We investigated the relationships between intelligence, alcohol consumption, and adverse consequences of drinking in young men from Norway (a neighboring Scandinavian country) born in the late 1970s.Research suggests that intelligence is positively related to alcohol consumption. However, some studies of people born around 1950, particularly from Sweden, have reported that higher intelligence is associated with n = 1126). Measures included self-reported alcohol consumption/intoxication, alcohol use disorders (AUDIT), and a scale measuring adverse consequences of drinking. Controls included family background, parental bonding, and parents’ and peers’ drinking. Intelligence test scores—scaled in 9 “stanines” (population mean of 5 and standard deviation of 2)—were taken from conscription assessment.This analysis was based on the population-based Young in Norway Longitudinal Study. Our sample included young men who had been followed from their mid-teens until their late 20s . Intelligence was not associated with intoxication frequency at any age and did not moderate the relationships between drinking frequency and adverse consequences.Our results suggest that the relationship between intelligence and drinking frequency is age dependent. Discrepancies with earlier findings from Sweden may be driven by changes in drinking patterns. In otsumption -14. We aAs noted, several studies have found a positive association between intelligence and alcohol consumption. In two different UK cohort studies, high childhood intelligence predicted more frequent drinking  and life11Some studies have produced null, mixed, or contradicting results , 15-18. 15. 1518. AThus, reported exceptions to the positive association between intelligence and alcohol use seem to fall into two partially overlapping categories. One comprises Swedish studies based on cohorts born around 1950 who entered their late teenage years and young adulthood in the late 1960s and early 1970s -21. The One reason why studies from Sweden have reported findings contrary to those from other parts of Europe and the USA may be that the association between intelligence and alcohol consumption is contingent on context and drinking culture. In the late 1960s and early 1970s, when these cohorts started to drink, both Sweden and Norway were “unintegrated” drinking cultures, with low population levels of drinking but high levels of binge drinking, alcohol-related harm, violence, and arrests for public drunkenness during weekends . SwedishConsidering why some studies have found negative associations between intelligence and adverse consequences of drinking, we speculate that the presumably protective role of intelligence may function differently with regard to alcohol than to other aspects of health behavior. Unlike many other health risk factors, many adverse consequences related to alcohol consumption are short term and relate to people’s behavior while under the influence  rather than the long-term physiological or psychological effects of consumption. If intelligence generally is protective against health risks, intelligence may perhaps moderate people’s behavior while drinking, rather than moderating the amount and frequency of drinking itself. Higher intelligence may be associated with a greater degree of self-control over intake, behavior while under the influence, and/or exposure to risky situations. If so, drinkers with higher intelligence may more often avoid adverse consequences of their drinking. Such a pattern of positive correlations with consumption but negative correlations with alcohol-related harm has previously been observed for socioeconomic status, and the notion that high SES groups consume more alcohol but experience less alcohol-related problems has been denoted the “Alcohol Harm Paradox” .We investigate the association between intelligence and drinking patterns among young Norwegian men born in the 1970s. This is a relevant group due to their relatively high levels of alcohol-related problems, and because drinking patterns and lifestyles often consolidate in early adulthood . A positn = 2923). Data collection for T3 was postal. Only participants who had completed the questionnaire in school at T2 (i.e. those who had not left school or changed schools between T1 and T2) were contacted to participate at T3. At T4, 2890 participants completed the questionnaire (response rate: 82.4%).We use data from the Young in Norway (YiN) panel study . Particin = 968) and T4 (n = 1126). Since intelligence and several other variables are measured only once, we cannot exploit the panel structure of our data and therefore conduct separate analyses for our outcome variables at T3 and T4. Written consent, and parental consent for those below age 15, was obtained from all participants at T1. The YiN study was approved by the Norwegian Data Inspectorate and the Regional Committee for Medical Research Ethics .Survey data were linked to register data on parents’ educational level and intelligence test scores from the Norwegian Armed Forces’ conscript assessment . Some men in our sample did not complete this test, likely including those deemed unfit for service before the assessment took place. In our analyses, we included all male respondents with valid IDs and non-missing conscription test scores at T3 , which was measured numerically. We top-coded all values above 28. We also measured 12-month frequency of intoxication by asking respondents to indicate how often they had drunk so much that they clearly felt drunk. The response options were “0 times,” “1 time,” “2–5 times,” “6–10 times,” “11–15 times,” and “more than 50 times.”Supplemental Material (S1). From these, we calculated the mean score . In the analyses, we standardized this scale, which we called the “adverse consequences scale,” to have a mean of 0 and a standard deviation (SD) of 1.At T3 we used five items from the Rutgers Alcohol Problem Index (RAPI) , which iSupplemental Analyses (S2) where drinking frequency was included as an independent variable.At T4, when participants were aged around 28 years, we used the 10-item Alcohol Use Disorders Identification Test (AUDIT)  to measuIntelligence was measured by the General Ability scores from the Norwegian Armed Forces’ conscript assessment, which is a composite score calculated from the three timed tests in Arithmetic 30 items), Figures (36 items), and Word Similarities (54 items). The Arithmetic and Word Similarities tests are similar to the Arithmetic and Vocabulary Test in the Wechsler Adult Intelligence Scale (WAIS). The Figures test is comparable to the Raven Progressive Matrices. Scores from the three tests were combined and then transformed into stanine scores . The reliability has been reported to be high, and the correlation between the General Ability score and the WAIS IQ has been reported as .73 in a sample of 48 Norwegian conscripts [ items, FParents’ educational level comprised four categories, linked from administrative register data. These indicated the highest educational level among parents when the respondent was 16 years old, plus an indicator of missing data. We included each parent’s employment status (six categories) and occupational class . We also included a dummy variable to indicate whether the respondent lived with both parents to capture having experienced parents’ early divorce or living with a single parent. The Parental Bonding Instrument (PBI) is a 10-item additive scale version of the original , measuriWe ran separate ordinary least-squares (OLS) regression models for each outcome at T3 and T4, and introduced our control variables in a stepwise fashion. For each outcome and survey wave, we ran four models:Model 1 included intelligence and age.Model 2 added controls for family background indicators.Model 3 added controls for the PBI and parents’ drinking at T1.Model 4 added controls for conduct problems and friend’s drinking.We treated intelligence score stanines  and frequency of intoxication  to 5 (“50 times or more”)) as continuous variables in OLS regressions. Ordered logistic regression, in which these were treated categorically, produced substantively similar results (not shown). To make results comparable across models, we restricted the sample in each analysis to respondents without missing values on any variable included Model 4. This restriction did not substantively affect our results.Supplemental Material (S2) along with a visualization of the distribution of the main variables (S3). Overall, the respondents drank slightly less frequently at T3 when the respondents were around 22 years old (mean: 4.05 times during the last four weeks) than at T4 when respondents were around 28 years old (mean: 4.73 times) but were intoxicated slightly more often during the last year at T3 than T4  to 5 (“more than 50 times”)).Descriptive statistics  for the main variables are shown in To examine the relationship between intelligence and alcohol consumption, we regressed 4-week drinking frequency on intelligence scores while controlling for age (Model 1). The results are shown in We then regressed 12-month intoxication frequency on intelligence scores, separately by survey wave . We founRegressing the adverse consequences scale on intelligence revealed a weak, negative association when respondents were around age 22 , Panel 1p > .05). These results are provided as Supplemental Material (S4) and provide little support for the notion that intelligence moderates the relationship between frequent drinking and experiencing adverse consequences from drinking.Finally, we investigated whether intelligence may moderate the relationship between frequent drinking and experiencing adverse consequences of drinking. In this analysis, we regressed the adverse consequences scale at T3 (aged around 22) and the AUDIT instrument  at T4 (aged around 28) on drinking frequency and intelligence score and their interaction. The results showed only small, non-significant interaction terms  association between intelligence and drinking frequency. However, to disentangle these relationships, more research into changes in drinking patterns over time in relation to intelligence is necessary.The positive association between intelligence and drinking frequency in the late 20s, when most men have entered the labor market, is also consistent with the notion that selection into longer educational programs or high-status jobs may be relevant to this association. Our findings also indicated a small, negative association between intelligence and alcohol related problems at around 22 years of age. While one possible interpretation of this finding is that high intelligence may protect against adverse consequences from drinking, additional analyses (not shown) indicate that the negative association is driven solely by higher adverse consequences scores in the two lowest stanines. Finally, our results do not support the notion that intelligence moderates the relationship between drinking frequency and adverse consequences of drinking.Our study has several limitations. We cannot rule out the importance of selective attrition, measurement error, and similar survey-related issues. If people with greater cognitive abilities are more reflexive of, and concerned with, potentially adverse consequences of their drinking, they may be more likely to report alcohol use and related problems accurately in surveys , which mIn conclusion, studying young Norwegian men born in the 1970s, our findings suggest that the association between intelligence and alcohol consumption is only positive when they are in their late 20s, not when they are in their early 20s. In other words, the association appears to be age dependent. This finding also contrasts with Swedish findings from older cohorts, suggesting that the relationship may also be context-dependent. Our results also suggest that intelligence does not moderate the relationship between frequent drinking and adverse consequences.Click here for additional data file.Supplemental material, SJP944719_Supplemental_Material for Intelligence, alcohol consumption, and adverse consequences. A study of young Norwegian men by Adrian F. Rogne, Willy Pedersen and Tilmann Von Soest in Scandinavian Journal of Public Health


