−28), and TMEM258 (P = 1.00 × 10−56).To further clarify the possible functional mechanisms underlying the identified m tissues . The 5 Iy motifs   (IRF6 and NDST1 were affected by rs2013162 (P = 6.30 × 10−23) and rs2273235 (P = 3.23 × 10−7) according to eQTL data from HaploReg, respectively. It means that these 2 m6A-SNPs may affect IS through altering the local gene.For the genes which were presented in  < 0.05) . Among tPSMR < 5 × 10−6), and there was no significant heterogeneity underlying the eQTL signals (PHEIDI > 0.05) (P < 3.33 × 10−3 were considered significant) identified that three genes  were significantly associated with AIS, IL16 was associated with LAS and NDST1 was associated with CES (PHEIDI > 0.05).The SMR analysis identified several genes as potential causal genes underlying IS GWAS association ( > 0.05) . For thewith CES . All of IRF6 and NDST1 showed more convincing evidence. For example, the IFR6 SNPs achieved suggestive evidence of association with AIS (P < 5 × 10−5) (P = 1.27 × 10−4) m6A-SNP rs2013162 may affect IRF6 expression (P = 6.30 × 10−23), IRF6 was differentially expressed between IS cases and controls (P = 6.15 × 10−3) and showed a causal association with AIS . The association between NDST1 SNPs and CES did not reach the genome-wide significance level but many SNPs that were in linkage disequilibrium showed suggestive evidence (P = 3.99 × 10−4) m6A-SNP rs2273235 may affect NDST1 expression (P = 3.23 × 10−7), NDST1 was differentially expressed between IS cases and controls (P = 2.65 × 10−2) and showed a causal association with CES . So subsequently, we tested the association between rs2013162 and rs2273235 and mRNA expression levels of IRF6 and NDST1, respectively. The MAF for rs2013162 and rs2273235 were 0.3736 and 0.4821 in Europeans  (We noticed that  × 10−5) . The AISevidence . The CESuropeans , and 0.3 0.0007) , 3, resp6A-SNPs data, gene expression data and genetic association data from large scale GWAS. We found out several m6A-SNPs  which may be putative functional variants for IS. Moreover, we also showed that these SNPs have potential to affect transcription regulation and were associated with expressions of the local genes . These genes may be potential causal genes for IS.This study represents the first effort to identify putative functional variants and causal genes for IS by integrating m6A-SNPs may be causally associated with IS by influencing gene expression. Studies have shown that m6A modification plays a pivotal role in the regulation of downstream molecular events such as nuclear export, stability, translatability, splicing, and miRNA processing  in public and in-house data. This finding supported our hypothesis that m6A-SNPs may be causally associated with IS by influencing gene expression.It seems that mocessing . So one IRF6 gene encodes a member of the IRF family of transcription factors which play roles in cardiovascular diseases  has been well studied on disease. This SNP was not reported in the original METASTROKE GWAS (P = 3.26 × 10−4) (P = 9.34 × 10−3)   and largAS P = 3. × 10−4  was associated with CES in two GWAS  and the effects of the large amounts of m6A-SNPs on IS should be evaluated in larger GWAS datasets. Finally, the functionalities of the detected SNPs, especially effects on m6A modification, have not been experimentally validated. Further experiments are needed to determine their functions.This study has several limitations. First, no genome-wide significant associations between m6A-SNPs, and showed that these SNPs may affect the expressions of the local genes which might be potential causal genes for IS. This study increases our understanding on the regulation patterns of SNP. Although we found supplementary functional information to support the significant findings, further functional studies were needed to elucidate the mechanisms.In summary, the present study found out several IS-associated mAll datasets generated for this study are included in the manuscript and/or the This study was carried out in accordance with the recommendations of Soochow University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Soochow University.X-BM, S-FL, Y-HZ, and HZ contributed at all stages of manuscript preparation. HZ critically appraised and approved the final manuscript. All authors were involved in data recording and discussed the results.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.


Ureaplasma urealyticum (UU), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and herpes simplex virus (HSV) from 1 January 2013 to 31 December 2016 in Beijing, China. Results indicated that a decrease trend  in male and an increase trend  in female were observed during the period. Patients aged 20–39 years old were mostly affected by these pathogens, while the prevalence in patients aged 20–29 years old was the highest, The prevalence of UU in male was significantly lower than in female , while the prevalence of NG in male was significantly higher than in female . In patients with co-infections, 60.6% of male and 71.4% of female were co-infected by UU + CT. In total, 11.9% and 88.1% of patients with HSV infections were confirmed to be infected by HSV-1 and HSV-2. This study could contribute to a better understanding of the current epidemiological features of UU, NG, CT and HSV among symptomatic patients attending an outpatient clinic in Beijing, China, and thus facilitate to develop more effective intervention, prevention and treatment of STI.The prevalence of sexually transmitted infection (STI) pathogens in Beijing, China, is rarely reported. In this study, 34 911 symptomatic outpatients with suspected genital infections who attended outpatient clinics in a tertiary care hospital were included to investigate the updated prevalence of  Although some infections are curable , they can cause acute conditions such as cervicitis, urethritis and genital ulceration, even lead to severe complications and long-term sequelae including pelvic inflammatory disease, ectopic pregnancy, infertility, neonatal death, premature delivery, blindness and cervical carcinoma [Ureaplasma urealyticum (UU) is also confirmed to be an aetiological pathogen in nongonococcal urethritis (NGU) [Sexually transmitted infections (STIs), also referred to as sexually transmitted diseases or venereal diseases, are infections caused by pathogens which are commonly transmitted by sex, especially vaginal intercourse, anal sex or oral sex. According to the WHO data updated in August 2016, there are about 357 million new infections with chlamydia, gonorrhoea, syphilis or trichomoniasis, more than 500 million genital infections caused by herpes simplex virus (HSV), and more than 290 million women infections caused by human papillomavirus (arcinoma . Ureaplais (NGU) .Neisseria gonorrhoeae (NG) resistance rates to penicillin, tetracycline and ciprofloxacin are also high in China [Based on the prevalence data in general population from 2005 to 2012, the estimated global prevalence of chlamydia, gonorrhoea, trichomoniasis and syphilis is 4.2%, 0.8%, 5.0% and 0.5% in female, and 2.7%, 0.6%, 0.6% and 0.48% in male, respectively . Before in China . Howeverin China . The preChlamydia trachomatis, CT; NG; HSV) according to our retrospective analysis of outpatients from a tertiary care hospital in Beijing, China.The aim of this study was to characterise the prevalence of four common STI pathogens  with suspected genital infections who attended the outpatient