A correlation between the use of antibiotics in early life and the excessiveweight gain in later childhood has been shown in several large cohort studiesall over the world. One hypothesis explaining this association is the pervasiveimpact that antibiotics may have on the intestinal microbiota, and this has beensupported by recent mouse studies. Studies have shown dramatic changes in theintestinal microbiota of adults in response to oral antibiotic treatments.However, little is known about the impact of antibiotics on the intestinalmicrobiota of children, although antibiotics account for the majority of themedication prescribed to children in Western countries. In production animals antibiotic use increases weight gain at least partly by suppressingsubclinical infections. In laboratory mice, living in clean and infection-freeconditions, the antibiotic-induced weight gain was recently demonstrated by the group ofMartin Blaser in New York, USA to be associated with an altered gut microbiome.Epidemiological studies have confirmed the positive relationship between antibiotic useand weight gain in humans and indicated that even prenatal antibiotic exposurepredisposes to childhood overweight. Pre- and perinatal maternal and environmentalfactors are being recognized as important contributors to the long-term metabolicprogramming and weight development of infants, and multiple lines of evidence indicatethat childhood overweight may be strongly dependent on early-life exposures. Theintestinal microbiota, acquired initially during birth from the mother and nurtured bybreast milk, are emerging as an important modulator of early metabolic programming, withlong-lasting health consequences.Bifidobacterium and Bacteroides after a macrolidecourse, which normalized within 24 months. In addition, a likely causal relation betweenthe use of macrolide antibiotics and macrolide resistance was established, both at thegenetic and phenotypic levels. Overall, macrolide use was associated with changes in themicrobiome that have previously been linked with increased BMI, adiposity, obesity ormetabolic diseases in children or adults: low overall richness, reduction of bacterialbile-salt metabolism, and reduction in bifidobacteria andChristensenellaceae, as well as increase inBacteroides and Erysipelotrichaceae.Our recent study addressed the association between antibiotic use and intestinalmicrobiota composition in healthy children. We studied a well-controlled cohort of 142day care-attending children aged 2-7 years, collected data on their antibiotic use andhealth parameters, and coupled these too deep and global analysis of the intestinalmicrobiota and their functions. The microbiota composition in children exposed topenicillin or macrolide–type antibiotics during the previous 2 years was compared to thecomposition in children with no recent antibiotics and low lifetime antibiotic use. Ourresults confirmed a strong association between lifetime antibiotic use and BMI inchildren, and refined this observation by showing that the effect was evident only whenthe child had received macrolide antibiotics in early life. Remarkably, a similar strongeffect of macrolides was detected on the intestinal microbiota. The most dramaticapparent effect of antibiotic treatments was a shift in the relative abundance ofAkkermansia spp., the dedicated mucus degrader that had earlierbeen discovered by our colleagues at Wageningen University, The Netherlands and protectsmice from diet-induced obesity.Earlier animal experiments of the Alimentary Pharmabiotic Centre in Cork, Ireland haveshown that increasing the level of bile salt hydrolase decreased diet-induced obesity inmice. Further support to the antibiotic-induced disruption of bile-salt metabolism comesfrom a study by the group of Brett Finlay in Vancouver, Canada showing a reduction inbile-salt metabolism in mice experimentally treated with streptomycin. The microbiotachanges associated with macrolide treatment may explain the obesogenic effect of early-lifebroad-spectrum antibiotic courses. Moreover, we found a number of bacterial taxaassociated with high BMI: a combination of four genera differentiated obese vs.age-matched normal-weight children . These included several bacterial groupsthat had been implicated in obesity development, such as a decreased level ofBifidobacterium spp. in infancy and later BMI development. In adulthumans, obesity and related metabolic markers have been shown to correlate negativelywith the abundance of bifidobacteria. Work by the group of Nathalie Delzenne atBrussels, Belgium has shown that in rodents and to some extent in humans, bifidobacteriaprotect against diet-induced obesity and related metabolic effects. The beneficialmetabolic effects of these species are thought to arise at least partly from improvedgut barrier function, which reduces metabolic endotoxemia, i.e., abundance ofbacterially produced LPS in the circulation. Circulating LPS induces inflammation,insulin resistance, and weight gain, and is considered an important component in thedevelopment of obesity and the related conditions. Bifidobacteria reduce the leakage ofLPS from the gut presumably by up-regulating tight-junction proteins and can therebyimprove the metabolic health of the host and counter-act diet-induced weight gain. Ourstudy showed that macrolide treatment was associated with a reversion of theBifidobacterium-to-Bacteroides balance, thusincreasing the abundance of LPS-producing organisms and decreasing the abundance ofgut-barrier-improving organisms. Frequent macrolide use may therefore involve recurrentLPS surges, similar to the effects of a high-fat diet, which could contribute to theassociation between antibiotic use and weight gain . A full recovery of themicrobiota from an antibiotic course appeared to take longer than the average intervalbetween courses, which suggests that many children may not fully recover from antibioticdisturbance, but are in a continuously disrupted state throughout their early life . Whether this has any permanent effects on the microbiota composition or function iscurrently not known, but mouse studies indicate that even temporary disruption of themicrobiota in early life has long-term metabolic consequences.Our results, supported by earlier experiments with mice suggest that the early-lifemicrobiota is integrally involved in the long-term metabolic programming of infants.Increasing evidence is implying that bifidobacteria may have a key role in the metabolicprogramming of children. Finnish studies by the group of Erika Isolauri at Turku,Finland have noted a negative association between abundance ofWhile antibiotics should be used when needed, our study indicates that it is important toconsider the pervasive effects that macrolide use has on the microbiota as compared tothat of penicillin-type antibiotics. Moreover, our present work provides further supportin human for the hypothesis that the intestinal microbiota are involved inantibiotic-induced obesity and hence opens avenues to detect this in early stages andprovide corrective therapies that target the intestinal bacteria.


The poor prognosis of hepatocellular carcinoma (HCC), one of the most devastating cancers worldwide, is due to frequent recurrence and metastasis. Among the metastatic factors in the tumor microenvironment, hepatocyte growth factor (HGF) has been well known to play critical roles in tumor progression, including HCC. Therefore, c-Met is now regarded as the most promising therapeutic target for the treatment of HCC. However, there are still concerns about resistance and the side effects of using conventional inhibitors of c-Met, such as tyrosine kinase inhibitors. Recently, many alternative strategies of c-Met targeting have been emerging. These include targeting the downstream effectors of c-Met, such as hydrogen peroxide-inducible clone 5 (Hic-5), to block the reactive oxygen species (ROS)-mediated signaling for HCC progression. Also, inhibition of endosomal regulators, such as PKCε and GGA3, may perturb the c-Met endosomal signaling for HCC cell migration. On the other hand, many herbal antagonists of c-Met-dependent signaling, such as saponin, resveratrol, and LZ-8, were identified. Taken together, it can be anticipated that more effective and safer c-Met targeting strategies for preventing HCC progression can be established in the future. The poor prognosis of hepatocellular carcinoma (HCC), one of the most devastating cancers worldwide, is due to frequent recurrence and metastasis after surgical resection. In the tumor microenvironment, growth factors and cytokines, such as hepatocyte growth factor (HGF) ,2,3,4,5 The receptor of HGF, c-Met, is a RTK consisting of a disulphide-linked heterodimeric complex with an extracellular portion for ligand binding, a membrane spanning segment, a juxtamembrane domain, a catalytic domain, and a C-terminal docking site . BindingThe therapeutic strategy aiming at HGF-c-Met signaling for the prevention of tumor progression of HCC was intensively investigated decades ago. Previously, many preclinical studies had strengthened the feasibility of targeting HGF-c-Met  for There are many reports demonstrating the mechanisms for resistance to c-Met targeting, including the cross talk of c-Met with epidermal growth factor receptor EGFR , gene amThe toxicity of c-Met inhibitors has been demonstrated in recent studies. In animal experiments, the highly selective c-Met inhibitor GEN-203 and compound 8 may cause liver and bone marrow toxicity in mice  and myocThe aforementioned side effects caused by c-Met inhibition may be associated with the biological functions of c-Met. HGF and c-Met are broadly expressed in epithelial cells of many organs, playing essential physiological roles . ImportaThere are strategies that may address the aforementioned issues in c-Met target therapy. One of them is the targeting of downstream effectors of c-Met specifically involved in tumor progression. Previously, polypeptides competing with downstream effectors of c-Met were developed. For example, antagonists of the Grb2 SH2 domain were used to prevent metastasis mediated by c-Met . MoreoveOne of the c-Met downstream effectors that is more feasible as a therapeutic target may be the hydrogen peroxide-inducible clone-5 (Hic-5), which was first identified in a screen for TGFβ1 and hydrogen peroxide-inducible genes . Hic-5 bRecently, the role of endosomal signaling in tumor progression triggered by metastatic growth factors, including HGF, has been highlighted . ReceptoPreviously, c-Met endocytosis had been found to be critical in breast cancer tumorigenesis, which can be prevented by two endocytosis blockers, ConA and dynasore . RecentlAnother strategy for avoiding the problems of HGF-c-Met target therapy is the use of natural herbal drugs instead of synthetic chemical agents. For example, Timosaponin AIII (TAIII), a steroidal saponin, may suppress HGF-induced invasive activity . MoreoveTarget therapy aimed at the HGF/c-Met pathway is promising for the suppression of HCC progression. However, the problems of resistance and side effects in conventional kinase-based c-Met targeting remain unresolved. Many alternative strategies are emerging which aim to be more effective and safer. These include targeting the downstream effector of c-Met and using herbal drugs as safer antagonists of c-Met (summarized in