Treatment of ruptured high-grade Spetzler-Martin (S&M) arteriovenous malformation (AVM) is challenging and requires a multidisciplinary treatment approach. Here, we report a case of ruptured giant callosal Grade V AVM in a child initially treated with stereotactic radiotherapy followed by endovascular embolization with Onyx; a management approach recently described in a few reports on the “postradiosurgical embolization” method. Complete obliteration was achieved 20 months after stereotactic radiotherapy and embolization. In this article, we discuss the usefulness and significance of postradiosurgical embolization, particularly for high-grade AVMs. To our knowledge, this is the first case with a giant Spetzler-Martin Grade V AVM treated with a postradiosurgical embolization method. The treatment of ruptured pediatric high-grade AVMs is challenging, and until now, there are no agreed-upon protocols for its management. A multidisciplinary approach with the combination of radiotherapy, embolization, excision, and clinical follow-up is commonly practiced ,2. In hiRecently, a few reports suggested a different approach to manage AVMs, the postradiosurgical embolization method, where embolization is performed after radiation therapy. This approach has been developed to overcome the disadvantages of preradiosurgical embolization . Here, fClinical presentation and diagnosisA 12-year-old girl presented with headaches and mild disorientation [Glasgow Coma Scale (GCS): E3V5M6]. A plain brain CT scan showed an intraventricular hemorrhage Figure , and theCerebral angiography showed that the main feeder was from the left anterior cerebral artery (Lt. ACA). Furthermore, flow came from the right anterior cerebral artery (Rt. ACA) and left posterior cerebral artery (Lt. PCA). The drainer was deep, mainly towards the inferior sagittal sinus and basal vein of Rosenthal. The AVM’s maximum diameter was 7.5 cm, and its Spetzler-Martin (S&M) grade was V  Figure .Because of the AVM location, which is widely extended to a large area of the corpus callosum, surgical excision was expected to cause a significant neurological deficit for the patient. Furthermore, with a nidus volume up to 17.3 ml, achieving a complete cure by radiotherapy alone was expected to be challenging. Therefore, we decided to treat the patient with CyberKnife M6  radiotherapy followed by Onyx embolization, the so-called postradiosurgical embolization method.TreatmentAfter 135 days from the onset of symptoms, stereotactic radiation of the AVM was done with a gross tumor volume (GTV) of 17.3 ml using CyberKnife M6 with 35 Gy in five fractions and an isodose line 77%. In order to reduce the blood flow to the nidus, we applied 35 Gy focusing on the area where the feeder flows into the nidus  into the main feeder, the Lt. ACA, and injected Onyx18  while the balloon of the microcatheter was inflated Figure . In the A total of 9 ml of Onyx18 agent was injected into the AVM, and approximately 50% of the nidus was embolized. The residual lesions of AVM nidus were on the anterior-inferior, right-center, and left posterior .In a meta-analysis of 3923 cases of cerebral AVMs, a history of hemorrhage, deep brain localization, drainage only into deep veins, and presence of aneurysm were shown statistically predominant risk factors for bleeding . In our Although there is a lack of a guideline, several reports recommend a multimodality approach treatment for large AVM ,12. ChanVarious radiotherapy methods have been reported large and challenging to treat AVM. The commonly used methods are single-stage stereotactic radiosurgery (SS-SRS), volume staged SRS (VS-SRS), and dose staged SRS (DS-SRS). The most common radiotherapy methods for large AVM are VS-SRS and DS-SRS. For VS-SRS, a gamma knife is basically used, and the mean marginal dose varies from 15 to 20 Gy, and the number of stages of irradiations is often reported to be two to four stages ,15. For Veznedaroglu et al. compared the outcome of seven AVM cases with a mean nidus volume of 23.8 ml who received 42 Gy in six fractions of 7 Gy with 23 AVM cases with a mean nidus volume of 14.5 ml of nidus who received 30 Gy in six fractions of 5 Gy. The complete occlusion rate obtained after more than five years of embolization was 83% in the 7 Gy group versus 22% in the 5 Gy group. However, complications associated with irradiation were 28% in the 7 Gy group and 4.3% in the 5 Gy group. This study concluded that for large AVM, high conformality is necessary for fewer complications and higher cure rates . A margiThe current case’s AVM was 7.5 cm in diameter. Accordingly, we irradiated it with a 35 Gy in five fractions, 77% isodose line, with a minimum marginal dose of 30 Gy. Firlik et al. introduced the method of staged volume radiosurgery for giant AVM, and irradiation was performed from at feeder side to avoid pressure rise in the nidus . The samRegarding the time to treat an AVM, Maruyama et al. found that there was no benefit in postponing treatment until hematoma resorption because the number of bleeding events was predominantly higher in patients with a median follow-up of six months or longer . In our Large AVMs are often challenging to treat with embolization alone or radiation alone, and the traditional strategy has been first to reduce the size of the AVM with embolization and then add radiation therapy. This will help to reduce the amount of radiation exposure to surrounding structures. However, many reports show lower obliteration rates in the embolization followed by the radiotherapy group compared to the radiotherapy alone group ,9. In geKim et al. compared 81 patients treated with only Gamma knife radiosurgery (GKRS) with 17 patients treated with GKRS followed by embolization for ruptured AVM. In the ruptured S&M Grade III and IV AVM cases, the rate of occlusion was higher in the group treated with GKRS followed by embolization than in the group treated with GKRS alone . In the We present a ruptured giant high-grade callosal AVM in a child cured by postradiosurgical embolization for the first time. Postradiosurgical embolization can be an effective treating strategy for high-grade ruptured AVMs. This method enables accurate radiation planning, early cure, and a low recurrence rate. Further studies of postradiosurgical embolization in larger samples remain necessary to validate the usefulness and applicability of this method.


Plasmodium malariae has a wide geographic distribution, but mainly at very low parasitemias and in co-infections, leading to an underestimated prevalence of this species. Studies for the detection of antibodies against Plasmodium recombinant proteins are increasingly used to map geographical distributions, seroprevalence and transmission intensities of malaria infection. However, no seroepidemiological survey using recombinant P. malariae proteins has been conducted in Brazil. This work evaluated the antibody response in serum samples of individuals from endemic regions of Brazil (the Amazon region and Atlantic Forest) against five recombinant proteins of P. malariae merozoite surface protein 1 (MSP1), and the MSP1 C-terminal portions of P. vivax and P. falciparum, in a multiplex assay. The positivity was 69.5% of samples recognizing at least one MSP1 recombinant protein. The mean of the Reactivity Index for the C-terminal portion of the P. falciparum was significantly higher compared to the other recombinant proteins, followed by the C-terminal of P. vivax and the N-terminal of P. malariae. Among the recombinant P. malariae proteins, the N-terminal of P. malariae showed the highest Reactivity Index alone. This study validates the use of the multiplex assay to measure naturally acquired IgG antibodies against Plasmodium MSP1 proteins and demonstrate that these proteins are important tools for seroepidemiological surveys and could be used in malaria surveillance. Plasmodium malariae is one of the oldest parasites that cause malaria in humans but is also one of the most neglected, probably due to their lower prevalence and disease severity, as compared to other human Plasmodium spp. [P. malariae is widespread throughout sub-Saharan Africa, Southeast Asia, Indonesia, South America and on islands of the western Pacific [P. malariae infections in the last two decades, showing an overall seroprevalence of 2%, with the highest prevalence (3.16%) in the African region and the lowest prevalence (0.06%) in the Eastern Mediterranean region, whereas the American region had the second highest P. malariae prevalence (2.94%) [ium spp. . However Pacific . Recentlevalence .16% in tPlasmodium malariae also occurs more likely in co-infections than mono-infections [P. malariae is rarely apparent, and low-level or subpatent infections are revealed only by molecular techniques [P. malariae may be underestimated due to its misrecognition in thick blood smears, often leading to a mistaken identification as P. vivax [fections ,4. Howevchniques . MoreoveP. vivax .P. malariae, suggesting that asymptomatic infections with this species may be frequent [P. malariae is known to produce low parasitemia infections that are frequently non-symptomatic and long-lasting [Despite the minor number of cases reported in many parts of the world, a significant part of the population shows serologic evidence of recent exposure to frequent ,7,8,9. I-lasting , possibl-lasting  and pref-lasting .P. falciparum and P. vivax malaria [P. malariae [In a general way, subclinical malaria arises due to naturally acquired immunity, directly related to both the age and the many years of exposure of the individual to the malaria parasites ,14. Asym malaria . Their dmalariae . Thus, iPlasmodium merozoite surface protein 1 (MSP1) in human serum samples using a multiplex serological assay [P. vivax, P. falciparum and P. malariae occurs predominantly in the Amazon region, where it is a serious public health problem [P. vivax and P. malariae are transmitted and maintained in small foci of transmission with very specific characteristics [P. malariae , P. falciparum (PfMSP119) and P. vivax (PvMSP119), and the sera of individuals from Acre and Rondônia, in the Amazon region, and Intervales, in the Atlantic Forest, to establish the prevalence of antibodies against these parasite species in different endemic areas.Serological assays can measure past exposure identifying at-risk populations and help to produce risk maps to improve the targeting of interventions . Therefoal assay . MSP1 isal assay  and has al assay . In Braz problem . In the eristics . A multiThe efficiency of the coupling of each protein was determined by the analysis in singleplex, and later evaluated in a multiplex bead assay (MBA), where it played the role of quality control in all of the sample plates tested. For this, beads were covalently coated with the different recombinant proteins and tested with a biotinylated anti-GST antibody. The graph in 19 (~380 MFI).To determine the cut-off values for each recombinant protein in each plate, sera from uninfected individuals were used in duplicate to obtain the MFI values. The cut-off values are presented as the geometric mean of the values, obtained with a panel