clinics of department of gynaecology and department of urology in our tertiary care hospital (1500 beds) from 1 January 2013 to 31 December 2016. All patients complained of one or more urethral syndromes like vaginitis, cervicitis, dysuria, urinary frequency and pelvic inflammatory disease. After laboratory tests, patients could obtain effective symptomatic treatments accordingly. Patients who were tested twice or more times during the treatment process were only calculated for once as following: negativity was considered if all tests were negative, while positivity was considered if one test was positive.g for 5 min at room temperature. The supernatant was discarded and the pellet was washed twice with normal saline. After the second washing, the pellet was resuspended in 100 µl extraction buffer and heated at 100 °C for 10 min. After another centrifugation at 13 000 g for 5 min, the supernatants were collected and used as DNA extracts.The genital swabs were collected from outpatients by strictly aseptic manipulation. Swabs were placed into 2 ml of sterile normal saline and the tubes were vortexed. CT real-time polymerase chain reaction (PCR) kit, NG real-time PCR kit, UU real-time PCR kit and HSV I&II real-time PCR kit (Liferiver Biotech) were used for detection as per the manufacture's instruction. After September 2015, HSV I&II typing real-time PCR kits (Liferiver Biotech) were used for detection and typing. One millilitre of each sample was centrifuged at 13 000 ® 480 system in the following conditions: 94 °C 2 min, 40 cycles of 93 °C 15 s and 60 °C 60 s.The reaction mixtures were prepared as described by the manufacturer's instructions. The amplifications were performed on a Roche LightCyclerχ2 analysis. P values of less than 0.05 were considered statistically significant.All statistical analysis was performed using the SPSS software package version 20.0. Overall prevalence was calculated by dividing the number of positive patients by the number of tested patients. The age-adjusted prevalence in male or female was calculated according to the updated Beijing census population data (2010). Age was stratified into seven categories: 10–19, 20–29, 30–39, 40–49, 50–59, 60–69 and 70–79. The confidence intervals (CI) were calculated at the 95% level. Frequencies were compared using Pearson's A total of 34 911 outpatients  were included. The overall prevalence of UU, CT, NG and HSV was 35.5% (8481/23 860) (95% CI 34.9%–36.2%), 7.7% (1827/23 860) (95% CI 7.3%–8.0%), 2.7% (654/23 860) (95% CI 2.5%–3.0%) and 1.2% (277/23 860) (95% CI 1.0%–1.3%) in male, and 58.0% (6414/11 051) (95% CI 57.1%–59.0%), 5.9% (650/11 051) (95% CI 5.4%–6.3%), 0.6% (66/11 051) (95% CI 0.5%–0.7%) and 1.1% (124/11 051) (95% CI 0.9%–1.3%) in female, respectively. The prevalence of co-infections (two pathogens or more) in male and female was 5.0% (1195/23 860) (95% CI 4.7%–5.3%) and 6.3% (695/11 051) (95% CI 5.8%–6.7%), respectively. The prevalence of mono-infection with UU in male and female was 31.8% (7593/23 860) (95% CI 31.2%–32.4%) and 52.5% (5806/11 051) (95% CI 51.6%–53.5%), respectively.P < 0.05).In total, 3073, 7741, 8644 and 4402 male patients, as well as 629, 1933, 4501 and 3988 female patients, were tested from 2013 to 2016, respectively. As the adjusted prevalence showed in According to seven age categories of 10–19, 20–29, 30–39, 40–49, 50–59, 60–69 and 70–79, there were 485, 9970, 7612, 3654, 1549, 523 and 67 male patients, as well as 117, 3723, 3821, 2077, 1069, 209 and 35 female patients, were included in this study, respectively. The majority of patients infected by UU, CT, NG and HSV were those aged 20–39 years old, which was characterised by 73.5% (6234/8481) (95% CI 72.6%–74.4%), 79.5% (1452/1827) (95% CI 77.6%–81.3%), 77.5% (507/654) (95% CI 74.3%–80.7%) and 68.2% (189/277) (95% CI 62.7%–73.8%) for male, 71.7% (4598/6414) (95% CI 70.6%–72.8%), 80.2% (521/650) (95% CI 77.1%–83.2%), 78.8% (52/66) (95% CI 68.7%–88.9%) and 72.6% (90/124) (95% CI 64.6%–80.5%) for female, respectively. As shown in vs. 49.3%, P < 0.05). Contrarily, the adjusted prevalence of NG in male was significantly higher than that of female  (P > 0.05)  . There w > 0.05) .Fig. 3.vs. 27.8%, 8.9% vs. 4.2%; P < 0.05), while the adjusted prevalence of CT + NG in female was significantly lower than those in male  (P > 0.05)  (95% CI 57.8%–63.4%) of male and 71.4% (496/695) (95% CI 68.0%–74.7%) of female were co-infected by UU + CT. The adjusted prevalence of UU + CT and UU + HSV in female was significantly higher than those in male (44.0%  < 0.05) . There w > 0.05) .Fig. 4.From September 2015 to December 2016, the HSV I&II typing real-time PCR kit, instead of the universal HSV I&II real-time PCR kit, was used to distinguish infections caused by HSV-1 or HSV-2. In the 12 218 tested patients, HSV types were confirmed in 118 patients which was characterised by 11.9% (14/118) (95% CI 5.9%–17.8%) of HSV-1 and 88.1% (104/118) (95% CI 82.2%–94.1%) of HSV-2.STIs remain major significant public health problems worldwide . In ChinA National System of STIs Surveillance was initiated to monitor the magnitude of STIs and its trend in China in 1988. Based on data retrieved from the surveillance system, the epidemiology of STIs from 1989 to 1998 is described in a previous study which shows an increasing prevalence of gonorrhoea, syphilis, genital warts and NGU in patients . HoweverFor both male and female, a strong risk factor associated with CT and NG infections is young age , 16. OurGenital herpes is a common sexually transmitted disease caused by HSV-1 or HSV-2. Typically, HSV-1 causes non-sexually-transmitted oral herpes infections. However, oral sex can increase the possibility and incidence of primary genital infections by HSV-1 –19. In tUU is confirmed to be an aetiological pathogen in NGU . In thisOur study has three limitations. First, a weakness of our study is that it comes from a single hospital. Larger sample and multi-centres  studies including more STI pathogens are needed to investigate the epidemiology more rigorously. Second, because pharyngeal and rectal swabs are not available in this study, the NG prevalence might be underestimated due to higher prevalence in the risk population like MSM and sex workers. Third, this is a retrospective study and all symptomatic individuals are outpatients from department of gynaecology and department of urology. No interviews or questionnaires are completed to obtain patients’ information like marriage, education, condom usage, sexual history, sexual partners, sexual debut, sexual orientation, STI history, and drug usage, which might bias the prevalence estimates presented here. In addition, the demographic information could be used to identify more risk factors for these STI pathogens in Chinese.In conclusion, youth between 20 and 39 years old are mostly affected by the four common STI pathogens , while the prevalence of those between 20 and 29 years old is the highest; NG infections are biased towards male and UU infections are biased towards female; HSV-1 has become a common STI pathogen in Beijing, China; as to the prevalence of UU, CT, NG and HSV from 2013 to 2016, there is a decrease trend and an increase trend in male and female, respectively . This study could contribute to a better understanding of the current epidemiological features of four common STI pathogens  in Beijing, China, and thus facilitate to develop more effective intervention, prevention and treatment of STI.