Internationally, overdose is the primary cause of death among people injecting drugs. However, since 2001, heroin-related overdose deaths in the United States (US) have risen sixfold, paralleled by a rise in the death rate attributed to synthetic opioids, particularly the fentanyls. This paper considers the adaptations some US heroin injectors are making to protect themselves from these risks.n = 22; Chicago, n = 24; Massachusetts and New Hampshire, n = 36; San Francisco, n = 9). Experience injecting heroin ranged from < 1–47 years. Eight participants, who were exclusively heroin snorters, were also interviewed. Data were analyzed thematically.Between 2015 and 2016, a team of ethnographers collected data through semi-structured interviews and observation captured in field notes and video recording of heroin preparation/consumption. Ninety-one current heroin injectors were interviewed  to injecting a partial dose and waiting. Partial injection took different forms: a “slow shot” where the user injected a portion of the solution in the syringe, keeping the needle in the injection site, and continuing or withdrawing the syringe or a “tester shot” where the solution was divided into separate injections. Other techniques included getting feedback from others using heroin of the same batch or observing those with higher tolerance injecting heroin from the same batch before judging how much to inject themselves. Although a minority of those interviewed described using these drug sampling techniques, there is clearly receptivity among some users to protecting themselves by using a variety of methods.The use of drug sampling as a means of preventing an overdose from injection drug use reduces the quantity absorbed at any one time allowing users to monitor drug strength and titrate their dose accordingly. Given the highly unpredictable potency of the drugs currently being sold as heroin in the US, universal precautions should be adopted more widely. Further research is needed into facilitators and barriers to the uptake of these drug sampling methods. Heroin can be taken into the human body in a wide variety of ways, including snorting or sniffing powder or heroin solution , inhalation of the heated vapors (“chasing”), orally and as anal suppositories (“plugging”). Injecting, whether intravenous, subcutaneous or intramuscular, is the method of administration carrying the highest risk for multiple types of infections, overdoses and their complications –15. HoweHeroin “source form”—its chemical characteristics based on country of origin—also influences its mode of use , 19. BioAnother non-injection option to chasing/smoking is intranasal use. Intranasal heroin administration has been found to be an effective drug delivery route. It has been theorized that the lower price of US heroin in the early 1990s rather than its higher purity—as well as fears of contracting HIV—may have led to increased usage of intranasal administration . US dataFor many decades, overdose has been the primary cause of deaths among people injecting drugs , 29–32, In the face of overdose-related morbidity and mortality, numerous public health interventions have tried to address the risks of overdose over the last 20 years. These strategies include overdose education and peer naloxone distribution –44. PromOne study of UK-based “chasers” and injectors found that while preferred administration routes were likely to persist for years and multiple route transitions were uncommon, 16% of their sample had reverse transitioned . AnotherGiven the low rate of “chasing” among US heroin users, one practice some injectors have adopted to reduce the risk of overdose is the use of “tester shots” or “test shots.” This entails injecting a small quantity of a drug sample in order to assess its potency qualitatively before deciding whether to inject the remainder of the dose –52. A teCircumventing the multiple injection problem of the tester shot, “slow shots” allow the injector to insert the needle, release the tourniquet, and very slowly inject while assessing the embodied effect with the needle still in the vein . A furthThere is a dearth of qualitative research on behavioral adaptions that current heroin injectors are making with respect to the ongoing fentanyl adulteration crisis in the US. In this paper, we present findings from ethnographic fieldwork trips in 2015 and 2016 to Baltimore, Maryland; Worcester, Lowell, and Lawrence, Massachusetts; Nashua, New Hampshire; San Francisco, California; and Chicago, Illinois on embodied methods of gauging opioid strength that injection drug users in these areas are taking to prevent overdose. With the exception of California, where solid black tar heroin dominates, all these states have powder sourced from Mexico or Colombia and are suffering rising heroin- and fentanyl-related deaths.In 2016, Baltimore lost 454 people to heroin-related overdoses, up from 260 the previous year, and 419 people to fentanyl-related overdoses, up from 120 in 2015 , 57. TheThe “Heroin in Transition” (HIT) study is conducting “hotspot” research where ethnographers are dispatched to locations around the country upon receiving reports of unusual or dangerous heroin or high levels of overdose . HIT’s aHIT uses highly focused ethnography to investigate new and evolving heroin forms and users’ responses, their methods and contexts of use . The pauUnlike most rapid assessment, the HIT model is chiefly qualitative, with quantitative questions explored by the Heroin in Transition study outside the rapid assessment to inform the overall picture. Our research model aims to represent “from below” the experiences and perceptions of users in the current opioid epidemic. These data are placed alongside observations by the ethnographers based on their own experience of observing heroin and its use in other locations. Data are not formally triangulated, but we hope that a composite picture emerges from the multiple perspectives of users and ethnographers. Further, rapid ethnography allows us to generate hypotheses to be tested by quantitative research methods. Between 2015 and 2016, the ethnographic team visited Baltimore, Maryland (November 2015 and March 2016); San Francisco, California (February 2016); the Massachusetts cities of Lawrence, Lowell, Worcester and Nashua, New Hampshire (June 2016); and Chicago, Illinois (September 2016).Following information about fentanyl-laced and fentanyl-substituted heroin in Baltimore, New Hampshire and Massachusetts and high levels of overdose in Chicago, contact was made with harm reduction service providers in these locations. In San Francisco, the ethnographic team used personal contacts to arrange interviews with users and also carried out recruitment on the street. The study protocol was also approved by the University of California, San Francisco Institutional Review Board. The data and its collection are protected by a US Federal Certificate of Confidentiality issued by the National Institutes of Health/National Institute on Drug Abuse.Data were collected through semi-structured interviews, ethnographic observation captured in collaborative field notes and video recording of heroin preparation and consumption. With its observation in the field and long established “ground-up” approach to data collection, ethnography is a method well suited for examining the experiences and phenomena of everyday life—and has been used widely in research among drug users , 70. RapTo be eligible for the study, participants had to be at least 18 years of age and self-reported current injectors whose primary drug was heroin. Exclusion criteria covered individuals who were intoxicated or otherwise unable to give informed consent or answer questions reliably. Researchers approached users attending needle and syringe program sites, explained the study to them, and obtained their consent. Some snowball sampling was also conducted . Only veThe semi-structured interviews were carried out by the ethnographers , project director (SM), and PI (DC) immediately upon recruitment at the needle and syringe program locations, in rental cars, cafés, users’ homes or drug-using locations. An interview guide provided a general structure to the conversation, including questions on the respondents’ life course of drug use, a typical day in their life involving drug acquisition and use, perceived changes in the heroin supply, knowledge of fentanyl, preferences for heroin vs. fentanyl, methods of use, perceived physiological effects and experiences of overdose.After our initial visit to Baltimore indicated some tester shot usage among the population, focused questioning on drug sampling was incorporated into the interview guide for subsequent sites. All interviews were audio-recorded and transcribed in their totality and verified (by JO) against the audio recording. Field notes were drafted collaboratively each research day and finalized after the trips. The PI (DC) is a physician and can address participants’ medical concerns or questions or that arise during the flow of this research. As data is analyzed, findings are fed back to local service providers to assist in the development of tailored harm reduction responses.n = 22; Chicago, n = 24; Massachusetts and New Hampshire, n = 36; San Francisco, n = 9), of whom 62 were male and 29 were female. The experience injecting heroin ranged from less than a year to 47 years. Eight participants, who were dedicated heroin snorters, were also interviewed. Names have been changed to protect confidentiality.This was a non-random convenience sample. Across all sites, 91 current heroin injectors were interviewed The unpredictability of heroin potency is a long-established problem of its illicit supply, particularly for injectors whose dose is delivered in a single “bolus,” putting them at increased risk of overdose relative to those who insufflate  or smoke heroin. Users become aware of these risks in a number of ways, including personally experiencing or witnessing overdoses or losing friends or partners. The research study’s first encounter with drug sampling was with a young man in San Francisco who, after experiencing an overdose, had started smoking the strong black tar heroin known locally as “point dope” before judging how much to inject:[…]when I get stuff I don’t know what it is, I do a little bit before I do something that I feel. Like I want to kind of scale out how much I want to do. Because I don’t want to die. But these people are just doing a gram shot and just… my friend just died two days ago.Like Riley, some interviewees mentioned adopting drug sampling as a result of experiencing overdose firsthand, but others did so after observing others. Liz from Lawrence, MA, a woman in her 20s who had been injecting for 1 year and had never personally overdosed, explained that she had adapted her behavior with fentanyl-laced heroin, “Because you learn from everybody else being stupid”:Q: Do you ever taste a new bag?A: No, because I’ve been going through the same guy and it’s just always consistent. His is always, you know, straightforward. You know what you’re going to get. So I trust it. But if I were to go to somebody else that I hadn’t gone to before I would definitely taste it, snort a little bit, or you know if I were to shoot it, I would shoot a small amount. Because you can always shoot more, you can never shoot less.While some referred to these methods as their regular practice, others sampled in particular circumstances such as after periods of abstinence or when the heroin or its source were unfamiliar. In Chicago, David, who was in his 30s and had been using for 7 years, generally relied upon a regular dealer to regulate the quality and strength of his heroin, as well as keeping his doses small, but he snorted heroin prior to injecting when buying from an unfamiliar source:Users described a wide variety of methods for drug sampling, sometimes creatively combining them together, from using non-injecting routes of administration such as snorting, smoking or tasting a small amount prior to injection to injecting a partial dose and waiting. Partial injection could take different forms: either a “slow shot” where the user injected a portion of the solution in the syringe, keeping the needle in the injection site, and then either continuing or withdrawing the syringe or a “tester shot” where the solution was divided between two or more separate injections. Other techniques included getting feedback from other users who were using heroin of the same batch or observing other users with higher tolerance injecting heroin from the same batch before judging how much to inject themselves.In Chicago, where the powder heroin can be snorted rather than smoked, several injectors described snorting their heroin before injecting. Ray, in his 50s and using for 25 years, explained that this not only gave an indication of its strength but also a taste at the back of the throat which, he believed, was indicative of its ingredients:A: […] I’m not the only one that do this. There’s a lot of my buddies that do heroin too, will toot [snort] it first before we even start cooking it up to see what’s it about. […]See, when you toot dope it’s supposed to go down your nose and it’s supposed to go down smooth without no burn or none of that… Now, once you get that drain, it’s the taste. It tastes like dope. Because see, some dope that tastes like aspirin ain’t dope…. So you get familiar with the taste of dope too once