The aim of this research was to assess university students’ health-related quality of life whether they use some contraceptive method or not.This research is a cross-sectional study. Female participants who studied a degree in health at the University of Seville (Spain) were recruited. Respondents completed a demographic questionnaire and health-related quality of life was measured with validated instrument SEC-QoL  in Spanish that measured five dimensions: sexual, social, breast, menstrual and psychosocial.A total of 992 women aged 21.37 (3.6) years old participated in this study. Women who used a contraceptive method reached higher scores at the SEC-QoL questionnaire 47.09 (17.04) and 46.91 (18.73) than those that didn’t. Likewise, women who chose a hormonal method showed a better overall health-related quality of life, compared to those who used a non-hormonal method. Participants who used hormonal contraceptives obtained higher scores in all health-related quality of life domains , except psychological domain when compared to those who used a non hormonal method or none. Furthermore, a moderated mediation model showed that the effect of the current contraceptive method on health-related quality of life was partially explained by the moderated mediation of the time using this method, the reason for using it and the existence of a partner.The usage of hormonal contraceptives increases health-related quality of life in young women. Several variables regarding the experience with contraceptive methods should be considered in order to examine the effect on health-related quality of life in undergraduate women. Contraceptives present benefits for young women beyond avoiding a pregnancy. This research was designed to assess the impact of contraception on health-related quality of life. A cross-sectional study was carried out with a sample of 992 female university students from the University of Seville using SEC-QoL  scale validated by the Spanish Society of Contraception. The use of any contraceptive method improves women’s quality of life measured. Women who used a hormonal contraceptive had higher health-related quality of life scores. The use of hormonal methods improves all domains in young women, except for the psychological domain at SEC-QoL scale. Several variables about the use of contraceptive methods should be considered in order to examine the effect on health-related quality of life.Contraceptive methods present benefits besides contraception . NeverthPremenstrual symptoms are common in reproductive-aged women. PMS refers to the cyclic occurrence of a set of disturbing physical, emotional or behavioral alterations that are of sufficient severity to interfere with interpersonal relations and routine life, affecting quality of life . EpidemiPrevious research has demonstrated that using hormonal contraceptives can theoretically reduce PMS symptoms in all forms of hormonal contraception . These eThere are studies where women’s health in relation with their quality of life has been measured using standard questionnaires such as EuroQol-5D (EQ-5D) , SF-36 , 21, WorThis study tries to assess associations between contraceptive choice and women’s health-related quality of life in a sample of university students.We conducted a cross-sectional study among students at the University of Seville from June to October 2015. Participants answered a self-administered questionnaire based on SEC-QoL, sociodemographic variables and questions about the use of contraceptives and sexual habits: a) current contraceptive method being used; b) contraceptive methods used throughout life; c) partner; d) time with a relationship; e) number of sexual partners in the last year; f) frequency of sexual relations; g) age at first sexual relation; h) motivation to use current contraceptive method.Women were eligible to participate in this study whether they were: a) older than 18 years; b) having menstrual cycle monthly . Women who reached menopause were excluded.Quality of life was assessed with the SEC-QoL questionnaire see . The queThis is a self-administered questionnaire consisting of 19 items and five domains: social (consisting of 5 items), menstrual symptoms (4 items), breast symptoms (3 items), psychological (4 items) and sexual (3 items). Each item is scored from 1 to 5 likert response choices . The total score range is 0–100.This questionnaire showed a good internal consistency with an overall Cronbach’s alpha of 0.88 and a value higher than 0.70 in all domains, except for the sexual domain (0.55). The overall intra-class correlation coefficient was 0.82, which was higher than 0.70 in all domains, thus indicating the good test-retest reliability of the questionnaire (25).Standardization is obtained as follows: (current score- minimum score)/ (maximum score-minimum score) *100.The study was approved by the Ethical committee at the University of Seville and written informed consent was obtained from each participant. The study protocol conformed to the ethical guidelines of the 1975 Helsinki declaration.p value of less than 0.05 was considered as indicating significance for these analyses. 95% confidence intervals were calculated assuming a Poisson distribution.A descriptive analysis of the sociodemographic characteristics was performed in IBM Corp. Released 2013. IBM Statistical Package for the Social Sciences (SPSS) for Windows, Version 21.0. Armonk, NY: IBM Corp. We compared the characteristics of patients who used any contraceptive method to patients without recent contraceptive use and users of a hormonal method and others. Data were expressed as absolute frequencies, average and percentages. The differences in SEC-QoL scores between hormone users and non-users were calculated by analysis of variance and by chi-square test. A Finally, some mediation and moderation analyses were performed to further explore the type of relationships among the study variables. These analyses were carried out following the recommendations described by Preacher et al. , based oThis study was carried out within a cohort of university students. Population included 1007 women aged between 17 and 54 years old who were enrolled in any of the degrees at the Faculty of Nursing, Physiotherapy and Podiatry from the University of Seville (Spain). A total of 992 (98.5%) women aged between 18 and 50 years old answered the questionnaire, two of which did not meet inclusion criteria because of menopause. Sociodemographic characteristics of participants are shown in Table The age of participants was 21.37 years old (3.60). All women included were studying a degree in health science at university.Participants who had a partner were 67.60% (665) and were engaged in a relationship that lasted an average of 36.27 (32.02) months. The initiation of sexual intercourse among women who answered was at 16.71 (1.78) years old. Most common frequency of sex was once-twice a week 40.62% (403), more than twice a week 21.37% (212); once-twice a month 19.10% (189) and once-twice a year 4.58% (48). Female students that had not had sex before were 9.90% (98).Contraceptive use in women who participated in this study is 90.6%. Students who had used a condom at least once in their lives were 84.57% (839); coitus interruptus by 24.39% (242), hormonal contraceptive pills by 42.54% (422), hormonal vaginal ring by 10.18% (101); contraceptive patches by 1% (10) and subdermal implant by 0.10% (1).The current most widely used contraceptive method is the condom: 47.07% (467), followed by contraceptive pills: 23.28% (231), and 12.2% (115) do not use any method. However, only a 9.90% (98) referred that they had not had sex before, so 2.52% had sex without using any contraceptive method. Timing using the current method is 24 months (p25 (12)-p75 (48)).p < 0.001)Motivation to choose a method is based on security (55.14% (547)), prevention of sexual transmission infection (STI) (14.61% (145)), ease of use (6.35% (63)), price (1.91% (19)), availability (1.71% (17)). Women who chose a method to prevent STI used a condom more often (p < 0.001).Participants´ mean score (SD) was 54.57 13.26), (95% CI 53.74 to 55.39) at global SEC-QoL score. If we analyze women who use a contraceptive method in comparison to women who do not, mean scores are 47.09 (17.04) and 46.91 (18.73) respectively. If we detail each score depending on the method we find that hormonal methods show higher scores as it is shown in Table 6, . Time using a contraceptive method is not related to higher SEC-QoL scale scores (p > 0.05). In bivariate analysis women who have been using hormonal contraceptives longer did not show better scores (p > 0.05). However, significant differences were found between women using hormonal contraceptives who had sex more frequently than women who did not (p < 0.001).Health-related quality of life by age did not show any differences among groups under and over 30 years old (46.85 (17.12) vs 47.88 (27.72), p = 0.004, 95% CI ), so that higher scores in SEC-QoL were found in participants who used hormonal methods , while lower scores were observed in those who used non-hormonal methods , F = 8.69, p < 0.001, η2p = 0.02. Second, these differences on SEC-QoL were partly related to the time using that method. Moreover, the reason for using the contraceptive method and having a partner were proposed as moderators in that partial mediation. Results showed, on the one hand, some differences in the time using the current contraceptive method according to the specific method  than those who used hormonal methods , which is expectable on the basis of participants’ sexual life course. Moreover, the relationship between the current contraceptive method and the time in months using that method was found to be moderated by the reason for using it and having a partner or not , F = 3.16, p = 0.003, R2 = 0.04. Thus, among participants who used non-hormonal contraceptive methods because of its security, those who had a partner reported more months using it  than those without a partner . Moreover, among participants who used non-hormonal contraceptive methods because of its availability, those who had a partner reported more months using it  than those without a partner . On the other hand, results indicated that the motive to use the current contraceptive method also moderated the relationship between the time using it and the scores in SEC-QoL , F = 4.73, p = 0.002, R2 = 0.02. Among participants who had used the current contraceptive methods for more than 24 months, the motives of availability  and price  were associated with higher health-related quality of life compared to those participants with less than 24 months using that method . Moreover, greater health-related quality of life was also found in undergraduate women that used the current contraceptive method for more than 24 months in order to prevent an STI , compared to those who shared this same motive but had used the method for less time . However, no significant association was observed between the time using the contraceptive method and health-related quality of life scores  that allowed us to find differences related to the use of contraceptives. There have been few studies assessing the impact of contraceptive use on women’s quality of life with such a specific questionnaire as SEC-QoL –28.This study found that women who use any contraceptive method have higher scores of SEC-QoL than those who use no contraceptive. When comparing different methods of contraception, women who used hormonal methods had higher scores. Actually, women who use any form of contraception may have an improved health-related quality of life and different contraceptive methods have varying degrees of impact on women’s. However, it is difficult to determine the exact impact of contraception on health-related quality of life  so this We asked about the motivation for using contraceptives. Some may use them to prevent a pregnancy and others for non-contraceptive benefits. The main reason is to avoid unplanned pregnancies. In other studies, with young women, the main reason for choosing a method is to plan a pregnancy .Women obtained more benefits other than contraceptive effects. Menstrual symptoms’ dimension of SEC-QoL was shown to be less severe in women who chose hormonal contraceptive methods. In our study, we observed an improvement in these domains of SEC-QoL in those women who chose hormonal contraceptive method: breast tenderness, social, menstrual and sexual symptoms. These results are consistent with previous studies , 35. LikThere is a lack of knowledge about how contraceptives affect sexual function. Zethraeus et al. showed no negative impact of oral contraceptives on overall sexual function . In thisThis sample is formed by women in their twenties, so we do not have users of definitive methods or intrauterine dispositive, more common in older women. Most women in this study reported using hormonal methods or a condom as in others studies carried out in Spain , 40. We The amount of time using a method is not directly related to higher scores, so we could presume that positive effects are felt sooner. Students who have been using contraceptives longer did not show higher scores on health-related quality of life. Zocal’s study showed that after 6 months SEC-QoL global and dimension scores had greater increases from baseline in women who chose to switch to combined oral contraception based on natural estrogen . A revisAs a limitation of this study, we state that we did not measure the longitudinal effect of contraception on premenstrual syndrome from a baseline. We asked women about their perception of health-related quality of life at one specific point and in retrospect by applying a questionnaire. Other limitation that should be acknowledged is the need to assess other relevant variables, such as beliefs about premenstrual symptoms , and a fThe achieved results suggest higher scores of SEC-QoL in women using hormonal contraceptive methods. Overall, women reported better menstrual health-related quality of life, lower breast tenderness and a better sexual life.


