BACKGROUND: There have been few prospective studies on diet and glucose abnormalities as determined by oral glucose tolerance test.METHODS: To investigate the impact of dietary factors on the development of glucose intolerance including diabetes and impaired glucose tolerance, we performed a follow-up survey of 1,075 subjects aged 40-74 years of normal glucose tolerance from 1988 through 1993/1994 by repeated 75 g oral glucose tolerance test and dietary survey. Information on habitual food consumption was obtained using a semiquantitative food frequency method.RESULTS: Of the total subjects studied, 119 (11.1 %) developed impaired glucose tolerance and 24 (2.2 %) developed diabetes during the follow-up. At baseline, the age-adjusted amount of alcohol intake was significantly higher in males who developed glucose intolerance than in those who did not , while the polyunsaturated/saturated fatty acids (P/S) ratio was significantly higher in females with future glucose intolerance . Among the female subjects who developed glucose intolerance, the intake of animal fat less decreased during the follow-up period compared with normal subjects, resulting in a significant decrease in the P/S ratio . In a multiple logistic regression analysis, alcohol intake at baseline for males and decreased P/S ratio during the follow-up for females remained a significant risk factor for glucose intolerance independent of other dietary and non-dietary factors as well.CONCLUSIONS: These results suggest that a high intake of alcohol and a decreased P/S ratio contribute to the risk of glucose intolerance in contemporary Japanese. In the literature, a number of studies have presented cross-sectional associations of dietary intake with glucose levels6,7,8,9 and hyperinsulinemia,6,10,11,12 but there have been very few studies of Japanese. Moreover, only a few prospective studies on diet and glucose abnormalities have been reported,13,14,15 and those using an oral glucose tolerance test (OGTT) have been fewer still even in western countries.16,17Diabetes mellitus appears to confer an excess risk of cardiovascular disease and premature death. In Japan, the prevalence of diabetes has increased over the past several decades.18 The present follow-up study used OGTT and nutritional surveys to investigate the longitudinal association between dietary factors and the development of diabetes and IGT among subjects having normal glucose tolerance at baseline.In 1988, we performed a prevalence study of diabetes and impaired glucose tolerance (IGT) using OGTT in a defined general Japanese population, Hisayama.18,19 Full community surveys of the residents aged 40+ years were repeated since 1961.The town of Hisayama is a suburban community adjoining the city of Fukuoka, a metropolitan area on Kyushu Island in Japan. The population of the town is approximately 7,500 and has scarcely changed over the last 40 years. According to the census information for this period, the population of the town, in terms of age, sex, and occupational distribution, reflects the population of Japan as a whole.18 Briefly, among the total of 3,227 Hisayama residents aged 40 to 79 years, 2,587 (80.2%) took part in the examination. After excluding 82 non-fasting subjects, 10 subjects under insulin therapy, and 15 subjects who could not complete the OGTT due to complaints of nausea or general fatigue during ingestion of a glucose solution, 2,480 remaining subjects  completed the OGTT and the nutritional survey. In accordance with the 1998 World Health Organization criteria,20 these participants were classified into three groups: a normal glucose tolerance, an IGT, and a diabetes mellitus group. The criteria are as follows: for diabetes, fasting plasma glucose 7.0+ mmol/L or 2 hr plasma glucose 11.1+ mmol/L in the OGTT; for IGT, fasting plasma glucose <7.0 and 2 hr plasma glucose 7.8-11.0 mmol/L; for normal glucose tolerance, fasting glucose <7.0 mmol/L and 2 hr glucose <7.8 mmol/L.The diabetes prevalence study was performed by simultaneously using a 75 g OGTT and a nutritional survey in a screening examination from June 29th through November 14th in 1988. A detailed description of this survey was published previously.Among the 1,625 persons  aged 40 to 74 years found to have normal glucose tolerance in 1988, 1,075  also participated in the follow-up examination performed in 1993/1994, in which we repeated the OGTT and nutritional survey in the same manner as in the initial screening . Among t21 and the nutritional elements were adjusted for energy intake using the method of Willet and Stampfer.22 A self-administered questionnaire concerning food intake over the previous year, which consisted of 70 food items, was completed prior to the start of the study by each participant and was checked by experienced dieticians and nutritionists by showing food models of actual size in the survey. The average food intake per day was estimated based on detailed descriptions of the frequency of eating and the quantity of each food. Nutritional intake was calculated using the fourth revision of the Standard Tables of Food Composition in Japan23 and its follow-up version for fatty acids, cholesterol, and vitamin E.24 In regard to foods which were not included in these Tables, we estimated the amounts of nutritional elements by using the similar foods included in the Tables.The dietary survey was conducted using a