Metallosphaera sedula is a thermoacidophilic archaeon and has an incomplete TCA/glyoxylate cycle that is used for production of biosynthetic precursors of essential metabolites. Citrate synthase from M. sedula (MsCS) is an enzyme involved in the first step of the incomplete TCA/glyoxylate cycle by converting oxaloacetate and acetyl-CoA into citrate and coenzyme A. To elucidate the inhibition properties of MsCS, we determined its crystal structure at 1.7 Å resolution. Like other Type-I CS, MsCS functions as a dimer and each monomer consists of two distinct domains, a large domain and a small domain. The oxaloacetate binding site locates at the cleft between the two domains, and the active site was more closed upon binding of the oxaloacetate substrate than binding of the citrate product. Interestingly, the inhibition kinetic analysis showed that, unlike other Type-I CSs, MsCS is non-competitively inhibited by NADH. Finally, amino acids and structural comparison of MsCS with other Type-II CSs, which were reported to be non-competitively inhibited by NADH, revealed that MsCS has quite unique NADH binding mode for non-competitive inhibition. Metallosphaera sedula belongs to the sulfolobaceae family. It is a thermoacidophilic archaea with optimum growth conditions of 73°C and pH 2.0 [M. sedula grows chemolithoautotrophically on metal sulfides or molecular hydrogen, and obtains reducing power by biologically catalyzing iron oxidation and metal sulfide oxidation [M. sedula gains access to a carbon source by immobilization of bicarbonate using the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle [M. sedula (MsCS) catalyzes the first step of the incomplete TCA/glyoxylate cycle in this strain.d pH 2.0 –3. M. seB) cycle . The triB) cycle . CitrateCSs (EC 2.3.3.1) catalyze conversion of oxaloacetate and acetyl-CoA into citrate and coenzyme A and are well-conserved enzymes in most organisms –10 Fig . In the MsCS in complex with citrate at 1.7 Å and in complex with oxaloacetate at 2.1 Å. The findings reveal that the protein functions as a dimer, similar to other Type-I CSs. Based on the complex structures with its substrate and product, we elucidated the substrate binding site formation and structural changes upon substrate binding. Interestingly, enzyme inhibition kinetic studies revealed that, unlike other Type-I CSs, MsCS is non-competitively inhibited by NADH, which is a unique biochemical property of the archaeal MsCS.CSs comprise Type-I and Type-II depending on their oligomeric status and an additional β-sheet at the N-terminus. Type-I CSs are homo-dimeric and found in gram-positive bacteria, archaea, and eukaryote . On the MsCS was amplified from chromosomal DNA of M. sedula by polymerase chain reaction (PCR). The PCR products were digested by NdeI and XhoI restriction enzymes, and sub-cloned into the pET-30a expression vector, which contained a 6×His tag at the C-terminus of the target protein. The resulting expression vector pET-30a:MsCS, was transformed into a Escherichia coli BL21(DE3)-T1R strain, which was grown to an OD600 of 0.7 in fresh LB medium containing 50 mg L-1 kanamycin at 310 K, and MsCS protein expression was induced by 0.5 mM 1-thio-β-D-galatopyranoside (IPTG). After 20 h at 293 K, the cells were harvested by centrifugation at 4,000 × g for 15 min at 277 K. The cell pellet was resuspended in ice-cold buffer A  and disrupted by ultrasonication. The cell debris was removed by centrifugation at 13,000 × g for 30 min, and the lysate was applied onto a Ni-NTA agarose column (Qiagen). After washing with buffer B , the bound proteins were eluted with buffer C . Finally, the trace contaminants were removed by size-exclusion chromatography using a HiPrep 26/60 Sephacryl S-300 HR column  equilibrated with buffer A. The eluted protein had a molecular weight of approximately 85 kDa, indicating a dimeric structure. The protein was concentrated to 54 mg mL-1 using spin column , and kept at 193 K for further experiments. All purification steps were performed at 277 K.The gene coding for MsCS protein was initially performed with commercially available sparse-matrix screens, including Index, PEG ion I and II (Hampton Research), and Wizard Classic I and II (Rigaku Reagents) using the sitting-drop vapor diffusion method with MRC Crystallization plate (Molecular Dimensions) at 295 K. Each experiment consisted of mixing 1.0 μL protein solution  with 1.0 μL reservoir solution and then equilibrating against 50 μL of reservoir solution. MsCS crystals were observed from several crystallization screening conditions. For crystal improvement, each experiment consisted of mixing 1.0 μL protein solution with 1.0 μL reservoir solution and then equilibrating it against 500 ml of the reservoir solution by the hanging-drop vapor-diffusion method at 295 K. The crystals of the best quality appeared in 0.8 M sodium phosphate monobasic, 1.2 M potassium phosphate dibasic, and 0.1 M sodium acetate pH 4.2. The actual pH condition of 0.8 M sodium phosphate monobasic, 1.2 M potassium phosphate dibasic, and 0.1 M sodium acetate pH 4.2 is pH 6.65. The crystals were transferred to a cryo-protectant solution containing 0.8 M sodium phosphate monobasic, 1.2 M potassium phosphate dibasic, 0.1 M sodium acetate/acetic acid, pH4.2 and 25% (v/v) glycerol. The crystals were harvested with a loop larger than the crystals, and flash-frozen in a nitrogen gas stream at 100 K. Data were collected to a resolution of 1.7 Å at 7A beamline of the Pohang Accelerator Laboratory , using a Quantum 270 CCD detector . All data were indexed, integrated, and scaled together using the HKL2000 software package [MsCS in complex with citrate belonged to space group P1 with unit cell parameters a = 50.18 Å, b = 53.52 Å, c = 76.28 Å, α = 93.56°, β = 105.73°, and γ = 102.16°. Assuming two MsCS molecules in the asymmetric unit, the crystal volume per unit of protein mass was 2.12 Å3 Da-1, which indicates a solvent content of approximately 42.15%. MsCS in complex with oxaloacetate was crystallized with the same method except for the addition of 20mM oxaloacetate to the protein solution . The crystals of the best quality appeared in 22% polyethylene glycol 3350 and 0.2 M potassium sodium tartrate tetrahydrate supplemented with 20mM of oxaloacetate. The actual pH condition of 22% polyethylene glycol 3350 and 0.2 M potassium sodium tartrate tetrahydrate is pH 7.24. Data were collected to a resolution of 2.0 Å at 7A beamline of the Pohang Accelerator Laboratory , using a Quantum 270 CCD detector . All data were indexed, integrated, and scaled together using the HKL2000 software package. Crystals in complex with oxaloacetate belonged to space group P1 with unit cell parameters a = 50.13 Å, b = 55.97 Å, c = 76.97 Å, α = 83.73°, β = 73.94°, and γ = 72.24°. Assuming two molecules of MsCS per asymmetric unit, the crystal volume per unit of protein mass was 2.35 Å3 Da-1, which corresponds to a solvent content of approximately 48% [Crystallization of the purified  package . CrystalMsCS in complex with citrate and with oxaloacetate was determined by molecular replacement with the CCP4 [Sulfolbus tokodaii (PDB code 1VGP) as a search model. Further model building was performed manually using the WinCoot [−3, 1 sigma at distance between 2.5–3.5 Å under 2Fo-Fc map. The refined models of MsCS in complex with citrate and oxaloacetate were deposited in the Protein Data Bank [The structure of the the CCP4  version the CCP4 , using t WinCoot , and ref WinCoot . The watata Bank  with PDBMsCS was determined by measuring the increase of absorbance at 412 nm (extinction coefficient of 1.415 × 104 M-1 cm-1). The enzyme reactions were performed with reaction mixtures of 0.5 mL total volume at 298 K. For the enzymatic activity curve versus pH, relative activity of MsCS was measured under pH 6.0 to pH 10.0. Each reaction mixture contains 10 μM MsCS protein, 50 μM acetyl-CoA, 0.1 mM oxaloacetate, 100 mM buffer , 150 mM NaCl, and 50 μM DTNB. For the kinetic analysis of oxaloacetate, reaction mixtures containing 0.5 mM acetyl-CoA, 100 mM potassium phosphate, pH 8.0, 150 mM NaCl, 50 μM DTNB, and various concentration of oxaloacetate (0.01 to 5 mM) were used. For the kinetic analysis of acetyl-CoA, reaction mixture containing 100 mM potassium phosphate pH 8.0, 150 mM NaCl, 50 μM DTNB and various concentrations of acetyl-CoA (10 to 200 μM) were used. All reactions were initiated by the addition of enzyme to a final concentration of 100 nM. In order to measure the enzyme inhibition, kinetic analysis was performed using various concentrations of citrate, ATP, and NADH. The kinetic statistics of the enzyme were calculated using Origin software.The activity of Metallosphaera sedula (MsCS), we determined its crystal structure at 1.7 Å resolution. The overall refinement and validation statistics are presented in MsCS consists of seventeen α-helices (α1-α17) and four β-strands (β1-β2), with distinct large domain and small domains  and three loop regions . In the large domain, Asn182 interacts with the C2 and C3 carboxyl-groups and Arg332 stabilizes the C3 carboxyl-group of oxaloacetate via a salt bridge. In addition, His253 from the small domain interacts with the C3 carbonyl-group and Arg262 is associated with the stabilization of C3 carbonyl- and carboxyl-groups of oxaloacetate through hydrogen bonds -1, respectively. It has been known that the CS enzymes are inhibited by various molecules including citrate, ATP, and NADH [MsCS, we measured inhibition kinetics using citrate, ATP, and NADH. When we measured the inhibition kinetics of citrate, the Km values increased while the Vmax values remained constant, as the concentration of citrate increased  in complex with the NADH inhibitor elucidated the binding mode of the NADH inhibitor Click here for additional data file.