you do it, once you had done it so long with me…. So once you get all that in perspective, […] the taste, the smoothness going down, the drain, you’ll start feeling a nice, warm sensation. So now I know it’s straight [heroin] so it’s time for me to party now.A: […] I’m very cautious on my heroin intake. I like to know how much I’m doing. And if anything, I always do a little bit less and sometimes I get pissed off because I don’t get high from the little shot.Q: So you take care of yourself, testing a little bit.A: I always test the waters before. You don’t just jump in with heroin.Several of our research participants indicated that they had begun injecting a smaller amount of heroin to assess qualitatively its embodied effects before injecting a larger dose. Johnny in Chicago, in his 20s and using for 6 years, was among those who endorsed this method:Q: How long are you waiting to judge how strong it is?A: I’d say anywhere from 5 to 15 minutes.Q: 5 to 15 minutes. You’re a patient guy. I imagine a lot of other people aren’t so patient.A; Well, it’s… pays off in the end, I guess.Glen, in his 20s, using for approximately 1 year in Baltimore, described his method of tester shot:Q: So what happens if you’re feeling like that half shot is really good, do you have enough self-control to take the rest of it out and not do it?A: Yeah, I can. And I also like to have it where I can put the rest of it in me, because I feel as though my intake can allow me to take the rest of that there.Some of those interviewed had managed to avoid overdose entirely, perhaps as a result of using drug sampling methods. Larry, a Baltimore man in his 60s who had survived over 40 years of heroin use without overdosing, explained how he injected half his shot of heroin, waited a short time, and then decided whether to inject the rest. If he decided to inject the rest of the shot after waiting, he registered to check whether he was still accessing the vein first. We asked him about how he managed the decision not to use the whole shot:A: […] I don’t, what they call, slam it and put the whole thing in me. I do it little by little just to see what the effects is and stuff.Q: So do you like have one syringe and you use a little bit of it or do you like have several syringes?A: No, just one syringe and one syringe I put what we say 80 on the height. So once I cook it up and do what I’m going to do with it, and most of the time I’ll speedball, you know, cocaine and heroin. And I will take 40 of it and I’ll take the other 40 and put it to the side and then when I hit myself I’ll just put 20 in it just to see, you know, what the effects is …James, a Baltimore man in his 50s who had been injecting for half his life and never overdosed, had recently experienced a number of friends dying from overdoses. His method involved loading the syringe with half the dose and then further dividing the half doses for a slow shot:Q: … Since you don’t know whether it’s going to be weak or strong how do you manage that?A: What I do is I get a reading from someone else because I know myself.Q: A reading?A: Meaning they tell me how it is for them. And that determines how much water I’ll put on mine. Like my girlfriend might say, 'Girl, that dope is good as a motherfucker.' I might put 40 on it and I’ll put a 60. […]Q: You dilute it if the strength is good?A: Yes.Q: All right. Then how do you inject that 60? […]A: 30.Q: You’ll do 30?A: I’ll do 30, wait for it to hit me and see if I can handle it or do I need to come out.Q: How long you waiting for?A: As soon as it hit me. Which usually takes I’d say 20 seconds.Q: 20 seconds, okay. And then you just decide to pull out or --?A: Or go ahead with it.Janice, also a long-term user in Baltimore, in her 50s, who had also never overdosed, used a combination of approaches to regulate her intake. This involved getting an oral report from a friend, gauging how much to dilute the heroin depending on her friend’s reaction, and then taking a half-dose slow shot, keeping the needle in place before deciding whether to use more.Relying on friends’ experiences of a single batch was also something we observed in Baltimore. Glen and Scott are running buddies sharing resources including heroin . Glen haI almost overdosed, I almost died. I almost went out. And I didn’t do much, it was kind of like a tester shot and it was really powerful. I just thought it was really good dope, you know? I didn’t know it was fentanyl until it started getting around that the fentanyl was floating around…However, there are limits to the drug sampling strategies just described, especially in the age of fentanyl. Harry, a user in his 40s from Lowell, MA, using for 20 years, explained that even with a tester shot, he had almost overdosed from heroin he later believed to contain fentanyl:I’m taking my time because if I’m gonna mix it with 65 [units of water], I’m gonna put 10 or 15 in me to see the quality of what I’m putting in me, because you can always put more in, but can’t take it out. I’m a firm believer in that.—Doug, Baltimore, in his 40s, using for 32 yearsWhether users developed these methods themselves, learnt them from peers, publicity campaigns or harm reduction workers was often unclear. However, an awareness of the irreversibility of injection was a recurring theme.I’ll do a little bit at a time just to make sure I’m not – that I’m cool. And then you can always give yourself more but you can’t take back.—Joanie, Chicago, in her 30s, using for 14 yearsQ: Do you ever do a tester shot or do you use the whole thing in one shot?A: It depends on the drug, sometimes I don’t do it all at once. You have to respect dope; if you know it’s strong, you have to use it carefully.—Gonzalo, Nashua, NH, in his 50s, using for 20 yearsQ: And you were saying a bit about how you avoid ODing yourself?A: Oh yeah, because I don’t, you know, push the whole thing in me at one time, because like I said, there is no pulling it back […] once you push it in you, ain’t no trying to pull it back […]—James, BaltimoreThe presence of overdose as an imminent danger was especially pervasive among users in Baltimore and Massachusetts, many of whom had lost friends, relatives or acquaintances. Although a minority of those interviewed described using these drug sampling techniques, there is clearly receptivity among some users to protecting themselves by using a variety of methods.Our ethnographic research shows that in the five states visited for this study, some people using heroin have incorporated tester shots and other drug sampling strategies into their injection practices. These included a wide range of approaches, sometimes in combination, in which people used their own or others’ embodied experiences to judge the strength of the drug qualitatively before deciding on their dose. While these methods do not guarantee safety from an overdose, particularly in the cases of some of the more powerful fentanyls, it would be valuable to test epidemiologically whether those using drug sampling methods report experiencing fewer overdoses.Peer advocacy for tester shots and drug sampling may be the most effective means of expanding the use of these methods. A number of research studies conducted in the US and internationally have demonstrated the connection between social norms, peer adoption, self-efficacy and perceived acceptability in the successful adoption of harm reduction strategies –77. OthePsychologically informed approaches may also shed some light on how best to encourage the use of drug sampling. One survey of the self-efficacy of harm reduction practices, including tester shots, found that adherence to all harm reduction strategies surveyed was markedly lower when respondents were asked to imagine being in withdrawal . A laterIn order for tester shots and other drug sampling strategies to be effective at preventing overdose, people who inject heroin must be willing to use them consistently, since risk is difficult to determine at the point of use. Other interventions that may be effective aids in assessing overdose risk include point-of-use testing, also known as drug checking, and qualitative user discernment of street heroin. Recent research has shown a high willingness among younger, US-based injection drug users to utilize fentanyl test strips , but theThis study is explorative qualitative research based on a convenience sample of heroin injectors in five US states. Due to the non-random sampling methods and semi-structured interview format, quantitative comparisons of drug sampling patterns across the research locations or by sample characteristics should not be made. The data comprise a snapshot of drug consumption behavior that can generate hypotheses but not conclusive findings. Interviews carried out at needle and syringe programs and other public health settings may be influenced by social desirability bias. Some interviewees were recruited outside of these environments, however, in an effort to mitigate this bias.Harm reduction services are central in the promotion of safer drug use techniques. Bearing in mind the non-random study sample, we found heroin sampling methods to be most common in Baltimore and Chicago, where robust harm reduction services have been established for more than two decades . In Massachusetts, two of the three cities visited had no official needle and syringe programs, while Worcester’s had only started in 2016, and few users mentioned using tester shots or other drug sampling methods. In Chicago, where most of those using tester shots and snorting were interviewed, the local harm reduction service, Chicago Recovery Alliance , has beeThe use of tester shots and other drug sampling methods as a means of preventing an overdose from injection drug use reduces the quantity absorbed at any one time, allowing users to monitor for drug strength and titrate their dose accordingly. In the face of greater perceived susceptibility to and severity of opioid-related overdose due to the rising presence of fentanyl-adulterated and -substituted heroin , this co


In order to ascertain the fire resistance performance of recycled aggregate concrete (RAC) components with different concrete compressive strengths, four full-scaled concrete columns were designed and tested under high temperature. Two of the four specimens were constructed by normal concrete with compressive strength ratings of C20 and C30, respectively, while the others were made from recycled coarse aggregate (RCA) concrete of C30 and C40, respectively. Identical constant axial forces were applied to specimens while being subjected to simulated building fire conditions in a laboratory furnace. Several parameters from the experimental results were comparatively analyzed, including the temperature change, vertical displacement, lateral deflection, fire endurance, and failure characteristics of specimens. The temperature field of specimens was simulated with ABAQUS Software  and the results agreed quite well with those from the experiments. Results show that the rate of heat transfer from the surface to the interior of the column increases with the increase of the concrete’s compressive strength for both RAC columns and normal concrete columns. Under the same initial axial force ratio, for columns with the same cross section, those with lower concrete compressive strengths demonstrate better fire resistance performance. The fire resistance performance of RAC columns is better than that of normal concrete columns, with the same concrete compressive strength. Statistics show that the most common and serious losses in connection with fires occur during building structure fires. Reinforced concrete (RC) construction accounts for a large proportion of buildings; during a building fire its structural performance is degraded, structural deformation increases, and the load-carrying capacity declines. Lots of studies focusing on the fire performance of concrete components were conducted. In the 1990s, Lie et al.  and othe[ et al. ,4,5 and [ et al.  investig[ et al.  studied  et al. [ et al. [ et al. [ et al. [ et al. [Recycled aggregate concrete (RAC), a new type of environmentally friendly material using waste concrete as recycled aggregates to substitute for natural aggregates, is made partly or totally by processes of washing, crushing, grading, and proportional mixing. Although RAC is no longer considered a new technology and many studies have focused on its