of eight negative control sera, plus three standard deviations. The averages of the cut-off values are shown in F1, PmMSP1F2, PmMSP1F3, PmMSP1F4, PmMSP119, PfMSP119 and PvMSP119). The overall seroprevalence of IgG antibodies against at least one of the recombinant proteins was 69.5% (289 samples). Sera reactive to P. vivax PvMSP119 were the most frequent (52.8%), followed by those positive to P. falciparum PfMSP119 (21.5%), whereas the percentages of positivity were very low for the five recombinant proteins of P. malariae  and the Atlantic Forest , were analyzed against seven recombinant proteins  compared to the other recombinant proteins , followed by the C-terminal of P. vivax (PvMSP119) and the N-terminal of P. malariae (PmMSP1F1), the latter being the most reactive among all P. malariae recombinant proteins tested. However, the RI value for the PmMSP1F1 was non-significantly higher  when compared to the other P. malariae recombinant proteins , which denotes the ratio between the MFI of the samples and the cut-off values. In a general analysis with sera from all of the three locations together, the RI value was significantly higher for the C-terminal portion of the proteins B.P. vivax had a high RI in all of the three locations    C–E. Howectively) D–E. F1 and PmMSP1F3 stood out with high RIs , and three samples were found that recognized PfMSP119 with an RI close to 1.0 .In the Atlantic Forest region, sera reactivity against the recombinant proteins PmMSP1e to 1.0 C. In Ramlciparum D. In Poranalyses E, with a19-specific antibodies  were evaluated, in order to prevent bias towards P. malariae. Thus, Plasmodium MSP119 antibody prevalence in serum samples from Brazil according to the recognition for the three recombinant proteins in each locality analyzed   B. Data ato Velho B. The prto Velho B. The si Granada B. The ovn region C and P. c Forest D. P. malariae proteins, PmMSP1F1, PmMSP1F2, PmMSP1F3, PmMSP1F4 and PmMSP119 were recognized by 41.2%, 29.4%, 52.9%, 20.6% and 38.2% of the P. malariae positive sera (n = 68), respectively. A comparison was performed by the eighteen distinct combination patterns obtained, the frequencies of which are shown in F3 was recognized distinctly with a frequency of 27.9%, whereas 16.2% of the sera were solely positive for PmMSP1F1. The five P. malariae recombinant proteins together  were recognized in 8.8% of the P. malariae-positive samples, showing RI values considered high in our sample set scale (F1) and also the C-terminal portion (PmMSP119), with the highest RI values obtained for these proteins   .Plasmodium MSP1 proteins are increasingly used to map geographical distributions, seroprevalence and transmission intensities of malaria infection [19) is a promising candidate for the malaria vaccine, as it is highly immunogenic [P. vivax and P. falciparum infections [P. malariae MSP1 [P. malariae in epidemiological studies [P. malariae , P. falciparum (PfMSP119) and P. vivax (PvMSP119).Studies for the detection of antibodies against nfection ,23,24. Tunogenic ,26, therunogenic ,26,27,28fections ,30,31,32iae MSP1 , no sero studies . Here, w19 were identified in more than half of our sample set, followed by those for PfMSP119, and in smaller percentages by those against the different recombinant proteins of P. malariae , corroborating with the data obtained in Brazilian historical data of malaria that have demonstrated the predominance of P. vivax over P. falciparum over the years [19 and P. malariae, mainly for PmMSP1F1 and PmMSP1F3 in the Atlantic Forest region, a documented area for the presence of P. malariae infections [Antibodies against PvMSP1he years ,36. Howefections . P. falciparum mainly in Porto Velho, a known hotspot for this species in Brazil [P. falciparum MSP1 protein, PfMSP119, has already shown to be related to the acquisition of clinical immunity, which is probably cumulative, with the expansion and refinement of a repertoire of antibodies, protecting the individual from severe malaria, which is a rare event in the Amazon region [The highest Reactivity Index for IgG antibodies among the three species was found for n Brazil . The C-tn region .P. vivax or P. malariae [P. malariae  compared to other locations, reflecting a cumulative transmission profile. This suggests that these individuals may be frequently exposed to infections caused by this species or even maintain these infections for long periods, since P. vivax infections induce long-lasting memory B-cell responses even in settings with very low transmission [In the Atlantic Forest, although only a small number of clinical cases of malaria are registered, a large portion of the population shows serological evidence of recent exposure to malariae ,39, suggmalariae ,40,41. Ismission . P. falciparum (PfMSP119) with very low RI, in concordance with previous reports [P. falciparum in the Atlantic Forest has been previously detected by PCR in individuals from Intervales, who did not present classical symptoms and reported not having travelled to malaria-endemic areas, such as the Amazon region [P. falciparum in humans with malaria [P. falciparum in a silent cycle, detected only by molecular methods in asymptomatic individuals [Interestingly, three samples from the Atlantic Forest recognized the C-terminal of  reports ,43,44. In region . A cross malaria . Recent ividuals .19 in the Brazilian Amazon was in line with other reports from this endemic area, where nearly 70% of the studied population carried PvMSP119 antibodies [P. falciparum also showed a high RI of IgG antibodies in the Amazon region, showing a profile of constant exposure to parasites, as demonstrated in previous studies [The overall seroprevalence of the highly immunogenic PvMSP1tibodies . Similar studies ,47.19, endorsing the idea that IgG antibody responses to the utilized malaria MSP1 antigens appear to be species-specific [It is important to note that 60% of our study participants with positive results harbored single-species antibodies against MSP1specific .P. malariae. Interestingly, the recognition of the five fragments combined was formed by samples with average Reactivity Indexes. However, PmMSP1F1 and PmMSP1F3 were recognized singly in a high frequency, showing the need to use the various domains of PmMSP1 in combination during serological surveys focusing on this species. Analyzing the eighteen recognition patterns that were obtained, the PmMSP1F1 protein is present in 50% of them, which may indicate the potentially high immunogenicity of this MSP1 region of P. malariae, as has already been demonstrated for P. vivax and P. falciparum MSP1 [The possible combinations of antibodies were analyzed in order to obtain information on the combined occurrence of specific antibodies against rum MSP1 ,31,32,41P. malariae cases are rarely identified [P. vivax and still a certain prevalence of P. falciparum, combined with low parasitaemia and all of the difficulties in diagnosing P. malariae, it is not surprising that P. malariae infections have been overlooked in this area. Though not as clinically relevant, it is becoming more appreciated that donors with asymptomatic P. malariae infection could be reservoirs of transfusion-transmitted malaria [P. malariae in routine malaria diagnosis by rapid diagnostic tests (RDTs), which currently target P. falciparum, and other Plasmodium species are identified as “pan-species” [In the Amazon region, entified . However malaria , and serspecies” . All of the samples used in this study were collected during research projects that have been published previously ,36,38 anP. vivax [Sera from the Amazon region were received from two different localities in the Western Brazilian Amazon: (i) 238 samples from an ongoing population-based cohort study accomplished in an agricultural settlement , collected from March 2004 to May 2005 ; (ii) 52P. vivax .P. vivax malaria and few, but not insignificant, infections by P. malariae [Sera from the Atlantic Forest were collected in Intervales State Park (126 samples), district of Guapiara, São Paulo state, in January 2002 . This Vamalariae .P. malariae, representing the polymorphic N-terminal (PmMSP1F1), the central  and the conserved C-terminal (PmMSP119) regions, were used in parallel with the GST protein alone (as a control), for detecting anti-MSP1 antibodies in the human serum samples. Moreover, C-terminal of P. vivax (PvMSP119) and C-terminal of P. falciparum (PfMSP119) previously produced [GST-fusion proteins of produced , were alPlasmodium spp. (GST-MSP1) to Bio-Plex carboxylated beads in MBAs were performed using a rabbit anti-GST (Biotin) polyclonal IgG antibody  to detect bead-coupled fusion protein.The overall efficiency of Anti-GST antibody was used 1:1000 in the assay buffer  (50 µL/well). Bound anti-GST antibody was detected with Streptavidin-R-Phycoerythrin (2 µg/mL)  and fluorescence was measured on the Bio-Plex 200 instrument , as described below. The system includes two lasers: the classification laser (635 nm excitation) for identifying the bead signatures, and the reporter laser (532 nm excitation) for detecting the target.The recombinant proteins were covalently linked to Bio-Plex Pro Magnetic COOH Beads using the Bio-Plex Amine Coupling Kit  following the manufacturer’s instructions. The coupled beads were then used for the analysis of the samples of individuals as described , with moSera were tested in two replicates and assessed by the MFI values of antibodies binding to the recombinant proteins, minus the MFI value of the same serum for GST alone. Cut-off values are presented as the geometric mean of values, obtained with a panel of eight negative control sera, plus 3 × standard deviations. Mean cut-off values are shown in Quantitative results were obtained by normalizing the data using the Reactivity Index (RI). The RI was established using the fluorescence values expressed as MFI for each sample and divided by the cut-off of each plate. Samples with an RI ≥ 1 were considered positive.Plasmodium species  in two Brazilian areas: in the Amazon region, where malaria is a serious public health problem, and in regions that record a low but persistent number of autochthonous cases, as is the case for the Atlantic Forest, in the extra-Amazonian region. The high rates of IgG antibody reactivity against P. vivax recombinant proteins in all localities, P. malariae in the Atlantic Forest region and P. falciparum in the Amazon region, support the hypothesis that there is a frequent circulation of the parasites in asymptomatic infections that can play the role of potential reservoirs.The current study provided the first malaria seroprevalence data for the three circulating P. malariae recombinant MSP1 proteins in humans and demonstrate that these proteins are important tools for seroepidemiological surveys that must be considered in malaria surveillance and elimination programs.Our results also validate the use of the MBA to measure naturally acquired IgG antibodies against P. vivax and P. falciparum transmission. Current P. falciparum transmission is basically restricted to the same hotspot areas as in 2000 to 2010, when sera were obtained for this study. Similarly, malaria reported in the Atlantic Forest comprises autochthonous P. vivax and P. malariae transmission without significant modifications in its epidemiology during this period mentioned above [P. malariae were used. Other antigenic surface proteins from the parasite, such as the Circumsporozoite protein (CSP), thrombospondin-related anonymous protein (TRAP), Duffy-binding protein (DBP) and apical membrane antigen 1 (AMA1), may also produce valuable results.The incidence of malaria in Brazil has dropped in the last decade; however, 99% of all malaria cases registered in the country are still stemming from the Amazon region, which is largely due to autochthonous ed above . Another