MicroRNA (miRNA) are key players in regulating expression of target genes at post-transcriptional level. A number of miRNAs are implicated in modulating tolerance to various abiotic stresses. Waterlogging is an abiotic stress that deters plant growth and productivity by hypoxia. Dozens of reports mention about the miRNAs expressed in response to waterlogging and hypoxia. Despite the fact that tomato is a model vegetable but waterlogging sensitive crop, the role of miRNAs in hypoxia tolerance is poorly understood in tomato.In this study, we investigated the differentially expressed miRNAs between hypoxia-treated and untreated wild tomato root by using high-throughput sequencing technology. A total of 33 known miRNAs were lowly expressed, whereas only 3 miRNAs showed higher expression in hypoxia-treated wild tomato root compared with untreated wild tomato root. Then two conserved and lowly expressed miRNAs, miR171 and miR390, were deactivated by Short Tandem Target Mimic (STTM) technology in Arabidopsis. As the results, the number and length of lateral roots were more in STTM171 and STTM390 transgenic lines compared with that of wild type plant, which partly phenocopy the increase root number and shortening the root length in hypoxia-treated wild tomato root.The differentially expressed miRNAs between hypoxia-treated wild tomato and control root, which contribute to the auxin homeostasis, morphologic change, and stress response, might result in reduction in the biomass and length of the root in hypoxiated conditions.The online version of this article (10.1186/s12870-019-1698-x) contains supplementary material, which is available to authorized users. Tomato is an important vegetable crop and it is highly sensitive to waterlogging . As an iPlant microRNAs (miRNAs) are 18–24 nucleotides (nt) long noncoding small RNAs. Generally, miRNAs negatively regulate their target genes expression by binding to mRNA sequences, leading to mRNA cleavage or translation repression . RecentlDozens of studies have been done upon waterflooding and hypoxia-responsive miRNAs. A previous study indicated that 46 miRNAs from 19 families and three tasiRNA families were differentially expressed in Arabidopsis after 5 h of hypoxia treatment . HoweverIn this study, high-throughput sequencing technology was employed to detect the differentially expressed miRNAs between hypoxiated and control wild tomato roots. The results showed that a total of 33 known miRNAs were lowly expressed, while 3 known miRNAs were highly expressed in hypoxia-treated wild tomato root compared with that of the control plant. Subsequently, to phenocopy the shorter but more roots phenotype of hypoxia-treated wild tomato root, two lowly expressed conserved miRNAs, miR171 and miR390, were blocked by using short tandem target mimic (STTM171 and STTM390) in Arabidopsis. As expected, more and longer roots were observed in STTM171 and STTM390 transgenic lines. All these results indicated that the phenotype of hypoxia-treated wild tomato root may result from the differentially expressed miRNAs.S. habrochaites 178 ) and cultivar tomato S. lycopersicum Fenzhenzhu  were used as the material and cultivated under hydroponics. For Arabidopsis, Columbia-0 (Col-0) was used as the wild type (WT), all plants were grown in long day condition with 16 h light/8 h dark at 23 °C in an incubator.Hypoxia sensitive wild tomato − 1 electrical conductivity for two days prior to treatment. After that, two treatments, hypoxia and control at rhizosphere, were conducted. The treatments followed the protocols of Gasch et al., [− 1 and 7.0–8.0 mg L− 1 for hypoxia and control at rhizosphere, respectively. For hypoxia at rhizosphere, N2 was used as source of the air pump, while for the control plant (CK), air was used as a source of the air pump. The liquid media was replaced every week. The materials were collected at 12 days after treatment. Within each treatment, half of roots from 10 plants were placed in liquid nitrogen and stored at − 80 °C for RNA extraction, and half were collected and used to measure the phenotype.The seeds of tomato were sowed on the solid medium (vermiculite: peat: pearlite = 2:1:1) after germination. 27 days old seedlings (counted from sowing) were transferred to plastic pot (65 cm × 40 cm × 15 cm) covered by a PVC plastic board, which was drilled holes with the spacing of 7 cm × 7 cm. The seedlings were then put into the 1/2 Hoagland liquid media with 6.1 ± 0.2 pH and 2.2–2.5 ms cm et al., . SpecifiRegarding to morphological index measurement, root number, length and diameter, dry weight of root and shoot were assayed with 6 repeats. In detail, root number was counted manually, root diameter and length were respectively measured by vernier caliper and ruler, dry weight of root and shoot were determined by electronic scales after the samples thoroughly dried in drying oven. In addition, the data of root number and diameter were showed by the average, while root length and dry weight of root and shoot were presented by the average of per plant.-ΔΔCT method.To detect the expression of miRNAs in hypoxia-treated and control plant. Total RNA of hypoxia-treated and control plant roots was isolated by Trizol (Invitrogen) following the manufacturer’s instructions. Stem-loop qRT-PCR was used to measure the targeted miRNA expressional level, and was followed the procedure of Peng et al. . In brieTotal RNAs were extracted from the roots of hypoxia-treated and the control plants for deep sequencing. Small RNA sequencing and data analyses were performed as described previously . BrieflyTo compare with the abundance of known miRNAs, only the tags meet with the following criteria were defined as know miRNAs : (1) seq(I) Normalization formula:After normalization, we set the expression to 0.01 for miRNAs that were not expressed in one of the libraries. In addition, the miRNA whose expression was lower than 1 transcript per million (TPM) in both the hypoxia-treated and control library were filtered, and the remainder miRNAs were used for differential expression analysis.P-value from the normalized expression.(II) Calculate the fold-change and Fold-change formula:P-value formula:1 is the total number of clean tags in hypoxia-treated root, x is the number of miRNAs surveyed, N2 is the total number of clean tags in control, and y is the number of homologous miRNAs in hypoxia-treated root.P-value < 0.05, represents this miRNA differentially expressed, while a P-value < 0.01 indicates that the miRNA was significantly different between hypoxia-treated and control wild tomato root.If the |fold-change| of the miRNA is ≥0.6, simultaneously 0.01 ≤ https://sourceforge.net/projects/mireap/) to identify the novel miRNA candidates. Finally, the criteria proposed by Meyers et al. [To identify novel miRNAs, the following analyses were carried out as described by Peng et al. . In bries et al.  were useSolanum lycopersicum (tomato), transcript, cDNA library, version 2.4 was used as the genomic library for the target search.The potential miRNA targets were predicted and validated using psRNATarget  and DPMITo study the function of differentially expressed miRNAs identified from hypoxia at rhizosphere, a popular technique, short tandem target mimic (STTM), was employed to block miR171 and miR390’s expression in Arabidopsis. STTM vectors construct were generated by the following procedures described by Tang et al. . In brieContents of IAA was assayed by high-performance liquid chromatography-tandem mass spectrometry (ESI-HPLC-MS/MS) as previously reported . BrieflyP < 0.01) and 14.20% (P < 0.01), respectively. And the decreased range of root length, dry weight of root, and dry weight of shoot were 285.77% (P < 0.01), 65.35% (P < 0.01), and 24.69% (P < 0.01), respectively. On the contrary, higher auxin content was presented in hypoxia-treated wild tomato root Additional file 2:Phenotype of 41 days old cultivar tomato plant at root rhizosphere under 12 days hypoxia treatment and control condition. (DOCX 176 kb)Additional file 3:The miRNAs and their expression detected in hypoxiated and control wild tomato root by high-throughput sequencing technique. (XLSX 14 kb)Additional file 4:The differentially expressed miRNAs between hypoxiated and control wild tomato root detected by high-throughput sequencing technique. (XLSX 13 kb)Additional file 5:The expression of differentially expressed miRNAs verified by stem-loop qRT-PCR. Actin served as internal control. (DOCX 147 kb)Additional file 6:Predicated targets of differentially expressed known miRNAs between hypoxiated and control wild tomato root by psRNATarget. (XLSX 84 kb)Additional file 7:Validated targets of differentially expressed known miRNAs between hypoxiated and control wild tomato root by DPMIND. (XLSX 23 kb)Additional file 8:The expression of differentially expressed miRNA targets detected by qRT-PCR. Actin served as internal control. (DOCX 99 kb)Additional file 9:Predicted targets of novel miRNAs identified in this study by psRNATarget. (XLSX 11 kb)Additional file 10:Comparison of lateral root number and length of Arabidopsis STTM171, STTM390 transgenic lines and wild type (WT). (DOCX 85 kb)


Context:Stachys pilifera Benth (Lamiaceae) has long been used to treat infectious diseases, respiratory and rheumatoid disorders in Iranian folk medicine. Antitumor and antioxidant activity of the plant have been reported.Objective: The study was designed to assess the hepatoprotective activity of ethanol extract of Stachys pilifera in carbon tetrachloride (CCl4)-induced hepatotoxicity in rats.Materials and methods: The rats were randomly divided into six equal groups (n = 7). Group I was treated with normal saline; Group II received CCl4  for 60 consecutive days; Groups III, IV and V were given CCl4 plus Stachys pilifera ; Group VI received the extract . Histopathological analysis and measurement of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), malondialdehyde (MDA), total protein (TP) and albumin (ALB) were performed.Results: CCl4 caused a significant increase in the serum levels of AST, ALT, ALP and MDA as well as decreased ALB, and TP serum levels (p < 0.001). The extract (200 and 400 mg/kg/d) significantly normalized the CCl4-elevated levels of ALT, AST, ALP and MDA (p < 0.001). The extract (200 and 400 mg/kg/d) also increased the serum levels of TP compared to CCl4 group (p< 0.01). The extract (200 and 400 mg/kg/d) also decreased the histological injuries (inflammation and fatty degeneration) by CCl4.Discussion: The results revealed that the Stachys pilifera extract could provide considerable protection against CCl4 hepatotoxicity in rats that may be related to its antioxidant properties. The liver is the main organ involved in metabolic functions. Hepatic damage is associated with alteration of these metabolic functions. Liver dysfunction induces by habitual repeated alcohol consumption, exposure to some xenobiotics, and/or drug interactions. Management of the liver disorders remains a controversial subject  grows in tropical and subtropical countries. This genus is represented in Iran by 34 species that 13 of them are endemic -induced hepatotoxicity in rats. Zargari . Presenc Zargari . AntioxiStachys pilifera including stems and leaves were collected from Kakan in Yasuj, Iran at the end of spring of 2015. The plant was authenticated by Dr. A. Jafari from Department of Botany, Center for Research in Natural Resource and Animal Husbandry, Yasuj University, Yasuj, Iran, where a voucher specimen (herbarium No. 1897) was deposited. The plant leaves were dried far from direct light, and then powdered. The powder was kept in a closed container in 4 °C.Aerial parts of 2O (70:30) at 400 °C by maceration for four days. After this time, the extract were filtered, the solution was evaporated by rotary evaporator in 40 °C. The rest was kept at 4 °C prior to being tested  was extracted three times with 1500 mL mixture of EtOH-H4) diethyl ether and other solvents were obtained from Merck, Germany. The assay kits for the determination of ALT, AST, ALP, ALB and TP were purchased from Pars Azemun, Tehran, Iran.Trichloroacetic acid (TCA), tiobarbituric acid (TBA),  and a 12 h dark/light cycle and were allowed to take standard laboratory feed and tap water. The animals were kept fasted 2 h before and 2 h after drug administration.The animals were randomly assigned to six groups, each consisting of 7 rats as follows:Group I: the animals received intraperitoneal injection (i.p.) of olive oil  and normal saline  for 60 consecutive days.4 as a 50% solution in olive oil (1 mL/kg twice a week) for 60 consecutive days and 0.5 ml normal saline  for 60 consecutive days.Group II: the animals received i.p. injection of CCl4 as a 50% solution in olive oil  and the Stachys pilifera extract  for 60 consecutive days.Group III: the animals received i.p. injection of CCl4 as a 50% solution in olive oil  and the Stachys pilifera extract  for 60 consecutive days.Group IV: the animals received i.p. injection of CCl4 as a 50% solution in olive oil  and the Stachys pilifera extract  for 60 consecutive days.Group V: the animals received i.p. injection of CClStachys pilifera extract  for 60 consecutive days.Group VI: the animals only received the Stachys pilifera were selected according our previous study , alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the levels of total protein (TP) and albumin (ALB). The assays were performed by a colorimetric method using commercially available kits  . Finally the absorption of supernatant was determined at 535 nm against a blank that contained all reagents except the serum sample. Serum MDA concentration was expressed as nmol/mL  and cut into 3–4 mm slices using a rotary microtom. The slices were then stained with Hematoxylin-Eosin dye and studied for histopathological changes  (SPSS 21 for windows) followed by Dunnett and Tukey post-test. 4 caused a significant increase in the serum levels of ALT, AST, and ALP compared to the saline group (p < 0.001). Pretreated with the ethanol extract of Stachys pilifera, at the doses of 200 and 400 mg/kg/d, significantly decreased the CCl4-elevated serum levels of ALT, AST and ALP (p < 0.001). Stachys pilifera at the dose of 100 mg/kg/d also considerably reduced the serum levels of ALT, and ALP (p < 0.01). The serum levels of TP and ALB was considerably reduced due to intoxication with CCl4 (p < 0.01). Stachys pilifera extract, at doses of 200 and 400 mg/kg/d markedly normalized the serum levels of TP and ALB compared to CCl4 group (p < 0.001), while 100 mg/kg/d of the extract was observed to non-significantly reduced the elevated levels of the indicated parameters. Analysis of the serum levels of TP and ALB and activities of ALT, AST, ALP confirmed no significant differences in the biochemical parameters between the control group and the animals treated with the extract at a dose of 400 mg/kg/d (p > 0.05).As presented in 4 compared to the saline group (p < 0.001). According to the results, the extract at doses of 200 and 400 mg/kg/d, considerably reduced the MDA levels in the rats that received CCl4 (p < 0.001). There was no difference in the MDA levels in the rats treated with Stachys pilifera (400 mg/kg/d) compared to the saline group (p > 0.05).As shown in 4 caused the normal architecture of liver to be entirely lost. CCl4 poisoning resulted in excessive formation of fatty acid, necrosis of Kupffer cells around the central vein and netrophil infilteration  reversed the elevated levels of ALT, AST and ALP induced by CCl4 toxicity. In this regard, we have shown that the extract significantly increased the abnormal plasma levels of TP and ALB. The findings are consistent with a previous study of that showed methanol extracts of four Stachys species seeds reduced the levels of ALT, AST and ALP in paracetamol or CCl4-induced hepatotoxicity , to initiate lipid peroxidation  considerably normalized the abnormal elevating serum levels of MDA in the CCl4-induced hepatotoxic rats.It has been reported that lipid peroxidation, reducing activity of antioxidant enzymes and generation of free radicals are the primary reasons of CCl4 induced a variety of hepatic histological changes including congestion in central vein, destroyed lobular structure and leukocyte infiltration. These changes significantly inhibited by 200 and 400 mg/kg/d Stachys pilifera.Histopathological examination of liver biopsies confirmed our biochemical findings. Injection of CClStachys pilifera in the model of CCl4-induced liver injury is not clear, but it is important to note that the plant is rich in phenolic compounds (Sadeghi et al. Stachys pilifera ethanol extract reduced the elevated levels of MDA. Therefore, it is possible the Stachys pilifera extract exerted its protective effects through the antioxidant effect or scavenging free radicals.According to the results the exact underlying mechanisms for this protective effect of Stachys pilifera ethanol extract in CCl4-induced hepatic damage in rats. These protective effects may be, at least in part, related to antioxidant properties of the extract.In conclusion, the results of the present study clearly demonstrated hepatoprotective effects of the


We retrospectively compared the prognostic value between the AJCC 8th edition anatomic (AS) and prognostic staging (PS) system for triple negative breast cancer (TNBC) in a cohort from two involved institutions and a large population database.Clinicopathological data of TNBCs were identified in two involved institutions (SYSUCC-PWH cohort). Data from SEER database during 2010–2015 was also accessed. We restaged all cases into AS and PS group according to the AJCC 8th staging system.p = 0.049, p < 0.001).A total of 611 and 31,941 TNBCs were identified in two cohorts, with a median follow-up of 53.5 and 27 months respectively. PS upstaged 46.1% of patients in SYSUCC-PWH cohort, and 62.4% in SEER cohort. No significant difference was observed in C index between AS and PS models for disease-specific survival (DSS), progression-free survival (PFS) or overall survival (OS) in either cohort. χ2 statistic and Hazard Ratio for PFS, DSS and OS showed better discrimination between IA and IB, IIB and IIIA, IIIA and IIIB in AS model than PS model. Besides, patients with IIIC unchanged stage showed worse PFS compared to those with AS IIIA or IIIB upstaged to PS IIIC in both cohorts(Our findings demonstrated that prognostic staging system did not provide better discriminatory ability in predicting TNBCs prognosis than anatomic staging system. Cancer staging helps clinicians to determine prognosis and design treatment plans for individual patient. Since 1977, the American Joint Committee on Cancer (AJCC) staging system for breast cancer (BC) has assigned stage based on anatomical parameters, including tumor size and extent of tumors (T), Lymph node involvement (N) and the presence or absence of distant metastasis (M). Once the T, N, and M are determined, they are combined for assigning the overall anatomic stage (AS) of 0, I, II, III, IV. However, a significant limitation of AS for BCs is that it does not include the biologic factors and may not represent the BC biologic behavior sufficiently.Identification of BCs molecular subtypes and significant progress in genomics studies have led to a better understanding of BCs biologic behavior, which provides valuable information for the individualized treatment of BCs. Therefore, AJCC 8th Edition Cancer Staging Manual has updated with a prognostic stage (PS) group for breast cancer by incorporating traditional TNM anatomic parameters and additional biologic factors, including estrogen receptor (ER) and progesterone receptor (PR) status, HER2 status, tumor grade and Recurrence Scores (Oncotype Dx), which are known to have predictive and prognostic value , 2. PrevIn this study, we retrospectively compared the prognostic value between the AJCC 8th edition AS and PS for TNBCs in a large local cohort  and performed a SEER population-based analysis. We aim to examine whether this novel PS system provide more accurate risk stratification in overall survival, disease-specific survival, progression-free survival than the AS system.Clinicopathologic data, including tumor histologic grade, pathologic T and N categories, and ER, PR, and HER2 status, for patients who underwent surgery as the initial intervention with pathological confirmed invasive breast cancer in Sun Yat-sen University Cancer Center (SYSUCC) during 2005–2013 and Prince of Wales Hospital (PWH) during 2002–2008 were recorded. Patients receiving neoadjuvant therapy, and those with stage IV disease, re-operation for local recurrence,or incomplete or unknown clinicopathologic data were excluded.All pathologic specimens were reviewed by experienced pathologists to confirm the clinicopathologic characteristics including the tumor size, axillary nodal status, resection margin. ER, PR, and HER2 status were determined according to immunohistochemical (IHC) staining. For patients before 2010, ER and PR status was reclassified as negative using a cut-off of 1% according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines . HER2 stThe SEER 18 Registry Research Data was accessed through the SEER*stat (Version 8.3.5) to extract cases in this cohort . Since tPS and AS were determined for cases in both SYSUCC-PWH cohort and SEER cohort according to the AJCC 8th edition staging manual. Cases with histologic grade of grade III (poorly differentiated) and IV  in SEER cohort were both classified as G3 in PS system. Distributions of AS and PS were compared in each cohort. We evaluated the number of patients with alterative stage by the PS system. PFS was define as the duration between date of diagnosis to date of disease progression or last contact. DSS was define as the duration between date of diagnosis to date of death because of breast cancer progression or last contact. OS was define as the duration between date of diagnosis to date of death or last contact. PFS and DSS according to AS and PS were compared for SYSUCC-PWH cohort. OS and DSS between the stages were compared for SEER cohort.t test. The χ2 statistic of the log-rank test was used to further calculate the discrimination between groups. A larger χ2 statistic indicates further distance between survival curves, and its P value reflects the statistical significance of this distance. The relationship of AS and PS with DSS, PFS or OS was modeled using a Cox proportional hazards regression model. The results were expressed in hazard ratios (95% CIs). Two-tailed p value < 0.05 was considered statistically significant, and IBM SPSS (Version 21.0) was used for analysis.The log-rank test was used to compare differences of survival rates between groups. The Harrell concordance index (C index) was calculated using SAS (Version 9.4) to measure the model’s predictive performance for the AS and PS models. A higher C index indicates a better predictive performance. Significance between the C index of AS and PS models was determined using A total of 611 patients with stage I to IIIC TNBC were identified in the SYSUCC-PWH cohort, of which 427 and 184 patients were from Sun Yat-sen University Cancer Center (SYSUCC) and Prince of Wales Hospital (PWH) respectively. All patients were assigned a PS. Compared to AS, PS upstaged 282 patients (46.1%) and, unchanged stage was in 329 patients (53.9%). No patient showed downstaged PS in this cohort. For those with upstaged PS, 232 patients  showed one stage up  and 50 patients  showed two stages up (IIIA to IIIC).A total of 31,941 patients with stage I to IIIC TNBC were identified in the SEER cohort. Compared to AS, PS upstaged 19,924 patients (62.4%), and unchanged stage was in 12,009 patients (37.6%). Eight patient showed downstaged PS (IIIC to IIIB) in this cohort. For those with upstaged PS, 17771 out of 19,924 patients (89.2%) showed one stage up  and 2153 patients (10.8%) showed two stages up (IIIA to IIIC). Clinicopathologic characteristics and staging alterations for patients in both cohorts are listed in Additional file p = 0.943) or PFS (p = 0.887) than AS model. Similar outcomes were observed in SEER cohort, the C index for AS was 0.86 (95% CI 0.81–0.89) in DSS and 0.90 (95% CI 0.87–0.92) in OS. The C index for PS was 0.85 (95% CI 0.81–0.89) in DSS and 0.90 (95% CI 0.87–0.92) in OS. No significant difference of C index between AS and PS models again in either DSS (p = 0.95) or OS (p = 0.98).Survival analyses were conducted in both cohorts and survival curves are shown in Fig. 2 statistic was applied to evaluate the discriminatory power between different stages among AS and PS in both cohorts. In the SYSUCC-PWH cohort, χ2 on PFS between IIA and IIB for AS was 10.36 (p = 0.0013) and that for PS was similar, showing a χ2 of 10.05 (p = 0.0015). Comparing between IIB and IIIA, χ2 on PFS was 5.765 (P = 0.0163) for AS while that for PS was 0.6587 (P = 0.417). Similarly, between IIIA and IIIB, AS