use, currently most of the experimental studies on RAC fire resistance performance have been confined to RAC material rather than structural components ,12,13,14 et al.  analyzed[ et al.  conducte[ et al.  studied [ et al.  investig[ et al.  designedConsidering most columns used in low-rise buildings are concrete columns without an outer steel tube, it is essential to study their fire resistance performance. For this paper, in order to ascertain the fire resistance performance of RAC-constructed components with different concrete compressive strengths, the fire resistance performance of two RAC columns and two normal RC columns with different concrete compressive strengths was analyzed based on experimental data, with the aim of providing an experimental basis for fire resistance design using RAC columns.Four full-scale concrete columns were constructed: NCC-1, a normal RC column constructed from C20-specified concrete; NCC-2, a normal RC column from C30-specified concrete; RCC-1, a 100% RCA replacement RAC column with C30-specified concrete; and RCC-2, a 100% RCA replacement RAC column using C40-specified concrete. All specimens were constructed outside in winter. The geometry and reinforcement details of the four columns were identical, each with a cross section of 450 mm × 450 mm, a height of 3640 mm, longitudinal bars of 8D20, a ratio of reinforcement of 1.24%, D10-150 stirrups, and a volume stirrup ratio of 0.6%, as shown in 3. Its water absorption was 3.23%.These fine natural aggregates (FA) and coarse natural aggregates (NA) are typical in ready-mix concrete construction in North China. The RCA used in the research came from a local construction and demolition recycling plant. It was made from concrete columns and beams of an old building demolished at Guangqumen, Beijing, China; the test cubic compressive strength was 32.43 MPa. Since the RCA came from demolition, components such as bricks were inevitable (about 1.62 wt%). The RCA used in the research had an aggregate content of 40.1%, mortar aggregate content of 34.9%, residual mortar content of 23.42%, and deleterious material content of 1.62%. The density of the RCA was 2290 kg/mThe direct mass replacement method was used to replace NA with RCA. 5 MPa, respectively, and those of bar D10 are 433 MPa, 497 MPa, and 1.99 × 105 MPa, respectively.Each grade of concrete after 28 d was tested to ascertain its mechanical properties prior to heating—these results are shown in n, can be calculated by the following equation:N is the vertical axial load, which means the vertical load on the two ends of a column herein; Ac is the cross sectional area of a column and is assumed to remain unchanged unless concrete separates from the column during heating; and fc is the design value for concrete axial compressive strength, which is obtained by multiplying the experimental concrete cubic compressive strength by 0.478 according to GB50010-2010 [fc, in Equation (1), is also listed in The vertical load on a building during a fire usually remains constant most of the time. As the vertical load is the main concern in the design of a column, the same axial force ratio was used during the experiments to compare the fire resistance performance of columns with different concrete strengths. The vertical load on the columns was applied using a vertical loading device, which was set under the column and kept static during the fire test. The fire resistance test equipment used limits test duration to a maximum of 4 h, so in order to obtain a complete range of data during the heating process, a high axial force ratio was adopted to accelerate the process of column damage. The axial force ratio, The test was carried out in the test column furnace at the Testing Center of the National Fixed Fire Extinguishing System and Fire-Resistant Component Quality Supervision, Tianjin Fire Research Institute, China Ministry of Public Security . The test furnace is shown in T = T0 + 345lg(8t + 1) was used to control the heating. Here, T is the heating temperature in Celsius, T0 is the initial temperature in Celsius of the furnace, and t stands for the heating time in minutes. The actual heating curve, as shown in According to International Standard ISO834 , the heaThe specimens were allowed to cool down naturally after the heating test. The thermocouples used were of nickel-chromium or nickel-silicon, and chromel construction; three of these were arranged in the middle cross section along the height of the column, with a distance from the outer side of 15 mm, 112 mm, and 225 mm, respectively, as shown in H/100 (mm), or when the rate of the axial deformation reaches 3H/1000 (mm/min). H is the valid height of a column on fire, which is 3000 mm here. In this instance, the fire resistance duration of a column was determined to be when the axial deformation is greater than or equal to 30 mm, or when the rate of the axial deformation reaches 9 mm/min.The initial vertical load was determined by a fixed axial force ratio and remained unchanged during the heating test. The heating curve from ISO834 was adopted inside the furnace. According to Chinese Standard GB/T 9978-2008 , the firThe experimental fire resistance duration of specimens NCC-1, NCC-2, RCC-1, and RCC-2 is 227 min, 78 min, 118 min, and 39 min, respectively. All of the specimens failed because of the large axial deformation. The concrete cubic specimens were put into the furnace with the columns at the same time and subjected to International Organization for Standardization (ISO) fire exposure. The cubic compressive strength after fire was also tested; nloss after fire tests.The initial temperature inside the furnace was 20 °C. TNCC-1, TNCC-2, TRCC-1, and TRCC-2 represent the measured temperature of specimens NCC-1, NCC-2, RCC-1, and RCC-2, respectively.In order to compare the temperature at a given time in the columns as a function of the concrete class, the temperatures during the 39 min heating are listed in (1)Before the 39 min of heating, the curves of “measured temperature to time” of the four specimens appear very similar. The temperature variation at the cross section for both the normal RC columns and the RAC columns shows no significant difference.(2)The heating rate is slower and the fire resistance duration is longer for RAC column RCC-1 compared to normal concrete column NCC-2, which has almost the same concrete strength rating. This could be attributed to the greater porosity and water absorption in RCAs, causing greater moisture content, which may in turn consume more heat through water evaporation .(3)The fire resistance performance of lower concrete strength columns with the same cross section is better than that of higher concrete strength columns under the same initial axial force ratio. Because of the lower permeability with increased strength of concrete, higher concrete strength is more susceptible to spalling and results in lower fire resistance .(4)The variation tendency of heating curves from Measure Point 1, which is very close to the outside of the columns, is similar to that from ISO834  in Figur(5)Due to the rapid proliferation of internal cracks inside the columns through heating, at around 40 min heating, the heating rate at Measure Point 2 for normal concrete specimen NCC-1 and NCC-2 increases, while at Point 2 around 120 min and at Point 3 around 100 min for specimen NCC-1 the increase accelerates.(6)TNCC-2/TNCC-1 and TRCC-2/TRCC-1 in total for four specimens, with five of them greater than 1. The temperature for higher concrete strength columns at the same measure points and same moment is slightly higher, but the difference is small.As shown in The analysis in l is the axial displacement of columns, positive values indicate columns in a state of axial compression, and negative values indicate a state of extension. It can be seen from (1)The normal RC column NCC-2 shows axial extension before 48 min, while the columns NCC-1, RCC-1, and RCC-2 do not. The expansion of inner air voids in the vertical axial direction under the pressure of trapped expanding steam during heating made the axial extension for column NCC-2. Similar axial extension happened for columns under heating test in Miao’s paper . The ext(2)N under the similar axial force ratio n; N can be seen in N in Equation (1) remained constant. The actual area of the cross section of columns reduced during the test because the concrete started to crack and some material was shed under heating, which means fc and Ac in Equation (1) became smaller and the actual axial force ratio n became greater. This is the key factor in the sharp deformation of columns—the damage which occurred under the combined effect of heating and axial load.The RAC columns RCC-1 and RCC-2 exhibited no axial extension. The fire resistance performance of RCC-1 is better than that of RCC-2: Specimen RCC-2 deformed sharply and was seriously damaged at 39 min heating, while this happened 79 min later (at 118 min) for RCC-1. The cracks on the column with higher concrete strength started earlier because of its greater axial compression force (3)The concrete strength of the normal RC column NCC-2 is very close to that of the RAC column RCC-1. The axial displacement was only 2.3 mm at 78 min for NCC-2, then the axial deformation increased sharply and the column exhibited sudden damage. The same occurred to RCC-1 at 118 min, with an axial displacement of 15 mm. The duration of effective fire resistance for specimen RCC-1 is therefore 51.28% longer than that of NCC-2. The greater porosity in the RCAs enables the column RCC-1 to exhibit greater deformation under heating. This clearly shows that the RAC column has better fire resistance performance than the normal RC column.In order to measure the lateral deflection of the columns in horizontal direction, two asbestos-wrapped molybdenum wires were attached at the bottom and the middle of the columns, as shown in f is the lateral deflection, f1 and f2 are the lateral deflection at the middle and the bottom, respectively. The lateral deflection at the middle height of column NCC-2 reached 198.5 mm during failure.(1)The developing rate of lateral deflection of all the specimens became faster with the increase of heating time.(2)f1 to f2 increased more quickly at the later period with the increase of heating time.The ratio of (3)Compared to the normal RC column NCC-2 with a similar concrete strength, the lateral deflection of RAC column RCC-1 was smaller at the earlier heating and the deformation developing was relatively stable because more heat needed to be consumed by the greater moisture content in recycled aggregates; this induced less severe deformation in the specimen.(4)The concrete on the top part of column RCC-1 cracked and the column failed while there were no lateral displacements during the first 90 min of the fire at the bottom and middle part of the column.Cracks developing at the lower part of the columns on fire could be observed through an observation port on the furnace. We observed the concrete spalling on NCC-2 and RCC-2 because it occurred just at the area we could see. Unfortunately, we could not see spalling on columns NCC-1 and RCC-1 because it happened at the top area of the columns, as shown in (1)Specimen NCC-1. The observed cracks occurred at about 39 min. It could be observed through an observation port on the furnace that the dense cracks appeared at about 60 min. The bared coarse aggregate turned whiter and then the column failed at 227 min. There was a vertical crack with a maximum width of 2 mm running through the height of the column on the four sides. The concrete dropped from a large area, about 300 mm to 130 mm along the height from the top. The steel bars buckled to 50 mm at this position, which is 600 mm from the top.(2)Specimen NCC-2. The observed cracks occurred at about 28 min. Dense cracks appeared at about 40 min. The lateral deflection started to accelerate at 60 min and the column failed at 78 min. The maximum lateral deflection reached 198.5 mm. The concrete dropped at the area of 200 mm to 1110 mm from the bottom. The steel bars were exposed and buckled to 60 mm.(3)Specimen RCC-1. The observed cracks occurred at around 50 min. The cracks stretched visibly at about 90 min, and the specimen failed at 118 min. The concrete cover, which was 700 mm from the top, separated from the column and the inside steel bars were exposed. A vertical crack with a maximum width of 2 mm ran along the height of the column. The steel bars buckled to 50 mm, 350 mm down from the top of the column.