The objective of this article is to gain insight into how individuals who frequent open illicit drug scenes experience opioid maintenance treatment (OMT) and investigate how this appears to affect their recovery processes.By means of the ethnographic method, one of the researchers spent time in an open illicit drug scene over a 1-year span, and gathered data on individuals who frequent the scene on a regular basis, and their experiences with OMT. The data are based on field notes and audiotaped interviews.Four themes emerged as relevant for the participants’ experiences with OMT: 1) the loss of hope, 2) trapped in OMT, 3) substitution treatment is not enough, and 4) stigmatization of identity.The participants found the OMT to be overruling and degrading. Several of the individuals from the illicit drug scene are part of the OMT programme, but as the treatment does not remove painful emotions, they supplement OMT with illegal substances, violate the OMT regulations, and run the risk of being excluded from the programme. In fear of losing the replacement opioid, they conceal parts of the addiction they seek treatment for and end up lying and cheating instead of exploring strategies for reducing and managing the addiction. The patients’ relation to the OMT personnel is negatively affected by the need to hide a large portion of their issues. The result is a feeling of hopelessness, increased stigmatization, lack of control and being trapped between two worlds—in limbo, an intermediate state which interferes with the recovery process. In a study of people in an open illicit drug milieu, “the Bench”  carried out an evaluation of OMT. They found that there were large regional differences in practice, where practice often is more influenced by habits and ideology than actual research and also that there was too much focus on the medicinal side of treatment and less on rehabilitation Another tradition in drug treatment, which started in Britain in 1926, is harm reduction, the so-called “British system.” Heroin addiction was defined as a disease and legitimized the prescription of injectable heroin on a maintenance basis for people addicted to heroin  also for persons for being stigmatized and identified in an open drug scene. In a systematic review and modified narrative synthesis of the term recovery, five-core elements were identified as important to facilitate the recovery process: 1) empowerment and reclaiming control over one's life, personal ties, 2) hopes and beliefs for the future, 3) rebuilding positive personal identity and overcoming stigmas, 4) finding meaning in life, and 5) connectedness to adores  the loss of hope, 2) trapped in OMT, 3) substitution treatment is not enough, and 4) stigmatization of identity.I'm fed up. I'm just excited to get into OMT and start taking methadone and have a normal life and start working again. He is divorced from his wife but is very close to his two sons, who mean the world to him. The need for heroin and the struggle to finance his drug use have been interfering with their relationship, and this has been hard on him. Talking about the anticipated treatment brings a smile to his face.The leading motivation for joining the OMT programme and giving up heroin was that the individuals are worn out and fed up with their living conditions and the constant hunt for heroin, as well as longing for a “normal” life. “Arne” (40) has been using heroin since the age of 21 and has lately been taking a gram of heroin every day, just to stay “healthy,” as he puts it. When interviewed a few days before commencing OMT, he says: I'm hooked on methadone and everything else I can get hold of. This is going to kill me soon (“Arne”). In addition to the methadone, Arne is still using all the drugs he can get hold of. He is no longer expressing any hope for the future, he is staring at the ground with a blank expression on his face.Two years later, the researcher meets Arne again. He is wearing dirty clothes, has several smaller wounds and infections, and seems to be excessively intoxicated. He tells that he does not share his problems with anyone in the OMT programme, he just gets his daily dose, and leaves. I wanted a clear head and to live my life, I had dreams, wanted a family. I had a lot of goals … I then confronted my partner, hey, you're lying to me, I know what's going on, and that it's been going on for a long time. … I was so embarrassed, so ashamed.“Lene” (45) conveys a similar loss of hope. She joined the OMT programme with her partner. While hoping to get her life sorted, she discovered that her partner had “cracked,” taken up heroin, and put them in debt.Lene sought help from the OMT staff, but did not feel as though she received the understanding and support she needed. She talked to a social worker on the phone but was not offered any further assistance.Part of the discourse in the drug community focused on being worn out and tired of the constant hunt for heroin and risk of imprisonment. Most of the drug users in this study wanted a change for the better: a stable life with a job, a family, and of course a “clear head” free of intoxication, just like “Lene.” Even though they had struggled with abuse for years, and had seen OMT fail, they expected the methadone to straighten out their lives. When their expectations were not met, or they themselves did not manage to live up to their own expectations their hope was challenged. They then blamed OMT for loosing hope, and expressed that they felt betrayed because the OMT programme offered no assistance in maintaining the hope.I cracked and took pills when the methadone gave me cravings, and I wanted to tell them that they couldn't give me methadone …. I remember how similar it was to heroin, if I just take a little more it'll feel just like heroin. Today I want to take more to make it feel like heroin, why not? I feel like shit anyway. I stopped doing everything I used to do and sat around at home, so I ended up taking more methadone , hoping that it would give me the last finish. But it never did. It's not heroin, you know. (Lene)The participants we were in contact with had been using the replacement opioids methadone or Subutex, or both. When “Lene” began taking methadone, she started craving the substance, and bought pills and illegal methadone in addition to the dose she received from the OMT.Subutex makes you ill for 5 months. I'm not kidding—it's insane. If you want to quit, you can't curb it with any pill. There's nothing like it. It's a suffering that can't be described. (Leif)Expressed in the above quote, the replacement opioids initially give a high similar to that of heroin, but most users experience withdrawal symptoms if they try to quit. It is in fact so difficult to quit that the Norwegian Directorate of Health advise against it unless the person can manage without opioids. There is a high risk of relapse, overdose, and death when this is not the case During the course of the field study, the researcher met several people who used Subutex illegally as their main intoxicant, people who were not part of the OMT programme. To prevent the misuse and leakage of replacement opioids, strict control routines have been introduced for the consumption of opioids and additional drug abuse. One such measure is the regular submission of urine samples to reveal additional drug abuse. The samples must be submitted under supervision, an action which is perceived as a violation of privacy. “Helge” (47):I had been “clean” for a long time, and felt as though I was getting my life back on track. Then I took a little amphetamine, which showed up in the urine sample, and now they refuse to give me methadone. I don't understand the logic. The methadone is for heroin addiction, and I haven't taken any heroin. The methadone isn't for the amphetamine. (Field notes)“Helge” used heroin for many years but is now part of the OMT programme. The fact that he tries to laugh off the supervised urine submission can be seen as a reaction to a degrading situation, an attempt to maintain his dignity. The urine sample must be “clean,” or else he runs the risk of getting his methadone dose reduced or even kicked out of OMT, like “Reidar” (40) describes below:If Reidar is right, this is not entirely in keeping with the OMT regulations, which allow some abuse of illicit substances on the side. Nevertheless, there were several other participants with similar accounts, where what they regarded as “minor slips” brought on major consequences. It may thus seem as though the user's opinion is that OMT practice is stricter than the regulations allow.Some individuals still had positive experiences with OMT. According to “Nina” (28), she needed someone to supervise her in order to stay off heroin. However, she did not get a say in the size of the Subutex dosage. She received 16 mg Subutex and wanted 8 mg. She was also of the opinion that she could manage without Subutex, it was the aspect of control that kept her from relapsing. Nina needed someone to keep an eye on her. The human contact with someone who sees you was according to her the most important aspect.I don't experience any support in these meetings, I just get told off. It's a way of making me do what they want. I always bring my mother for support, but everyone doesn't have a mother to take with them. (Field notes)The national guidelines for OMT state that the service shall be coordinated between the different authorities the person is in contact with, such as NAV (the Norwegian Labour and Welfare Administration), outpatient clinics, etc. An important part of this is meetings about the patients individual plan. These meetings are meant to assist the patient, but many experience them as another form of control. “Rune” (52) started using heroin after a work place accident left him on disability benefits. He has been part of the OMT programme for several years but lives in constant fear of being kicked out of the programme. A few days prior to a group meeting, he relates that he always dreads these meetings.Rune explains that if he gets kicked out of OMT, he will have to spend his disability benefits on expensive illicit substances, as he does not think he can manage without. If this happens, he would risk missing the mortgage payments on his house and end up on the street. He therefore feels that OMT has great power over him.I've tried everything to get clean. Rehab institutions, OMT etc., but nothing works. I'm so fed up with the drugs, but I can't handle the OMT routines. There has to be something that works for me? Like in Switzerland, where they hand out heroin as part of the treatment. Then I could have done it.  (Field notes)The control and demand for abstinence from illicit substances cause some to give up and lose faith in the programme:Replacement opioids are addictive in the same way as heroin, but the withdrawal period is longer, which makes it harder to quit Another emerging theme was how the replacement opioids did not have the same effect as heroin. “Atle” (42) visits the OMT office a few times a week to pick up methadone. He keeps away from the drug scene and has never really identified with the community. He is freshly shaved, dressed in clean, modern clothes, and stands out from the regular crowd awaiting the distribution. He explains that he was depressed all throughout childhood and shows the scars on his arms from several suicide attempts. He started using hash, amphetamine, and cocaine as a way of self-medicating, but experienced terrible comedowns between the periods of intoxication and gained little from it. While in Oslo to buy drugs, he tried heroin for the first time:The methadone took away the cravings, but it didn't replace the heroin. I was constantly struggling with severe depression. (Atle)He used heroin every day for 15 years and financed the consumption by working overtime in his own firm. When the drug abuse was discovered, his wife separated from him and he lost touch with her and the children. He has now been clean for years and has started a new family. One year ago, he tried to resume his job but was unable to because of the painful emotions that returned when he quit heroin, and he now receives disability benefits.When you're on heroin, you get all “never mind”—properly “never mind”.Researcher: Can you get hooked on heroin after one shot?Leif: Nonsense, that's impossible (laughs). You're not hooked until the nerves kick in. When you wake up in the morning with your nerves on edge—then you're hooked. … You can do heroin for weeks without getting hooked, unless you have something bad in the back of your mind and the shot killed the bad thought. But if you've been fucked as a child and the shot killed the bad thought, you'll be hooked the next day, no question.Many of the people in the community described how nothing made you relax and indifferent to your surroundings like heroin. “Leif,” who has been using heroin for over 30 years, gives the following account:The participants have different explanations as to why heroin is so important to them, but a great deal of them use heroin as a coping strategy for painful, traumatic childhood memories, as well as a protection against the guilt they feel about their actions while intoxicated. It seems like heroin is good in cowering the underlying problems who rose to the surface when they stopped using heroin. The replacement opioids did not remove the painful emotions as successfully as the heroin did, and this led many to seek other ways of removing or reducing the pain.Suddenly I'm all alone, sober, and my children have been taken from me … The kids, the loneliness, the community that tends to keep its distance because you're a drug addict, that's when stuff happens. Exclusion. … You know what? I'll tell it from a different angle. What they  do is they lower your expectations to yourself. They don't think an addict can achieve anything, and so that's what happens, because everyone wants to be as comfortable as possible. (Nina)Other reasons for staying in the drug community include the loneliness and exclusion many face when attempting to leave and enter ordinary society. “Nina” has four children who are in foster care. She used heroin on occasion in her youth, but stopped when she got married. During her last child birth she was given morphine, which stirred up the old cravings. She then kept her drug use hidden for a long time. While experiencing heroin withdrawal, she was admitted to hospital with a suspected food poisoning, and the addiction was discovered. She was then introduced to OMT. Nina, who is in her mid-30s, describes what it was like to get “clean” with the OMT. She was involuntarily discharged from the programme and now uses any substance she can get hold of. She speaks of her time in the programme:When family and friends found out about her drug use and the OMT programme, the rumour spread quickly in the neighbourhood. The neighbours and the other mothers at the nursery stayed away from her, even when the children were still in her custody. Her adult siblings stopped giving her lifts, and no one would lend her money. She was no longer included, her surroundings stopped counting on her. This was frustrating to her, but it also allowed her to relax and slack off. The result was that her identity as a wife, neighbour, sister, and mother was exchanged for the identity of a drug addict, and she felt rejected from the community she used to be part of.I think it's because everything is so levelled (when using heroin). We didn't care about anything. And then there's the feeling of being included, you had an identity and respect. When you enter the other life, you're nobody. At first people pat you on the back, but it goes away despite that I've said I need to be seen, or else I'll be lost. I've done it before. If you haven't found your identity, you have to search for it. I'm still searching, I think. Even after being clean for several years, the reactions from the surroundings can complicate the establishment of a new identity. The person quoted below has not been involved with illicit substances for 10 years. She nonetheless has to submit weekly urine samples and pick up her replacement opioids. This is her account of the transition from heroin to OMT:someone. When she joined the OMT programme, she felt as though the things about her that were appreciated in the drug community lost their value, and she became no one. To be no one was comfortable as it involved lower expectations, but also dangerous because it left her vulnerable. She needed to be supervised by someone in OMT, to be supported, and cheered on in order to refrain from slipping back to the drug scene, as she had done on previous occasions.When she was on heroin, she didn't care about the reactions from the surroundings, because she was a part of the drug scene where she felt respected and included. She had an identity, she was no one and were left vulnerable and at risk of seeking the communities where they were recognized and felt a sense of belonging.Individuals who join the OMT programme have been using heroin for a long time, and the surroundings have assigned them a stigmatized identity with negative qualities that overshadow all the other qualities which also define the person (master status). They also lower their expectations to themselves, a kind of self-stigmatization. This is what “Nina” experienced. Although many of the participants described the drug scene as rotten and deceitful, they still felt included, respected, and to a certain degree appreciated. In ordinary society, they became The dream is at risk of being shattered when the ex-addict tries to enter the ordinary world . Weingarten Most of the participants were motivated to join OMT by a combination of being tired of life on the drug scene with its constant hunt for heroin, and wanting a normal life with a home, a job, and a family. Even individuals well into their 40s, who had been addicts almost half their lives, hoped for a normal life when they started on methadone. This hope of a “normal” life appears unrealistic, as the hope has been shattered several times before, and as it is a known fact that integration in the greater society is challenging for people with an extensive substance abuse and connection to the drug scene. Even so, it seems that one clings to the hope of a better life no matter how unrealistic it may be. In an ethnographic study of the drug scene in Malmö, Sweden, Svensson  relates:ingarten  stated tWhen the replacement opioids did not have the anticipated effect, they compensated with illicit substances, which is in line with the research of Sælør, Ness, Holgersen, and Davidson . It is aWhen the replacement opioids did not have the effect the participants hoped for, and OMT did not provide sufficient support and help to handle the transition to “normal” life, the hope was lost. And when hope is the main catalyst of the recovery process  which reduces the pains incurred in meeting the demands of a powerful other. (p. 35)Quite a few of the individuals we were in contact with used the replacement opioids to keep the withdrawal symptoms in check, and further supplemented with other remedies/substances to endure painful emotions. It is well known that depression among heroin addicts in OMT is highly prevalent  would only be a subject or a way into a conversation about what it is all about.


This study assessed the long-term cost-effectiveness of saxagliptin+metformin (SAXA+MET) versus acarbose+metformin (ACAR+MET) in Chinese patients with type 2 diabetes mellitus (T2DM) inadequately controlled on MET alone.Systematic literature reviews were performed to identify studies directly comparing SAXA+MET versus ACAR+MET, and to obtain diabetes-related events costs which were modified by hospital surveys. A Cardiff Diabetes Model was used to estimate the long-term economic and health treatment consequences in patients with T2DM. Costs (2014 Chinese yuan) were calculated from the payer’s perspective and estimated over a patient’s lifetime.SAXA+MET predicted lower incidences of most cardiovascular events, hypoglycemia events and fatal events, and decreased total costs compared with ACAR+MET. For an individual patient, the quality-adjusted life-years (QALYs) gained with SAXA+MET was 0.48 more than ACAR+MET at a cost saving of ¥18,736, which resulted in a cost saving of ¥38,640 per QALY gained for SAXA+MET versus ACAR+MET. Results were robust across various univariate and probabilistic sensitivity analyses.SAXA+MET is a cost-effective treatment alternative compared with ACAR+MET for patients with T2DM in China, with a little QALYs gain and lower costs. SAXA is an effective, well-tolerated drug with a low incidence of adverse events and ease of administration; it is anticipated to be an effective second-line therapy for T2DM treatment. Diabetes mellitus is a huge and growing health problem in the 21st century, and the costs to healthcare systems and society are high and escalating. It is estimated by the International Diabetes Federation that 387 million people worldwide had diabetes in 2014; by 2035, this number is expected to rise to 592 million [In light of poor management and high numbers of diabetes-related deaths, a series of antidiabetic drugs have been successively introduced into the Chinese pharmaceutical market in recent decades. Despite the proven efficacy of these antidiabetic drugs, their nonnegligible adverse effects  –6 have iPatient compliance is a major problem in T2DM treatment. Negligible or suboptimal adherence to diabetes medications is associated with poor blood glucose control, increased risk of hospitalization, and mortality –16. ConvAcarbose (ACAR) and saxagliptin (SAXA) are both recommended as second-line therapies for T2DM treatment in China . ACAR, aSaxagliptin is a new dipeptidyl peptidase-4 inhibitor (DPP-4i) that inhibits the breakdown of the incretin hormones GLP-1 and glucose-dependent insulinotropic polypeptide, resulting in increased glucose-dependent insulin secretion and suppression of glucagon secretion . As monoChoice of antidiabetic drugs should evaluate individual patient characteristics, preferences, and values and balance the need to optimize blood glucose control with the need to minimize adverse events . As an eTo our knowledge, there are no Chinese or international studies that directly compare both long-term benefit and cost aspects of SAXA with ACAR as second-line add-on therapy to MET, and there are only few short-term head-to-head Chinese clinical trials comparing the efficacy of SAXA with ACAR as an add-on to MET identified. From the health insurance payer’s perspective, this study aims to estimate the long-term cost-effectiveness of SAXA+MET compared with ACAR+MET in T2DM patients with glucose inadequately controlled on MET alone in China.We used a previously published and validated simulation model, the Cardiff Diabetes Model, which is designed to estimate the long-term economic and health impact of comparable medical therapies in patients with diabetes –36. The Hypoglycemia and other adverse events were modeled depending on therapy-specific incidence rates. Hypoglycemia was separated into symptomatic and severe events in the model, and severe events  were correlated with healthcare costs ,40. The A literature review was conducted to evaluate the arts of the disease state and to collect patient profiles, clinical data for each