semiquantitative food frequency method,Blood samples were collected from an antecubital vein after an overnight fast for the determination of the serum insulin, serum lipid, and plasma glucose levels. After the fasting blood specimen was taken, each subject ingested a 75 g glucose-equivalent carbohydrate load  from 8:00 a.m. through 10:30 a.m. At 120 min after glucose loading, second blood sample was obtained to determine the postloading plasma glucose. Subjects receiving oral hypoglycemic agents were requested to avoid taking their medication until completion of the OGTT. The blood specimens were transferred immediately in ice-cooled containers to the central study laboratory  and were analyzed within 24 hours. The plasma glucose level was measured by the glucose-oxydase method using a glucose autoanalyser . The serum insulin level was determined by a commercial double-antibody solid-phase radioimmunoassay . The total cholesterol, HDL-cholesterol, and triglycerides were all measured enzymatically using an autoanalyser .2) was calculated. A questionnaire concerning physical activity during leisure time and smoking habits were also administered for all subjects. Those subjects engaging in sports at least once a week during their leisure time made up physically active group. Finally, subjects were classified into two groups according to their smoking habits: a habitual use and a non-habitual use group.Body height and weight were measured with subjects wearing light clothing without shoes, and the body mass index . In females, subjects without follow-up examination had a lower mean value of baseline body mass index  and a higher mean value of systolic blood pressure  than others, but such differences were not observed in males. In regard to dietary factors, baseline energy intake was lower in subjects without follow-up examination than in those with follow-up examination for both sexes . Males without follow-up examination had lower intake of carbohydrates compared with those with follow-up examination , while females without follow-up examination had lower intake of protein . However, these facts make it unlikely that this selection bias invalidates the findings of the present study.First, it is probable that some heavy drinkers among our subjects underreported their alcohol intake. However, the findings of our study do not seem to be results of this under-reporting, since all subjects were of normal glucose tolerance at baseline, and under-reporting is considered to have occurred randomly in both groups irrespective of future glucose tolerance status. Second, the information regarding nutrient intake was derived from a semi-quantitative food frequency questionnaire that asked about average intake over the previous year. The limitations of this method are well known,The results show that, in addition to fasting insulin, nutritional factors also predict the development of GI. A decreased P/S ratio is an independent risk factor for GI in females, as is alcohol intake in males. For the primary prevention of diabetes, of which the occurrence has increased steeply in Japan, it is important to restrict the westernization of dietary habits in addition to decreasing the incidence of both a sedentary lifestyle and obesity.


However, arsenic significantly enhanced ARG accumulation in earthworm casts in a dose-dependent manner. Moreover, vermicomposting increased the percentage of Bacteroidota in the converted products. Furthermore, arsenic exposure at low concentrations promoted the proliferation of Proteobacteria, whereas high concentrations had little effect on Proteobacteria. Our study provides valuable insight into the changes in the antibiotic resistome and related microorganisms during vermicomposting of arsenic-amended cow manure, and it is crucial to explain the environmental impact of earthworms and improve our understanding of the reciprocal benefits of soil invertebrates.Antibiotic resistance pollution in livestock manure is a persistent issue that has drawn public attention. Vermicomposting is an ecofriendly biological process that can render livestock manure harmless and resourceful. However, little is known about the impact of vermicomposting on antibiotic resistance in livestock manure under stress caused by potentially toxic arsenic levels. Herein, lab-scale vermicomposting was performed to comprehensively evaluate the shift in antibiotic resistance genes (ARGs) and related microorganisms in fresh earthworm casts as well as vermicompost product health  when they were fed on arsenic-contaminated cow manure. The results showed that the earthworms’ interaction with cow dung led to a significant reduction in ARG concentrations, especially for tetracycline ARGs ( In animal nutrition, arsenic, similar to selenium, molybdenum, iodine, and fluorine, although an essential trace element in the animal body, is also a potentially toxic one. Arsenic has been widely used in livestock and poultry breeding for a long time because of its ability to promote animal growth and improve feed efficiency . HoweverWidespread use of veterinary antibiotics fuels the contamination of livestock waste with ARGs, which seriously threatens animal safety and human health ,7,8,9. DVermicomposting is a biochemical decomposition process of livestock waste that involves the interaction between earthworms and microorganisms . VermicoEisenia fetida was chosen as the