(4)Specimen RCC-2. The observed cracks occurred at about 30 min. The cracks stretched visibly at about 35 min. At 36 min 51 s, concrete spalling occurred on the column over a large area, 50 mm to 1100 mm up from the bottom, and the inside steel bars were exposed. Due to the non-homogeneity of concrete, which gives each specimen a unique material structure and a unique distribution of voids and moisture inside the concrete mass, the damage process of each specimen under heating is different and can be described as follows:c; and specific heat capacity, cc. The fire temperature changes with the heating time, and the thermal performance parameters of the material change with the fire temperature. The temperature field analysis of a structure is a nonlinear transient heat conduction problem.The thermal performance parameters of concrete include bulk density, ρ; thermal conductivity, λFor this paper, the cross section analysis of the temperature field was independently analyzed according to . The parc, W/(m·°C).Thermal conductivity λWhen the RCA replacement is 0%:When the RCA replacement is 100%:cc, J/(kg·°C).Specific heat capacity When the RCA replacement is 0%:When the RCA replacement is 100%:3).Bulk density ρ, , the thermal conductivity of the material was set in software, and the mass density and specific heat parameters were determined thereby. The measured furnace temperature was used as a heating parameter. The four lateral sides of columns were set as the fire surfaces, while the top and the bottom sides were set as the adiabatic surfaces. The eight-node continuous heat conduction 3D solid element DC3D8s were used and meshed as shown in Analysis from the calculated results shows that: (1) the temperature field at the four corners of the cross section exhibits arc-shaped distribution due to the bi-directional nature of the fire source; (2) a significant temperature gradient exists between the (hot) surface and (cooler) interior of the cross section; and (3) the area exhibiting higher temperatures spread inward gradually as the fire time increased.(1)The rate of heat transfer from the surface to the inside of the column increases with compressive strength for both RAC columns and normal RC columns during fire conditions.(2)Under the same initial axial force ratio, for both RAC columns and normal RC columns with the same cross section, those with lower concrete compressive strengths demonstrate better fire resistance performance. The fire resistance performance decreases as the concrete compressive strength increases, due to its higher density and lower porosity, which cause cracks to appear earlier and the material to degrade more rapidly.(3)The fire resistance performance of RAC columns is better than that of the normal concrete columns with the same concrete compressive strength. This is because the rate of temperature penetration for RAC columns is lower than that of normal RC columns under nominal fire condition heating.(4)Finite element method (FEM) analysis on the temperature fields of both the normal concrete columns and RAC columns, which showed almost the same results as the experiments, is able to be used to avoid the expensive test cost.(5)The RAC columns may be applied in practical projects.


The major therapeutic strategy used to treat exacerbated cystic fibrosis (CF) is antibiotic treatment. As this approach easily generates antibiotic-resistant strains of opportunistic bacteria, optimized antibiotic therapies are required to effectively control chronic and recurrent bacterial infections in CF patients. A promising future for the proper use of antibiotics is the management of lung microbiota. However, the impact of antibiotic treatments on CF microbiota and vice versa is not fully understood. This study analyzed 718 sputum samples from 18 previous studies to identify differences between CF and uninfected lung microbiota and to evaluate the effects of antibiotic treatments on exacerbated CF microbiota. A reference-based OTU  picking method was used to combine analyses of data generated using different protocols and platforms. Findings show that CF microbiota had greater richness and lower diversity in the community structure than uninfected control (NIC) microbiota. Specifically, CF microbiota showed higher levels of opportunistic bacteria and dramatically lower levels of commensal bacteria. Antibiotic treatment affected exacerbated CF microbiota notably but only transiently during the treatment period. Limited decrease of the dominant opportunistic bacteria and a dramatic decrease of commensal bacteria were observed during the antibiotic treatment for CF exacerbation. Simultaneously, low abundance opportunistic bacteria were thriving after the antibiotic treatment. The inefficiency of the current antibiotic treatment against major opportunistic bacteria and the detrimental effects on commensal bacteria indicate that the current empiric antibiotic treatment on CF exacerbation should be reevaluated and optimized. Pseudomonas aeruginosa and Staphylococcus aureus, induce intense bronchial neutrophilic inflammation. As a result of chronic polymicrobial infection and inflammation, CF is a devastating life-threatening disease with the majority of mortalities due to respiratory failure [exacerbated and clinically stable. Pulmonary exacerbation is the hallmark of CF and is characterized by aggravated pulmonary symptoms and decreased pulmonary function. A clinically stable condition is the stationary phase before or after pulmonary exacerbation.Cystic fibrosis (CF), as an inherited disease of the secretory glands, is caused by defective mutations of the CF transmembrane conductance regulator (CFTR) gene –3. Thick failure , 7. OverStreptococcus, Veillonella, Prevotella, and Actinomyces. [Unlike the traditional view of a sterile lung, accumulating evidence shows that a microbial community resides in the lung tissue; this is referred to as the lung microbiota –11. Healnomyces. , 12–14. nomyces. , 16. Decnomyces. –20. A sunomyces. , 21–23.Chronic and recurrent colonization of opportunistic bacteria causes devastating lung damage and respiratory decline. Intravenous (IV) injection of broad-spectrum antibiotics is the leading therapy for treating pulmonary exacerbation. A typical IV-antibiotic treatment usually requires a duration of two weeks and is inefficient at controlling infections because of the easily generated antibiotic-resistant bacterial strains –29. ThusManagement of lung microbiota provides a promising alternative for the treatment of CF with pulmonary exacerbation , 10. ProThis study analyzed the largest number of airway microbiota from CF patients and uninfected controls from published studies to evaluate the differences between uninfected and CF-derived microbiota. The efficacy of the antibiotic treatments in regulating the composition of lung microbiota in CF patients was also evaluated. In general, intravenous antibiotics treatment is the most popular strategies for CF exacerbation, while oral intake of antibiotics is commonly used to control seizures. The antibiotic treatment mentioned in this study refers to the large overdose intravenous therapy for CF exacerbation. Overall, our findings show that CF microbiota were richer and had a lower diversity in the community structure than NIC microbiota The obtained data provide insights necessary to evaluate the current therapeutic strategy.et al [The items used in this study were the same as those defined by Rabin et al  with somHigh-throughput sequencing data of 16s rRNA were collected from public databases . BacteriTo compare sequences from different regions of the 16s rRNA gene, a reference mapping protocol –39 was uhttp://bioinfogp.cnb.csic.es/tools/venny/index.html). Alpha diversity measurements of community richness and diversity were analyzed by estimators of Chao1 and Shannon indices, respectively. Default parameters were applied and boxplots were created using the “Phyloseq” in R package [http://qiime.org/tutorials/tutorial.html).Shared OTUs among different sample groups were plotted in Venn diagrams using Venny software  with accession numbers listed in The 16s rRNA sequencing data and corresponding metadata used in this study are available in publically accessible databases  tests were performed to assess OTUs that significantly differed between sample categories. T-tests were used to determine α-diversity significance. Analysis of similarities (ANOSIM) tests were performed to determine β-diversity significance.p < 0.01, Alpha diversity measurements were used to assess the variation in community structure between the microbiota of CF patients and NICs. Specifically, Chao1 and Shannon indices were employed to analyze community richness and diversity, respectively . These rAs the microbiota of the two CF stages and NICs exhibited divergence in the alpha diversity measurements, beta diversity indices were then calculated to depict this divergence. The unweighted UniFrac-PCoA analysis demonstrated that the microbiota of NICs can be easily distinguished from that of CF patients . No markVenn diagrams were then generated to analyze the differences in the bacterial composition between the CF and NIC microbiota . The NICPseudomonas was the major component of CF-Base and CF-Exa microbiota with a relative abundance of approximately 30%  and slightly increased community diversity (p = 0.24) compared to the microbiota of Exa-BeforeTreat. These findings demonstrate that antibiotic treatment plays an important role on the community structure of the microbiota in patients of CF with pulmonary exacerbation. However, the community richness and diversity of Exa-AfterTreat microbiota returned to levels similar to those of Exa-BeforeTreat microbiota , the structure of the bacterial community in the NIC microbiota was completely different from that found in the CF microbiota. CF microbiota had greater community richness and lower diversity compared to NIC microbiota, indicating that the colonization of opportunistic bacteria had important effects on the community structure of the lung microbiota. These differences were further supported by beta diversity analyses and were consistent with previous reports [Healthy microbiota in the airway mucosa is essential to properly shape the immune response. Changing a well-balanced “healthy” microbiota to an unhealthy restricted microbiota renders the airway mucosa increasingly susceptible to pathogens such as g injury , 45, 46.g injury , asthma g injury , interstg injury , etc.),  reports , demonstMany studies have long searched for a correlation between certain lung microbes and the severity of CF disease. To date, only the loss of microbial diversity has been associated with severe lung disease in CF ; this noPseudomonas was not significantly affected by antibiotic treatment (Pseudomonas is the major opportunistic bacteria found in CF patients with pulmonary exacerbation, more effective antibiotics targeting Pseudomonas species are required to control these bacteria. Interestingly, the relative abundance of Staphylococcus, another important opportunistic bacteria, was dramatically reduced by antibiotic treatment but these abundance levels returned to their original levels after the withdrawal of antibiotics. This noteworthy phenomenon has also been reported in a previous study [Although intense empiric antibiotic treatment is commonly used to control CF with pulmonary exacerbation, its effects on CF-Exa microbiota must be evaluated to further optimize this strategy , 33, 52.reatment , which ius study  and demoIn summary, this study employed microbiota data from a collection studies to analyze the differences between the microbiota of uninfected healthy individuals and CF patients and to evaluate the impact of antibiotic treatments for CF. CF-Exa microbiota had comparable richness but lower diversity compared to NIC microbiota. Antibiotic treatments for CF patients with pulmonary exacerbation slightly reduced the presence of opportunistic bacteria, but markedly reduced the abundance of commensal bacteria and allowed the proliferation of untargeted opportunistic bacteria. Thus, this study highlights the deleterious influences of antibiotic treatment to microbiota of CF exacerbation, which emphasizes the need to optimize empiric antibiotic treatments.