model also displayed a larger χ2 statistic than PS on DSS . χ2 statistic and P value on pair-wise stage comparison of the cohort are shown in Table The χ2 statistic on DSS of AS model showed a larger and statistically significant difference between IA and IB , IIA and IIB , IIB and IIIA  as well as IIIA and IIIB . However, PS model displayed larger χ2 statistic on DSS than AS model between IB and IIA  as well as IIIB and IIIC . Similar results were also observed on the discrimination between stages for OS. χ2 statistic and P value on pair-wise stage comparison of the cohort are shown in Table In the SEER cohort, compared to PS model, χp = 0.049, Fig. p = 0.515, Fig. p < 0.001, Fig. p = 0.0083, Fig. p value are shown in Additional file Furthermore, we also compare survival data between patients with unchanged stage and patients with upstaged PS. In SYSUCC-PWH cohort, patients with IIIC unchanged stage showed worse survival outcome than those patients with AS IIIA or IIIB upstaged to PS IIIC in PFS  for DSS, PFS or OS by stage in both cohorts. The hazard ratios for anatomic and prognostic stages in both cohorts are summarized in Additional file The greatest change of AJCC 8th edition cancer staging system is the incorporation of tumor grade, and ER, PR, and HER2 status, as well as genomic assays, which are generally accepted as important predictive and prognostic factors in clinical practice –14. ThisPrevious studies have demonstrated that the PS model provided a better prognosis value than the AS model in individuals with breast cancer , 6, 15, Weiss, et al.  found thUpstaged TNBCs by PS in the present study are mostly from AS IA, IIB, IIIA and IIIB, however, we noticed that those upstaged cases do not demonstrate the relevant worse survival outcomes. These findings are consistent with previous study by Liu, et al. . On the The mainstay treatment of TNBC remains to be chemotherapy. Despite the upstages in PS, the clinical treatment decision will unlikely be changed for the time being , 20. So Our findings demonstrated that prognostic staging system did not provide better discriminatory ability in predicting TNBCs prognosis than anatomic staging system. The prognosis staging system should be further modified following the development of novel clinical therapies in TNBCs.Additional file 1: Figure S1. Kaplan-Meier curves of DSS (A) and PFS (B) for the SYSUCC-PWH cohort between patients with IIB unchanged and those with anatomical stage IIB upstaged to prognostic stage IIIA.Additional file 2: Figure S2. Kaplan-Meier curves of OS (A) and DSS (B) for the SEER cohort between patients with IA unchanged and those with anatomical stage IA upstaged to prognostic stage IB; Kaplan-Meier curves of OS (C) and DSS (D) for the SEER cohort between patients with IIB unchanged and those with anatomical stage IIB upstaged to prognostic stage IIIA.Additional file 3: Table S1. Clinicopathologic characteristics of TNBCs included in SYSUCC-PWH cohort (N = 611) and SEER cohort . Table S2. Alteration from Anatomic to Prognostic Stage of TNBCs included in SYSUCC-PWH cohort (N = 611) and SEER cohort . Table S3. Hazard Ratio for Disease-Specific Survival and Progression-Free Survival by Stage of TNBCs included in the SYSUCC-PWH cohort (N = 611). Table S4. Hazard Ratio for Disease-Specific Survival and Overall Survival by Stage of TNBCs included in the SEER cohort .


Acute kidney injury (AKI) is a common complication in patients undergoing liver transplant (LT) and is associated with high morbidity and mortality. We aim to evaluate the pattern of urine and plasma neutrophil gelatinase-associated lipocalin (NGAL) elevation during the perioperative period of LT and to assess it as a prognostic marker for AKI progression, need for dialysis and mortality.We assessed NGAL levels before induction of anesthesia, after portal reperfusion and at 6, 18, 24, and 48 h after surgery. Patients were monitored daily during the first week after LT.Of 100 enrolled patients undergoing liver transplant, 59 developed severe AKI based on the KDIGO serum creatinine (sCr) criterion; 34 were dialysed, and 21 died within 60 days after LT. Applying a cut-off value of 136 ng/ml, UNGAL values 6 h after surgery was a good predictor of AKI development within 7 days after surgery, having a positive predictive value (PPV) of 80% with an AUC of 0.76 (95% CI 0.67–0.86). PNGAL at 18 h after LT was also a good predictor of AKI in the first week, having a PPV of 81% and AUC of 0.74 (95% CI 0.60–0.88). Based on PNGAL and UNGAL cut-off criteria levels, time to AKI diagnosis was 28 and 23 h earlier than by sCr, respectively. The best times to assess the need for dialysis were 18 h after LT by PNGAL and 06 h after LT by UNGAL.In conclusion, the plasma and urine NGAL elevation pattern in the perioperative period of the liver transplant can predict AKI diagnosis earlier. UNGAL was an early independent predictor of AKI development and need for dialysis. Further studies are needed to assess whether the clinical use of biomarkers can improve patient outcomes.clinicaltrials.gov) in March 24th, 2014 by title “Acute Kidney Injury Biomarkers: Diagnosis and Application in Pre-operative Period of Liver Transplantation (AKIB)” and identifier NCT02095431, retrospectively registered.Registered at Clinical Trials ( Acute kidney injury (AKI) is a common complication in patients undergoing liver transplant (LT) . Recent The etiology of AKI after LT is multifactorial and includes recipient, graft, perioperative, and postoperative risk factors . Pre-opeDespite improvements in organ preservation, surgery techniques, and immunosuppression protocols, the incidence of AKI after LT continues to be high, reaching 50% in some studies , 3. In tChallenges in risk assessment and early management in patients developing AKI significantly contribute to worse prognoses in these patients. The limitations of serum creatinine (sCr) as a diagnostic marker for acute kidney injury are more evident in patients with liver disease due to malnutrition and decreased muscle mass –17. TherSeveral biomarkers of early kidney injury have been identified and, although these novel AKI biomarkers have primarily been assessed in general intensive-care populations –21, theyIn this study, we aimed to determine whether the pattern of NGAL urinary and plasma elevation in the perioperative period of LT could be a prognostic tool for determining AKI severity, progression, need for RRT and mortality.https://clinicaltrials.gov, with identifier NCT02095431. Patients were enrolled into the study after written informed consent was obtained from all participants as per the guidelines of the Institution’s Ethics Committee.The University of Sao Paulo Ethics Committee approved the study under protocol numberCAAE:06636513.4.0000.0068. All clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul and with the Declaration of Helsinki. The protocol is registered among the clinical trials available at During a 24-month period from June 2013 to June 2015, a total of 189 liver transplants were performed and 139 recipients were eligible for our study : hepatitis-B or -C cirrhosis, alcoholic cirrhosis, cryptogenic cirrhosis, acute hepatitis and miscellaneous causes. The miscellaneous group included patients with non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, hemochromatosis, Budd–Chiari syndrome, biliary atresia, Wilson’s disease, primary sclerosing cholangitis, polycystic disease, biliary cirrhosis, and hepatocellular carcinoma.The standard triple-drug immunosuppression regimen of tacrolimus with mycophenolate mofetil and steroids was used to prevent allograft rejection.We recorded baseline kidney function and comorbid history from electronic medical records (EMRs). The following perioperative variables were included: main patient characteristics, intra-operative data, clinical follow-up in the first week after LT, need-for-RRT data, and outcomes.The functional Model for End-stage Liver Disease (MELD) was calculated on the basis of serum bilirubin, international normalised ratio (INR) and serum creatinine, by the following formula:The liver transplant (LT) MELD was the sum of the functional MELD and the point score in special situations of transplant priority .Blood and urine samples were collected simultaneously in the perioperative period of LT before induction of anesthesia, after portal reperfusion and at 6, 18, 24 and 48 h after surgery. We recorded vital signs, process of care, and lab results daily for 7 days after LT. Outcomes including maximum AKI severity stage, need for RRT and mortality were assessed in the intensive care unit (ICU), on hospital discharge and 60 days after enrolment.Blood samples were collected from central venous catheters or arterial lines, and urine through indwelling catheters. After collection, samples were immediately centrifuged – blood samples at 3000 rotations per minute (rpm) for 15 min and urine at 1000 rpm for 10 min – and stored at − 80 °C prior to analysis.Quantitative NGAL levels were measured using particle-enhanced turbidimetric immunoassay (PETIA) for NGAL determination, according to the manufacturer’s instructions. The test uses an immunoturbidimetric method, in which polystyrene particles are coated with anti-NGAL antibody and, upon contact with the analyte in the sample, this results in turbidity proportional to the concentration of NGAL. Tests were performed in Labmax 560 equipment. Perioperative serum creatinine from the same samples as the NGAL measurement was analysed by chemiluminescence. All other serum creatinine values recorded from medical records were analysed by Jaffe method in the hospital’s central laboratory.The primary outcome was AKI development during the first week of LT. Baseline renal function was defined as the lowest value in the prior three months and was used to assess renal recovery. AKI diagnosis was based on Kidney Disease: Improving Global Outcomes (KDIGO) . For theP values were two tailed, and P < 0.05 was considered significant. Conventional receiver operating characteristic (ROC) curves were generated, and the area under the curve (AUC) was used to assess the ability of continuous variables to distinguish the categorical state: AKI development, need for dialysis and non-survival. The optimal cut-offs were determined by the best point of sensitivity versus specificity of the AUC (Youden index).Continuous variables are presented as mean ± standard deviation (SD) or median (25th–75th percentiles) and were compared using one-way ANOVA or Kruskal–Wallis testing according to the Gaussian distribution. Categorical variables are presented as absolute numbers and percentages and were compared by the Chi-square test. We compared time to AKI diagnosis based on biomarkers versus creatinine analyzed by chemiluminescence in the same sample collection. Time to diagnose based on sCr criterion was assessed by KDIGO stage 1 definition and biomarkers were determined by the cut-off value determined for plasma NGAL (PNGAL) and urine NGAL (UNGAL).P < 0.01 were used to construct a multivariable model, in which the impact of each comorbidity or covariate was adjusted. In each model, UNGAL or PNGAL was included as a covariate. The SPSS  version 20  software was used for the statistical analysis.The perioperative covariates were tested in univariate analysis for their impact on AKI development, need for dialysis and non-survival. Factors associated with the outcomes at Of 189 adult patients undergoing LT within the study period, 138 were eligible, and 100 were enrolled in the study. Figure Based on the baseline ambulatory values, 34 patients were classified with acute kidney disease before surgery, of whom 23 (67.6%) were KDIGO stage 1. Based on the reference sCr, 85 patients developed AKI (stage 1/2/3). Patients developing no AKI or stage 1 AKI within the first 7 days of LT were summarized as the no-AKI/mild-AKI group 41 (41%), whereas patients meeting the criteria for stage 2 or 3 AKI group within the first 7 days of LT were summarized as the severe-AKI 59 (59%) .Patients developing severe AKI were younger, had a higher functional MELD before surgery and higher Sequential-Organ--Failure (SOFA) scores on ICU admission Table . DuratioSix hours after LT, severe-AKI patients had 10.6-fold higher UNGAL levels than those in the no-AKI/mild-AKI group, followed by a sustained significant difference for 24 h after surgery . Eighteen hours after LT, PNGAL levels were approximately 2-fold higher in