Hypertension increases the likelihood of cardiovascular diseases (CVDs). Cytochrome P450 1A2 (CYP1A2) single nucleotide polymorphism (SNP) is related to caffeine metabolism and the risk of CVD among coffee drinkers. CYP1A2 rs762551 influenced the risk of stroke among hypertensive patients. We examined the relationship between hypertension and coffee drinking based on CYP1A2 rs762551 SNP in Taiwanese adults.We used data contained in the Taiwan Biobank database (2011–2018) and included 19,133 participants having complete information on hypertension, rs762551 polymorphism, coffee intake, etc. The risk of hypertension was determined using multiple logistic regression.p value = 0.0303). After stratification by rs762551 genotypes, the inverse coffee drinking-hypertension association was retained, but significant results were observed only in those with the AC + CC genotype . According to the combination of coffee drinking and rs762551 genotypes (reference group: no coffee drinking and rs762551 AA), the coffee drinking-AC + CC group had a lower risk of hypertension .Coffee intake was significantly associated with a lower risk of hypertension. The odds ratio (OR), 95% confidence interval (CI), and p-value were 0.877, 0.807–0.954, and 0.0032, respectively. CYP1A2 rs762551 was not significantly associated with the risk of hypertension, but it had a significant interactive association with coffee drinking (Coffee drinking, particularly among individuals with the CYP1A2 rs762551 AC + CC genotype was associated with lower odds of hypertension. Hypertension is a significant public health burden. Its prevalence among adults has been increasing. In 2000, about 972 million adults in the world were believed to be hypertensive . HoweverCoffee consumption has been increasing in Taiwan and the world , 11. DieSo far, findings from epidemiological research on coffee drinking and hypertension have been controversial . Some shGenetic factors contribute to variability in the effect of coffee on cardiovascular health . SpecifiWe conducted this study using data contained in the Taiwan Biobank database (2011–2018). The biobank contains genetic data as well as information like medical history, sex, age, diet, and other lifestyle factors. Enrolment into the Taiwan Biobank project is restricted to Taiwanese adults between 30 and 70 years without a personal history of cancer.2. Underweight was defined as BMI < 18.5 kg/m2, normal BMI as 18.5 ≤ BMI < 24 kg/m2, overweight as 24 ≤ BMI < 27 kg/m2, and obesity as BMI ≥ 27 kg/m2. Participants were considered non-smokers if they had no history of cigarette smoking for continuously 6 months or more. Former smokers were those who smoked cigarettes continuously for at least 6 months but were not smoking during data collection. Current smokers were those who had a history of continuous cigarette smoking for at least 6 months and were still smoking during data collection. Participants were considered non-drinkers if they had no history of continuous drinking for 6 months or drank less than 150 cc of alcohol per week. Former drinkers were those who abstained from alcohol for more than 6 months while current drinkers were those who drank at least 150 cc of alcohol per week continuously for 6 months. Exercise was defined as regular exercise taken 3 times a week (with each session lasting over 30 min). This study included 3411 hypertensive and 15,722 non-hypertensive individuals. All participants signed an informed consent letter. We obtained ethical approval from the Institutional Review Board of Chung Shan Medical University .Variables included hypertension (yes/no), coffee drinking (yes/no), rs762551 genotypes (AA/AC + CC), tea drinking (yes/no), sex (men/women), age, body mass index or simply BMI , cigarette smoking (never/former/current), alcohol drinking (never/former/current), and exercise (no/yes). Hypertensive cases were those who confirmed through questionnaires to have ever been clinically diagnosed with the disease. Coffee drinking was defined according to self-reports on weekly consumption frequency. The coffee-drinking group included individuals who self-reported a habit of drinking coffee at least thrice per week while the non-drinking group included those who habitually drank coffee less than three times per week. Tea drinking was defined based on daily consumption frequency. The tea-drinking category included those who self-reported a habitual daily tea intake of at least one time while the non-drinking category included those who had a habit of not drinking tea every day. Sex and age were self-reported while BMI was derived from weight (kg) and height (m) using the standard formula: weight divided by (height)p > 0.001, a minor allele frequency ≥ 0.05, and call rate ≥ 0.05. Quality control was performed using PLINK version 1.9. Chi-square test was used to differentiate the categorical variables while Student’s T test was used to differentiate the continuous variables between cases and non-cases. Categorical variables were presented as n (percentage) and continuous variables as mean ± standard error. Multivariate logistic regression was used to evaluate the association of coffee drinking and rs762551 with hypertension and also the interaction between coffee drinking and rs762551. Statistical analyses were performed using SAS version 9.4 , and p values less than 0.05 were considered to be statistically significant.CYP1A2 rs762551 was validated using quality control measures including, Hardy–Weinberg equilibrium p value < 0.0001) but not according to rs762551 genotypes . Coffee drinkers and non-drinkers also differed in terms of tea drinking , sex , age, BMI, cigarette smoking, and alcohol drinking  but not exercise .Table p value = 0.0032) while the rs762551 variant was associated with a higher but not significant risk of hypertension .Table p value = 0.0303) but not with tea  as seen in Table p value  = 0.0001). Further stratification of coffee drinking revealed a significant dose–response association  between coffee drinking and hypertension in those carrying the AC + CC genotype .According to the combination of coffee drinking and the rs762551 genotypes  and coffee drinking (data not shown). After multivariate logistic regression analysis, coffee drinking had a significant inverse association with hypertension. CYP1A2 rs762551 was not significantly associated with the risk of hypertension but had a significant interaction with coffee drinking. After stratifying participants by rs762551 genotypes, the inverse coffee-hypertension association was retained only among those with the AC + CC genotype. Combining coffee and rs762551 genotypes revealed a lower risk of hypertension among coffee drinkers with the AC + CC genotype. The inverse association between coffee drinking and hypertension in the AC + CC group was in a dose-dependent manner.The non-significant association between coffee drinking and rs762551 genotype observed in the current study is in concordance with previous reports among Costa Ricans  and ItalThe relationship between coffee and hypertension has been controversial. For instance, a meta-analysis of randomized clinical trials demonstrated a positive but weak association between coffee intake and hypertension . In a foThe observed differences in the relationship between coffee drinking and hypertension are in part due to genetics . Coffee Despite the large sample size, our study is limited in that the TWB questionnaires contained no information on caffeine intake and coffee type. Therefore, we could not determine the interaction between CYP1A2 polymorphism and caffeine or decaffeinated coffee on hypertension risk. Moreover, we defined hypertension based on the Taiwan Biobank questionnaires (as described in the Methods section). This might have resulted in information bias. However, it should be noted that in our initial analyses, we merged the Taiwan Biobank database with the National Health Insurance Research Database (NHIRD) using encrypted personal identification numbers with the resources at the Health and Welfare Data Science Center (HWDC). The NHIRD contained diagnostic codes based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). Hypertension was identified based on either two outpatient visits or one-time hospitalization between 1998 and 2015 using the diagnostic codes ICD-9-CM 401-405, A260, and A269. Even though we defined hypertension based on self-reports, all cases were those whose diagnosis was confirmed by a medical doctor. Noteworthy, the results observed are similar to those observed when hypertension was defined using ICD codes.Generally, coffee drinking was associated with a lower risk of hypertension, while the AC + CC genotype was associated with a higher but non-significant risk. Notwithstanding, coffee drinking and rs762551 had a significant interactive relationship. Grouped analyses revealed an inverse association between hypertension and coffee drinking, particularly among individuals with the CYP1A2 rs762551 AC + CC genotype. Our results indicate the possibility of a protective effect of coffee on hypertension and confirm the joint role of coffee and CYP1A2 rs762551 polymorphism on hypertension.


Clostridium paraputrificum ATCC 25780. From six candidate C. paraputrificum ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12β-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12β-HSDH, leading to validation of two additional bile acid 12β-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12α-HSDH, we identified the first potential C-12 epimerizing strains: Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12β-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso- (7α-HSDH & 7β-HSDH) and iso- (3α-HSDH & 3β-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12β-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12β-bile acid denoted epiDCA; however, a gene(s) encoding this enzyme has yet to be identified. We confirmed 12β-HSDH activity in cultures of  Once bile acids reach the terminal ileum, high affinity transporters  actively transport both conjugated and unconjugated bile acids from the intestinal lumen into ileocytes where they are bound to ileal bile acid binding protein (IBABP) and exported across the basolateral membrane into portal circulation and returned to the liver.3 This process of recycling bile acids is known as enterohepatic circulation (EHC) and is responsible for recirculating the ~2 g bile acid pool 8–10 times daily. While ~95% efficient, roughly 600–800 mg bile acids escape active transport and enter the large intestine.2The human liver produces all 14 enzymes necessary to convert cholesterol into the dihydroxy bile acid chenodeoxycholic acid  and the trihydroxy bile acid cholic acid .2 Conjugated bile acids are hydrolyzed, releasing the amino acids, by bile salt hydrolases (BSH) in diverse gut bacteria representing the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as the domain Archaea.4 In contrast, the unconjugated primary bile acids CA and CDCA are 7α-dehydroxylated by a select few species of gram-positive Firmicutes mostly in the genus Clostridium, forming deoxycholic acid  and lithocholic acid , respectively.5 The secondary bile acids DCA and LCA have increased hydrophobicity relative to their primary counterparts, which is associated with elevated toxicity.6 DCA and LCA have been causally linked to cancers of the colon,7 liver,8 and esophagus.9 Importantly, gut microbiota can produce less toxic oxo-bile acids and β-hydroxy bile acids as well.6Anaerobic bacteria adapted to inhabiting the large