target treatment, and relevant costs for diabetes-related events. A series of English-language databases  and Chinese-language databases  were systematically searched for relevant studies dating from "2009/01/01" to "2016/09/30" (Date of first authorization of saxagliptin in USA: 1st October 2009)  that proSearch terms included “saxagliptin”, “Onglyza”, “acarbose” or “Glucobay”, in combination with “type 2 diabetes”, “non-insulin-dependent diabetes mellitus” or “T2DM”, and “Chinese” or “China”  or ACAR+MET (control arm), and when patients in either arm failed to reach target HbA1c level, therapy escalation for insulin therapy would take place. In accordance with 2013 clinical guidelines from the Chinese Diabetes Society, the HbA1c threshold values for a switch in medication were defined as 7.0% . An alteThe clinical effects evaluated included treatment-induced impacts on HbA1c, body weight, SBP and cholesterol; and rates of adverse events, including hypoglycemia and GI adverse events, were also evaluated for each arm. These data were obtained from meta-analysis of the five head-to-head studies ,41–44. HInput costs were those related to drug acquisitions, diabetes-related complications, adverse events, and body weight changes. From the payer’s perspective, only country-specific direct medical costs were considered in this study, and all costs were inflated to 2014 values (Chinese yuan) using the Chinese Consumer Price Index .Annual treatment costs of target drugs were calculated using the highest retail price from the most recent official drug price list –50 and dCosts associated with diabetes-related complications were split into fatal or nonfatal costs, which were applied in the cycle in which the event occurred. For those surviving the event, maintenance costs were applied in all subsequent years until patient death or the simulation ends. The costs were estimated based largely on Gao et al ; when thThe treatment cost of severe hypoglycemia (¥3829.96) was abstracted from Zheng et al, which investigated direct medical costs for episodes of hypoglycemia in China ; treatmeBecause there were no country-specific utility decrements for diabetes-related events in China, we mainly adopted data from the UKPDS 62 study , excludiThe impacts of uncertainty and variability around the model inputs were tested by both a series of univariate sensitivity analyses and a probabilistic sensitivity analysis (PSA). Various assumptions about parameters, including baseline demographics, costs and utility decrements associated with diabetes-related complications, body weight changes and other variables, were assessed in univariate sensitivity analyses. All sensitivity analyses were conducted for 1000 patients over 40 years, and a scatter plot of the incremental cost-effectiveness ratios (ICERs) and a cost-effectiveness acceptability curve (CEAC) were generated in the PSA.In the base case analysis, the SAXA+MET cohort predicted lower incidences of most cardiovascular events, hypoglycemia events and fatal events as compared with that of the ACAR+MET cohort. Consistent with the differences in cases of diabetes-related events, the costs for most of these events were lower in SAXA+MET than that of ACAR+MET, except for congestive heart failure, stroke and nephropathy which were lower in ACAR+MET. Although drug treatment costs were higher in SAXA+MET, this disadvantage was offset by its much lower BMI-related prescription costs and hypoglycemia costs as compared with ACAR+MET. Overall, SAXA+MET was associated with lower total costs than that of ACAR+MET . The timFor an individual patient, the total discounted costs accumulated over the lifetime on SAXA+MET was ¥18,736 lower than ACAR+MET; but the QALYs gained with SAXA+MET was 0.48 more than ACAR+MET. This resulted in a cost saving of ¥38,640 per QALY gained with SAXA+MET , which indicated that SAXA+MET would lead to better utility and decreased costs for patients .In the sensitivity analyses, an initial tornado model, run to explore factors with the greatest influence on the cost-effectiveness results, suggested the primary importance of BMI, HbA1c and utility . SubsequWhen the baseline HbA1c was decreased by 20%, the reported ICER was −¥67,630/QALY, showing that SAXA+MET still dominant over ACAR+MET. Alternative HbA1c threshold value for therapy switch was also investigated, reporting an ICER of −¥60,662/QALY. Further, utility decrement and cost associated with per unit BMI gain were varied. In the scenario in which the utility decrement per unit BMI gain halved, the incremental QALYs decreased from 0.48 to 0.27, but SAXA+MET kept dominant over ACAR+MET with a reported ICER of −¥69,725/QALY. In the scenario in which an alternative utility decrement per unit BMI change  was used, SAXA+MET was still cost-effective compared with ACAR+MET, reporting an ICER of −¥105,897/QALY which was 174% higher than that of base case . As expected, a decrease in BMI-related prescription costs would have a negative effect on ICER. When the BMI-related prescription cost was halved, the incremental cost saving decreased from ¥18,736 to ¥8,762, with an ICER of −¥18,070/QALY. Only when the BMI-related prescription cost was excluded from the model, the converse occurred: SAXA+MET cost ¥1,212 more than ACAR+MET with an ICER of ¥2,500/QALY, within the acceptable range of 2014 GDP per capita of China  . Table .Drug treatment costs played an important role in the total costs of both arms in the base case analysis and further influenced ICER result. Scenarios analyses demonstrated that either by adjusting the annual treatment cost of SAXA or ACAR , the cost saving increased from ¥38,640/QALY to ¥42,381/QALY, ¥45,995/QALY or ¥44,219/QALY gained with SAXA+MET, respectively; SAXA+MET gained more dominance over ACAR+MET as compared with that of the base case. GI adverse events and hypoglycemia were commonly observed in the treatment of T2DM, which might have an effect on both cost and utility. Alternative treatment costs of GI events or hypoglycemia in the sensitivity analyses, resulting in a little changes in cost saving gained by SAXA+MET compared to that of base case. When GI adverse events in ACAR+MET doubled or hypoglycemia of SAXA+MET equal to ACAR+MET, the incremental QALYs gained by SAXA+MET increased from 0.48 to 0.49. Alternative annual discount rate for costs and benefits and alternative costs of diabetes-related complications also had some influences on the magnitude of the cost-effectiveness results but did not change the results; SAXA+MET kept dominant over ACAR+MET with better utility and lower cost .In the PSA, the incremental QALYs gained for SAXA+MET versus ACAR+MET was lower than that of the base case analysis (0.47 versus 0.48); but the incremental cost saving was higher than that of the base case . Thus reporting a cost saving of ¥46,815/QALY gained for SAXA+MET versus ACAR+MET, higher than that of the base case. This is the first economic evaluation study using the Cardiff Diabetes Model to determine the long-term economic and health impact of SAXA versus ACAR as add-on therapy to MET for Chinese patients with T2DM who were inadequately controlled following MET monotherapy. The results indicated that the combination therapy of SAXA+MET was dominant over ACAR+MET, with a little QALYs gain and lower costs. The results remained consistent under a series of assumptions in the sensitivity analyses.Because there is a lack of published studies estimating either the cost or benefit of SAXA compared with ACAR in treating T2DM, we reviewed available literature on the topic of DPP-4i drugs versus ACAR, regardless of the ongoing dearth of cost-effectiveness studies. A 24-week, multi-center, double-blind, randomized trial comparing the DPP-4i vildagliptin (100mg/d) with ACAR (up to 300mg/d) monotherapy in patients with T2DM demonstrated that vildagliptin had similar glycemic efficacy to ACAR but with fewer GI adverse events and better tolerability . BecauseA patient-centered treatment approach for T2DM should take into account the individual patient’s characteristics, preferences and values and balance the need to optimize efficacy with the need to minimize adverse events . In thisMedication nonadherence or suboptimal adherence is one of the leading public health challenges, particularly in the case of medications for chronic diseases like diabetes –69. NonaWith the growth of national economies and concomitant increase in income levels, people are more focused on QOL and may be willing to spend more money for convenient drug administration and reduced drug adverse effects. Although the use of SAXA is somewhat limited by its slightly high cost ,6, our sSAXA is a well-tolerated drug that effectively controls blood glucose levels and has a low risk of hypoglycemia, weight gain, and GI adverse events, making it possible to increase patient QOL and allow for better medication adherence ,28–31. GThere were several limitations to our study. First, there is a paucity of long-term follow-up data from well-designed clinical or epidemiologic studies directly comparing the treatment effects of SAXA+MET versus ACAR+MET in patients with T2DM in China; only five short-term head-to-head trials on this topic was identified. We therefore used the aforementioned trials to project long-term outcomes of both treatments using UKPDS 68 equations, which might cause uncertainty in the input parameters and bias to real-world settings. Second, only direct medical costs were investigated in our study, which neglected the considerable indirect costs of diabetes-related events  on productivity. Moreover, total costs in the ACAR+MET cohort were underestimated because of the unavailability of expenses related to GI adverse events, which may have undermined the comparability of the treatment arms to some extent. Third, because there were no country-specific utilities for diabetes-related complications and adverse events in China, utilities from the UKPDS 62 and other published studies of foreign populations were used, which might introduce a bias.This study showed from a payer’s perspective that SAXA+MET is a cost-effective treatment alternative compared with ACAR+MET for patients with T2DM inadequately controlled on MET monotherapy in China, with a little QALYs gain and lower costs. SAXA is an effective, well-tolerated drug with a low incidence of adverse events and ease of administration. It is anticipated to favor patients who wish to avoid the treatment-induced adverse events and inconvenient administration of other antidiabetic drugs, with the goal of improving patient with effective second-line therapy for T2DM treatment.S1 Appendix(PDF)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Diabetes is one of the most common chronic and costly diseases worldwide and type 2 diabetes is the most common type which accounts for about 90% of cases with diabetes. New medication-therapy regimens such as those containing linagliptin alone or in combination with other medications (within the category of DDP-4 inhibitors) must be evaluated in terms of efficacy and compared with other currently used drugs and then enter the medication list of the country. Hence, this study aimed to compare the clinical efficacy of the two drugs, i.e. linagliptin and sitagliptin, in patients with type 2 diabetes.A systematic review was conducted to identify all clinical trials published by 2015 which compared the two drugs in patients with type 2 diabetes. Using keywords such as “linagliptin”, “type 2 diabetes mellitus”, “sitagliptin” and related combinations, we searched databases including Scopus, PubMed, and Web of Science. The quality of the selected studies was evaluated using the Jadad score. Considering