severe-AKI patients; median 326 ng/ml in AKI vs. 170 ng/ml in no-AKI /mild-AKI , sensitivity of 87% and a specificity of 83%. Using the cut-off value of 286 ng/ml, UNGAL was able to predict the need for RRT with the best performance at 6 h after LT, a cut-off of 210 ng/ml determining an AUC 0.85 (95% CI 0.77–0.93), with a sensitivity of 86% and the specificity of 80% . The main causes of death were septic shock 07 (33.3%), primary graft dysfunction 03 (14.3%) and hemorrhagic shock 03 (14.3%). The best time point for using PNGAL levels to predict mortality was at 18 h after surgery, with an AUC of 0.76 (CI95% 0.60–0.92) for levels higher than 483 ng/ml. UNGAL was significantly higher in non-survivors even before surgery, in the pre-operative period. The median was 33 ng/dl in survivors versus 220 ng/dl in non-survivors (P = 0.002). Six hours after LT, a value higher than 242.06 ng/ml had an AUC of 0.81 (CI 0.65–0.97) for predicting mortality with sensitivity 86% and specificity 64% ; 03 (14.3%) in no-AKI/mild-AKI group and 18 (85.7%) in the severe-AKI group . This finding suggests that early peak levels can be useful in deciding on early RRT initiation and influencing RRT method, e.g., continuous therapy in high-risk patients.The high mortality rate in our cohort can be partially explained by the overall severity of patients, their older age and presence of comorbidities , higher MELD , 24, 33,Despite not being a multicentre study, this is the largest cohort for evaluating a biomarker perioperative of liver transplant. In this analysis, we used serum creatinine as a sole performance comparator for diagnosis of AKI, not applying urine output criteria. It is possible that some patients would have an earlier diagnosis based on urine volume and the delay in AKI diagnosis by KDIGO system decreased. Our incidence of AKI was high: 85% and 59 were AKI stage 2/3. Previous studies applying the RIFLE criteria have not considered 0,3 mg/dl serum creatinine criteria to define AKI , 29, 32.NGAL is a promising biomarker for predicting AKI severity in patients undergoing liver transplant. The pattern of plasma- and urinary-NGAL elevation in the period perioperative of liver transplant allows earlier AKI diagnosis than KIDGO criteria based on serum creatinine. UNGAL was an independent predictor of AKI development and need for dialysis. Future studies should evaluate whether the clinical use of these biomarkers could improve patient outcomes.


Multi-drug resistant organisms are an increasingly important cause of neonatal sepsis.This study aimed to review neonatal sepsis caused by multi-drug resistant Enterobacteriaceae (MDRE) in neonates in Johannesburg, South Africa.This was a cross sectional retrospective review of MDRE in neonates admitted to a tertiary neonatal unit between 1 January 2013 and 31 December 2015.p = 0.01), lower birth weight (p = 0.04), maternal HIV infection (p = 0.02) and oxygen on day 28 (p < 0.001). The most common isolate was Klebsiella pneumoniae (66.2%). Total MDRE isolates increased from 0.39 per 1000 neonatal admissions in 2013 to 1.4 per 1000 neonatal admissions in 2015 (p < 0.001). There was an increase in carbapenem-resistant Enterobacteriaceae (CRE) from 2.6% in 2013 to 8.9% in 2015 (p = 0.06). Most of the CRE were New Delhi metallo—β lactamase- (NDM) producers.There were 465 infections in 291 neonates. 68.6% were very low birth weight (< 1500 g). The median age of infection was 14.0 days. Risk factors for MDRE included prematurity (p = 0.003), necrotising enterocolitis (p < 0.001) and mechanical ventilation (p = 0.007) were significantly associated with mortality. Serratia marcescens was isolated in 55.2% of neonates that died.The all-cause mortality rate was 33.3%. Birth weight (There was a significant increase in MDRE in neonatal sepsis during the study period, with the emergence of CRE. This confirms the urgent need to intensify antimicrobial stewardship efforts and address infection control and prevention in neonatal units in LMICs. Overuse of broad- spectrum antibiotics should be prevented. Klebsiella pneumoniae and Escherichia coli isolated from a group of preterm infants in Malaysia were MDRO [Sepsis remains a major cause of morbidity and mortality in preterm infants . There here MDRO .Infections with multi-drug resistant gram-negative organisms, especially Enterobacteriaceae, are of concern in preterm infants. Neonatal sepsis caused by these pathogens is increasing and there are limited choices available for treatment . InfectiKlebsiella pneumoniae carbapenemase (KPC) [Carbapenem-resistant organisms were already described as a cause of neonatal septicaemia in India in 2007 . It is nse (KPC) . There i{Magiorakos, 2012 #2052} NDM and KPC (KPC-2) were first described in adult patients in Johannesburg in 2011 . The aimblaNDM, blaKPC, blaOXA-48 and its variants, blaGES, blaIMP and blaVIM; LightMix Modular kits, Roche Diagnostics, Basel, Switzerland) was performed on a subset of the CRE isolates. Typing of isolates was not performed.This is a retrospective descriptive cross-sectional study. All newborn neonates admitted to the neonatal unit between 01 January 2013 and 31 December 2015 were eligible for inclusion. The study group included all neonates with culture proven blood stream infection (BSI) caused by MDRE. A control group of 30% of all neonates without infection admitted to the neonatal unit during the study period was randomly generated from the neonatal database using SPSS IBM 24. Subjects were identified through the laboratory information system of the National Health Laboratory Service (NHLS). Patient characteristics were obtained from the neonatal computer database. Information was obtained from hospital records on discharge of each neonatal patient and was entered into a computerised database for the purpose of quality control. Data was managed using Research Electronic Data Capture (REDCAP), hosted by the University of the Witwatersrand . Maternap value of 0.05 was considered to be statistically significant. Adjusted odds ratios were determined through binary logistic regression for significant associations with mortality and MDRE infection respectively.IBM SPSS 24 was used to analyse the data.. Maternal and neonatal characteristics were described for each patient . Microbiological information  was analysed for each bacterial isolate. Mean and standard deviation or median and range, were used to describe central tendency in continuous variables, depending on the distribution of the data. Categorical variables were described using frequency and percentages. Only valid cases were analysed for each variable (i.e. missing cases were excluded). Two comparisons were performed. Firstly, survivors and non- survivors within the MDRE group were compared to determine risk factors for mortality. Secondly, the MDRE group and control group were compared to establish associations with MDRE infection. Frequencies were compared using Chi Square analysis, while unpaired t tests were used to compare continuous variables, as the data was normally distributed. A Ethics clearance was obtained from the Human Research Ethics Committee of the University of the Witwatersrand (Certificate M 151108). Permission was obtained to access the Laboratory information system from the NHLS.Early-onset sepsis (EOS) was defined as culture proven sepsis within the first 72 h of life, while late onset sepsis (LOS) was referred to as culture proven sepsis after 72 h of life . Multidrp < 0.001). Neonates with MDRE sepsis were significantly more preterm than controls − 30.4 weeks (SD 4.0) vs 32.9 weeks (SD 4.8) (p < 0.001) The all-cause mortality rate was higher in MDRE neonates than controls − 97/291 (33.3%) vs- 49/2146 (2.2%)) (p < 0.001). Most of the neonates with MDRE sepsis  were very low birth weight. The median age at presentation with MDRE was 14.0 days (IQR 20). Although more than one third of neonates with MDRE sepsis were HIV exposed, there was only one neonate  with a positive HIV polymerase chain reaction at birth. Adjusted odds ratios for those variables significantly associated with MDRE are shown in Table There were a total of 465 MDRE infections in 291 neonates and 2146 control neonates without infection. The comparison between neonates with MDRE and controls is shown in Table Klebsiella pneumoniae , followed by Enterobacter cloacae. , Escherichia coli ; Serratia marcescens , Klebsiella spp. (Klebsiella spp. other than K. pneumoniae) , Proteus mirabilis , Citrobacter freundii , Citrobacter koseri  and Salmonella spp. .There were 41/465 (8.8%) of EOS caused by MDRE. Most neonates  had a single episode of infection, 18.2% (53/291) had two episodes of infection and 14.8% (43/291) had three or more episodes. The most common isolate was Resistance patterns of all isolates are shown in Table p < 0.001). Monthly variation in isolates of K. pneumoniae, E. cloacae, S. marcescens and E. coli is shown in Fig. The variation in annual MDRE isolates is shown in Fig. C. freundii and Klebsiella oxytoca and two E. cloacae. The remaining 22 isolates were K. pneumoniae. CRE isolates per year are shown in Fig. p = 0.06). One Klebsiella pneumoniae isolate produced Verona Integron-Mediated Metallo-β-lactamase (VIM) and fourteen produced NDM. The K. oxytoca produced VIM and the C. freundii produced NDM. One isolate was negative for CPE genes and 8 isolates were not tested. Ninety-two percent (24/26) and 62% (16/26) of the CRE isolates were ciprofloxacin and amikacin susceptible, respectively.There were 26 CREs isolated from 24 neonates shown in Table p = 0.003), NEC  and mechanical ventilation . Mortality rate by isolate is shown in Table S. marcescens BSI (55.2%) (p < 0.017). Neither ESBL production nor carbapenem resistance was significantly associated with mortality.All-cause mortality in the neonates with MDRE sepsis was 33.3% (97/291). Adjusted odds ratios for variables significantly associated with mortality were birth weight  for empiric treatment of presumed sepsis in neonates. This compounds the problem of resistant organisms as widespread use of broad-spectrum antibiotics is an important mechanism in the development and spread of MDRE [Klebsiella infections in neonates [Klebsiella spp.) in the current study, there was no evidence of a seasonal variation.Most of the MDRE isolates in the current study were resistant to the penicillins and cephalosporins; the most common mechanism of resistance being production of ESBLs, particularly in  of MDRE , 21, 25. of MDRE . Treatme of MDRE . The lacS. marcescens in the present study. S. marcescens is an important nosocomial pathogen, especially in neonatal intensive care units (NICU) where several outbreaks have been described globally. It causes serious infections, including bacteremia, pneumonia, urinary tract infections and meningitis with significant morbidity and mortality rates among newborns. Risk factors for acquisition of nosocomial infections caused by S. marcescens in NICUs are low birth weight, long duration of hospitalization and receiving of critical care [S. marcescens infections, we did not analyze possibly confounding variables such as the severity of illness related to prematurity in these infants.The mortality rate was highest in neonates infected with cal care . AlthougLactobacillus plantarum and Bifidobacterium breve, can have a protective effect by exhibiting antimicrobial activity against gut pathogens [Prevention of MDRE infections includes screening for colonization, antibiotic stewardship, and stringent infection control practices including, hand hygiene practices, use of appropriate personal protective equipment, and decreased use of invasive devices , 25. TheThis was a retrospective review of an existing database, so timing of complications and antibiotic therapy for BSI could not be evaluated. In addition, many possible risk factors for sepsis, such as catheterization, time to the onset of feeding, and total parenteral nutrition had not been captured and were therefore not available for analysis.The present study confirms that there is an alarming increase in MDRE with the emergence of CRE. This confirms the urgent need to intensify antimicrobial stewardship efforts and address infection control and prevention in neonatal units in LMICs. In particular, the overuse of broad-spectrum antibiotics should be discouraged. In addition, other treatment and prevention modalities, such as strategies targeting the neonatal gut microbiome, should be investigated.