intestine have evolved enzymes to modify the structure of host bile acids.10 HSDH enzymes that recognize bile acids are regio- (C-3 vs. C-7) and stereospecific (α vs. β) for hydroxyl groups decorating the steroid nucleus. Thus, bile acid 12α-HSDH reversibly converts the C-12 position of bile acids from the α-orientation, such as on DCA, to 12-oxo bile acids, such as 12-oxolithocholic acid (12-oxoLCA).11–14 Bile acid 12β-HSDH completes the epimerization by interconverting 12-oxo bile acids to the 12β-configuration, forming epi-bile acids. We recently identified and characterized NAD(H)- and NADP(H)-dependent 12α-HSDHs from Eggerthella sp. CAG:29815, Clostridium scindens, C. hylemonae, and Peptacetobacter hiranonis (formerly Clostridium hiranonis).10 In addition to these recently reported 12ɑ-HSDHs, multiple genes encoding enzymes in the urso- (7α- & 7β-HSDH) and iso- (3α- & 3β-HSDH) bile acid pathways have been described to date  distributed across the major phyla including Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, as well as methanogenic archaea. to date .5 Howeve16–18 Edenharder and Schneider (1985) reported 12β-dehydrogenation of bile acids by Clostridium paraputrificum, and epimerization of DCA by co-culture with E. lenta and C. paraputrificum.19 Thereafter, Edenharder and Pfützner (1988) characterized crude NADP(H)-dependent 12β-HSDH activity from C. paraputrificum D 762–06.20 However, little is known about the potential diversity of gut bacteria capable of forming 12β-hydroxy bile acids that molecular analysis is predicted to yield. Here, we report the identification of a gene encoding NADP(H)-dependent 12β-HSDH from C. paraputrificum ATCC 25,780 and characterization of the recombinant gene products purified after heterologous expression in E. coli from C. paraputrificum. We also identify novel taxa encoding bile acid 12β-HSDH by phylogenetic analysis, confirmed by a synthetic biology approach.The first indication that gut bacteria may encode 12β-HSDH was suggested by the detection of 12β-hydroxy bile acids in human feces.C. paraputrificum ATCC 25780 because previous studies reported bile acid 12β-HSDH activity in other C. paraputrificum strains, but did not identify the gene(s) responsible.20 The epi-bile acid pathway of DCA involves the reversible conversion of DCA  to 12-oxoLCA  through the action of 12α-HSDH, and 12-oxoLCA to epiDCA  by 12β-HSDH  consistent with DCA (392.57 amu), demonstrating bile acid 12α-HSDH activity. In addition, reaction with DCA resulted in a peak at 15.57 min and 391.28 m/z along with a peak agreeing with 12-oxoLCA at 14.34 min and 389.27 m/z. When epiDCA was incubated with cultures of C. scindens ATCC 35704, we did not observe formation of 12-oxoLCA.We first investigated the bile acid metabolizing capability of 12β-HSDH . C. paraion mode . This isC. paraputrificum ATCC 25780, its genome was searched for genes encoding proteins annotated as oxidoreductases within the NCBI database. HSDHs are NAD(P)-dependent and often members of the large and diverse SDR (short-chain dehydrogenase/reductase) family.21 Five SDR family oxidoreductase proteins and one aldo/keto reductase were identified as 12β-HSDH candidates in the C. paraputrificum ATCC 25780 genome and pursued for further study. These six genes were amplified from genomic DNA of C. paraputrificum ATCC 25780, cloned into the pET-28a(+) vector, and overexpressed in E. coli (Table S1). The N-terminal His6-tagged recombinant proteins were purified by metal-affinity chromatography and resolved by SDS-PAGE  were not soluble and bands at the expected molecular masses were apparent in the membrane fraction by SDS-PAGE. These proteins were not explored further. The other four 12β-HSDH candidates  were soluble and visualized by SDS-PAGE. The four soluble recombinant proteins were then screened for pyridine nucleotide-dependent bile acid 12β-HSDH activity by TLC and spectrophotometric assay. Screening reactions were prepared with 12-oxoLCA and NADPH, or epiDCA and NADP+, DCA and NADP+, and no substrate control were subjected to LC-MS. In the presence of purified recombinant WP_027099077.1 and NADPH, 12-oxoLCA was reduced quantitatively (2 hydrogen addition) to a product that eluted at 13.12 min with 391.28 m/z in negative ion mode. This is consistent with the 392.57 amu formula weight and elution time for epiDCA based on the substrate standard. Additionally, epiDCA was oxidized to a product with an elution time of 13.40 min at 389.27 m/z, agreeing with the retention time and formula weight of 390.56 amu for authentic 12-oxoLCA. DCA (392.57 amu) was not converted by WP_027099077.1 as the sole peak observed matched DCA standard at 14.60 min and 391.29 m/z. The interconversion of 12-oxoLCA and epiDCA, but no activity with DCA, indicates stereospecificity for the 12β-hydroxy position. Thus, DR024_RS09610 has been identified as the first gene reported that encodes bile acid 12β-HSDH (WP_027099077.1).Only WP_027099077.1 exhibited 12β-HSDH activity by TLC and spectrophotometric assay, which was also confirmed by LC-MS . ReactioC. paraputrificum WP_027099077.1, hereafter referred to as Cp12β-HSDH, had a theoretical subunit molecular mass of 27.4 kDa. The observed subunit molecular mass was 26.4 ± 0.5 kDa by SDS-PAGE, calculated from three independent protein gels. WP_027099077.1 is predicted to be a cytosolic protein that is not membrane-associated by TMHMM v. 2.0.22Recombinant + as co-substrate was pH 7.5. In the reductive direction where 12-oxoLCA was the substrate and NADPH the co-substrate, the optimum pH was 7.0.The approximate native molecular mass of Cp12β-HSDH was determined by size-exclusion chromatography. Cp12β-HSDH exhibited an elution volume of 15.04 ± 0.02 mL, corresponding to a 54.67 ± 0.79 kDa molecular mass relative to protein standards . The sizmK value for 12-oxoLCA at 18.76 ± 0.40 µM which was similar to that of NADPH . The mK value in the oxidative direction with epiDCA as substrate was about twice the mK determined for 12-oxoLCA. The mK for NADP+ was 36.84 ± 0.55 µM. The maxV and catk were greater in the oxidative than the reductive direction. However, the catalytic efficiency (cat/Kmk) of 12-oxoLCA as substrate was greater than the oxidative direction with epiDCA as substrate.Michaelis-Menten kinetics were performed at the pH optimum for each direction. In the reductive direction, Cp12β-HSDH displayed a + and NADH were not co-substrates for Cp12β-HSDH. DCA  as well as CA  were not substrates, which is expected because they are 12α-hydroxy bile acids not 12β-hydroxy bile acids. CDCA  lacks a 12-hydroxyl group, and as expected was not a substrate. The CA derivatives 12-oxoCDCA  and epiCA  had ~12% and 27% activity, respectively, relative to bile acids lacking a 7α-hydroxyl group. The activity of 3,12-dioxoLCA was ~19% compared to 12-oxoLCA. Altogether, the results suggest Cp12β-HSDH is specific for NADP(H) and favors 12-oxoLCA and epiDCA over their 7α-hydroxy counterparts.Pyridine nucleotide cofactor and bile acid substrate-specificity of Cp12β-HSDH were determined by relative activity compared to either 12-oxoLCA or epiDCA through spectrophotometric assay . NAD+ anC. paraputrificum ATCC 25780 (WP_027099077.1) was used in a BLASTP search against the NCBI non-redundant protein database in order to determine its prevalence across bacteria. A maximum likelihood phylogeny of 5,000 sequences was constructed, revealing that many sequences most similar to Cp12β-HSDH are found in Firmicutes and Actinobacteria . Within the 5,000-member phylogeny, a subtree (highlighted gray) of the most closely related proteins to Cp12β-HSDH was selected for closer inspection . These sequences are encoded by C. paraputrificum strains isolated from preterm infants, namely strain LH025, LH141, and LH058,23 or isolated from feces (Gcol.A11).24The Cp12β-HSDH sequence from spection . Cp12β-HEisenbergiella, Ruminococcus, and Coprococcus. To determine if other organisms within the tree have bile acid 12β-HSDH activity, the gene encoding WP_118677302.1 from Eisenbergiella sp. OF01-20 was synthesized by IDT in the codon-usage of E. coli (Table S1), cloned into pET-28a(+), overexpressed in E. coli, and purified by affinity chromatography . The three 12β-HSDH genes did not appear to be organized within an operon nor was the genomic context conserved across these organisms.The genomic context of 12β-HSDH genes from Collinsella tanakaei and Collinsella stercoris  and 12β-HSDH (WP_009140706.1) are encoded by the genes HMPREF9452_RS06335 and HMPREF9452_RS03390, respectively. Collinsella stercoris DSM 13279 also contains both putative 12α-HSDH  and 12β-HSDH .10 While the paired activity of 12α/12β-HSDH has not been tested in culture, these organisms may be novel epi-bile acid epimerizing strains that convert bile acid 12α-hydroxyl groups to the epi-configuration. To our knowledge, these are the first strains identified with C-12 epimerizing ability.Two organisms present in the 12β-HSDH subtree, 30–33 using reference sequences from the 12β-HSDHs characterized in this paper  search was performed against metagenome assembled genomes (MAGs) from four publicly available cohortsis paper . Putativis paper . Twenty-Lachnospira eligens (formerly Eubacterium eligens)  was the only member of phylum Bacteroidetes with the gene.Two hundred twenty microbial genomes contained putative 12β-HSDH genes among 16,936 total available genomes. Putative 12β-HSDH genes were most often identified in the phylum Firmicutes, which was dominated by genes in eligens) . The gen subtree . LachnosTable S2).15 The sequences included span the known HSDH functional capacities, with some recognizing bile acids and others recognizing steroids like cortisol or progesterone. Most members of each HSDH class are clustered together, which is apparent by each highlight color encompassing more than one HSDH of the same known function. Furthermore, most bacterial HSDHs grouped separately from their eukaryotic counterparts.Next, the phylogenetic relationship between Cp12β-HSDH (WP_027099077.1) and other regio- and stereospecific HSDHs was explored. To accomplish this, we updated the HSDH phylogeny presented by Mythen et al. (2018) by including additional bacterial or archaeal HSDH sequences of known or putative function along with representative eukaryotic sequences 36 and Eggerthella (CDD59475.1).15Collinsella aerofaciens (EBA39192.1), which has been reported to express bile acid 12α-HSDH activity,25 grouped with the known bile acid 12α-HSDHs along with two human gut archaeal sequences from Methanosphaera stadtmanae and Methanobrevibacter smithii.Prokaryotic sequences were interspersed among the eukaryotic with some exceptions in grouping by HSDH function. Cp12β-HSDH, the two other confirmed bile acid 12β-HSDHs , and additional similar sequences from across our bile acid 12β-HSDH subtree formed their own cluster. These sequences shared a branch with bacterial bile acid 12α-HSDHs as well as eukaryotic 3β-HSD/Δ37 clustered separately from those expressed by E. coli (BAA01384.1)38 and those predicted in Bacteroides (gram-negative), similarly to the Mythen et. al. (2018) phylogeny. Bile acid 7β-HSDHs did not closely cluster with other classes of bacterial HSDHs. Instead, the nearest neighbors to the three known bile acid 7β-HSDHs 40 included in this tree were eukaryotic steroid 11β- and 17β-HSDs.Clostridial (gram-positive) bile acid 7α-HSDHs (AAB61151.1 etc.)Eggerthella lenta (ACV54671.1).41 Within the bile acid 3α-HSDH group, four enzymes predicted with this function formed a separate branch apart from the confirmed bile acid 3α-HSDHs. Likewise, three known bile acid 3β-HSDHs grouped together ,41 while one Eggerthella sequence was most closely related to putative bile acid 3β-HSDHs from Lactobacillus spp. identified by BLAST search in Mythen et. al (2018).Bacterial 3α-HSDHs clustered together, excluding one outlier from 43 grouped with putative sequences from both gut and urinary tract isolates. To date, only one steroid 20α-HSDH sequence, which interconverts cortisol and 20α-dihydrocortisol, has been reported (C. scindens EDS07887.1).45 Therefore, we performed a BLASTP search and found two sequences with high similarity, WP_145772308.1 from Denitratisoma oestradiolicum DSM 16959 and WP_107631222.1 from Intestinibacillus sp. Marseille-P4005. D. oestradiolicum DSM 16959 was isolated from sludge in a municipal wastewater treatment plant and can use 17β-estradiol as a sole carbon and energy source.46Intestinibacillus massiliensis strain Marseille-P3216, a close relative to Intestinibacillus sp. Marseille-P4005 found in our tree, was isolated from the human colon and is most closely related to the species Butyricicoccus desmolans (formerly Eubacterium desmolans) by 16S rRNA gene sequence.47 Interestingly, B. desmolans ATCC 43058 encodes a 20β-HSDH (WP_051643274.1).43Bacterial steroid 20β-HSDHs convert the glucocorticoid cortisol to 20β-dihydrocortisol. Two experimentally confirmed 20β-HSDHs Homo sapiens 11β-HSD type 1 (NP_861420.1) was closely related to Rattus norvegicus 11β-HSD type 1 (NP_058776.2) and more distantly related to Homo sapiens 11β-HSD type 2 (NP_000187.3). 