experimental organism to study the effect of arsenic on ARGs and their host bacteria during vermicomposting via lab-scale vermicomposting with the addition of arsenic to livestock manure . The objectives of this study were to: (1) determine the effect of earthworms (Eisenia fetida) on ARGs in cow manure by investigating the growth of ARGs in cow manure with the presence of earthworms, (2) explore the response of ARGs and related microorganisms to arsenic cow dung during vermicomposting at two concentration levels, and (3) identify the main factors influencing the variation in ARGs in the process of vermicomposting.In this study, Eisenia fetida with a well-developed clitellum were used as the subject earthworm species. Trisodium arsenate (Na3AsO4·12H2O) was purchased from Shanghai Chemical Reagent Purchasing and Supply Station. The earthworms were purchased from Tianjin Baiming Technology Development Co., Ltd.  Cow dung was collected from a dairy farm in Tianjin, China. Before the experiment, to improve the palatability of earthworms to cow manure, the cow dung was naturally stacked for 30 days to decompose.Mature specimens of 10, and T50, respectively. Based on 1 kg cow manure and different arsenic concentration treatments , the corresponding Na3AsO4 was weighed and dissolved in 600 mL deionized water. Then, the solution was sprayed evenly on the cow dung, which was stirred while spraying. Fermented cow dung (1.0 kg) was then added to each reactor. The reactor was an earthworm breeding box with dimensions of 28 cm × 19.5 cm × 11.5 cm and was drilled on the bottom to remove excess lixiviate. To prevent the earthworms from escaping, the top of the box was covered with gauze. About 24 h before exposure, the worms were removed from the cultures, rinsed with water, and kept on damp filter paper in darkness at 20 °C to obtain fresh casts. After depuration, 300 clitellum-banded Eisenia fetida of similar weight were placed in each plastic box. The experiment was conducted at room temperature (22–24 °C), and distilled water was sprinkled once every 3 days to keep the moisture content at 65–75%. The experimental period lasted 14 days. Before and after vermicomposting, the earthworms and the conversion product were collected from each reactor for measurement. Earthworms were counted every two days, and their weight was measured at the beginning and after vermicomposting of the experiment.Nine parallel vermicomposting reactors were set up that were randomly divided into three groups, each with three replicates. According to the actual residual range of arsenic in cow dung, the supplemental concentrations of arsenic in the three groups were set at 0, 10, and 50 mg/kg, and the groups were named CK, TThe obtained conversion product was air-dried naturally. The dry samples were ground and sifted through a particle size of 20 mesh (0.85 mm) for subsequent chemical analyses. All chemical analyses were based on Chinese organic fertilizer standards (NY/T 525-2021). First, 5.0 g of air-dried sample was added to 50 mL of deionized water, and the pH of the solution was measured at 25 °C with a pH meter. Next, organic matter (OM) was measured according to the instructions contained in the Soil Organic Matter (SOM) Kit  based on the potassium dichromate volumetric method ; the totThe earthworms were removed and placed in a dark, damp petri dish for 24 h, and the intestinal contents were emptied and retained. DNA samples from earthworm intestinal contents were extracted using the FastDNA Spin Kit as per manufacturer’s instructions . The same method was used to extract DNA from the cow manure and earthworm casts. The obtained DNA samples were resuspended in DNase/RNase-free water, and then the DNA concentration was determined by Qubit 2.0 fluorescence meter . All DNA extracts were stored at −20 °C before analysis.sul1, sul2, tetL, tetX, tetQ, tetO, tetW, blaampC, blaTEM-1, blaOXA-1, blaNDM, aadA, fexA, cfr, ermC, ermB, qnrB, qnrS, oqxB, strA, strB), two key integrase genes , and the 16S rDNA gene  in samples was quantified by real-time qPCR. Additional details of the primers used are provided in the The expression of 21 specific subtypes of ARGs  and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) in a thermocycler system. PCR amplification was performed using the following program: denaturation at 94 °C for 5 min, followed by 30 cycles of 30 s at 94 °C, annealing at 50 °C for 30 s, elongation at 72 °C for 60 s, and a final extension at 72 °C for 7 min. PCR amplification was performed in triplicate, with 25 μL reaction mixture containing 12.5 μL of 2× Taq Plus Master Mix, 3 μL of BSA (2 ng/μL), 1 μL of each primer (5 μM), 7.5 μL of ddH2O, and 30 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel and further purified. Purified amplicons were pooled in equimolar amounts and paired-end sequenced (PE 300) on an Illumina MiSeq platform  at Allwegene Technology, Beijing, China.t-test and ANOVA were conducted by SPSS software  for multiple comparisons to determine statistical significance at the p = 0.05 level.The abundance and mean values of the target genes were calculated using Microsoft Excel 2020. Heat maps and histograms were plotted using OriginPro 2020 software . Alpha diversity indices, including observed_species, chao1, and Shannon, were calculated using QIIME (Version 1.7.0) and displayed with R software (Version 4.1.2), as well as statistical