primary and secondary outcomes extracted from the reviewed studies, a network meta-analysis was used to conduct a systematic comparison between the two studied drugs.n = 8, Sitagliptin vs PLB: n = 13, Linagliptin + MET vs PLB + MET: n = 4, and Sitagliptin + MET vs PLB + MET: n = 7) and a total of 13,747 patients. The results showed no significant difference between linagliptin and sitagliptin in terms of key efficacy and safety outcomes such as HbA1c changes from baseline, body weight change from baseline, percentage of patients achieving HbA1c <7, and percentage of patients experiencing hypoglycemic events (p > 0.05). The results showed that the efficacy of the two drug regimens was the same.This network meta-analysis included 32 studies  contains supplementary material, which is available to authorized users. The incidence and prevalence of diabetes has been rapidly increasing in the last century and morbidity and mortality from these two pandemic diseases have caused massive problems for the health of human communities , 2. DiabAmong different types of diabetes, type 2 diabetes is the most common type which accounts for about 90% of the cases . AlthougDiabetes has many complications which can severely affect the quality of life of the patients and impose a high economic burden on individuals and community , 10.Among available diabetes treatments, DPP4 inhibitors are one of the new generation classes with good efficacy and safety profile –13. As tAlso, According to published clinical studies, linagliptin in this class seems to be more advantageous for patients with renal insufficiency . Using eIn order to evaluate the efficacy of the two drugs, linagliptin and sitagliptin, we conducted a systematic review of the studies published by the end of 2015. Using keywords such as “linagliptin”, “type 2 diabetes mellitus”, “sitagliptin” and related combinations, we searched databases including Scopus, PubMed, and Web of Science , and had the same methodology were entered into the network meta-analysis.After searching and investigating the mentioned databases, we did not find any study which directly compared the two drugs; hence, we decided to find and review clinical trials which investigated DPP-4 drugs, extract the data on their efficiency, and compare the data extracted from independent studies. Therefore, in order to integrate the results of the studies, we used network meta-analysis. To carry out the analysis, we used STATA and Excel softwares.Thus, as noted above, in this study first we used a systematic review approach to collect studies on the efficiency of the two drugs, i.e. sitagliptin and linagliptin, which are among DPP-4 category. Then, using network meta-analysis, we analyzed the data and compared the efficacy of the drugs. In this study, we not only evaluated the efficacy of the monotherapy using each of the drugs, but also assessed the efficacy of combination therapy using the drugs together with metformin. However, due to the lack of sufficient studies, we did not assess combination therapy with other drugs, such as sulfonylureas, insulin, and glitazones.Moreover, in order to include a study in meta-analysis, it had to meet the following PICO criteria:P (Population): Patients with type 2 diabetes.I (Intervention): linagliptin.C (Comparators): sitagliptin.O (Outcomes): The desired outcomes were: “HbA1c change from baseline”, “Percentage of patients achieving HbA1c < 7%”, “Percentage of patients experiencing hypoglycemic events”, and “Body weight change from baseline”.To carry out this network meta-analysis, we analyzed the data using random effect approach. In this approach, first the researcher conducted a meta-analysis of the existing comparisons and combined the results via indirect meta-analysis and finally made its own comparison.Lina,PLB) and PLB relative to sita :For calculating indirect effect, Bucher et al. method was used . In thisLina,Sita is the sum of the direct estimators variances:The indirect estimator variance of EffectIn this study, two tables were provided for every outcome. In the first table, it was considered that the monotherapy and combination therapy studies were not similar, while in the second table the effects of monotherapy and combination therapy were considered to be similar. For each parameter in the first table, we combined the results of the meta-analysis of the existing combinations and indirectly compared linagliptin 5 mg with sitagliptin 100 mg and also compared LIN 5 mg + MET with SIT 100 mg + MET. In the second table, it was assumed that the results of comparisons between drugs with and without MET were the same; thus, we compared linagliptin 5 mg (with and without MET) with sitagliptin 100 mg (with and without MET). For the first two outcomes, we compared the changes from the baseline and for the second two outcomes we compared the Log of odds ratios.After searching the electronic databases and reviewing the references of articles, a total of 3711 articles was obtained. All the obtained articles were carefully assessed to find the articles which met the intended criteria and finally a total of 32 articles which had an acceptable level of quality were selected for the meta-analysis. Table Network meta-analysis was used to compare different groups in terms of HbA1c changes from baseline, body weight change from baseline, percentage of patients achieving HbA1c <7, and percentage of patients experiencing hypoglycemic events. Figure Based on the results of the meta-analysis presented in Table Table Then, the results of reported meta-analyses were combined and an indirect comparison showed no significant difference between linagliptin and sitagliptin groups in terms of body weight changes from baseline (comparison 5). However, it showed a significant difference in the LIN 5 mg + MET group as compared with the SIT 100 mg + MET groups, so that LIN 5 mg + MET decreased body weight by 2.288 units, as compared with SIT 100 mg + MET (comparison 6).As shown in Table As shown in Table As shown in Table Comparing the groups in terms of body weight changes from baseline showed that there was no significant difference between the linagliptin and sitagliptin groups (with and without MET), as compared with the placebo group (with and without MET) (comparisons 7 and 8). In addition, finally, there was no significant difference between the linagliptin group (with and without MET), as compared with the sitagliptin group (with and without MET) in terms of body weight changes from baseline (comparison 9).Comparing the groups in terms of the percentage of HbA1c <7 revealed that there was a significant difference between the linagliptin and sitagliptin groups (with and without MET), as compared with the placebo group (with and without MET) as the odds ratio of HbA1c in the linagliptin and sitagliptin groups, respectively, was 2.03 and 1.67, as compared with the placebo group (comparisons 7 and 8). However, finally, there was no significant difference between the linagliptin group (with and without MET), as compared with the sitagliptin group (with and without MET) (comparison 9).Comparing the groups in terms of hypoglycemic events showed that there was a significant difference between the linagliptin and sitagliptin groups (with and without MET), as compared with the placebo group (with and without MET) as the odds ratio of hypoglycemic events in the linagliptin and sitagliptin groups, respectively, was 0.28 and 0.47, as compared with the placebo group (comparisons 7 and 8). However, there was no significant difference between the linagliptin group (with and without MET), as compared with the sitagliptin group (with and without MET) (comparison 9).Using meta-analyses approach, we performed a direct-comparison between the drugs and placebo that the results were different. The results of the meta-analysis showed a significant difference in HbA1c changes from baseline in the linagliptin group and sitagliptin group (with and without MET), as compared with the placebo group, because HbA1c was reduced by these drugs, as compared with the placebo. It is in the same line with the results of the study of Gross JL et al.  However,Considering body weight changes from baseline revealed that there was no significant difference between the linagliptin and placebo groups and between the sitagliptin groups (with and without MET) and placebo group. However, it showed a significant difference in the LIN 5 mg + MET group, as compared with the PLB + MET group, so that the drug reduced body weight, compared with the PLB + MET group.As to the percentage of patients achieving HbA1c <7, it significantly increased in the linagliptin group, as compared with the placebo group; the odds ratio of HbA1c <7 in this group was two times more than that of the placebo group. However, there was no significant difference in the LIN 5 mg + MET and sitagliptin groups (with and without MET), as compared with the placebo group. Moreover, as to the percentage of hypoglycemic events, there was a significant difference in the linagliptin (with and without MET) and sitagliptin groups, as compared with the placebo group. The odds ratio of hypoglycemic events in the two groups was less than that in the placebo group. Nevertheless, there was no significant difference in the SIT 100 mg + MET group, as compared with PLB + MET group.The results of network meta-analysis showed no significant difference in HbA1c changes from baseline, body weight changes from baseline, percentage of HbA1c <7, and percentage of hypoglycemic events in the linagliptin group (with and without MET), as compared with the sitagliptin group (with and without MET); thus, the efficiency of the two drugs are identical. Therefore, the results of this study are consistent with those of the studies conducted in other countries –22.As noted in the results, in certain cases linagliptin has a relative advantage in pharmacokinetic superiority over sitagliptin; thus, it can be considered as a suitable alternative. Non-renal clearance is one of the pharmacokinetic features of linagliptin which makes it different from other gliptins available on the market. Therefore, its use in patients with renal insufficiency is safe and does not have any restriction . TherefoAlthough the efficacy of linagliptin and sitagliptin is the same, because the use of linagliptin has no restrictions in patients with renal dysfunction, and this drug is a better alternative for patients with renal dysfunction, it is necessary to make it accessible in Iran pharmaceutical market along with sitagliptin so that the physicians have an opportunity to prescribe thesedrugs for patients with different background diseases.The results of meta-analysis can be different from those of indirect comparisons; however, we should be more cautious when interpreting the results of indirect comparisons. Systematic reviews and evaluation of the quality of clinical trials can help reduce the errors in the results of network meta-analysis but when there is a difference between the results of direct-comparison meta-analysis and indirect meta-analysis, it is necessary to re-examine the validity and generalizability of clinical trials to find the reason for the error. On the other hand, recent experimental results indicate the match between these two types of comparisons . When usThe results showed no significant difference between linagliptin and sitagliptin in terms of clinical efficacy and they had the same effect. However, as there is no restriction on the use of linagliptin in patients with renal dysfunction, it might be considered as a treatment of choice. Hence, it is recommended to include this drug, along with sitagliptin, in the list of pharmaceuticals in Iran.