Gastric cancer remains a major health problem worldwide. Natural products, with stronger antitumor activity and fewer side effects, are potential candidates for pharmaceutical development as anticancer agents. In this study, quercetin and curcumin were chosen for testing and were applied separately and in combination to human gastric cancer MGC-803 cells. The MTT assay was used to evaluate cell growth inhibition. Annexin V-FITC/PI was carried out to measure apoptosis rate. Flow cytometry was performed to analyze mitochondrial membrane potential levels. Western blots were applied to detect expression of cytochrome c, total and phosphorylated ERK and AKT. Combined treatment with curcumin and quercetin resulted in significant inhibition of cell proliferation, accompanied by loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c and decreased phosphorylation of AKT and ERK. These results indicate that the combination of curcumin and quercetin induces apoptosis through the mitochondrial pathway. Notably, effect of combined treatment with curcumin and quercetin on gastric cancer MGC-803 cells is stronger than that of individual treatment, indicating that curcumin and quercetin combinations have potential as anti-gastric cancer drugs for further development. Gastric cancer remains a leading cause of cancer deaths worldwide. Although mortality rates of gastric cancer have declined over the past few decades, the disease still has a poor prognosis and remains a major health problem , as manyCurcuma longa (commonly known as turmeric). It has been shown to suppress multiple signaling pathways and inhibit cancer cell proliferation, invasion, metastasis and angiogenesis [Natural products, with stronger antitumor activity and fewer side effects, have gained increasing scientific attention in recent years. Curcumin , a yelloogenesis . The cliogenesis ,5. To ovogenesis . In partogenesis ,8. Herei50 values of curcumin and quercetin against MGC-803 cells were 9.32 ± 1.06 μM and 23.35 ± 2.14 μM. Growth inhibitory rates were 12.26% ± 3.09%, 40.98% ± 4.33%, 51.44% ± 3.90%, 76.99% ± 3.06% and 84.37% ± 4.99% after cells were treated with 10.0 μM quercetin, 5.0 μM curcumin, 10.0 μM curcumin, 10.0 μM quercetin + 5.0 μM curcumin and 10.0 μM quercetin + 10.0 μM curcumin, respectively.The cytotoxicity of curcumin and quercetin individually and in combination was determined by the MTT assay to evaluate their antitumor activity. It was shown that the combination of curcumin and quercetin exhibited stronger anticancer activity than individual treatment. The ICin vitro and in vivo through a variety of mechanisms. It inhibits proliferation and induces apoptosis in a wide array of cancer cell types in vitro, including cells from cancers of the bladder, breast, lung, pancreas, prostate, cervix, head and neck, ovary, kidney, brain, bone marrow, and skin [Curcumin exhibits anti-cancer activities both and skin . In formand skin .Defects in apoptosis are critical in tumorigenesis and resistance to therapy . To clarApoptotic cell death can be induced through the extrinsic  or the intrinsic  signalling pathways. Within the mitochondrial pathway of apoptosis, chemotherapeutic drugs or other stimuli change the properties of the inner mitochondrial membrane and subsequently cause the loss of mitochondrial membrane potential (ΔΨm) and release of cytochrome c from the mitochondria into the cytosol. Collapse of ΔΨm and release of cytochrome c are biochemical markers in apoptosis through the mitochondrial pathway . To elucp > 0.05), 68.49% ± 3.18% (p < 0.01) and 48.14% ± 3.08% (p < 0.01) of the control, respectively  for control, 10.0 μM quercetin, 5.0 μM curcumin and combination group were 3.94% ± 1.50%, 20.16% ± 2.65%, 102.10% ± 2.73% and 144.09% ± 5.62%, respectively . The resp > 0.05). Densitometric ratios (%) of p-ERK/ERK for control, 10.0 μM quercetin, 5.0 μM curcumin and combination group were 87.01% ± 4.39%, 84.25% ± 3.32%, 34.89% ± 3.24% and 14.14% ± 2.18%, respectively. Densitometric ratios (%) of p-AKT/AKT for those were 122.04% ± 7.23%, 116.56% ± 3.09%, 60.98% ± 5.61% and 36.49% ± 2.85%, respectively. All treatments had no effect on total proteins levels. These results further indicate that the MAPK and PI3K pathways are also responsible for quercetin and curcumin-induced cell growth inhibition. To support further development of these two phytochemicals, investigation about the combined effect of quercetin and curcumin on MAPK, PI3K and other pathways is still in need.Given that cancer is a multi-step process, antitumor activities generally involve multiple pathways. For example, curcumin can modulate various molecular targets including transcription factors, growth factors and their receptors, cytokines, enzymes, and genes regulating cell proliferation and apoptosis . Deregulin vivo. In this paper, we reported studies of the combined effect of curcumin and quercetin on gastric cancer MGC803 cells and of the mechanism by which this effect occurs. Results show that the combination of curcumin and quercetin can inhibit the phosphorylations of AKT and ERK and induce apoptosis via mitochondrial pathway  or Santa Cruz Co. . Secondary anti-mouse and anti-rabbit IgG-HRP were products of KangChen Biotechnology Co. . All culture media and growth supplements were acquired from Gibco-BRL Co. . Other routine reagents used were of analytical or HPLC grade and purchased from local commercial sources.Quercetin and curcumin of 98% purity were purchased from Tianjin Shilan Technology Co. Ltd . 3,3ʹ-Dihexyloxacarbocyanine iodide (DiOC6(3)) and 3-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. . Annexin V-FITC/PI Apoptosis Detection Kit was purchased from KeyGEN Biotech . Primary antibodies against cytochrome 2 at 37 °C.MGC-803 cells (human gastric cancer cell line) were cultivated in RPMI 1640 medium containing 100 U/mL penicillin, 100 μg/mL streptomycin and 10% FBS in an incubator with a humidified atmosphere of 5% COMGC-803 cells were plated in 96-well plates and allowed to adhere 24 h. Cells were then incubated with varying doses of indicated compounds . After 3 days, MTT was added to each well and plates were incubated for 4 h. Formazan crystals were dissolved with 200 μL DMSO and absorbance at 540 nm was measured by microplate reader with 655 nm as reference filter. Cell survival was calculated as: survival (%) =  × 100% .Apoptosis rate was detected by the Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer’s instruction. Quercetin (10.0 µM) and curcumin (5.0 µM) for individual or combination treatment were cultured with MGC-803 cells for 36 h. Cells were then harvested and resuspended in 0.5 mL binding buffer containing Annexin-V (1:50) and 40 ng/sample of PI for 30 min at 37 °C in the dark. The samples were analyzed by a flow cytometer (BD FASCanto) and CellQuest software. Apoptosis rate (%) =  × 100% .ΔΨm levels were determined by flow cytometry with the mitochondrial tracking fluorescent DiOC6(3) as previously described . MGC-8036 cells/well. After 24 h incubation, cells were treated with curcumin and/or quercetin for 6–24 h. Then, cells were harvested and washed twice with ice-cold PBS.As previously described , MGC-803g for 5 min to discard the supernatant. The pellet was vortexed, and 100 μL of 1× loading buffer for every well was added. After heating at 100 °C for 20 min, the lysates in the Eppendorff were centrifuged at 15,000 g for 10 min and the supernatant was collected.For whole cell lylates, Eppendorff tubes (1.5 mL) containing cells was centrifuged at 110 g for 10 min at 4 °C. The supernatant was subsequently centrifuged at 15,000 g for 15 min at 4 °C, and the final supernatant was used as the cytosolic fraction. Then, 5 × loading buffer was added to the above obtained supernatant, and the mixture was boiled at 100 °C for 15 min.For the subcellular fraction, cells were suspended in Eppendorff tubes (1.5 mL) with a 5-fold volume of ice-cold cell extract buffer and incubated for 40 min at 4 °C. Then, the cells were centrifuged at 110 Proteins were then separated on 8%–12% sodium SDS-PAGE, electrotransferred to PVDF membranes  and probed with protein specific antibodies followed by HRP-conjugated secondary antibody. Protein bands were detected with a PhototopeTM-HRP Detection Kit  on Kodak medical X-ray processor . The cytochrome c, p-AKT, AKT, p-ERK and ERK protein were detected by specific primary antibody in the ratio of 1:1,000. HRP-conjugated secondary antibodies were diluted in the ratio of 1:2000.p ≤ 0.05 was considered statistically significant.All quantitative values are expressed as mean values ± SD of at least three independent experiments. Significant differences were determined by t-test or one-way ANOVA with SPSS 13.0 software .


Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters mK and maxV were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0–8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1–1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse “in natura”, sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable  Raw material processing generates tons of agricultural and industrial waste every year. Unfortunately, biomass accumulation and misuse of such materials cause serious environmental issues. In addition, increased energy consumption, depletion of fossil fuel sources, and the need to abate global warming have drawn special attention to a new generation of renewable energy ,2,3. In Lignocellulose consists mainly of cellulose 35–50%), hemicellulose (20–30%), and lignin (20–30%) –50%, hem,7. In faEndo-1,4-β-glucanases catalyze the initial attack on the cellulose fibrils and randomly cleave the β-1,4-glycosidic bonds present in the amorphous regions of the cellulose chain. The other two cellulases then act to convert the oligosaccharides released from this hydrolysis into glucose ,11. AccoBecause enzymes need to be exposed to extreme conditions during several industrial processes, stability to high temperatures within a wide pH range, as well as tolerance to inhibition by reaction products, are the most desired enzymatic properties at the industrial level ,12. BiomAspergillus fumigatus, a thermophilic fungus, secretes several CAZymes that can be further used in enzymatic cocktails. Therefore, this fungus plays an important role in biomass deconstruction [E. coli and characterized its function [P. pastoris offers many advantages, especially in terms of recombinant protein processing, folding, and post-translational modifications, which can influence enzyme functioning and even stability [truction ,7,14. Intability ,16.Af-egl7 cloning and expression in P. pastoris X-33 and on the characterization of the purified Af-EGL7. It shows that a recombinant GH7 endo-1,4-β-glucanase, with higher pH and thermal stabilities, is more active on complex biomasses. Our results highlight the industrial potential of the A. fumigatus recombinant endoglucanase Af-EGL7.This paper reports on the successful gene Af-egl7 (Afu6g01800) from the A. fumigatus cDNA and to clone it into the expression vector pPICZαA [Af-egl7 was transformed into E. coli DH10β for plasmid propagation. Sequencing was performed to analyze possible mutations in the Af-egl7 sequence and to verify if the sequence had been cloned in frame with α factor signal peptide, which is important to obtain the recombinant enzyme extracellularly in the culture broth.Here, the CPEC method was used to amplify the endo-1,4-β-glucanase ORF (Open reading frame)  pPICZαA . The recAf-egl7 was linearized with the restriction enzyme PmeI to enable its integration into the P. pastoris X-33 genome by homologous recombination, and transformed into this strain. After growth at 30 °C for three days, positive transformants were selected with the Zeocin resistance marker. The 24 selected positive transformants were screened in solid medium containing 1% (w/v) CM-Cellulose as a substrate, and 1% (v/v) methanol for protein induction (Pichia pastoris transformed with empty pPICZαA vector) emerged around all the tested colonies, except for number 4. This indicated expression of a functional enzyme that can degrade CM-Cellulose. Colony PCR also confirmed positive transformants.Next, the recombinant plasmid pPICZαA/nduction . After gP. pastoris X-33 transformant was selected, and Af-EGL7 was successfully expressed under control of the AOX-1 promoter after induction with 1.5% (v/v) methanol for six days. The culture supernatant was harvested by centrifugation and concentrated by tenfold before Af-EGL7 was purified by Ni2+-nitrilotriacetic (Ni-NTA) affinity chromatography. During protein elution, all fractions were collected and analyzed by SDS-PAGE  predicting four potential N-glycosylation sites in the Af-EGL7 structure, at the N96, N201, N205, and N379 residues [A previous study had indicated that the Af-EGL7 molecular weight is 48 kDa. However, the Af-EGL7 molecular weight estimated by SDS-PAGE herein was approximately 70 kDa. This difference suggested the presence of P. pastoris heterologous proteins, and to O-glycosylations, which usually occur in GH7 enzymes [Af-EGL7 treatment with Endoglycosidase H produced a protein with a molecular weight of approximately 60 kDa b. Howeve enzymes ,20.E. coli and found that this enzyme displays optimal activity at 55 °C and pH 5.0 [P. pastoris also presented optimal activity under these same conditions.In a previous work, our group characterized recombinant Af-EGL7 expressed in d pH 5.0 . Here, rmax, KM, and kcat kinetic parameters were determined at 55 °C by using CM-Cellulose, β-glucan, or Xyloglucan as substrates  occurred [2+ in lytic polyssacharide monooxygenase (LPMO) activity and the synergistic effect that LPMO and endoglucanases have on biomass hydrolysis, Af-EGL7 activity was also assessed at different Cu2+ concentrations . Addition of 5, 10 and 15 mM Cu2+ partially reduced Af-EGL7 activity to 85%, 49%, and 21%, respectively. These results showed that the association of Af-EGL7 with LPMOs (copper-dependent enzymes) in an enzymatic cocktail is possible, which could enhance cellulose hydrolysis.Af-EGL7 activity increased at low CuCAZymes (carbohydrate-active enzymes) glycosylation has been associated with higher enzyme thermal stability during natural and industrial processes, cellulose hydrolysis or binding, and protection from proteolysis ,24. In tAt 55 °C, the enzyme did not lose activity after incubation for 72 h. However, the enzyme started to lose activity at higher temperatures and retained 61% of its maximal activity after 24 h at 60 °C. After one hour at more extreme temperatures such as 70, 80 and 90 °C, the residual activities were 75%, 60% and 32%, respectively a.E. coli, stability at these temperatures decreased rapidly, and residual activity was absent after 30 min at 60–90 °C [P. pastoris, including post-translational modifications like glycosylation [The comparatively high temperature stability of the enzyme obtained here was surprising. When Af-EGL7 was expressed in 60–90 °C . Again, sylation ,16.MtEG7a and Ctendo7 from the thermophilic fungi M. thermophila and C. thermophilum displayed about 40% and 60% residual activity after eight and one hour at 80 °C, respectively [T. emersonii and N. fisheri retained more than 40% and 16% of their maximal activity after they were incubated at 70 °C for 1 h [Cellulases from thermophilic microorganisms have commonly been described as having a high thermostability ,26. The ectively ,11. Egl7 for 1 h ,27  can be used in brewing and biofuel production processes ,11,32. AAnalysis of the pH stability after incubation for 72 h did not show any Af-EGL7 activity loss. On the basis of these results, Af-EGL7 exhibited higher pH stability as compared to all the GH7 described so far, for which pH stability was only detected after shorter incubation intervals, as described in Cellobiose and glucose accumulation is one of the factors that limit biomass hydrolysis the most because it reduces enzyme catalytic efficiency and may increase process costs ,35,36.Previously, we described Af-EGL7 activity in xyloglucan . The xylAddition of glucose did not affect Af-EGL7 and even enhanced its activity at concentrations higher than 25 mM a. MoreovThese characteristics are highly important and desirable for the application of enzymes during biomass hydrolysis and make Af-EGL7 an enzyme of great commercial interest.