Although increased serum IgG4 level and tissue infiltration of IgG4-positive cells are key events in IgG4-related disease (IgG4RD), and nearly half of IgG4RD patients show hypocomplementemia, the role of IgG4 in the pathogenesis of IgG4RD remains unclear. Many reports show that altered IgG glycosylation, especially IgG with agalactosylated N-linked glycan (G0 N-glycan), have proinflammatory roles including complement activation, implicated in the pathogenesis of various inflammatory diseases. This study determined the concentration of N-linked glycans (N-glycan) released from serum IgG4 in IgG4RD patients and compared the difference of glycosylation changes to those in healthy controls. We also compared the concentration of each IgG4 glycoform between patients with and without hypocomplementemia and individual organ involvement  in IgG4RD.Map® Xpress methodology.We collected sera from 12 IgG4RD patients and 8 healthy controls. IgG4 was isolated from sera via Melon™ Gel IgG Spin Purification Kit followed by Capture Select IgG4 (Hu) Affinity Matrix. IgG4 N-glycans were analyzed by S-BIO GlycanSignificant increases of IgG4 G0 N-glycan and IgG4 fucosylated N-glycan (F1 N-glycan) concentrations were observed in IgG4RD compared with healthy controls. Although we observed decreased levels of IgG4 F0 glycan in IgG4RD with hypocomplementemia, there were no significant differences in the galactosylation and sialyation of IgG4 N-glycans. Furthermore, there were no significant differences in the glycosylation of IgG4 N-glycans between patients with and without individual organ involvement of IgG4RD.Although IgG4 has anti-inflammatory properties, IgG4 G0 and F1 glycans were increased in patients with IgG4RD. Our results suggest that decreased galactosylation of IgG4 is not related to complement activation and the differences of individual organ involvement in IgG4RD. IgG4 fucosylation change may be related to complement activation in IgG4RD. Further investigation is needed to clarify the role of IgG4 in IgG4RD. IgG4-related disease (IgG4RD) is an emerging clinical disorder characterized by increased serum IgG4 concentrations, dense lymphoplasmacytic infiltrate, IgG4-positive plasma cells, and storiform fibrosis , 2. IgG4in vivo to form antibodies with two different binding specificities that are monovalent [Among the subclasses of IgG, IgG4 is the least abundant subclass in human serum. IgG4 is distinguished from other IgG subclasses by its unique functional and structural properties. IgG4 lacks the ability to activate the classical complement pathway because of reduced binding capacity to complement component 1q (C1q)–17. IgG4novalent .Although IgG4 itself does not activate the classical complement pathway because of a low C1q binding capacity, nearly half of IgG4RD patients show hypocomplementemia, 20. SevProtein glycosylation is a post-translational modification affecting the structures and functions of many proteins required for normal immune function. Over the past decades, many studies have investigated the structural and biological roles of immunoglobulin G (IgG) glycosylation. In IgG molecules, two N-glycans are linked to heavy chains at asparagine (Asn) residues (CH2–84.4) in the CH2 domain of the Fc portion, 25. Fc-Recent studies revealed that IgG glycosylation, especially the degree of galactosylation, is associated with various physiological and pathological conditions. ClinicaAlthough recent studies revealed the importance of altered IgG glycosylation in the pathogenesis of various autoimmune and inflammatory diseases, little is known about the glycosylation of IgG4 in IgG4RD. In this study, we determined the occurrence (%) and absolute concentration of each glycoform released from IgG4 isolated from the sera of IgG4RD patients and healthy controls. We also compared the differences of glycosylation changes between patients with IgG4RD and healthy controls. The occurrence (%) and absolute concentration of each glycoform released from IgG4 isolated from the sera of IgG4RD patients were compared between patients with and without hypocomplementemia and individual organ involvement  for galactosylation, fucosylation, and sialyation in IgG4RD.Overall, 12 patients with IgG4 related disease (IgG4RD) who fulfilled the comprehensive diagnostic criteria for IgG4RD[g for 1 min to collect purified IgG. The concentration of purified IgG was measured by absorbance at 280 nm using an extinction coefficient (E2800.1%) of 1.4.IgG was isolated using the Melon™ Gel IgG Spin Purification Kit  according to the manufacturer’s instructions. Briefly, serum was dialyzed with Melon™ Gel purification buffer, and mixed with 20% slurry gel in a spin column. After end-over-end mixing for 5 min at room temperature (RT), the spin column was centrifuged at 5,000 ×2O, then concentrated using Amicon ultra-0.5 ml (Ultracel®-3K) . Protein concentrations were determined using the Bio-Rad Protein Assay . IgG4 purities were established by SDS-PAGE and western blotting.IgG4 was isolated via Capture Select IgG4 (Hu) Affinity Matrix  according to the manufacturer’s instructions. Briefly, purified IgG was loaded onto a 2 ml Capture Select IgG4 column in fully preserved PBS. Then, the column was eluted with 0.1 M glycine-HCl buffer (pH 2.8). The eluted IgG4 fractions were neutralized with 1/10 volume Tris-HCl (pH 9.0). Purified IgG4 was immediately buffer exchanged in ddHIsolated IgG4 were analyzed by western blotting assays for IgG subclass composition. Isolated IgG4 were resuspended in non-reduced sample buffer, subjected to 10% SDS-PAGE, and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane. After incubation with blocking buffer, the membrane was incubated separately with biotinylated monoclonal anti-human IgG subclass 1–4  diluted at 1:1000 in blocking buffer. After incubation at 4°C overnight, the membrane was washed with PBS-T, incubated with Vectastain ABC Reagent  for 30 min at RT, and subsequently developed with 3,3′-diaminobenzidine tetrahydrochloride. The color of the image was converted from brown to black by computer.Map® Xpress methodology based on methods previously reported[Each sample was analyzed to quantitate N-linked glycans using S-BIO proprietary Glycan reported–41. SeveMALDI-TOF mass spectrometry (MS) analysis was performed on an ultraflex III mass spectrometer  in the positive-ion, reflectron mode using S-BIO proprietary matrix composition. Each sample was spotted in quadruplicate, and spectra were obtained in an automated manner using the AutoXecute feature in flexControl software .Map® Xpress assay.Resulting mass spectra were analyzed using S-BIO proprietary bioinformatic programs, which correct for mass differences induced by the chemical derivatization inherent in the GlycanGlycan compositions were assigned to each peak based on m/z. The intensities from each of the four quadruplicated spectra were normalized to that of an internal standard with a known concentration and averaged. Data were also corrected for the inherent decrease in peak intensity as m/z increases in mass spectrometric measurements, yielding absolute concentrations that are comparable to those created by HPLC/fluorescent methods. Glycans representing less than 0.5% of the total glycan concentration were not reported.Glycan compositions were shown as a code of five digit. The code represents the number of hexose , Mannose (Man), or Glucose (Glc)), N-acetylhexosamine (N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine ), deoxyhexose (Fucose (Fuc)), N-acetylneuraminic acid (N-acetylneuraminic acid (Neu5Ac)), and N-glycolylneuraminic acid (N-glycolylneuraminic acid (Neu5Gc)) residues.U-test for the comparison of median values. Statistical analyses were performed using SPSS software, version 22 . P values less than 0.05 were considered statistically significant.Statistical analysis was performed using the Mann-Whitney The purity of IgG4 isolated from the sera of patients with IgG4RD was confirmed by SDS-PAGE and western blot analysis. We did not observe protein contamination by SDS-PAGE  S1 Fig)S1 Fig). We detected 23 glycans released from isolated IgG4 . ProposeResults are expressed as the percentage of each glycan group released from IgG4 isolated from the sera of IgG4RD patients and healthy controls. As shown in Based on the number of galactoses, N-glycans released from IgG4 (IgG4 glycan) were divided into 4 groups: glycans without galactose (G0 IgG4 glycan), with monogalactose (G1 IgG4 glycan), with digalactose (G2 IgG4 glycan), and with trigalactose (G3 IgG4 glycan) . AbsolutRegarding G1 IgG4 glycans, there was no significant difference in total G1 IgG4 glycan between IgG4RD and healthy controls, although 3 of 7 G1 IgG4 glycans  were significantly increased in IgG4RD compared with healthy controls .Regarding G2 IgG4 glycans, there was no significant difference in total G2 IgG4 glycan between IgG4RD and healthy controls, although 4 of 10 G2 IgG4 glycans  were significantly increased in IgG4RD compared with healthy controls .As shown in Based on the number of fucoses, IgG4 glycans were divided into 2 groups: glycans without fucose (F0 IgG4 glycan) and with fucose (F1 IgG4 glycan) . As showBased on the number of sialic acids, glycans detected in IgG4 were divided into 3 groups: glycans without sialic acid (S0 IgG4 glycan), with 1 sialic acid (S1 IgG4 glycan), and with 2 sialic acids (S2 IgG4 glycan) . As showIgG4 glycan level was compared between patients with and without hypocomplementemia and for individual organ involvement  for galactosylation, fucosylation, and sialyation in IgG4RD. As shown in This study reports significant increases of G0 IgG4 glycan concentrations in IgG4RD compared with healthy controls. Furthermore, F1 IgG4 glycan levels in IgG4RD were significantly increased in IgG4RD compared with healthy control. However, there were no significant differences in the levels of sialic acid or IgG4 glycan with bisecting arm between IgG4RD and healthy controls. Furthermore, we observed a significant decrease in F0 IgG4 glycans in IgG4RD with hypocomplementemia compared with those without hypocomplementemia, although the difference was small. There were no significant differences in the galactosylation and sialyation of IgG4 glycans between IgG4RD with and without hypocomplementemia. Regarding the individual organ involvement of IgG4RD , there were no significant differences in the galactosylation, fucosylation, and sialyation of IgG4 glycans between patients with and without each organ involvement.Recent studies strongly suggest that G0 glycans have proinflammatory nature, 37, 42.IgG subclasses other than IgG4 have proinflammatory properties that induce activation of the complement system as well as stimulating phagocytic or cytotoxic reactions. Unlike other IgG subclasses, IgG4 does not have the same proinflammatory properties, because IgG4 does not bind to C1q compared with FcγRI, RII, and RIIII. FurtherHowever, although IgG4 has anti-inflammatory properties, it does not mean that IgG4 is not pathogenic. Several reports showed that IgG4 autoantibodies were implicated in the pathogenesis of autoimmune diseases without requiring complement or other immune components. Huijhers et al reported that IgG4 autoantibodies to muscle-specific kinase cause myasthenia gravis by inhibiting the binding between muscle specific kinase and lipoprotein receptor-related protein, 48. In Elevated levels of G0 IgG were reported in the serum of patients with granulomatosis with polyangiitis . InteresIn this study, we observed a significant increase in F1 glycan concentration. Recently, Gornik et al. reported the increased level of serum IgG with F1 glycan in patients with RA. FurtherIn this study, although there were no significant differences, the absolute concentration of G1, G2, and F0 glycans tended to be higher in IgG4RD than in healthy controls; thus, the total concentrations of all IgG4 glycans were increased in IgG4RD. As a result, the percentages of G0 as well as F1 glycans were lower and there was no significant difference between IgG4RD and healthy controls. Thus, the increase in total IgG4 glycans might be caused by increased amounts of Fab glycans. Because we used whole IgG4 as a sample, we detected N-glycans including the Fc and Fab portion of IgG4. Recent studies revealed that between 15%–25% of the total IgG glycome originates from Fab, 59, andRecent studies show that agalactosylated IgG acts as an epitope for complement-activating mannose-binding lectins resulting in activation of the lectin complement pathway. FurtherRegarding the individual organ involvement of IgG4RD , there were no significant differences in the galactosylation, fucosylation, and sialyation of IgG4 glycans between patients with and without each organ involvement, which may suggest that the altered glycome of IgG4 is not related to the differences observed for individual organ involvement.Although the elevation of agalactosylated IgG levels are associated with several inflammatory and autoimmune diseases, the cause of the increase is not well understood. One possibility might be that reduced B-cell galactosyltransferase activity leads to lower galactosylation. Axford et al reported an association between reduced galactosyltransferase activity and serum agalactosylated IgG level in RA, 65. AnoThis study had several limitations. First, the numbers of patients and controls were relatively small because of our research budget. Second, it was impossible to differentiate N-glycans released from the Fab and Fc portions of IgG4. We used whole IgG4 as samples; therefore, we detected N-glycans from the Fc portion and the Fab portion of IgG4. Finally, because the number of IgG4RD patients and healthy controls were different, their age and gender were not matched. The proportion of agalactosylated IgG was reported to increase with age. AlthougAlthough we observed changes in IgG4 glycosylation isolated from the sera of patients with IgG4RD compared with healthy controls, it is not clear whether the increased IgG4 is the cause or a consequence of the inflammatory reactions associated with IgG4RD. Further studies of IgG4RD are needed to clarify the mechanism of IgG4 production and the role of IgG4 in the pathogenesis of IgG4RD.S1 FigOriginal uncropped gel.(TIF)Click here for additional data file.S2 FigOriginal uncropped blot.(TIF)Click here for additional data file.S3 FigOriginal uncropped blot.(TIF)Click here for additional data file.S4 FigOriginal uncropped blot.(TIF)Click here for additional data file.S5 FigOriginal uncropped blot.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


To determine the incidence of afferent limb failure of the in-hospitalMedical Emergency Team, characterizing it and comparing the mortalitybetween the population experiencing afferent limb failure and the populationnot experiencing afferent limb failure.HospitalPedro Hispano occurred from January 2013 to July 2015. A sampleof 285 activations was obtained after excluding incomplete records andactivations for patients with less than 6 hours of hospitalization. Thesample was divided into two groups: the group experiencing afferent limbfailure and the group not experiencing afferent limb failure of the MedicalEmergency Team. Both populations were characterized and compared.Statistical significance was set at p ≤ 0.05.A total of 478 activations of the Medical Emergency Team of Afferent limb failure was observed in 22.1% of activations. The causalanalysis revealed significant differences in Medical Emergency Teamactivation criteria (p = 0.003) in the group experiencing afferent limbfailure, with higher rates of Medical Emergency Team activation for cardiacarrest and cardiovascular dysfunction. Regarding patient outcomes, the groupexperiencing afferent limb failure had higher immediate mortality rates andhigher mortality rates at hospital discharge, with no significantdifferences. No significant differences were found for the otherparameters.The incidence of cardiac arrest and the mortality rate were higher inpatients experiencing failure of the afferent limb of the Medical EmergencyTeam. This study highlights the need for health units to invest in thetraining of all healthcare professionals regarding the Medical EmergencyTeam activation criteria and emergency medical response systemoperations. Despite post-resuscitation interventions, IHCA is usually associated with a worseprognosis than that of out-of-hospital cardiac arrest (CA). Furthermore, approximately 50% ofthese hospitalizations are potentially preventable because progressive clinical deterioration usuallyoccurs.,5In-hospital cardiac arrest (IHCA) occurs in up to 5:1,000 adulthospitalizations.-8 These systems have afferent and efferent limbs,regardless of the model. The afferent limb consists of clinical monitoring andsurveillance, the detection of patient clinical deterioration, the knowledge of theMedical Emergency Team (MET) activation criteria and its activation. The MET is theefferent limb. The healthcare professionals at each institution should be aware ofand familiar with the MET activation criteria in force in their hospital. Despitethe investment in training in this area, the literature recognizes the existence ofdeficient levels of knowledge and the persistence of barriers, which result indelayed MET activation. The MET is not activated in 30 to 78% patients with METactivation criteria; even when activation occurs, there is an average delay of 16hours.-13 This afferent limb failure (ALF) has a negativeimpact on prognosis andresults in obvious expenses for the health system.Several hospital units have developed medical emergency response systems (MERS) forpreventing CA by identifying and correcting factors in patients at risk for IHCA ina timely manner.Hospital Pedro Hispano has been in operation since 1998.Until 2002, the activation criteria were exclusively CA or respiratory arrest. From2002, the criteria were broadened, and teams experienced in the emergency care andmanagement of seriously ill patients are now activated using predefinedcriteria. Based on therecords of MET activations at Hospital Pedro Hispano, we aimed todetermine the incidence of in-hospital MET afferent limb failure, characterizing itand comparing the mortality of the population experiencing ALF with that of thepopulation not experiencing ALF.The MERS of Hospital Pedro Hispano, Unidade Local deMatosinhos (ULSM), E.P.E., which serves the municipality of Matosinhosand its 175,478 inhabitants. This hospital had 345 beds distributed according todifferent medical specialties .Hospital Pedro Hispano received patientsreferred from the Centro Hospitalar da Póvoa de Varzim/Vila doConde, which has a capacity of 145 beds serving a population of 143,000inhabitants.The study was performed at ® System .Data were collected from the internal emergency forms, which were available in theelectronic medical records and were filled out by the MET physician at the end ofeach activation. Some incomplete forms were filled out when possible to gather themissing data using the records of vital parameters, recorded by the nurses of theSClínicoAll MET activations from January 2013 to July 2015 were analyzed, totaling 478.Activations for outpatients (n = 29) and patients hospitalized for less than 6 hours(n = 48) and activations with incomplete forms (n = 116) were excluded. The finalsample consisted of 285 activations.The MERS afferent limb is based in the ICU, which, once activated, sends a teamtrained in emergency response and the management of seriously ill patients. Theafferent limb is composed of healthcare professionals working in wards and trainedin Basic Life Support (BLS) and MET activation criteria identification. A trainingprogram with regular recertification in BLS, Immediate Life Support (ILS) andAdvanced Life Support (ALS) for target populations selected based on the careresponsibilities of the professionals is also included in this model. The trainingprogram is held at an Emergency School of the ULSM, accredited by the PortugueseResuscitation Council. The MERS includes emergency carts with standardized contentthat are distributed at selected sites in the hospital to support responses toemergency situations. The MERS is preferentially contacted by dialing a directtelephone number and includes an audit program for system maintenance quality, withdesignated managers for system-wide management.The MET consists of a physician and a nurse, trained in ALS and experienced inresuscitation, who predominantly work in the ICU. The MET is available 24 hours aday and is activated when specific criteria are met by dialing a direct number(2211) to call