Dental demineralization was determined in patients at three time points during fixed orthodontic treatment. A multiple cross-sectional study included 108 patients divided into three different groups: (1) beginning of orthodontic treatment; (2) one year into treatment; and (3) two years into treatment. Demineralization was estimated using a DIAGNOdent pen. We obtained data from multiple tooth-by-tooth demineralization readings combined with salivary pH and patients’ oral hygienic and dietary behaviors. A t-test for independent samples and Spearman´s correlation were performed. No demineralizations differences were found between the initial stage and one year into treatment. Between one and two years small differences were observed, but demineralization increased between the initial stage and second treatment year, predominating in upper right central incisors (p = 0.056), upper left lateral incisors (p = 0.040), both upper canines  and first left premolars . We did not find associations between oral hygiene and dietary behaviours or salivary pH. In conclusion, demineralization occurred in first year of treatment and increased during second year, predominating in the upper arch and the left side mainly in upper right central incisors, upper left lateral incisors, both upper canines, and first left premolars. Orthodontic treatment is an elective procedure to improve the patient’s dentofacial appearance. A major barrier in achieving this goal is the risk to develop demineralization areas adjacent to orthodontic brackets. There is a well-established relationship between orthodontic fixed appliance use and enamel demineralization. The occurrence of enamel demineralization is a significant problem related to fixed orthodontic appliances. Demineralization varies from no perceptible change to white spots on the enamel, which is the first clinical sign of enamel demineralization, to cavitation. These areas may require further treatment after orthodontic therapy to mask or remove these spots. Many prior studies have reported that patients who use brackets have up to 85% risk of developing white spot lesionsIt has been shown that orthodontic treatment induces changes in the oral environment such as an increase in bacterial concentrations and alterations in pH, salivary buffering capacity, and salivary flow. Furthermore, all of these changes are closely associated with hygienic and dietary behaviours of the patient3The placement of orthodontic appliances increases retention areas, and irregular surfaces of brackets and bands provide protection to microorganisms from physical forces. Therefore, cleaning becomes more complicated, which favours plaque accumulation3Dietary behaviours significantly influence demineralization; if fermentable substances are continuously ingested, acid production by plaque exceeds neutralization by the saliva, and demineralization occurset al. showed that the correlation between DIAGNOdent and visual examination was 0.63 and reported that DIAGNOdent was a more objective and reproducible method than visual examination for detecting and long-term follow up of incipient carious lesions18There are technologies to help in early demineralization detection; one device is DIAGNOdent89109131415The aim of the present study was to determine the level of demineralization in patients during orthodontic treatment and its association with salivary pH and hygienic and dietary factors.This was a multiple cross-sectional study in 108 patients in three different stages of orthodontic treatment. Patients were in each group (n = 36): (1) at the beginning of treatment; (2) one year into treatment; and (3) two years into treatment. We did not use any sampling approach (convenience sampling). The patients in the first stage were free of caries lesions, since those lesions are one of the prerequisites for entry into the orthodontic treatment. The study was conducted in patients from the Orthodontic Clinic at the Research Center and Advanced Studies in Dentistry of the Autonomous University of State of Mexico (UAEM). All patients or their guardians consented to participation after obtaining information about the study.We also administered a face-to-face questionnaire in order to collect data regarding patients’ sociodemographic characteristics as well as their hygienic and dietary behaviours. This questionnaire was given to all patients and consisted of with nine closed questions: (1) gender; (2) occupation; (3) brushing time; (4) frequency of daily brushing; (5) use of dental auxiliaries; (6) frequency of changing toothbrush; (7) snacks between meals; (8) snacking time; and (9) frequency of daily snacks.Clinical data were taken from the patient while he/she was sitting on a dental chair under artificial light with all necessary infection control barriers. Demineralization was estimated using a DIAGNOdent pen 2190 . This device was previously calibrated, according to the manufacturer’s instructions. The results are shown as readings from 0 to 99 and appear on the digital display located on the back of the device. According to the manufacturer, 0–12 is considered low risk, medium is 13–24, and >25 is high for smooth surfaces; for proximal areas, 0–7 is low, medium is 8–15, and >16 is high.We analyzed six points on each tooth consisting of four points on buccal surfaces  and two points on interproximal areas  from premolar to premolar in both dental arches. In our study, we obtained the average of six measurements , because orthodontic appliances consisting of brackets; elastic chains, springs, and archwires and are continuous devices that prevent proper oral hygiene by the patient. These devices prevent the toothbrush bristles from reaching between the interproximal spaces. For this reason is important to include the degree of mineralization of these areas in order to have a representative measure of the condition of the entire tooth.The same DIAGNOdent pen was used throughout the study, and one examiner performed the measurements. The surface was dried with compressed air for 5 sec before each measurement. Each point was scanned two times with the pen, and the highest value from the two readings was registered. When all measurements were obtained, we then averaged the values to find a total value by each tooth. Subsequently, an average of demineralization values  by stage was obtained (at the beginning and at one and two years).The salivary pH was calculated using test strips , which were placed under the patient’s tongue for two minutes. For pH measurements, patients were requested not to eat for two hours before taking the sample at the same time of day, just before starting their orthodontic consultation, because ingested food, as well as the consumption of a drink and/or toothbrushing, can modify the salivary pH. For the collection of these samples, all participants were requested to be as quiet as possible. The test strips were immediately compared with a sampling table to determine degree of acidity or alkalinity.The information collected from questionnaires and clinical examination was analysed with SPSS version 19. We performed a descriptive analysis of the population by using central tendency and dispersion measurements for the quantitative variables and frequency and percentages for the qualitative variables. Subsequently, bivariate analyses were performed to determine if there was any difference using a t-test for independent samples and Spearman´s correlation. The statistical level of significance was set at 0.05.Written consent was obtained from all the patients/guardians. The protocol was approved by the ethics committee review board of the Research Center and Advanced Studies in Dentistry of the Autonomous University of State of Mexico. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Similarly, age, salivary pH, and total demineralization by each stage were correlated by Spearman’s analysis and no statistically significant differences between these parameters were found (p > 0.05) .The results of this study indicated that demineralization may be a significant problem during orthodontic treatment, increasing from the initiation of orthodontic treatment to one year and continuing to increase into the second year of treatment; a patient´s oral hygiene and dietary behaviours seemed to be irrelevant to the degree of demineralization.et al. reported that during orthodontic treatment the number of demineralization injuries increased significantly during the first six months and continued to increase up to 12 monthsTufekci In the study groups, demineralization predominated in the upper arch on the left side. About 90% of our population is right-handed. Claydon reported that in populations with poor oral hygiene, the most significant loss of the periodontal attachment is in the maxillary archIn this study, the teeth most affected by demineralization were the upper left central incisors, upper right lateral incisors, both upper canines, and first left premolars. The primary etiology of demineralization is bacterial acids, which may affect sites that are less protected by saliva such as the anterior teethet al. reported that the central and lateral incisors are the teeth most frequently affectedet al. reported that the first premolars are the teeth most affected by demineralization during orthodontic treatment. In our research, the left first premolars were presentedEnaia It has been reported that a majority of interproximal lesions are incipient, thus making clinical diagnosis difficult. It appears to be necessary to use other diagnostic tools in conjunction with clinical diagnosis.Literature studies report that salivary pH increases after placement of the orthodontic appliancesThe present study has some limitations, therefore its interpretation should be cautious and its design  was affected by temporal ambiguity. The cause and effect were measured at the same time. For that reason, this study precludes confirming causal relationships but favours associations only. In conclusion, demineralization occurs in first year of treatment and increases during the second year. The results obtained in this study allowed us to identify the teeth most frequently undergoing demineralisation. Predominance of demineralization was seen in the upper arch and on the left side, mainly affecting upper right central incisor, upper left lateral incisors, both upper canines, and first left premolars. Oral hygiene habits, dietary behaviours, and salivary pH did not show any significant associations with total demineralization. Demineralization patterns were the same even when the dietary and hygienic behaviours differed. It is important to implement specific preventive measures in patients who are undergoing orthodontic treatment.How to cite this article: Salmerón-Valdés, E. N. et al. Tooth demineralization and associated factors in patients on fixed orthodontic treatment. Sci. Rep.6, 36383; doi: 10.1038/srep36383 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Echinacea purpurea (L.) Moench and its endophytic communities on the production of alkamides is investigated. To mimic the in vivo conditions, we have set up an infection model of axenic in vitro E. purpurea plants inoculated with a pool of bacterial strains isolated from the E. purpurea stems and leaves. Here we show different alkamide levels between control (not-inoculated) and inoculated plants, suggesting that the alkamide biosynthesis may be modulated by the bacterial infection. Then, we have analysed the branched-chain amino acids (BCCA) decarboxylase gene  expression patterns. The expression profile shows a higher expression level in the inoculated E. purpurea tissues than in the control ones. These results suggest that the plant-endophyte interaction can influence plant secondary metabolism affecting the therapeutic properties of E. purpurea.The influence of the interaction(s) between the medicinal plant  Echinacea purpurea (L.) Moench (Asteraceae) is a medicinal plant with immune-modulatory and anti-inflammatory properties, whose roots and aerial parts are frequently used in Europe and North America for the preparation of therapeutic extracts for common cold1. It is rich in various phytochemicals including caffeic acid derivatives, alkamides and polysaccharides2. The concentrations of these bioactive compounds are species-specific and they may vary due to several factors such as plant material, cultivation techniques, plant tissue treatment, extraction methods and phytosanitary status3. Recently, the attention has been focused on the plant microbiota and its role in the production of secondary metabolites5. Many studies aim to investigate the influence of endophytic fungi on the production of plant bioactive molecules7 but the interest for the bacterial endophytes is considerably increasing8. Genomics and proteomics approaches have been applied to deepen the understanding of the plant-endophyte interaction9. Differential protein accumulations have been revealed in the proteome of in vitro-grown Zea mays10 and Chinese hybrid poplar clone 74111 inoculated or not-inoculated with Herbaspirillum seropedicae and Paenibacillus sp., respectively. In particular, the role of the endophytes is investigated related to agricultural aspects (e.g. plant-growth promoting and biocontrol) and very few studies have been conducted on medicinal plants12. E. purpurea root extracts are reported to stimulate macrophage TNF-α production but the extracts obtained from in vitro-grown axenic E. purpurea do not induce the same result, supporting the hypothesis that it is originated from the interaction with bacterial endophytes12. Our previous study shows that different compartments of E. purpurea, namely stems and leaves (SL), roots (R) and the rhizosphere (RS), share very few strains13. This finding has been shown to be related to the antagonism existing between strains inhabiting the different compartments14 and to the degree of resistance