® 1.5L) and 10 µg of thermostable Af-EGL7 and incubated at 55 °C and 1000 rpm for 24, 48, or 72 h. Interestingly, this enzyme showed a high degree of synergy with the cocktail in the deconstruction of all the tested substrates, except barley bagasse , rice straw, corncob, barley bagasse or bean straw was added to 0.009 FPU of cellulase cocktail  after 72 and 48 h of reaction, respectively.Finally, our data showed that Af-EGL7 was able to improve the hydrolysis of most of the evaluated agricultural residues, which have different compositions , was grown in yeast extract-agar-glucose (YAG) medium (2.0% (w/v) dextrose, 2.0% (w/v) agar, 0.5% (w/v) yeast extract, and 0.1% (v/v) trace elements) at 37 °C for two days to obtain a fresh conidium suspension. The conidia were inoculated to a final concentration of 2 × 106 per mL of YNB minimal medium  trace elements, and 0.05% (w/v) yeast extract) containing 1% (w/v) fructose and incubated at 37 °C and under shaking at 200 rpm for 16 h. Next, the mycelia were harvested, washed, and transferred to YNB medium containing 1% (w/v) sugarcane exploded bagasse (SEB) at 37 °C and 200 rpm for 24 h. Then, the mycelia were harvested for RNA extraction.Escherichia coli DH10β grown at 37 °C and 200 rpm in low salt Luria–Bertani medium supplemented with zeocin (50 µg·mL−1) was used to propagate the recombinant vector pPICZαA/Af-egl7. Pichia pastoris strain X-33  cells harboring the recombinant expression vector pPICZαA/Af-egl7 were used to produce heterologous protein. The employed growth conditions are described in the EasySelect™ Pichia Expression Kit manual .The plasmid pPICZαA  was used for gene cloning, sequencing, and expression. The low-viscosity substrate CM-Cellulose was purchased from Sigma . Medium-viscosity Barley β-glucan and Xyloglucan from tamarind seed were acquired from Megazyme . The natural substrate Sugarcane Exploded Bagasse (SEB) was kindly provided by Prof. Dr. João Atílio Jorge (University of São Paulo). Sugarcane bagasse “in natura” was provided by Prof. Dr. Delia Rita Tapia Blácido . Rice straw, bean straw, barley bagasse, and corncob were provided by Prof. Dr. Maria de Lourdes Teixeira de Moraes Polizeli .A. fumigatus mycelia were collected after growth, as described above. After freezing in liquid nitrogen and grinding into a fine powder, total RNA was isolated by using the Direct-zolTM RNA MiniPrep kit , according to the manufacturer’s instructions. cDNA was synthesized by using SuperScript® II Reverse Transcriptase .Phusion High-Fidelity DNA Polymerase  by using the follow thermocycling conditions: 98 °C for 30 s; 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min; and 72 °C for 10 min. The PCR product was analyzed by electrophoresis and purified from 1% (w/v) agarose gel by using the QIAquick Gel Extraction kit .Specific primer sequences containing overlapping regions between the vector and the insert were employed . The amplification reaction was performed with EcoRI and XbaI) by the Circular Polymerase Extension Cloning (CPEC) method [Phusion High-Fidelity DNA Polymerase ; the thermocycling conditions used were as follows: 98 °C for 30 s; 35 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 2 min 30 s; and 72 °C for 10 min. The cloning product was transformed into E. coli DH10β, and the resistant transformants were selected by zeocin (50 µg·mL−1). The recombinant expression vector pPICZαA/Af-egl7 was linearized with the restriction enzyme PmeI and transformed into P. pastoris X-33 competent cells by electroporation, according to the EasySelect™ Pichia Expression Kit manual .The ORF, without predicted signal peptide, was cloned into the vector pPICZαA (previously digested with the restriction enzymes ) method . The CPEw/v) low-viscosity carboxymethylcellulose (CM-Cellulose), zeocin (100 µg·mL−1), and 1% (v/v) methanol for induction. After incubation at 30 °C for 3 days, the plates were stained with 0.1% (w/v) Congo red solution for 20 min and de-stained with 1 M NaCl until pale orange hydrolysis zones appeared against an orange background (approximately 20 min).Recombinant transformants with high-level endoglucanase expression were screened in yeast extract-peptone-dextrose (YPD) plates containing 1% (P. pastoris X-33 harboring the vector pPICZαA/Af-egl7 was inoculated in buffered glycerol-complex medium (BMGY) (2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base, 1% (w/v) yeast extract, 1% (v/v) glycerol, 4 × 10−5 % (w/v) biotin, 100 mM potassium phosphate buffer, pH 6.0) and grown at 30 °C and 240 rpm until the culture reached log phase growth (O.D.600nm = 2–6). Then, the cells were harvested by centrifugation at 3000× g for 5 min and resuspended in buffered methanol-complex medium (BMMY) (2% (w/v) peptone, 1.5% (v/v) methanol, 1.34% (w/v) yeast nitrogen base, 1% (w/v) yeast extract, 4 × 10−5 % (w/v) biotin, 100 mM potassium phosphate buffer, pH 6.0) to an O.D.600nm = 1. Af-EGL7 expression was induced at 30 °C and 240 rpm for 6 days  under the control of the AOX1 promoter. Additional 1.5% (v/v) final concentration methanol was added to the medium every 24 h to maintain the expression levels. Once the Af-egl7 gene was fused with the α-factor signal sequence, the recombinant protein was obtained extracellularly.A single colony of recombinant g for 5 min and concentrated to the maximum by using an Amicon Ultra-15 Centrifugal Filter—10 kDa cutoff . Next, the concentrate was resuspended in 20 mM sodium phosphate buffer containing 500 mM NaCl (pH 7.4) and loaded onto Ni Sepharose 6 Fast Flow resin  pre-equilibrated with the same buffer. After incubation at 4 °C for 1.5 h under stirring, a linear gradient from 0 to 500 mM imidazole in 20 mM sodium phosphate buffer containing 500 mM NaCl (pH 7.4) was applied to the column to elute the His6-tagged recombinant endoglucanase. All the fractions were collected, and protein was analyzed by 10% (w/v) SDS-PAGE, stained with Comassie Brilliant Blue R-250 .After 6-day culture, the culture supernatant was collected by centrifugation at 3000× The fractions containing purified Af-EGL7 were mixed and submitted to buffer-exchange by using Amicon Ultra-15 Centrifugal Filter—10 kDa cutoff to remove excess imidazole prior to the subsequent enzymatic assays.w/v) CM-Cellulose in 50 mM sodium acetate buffer (pH 5.0) was incubated at 55 °C for 10 min. The enzyme action was stopped by adding an equal volume of the DNS reagent. The mixture was boiled for 5 min and cooled down, and the absorbance was measured at 540 nm. One unit of endoglucanase activity was defined as the amount of enzyme that released 1 µmol of reducing sugar from the substrate per minute. Each assay was carried out in triplicate. Protein concentration was determined by the Greenberg method [Af-EGL7 activity was determined by measuring reducing sugars from the reaction by the 3,5-dinitrosalicylic acid (DNS) method . The enzg method .2+ concentrations (from 0 to 15 mM) on the Af-EGL7 activity was also tested. The reactions were performed as described above.The effect of different CuPurified Af-EGL7 deglycosylation was accomplished by Endoglycosidase H  in both denaturing and non-denaturing conditions, according to the manufacturer’s instructions. In the first case, the recombinant protein was initially denatured at 100 °C for 10 min. Then, the deglycosylation reaction was performed at 37 °C for 24 h, as described by Meleiro et al. (2017) . ProteinThermostability was determined by pre-incubating 0.05 µg of purified Af-EGL7 in 50 mM sodium acetate buffer (pH 5.0) without substrate, at temperatures ranging from 55 to 90 °C for durations ranging from 30 min to 72 h. The residual activities were measured under standard conditions , as described in M, Vmax, and kcat) were determined when CM-Cellulose (2.5–30 mg·mL−1), β-glucan (0.5–15 mg·mL−1), or xyloglucan (1.0–8.5 mg·mL−1) were used as substrates. The reactions were performed in 50 mM sodium acetate buffer (pH 5.0) as previously described. The parameters were calculated by linear regression, by using the Lineweaver–Burk graphical method.The Af-EGL7 kinetic parameters  of both sugars by using the chromogenic substrate Azo-Xyloglucan from Tamarind . The enzymatic assays were performed in 85 mM sodium acetate buffer (pH 4.5) at 40 °C for 10 min, according to the manufacturer’s instructions, with slight modifications. The reactions were stopped by adding 1.7 volumes of absolute ethanol. The supernatants were harvested by centrifugation at 1000× w/v) of one of the following biomasses: sugarcane bagasse “in natura”, SEB, rice straw, corncob, barley bagasse, or bean straw.Lignocellulose enzymatic hydrolysis was carried out as described previously by Bernardi et al. (2018) with some modifications . The sac® 1.5L  and 10 µg of Af-EGL7, added per 10 mg of each biomass. The reactions were conducted at 55 °C and 1000 rpm for up to 72 h in a final volume of 1 mL. DNS was added to stop the reactions and to measure the released reducing sugars. Control experiments were conducted in the same way, in the absence of Af-EGL7. The reported results represent the means ± SD calculated from at least three experimental replicates.The reactions consisted of 0.009 FPU Celluclastp < 0.05).Data are expressed as the mean of replicates ± SD. Significant differences between the treatment groups were analyzed by using Tukey’s test (significance, All the data are the mean of at least three independent experiments and show consistent results.