analysis by one-way Tukey. The qvalue software package of R software (Version 4.1.2) was used to analyze the species difference at the phylum level. RDA and other plots were constructed using R software (version 4.1.2). Student’s p > 0.05), indicating that the addition of arsenic did not have a significant effect on earthworm survival, which is consistent with Tang’s study [In general, earthworm survival is closely related to their density, habitat environment, and food quality . As show’s study . The decMicroorganisms in the vermicompost products produce catalase, which breaks down hydrogen peroxide and is usually indicative of the size or activity of the vermicompost products . Therefop > 0.05), but the high concentration of arsenic resulted in a greater decrease in peroxidase activity in earthworms . In contrast, the activity of peroxidase increased after conversion compared with that before conversion, but the increase was much lower in the arsenic treatment group than in the control group. Although the difference between high and low concentrations was not significant, the catalase activity was slightly lower under high stress than under low stress. After cow dung conversion, SOD activity was higher in earthworms under low arsenic stress and lower in earthworms under high arsenic stress compared with that in the control group. Excessive accumulation of reactive oxygen species in organisms leads to lipid peroxidation (LPO) and DNA damage [Oxidative stress is thought to be one of the main processes of arsenic-mediated toxicity; thus, oxidative damage biomarkers and antioxidant enzymes have been widely used to evaluate the ecotoxicity of pollution . DramatiA damage . AntioxiA damage . MalondiA damage .sul-ARGs) were most abundant in the initial substrate of cow dung, followed by tetracycline resistance genes (tet-ARGs)  and after (vermicompost products) vermicomposting and fresh earthworm casts. The occurrence and variation in ARGs during vermicomposting are shown in et-ARGs) , which mbla-ARGs had the highest removal rate (23.4%). Vermicomposting also had removal effects on blaampC, oqxB, blaNDM, and qnrB, with removal rates ranging from 20.61% to 43.49% by vermicomposting. These results showed that vermicomposting could reduce ARG abundance in cow manure, similar to the results of Cui et al. [tetM encodes ribosomal protective proteins [sul1 at 3.4 × 107 copies/g; second, the concentration of the class I integrase gene intl1 was as high as 2.6 × 106 copies/g. However, it is now clear that the concentration of most ARGs in the fresh earthworm casts is generally lower than the abundance of the corresponding ARGs in earthworm casts over the same period. Furthermore, the majority of ARGs in the fresh earthworm casts are derived from feed microbes. The decrease in ARGs abundance after gastrointestinal passage of earthworms may be due to the loss and demise of ARG-containing bacteria, which further confirms the role of earthworms in reducing ARGs in the environment. Based on the study by Yang et al., it can be hypothesized that earthworm-associated symbiotic strains enhance the degradation of antibiotics and, thus, weaken the selection pressure of soil microorganisms to some extent, thus reducing the abundance of ARGs in the substrate [After vermicomposting, a decrease in the absolute abundance of most ARGs was observed in vermicompost products. Among the ARGs, i et al. . Previoui et al. , and tetproteins ,38, indiubstrate .Earthworms play a key role in organic decomposition, and epigeic earthworms directly influence the organic matter of soil decomposition through processes associated with the gut, that is, through the uptake, digestion, and absorption of organic matter and micro-organs, which are then released in earthworm feces . The ear10) and 0.13-fold (T50) compared with that in the control group, which indicated that the addition of arsenic could induce the proliferation of ARGs in cow manure. There was a significant decrease in the absolute concentrations of blaTEM-1, oqxB, blaampC, and blaNDM in the T10 treatment, with decreases of 31.86%, 25.99%, 22.77%, and 21.77%, respectively. Compared with untreated cow dung, the absolute concentrations of cfr and ermC increased by 16.88% and 12.99% after vermicomposting, respectively. In contrast, in the T50 group, there was a significant decrease in the absolute concentrations of oqxB and blaTEM-1 by 24.27% and 18.01%, respectively, and the concentration of tetX increased by only 8.64%. This result suggests that the response of different ARGs varies with the introduction of arsenic. The total absolute abundance of the T10 and T50 treatments increased significantly after vermicomposting compared with that in the control treatment. In addition, class 1 and class 2 integrons, as one of the most important MGEs, play an important role in the transfer and transmission of ARGs. Compared with untreated cow dung, the absolute concentrations of intl1 and intl2 increased by 8.87% and 3.80%, respectively, in the T10-treated group and increased by 6.90% in the T50-treated group. Furthermore, the absolute concentration of intl2 decreased by 1.84% compared with the control group. This demonstrates that the addition of As could increase the abundance of integrons, which might also accelerate the potential transmission risk of ARGs [The absolute abundance of ARGs in cow manure showed significant