to antibiotics15. Moreover, the presence of distinct bacterial communities in plant compartments could account for the different bioactive compounds found in the various plant organs. Therefore, E. purpurea represents an interesting and useful in vitro model for plant-bacterial interaction studies on the production of pharmacological relevant secondary metabolites as the alkamides.16. They are important bioactive compounds dissimilarly distributed in the different compartments of E. purpurea17. Indeed, a notable difference is reported for the total alkamide content between aerial parts and roots, which is mainly due to a larger presence of non-tetraene alkamides in roots than in other plant organs18. The main Echinacea spp. alkamides are the isomeric dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutylamides19. These compounds increase the TNF mRNA expression in macrophages and monocytes binding the cannabinoid CB2 receptor20. Furthermore, the alkylamides decrease mitogens-induced interleukin-2 secretion in Jurkat-T cells21 and show an in vitro inhibitory activity of the 5-lipoxygenase22 and the cyclooxygenase-1 and 2 enzymes23.Alkamides  are lipophilic compounds chemically composed of two moieties, an amine moiety acylated by a fatty acid-derived one24. Isotope labelling analyses have revealed the generation of isobutylamine and 2-methylbutylamine  from valine and isoleucine, respectively. PLP decarboxylase-like proteins have been identified in the proteome of E. purpurea through in silico analyses and their transcript levels correlated with alkamide accumulation patterns in E. purpurea tissues. Then, a valine decarboxylase (VDC), potentially involved in the generation of the amine moieties of the alkamides, has been identified.Recently, a pyridoxal phosphate-dependent (PLP) decarboxylating enzyme belonging to the Class II tryptophan synthase family that utilizes branched-chain amino acids (BCAA) as substrate has been suggestedE. purpurea plants in the regulation of bioactive compound  accumulation. To this purpose, we set up an in vitro model system in which axenic E. purpurea plants, in vitro germinated from sterilized seeds, are inoculated with a pool of selected endophytes previously isolated from the aerial compartment of E. purpurea plants cultivated in open field  as Colony Forming Units (CFU)/g into the host R and SL tissues. Data obtained revealed that the highest CFU/g was detected in the SL compartment (7.06 ± 6.50 log CFU/g), and the lowest one in the roots of infected plants . This finding could indicate that the endophytes tended to re-colonize the native niche (SL compartment). The absence of bacteria in the control plant tissues and in the washing solutions confirmed the use of an axenic plant model and a successful sterilization procedure, respectively.A pool of thirty-seven bacterial strains (Supplementary Table E. purpurea pooled plants were investigated by means of high performance liquid chromatography (HPLC) coupled to a photo diode array (PDA)/ultraviolet (UV) detector and electrospray ionization tandem mass spectrometry (ESI-MS/MS). The LC-PDA/UV chromatograms of both E. purpurea R and SL extracts showed different alkamide profiles  were not found in the R one. On the other hand, two R extracts showed the presence of the alkamides 13/14 (peak O) and 15 (peak P), which were absent in the SL ones. In all R and SL samples, the most representative alkamides were a mixture of the two co-eluting isomers dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (7) and dodeca-2E,4E,8Z,10E tetraenoic acid isobutylamide (8). During the alkamide chromatography of both R and SL extracts, polyacetylene amides  elute early, followed by tetraenes (peaks G and K), trienes (peak L), and finally dienes (N). Interestingly, the analysis of the alkamide content in the E. purpurea plant organs also revealed the presence of seven alkamides not previously reported , but ascribable to the alkamide class due to the characteristic absorbance at 260 nm and typical ESI-MS and MS/MS spectra (Supplementary Table P < 0.001). In particular, the R samples presented C12 diene-diyne alkamides (peak O and P), whilst the SL samples contained C11 diene-diyne alkamides (peak A and B). Both samples showed a prevalence of C12 tetraene alkamides (peak G and K). Furthermore, PCA showed a discrimination among control and infected samples both for R and SL extracts and suggested that such differences were mainly created by the amount of the most abundant alkamide isomers 7 and 8 (peak G). The comparison of means (Supplementary Table P < 0.001). A relative estimation of the alkamide isomers 7 and 8 in the R and SL infected samples was also performed in respect to the levels of the relative control samples showing that the infection resulted in an increase of alkamide levels of about 70% in SL and 87% in R infected samples compared to the controls.The alkamide profiles of both control and infected R and SL extracts of VDC gene expression in the SL and R tissues of the control and infected E. purpurea plants was calculated in respect to the ubiquitin E2 (UbE2) gene expression in the three biological replicates. The amplification efficiency was optimal as achievable by the R2 and slope values . As depicted in Fig. VDC gene expression levels were higher in the SL samples than the R ones (P < 0.01). The VDC transcription level in the infected SL samples was about 4 times more than in the SL control tissues (P < 0.001). Also, the expression level resulted increased in R infected tissues respect to the relative controls even if at a minor extent (P < 0.01). In parallel, the involvement of an enzyme  not decarboxylating valine or isoleucine  but utilizing the serine as substrate to generate ethanolamine24 was evaluated. The transcription of the SDC gene  appeared down-regulated in the infected SL samples respect to the SL control ones whilst the SDC expression levels were similar between control and infected R samples  and dodeca-2E,4E,8Z,10E tetraenoic acid isobutylamide (8) being predominant, according to previous studies performed on in vivo plants31. Moreover, seven alkamides  remained not identified, and among these one alkamide (peak F) was present only in the infected SL compartment. To our knowledge, these alkamides were not previously reported in E. purpurea.On the basis of data obtained in this work, the R and SL extracts displayed different alkamide profiles, with twelve and ten compounds, respectively identified comparing their HPLC elution orders, ESI-MS/MS, and PDA/UV spectra with data reported in the literatureE. purpurea roots contain more alkamides than the leaves2. However, the alkamide content in SL samples could be higher than the reported leaves amount since it was the sum of the leaves and the vegetative stems. In fact, in line with the literature32, the R and SL extracts were differentiated by the presence of the C12 and C11 diene-diyne alkamides (mainly reported in the stem), respectively. On the other hand, Qu et al.18 reported that the tetraene alkamides 7 and 8 accounted for the 75% in the aerial part and only for the 9% in the roots of E. purpurea plants cultivated into a field and these estimates resulted discordant with our results . To this concern, one possible explanation could be that, to the best of our knowledge, this study is the first to estimate the E. purpurea alkamide level in an in vitro model whose experimental conditions could influence plant genetics and/or biochemical synthesis. However, the estimated alkamide level was higher in both infected R and SL, with a relative increase of alkamides 7 and 8 about 87% and 70% respectively, when compared to the controls, suggesting that the alkamide biosynthesis was modulated by the E. purpurea endophyte infection.The estimated total alkamide level was higher in the SL samples than in R ones, apparently in contrast with the literature data reporting that the VDC gene expression profiles, the highest expression level was detected in the infected E. purpurea tissues. By comparing results concerning both the chemical profiling analysis and the VDC gene expression data, it could be observed that the use of the proposed in vitro infection model system allowed us to demonstrate that the infection of E. purpurea axenic plants with their endophytes influenced the alkamide levels. In fact, both alkamide content and VDC transcription level resulted increased after the infection. The transcriptional up-regulation in the infected R samples was lower than the expected one: this result could be explained considering that the increase of the alkamide quantity in the infected R might be due to the transcriptional regulation of other enzymes required for the biosynthesis of the alkamide ammine and fatty acid moieties24. Interestingly, the SDC enzyme did not seem to contribute to the increase of alkamide levels confirming the specificity of the enzymatic source for the amine moiety formation of the alkamides (i.e. the VDC decarboxylase) as reported by Rizhsky et al.24.Concerning the in vitro model to study the role of the interaction between E. purpurea and its endophytes in the modulation of the plant secondary metabolism. Bacterial communities differed substantially between E. purpurea organs13 probably exerting a host selectivity able to modulate the community structure as reported for endophytic fungi33. In fact, the E. purpurea endophytes inoculated in axenic plants tended to re-colonize the native niche whose specific properties probably were influenced in turn by the natural endophytes. Chemical profiles and VDC genetic expression resulted quantitatively different between the control and infected plants, in particular in the SL compartment. Therefore, despite the in vivo status during bacterium-host interactions was difficult to mimic in vitro, the infection with E. purpurea SL endophytes modulated the characteristics resulting in axenic conditions, at least to some extent.The main objective of this work was to develop an E. purpurea was influenced by the plant-endophyte interaction thus possibly contributing to the therapeutic properties of this medicinal plant.Consequently, the whole body of data obtained in this work strongly suggested that the secondary metabolism in E. purpurea plants grown at the “Il Giardino delle Erbe”, Casola Valsenio, Italy, as previously reported13. Stock cultures were grown at 30 °C on tryptone soy agar  solid medium or tryptone soy broth  liquid medium. E. purpurea seeds were provided by the “Il Giardino delle Erbe”.Bacterial endophytes were isolated from the aerial compartment (stem and leaves) of Seeds were surface sterilized in order to prevent any unwanted fungal or bacterial growth. Seeds were immersed in a 70% (v/v) ethanol for one minute and, subsequently, in a 5% sodium hypochlorite solution for eight minutes. They were then rinsed three times with sterile distilled water, kept overnight at 4 °C in the dark for growth synchronization and then germinated in De Wit Culture tubes  containing 5 ml of Linsmaier & Skoog Medium (LS) including vitamins  at 24 ± 1 °C in the dark. After root formation, the seedlings were transferred in Wavin flasks  containing 50 ml of LS solid medium, supplemented with 3% sucrose, for a photoperiod of 16 h light a day for a minimum of two months. In order to validate the sterility of the obtained model system, cultivable endophyte multiplication into host tissues was checked: both shoots and roots were separately collected, washed in saline solution , surface sterilized in 1% (v/v) hypochlorite for 8 min and rinsed three times with sterile distilled water. Both samples were homogenized in saline solution and five replications of 100 μl of the homogenates were plated on TSA medium. Bacterial growth was scored after three days of plate incubation at 30 °C.Inocula of bacterial endophytes, isolated from SL compartment of E. purpurea plants, were incubated for three days at 30 °C in horizontal position and in agitation. The bacterial suspensions were then adjusted to 8 × 108 CFU/ml (OD600 = 1). The optical density (OD) was measured in a biophotometer . The pool generated from 100 μl of each diluted 1:10 OD600 suspension cultures was then centrifuged at 4000 rpm for 20 minutes and the pellet suspended in a correspondent volume of 0.9% saline solution. Five 2-months old E. purpurea plants were infected with 100 μl of bacterial suspension culture. Five plants were used as control and were infected with 100 μl of sterilized saline solution. Plants were then incubated in the growth chamber at 24 ± 1 °C. After 45 days, SL and R samples from control and infected plants, were collected separately, firstly washed in saline solution and then sterilized in 1% (v/v) hypochlorite for 8 min. Then, both tissue samples were washed three times with sterile distilled water and separated in different aliquots. R and SL aliquots were weighed and dried at 60 °C to be used for n-hexane extract preparation. R and SL aliquots of fresh material were ground to a fine powder in liquid nitrogen and successively stored at – 80 °C for RNA extraction. Finally, 1.0 g of fresh R and SL tissues were immediately used for the in planta bacterial growth analysis. The experiment was performed in triplicate.