® 1.5L. Besides that, Af-EGL7 is a glucose-cellobiose-tolerant endoglucanase. All these properties make Af-EGL7 an important candidate for application in the improvement of commercial cocktails used in second-generation biofuel production as well as in other biorefinary processes.At the industrial level, it is essential that enzymes maintain their activity at high temperatures and high pH values. This study has shown a GH7 endoglucanase with favorable properties for industrial applications, such as high thermal and pH stabilities. In addition, Af-EGL7 can be considered as a good hydrolytic enzyme that can improve biomass degradation by Celluclast


Mycobacterium tuberculosis (Mtb) infection interferes in the antigen processing and presentation of BCG in inducing protective immunity against Mtb. Thereby, indicating that any vaccine that require extensive antigen processing may not be efficacious in TB-endemic zones. Recently, we have demonstrated that the vaccine candidate L91, which is composed of lipidated promiscuous MHC-II binder epitope, derived from latency associated Acr1 antigen of Mtb is immunogenic in the murine and Guinea pig models of TB and conferred better protection than BCG against Mtb.The clinical trials conducted at Chingleput India suggest that BCG fails to protect against tuberculosis (TB) in TB-endemic population. Recent studies advocate that non-tuberculous mycobacteria and latent Mtb antigens, respectively. These peptides were conjugated to the TLR-2 agonist Pam2Cys.In this study, we have used a multi-stage based bi-epitope vaccine, namely L4.8, comprising of MHC-I and MHC-II binding peptides of active (TB10.4) and latent (Acr1) stages of Mtb-burden and protected animals better than BCG and L91, even at the late stage of Mtb infection.L4.8 significantly elicited both CD8 T cells and CD4 T cells immunity, as evidenced by increase in the enduring polyfunctional CD8 T cells and CD4 T cells. L4.8 efficiently declined The BCG-L4.8 prime boost strategy imparts a better protection against TB than the BCG alone. This study emphatically denotes that L4.8 can be a promising future vaccine candidate for controlling active and latent TB.The online version of this article (10.1186/s12967-018-1653-x) contains supplementary material, which is available to authorized users. Mtb in TB-endemic population [Mtb [Mtb and TLR-2 agonist Pam2Cys [50 < 10 µM) to HLA class II alleles. Further, it chiefly elicited secretion of IFN-γ by T cells obtained from PPD+ healthy volunteers [Mtb [+ healthy volunteers [Vaccine is the best prophylactic measure to prevent and control any disease. Currently, potent vaccines are unavailable for many dreaded diseases, including TB. TB is a major global threat among the leading killer diseases. It kills 1.8 million individuals annually, despite of the availability of effective drug regime , 3. Therpulation . The majion [Mtb , 5–7. Thion [Mtb . Hence,  Pam2Cys , 10. Thelunteers , 12. TheMtb [Mtb persists in the host in distinct stages, namely active and latent. Therefore, for an improved vaccine, combination of multistage epitopes derived from active and latent form of disease is decisive. Henceforth, to achieve better protection, we improved our previous vaccine construct L91 by incorporating CD8 T cell epitope of the active form of Mtb antigen (TB10.4) (L4.8). In the current study, we observed that L4.8 efficiently activated both CD4 T cells and CD8 T cells and showed better protection than L91 and BCG vaccines. Thus, signifying that lipidated multistage epitopes based vaccine L4.8 may successfully confer protection against both the active and latent TB.Besides CD4 T cells, CD8 T cells also considerably contribute in protecting against Mtb –15. HencFemale BALB/c mice (6–8 weeks) were procured from Animal Facility of the ‘Institute of Microbial Technology’. The animals were housed in individually ventilated cages and food and water was provided ad libitum.Mtb conjugated to Pam2Cys, a TLR-2 agonist. The lipidated multi-stage and multi-epitope vaccine for mouse experiments L4.8 comprises of SEFAYGSFVRTVSLPVGADE and BALB/c restricted MHC-I binding peptide of sequence GYAGTLQSL of TB10.4 antigen of Mtb [Lipidated peptides, free peptides and Pam2Cys used in the study were synthesized, as described elsewhere , 10. Then of Mtb  linked tn of Mtb . F91 and6 CFU/animal). Twenty-one days later, mice were boosted twice with L4.8 and L91 (20 nmol and 10 nmol), at an interval of 2 weeks. Control groups were immunized with BCG alone, antigenically irrelevant lipidated hemagglutinin peptide (LH) of influenza virus and placebo (PBS). Ninety days after the last immunization, mice were aerosol challenged with Mtb  by aerosol machine Inhalation Exposure System . After 30 days of infection, mice were sacrificed, and cells were isolated from the lungs, spleen and lymph nodes. L4.8 specific T cell response was examined following in vitro incubation with L4.8 (1 nmol), F4.8 (1 nmol), Pam2Cys (50 ng/ml) and medium alone for 72 h. For protection studies, animals were sacrificed after 90 days of infection and lungs and spleen were excised and homogenates were prepared. The homogenates were plated on 7H11 medium plate to monitor CFUs. Histopathological analysis was done after staining of fixed lung sections with hematoxylin and eosin. The results of L4.8 vaccine were compared with L91, BCG and Pam2cys or unless otherwise mentioned.For short term immune response, mice were vaccinated subcutaneously (s.c.) at the base of tail with L4.8 (20 nmol) and 1 week later a booster was given with a half the dose (10 nmol) of peptide. One week later, animals were sacrificed, and cells isolated from spleen and lymph nodes were pooled. L4.8 specific T cell response was monitored following in vitro culturing with L4.8 (1 nmol), F4.8 (1 nmol), Pam2Cys (50 ng/ml) and medium alone for 72 h. For long-term immune response and protection studies, mice were immunized with a Danish strain of BCG (105/well) were added to 96 well U-bottom culture plates and incubated with L4.8 (1 nmol) and control cultures with F4.8 (1 nmol), L91 (1 nmol), Pam2Cys (50 ng/ml) and medium for 72 h/37 °C/5% CO2.Spleens and lymph nodes obtained from immunized mice were pooled and a single cell suspension was prepared by gently pressing through frosted slides. The lungs were perfused using chilled PBS and small pieces were prepared and digested with collagenase (2 mg/ml) and DNase (0.03 mg/ml) for 30 min/37 °C. Later, cells were passed through a sieve (70 μM). Viability was checked by trypan blue exclusion method. The cells (2 × 107 cells/100 μl PBS), and adoptively transferred in mice that were previously immunized twice with L4.8 or PBS at an interval of 1 week. After 16 h, mice were sacrificed, and single cell suspension of spleen cells was prepared. The percentage of cells expressing CFSEhi and CFSElo was enumerated through flow cytometry. The following formula was used to calculate specific lysis: ratio = (mean percentage of CFSElo cells/mean percentage of CFSEhi cells).In vivo target lysis