changes upon the addition of arsenic and earthworms, as shown in  of ARGs .50 group showed a greater decrease than that in the T10 treatment group. In the T10 and T50 treatment groups, the removal rate of nine and eleven ARGs, respectively, was higher than 10%. The removal efficiency of blaTEM-1 was the highest in the T10 treatment group, whereas that of oqxB was the highest in the T50 treatment group after earthworm treatment. β-lactam ARGs are known to pose a high risk to human health, and their removal can be improved at low concentrations of As contamination, which also provides a new direction for reducing the environmental risk of such ARGs. In the assay of wormcasts, it was found that the ARG concentration in the treatment group under high arsenic stress was generally lower than that in the treatment group with low arsenic addition. Studies have shown that arsenic has a toxic effect on earthworms [Overall, the absolute concentration of most ARGs decreased through vermicomposting, and the ARGs in the wormcast were more abundant than those in the fresh casts of earthworms . Furtherrthworms , the sevsul-ARGs, tet-ARGs, and str-ARGs.Overall, the presence of As contamination affected the reduction in ARGs during vermicomposting, with the absolute concentration of ARGs in the As treatment group in the substrate decreasing at a much lower rate than that in CK without As addition, and the absolute concentration of ARGs in fresh wormcasts was also generally higher than that in CK . HoweverNotably, arsenic has a toxic effect on earthworms, and the reduction in ARGs by earthworms in the treatment group with added arsenic is diminished by the weakening of metabolic functions . In addiIn the present study, 5073 operational taxonomic units (OTUs) were generated from all samples, and 4752 remained after leveling. Chao and Shannon indices are often regarded as ideal indicators of bacterial community richness and diversity, respectively . Figure Firmicutes after vermicomposting.Aspergillus in substrates exposed to arsenic and fresh earthworm feces increased significantly, which is similar to the findings of Wang et al. [In the low arsenic treatment group, the percentage of Bacteroidota in the substrate increased significantly, increasing from 9.45% to 16.20%, suggesting that Bacteroidota is resistant to arsenic. This was followed by Proteobacteria, which decreased from 33.37% to 28.66%. The low concentrations of arsenic contamination promoted the proliferation of Proteobacteria, whereas high concentrations did not have a significant effect on Proteobacteria. This was more prominent in fresh wormcasts of all treatment groups contaminated with arsenic; there were significantly higher percentages of Proteobacteria and Bacteroidota and lower percentages of Actinobacteria and Chloroflexi. However, this trend was more evident in the low-concentration treatment group rather than in the high-concentration one. The relative abundances of Bacteroidetes and g et al. . TreatmeNumerous studies have shown that the lower diversity and abundance of bacteria in wormcasts than in the surrounding soil is the result of a unique gut microenvironment ,56. MostAeromonas, Acinetobacter, and Flavobacterium were the three most abundant bacteria, but they were mainly positively correlated only with Bacteroidota and Proteobacteria, indicating the importance of these three bacteria in earthworm metabolism. In contrast, Acidobacteriota and Actinobacteria were positively correlated with other microorganisms. Thus, Acidobacteriota and Actinobacteria play a more important role in maintaining overall bacterial metabolism.In addition, network analysis was performed, showing correlations between four phyla, Actinobacteria, Bacteroidota, Proteobacteria, and Acidobacteriota, because these four gut bacteria are richer and/or more closely related than others. As the dot size indicates the count of bacteria, the more bacteria there are, the larger the dot size. Similarly, a line connecting two dots represents a connection between two bacteria. The thicker the line, the greater the correlation . As showTo further elucidate how microbial characteristics and environmental factors during vermicomposting affect the fate and behavior of ARGs, a redundancy analysis was performed. As shown in bla-ARGs. Furthermore, pH was significantly positively correlated with qnr-ARGs. The microbial community abundance index (Shannon index) for erm-ARGs was extremely high, indicating that erm-ARGs were mainly carried and spread by microorganisms in the environment.As shown in In order to identify the possible ARG hosts, the Spearman correlation between ARG-like levels and bacterial communities was investigated, as illustrated in (1)Vermicomposting reduced the abundance of most ARGs in cow dung and changed the structure of microbial communities in the related environment.(2)Arsenic residues in cow dung enriched ARG abundance and also reshaped microbial compositions in vermicompost products, significantly increasing the number of Bacteroides and Proteus.(3)The MGEs exhibited a trend similar to that of ARGs during vermicomposting of cow manure, which might be explained by the synchronous shift in ARGs and MRGs.(4)In the vermicomposting process, changes in physicochemical factors in cow dung played a direct or indirect role in ARG variation, and TP was the dominant factor driving the proliferation of ARGs.