−7/ml cells. Five replications of each dilution were plated on TSA medium. The washing solution and the distilled water after the last wash were also diluted to check the presence of bacterial cells on the surface of the tissues and the outcome of the sterilization procedure. Bacterial growth was scored after two, three and four days of incubation of the plates at 30 °C.In order to evaluate endophytes multiplication into host tissues, 1.0 g of each sample was homogenized in saline solution and 100 µl of the homogenate were serially diluted up to 10E. purpurea plants from three independent experiments were pooled and extracted at room temperature with n-hexane  as detailed in Supplementary Table n-hexane residue from R and SL samples were then prepared dissolving the respective n-hexane extract in an opportune volume of methanol and then centrifuging the mixture. Finally, 20 μl of each supernatant solution (2.0 mg/ml) were injected for HPLC-PDA/UV-ESI-MS/MS analysis. The experiment was performed in duplicate.Dried and powdered R and SL of control and infected m/z 150–1200. N2 was used as the sheath and auxiliary gas. PDA data were recorded with 200–600 nm range with preferential channel as the detection wavelength 260 nm.Qualitative HPLC-PDA/UV-ESI-MS/MS analyses were performed using a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan) equipped with Xcalibur 3.1 software. Analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Fusion-RP column (Phenomenex). The eluent was a mixture of methanol (solvent A) and a 0.1% v/v aqueous solution of formic acid (solvent B). A linear gradient of increasing 55% to 85% A was developed within 45 min. The column was successively washed for 15 min with methanol and equilibrated with 55% A for 10 min. Elution was performed at a flow rate of 0.8 ml/min with a splitting system of 2:8 to MS detector (160 ml/min) and PDA detector (640 ml/min), respectively. The volume of the injected methanol solutions was 20 μl. Analyses were performed with an ESI interface in the positive mode. The ionization conditions were optimized and the parameters used were as follows: capillary temperature, 270 °C; capillary voltage, 29.0 V; tube lens offset, 50.0 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 3.00 arbitrary units; spray voltage, 4.50 kV; scan range of E. purpurea tissues using the RNeasy Micro Kit  and quantified by Qubit® 2.0 fluorimeter. One μg of total RNA for each sample was reverse-transcribed using the Quantitect Reverse Transcription Kit according to the manufacturer’s instructions (Qiagen) including a treatment with 1X gDNA Wipeout Buffer to remove any remaining DNA. The relative abundance of the E. purpurea cDNA for BCAA decarboxylases  was determined on a QUANTSTUDIO 7 FLEX  using the QuantiNova SYBR Green PCR Kit . Ubiquitin E2 genetic expression was used as internal reference to normalize mRNA content. The quantification of the expression was measured by the comparative Ct (2−∆∆Ct) method34. Target gene expression was relative to the control SL cDNA, which has been adopted as calibrator. The experiment was conducted in triplicate. Primer3 software35 was used to design primers specific to SDC template (epa_locus_952_iso_8_len_1801_ver_2) as available in the Medicinal Plant Genomics Resource . The primer sequences for the VDC gene were reported in Rizhsky et al.24. All primers listed in Supplementary Table Total RNA from R and SL was extracted from 36. One-way analysis of variance followed by Tukey test was used to compare peak area values between control and infected plants and to identify the alkamides mainly responsible of the differences between the samples. Comparison of the qRT-PCR data of the three biological replicates was determined by 2-tailed t-test. P < 0.05 was considered significant . Error bars are shown as s.e.m. The analyses were performed by using the modules present in the PAST program, version 3.1537.Means and standard deviations of the bacterial TVC data of the three biological replicates were estimated and compared by one-way analysis of variance between R and SL samples. To evaluate whether the level estimations of the alkamides  were useful in reflecting the chemical relationships between R and SL samples (controls and infected ones), a PCA was performedThe Authors declare that all the data supporting the findings of this study are available within the manuscript and its Supplementary Material and from the Corresponding Author on request.Supplementary Figure 1Supplementary Figure 2Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5


Nanoparticles (NPs) can be used to overcome the side effects of poor distribution of anticancer drugs. Among other NPs, colloidal gold nanoparticles (GNPs) offer the possibility of transporting major quantities of drugs due to their large surface-to-volume ratio. This is while confining these anticancer drugs as closely as possible to their biological targets through passive and active targeting, thus ensuring limited harmful systemic distribution. In this study, we chose to use bleomycin (BLM) as the anticancer drug due to its limited therapeutic efficiency (harmful side effects). BLM was conjugated onto GNPs through a thiol bond. The effectiveness of the chemotherapeutic drug, BLM, is observed by visualizing DNA double strand breaks and by calculating the survival fraction. The action of the drug (where the drug takes effect) is known to be in the nucleus, and our experiments have shown that some of the GNPs carrying BLM were present in the nucleus. The use of GNPs to deliver BLM increased the delivery and therapeutic efficacy of the drug. Having a better control over delivery of anticancer drugs using GNPs will establish a more successful NP-based platform for a combined therapeutic approach. This is due to the fact that GNPs can also be used as radiation dose enhancers in cancer research. This isribution ,12,13,14ribution ,16,17. Tribution . The GNPribution . In addiribution .One of the most startling results of GNP uptake studies was the difference in size-dependence of GNP uptake at monolayer and multilayer (tissue-like) levels ,20. At t1Will the efficacy of the drug be compromised by conjugating them onto the GNP surface?2The action of the drug is through binding onto DNA. Does this mean NPs can also reach the nucleus?3Will drug conjugated GNPs (GNP-BLM) be more effective than the BLM alone (free drug)?In this study, we used smaller NPs for two main reasons: (a) smaller NPs will penetrate better in tumor tissue once they leave the tumor blood vessels; and (b) the improved delivery of drugs since the surface-to-volume ratio is higher for smaller NPs. We had two goals: one was to investigate whether the therapeutic efficacy of the drug changes once it is conjugated onto the GNP surface; and the second was to investigate whether NPs can reach the nucleus since the action of the drug is mainly in the nucleus. We used bleomycin (BLM) as the anticancer drug for our study. This drug could be easily conjugated onto the GNP surface using a thiol bond. BLM is one of the most potent natural anticancer drugs available and has been used for chemotherapeutic agents in clinical treatments of certain cancers ,37. HoweRGDMFG) in addition to BLM. The penta-peptide was used to stabilize the NP for conjugation with BLM and the RGD peptide. The RGD peptide was used to increase the NP uptake as discussed in the next section. Now onwards, GNPs conjugated with BLM along with the above mentioned two peptides will be referred to as GNP-BLM. The free drug, bleomycin, is referred to as BLM. The peak was red shifted to 519 nm for the GNP-BLM complex due to the presence of BLM and peptides. The shape of the spectrum remains the same up to 48 h post conjugation, which signifies that aggregation of the complex does not occur during this time period. The hydrodynamic radius was also measured using the dynamic light scattering technique (see The Ultraviolet-Visible (UV-Vis) spectra of citrate capped GNP had a peak of 517 nm A which cique see B. AddingThe hyperspectral imaging (HSI) technology is a novel technique that can be used not only for optical observation but also for spectral confirmation of NPs. vs. control (cells with no GNPs) using a clonogenic cell survival assay and DNA DSBs assay as illustrated in the vs. BLM (free drug).We first tested the viability of the cells in the presence of as-made (or citrate capped) GNPs vs. the GNP-BLM complex. The clonogenic assay measures the damage to cells over a period of two to three weeks while DNA DSBs and the apoptotic assay measures the damage to cell over a short period of time (24 h after the treatment). The survival fraction (SF) of cells was evaluated from clonogenic assay experiments   BLM see . This woBased on early apoptosis data, there were no significant differences in the percentages of late apoptotic cells for the different treatments see A. Severavs. BLM A RGD peptide on the GNP-BLM complex allows for the release of these GNP-BLMs from these vesicles into the cytoplasm . This wi(2)BLM is known to reach the nucleus and bind to DNA causing DNA DSBs. If a RGD peptide assists the transport of the GNP-BLM complex to the cytoplasm, it is also possible that we can even transport some GNPs into the nucleus with the help of BLM (if they are still available on the GNP surface). This would benefit GNP mediated combined therapeutic strategies of chemotherapy and radiation therapy in the near future.As shown in It is also important to use smaller NPs if we were to use them for nuclear delivery since nuclear pores are ~25 nm in dimension . It is avs. free BLM as illustrated in vs. free BLM. vs. free BLM (middle panel). Previously, Jain et al. treated MDA-MB-231 cells with 12 μM 1.9 nm GNPs and different concentrations of BLM in the μg/mL range. It was shown that there were improved therapeutics when BLM was used with GNPs (GNP-BLM) vs. BLM alone [We completed three assays to verify the effectiveness of the drug delivered using GNPs (GNP-BLM) LM alone . HoweverLocalization of the GNP-BLM within the nucleus was confirmed using the Hyper Spectral Imaging (HSI) technique, as shown in vs. BLM  was added to 30 mL of double distilled water and then heated on a hot plate while stirring. Once the solution began boiling, 1000 μL of 1% anhydrous citric acid (Sigma-Aldrich) was added while being stirred. The color of the solution slowly changed from dark purple to bright red. The solution was left to boil for five minutes and was then brought to room temperature while stirring.GNPs of ~10 nm size were synthesized using the citrate reduction method. Firstly, 300 mL of 1% HAuClGNP-BLM complexes were assembled through sequential conjugation of peptides and BLM onto the GNP surface. A penta-peptide (CALNN peptide) was first added according to the ratio of 300 peptides/GNP for stabilization of GNPs. This is referred to as GNP-PENT in 2. The concentration of GNP-BLM complexes used this study was 1 nM.MD-MB-231 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) at 37 °C in a humidified incubator with 95% air/5% COFollowing overnight incubation with GNPs, the cells were washed three times with PBS and were trypsinized for quantification of the number of GNPs present per cell. Cells were counted and then treated with aqua regia at 200 °C in an oil bath for ICP-AES analysis.After the treatment, the cells were trypsinized and seeded in 60 mm tissue culture dishes. The cells were left in the incubator for two weeks for colonies to form. Methylene blue (0.1%) was used for staining of the colonies. The survival fraction of cells was determined using the ratio of the number of colonies formed/number of cell seeded.Cells were grown in coverslips (#1.5 18 mm) in 6 well dishes. After the overnight treatment under different experimental conditions , the cells were rinsed three times with PBS. The cells were then treated with 2% paraformaldyhyde/PBS/0.2% and Triton X-100 for 20 min followed by treatment with 0.5% NP40 for 20 min. Cover slips were left in 2% BSA/1% donkey serum in PBS for 1 h. Cells were washed with PBS three times for 5 min between each treatment. Following this, the coverslips were fixed with primary antibodies (53BP1 Ser 1778 and Anti-phospho-histone H2Ax) overnight. The coverslips were then washed with 0.5% BSA/0.175% Tween 20/PBS (secondary wash) for 5 min three times before being treated with optically labeled secondary antibodies (αR Alexa 488 and αM Alexa 647) for 45 min. The coverslips were washed with the secondary wash before being treated with 0.1 μg/mL of DAPI for 10 min. The coverslips were then finally washed with PBS for 5 min three times and mounted onto glass slides after adding a drop of antifade solution. The edges were sealed and stored at 4 °C in the dark.The treated cells were washed with PBS and trypsinized. The cells were pelleted in a centrifuge 500xg for 5 min and the supernatant was removed. The cell pellets were re-suspended in the annexin staining buffer. Alexa 647 conjugated annexin V was added for 15–20 min. Propidium iodide (PI) was added to the sample immediately before cytometric measurement.The CytoViva technology in combination with dark field microscopy was used to image GNP distribution within cells. The illumination of the microscope system utilized oblique angle illumination to create high resolution dark-field images. This imaging system was designed so that in spite of NP interaction with cells or tissue, their spectra may still be confirmed because they are still optically observable. The microscope is a dark-field imaging system that uses oblique angle lighting. NPs appear bright due to high scattering cross-sections of GNPs. To confirm the spectra of GNPs, Spectral Angle Mapping (SAM) was conducted with the CytoViva hyperspectral imaging system . SAM determines the presence of GNPs in the input image by comparing unknown spectra in the acquired hyperspectral image to a user-defined spectrum, GNPs in these experiments. This hyperspectral imaging of GNPs in cells and tissues was practical since it does not require optical labeling of the GNPs. It is also possible to extract spectral information from each pixel for verification purposes. More information is provided in the We have succeeded in designing a GNP-based nano-platform (GNP-BLM) to deliver the anticancer drug BLM, while maintaining its cytotoxic activity. Furthermore, GNPs carrying BLM enabled interactions between the BLM and the nucleus, thus resulting in enhanced DNA DSBs. In light of these results, the GNP-based nano-platform proposed here has great potential for the delivery of BLM to tumors while improving its biodistribution.