Streptococcus mutans wild-type and lrgAB-mutant revealed that this organism undergoes dynamic cellular changes in the face of multiple exogenous stresses, consequently affecting its comprehensive virulence traits. In this current study, we further demonstrate that LrgAB functions as a S. mutans pyruvate uptake system.Our recent ‘-omics’ comparisons of S. mutans excretes pyruvate during growth as an overflow metabolite, and appears to uptake this excreted pyruvate via LrgAB once the primary carbon source is exhausted. This utilization of excreted pyruvate was tightly regulated by glucose levels and stationary growth phase lrgAB induction. The degree of lrgAB induction was reduced by high extracellular levels of pyruvate, suggesting that lrgAB induction is subject to negative feedback regulation, likely through the LytST TCS, which is required for expression of lrgAB. Stationary phase lrgAB induction was efficiently inhibited by low concentrations of 3FP, a toxic pyruvate analogue, without affecting cell growth, suggesting that accumulated pyruvate is sensed either directly or indirectly by LytS, subsequently triggering lrgAB expression. S. mutans growth was inhibited by high concentrations of 3FP, implying that pyruvate uptake is necessary for S. mutans exponential phase growth and occurs in a Lrg-independent manner. Finally, we found that stationary phase lrgAB induction is modulated by hydrogen peroxide (H2O2) and by co-cultivation with H2O2-producing S. gordonii.S. mutans with an alternative carbon source under limited growth conditions, as well as serving as a buffer against exogenous oxidative stress. Given the hypothesized role of LrgAB in cell death and lysis, these data also provide an important basis for how these processes are functionally and mechanically connected to key metabolic pathways such as pyruvate metabolism.Pyruvate may provide  Streptococcus mutans, withstand the limited resources or environmental fluctuations experienced in the oral cavity [S. mutans Cid/Lrg system, consisting of two dicistronic operons lrgAB (SMU.575c/574c) and cidAB (SMU.1701c/1700c) [Staphylococcus aureus cid and lrg mutants [S. mutans, the Cid/Lrg system is also involved in comprehensive virulence traits, including antibiotic resistance, autolysis, biofilm development, genetic competence, oxidative and heat stress responses [S. mutans and S. aureus. Direct evidence of their specific cellular functionality is still scarce.Development of a mature biofilm on the tooth surface is the central event in the pathogenesis of dental caries . This prl cavity , 3. An el cavity , 5. Towac/1700c) –11. Thesc/1700c) . Bacteric/1700c) . Further mutants –16. Notaesponses , 11, allS. mutans Cid/Lrg system is that expression levels of lrg and cid are counterbalanced throughout the growth cycle and in response to the availability of oxygen and glucose [lrg and cid operons were originally identified to be up- and down-regulated, respectively, in aerobically grown cells [lrg and cid to glucose levels is particularly remarkable. The lrg genes are highly induced in cultures containing lower levels of glucose (≤ 15 mM) but almost completely repressed in cultures containing glucose at concentrations of 20 mM and higher [cid expression is negligible when cells are cultured in the presence of lower glucose concentrations (≤20 mM) but increases at higher glucose concentrations (> 20 mM) [cid and lrg [lrgAB expression is also governed by the LytST two-component regulatory system (TCS), located immediately upstream of the lrgAB genes [S. mutans wild-type and isogenic ΔlrgAB mutant strains, using RNA-seq and label-free quantitative mass spectrometry, showing that a large number of genes and/or proteins involved in carbohydrate metabolism, ABC transporters, and oxidative stress adaptation were significantly altered in the lrgAB mutant under stress inducing culture conditions  [A hallmark finding for the  glucose , 8. In fwn cells . The resd higher . In cont> 20 mM) . Our rec and lrg , and lrgAB genes , 8. Theseatment) , 11.lrgAB promoter is highly active only in cells entering stationary phase, but not in cells growing exponentially, in the low-glucose condition [lrgAB induction pattern was also shown in our previous microarray data, comparing RNA expression profiles of wild-type and lytS-deficient strains between early- and late-exponential growth phases in BHI medium [lrgAB (SMU.575c-574c) was dramatically upregulated (about > 900-fold) at late-exponential phase, compared to early-exponential growth phase [Bacillus subtilis [lrgAB genes in S. mutans [ysbAB genes was regulated by LytSR/LytST, located upstream of ysbAB, as well as by CcpA [ysbAB was also maximal at stationary phase of growth [Esherichia coli also has two TCS (BtsSR and YpdAB) homologous to LytST that have been shown to regulate expression of pyruvate transporters in response to extracellular pyruvate [E. coli from the viable but non-culturable (VBNC) state [B. subtilis, suggest that LrgAB may also function as a pyruvate transporter in S. mutans.The fact that the ondition , impliesI medium . When thth phase . A four-th phase , suggestsubtilis , 19. Sim. mutans , 9, expr by CcpA , 21. Expf growth , 19 and f growth , 21. Eshpyruvate –24. InteS. mutans, as well as other oral streptococci. Pyruvate is the final product of glycolysis, as well as a major substrate for oxidative metabolism. Pyruvate is converted to acetyl-coenzyme (acetyl-CoA) by the Pdh complex or Pfl (pyruvate formate lyase), depending on the presence or absence of oxygen, or the limitation or excess of a preferred sugar (e.g. glucose) [S. mutans as an overflow metabolite, and is reimported into cells via LrgAB when the primary carbon source (i.e. glucose) becomes exhausted. We performed a series of pyruvate quantification and lrg promoter reporter assays in order to characterize the role and regulation LrgAB as a pyruvate uptake system. These experiments demonstrate that LrgAB expression and activity is tightly regulated at the transcriptional level and modulated by both external and internal metabolic conditions. We also show that due to the H2O2- scavenging activity of pyruvate, re-uptake of pyruvate by S. mutans may be influenced by interactions with H2O2-producing oral commensals, such as S. gordonii. Given the possible involvement of LrgAB in inducing cell death and lysis in a programed manner, the presented data provide a new basis for how cell death and lysis mechanisms in S. mutans may be functionally and mechanically connected to pyruvate, a metabolic signal which may modulate homeostasis and virulence of this organism.To date little is known about the role and regulation of pyruvate in glucose) , 26. AceStreptococcus mutans UA159 and its previously-constructed mutant derivatives [2O2, Fisher Scientific), as necessary. Antibiotics were used to supplement growth media in the following concentrations: spectinomycin (1 mg/ml), kanamycin (1 mg/ml), and erythromycin (10 μg/ml). Unless otherwise noted, cultures were grown at 37 °C in a 5% CO2, aerobic atmosphere. For aerobic growh, cultures were grown in an aerobic incubator. To achieve anaerobic conditions, sterile mineral oil was placed on top of the cultures [S. mutans. The optical density at 600 nm (OD 600) was measured at 37 °C at 30 min-intervals using a Bioscreen C growth curve analysis system.ivatives , 9 were ivatives  containicultures , 17, 28.S. mutans UA159 LrgA/B (SMU.574/575c), B. subtilis 168 YsbA/B (BSU_28900/28910), S. aureus MRSA252 LrgA/B (SAR_0259/0260), S. gordonii DL1 LrgA/B (SGO_1268/1269), and E. coli K12 LrgA/B  were retrieved from the National Center of Biotechnology Information (NCBI) Protein database or from Uniprot (www.uniprot.org). Alignments were then generated utilizing the T-COFFEE M-coffee protein alignment tool [https://embnet.vital-it.ch/software/BOX_form.html). Percent identity matrices were then produced using the same sequences using Clustal Omega [Amino acid sequences for S. mutans strains harboring Plrg-gfp gene fusion, previously constructed [600 = 0.5. At this point, these cultures were diluted 1:50 into 175 μl FMC in individual wells of a 96-well plate . To evaluate the possible role of pyruvate as an antioxidant against H2O2 in the oral cavity, the reporter strain was also cultivated with Streptococcus gordonii DL1, a H2O2-producing oral commensal, at a ratio of 1:1. The optical density at 600 nm (OD600) and green fluorescence were monitored  at 30 min intervals. The fluorescence of wild-type harboring plasmid without the reporter gene fusion was subtracted from fluorescence readings of the S. mutans strains harboring Plrg-gfp gene fusion. The results are representative of at least three independent replicates, each performed in triplicate.GFP activities of the structed , were obstructed , 31, 32.S. mutans UA159 wild-type and isogenic mutants  strains were grown in chemically defined FMC medium, supplemented with either 11 mM or 45 mM glucose. For time course measurements of extracellular pyruvate during growth, samples (200 μl) were taken at 1–2 h intervals and half of this volume (100 μl) was used to measure the OD600 in a spectrophotometer for monitoring growth. The other half (100 μl) was centrifuged for 2 min at 18,000 xg to remove the cells, and pyruvate and glucose concentrations of the supernatant were quantified with an EnzyChrom™ pyruvate assay kit  or glucose (HK) assay kit (Sigma-Aldrich), respectively, according to the manufacturer’s instructions. The results are average or representative of at least two independent replicates, each performed in duplicate.S. mutans LrgAB functions as a pyruvate transporter, we performed multiple sequence alignments alongside B. subtilis YsbA and YsbB, recently reported to be putative pyruvate transporters [S. aureus, E. coli and S. gordonii . In the alignment of LrgA homogues  and YsbB (30.0%), as well as to S. aureus LrgA (34.9%) and LrgB (30.0%), proposed to be involved in the regulation of cell death [S. aureus LrgA and LrgB. It is also notable that S. mutans lytST, known to regulate lrgAB [B. subtilis and S. aureus , allowing a strong stationary phase induction of lrgAB [2 atmosphere. As shown in Fig. lrgAB cultures, the level of excreted pyruvate was only slightly decreased without dramatic change  of pyruvate excreted during growth of the ΔlrgAB strain was about 50% elevated compared to wild type, and was reached after a delay to the onset of stationary phase, suggesting that lack of LrgAB may slow down pyruvate metabolism, consequently increasing the overflow of pyruvate. This is consistent with our recent RNA-seq data, showing that many metabolic pathway-related genes, including pfl, were significantly downregulated during growth of the ΔlrgAB strain [lytS strain  or overexpressing cidAB (UA159/184-cidAB) [, no obvious difference was observed (data not shown). Thus, we concluded that the Cid proteins are not likely involved in pyruvate uptake/utilization in S. mutans, thus no further investigation was performed on the cid operon in this study.Pyruvate is excreted as an overflow metabolite and re-consumed after other favorable carbon sources (i.e. glucose) are exhausted . Many basubtilis , 19 and [E. coli , 23 are of lrgAB , at 5% C glucose  mM, allo approx. 00 μM of of lrgAB . Given t approx. 00 μM of  approx. 