All patients received two sachets of placebo or probiotics twice/day for 15 days and were followed-up for 30 days. The operations were classified as neurosurgical, thoracostomies, laparotomies, orthopedics, and others; then, the SSI and the isolated pathogen were registered. A total of 23 (46.0%) and 13 (24.5%) infectious insults in 89 (50 placebo patients) and 88 (53 probiotics-treated) operations (p = 0.022) were recorded, the majority of them relating to osteosynthesis—17 and 8, respectively. The most commonly identified pathogens were Staphylococcus aureus and Acinetobacter baumannii. Our results support published evidence that the prophylactic administration of probiotics in MT patients exerts a positive effect on the incidence of SSI.Investigations that focused on the protective role of probiotics against Surgical Site Infections (SSI) in multiple-trauma (MT) patients are generally few, probably due to the complexity of the concept of trauma. We aimed to assess the efficacy of a four-probiotic regime to reduce the incidence of SSI in MT patients, with a brain injury included. MT patients, being intubated and expected to require mechanical ventilation for >10 days, were randomly allocated into placebo ( Surgical site infections (SSI) are currently one of the most challenging health care issues worldwide, being one of the most frequent types of nosocomial infections following any surgical procedure ,2,3. TodMoreover, the issue of critically ill patients has been well-studied with respect to the gut microbial diversity and richness; dramatic microbiome alterations were found to occur within hours  of even Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” via the restoration of commensal “healthy microbes” ; more sp11 cfu each): Pediococcus pentosaceus 5–33:3, Leuconostoc mesenteroides 32–77:1, Lactobacillus paracasei spp. paracasei 19, and Lactobacillus plantarum 2362 was administered in mechanically ventilated MT patients, resulting in a significant reduction in the overall infections and sepsis, mainly from Acinetobacter baumannii, as well as in the number of days on mechanical ventilation and ICU stay [Multiple trauma (MT) patients, and especially those also with a traumatic brain injury (TBI), are among the most vulnerable patients in trauma care. From a trauma cohort of 1277 patients, 1001 infection cases (multiple infections in a number of patients), mainly respiratory, were reported in a total of 580 (45.4%) patients, leading to an overall mortality of 14.7% . In an eICU stay . AdditioICU stay . In the recent ProVAP randomized clinical trial conducted by our study group , it was This post hoc analysis was based on the data relating to surgical site infections, which were prospectively collected for the ProVAP trial  . Briefly®  consisted of Lactobacillus acidophilus LA-5 (1.75 × 109 cfu), Lactiplantibacillus plantarum UBLP-40 (0.5 × 109 cfu), Bifidobacterium animalis subsp. lactis BB-12 (1.75 × 109 cfu) and Saccharomyces boulardii Unique-28 (1.5 × 109 cfu), while the placebo, looking identical to that of the probiotics, contained a powdered glucose polymer. Each patient received two sachets, twice daily, for 15 days, one by a nasogastric or gastrostomy tube into the stomach and the other as slurry to the oropharynx, hoping to be spread during passive swallowing towards the trachea and esophagus, as occurs with pathogens from the sinuses. Only the study participants who underwent surgical operations were included in this post hoc analysis.Patients eligible for participation, after written informed consent provided by a legal representative, were allocated to receive either a probiotic formula or placebo treatment, according to a computer-based randomization program blinded to the study investigators and the physicians in charge. The probiotic regime LactoLevureSSI was defined as any infection presenting within 30 days after surgery involving the skin and subcutaneous tissue of the incision  and/or the deep soft tissue (fascia or muscle) and necessarily accompanied by at least one of the following: (a) purulent drainage superficial or deep (fascia or muscle) but not from within the organ or space component of the surgical site; (b) pain or tenderness, localized swelling or redness, heat or fever, or several of these symptoms and the incision having been opened deliberately or by spontaneous dehisces; (c) abscess within the wound, clinically or CT-detected; and (d) the isolation of one typical pathogen ,28. OrgaAll patients who underwent an emergency operation upon admission, either to repair trauma or to investigate tissue damage, were initially recorded. Operations were classified according to the anatomic area into: (a) neurosurgery, mainly for cerebral decompression; (b) thoracostomies for pneumo/hemothotax; (c) exploratory laparotomy for the liver and/or spleen damage/hemorrhage or of other viscera injury; (d) orthopedics, for closed or open fracture reduction, and external or internal osteosynthesis; and (e) others, including severe facial fractures and vascular damage, related to open fractures.On the post-operative day on which the SSI was recognized and registered, the isolated pathogen was also recorded and correlated with the same or previous days hospital-acquired pathogens (if any) from cultures of the peripheral blood and/or the catheter tip.The number of patients having been operated on under a hemorrhagic shock status, either due to solid viscera rupture or due to a major fracture, was recorded and, finally, those with a history of diabetes mellitus and smokers.t-test was applied in order to compare the means between two independent samples with normally distributed data, whereas the Mann–Whitney U Test was used to compare the differences between two independent groups when the dependent variable was either ordinal or continuous and not normally distributed. Furthermore, the chi-square test was conducted for comparing the nominal data, and the odds ratio (OR) was calculated. Moreover, a Cox regression analysis was performed, and the Hazard Ratio of the incidence of SSI was calculated. Finally, statistical significance was set at p < 0.05.All the data were stored initially in a Microsoft Excel spreadsheet, while the statistical analysis was conducted with the help of the Statistical Package for Social Science (SPSS), Inc.  after them being extracted. The normality of the data’s distribution was assessed by the Shapiro–Wilk test. We described both groups of patients by their sociodemographic (sex and age) characteristics, comorbidities, and scores in different scoring systems for critically ill and trauma patients with absolute and relative frequencies. The results of continuous variables were presented as the means ± standard deviation when normality was assumed or as medians with their respective interquartile range (Q25–Q75) when the data was skewed. For categorical variables, counts and percentages are presented. The Independent Student’s The original intent-to-treat population analyzed 53 patients allocated to treatment with the placebo and 59 patients allocated to treatment with probiotics. In the present post hoc analysis dealing with SSI, only patients subjected to at least one emergency operation on the day of admission were included; thus, 3 and 6 more patients were additionally excluded from the above groups, thus leaving 50 in the placebo group and 53 in the probiotic group. The study flow diagram is presented in The patients were then analyzed in relation to the type and number of operations already performed before enrolment in the study , the majp = 0.61; 17 (34%) and 19 (35.8%); two operations, p = 0.84; and 11 (22%) and 8 (15.1%) three operations, p = 0.37; thus, 50 placebo-treated patients underwent 89 operations and 53 probiotic-treated patients in 88 operations. The combinations of the different types of operations are shown in Almost half of the patients, 22 out of 50 (44%) and 26 out of 53 (49%), underwent one operation, p = 0.022). One major concern is whether most of the protection provided by probiotics is for patients who were subject to just one intervention or to more than one. This is analyzed in p = 0.035), followed by facial fractures (5 out of 7 (71.4%) and 2 out of 4 patients (50.0%) (p = 0.477) in the placebo and probiotic groups, respectively), all in the one-operation sub-group. The OR for SSI under probiotics was 0.11  for patients undergoing one operation, 1.67  for patients undergoing two operations, and 0.72  for patients undergoing three operations. The time, post-operatively, to the appearance of SSI is presented in Twenty-three placebo-treated (46.0%) and 13 probiotic-treated patients (24.5%) experienced an SSI, the difference being statistically significant (p = 0.035), followed by facial fractures (5 out of 7 (71.4%) and 2 out of 4 patients (50.0%) (p = 0.477) in the placebo and probiotic groups, respectively), all in a one-operation sub-group. Quite similar, but not so excessive, were the findings in the two-operation group  and 2 out of 8 probiotic-treated (25.0%) (on group . The timp = 0.277). Another 14 and 3 patients (p = 0.029) in the two groups, respectively, were also found to have positive blood cultures at the time of SSI recognition, but the pathogen identified was different.The pathogens isolated in the two groups are tabulated in Finally, four patients from the placebo and three from the probiotic group experienced life-threatening hemorrhage; this was due to an open-book pelvic fracture in two cases, liver damage plus hemothorax in one, and a dual-hemothorax plus femur fracture in one, all four in placebo patients. Of equal severity were those in the probiotic group: an open, complicated fracture of a lower extremity leading, finally, to amputation plus facial fracture in one patient, spleen rupture and complicated femur fracture in one, and spleen rupture and hemothorax in one.L. acidophilus LA-5 1.75 × 109 cfu, L. plantarum 0.5 × 109 cfu, B. lactis BB-12 1.75 × 109 cfu, and S. boulardii 1.5 × 109 cfu was found to significantly reduce the incidence of SSI in severely ill, mechanically ventilated MT patients . In the main publication that emerged from our experimental protocols [p = 0.034), along with a delay in the onset of VAP, a shorter duration of ICU stay, and of hospital stay. In the present post hoc analysis, the early administration—within less than 6 h of admission—of a four-probiotic regimen containing rotocols , based oAlthough both studies contained data from the same research protocol, in no case should the present analysis be considered as a duplication of the first one. The first study (VAP) was exclusively focused on the incidence ventilator-associated pneumonia and its prevention by means of the early administration of probiotics and with no reference to other infections; the present post hoc analysis referred exclusively to surgical site infections following emergency operations . Additionally, SSI infections have occurred earlier than VAP; 20% of the SSI presented up to day 10, as opposed to 20% of VAP presented after day 10 . Additiop = 0.067. The number of infections, although significantly lower in probiotic-treated patients, sounds high for both groups, given that the patients were mostly young, healthy, and without comorbidities, but it clearly reflects the type of surgeries: most patients presenting with at least one open fracture requiring internal or external fixation, and we should not underestimate the significance of the fact that all patients suffered a brain trauma, the severity of which required emergency intubation and mechanical ventilation. Furthermore, a brain injury of any kind promotes a decrease in Bacteroidetes, Firmicutes, and Verrucomicrobia and an increase in the Clostridia and Enterococcus populations [In the present study, all included MT patients also suffering TBI were operated on upon admission: 22 (44%), 17 (34%), and 11 (22%) of the placebo group and 26 (49%), 19 (35.8%), and 8 (15.1%) patients of the probiotic group underwent one, two, or three emergency operations in a total of 89 and 88 operations, respectively. Fifty patients from 89 operations experienced 23 SSI (25.8%%), and 53 probiotic-treated patients from 88 operations developed 13 SSI (14.8%), ulations ,30,31. Aulations . When a ulations .Staphylococcus aureus infection (p = 0.096). Regarding fractures, the rate of infection after open reduction and internal fixation is generally recognized as 1–3% overall , whereasMuch more complicated remains the situation for patients also with a huge, even life-threatening, hemorrhage, needing thus to receive inotropes and massive blood