was determined by using standard protocol, described elsewhere . BrieflyMtb was cultured in 7H9 medium containing Tween-80 (0.05%) supplemented with albumin (10%), dextrose and catalase (ADC). Glycerol stocks of H37Rv were prepared and stored at − 80 °C, and later used for infection studies. BCG vaccine (TUBERVAC) used for immunization was purchased from Serum Institute of India, Pune, India. TUBERVAC  is a live freeze-dried vaccine derived from an attenuated strain of Mycobacterium bovis and meets the requirements of W.H.O. and I.P., when tested by the methods outlined in W.H.O., TRS. 745 (1987), 771 (1988) and I.P.H37Rv strain of Chemicals and reagents were purchased from Sigma Aldrich . Anti–mouse flurochrome labeled antibodies (Abs): CD4-PB, CD8-APC-Cy7, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, KLRG1-PE, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5 and Abs for ELISA were procured from BD Pharmingen ; CD27-FITC, CD43-PE, CXCR3-FITC and CCR6-APC from Biolegend  or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO . For culturing of cells, tissue culture grade plastic-ware was purchased from BD Biosciences .7 cells) were incubated with CFSE dye (1 μM) in PBS (4 ml) at 37 °C. Free CFSE was quenched with 2 ml of FCS and excess was removed by washing 3× with RPMI-FCS-10%. CFSE-labeled cells were cultured with either L4.8 (1 nmol) or control cultures with F4.8 (1 nmol), L91 (1 nmol), Pam2Cys (50 ng/ml) and medium for 72 h. The proliferation of CFSE-labeled cells was analyzed by flow cytometry.The cells  and ionomycin (10 μM) for 2 h followed by incubation with brefeldin A (5 µg/ml) for additional 4 h. Cells were then harvested; washed 2× with buffer (PBS+FBS 2%) and fixed with paraformaldehyde (1%) at 4 °C/30 min. These cells were then perforated with saponin (0.2%) and incubated with fluorochrome tagged anti-IFN-γ, IL-17A and TNF-α Abs at 4 °C/90 min. The cells were washed with saponin (0.2%), followed by wash buffer. For surface staining of CD4, CD8, CD44, CD62L, CD127, CD27, CD43, KLRG1, CCR6 and CXCR3, cells were incubated with either fluorochrome labeled Abs or biotinylated Abs/streptavidin-fluorochrome conjugates. Standard protocols of washing/incubation were followed at each stage. Flow cytometry was carried out using FACS-Aria III and data were analyzed using BD FACS DIVA software package .The cultures were set as mentioned in T cell proliferation assay. After 72 h, culture SNs were harvested and cytokines were estimated by standard sandwich ELISA, as described by the manufacturer .The statistical analysis was performed employing ‘one-way ANOVA’ test for multiple comparisons.p ≤ 0.05; CD4: p ≤ 0.005)  by CD8 T cells and CD4 T cells, as compared to Pam2Cys  and IL-17A (p ≤ 0.005) than L91 during recall response  and CD4 T cells (p ≤ 0.0001) obtained from L4.8 immunized animals displayed considerable enhancement in the expression of KLRG1, when in vitro challenged with L4.8; as compared to control cells incubated with either F4.8 or Pam2Cys or medium  higher in vivo target lysing capability of T cells isolated from L4.8 immunized animals, as compared to control placebo group    upon L4.8 stimulation   and effector memory (CD62LloCD44hi)  CD8 T cells and CD4 T cells in a group that was vaccinated with L4.8  and CD127+ (IL-7R)  memory population  or L91 (p ≤ 0.005) or placebo (p ≤ 0.0005)  and L91 (p ≤ 0.05) vaccinated animals, as evidenced by significant decrease in the CFUs in spleen  Fig. a. Furtheeen Fig. b. Furtheeen Fig. c. In agrMtb and AIDS pandemic [Mtb [Mtb [The fact remains that more people have been vaccinated with BCG than any other vaccine, yet TB continues to inflict scourge by killing 1.8 million people and infecting 10 million individuals annually [pandemic . Paradoxpandemic , 7, 33. pandemic . Consequpandemic . In addimic [Mtb . FurtherMtb [Mtb , 36–39. Mtb conjugated to TLR-2 agonist Pam2Cys (L91), activated both innate and adaptive arms of immunity [Mtb; we further introduced CD8 T cell epitope from an early stage antigen TB10.4 in the novel construct L4.8 to improve the efficacy of the vaccine. Besides CD4 T cells, CD8 T cells also play a crucial role in imparting protection against Mtb [Recently, we have demonstrated that promiscuous peptide of immunity . L91 bolimmunity . Thus, Limmunity –43. Furtinst Mtb . Therefoinst Mtb . The onlMtb.To monitor the efficacy of the construct, mice were vaccinated with L4.8 and following major findings emerged from the study regarding CD4 T cells and CD8 T cells: (i) substantial activation of both the subtype of cells; (ii) increase in the frequency of IFN-γ and IL-17A producing cells; (iii) robust expansion in the pool of multifunctional T cells, producing a combination of IFN-γ/TNF-α and IL-17A/IFN-γ cytokines; (iv) augmentation in the percentage of central and effector memory response; (v) elicitation of cytotoxic T cells; (vi) significant reduction in the bacterial burden in the lungs and spleen and thereby protection against Mtb burden in mice compared to BCG and L91, establishing the superiority of L4.8 over these vaccines. It is worth to mention here that the animals were rested for 180 days after vaccination, thus indicating L4.8 elicited long-lasting immunological memory response. The generation of the enduring immunity is a fundamental feature of any successful vaccine [Mtb [Mtb.The most striking findings of the study were that L4.8 significantly declined the  vaccine . Unfortu vaccine , 48. Howine [Mtb , 50. Furine [Mtb , 51. ConRegrettably, infants that are immunocompromised or infected with HIV cannot be vaccinated with BCG, since they are highly vulnerable to BCG dissemination. Thus, leaving a choice for search of an appropriate vaccine for this population –54. LipiMtb, etc. ready for clinical trials. However, they may encounter same problem of interference of non-tuberculous mycobacteria and helminth infestation in TB-endemic regions, as was associated with BCG failure. Consequently, peptide vaccines can overcome the aforesaid hurdles and may be a future choice of prophylactic measure to control TB.Currently, there are many whole cell-based vaccines including recombinant BCG, attenuated Additional file 1: Table S1. Description of lipidated and free peptides in the vaccine. Table shows the detail of the sequence of MHC class I and MHC class II binding promiscuous peptides and the presence or absence of Pam2Cys used in the vaccine constructs. mL4.8/mF4.8 are murine restricted epitopes. ‘+’: presence; ‘−’: absence.Additional file 2: Figure S1. Titration of peptide dose for in vitro stimulation. Mice were immunized with L91 or L4.8 (20 nmol) and boosted after 7 d (10 nmol). Splenocytes were obtained after 15 d of immunization and in vitro stimulated with different concentrations of (A) L91 and (B) L4.8 for 72 h. Splenocyte proliferation was assessed by 3H-thymidine incorporation assay and expressed as counts per minute (cpm). Figure S2. Gating strategy for CD4+ T cells and CD8+ T cells. The P1 gate was made on lymphocytes, P2 gate on SSC-A/CD4+ T cells and P3 on SSC-A/CD8+ T cells zones. Further, analysis of T cells was done on the gated cells. Figure S3. Gating strategy for monitoring the expression of KLRG1+ T cells and target lysis. (A) The P1 gate was made on lymphocyte zone. P2 gate on SSC-A and CD4+ T cells while P3 gate on SSC-A and CD8+ T cells. (B) For target lysis two gates were made in CFSEhi and CFSElo cells on SSC-A. Later, histogram was plotted for the analysis. Figure S4. Gating strategy for monitoring the expression of IFN-γ+ and TNF-α+ CD4 T cells.  The primary gate was made on lymphocyte zone and secondary gate on SSC-A and (A) CD4+ T cells; (B) CD8 T cells. The percentage of IFN-γ+/TNF-α+ was monitored on secondary zones . The isotype-matched control did not show any double positive T cell population.  Bar diagram represents the comparison of polyfunctional (C) CD4 T cells and (D) CD8 T cells (IFN-γ+ TNF-α+) obtained from BCG and BCG-L4.8 groups. The data are represented as mean ± SEM. *p ≤ 0.05; ****p ≤ 0.0001.