00 μM of  glucose  mM, allolrgAB was dramatically repressed in cultures containing glucose at concentrations of 20 mM and higher, although still detectable at concentrations of up to 45 mM [lrgAB culture  condition  condition, and under aerobic conditions leading to higher induction. We initially added 1 mM pyruvate into the medium at the beginning of growth, because about 400–800 μM pyruvate would be excreted in the same growth condition in the absence of pyruvate  of the pyruvate analog 3-fluoropyruvate (3FP), previously shown to compete with pyruvate and inhibit cell growth by binding to PDH complex in the cell [lrgAB was reduced in a dose-dependent manner by 3FP. Expression of lrgAB was obviously reduced in the presence of 0.01 mM 3FP  using a Bioscreen C plate reader. As shown in Fig. lrgAB strain, showing a more evident elongation of exponential phase of growth  or 0.004% (≈1.176 mM) H2O2, and its GFP production was monitored during growth. The strain displayed an expected increased lag phase in the presence of 0.002% H2O2  but triggers lrgAB induction at the stationary phase. The expression of lrgAB was remarkably reduced by co-cultivation with S. gordonii  is tightly regulated by glucose and oxygen levels [pdh and pfl genes, responsible for the conversion of pyruvate to acetyl-CoA, were highly upregulated at late-exponential phase [pdh genes were commonly upregulated in response to three stress conditions  previously tested [S. mutans to adapt to suboptimal conditions. Pyruvate is likely a common nutrient in the microbiome environment, such as oral cavity. But its utilization and regulation in S. mutans have been poorly characterized, especially with respect to how they are connected to the virulence network of this organism.The modeling –9, 11, wn levels , 8, 17. n levels , 43–45. n levels , 46. We al phase , suggestlrgAB induction. Growth in high levels of glucose abrogates stationary-phase response of lrgAB to extracellular pyruvate, suggesting that the response of lrgAB to extracellular pyruvate is subject to carbon catabolite repression. Supplementation of exogenous pyruvate was unable to induce earlier induction of lrgAB , reinforcing that uptake of pyruvate occurs only under limited growth, and is subject to tight metabolic control. Given that lrgAB expression requires activation by LytST TCS [lrgAB may be primarily due to inaccessibility of LytT to the promoter region of lrgAB. In our recent study, we found that the cre site for CcpA binding overlapped, in part, with a potential LytT binding site in the promoter region of lrgAB [lrgAB by LytT may be prevented by CcpA binding to the promoter region of lrgAB. However, repression of lrgAB was not released even in the absence of CcpA when the cell was cultivated in high-glucose medium [lrgAB. In fact, CcpA often works together with CodY to sense changes in nutrient availability and coordinate, directly and indirectly, the expression of hundreds of genes involved in carbon and nitrogen metabolism of Gram-positive bacteria [codY gene was several-fold upregulated at early-exponential phase, compared to late-exponential growth phase [lrgAB expression in response to environmental conditions and nutritional needs, which is currently under investigation.It seems that the re-uptake of pyruvate is tightly regulated by glucose levels and stationary growth phase ytST TCS , repressof lrgAB , suggeste medium , suggestbacteria , 47–49. bacteria . Accordith phase . Thus, ilrgAB to extracellular pyruvate was supported by the observation that supplementation of 10 μM 3FP had a profound effect on repressing the expression of lrgAB, likely by interfering with activation of the LytS sensor kinase by pyruvate. These results suggest that LrgAB may be responsible for facilitating the recovery from carbon starvation when concentrations of pyruvate are low. On the contrary, the finding that the stationary phase induction of lrgAB was alleviated by high concentrations of extracellular pyruvate (approx. > 20 mM), revealed the existence of a negative feedback regulation acting on LytST by the presence of high levels of extracellular pyruvate. This feedback regulation may contribute to balanced extracellular and intracellular pyruvate levels, although it remains to be elucidated. Given that the re-uptake of accumulated pyruvate is subject to a tight metabolic control, pyruvate may play a role as a potential metabolic signal to determine cellular fate under limited nutrient conditions. However, this regulatory model does not account for the observation that cells were still capable of utilizing extracellular pyruvate even in the absence of LrgAB, resulting in prolonged exponential growth, suggesting the existence of an additional pyruvate uptake system that operates in a LrgAB-independent manner and preferably when pyruvate concentrations are relatively high . Indeed, it has been suggested that E. coli uses one exporter and two uptake transporters to modulate the level of intracellular pyruvate [. It is also supported by the observation that S. mutans was also unable to grow in a medium containing high concentrations of pyruvate (up to 80 mM) as sole carbon source  of pyruvate. In a preliminary experiment, we found that stationary phase lrgAB induction was significantly inhibited by two hydrophobic protonophores, CCCP (carbonyl cyanide m-chlorophenyl hydrazine) and DNP  at 1 μM and 100 μM, respectively , suggesting that LrgAB may be specifically responsible for uptake of pyruvate. This theory is also supported by the observation that a synthetic analogue of pyruvate (3FP) effectively competed with pyruvate. This specificity also reinforces the role of pyruvate as a potential metabolic signal, because pyruvate is excreted together with other metabolic compounds. The detailed mechanism for how pyruvate is transported via LrgAB through the cell membrane is an important part of understanding the Cid/Lrg system and warrants further investigation.Although the data herein suggest that LrgAB is primarily responsible for uptake of pyruvate in 2O2, suggesting that the re-uptake of pyruvate may be influenced by interspecies interactions, especially with H2O2-producing oral commensals, such as S. gordonii and S. sanguinis [2O2 with pyruvate is presented as CH3-CO-COOH+H2O2 → CH3-COOH+H2O + CO2 [2 are the major by-products of pyruvate reaction with H2O2, with pyruvate being produced and excreted during exponential growth while glucose is converted to biomass. Acetate would be presumably also taken up into the cell in parallel with pyruvate under nutrient limited growth condition [pta-ackA pathway which generates acetate and ATP was reported to cause cell death in S. aureus [S. mutans [B. subtilis [2O2 [2O2 could be modulated by multiple metabolic products. It is an on-going study how the levels of H2O2 and pyruvate (or other organic acids) are changed in the interaction between S. mutans and S. gordonii (or S. sanguinis), and how the LrgAB pyruvate uptake system contributes to the protection from environmental H2O2 challenge.Another important finding of this study was that pyruvate directly reacts with Hanguinis , 54. The2O + CO2 . Indeed,ondition , 55. Int. aureus , and to . mutans  and B. ssubtilis . Therefosubtilis . It is alis [2O2 –42, suggS. mutans, and provide an important new basis for how the hypothesized role of LrgAB in modulating cell death and lysis are mechanistically connected to key metabolic pathways. Overall, excreted pyruvate may play several important roles in S. mutans physiology, such as a carbon source and/or metabolic precursor for amino acid/fatty acid biosynthesis under nutrient-limited and/or stationary phase growth conditions, as well as by buffering external sources of oxidative stress, such as H2O2, which primarily appears to be modulated by LytST-LrgAB. Therefore, it is possible that the trafficking and utilization of pyruvate may significantly contribute to shaping a homeostatic mechanism and composition of oral microflora, consequently influencing the development of caries. Nevertheless, due to highly variable in vivo conditions especially in respect to nutrient availability and unknown host factors, the actual effect and role of pyruvate remain to be elucidated and are currently under investigation. This study also opens the possibility of searching for additional pyruvate uptake system as well as examining the relationship between metabolite fluxes and ecological fitness. This will be important as we explore the possible mechanisms underlying stress tolerance and programmed cell death as a bacterial survival strategy at the community level.The results of this study demonstrate that LrgAB is the first identified pyruvate uptake system in Additional file 1: Figure S1. Schematic diagram of the lyt and lrg genetic loci in the genomes of S. mutans, B. subtilis, and S. aureus.Additional file 2: Figure S2. Growth of S. mutans wild type in chemically defined FMC medium containing increasing concentrations of pyruvate as the sole carbon source. Growth was monitored during growth in a Bioscreen C system that was set to shake for 15 s every 30 min. The results are representative of two independent experiments.Additional file 3: Figure S3. Measurement of extracellular pyruvate during growth of S. mutans ΔcidB mutant strain in low-glucose FMC medium. The strain was grown in a chemically defined medium (FMC) supplemented by 11 mM glucose. For time course measurements of extracellular pyruvate and growth, samples were taken at 1 or 2 h intervals . The concentration of pyruvate was determined using an EnzyChrom™ pyruvate assay kit, and growth was measured by the optical density at 600 nm (OD600). Bar indicates the concentration of extracellular pyruvate; line indicates growth curve. The results are average of two independent experiments.Additional file 4: Figure S4. Measurement of extracellular glucose during growth of S. mutans wild type and ΔlrgAB strains in the high-glucose media. The strains were grown in a chemically defined medium (FMC) supplemented by 45 mM glucose. For time course measurements of extracellular pyruvate and growth, samples were taken at 1 or 2 h intervals . The concentration of glucose was determined using an Glucose (HK) assay kit, and growth was measured by the optical density at 600 nm (OD600). Bar indicates the concentration of extracellular pyruvate; line indicates growth curve. The results are representative of two independent experiments.Additional file 5: Figure S5. Measurement of extracellular pyruvate during aerobic (A and B) or anaerobic (C and D) during growth of S. mutans wild-type (A and C) and ΔlrgAB (B and D) in the low-glucose media. The strains were grown in a chemically defined medium (FMC) supplemented by 11 mM glucose. For anaerobic growth, sterile mineral oil was placed on top of cultures. For time course measurements of extracellular pyruvate and growth, samples were taken at 1 or 2 h intervals . The concentration of pyruvate was determined using an EnzyChrom™ pyruvate assay kit, and growth was measured by the optical density at 600 nm (OD600). Bar indicates the concentration of extracellular pyruvate; line indicates growth curve. The results are average of two independent experiments.Additional file 6: Figure S6. The effect of 3-fluoropyruvate  on lrg promoter (PlrgA) activity in the presence of pyruvate. The PlrgA-gfp reporter strain was grown in a low-glucose (11 mM) FMC medium supplemented by 0 (A), 0.01 (B), 0.1 (C) or 1 mM (D) 3FP. Pyruvate was added into the medium at the concentration of 1 mM. Relative gfp expression (green square) and OD600  were monitored on a plate reader . The results are representative of two independent experiments.Additional file 7: Figure S7. The effect of 3FP on the growth of S. mutans wildtype. The strain was cultivated in 11 mM glucose FMC media containing different concentrations  of the pyruvate analog 3-fluoropyruvate (3FP). Growth was monitored during growth in a Bioscreen C system that was set to shake for 15 s every 30 min. The results are representative of two independent experiments.Additional file 8: Figure S8. The effect of protonophores CCCP and DNP on lrg promoter (PlrgA) activity. The PlrgA-gfp reporter strain was grown in a low-glucose (11 mM) FMC medium (A), supplemented by 1 μM CCCP  and 0.1 mM DNP . Relative gfp expression (green square) and OD600  were monitored on a plate reader . The results are representative of two independent experiments.Additional file 9: Figure S9. Change of lrg promoter (PlrgA) activity during growth in FMC medium supplemented by different concentrations of extracellular pyruvic acid. The PlrgA-gfp reporter strain was grown in a low-glucose (11 mM) FMC medium supplemented by 0 (A), 1 (B), and 2 mM (C) pyruvic acid (PA). Relative gfp expression (green squares) and OD600  were monitored during growth on a plate reader . The results are representative of two independent experiments.