11β-HSD type 1 and type 2 both interconvert steroids between active and inactive forms, such as cortisol and cortisone.48 However, 11β-HSD type 1 primarily acts as a reductase in many tissues while 11β-HSD type 2 functions as a dehydrogenase.48Eukaryotic HSDH sequences, typically denoted HSD, were spread throughout the phylogeny, but generally grouped with like sequences. The 17β- and 11β-HSD sequences did not form a group, instead clustering by type. For example, 49 Thereafter, 3β-, 7β- and 12α-HSDH activity was observed in cultures of various microbiota,1 including Eggerthella lenta (formerly Eubacterium lentum) which is capable of oxidizing CA and DCA at C-12 and epimerizing bile acids at C-3.50 In the mid-1980s, C. paraputrificum, C. tertium, and Clostridioides difficile each in binary cultures with E. lenta were shown to epimerize DCA via a 12-oxo-intermediate to epiDCA.18 Since then, HSDH genes encoding the iso- and urso-bile acid pathways and 12α-HSDH were identified, but not 12β-HSDH.5 In this work, we identified the first bile acid 12β-HSDH gene, completing the microbial epi-bile acid pathway.Microbial bile acid HSDHs have been studied since the early 1970s, with much of the original work focusing on 3α- and 7α-HSDHs.C. paraputrificum strain D 762–06, with differing results from our findings.19 Gel filtration analysis of crude extract from C. paraputrificum strain D 762–06 suggested a molecular mass of 126 kDa, whereas our current work with Cp12β-HSDH from ATCC 25780 is estimated at 54.6 KDa by gel filtration chromatography. The strain used in this study, C. paraputrificum ATCC 25780, was also isolated from feces.51 It is possible that these are the same NADP(H)-dependent enzymes by sequence from two different strains of C. paraputrificum and that the recombinant protein quaternary structure is unstable, resulting in a dimeric form in our study. Alternatively, these bacterial strains may have distinct versions of 12β-HSDH with different amino acid sequences, as we have shown previously for 12α-HSDH from Eggerthella lenta.52 Indeed, the 12β-HSDH from C. paraputrificum strain D 762–06 was reported to be partially membrane associated, whereas hydropathy prediction by TMHMM v. 2.0 found no evidence of transmembrane domains in Cp12β-HSDH. In addition, pH optima for the conversion of 12-oxoLCA between Cp12β-HSDH (7.0) and the native 12β-HSDH (10.0) from strain D 762–06 differed. Oxidation of epiDCA was optimal at pH 7.5 for Cp12β-HSDH, and reported as pH 7.8 for the crude native enzyme from strain D 762–06.19 Further work will be needed to determine if distinct bile acid 12β-HSDHs are present in C. paraputrificum strains.Edenharder & Pfützner (1988) initially characterized NADP(H)-dependent 12β-HSDH from crude extracts of the fecal isolate 53 or dimeric.55 Cp12β-HSDH was more specific for bile acids lacking a position 7-hydroxyl group: epiDCA and 12-oxoLCA, over epiCA and 12-oxoCDCA. Cp12β-HSDH also had lower activity with 3,12-dioxoLCA versus 12-oxoLCA. This indicates that both the 7-hydroxyl and 3-oxo groups hinder the ability of Cp12β-HSDH to convert the substrate. An x-ray crystal structure of Cp12β-HSDH may shed light on why this apparent steric hindrance occurs.Cp12β-HSDH exhibited a dimeric quaternary structure by size-exclusion chromatography under our experimental conditions. Although future crystallization of Cp12β-HSDH would better illustrate its true polymeric state, HSDHs are often either tetramericEisenbergiella sp. OF01-20 and Olsenella sp. GAM18, lending functional credibility to sequences throughout the subtree . Eisenbergiella spp. are often present at relative abundances of less than 0.1% in human fecal samples.57Olsenella sp. GAM18 was initially isolated from humans (IMG Genome ID: 2841219092). The relative abundance of Olsenella was shown to be about 2% within the gut microbiome of some individuals.58 Our subtree includes more abundant gut taxa such as Ruminococcus (relative abundance ~5%)60 and Collinsella (relative abundance ~8%),59 as well. Due to limitations in 16S rDNA sequencing depth, it is difficult to conclude if the species in our subtree are found at relevant levels in the human gut or if 12β-HSDH genes are present. Therefore, we performed a HMM search to assess the relative prevalence of 12β-HSDH genes. About 30% of subjects had putative 12β-HSDH genes, indicating the relevance of this gene in the human gut microbiome. The HMM search revealed that 220 microbial genomes out of 16,936 total contained putative 12β-HSDH genes. While concrete prevalence is difficult to establish, putative 12β-HSDH genes are less widespread than the ubiquitous bile-acid metabolizing gene, bile salt hydrolase,4 which was present in 2,456/16,936 total genomes in these cohorts. These data expand the limited metagenomic work that has focused on bile acid HSDH genes in the human gut.61Phylogenetic analysis of Cp12β-HSDH coupled with synthetic biological “sampling” and validation at different points along the branches revealed shared 12β-HSDH function among  subtree ; Table 210 Putative proteins WP_009140706.1 (12β-HSDH) and WP_009141301.1 (12α-HSDH) are both present in Collinsella tanakaei YIT 12063 and are encoded by the genes HMPREF9452_RS03390 and HMPREF9452_RS06335, respectively. Similarly, Collinsella stercoris DSM 13279 encodes both putative 12β-HSDH  and 12α-HSDH .10 Although the dual 12α/12β-HSDH activity is untested in culture, we predict these strains are novel C-12 epimerizers. Epimerizing strains have been identified for the C-341 and C-7 hydroxyl62 positions, however, this is the first indication of bacteria capable of C-12 epimerization.Two organisms from our 12β-HSDH subtree were also identified in a previous 12α-HSDH phylogeny from Doden et al. (2018).Novosphingobium sp. AP12, whose recombinant enzyme product did not exhibit bile acid 12β-HSDH activity with the substrates tested, may be specific for aerobic bile acid degradation products. Environmental microorganisms, such as Comamonas testosteroni TA441 and Pseudomonas sp. strain Chol1, encode a CA degradation pathway involving conversion of a 12-oxo-intermediate to 7α,12β-dihydroxy-androsta-1,4-diene-3,17-dione (12β-DHADD).64 Thus, sequences in the extension of the subtree may have 12β-HSDH activity, but with specificity for side-chain cleaved steroids rather than bile acids.The sequence WP_007678535.1 from 1 Bile acid 12α-HSDH activity has been detected in Eggerthella species52 in the phylum Actinobacteria and various clostridia36 in the phylum Firmicutes. Similarly, 3α- and 3β-HSDH are widespread among Firmicutes,65 and 3α-HSDH has also been reported in Eggerthella species.41 7α- and 7β-HSDH were shown in numerous Firmicutes65 and the Actinobacteria Collinsella aerofaciens.24 Along with these bile acid-specific HSDHs, the glucocorticoid 20α- and 20β-HSDHs are evident in both Firmicutes45 and Actinobacteria such as Bifidobacterium adolescentis. Until this study, there were no reports of genes encoding 12β-HSDH and the activity had only been shown in C. paraputrificum, C. tertium and C. difficile.18 Thus, our phylogenetic analysis revealed hitherto unknown diversity for bile acid 12β-HSDHs within the Firmicutes and Actinobacteria. Bacteroidetes sequences were notably absent within our 12β-HSDH subtree and only one sequence was identified in our HMM search, although Bacteroidetes have been shown to encode multiple other HSDHs.49 Interestingly, C. tertium and C. difficile enzymes were also not present in our phylogenetic analysis even though this activity has been reported for strains of these clostridia,18 indicating that genes encoding other forms of bile acid 12β-HSDH are present in the gut microbiome.Indeed, this function joins the vast repertoire of HSDHs already studied in many Firmicutes and Actinobacteria.66 HSDH enzymes may thus act in interkingdom-signaling, a hypothesis that has recent support based on the effect of oxidized and epimerized bile acids on the function of regulatory T cells.68The distribution pattern of microbial HSDHs is becoming increasingly clear , althougClostridium limosum (now Hathewaya limosa) expresses both bile acid-inducible NADP-dependent 7α- and 7β-HSDH capable of converting CDCA to UDCA.69 CDCA is more hydrophilic and more toxic to bacteria than UDCA.70 Indeed, treatment with UDCA increases the hydrophilicity of the biliary pool, reducing cellular toxicity and improving biliary disorders.71 Similarly, strains of Eggerthella lenta41 and Ruminococcus gnavus41 express both NADPH-dependent 3α- and 3β-HSDHs capable of forming 3β-bile acids (iso-bile acids). Iso-bile acids are also more hydrophilic and less toxic to bacteria than the α-hydroxy isomers.41 At least some strains of R. gnavus also express NADPH-dependent 7β-HSDH, contributing to the epimerization of CDCA to UDCA.39 It may be speculated that R. gnavus HSDHs function in detoxification of hydrophobic bile acids such as CDCA and DCA; however, further work is needed. Analogous to E. lenta and R. gnavus, C. paraputrificum is another example of a strain encoding multiple HSDHs that favor formation of β-hydroxy bile acids.19C. paraputrificum strains encode the iso-bile acid pathway as well as NADPH-dependent 12β-HSDH.19 While little is known about the biological effects of 12β-bile acids (epi-bile acids), the physicochemical properties relative to 12α-hydroxy bile acids should approximate that of iso- and urso-derivatives.70 An important question emerging from these observations is whether one particular epimeric product rather than another has important consequences on the fitness of the bacterium generating them, or if the increased hydrophilicity and reduced toxicity are the key factors.The concerted action of pairs of HSDHs result in bile acid products with reduced toxicity for microbes expressing the HSDH(s) or for an important inter-species partner, which was likely a factor in the evolution of these enzymes. Examples of strains of species capable of epimerizing bile acid hydroxyl groups are found in the literature, and the physicochemical properties and reduced toxicity of β-hydroxy bile acids are known, providing hypotheses for physiological function. 