Infection of the hip after implant fixation is an uncommon yet devastating complication that results in poor long-term outcome. The gold standard treatment for chronic infection after hip arthroplasty is a two-stage protocol: eradication of infection, follow by re-implantation arthroplasty. The use of interim antibiotic-laden cement spacer has become a popular procedure to maintain hip joint function and provide antibiotic elution simultaneously before re-implantation. However, antibiotic cement spacer is mechanically weak and breaks if overloaded. Therefore, we designed a cement mould with metallic endoskeleton with the aim of creating a stronger, inexpensive, antibiotic-impregnated spacer resembling a unipolar arthroplasty. We report two cases of severe hip joint infection after implant fixation . Both patients had undergone first stage surgery of debridement and articulating antibiotic cement insertion using our design. Although the second stage surgery was planned for these patients, both patients delayed the operation in view of good functional status after a year walking with the antibiotic cement spacer. These cases showed that the mechanical property of the new antibiotic cement spacer was promising but further mechanical studies upon this new endoskeleton design are required. Antibiotic-laden articulating spacers achieve infection control by local tissue antibiotic drug elution with an added advantage of preserving limb length, joint mobility, and preventing joint contracture2. However, cement spacers are mechanically weak and may fracture while awaiting revision surgery3. The presence of an endoskeleton and standardization of antibiotic cement preparation are designed to overcome this mechanical failure of the articulating spacer4. Albeit various metallic endoskeletons are available in the market, it is unclear whether different shape and material used for endoskeleton affects the function, drug elution and mechanical properties of the cement spacer5. We present a novel design of an antibiotic cement mould with an endoskeleton to create an inexpensive antibiotic-laden cement spacer mechanically strong enough to be used in two cases with implant infection involving the hip as an interim arthroplasty replacement.Implant infection in a hip joint is a rare yet debilitating complication, with a prevalence of 0.5% to 3%. The gold standard treatment for chronic infection of the hip joint after hip arthroplasty is a two-stage revision. First stage involves implant removal, appropriate antibiotic therapy and an interim antibiotic-laden cement hip spacer. The second stage is reimplantation surgery once infection is eradicatedA 76-year old lady, with underlying diabetes mellitus and hypertension, gave a history of a fall in 2014 and had sustained fracture of the right neck of femur. She was treated surgically with bipolar hemiarthroplasty and recovered uneventfully. However, five months into rehabilitation, patient had sudden onset of debilitating right hip pain associated with fever and swelling over the previous operative scar. A diagnosis of surgical site infection extending up to the implant was made. Meticulous surgical site debridement with temporary antibiotic cement insertion was performed as first stage surgery . There wAt follow-up after two years, patient was ambulating with walking stick and applying full weight over the affected limb. She was able to maintain an adequate ROM of the hip: flexion 0-90 degrees, internal and external rotations of 45 degrees. With the eradication of the infection as evident by absence of symptoms and significant reducing trend of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Radiograph of the hip showed resolution of OM of the acetabulum. We proposed total hip replacement to the patient but she refused any intervention in view of her age and satisfaction of her current state.A 27-years old male was involved in a devastating motor vehicle accident in June 2015 and had sustained closed fracture left neck of femur, open comminuted fracture (Gustilo – grade 3B) of the midshaft of the tibia and fibula and closed fracture distal third of the right tibia. He was treated surgically with screw fixation of the left neck of femur and wound debridement with Illizarov external fixator of left tibia after the accident . The rigCT scan and bone scan of left hip confirmed destruction of the femoral head. Hence wound debridement, removal of screws of left femoral head with temporary antibiotic cement rod insertion was performed. Intra-operatively, about 50 cc pus was found extending up to hip joint with biofilm surrounding all three screws and osteomyelitis with femoral head collapse . This waOn follow up after a year, patient could walk with crutches, partial weight bearing over his left lower limb. He achieved adequate ROM of left hip: flexion 0-110 degree, internal and external rotations of 60 degree. His infective markers  were within normal range. In view of his young age, he was given the surgical option of hip fusion or replacement later in life when there was unbearable hip pain affecting the quality of life. He was content with the functionality provided by the antibiotic cement arthroplasty for now and wished to delay the operation.5. The cement mixture was poured on both sides of the plate and the metal endoskeleton was placed in the center  to fabricate a prototype cement mould using stainless steel plates based on the Thompson unipolar hemiarthroplasty model. The newly design metallic endoskeleton was also made from stainless steel . First, e center . Next, be center . Once the center . Finallye center .1. They have been successfully utilized in the treatment of proximal femur infections after osteosynthesis. Articulating antibiotic-impregnated spacers: (i) provide high dosage of antibiotic locally to the surrounding tissue by targeting the source of infection, (ii) preserve limb length and joint mobility, (iii) limit soft-tissue contracture and scarring, (iv) facilitate second-stage arthroplasty replacement1.Periprosthetic hip infections impose a significant burden not only to the patients, but also to the healthcare system. Recently, antibiotic-laden hip spacers have become indispensable in the treatment of hip joint infections2. The aim of the first stage surgery was to sterilize the infected joint after removal of the prosthesis through meticulous surgical debridement, identification of the infecting organism and antibiotic sensitivity from tissue biopsies, and administering targeted antibiotic therapy before re-implantation2. However, the timing of the second stage was variable but essentially depended on the resolution of infection as evident by improved hip mobility, absent clinical signs of infection, and ESR and CRP levels returning to normal values.A two-stage protocol with the temporary insertion of an antibiotic-laden cement spacer was suggested as the gold standard treatment for chronic infections with a successful eradication rate of up to 95%et al3. Mainly of prosthetic spacers fracture dislocation. Therefore, the logical way to improve the mechanical stability of the spacer would come from the introduction of a metal endoskeleton. Thielen et al showed that presence of endoskeleton doubled the strength of the spacer as compared to the spacer without. It could withstand hip joint forces up to 6.0 kN, sufficient for protected daily activities. The study also showed that all failures occurred in the upper third of the stem4.Despite the popular use of cement spacers, they still pose a significant risk of mechanical complications up to 50% as reported by Jung 5. An alternative method was re-using the excised prosthetic components or femoral bone after intraoperative sterilization and adding extra support with PMMA or metallic implant such as K-wires, Rush rod, etc5. The disadvantage of this method was that it was time consuming and with higher risk of fractures due to cement heterogeneity and inconsistencies in mixing.Commercially, PROSTALAC is a prefabricated mould with fixed low-dose antibiotic content with different sizes available for making cement spacer. However, this device limits the size of cement spacer and dosage of antibiotic used, not to mention the high cost as wellThe method proposed in these two cases is cost effective as PMMA and antibiotic are the only consumables. It allows customization of the formula of bone cement and various antibiotics. The newly design metallic endoskeleton has improved the sturdiness of the articulating cement spacer evenly. Patients achieved remarkable ROM of the affected hip and functional outcome thus far. However, the prefabricated cement size is fixed and it is unclear whether different designs of endoskeleton might affect the results of interim function, infection control and mechanical complications. Currently, further mechanical studies upon this new endoskeleton design are required.Fabrication of the cement mould and stainless steel endoskeleton was sponsored by Leonix, Penang, Malaysia.