Search results were screened, and publications comparing oncologic outcomes of T1N0M0 glottic carcinoma treated with TLM or RT were included. Data was extracted independently by two authors, and publication quality was graded according to the Oxford Centre for Evidence-based Medicine. Meta-analysis was performed for overall survival, disease specific survival, laryngeal preservation, and local control.P = 0.02), disease specific survival , and laryngeal preservation . There was no difference in local control between TLM and RT in T1 glottic cancer .Sixteen studies were included in the meta-analysis, the majority being retrospective cohort studies with two prospective cohort studies. Included studies were rated as either Level II or III evidence. Meta-analysis favoured treatment with TLM for T1 glottic carcinoma patients for the following outcomes: overall survival (odds ratio [OR], 1.52; 95% confidence interval [CI], 1.07–2.14; Our study provides a current and thorough comparison of TLM and RT outcomes in T1 glottic carcinoma. Limitations of our study include lack of randomized control trials, and non-randomized allocation of patients to treatment groups. Our meta-analysis suggests that TLM is the superior modality in terms of overall survival, disease specific survival, and laryngeal preservation. Future prospective randomized controlled studies are required for confirming these findings and developing appropriate clinical practice guidelines.2A; as per the Centre of Evidence Based Medicine. Cancer of the larynx is the most common head and neck malignancy, with an estimated 1150 new cases in Canada in 2017 [In the past, laryngeal cancers were traditionally treated with radiotherapy (RT) or open partial laryngeal surgery. Transoral laser microsurgery (TLM) for glottic cancer was first described in 1972 by Strong and Jako . CurrentTo our knowledge, an international standard guiding therapeutic selection in early glottic carcinoma does not exist. In order to improve clinical decision making, we systematically reviewed the literature for studies directly comparing TLM and RT in T1 glottic carcinoma and performed a meta-analysis of critical oncologic outcomes including overall survival, disease-specific survival, laryngeal preservation, and local control. Although previous reviews have investigated this question, our systematic review provides an updated analysis. For instance, Mo et al. in 2017  includesLiterature searches were conducted independently by authors MFV and CAM in the following electronic databases: Medline/PubMed, Web of Science, EMBASE, and the Cochrane Library. These databases were selected to provide a broad search of high quality medical research and allowed for a thorough scope of the available literature. Searches were designed to combine disease specific terms , site specific terms , and treatment specific terms for TLM , and RT . Searches in separate databases were similarly constructed, using the Boolean operator ‘AND’ to combine disease, site, and treatment specific term categories. All searches were conducted between March and May 2017.Search results for each database were imported in EndNote X8.2 software by Clarivate Analytics. According to currently accepted practises, duplicate results between databases were retrieved using the “Find Duplicates” function in EndNote X8.2, and manually reviewed by author MFV prior to removal . Titles Full texts of eligible studies according to the above inclusion criteria were retrieved and carefully searched independently by authors MFV and CAM. For each text, study characteristic data was extracted and reported in Table The search strategy retrieved 6502 unique studies, whose title, keywords, and abstract if necessary were reviewed independently by MFV and CAM. A total of 120 abstracts were selected for analysis and were subsequently narrowed to 41 full-text articles (including 19 literature reviews). After comprehensive review, we found only 14 eligible studies from our search, with two additional studies retrieved from analyzing references of literature reviews. The final result was 16 eligible studies that met inclusion criteria , 18–28, Of the sixteen eligible studies in our meta-analysis, ten were published within the last decade, and of these only six –15, 20 pIn terms of glottic cancer staging and treatment, just seven studies reported use of a specific staging system, of which five studies , 25, 28 The majority of studies reported oncologic outcomes for T1a glottic cancer, with five studies , 21, 24 A methodological quality assessment of eligible studies was performed using a grading tool published by Oxford Centre for Evidence-based Medicine  as per pP < 0.1. Included studies are considered to have low heterogeneity (or be homogeneous) if I [P < 0.1) or I [z statistic with statistical significance set at P <0.05.Event numbers of oncologic outcomes from both TLM and RT treatment arms were pooled from eligible studies. Meta-analysis was performed using RevMan 5.3, an open-source statistical analysis software . Heterogeneity between studies was assessed via a chi-square analysis and the I  test, wius) if I  is less us) if I  is 25 toOncologic outcomes for included studies are reported as number of events with sample size for reference in Table 2 = 2.20, P = 0.97, I2 = 0%), and the fixed effect model was applied. The pooled analysis significantly favoured TLM for overall survival of T1 glottic cancer patients, with an OR of 1.52  and a Z score of 2.36 (P = 0.02).Of the sixteen studies included in our meta-analysis, ten , 25, 26 2 = 5.13, P = 0.82, I2 = 0%), and the fixed effect model was applied. Meta-analysis significantly favoured TLM for disease-specific survival in T1 glottic cancer, with an OR of 2.70  and a Z score of 2.71 (P = 0.007).Twelve of sixteen studies , 26, 28 2 = 11.22, P = 0.67, I2 = 0%), and the fixed effect model was applied. Meta-analysis significantly favoured laryngeal preservation with TLM, with an OR of 6.31  and a Z score of 7.00 (P < 0.00001).Fifteen of sixteen studies , 25–28 r2 = 22.76, P = 0.04, I2 = 43%), and the random effects model was applied. Meta-analysis demonstrated no difference in local control with TLM or RT as primary therapy, with an OR of 1.19  and a Z score of 0.84 (P = 0.40).Fourteen of sixteen studies , 25–28 rSince TLM was introduced in 1972 [Meta-analysis revealed the absence of heterogeneity in all oncologic outcomes with the exception of local control. This was mitigated by the use of a random effects model rather than a fixed effects model in our meta-analysis. To our knowledge we are the first systematic review comparing TLM and RT in early glottic cancer to demonstrate a significant improvement in disease-specific survival with TLM Fig. . In addiThere were discrepancies between our analysis of oncologic outcomes of TLM versus RT in early glottic cancer and those reported by earlier systematic reviews. As previously highlighted, this may be the result of inclusion of new literature or methodological differences. Thus, if we consider just the recent systematic reviews that analyzed two-armed studies , 6, 32, Inherently, the quality of a systematic review is limited by the quality of available literature. There were no available RCTs that met our inclusion criteria. All included studies were observational non-randomized cohort studies, the vast majority retrospective, with two prospective by design. Non-randomization may produce selection bias when allocating patients to treatment cohorts. Nine studies included in our review specified potential sources of selection bias within their methods Table . Most noVariability was also present in treatment administration. For instance, there was little consensus among reported ELS types of cordectomies, poor reporting on the use of specific cancer staging systems, and just four studies , 24, 25 The optimal therapy in early glottic cancer will ultimately have superior survival outcomes, functional outcomes, and favourable cost utility. Here we demonstrate that TLM has favourable survival outcomes, however we do not assess functional outcomes or cost utility. A previous randomized trial of 60 men with T1 disease found that RT had favourable voice-outcomes compared to TLM, including less-hoarseness-related inconvenience at 2 years follow-up . In contAdditional research, specifically prospective RCTs will be required to address these uncertainties. Unfortunately, performing an RCT that directly compares TLM and RT may be difficult due to a number of factors, including ethical considerations and inherent institutional bias. Several attempted RCTs investigating TLM vs RT have been abandoned due to poor patient recruitment . If RCTsOur systematic review and meta-analysis examined studies that directly compared oncologic outcomes in T1 glottic cancer with a primary treatment of TLM or RT. There were no eligible RCTs, and all studies included in our analysis were observational cohort studies, with a level II to level III evidence rating. In addition, there was some variability in treatment methods and non-randomized patient allocation to treatment groups, creating bias. As a result, these results should be interpreted with a degree of caution.Despite limitations, our findings demonstrated that TLM was associated with favourable outcomes in terms of overall survival, disease-specific survival and laryngeal preservation. There was no difference in associated local control between TLM and RT after the initial TLM procedure. The ability to salvage patients who recur after their initial TLM procedure with repeat TLM or radiation may explain the associated improvement in overall survival, disease specific survival and laryngeal preservation despite the associated equivalence in initial local control.Overall, our analysis has contributed to improving our understanding of optimal management in early glottic cancer. Determining optimal therapeutic management in glottic cancer must also consider the availability of the therapy, patient preference, cost utility, and inherent advantages of each modality. Ultimately, well-designed prospective and multicentre RCTs or studies using statistical modelling will be required to provide higher quality evidence in addressing the remaining uncertainties, corroborating the efficacy of TLM in early glottic cancer, and establishing clinical guidelines.