15–17 the measurement of bile acid metabolomes in clinical samples has become commonplace,72 yet few studies measure or report epi-bile acids. Recently, 12β-hydroxy and 12-oxo-bile acids have been quantified in human feces by Franco et. al. (2019). 12-oxoLCA was the most abundant oxo-bile acid in feces at concentrations of about one half that of DCA in stool. While epiDCA itself was not measured, 3-oxo-12β-hydroxy-CDCA was shown at 12 ± 4 µg/g wet feces.73 Additionally, epiDCA has been reported in biliary bile of angelfish, likely produced from bacterial origin, so the 12β-HSDH gene may be widespread among resident microbiota of diverse vertebrate taxa.74 A critical limitation to the study of epi-bile acids is the absence of commercially available standards, although there are methods available for chemical synthesis.76 The newly identified bile acid 12β-HSDHs could be employed for the enzymatic production of epi-bile acid standards from oxo-intermediates.Since the initial detection of epi-bile acids by Eneroth et. al. and Ali et. al., 77 Another study reported increased 12-oxoLCA levels in rats with high tumor incidence when they were fed a high safflower oil or corn oil diet.78While the toxicity of epi-bile acids has not yet been tested relative to the secondary bile acids DCA or LCA, both 12-oxoLCA and epiDCA are less hydrophobic than DCA by LC-MS . E. coli DH5α (Turbo) competent cells from New England Biolabs  and NovaBlue GigaSingles™ Competent cells from Novagen  were used for cloning, and E. coli BL21-Codon-Plus (DE3) RIPL was purchased from Stratagene  and used for protein overexpression. 5β-Cholanic acid-3α, 7α, 12α-triol (CA), 5β-cholanic acid-3α,12α-diol (DCA), and 5β-cholanic acid-3α,7α-diol (CDCA) were purchased from Sigma-Aldrich . Authentic 5β-cholanic acid-3α,12β-diol (epiDCA) and 5β-cholanic acid-3α,7α,12β-diol (epiCA) were generously obtained from Lee R. Hagey . Other bile acids were purchased from Steraloids . All other reagents were of the highest possible purity and purchased from Fisher Scientific .C. paraputrificum ATCC 25780 and C. scindens ATCC 35704 were cultivated in anaerobic brain heart infusion (BHI) broth for 24 hrs. Two mL anaerobic BHI was inoculated with 1:10 dilution of either organism along with 50 μM bile acid substrate and incubated at 37°C for 24 hours. The bacterial cultures were centrifuged at 10,000 × g for 5 min to remove bacterial cells and the conditioned medium was adjusted to pH 3.0. Solid phase extraction was used to extract bile acid products from bacterial culture. Waters tC18 vacuum cartridges (3 cc)  were preconditioned with 6 mL 100% hexanes, 3 mL 100% acetone, 6 mL 100% methanol, and 6 mL water (pH 3.0). The conditioned medium was added to the cartridges and vacuum was applied to pull media through dropwise. Cartridges were washed with 6 mL water (pH 3.0) and 40% methanol. Bile acid products were eluted with 3 mL 100% methanol. Eluates were then evaporated under nitrogen gas and the residues dissolved in 200 μL 100% methanol for LC-MS analysis.LC-MS analysis for all samples was performed using a Waters Acquity UPLC system coupled to a Waters SYNAPT G2-Si ESI mass spectrometer . LC was performed with a Waters Acquity UPLC HSS T3 C18 column  at a column temperature of 40°C. Samples were injected at 1 μL. Mobile phase A was water and B was acetonitrile. The mobile phase gradient was as follows: 0 min 100% mobile phase A, 0.5 min 100% A, 25 min 0% A, 25.1 min 100% A, 28 min 100% A. The flow rate was 0.5 mL/min. MS was carried out in negative ion mode with a desolvation temperature of 300°C and desolvation gas flow of 700 L/hr. The capillary voltage was 3,000 V. Source temperature was 100°C and cone voltage was 30 V. Chromatographs and mass spectrometry data were analyzed using Waters MassLynx software .C. paraputrificum ATCC 25780 using the Fast DNA isolation kit from Mo-Bio  according to the manufacturer’s protocol for polymerase chain reaction and molecular cloning applications.Genomic DNA was extracted from C. paraputrificum ATCC 25780 genomic DNA or genes synthesized in E. coli K12 codon usage . Cloning primers and genes created by gene synthesis are listed in Table S1. Inserts were amplified using the Phusion High Fidelity Polymerase  and cloned into pET-28a(+) after insert and vector were double digested with the appropriate restriction endonuclease and treated with DNA Ligase, or annealed into pET-46 Ek/LIC after treatment with T4 DNA Polymerase. Recombinant plasmids were transformed via heat shock method, plated, and grown overnight at 37°C on lysogeny broth (LB) agar plates supplemented with antibiotic (50 µg/mL kanamycin or 100 µg/mL ampicillin). Vectors were either transformed into chemically competent E. coli DH5α cells and grown with kanamycin (pET-28a(+)) or transformed into NovaBlue GigaSingles™ Competent cells and grown with ampicillin (pET-46 Ek/LIC). A single colony from each transformation was inoculated into LB medium (5 mL) containing the corresponding antibiotic and grown to saturation. Recombinant plasmids were extracted from cell pellets using the QIAprep Spin Miniprep kit . The sequence of the inserts was confirmed by Sanger sequencing .The pET-28a(+) and pET-46 Ek/LIC vectors were obtained from Novagen . Restriction enzymes were purchased from NEB . Inserts were generated by PCR amplification with cloning primers from Integrative DNA Technologies  of E. coli BL-21 CodonPlus (DE3) RIPL chemically competent cells by heat shock method and cultured overnight at 37°C on LB agar plates supplemented with ampicillin or kanamycin  and chloramphenicol (50 µg/ml). Selected colonies were inoculated into 10 mL of LB medium supplemented with antibiotics and grown at 37°C for 6 hours with vigorous aeration. The pre-cultures were added to fresh LB medium (1 L), supplemented with antibiotics, and aerated at 37°C until reaching an OD600nm of 0.3. IPTG was added to each culture at a final concentration of 0.1 mM to induce and the temperature was decreased to 16°C for a 16-hour incubation. Cells were pelleted and resuspended in binding buffer . The cells were subjected to five passages through an EmulsiFlex C-3 cell homogenizer , and the cell debris was separated by centrifugation.For protein expression, the extracted recombinant plasmids were transformed into https://imagej.nih.gov/ij/docs/faqs.html). TMHMM v. 2.0 was used to predict transmembrane helices.21The recombinant protein in the soluble fraction was then purified using TALON® Metal Affinity Resin  per the manufacturer’s protocol. The recombinant protein was eluted using an elution buffer composed of 20 mM Tris-HCl, 300 mM NaCl, 10 mM 2-mercaptoethanol, and 250 mM imidazole at pH 7.9. The resulting purified protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The observed subunit mass for each was calculated by migration distance of purified protein to standard proteins in ImageJ (−1.cm−1) continuously for 1.5 min on a NanoDrop 2000c UV-Vis spectrophotometer  using a 10 mm quartz cuvette . Additional reactions were incubated overnight at room temperature and extracted by vortexing with two volumes ethyl acetate twice. The organic layer was recovered and evaporated under nitrogen gas. The products were dissolved in 50 μL methanol and LC-MS was performed as described above or used for thin layer chromatography.Pure recombinant 12β-HSDH reaction mixtures were made using 50 μM substrate, 150 μM cofactor and 10 nM enzyme in 150 mM NaCl, 50 mM sodium phosphate buffer at the pH optima 7.0 or 7.5. Reactions were monitored by spectrophotometric assay measuring the oxidation or reduction of NADP(H) aerobically at 340 nm , 50 mM sodium phosphate (pH 6.5 to 7.5), and 50 mM Tris-Cl (pH 8.0). Substrate specificity was performed according to the above reaction conditions at the optimal pH.+ when epiDCA concentrations were changed or 100 µM epiDCA when NADP+ was varied. Kinetic parameters were estimated with GraphPad Prism  to fit the data using nonlinear regression to the Michaelis-Menten equation.The reaction mixtures for kinetic analysis were 10 nM enzyme, sodium phosphate buffer (pH 7.0), and 150 µM NADPH for varying concentrations of 12-oxoLCA or 80 µM 12-oxoLCA for varying NADPH concentrations in the reductive direction. The oxidative reaction mixture contained 10 nM enzyme, sodium phosphate buffer (pH 7.5), and 300 µM NADP79Reaction mixtures were made using 50 μM substrate, 150 μM cofactor and 10 nM enzyme in 150 mM NaCl, 50 mM sodium phosphate buffer at pH 7.0. Reactions were incubated overnight at room temperature and extracted by vortexing with two volumes ethyl acetate twice. The organic layer was recovered and evaporated under nitrogen gas. The products were dissolved in 50 μL methanol and spotted on a TLC plate . The steroids were separated with a 70:20:2 toluene–1,4-dioxane–acetic acid mobile phase and visualized using a 10% phosphomolybdic acid in ethanol spray and heating for 15 min at 100°C.12.Size-exclusion chromatography was performed using a Superose 6 10/300 GL analytical column  connected to an ÄKTAxpress chromatography system  at 4°C. The column was equilibrated with 50 mM Tris-Cl and 150 mM NaCl at a pH of 7.5. The purified protein was loaded onto the analytical column at a concentration of 10 mg/mL and eluted at a flow rate of 0.3 ml/min. The native molecular mass of 12β-HSDH was determined by comparing its elution volume to that of Gel Filtration Standard proteins : thyroglobulin, γ-globulin, ovalbumin, myoglobin, vitamin BC. paraputrificum 12β-HSDH protein (accession number WP_027099077.1) was used as query for a similarity search against the NCBI non-redundant protein database by BLASTP,80 with a maximum E-value threshold of 1e-10 and a limit of 5,000 results. Retrieved sequences were aligned with Muscle v. 3.8.155181 and analyzed by maximum likelihood with RAxML v. 8.2.11.82 Selection of the best-fitting amino acid substitution model and number of bootstrap pseudoreplicates were performed automatically, and substitution rate heterogeneity was modeled with gamma distributed rate categories. The resulting phylogenetic tree was formatted by Dendroscope v. 3.5.1083 and further cosmetic modifications were performed with the vector editor Inkscape, v. 0.92.4 (https://inkscape.org).The sequence of the C. paraputrificum ATCC 25780 12β-HSDH, sequences from the well-supported subtree where this sequence is located in the 5,000-sequence tree, plus an outgroup, were reanalyzed for confirming the relative placement of all sequences nearest to Cp12β-HSDH. The methods used were the same as described above for the full tree.For closer analysis of the phylogenetic affiliation of 35 with the addition of eukaryotic, archaeal, and other bacterial sequences deposited in the public databases. Phylogenetic inference methods were the same as described above.A maximum-likelihood tree of representative HSDH sequences was inferred by selecting sequences from each HSDH subfamily, based on the tree from Mythen et al. (2018),29–32 MAGs were filtered for genome completeness, quality, and contamination as described.84 For generation of the custom 12β-HSDH profile, reference sequences from the 12β-HSDHs characterized in this paper were aligned with MAFFT, manually trimmed, and constructed using hmmscan.85 The MAG database was searched using HMMSearch version 3.3.085, using an individually identified cutoff of 350.00. Resulting hits were then filtered to remove results less than 70% completeness and closest matched species were recorded. The HMM search file is publicly available at: https://github.com/AnantharamanLab/doden_et_al_2021.A Hidden Markov Model (HMM) search was performed using a custom HMM profile against a concatenated file of metagenome assembled genomes (MAGs) from four publicly available cohorts.Click here for additional data file.