the team at the ICU.The existence of a MET activation delay longer than 15 minutes, in the presence ofthe activation criteria, was considered an ALF. This time period was measured usingthe MET activation time and the time of the measurement of vital signs or clinicalrecords meeting the activation criteria. The sample was divided into two groups: theMET ALF group and the MET non-ALF group. The activation criteria are outlined inStatistical analysis was performed using the program Statistical Package for theSocial Sciences (SPSS), version 17.0 . The statistical description was performed using the counts andpercentages for categorical variables and the median, maximum and minimum valuesfor continuous variables. Comparisons between samples were performed using thechi-squared test. We considered the results to be statistically significant whenp < 0.05.Hospital Pedro Hispanofrom January 2013 to July 2015. The final sample is outlined in A total of 478 MET activations occurred at Of a total of 285 activations, 58.6% were for female patients. The patient agesranged from 11 to 94 years, with a median of 75 years. Most activations occurred forpatients older than 75 years (55.1%). Regarding the MET activation site, most(52.3%) activations occurred for patients hospitalized in surgical wards ,compared with 43.5% of cases hospitalized in medical wards . In 4.2% of cases, the patients were in the imaging unit. Of the 285activations, 10.9% were for patients who had invasive procedures performed in theprevious 24 hours ; in 5.6% of cases, the patients had been in the ICU, the MICU or theemergency room in the previous 24 hours.The most common MET activation criterion was an altered state of consciousness(43.2%), followed by CA (33.0%) and a threatened airway (19.2%). Treatmentlimitation decisions occurred in 10.9% of cases, and do-not-resuscitate (DNR) orderswere documented in 4.2% of cases. At the end of the MET intervention, 28.4% ofdeaths were recorded; DNR orders were followed in 5.6% of cases, and treatmentlimitation decisions were noted in 8.8% of cases. The overall hospital mortality was51.2%. Multiple MET activations in the same hospitalization episode occurred in ninepatients (3.15%).Afferent limb failure was observed in 22.1% of activations. This cross-sectional retrospective study evaluated data for patients treated by theMET. One in five activations were triggered by situations whose subsequentevaluations identified the existence of MET activation criteria in the previous 6hours, thus indicating MET ALF, which resulted in an increased number of activationsfor CA and increased mortality. who found ALF,defined as the presence of MET activation criteria from 15 minutes to 24 hoursbefore MET activation, CA or ICU admission. The prevalence of ALF was 22.8% amongthe 575 events recorded. The number of ALF cases identified in our study was similar(22.1%), and this high incidence justifies a reflection aimed at identifyingpossible causes to be corrected and starting urgent corrective interventions.This problem has already been identified in the literature in the study by Trinkle etal., reported that ALFis independently associated with an increase in hospital mortality.In our study, we observed that higher percentages of cases of CA and cardiovasculardysfunction and higher immediate mortality rates and higher mortality rates athospital discharge were observed for patients in the ALF group than for patients inthe non-ALF group, but the differences were non-significant. These results arecorroborated in the literature. An observational prospective study by Tirkkonen etal.At this point, we must analyze several factors, including MET maturity and thetraining of healthcare professionals working in wards and hospital units withdifferent levels of patient monitoring, including ICUs, intermediate care units andthe operating room. Situations of DNR orders and treatment limitation decisions,albeit not explicitly assumed and which may cause inappropriate MET activations,must also be analyzed. Another factor that may have contributed to this high ALFrate is the sensitivity of the activation criteria used. report thatthe MET of Melbourne Hospital in Australia also consists of a physician and a nursespecifically trained in ALS and resuscitation. The MET operates 24 hours a day, andelectronic medical records are completed for all activations at the end of eachactivation, similar to our MERS. The MET may be activated for any person in thepresence of MET activation criteria. Calzavacca et al. compared two cohorts ofpatients with similar characteristics: a recent cohort receiving a MET review andanother cohort receiving a MET review 5 years earlier at the start of itsimplementation, concluding that decreased ALF was observed in the group with the5-year team and explaining that team maturation leads to decreased ALF.The studied MET characteristics are similar to those reported for other METs.Calzavacca et al. Thus, at this point in time, we may assume that the teamand the entire system are presently mature and that no AFL would occur.The MET analyzed in this study has existed in the hospital since 1998. From 1998 to2002, the MET activation criteria were exclusively CA or respiratory arrest; thecriteria have been extended since 2002. A study conducted in our hospital includedall MET activations for patients hospitalized from 2002 to 2006 and found a decreasein CA correlated with the increase in MET maturity, combined with an integratedtraining program for healthcare professionals.,19,20 There is a need to strengthen the knowledge of METactivation criteria for those who assess and register patient monitoring parametersby implementing training sessions and presenting the data shown in this study. Inthe specific case of our in-hospital emergency system, training in BLS has decreaseddue to budget constraints, which may be correlated with this increase in ALF amongALS teams.In hospitals, new professionals are constantly hired, including for nursing teams.The new staff members may not be adequately informed and familiar with the METcriteria and method of activation or may still fear performing an unnecessaryactivation, causing an increase in MET ALF. Several studies suggest that increasingand improving training, familiarization, the continuous training of healthcareprofessionals and the existence of audits and feedback of METs are essential for METsuccess and ALF reduction.Another factor that may have contributed to the ALF values observed is that patientshad been previously admitted to different hospital units, including the operatingroom, the ICU, the MICU and the emergency room, during the same hospitalization for5.6% of the MET activations. Early discharges from those hospital units may haveoccurred. Therefore, greater investment towards increasing the sensitivity andknowledge of healthcare professionals regarding procedures and pathologies requiringlonger hospitalizations in different units, with higher levels of clinicalsurveillance and monitoring, should be considered. However, continuous monitoring isassociated with a better outcome in patients with prior IHCA,22 because less frequent monitoring may delay the detection ofinstability and may contribute to increased MET ALF. Other studies do not supportthis premise, including one study reporting that 40.6% of anomalies found incontinuous monitoring resulted from artifacts.The level of patient monitoring depends on the hospital unit in which patients arehospitalized. In our study, most activations occurred for wards in which monitoringis performed with standardized assessments of vital signs every 6 hours andaccording to the recommendations from the Rapid Response Team Consensus Conference,which advocates that vital signs should be assessed every 6 to 12 hours.Another factor that may have contributed to bias in the MET ALF rate found in ourstudy is the existence of activations for patients with prior DNR orders (4.2%) andtreatment limitation decisions (10.9%). In these cases, tolerance and delayed METactivation occur more frequently in the presence of abnormal vital signs. Thepercentage of cases found in our study was similar to that of cases described by theMERIT multicenter study (7.85%). reported that DNR orders existed in 6.2% of casesbut were not documented or had not been conveyed to the MET. The DNR/treatmentlimitation process requires time to communicate with the patient and relatives andamong clinicians involved in treating the patient. MET activation may be a valuableopportunity to start this process. In the case of our study, higher rates of DNR orders (6.35%)and treatment limitation decisions (14.3%) were found in the group of ALF patientsthan in the group of non-ALF patients at the end of the MET intervention.In addition to cases with previously documented DNR orders, it should be noted thatthere could be cases of DNR orders that were not documented or conveyed to thenursing team of the ward or the MET. Mitchell et al.Strategies rendering DNR orders and their documentation and communication to theother elements of the team more effective must be developed.,27Another factor that may have contributed to this high MET ALF rate is the use ofrelatively few sensitive activation criteria in our system. In-hospital emergencysystems using more sensitive criteria, including the National Early Warning System(NEWS) and the Modified Early Warning System (MEWS), which use scoring and alertsystems based on a set of criteria, including temperature and oxygen saturation, inaddition to urinary output, have been reported in the literature.This study was conducted in only one center, and the results may not apply toother settings. Furthermore, the analysis of the activation criteria present 6hours before the actual MET activation had some incomplete records. Thus, thosepatients were not included the analysis, which may have led to bias in thesample.The timely identification of patients with clinical deterioration does not alwaysoccur in hospital units. An increased incidence of cardiac arrest and highermortality rates are observed in patients for whom this identification fails.This study showed the need for hospital units to invest in training and familiarizingall health professionals with the Medical Emergency Team activation criteria andin-hospital emergency system operations.