Noise exposure leads to a reduction in cognitive abilities in diverse settings, however, only limited data exist examining the effects of environmental ICU noise on the cognitive performance of ICU professionals. A frequently occurring and demanding retention task in ICUs are ward rounds, which are considered key for the provision of medical care. Here, we investigate the influence of noise on information retention in a simulated ward round.ICU professionals were exposed to a recorded, ICU ward round, simulated partly with and partly without environmental ICU noise. Ward rounds were followed by specific questions about previously provided information.P < 0.001) in the ward round test performance when participants were exposed to environmental ICU noise. Furthermore, advanced age was associated with reduced retention , questions containing important information performed better , and higher stress led to better performance in retention .56 ICU professionals (aged 26–59 years) were included. A logistic mixed model showed a reduction of 27% (Our data showed a considerable negative influence of environmental ICU noise during a simulated ward round. Therefore, reduction of environmental ICU noise is recommended. The influence of additional factors, including stress, priorities, and demographic factors should be pursued in subsequent investigations.The online version contains supplementary material available at 10.1186/s40635-022-00430-1. Intensive Care Unit (ICU) professionals are exposed to a lot of noise in their ICU work environment , 2. ThisIn the ICU, examples of the most common sound sources are equipment , patient care (e.g. patient transfer), patients themselves (e.g. coughing), the immediate work environment (e.g. staff conversations), and general background noise . When coThe impact of background noise in a non-hospital setting has been widely discussed in the literature and reduced performance in cognitive functions has been shown, such as in working memory , recall The effect of noise could be especially important during ICU ward rounds, which are used to communicate between shifts . Ward roThe aim of the simulated ward round was to assess the influence of ICU noise on the retention ability of ICU professionals, in an experimental setting. Potential influencing variables were also included for a more precise influence analysis of the retention ability after ward rounds.Participants were ICU professionals, namely nurses and physicians, working in the Department of Intensive Care Medicine at the University Hospital Bern in Switzerland. They were invited via word-of-mouth during their working shift. Professionals were excluded if any of the following criteria were met, age under 18 years, pre-existing known neurological disorder or auditory impairment, or use of prescribed medication that could alter the level of alertness. Participation was voluntary and all participants signed a written informed consent. The study was approved by the local ethics committee at the University of Bern . The study was performed in adherence to the Declaration of Helsinki.This pilot study took place in a quiet room in the hospital, where a maximum of two ICU professionals could participate at a time. When there was more than one participant at a time, they sat with their backs to each other while wearing noise-cancelling over-ear headphones. Each participant listened to eight different audio files . The participants then listened to half of the cases in the Speech Condition and the other half in the Noise Condition. The starting condition and the order in which the eight patient cases were presented to the participants were randomised, while the conditions (i.e. Speech Condition vs Noise Condition) were presented alternately. Directly after listening to the eight audio files, participants’ demographics were collected, and the ward round test  and were played at a weighted sound pressure level of 70 dB SPL. In the Noise Condition simulated ICU noise was added. The simulated noise was manually constructed by adding different ICU sound sources, such as alarms and pagers, to ICU background noise recorded in a real ICU environment. Due to the limited effect of alarms on overall sound levels  and 17 were male, 38 were female, and 1 was not specified .Descriptive analyses showed the percentage of the correct responses given in the ward round test per case. Percent correct answers in the ward round test showed higher retention performance in the Speech Condition in six of the eight patient cases presented during the ward round Table . Due to P < 0.001, Table P < 0.001), more important questions performed better , and higher perceived stress in the past week led to better performance in the ward round test .In the mixed logistic regression analysis, the Noise Condition led to a decrease of 27% , which were found to be important, too. In further studies, however, a deeper focus on these aspects is necessary. Especially, the inclusion of other memory tasks might lead to complementary results.Moreover, due to the investigatory nature of a pilot study ICU professionals were grouped in this first analysis, however, future work would need to differentiate between these two groups.Finally, absence of real patients in the experimental setting could be a limitation. On the one hand, the presence of patients may be important, because they could support the memorisation of information as it is being received both visually and auditory , on the Environmental noise in the ICU during ward rounds negatively influences retention of ICU professionals. Our data lend to the fact that reducing noise, e.g. by means of targeted interventions, should be investigated. Ideally these interventions will target age, stress, and the influence of important questions.Environmental ICU noise reduced retention in ICU professionals by about 27%.Younger age, importance of information, and stress level positively influenced the retention performance of ICU professionals.Additional file 1. Part 1. Cases. Part 2. Full Demographic Questionnaire. Part 3. Ward round questionnaire. Part 4. Audio files.


Kinetics studies of the copolymerization of the fluoroalkenes copolymers were monitored by GPC and 19F NMR with molar masses up to 12,200 g/mol and dispersities (Đ) ranging from 1.33 to 1.47. Such an OMRcP behaves as a controlled copolymerization, evidenced by the molar mass of the resulting copolymer-monomer conversion linear relationship. The reactivity ratios, ri, of both comonomers were determined by using the Fineman-Ross and Kelen-Tüdos fitting model leading to rVDF = 0.384 ± 0.013 and r1234yf = 2.147 ± 0.129 at 60 °C, showing that a lower reactivity of VDF integrated in the copolymer to a greater extent leads to the production of gradient or pseudo-diblock copolymers. In addition, the Q (0.03) and e (0.06 and 0.94) parameters were assessed, as well as the dyad statistic distributions and mean square sequence lengths of PVDF and P1234yf.New copolymers based on vinylidene fluoride (VDF) and 2,3,3,3-tetrafluoroprop-1-ene (1234yf) were synthesized by organometallic-mediated radical copolymerization (OMRcP) using the combination of bis( All copolymerizations were conducted under similar conditions ([VDF] 19F NMR  and GPC  19F NMR a. At thie chains .D) signals since the nD increment of PVDF (and probably poly(1234yf)) in DMF is negative 0/[1234yf]0 molar ratios ranging from 90/10 to 16/84 in DMC at 60 °C using a predetermined amount of P16 and Co(acac2) of which the molar ratio was always maintained at 2/1. The copolymer conversion was limited to less than 10–15% to minimize the drift of copolymer formation. The VDF molar fraction contained in the copolymer was assessed from 19F-NMR spectra based on Equation (1)  , Table 2VDF > FVDF). This result clearly indicates that there is a significant difference in the reactivity ratios between the fluoromonomers. The reactivity ratios of VDF and 1234yf were determined by applying the Fineman-Ross 0/[Co(acac)2]0 = 2/1, T = 60 °C), leading to P(VDF-co-1234yf) copolymers with relatively low dispersities (Ð < 1.47) until conversion up to 33%. The molar masses of such copolymers, determined by GPC, increased linearly with the monomer conversions. The reactivity ratios of both comonomers were calculated based on Fineman-Ross and Kelen-Tüdös models where similar ratios were obtained in both cases: rVDF = 0.384 ± 0.013 and r1234yf = 2.147 ± 0.129 at 60 °C, denoting that 1234yf is more reactive than VDF, thus likely favoring the formation of gradient copolymers. The result is in agreement with the dyad statistical distribution in the copolymers. Finally, the Q-e values could also be calculated for the first time. The above results demonstrate that Co(acac)2-mediated controlled radical copolymerization of VDF and 1234yf initiated by P16 is possible. Finally, future works will look more closely into further optimization of this synthetic strategy which could open the route towards the synthesis of well-defined block terpolymers that will offer the desired combination of properties for advanced applications.For the first time, the OMR copolymerization of VDF and 1234yf initiated by the presence of P16 using Co(acac)