transfusion, and it is well-known that both blood cells themselves and the iron content promote pathogen growth . In the L. plantarum and L. fermentum were shown to induce the phagocytic activity of PMNs in the peripheral circulation, which is related to the induction of granulocyte–macrophage colony-stimulating factor (GM-CSF) production [L. reuteri and L. acidophilus were shown to increase the absorption of dietary vitamins D and E, known to be important for wound healing [Probiotics, although consumed for centuries by healthy individuals for GI health, are now gaining wide attention for use in hospital settings for the prevention of infectious complications. In recent years, a considerable number of studies have shown that the perioperative administration of probiotics and/or synbiotics, as a strategy to reduce dysbiosis, significantly diminishes the risk of infectious complications following surgery, with the magnitude of this risk reduction approaching 50% ,20,21,39oduction ; (ii) th healing ; and , while Poutahidis et al. [Lactobacillus reuteri-treated mice, the cutaneous wound healing process was accelerated two-fold due to the upregulation of the neuropeptide hormone oxytocin by a vagus nerve-mediated pathway.Besides the anti-inflammatory and immune-modulatory effects, another benefit of probiotics is the improvement of tissue repair by means of positive stimulation of the wound healing process. Although this process has not yet been completely investigated in relation to surgical trauma, this is of high priority, since speedy trauma repair is vital to prevent the entrance of harmful microorganisms to avoid blood loss and body dehydration and mainly to promptly restore skin barrier function. In a recent experimental study of our group , the tops et al.  reportedThe study protocol did not include a skin microbiome analysis or surgical wound closure inspection. However, there were significantly fewer surgical infections in the probiotic-treated patients, which is hard to attribute to other than the improvement of the total body microbiome diversity. It is also possible that probiotic administration also leads to earlier trauma re-epithelialization and collagen deposition. The increased immune response of the patent/host results in faster wound healing as a consequence of the trauma remaining uninfected or as a positive effect of the probiotics on the intestinal microbiome, leading to accelerated wound healing.Finally, it is noteworthy that no patient in either group experienced an infection related to probiotics treatment, as Hempel et al.  also conOur study had some limitations: (i) it was a post hoc analysis; after we published the results on VAP prevention, we realized that there was a gap in the literature regarding the influence of probiotics on surgical site infections after emergency operations in multiple traumatized patients; (ii) the study was powered as the primary outcome being the reduction of the incidence of ventilator-associated pneumonia; and (iii) there is a statistically significant difference in the number of osteosyntheses performed, either as an external or internal fixation, as well as the number of fractures per patient.The results of the present study support published evidence that the prophylactic administration of probiotics exerts a positive effect on the incidence of surgical site infections in severe multiple trauma—involving injury of the brain and at least one more organ system—patients being urgently intubated and under ventilatory support. The four-probiotics regime used continues to be a safe measure to fight microbial invasions in surgical traumas in patients experiencing microbial dysbiosis after stressful stimuli, as is an acute multiple trauma plus surgical stress for restoration. The evidence indicates that probiotics should hold a strong position in the treatment regime for the management of critically traumatized patients.


In recent years, the preferentially expressed antigen in melanoma (PRAME) has also been used in the histopathological diagnosis of melanocytic lesions, in order to understand if it could constitute a valid, inexpensive, and useful resource in dermatopathological fields. We performed a double-center study to evaluate whether the data on the usefulness and possible limitations of PRAME could also be confirmed by our group. From 1 December 2021 to 29 March 2022, we collected 275 cases of melanocytic lesions that were immunostained with PRAME (Ab219650) and rabbit monoclonal antibody (Abcam). To better correlate the PRAME expression with its nature , we categorized PRAME tumor cells’ percentage positivity and intensity of immunostaining in a cumulative score obtained by adding the quartile of positive tumor cells  to the PRAME expression intensity in tumor cells . Of these 275 lesions, 136 were benign, 12 were of uncertain potential for malignancy (MELTUMP or SAMPUS or SPARK nevus), and 127 were malignant. The immunoexpression of PRAME was completely negative in 125/136 benign lesions (91.9%), with only a few positive melanocytes (1+) and intensity 1+ in the remaining 11 cases (8.1%). Of the 127 cases of melanoma , PRAME was strongly positive in 104/127 cases (81.8%) with intensity 4+ and 3+. In 17 cases , PRAME was positive in percentage 2+ and with intensity ranging from 2+ to 3+. In 7 cases (5.5%) of desmoplastic melanoma, PRAME was 1+ positive and/or completely negative. Of the 12 cases of lesions with uncertain potential for malignancy, the immunoexpression of PRAME was much more heterogeneous and irregularly distributed throughout the lesion. These data are perfectly in agreement with the current literature, and they demonstrate that the reliability of PRAME is quite high, but its use cannot cause physicians to disregard the morphological information and the execution of other ancillary immunohistochemical stains such as Melan-A, HMB-45, MiTF, and SOX-10. Allen–Spitz criteria, various ancillary immunohistochemical techniques have become more and more widespread, allowing an additional possibly helpful resource in challenging melanocytic lesions Allen–Spi lesions , and, re lesions . In the  lesions ,12,13. IWe consulted the archives of two pathological anatomy departments planning to extrapolate their cases from 1 December 2021 to 29 March 2022, on which it was necessary, in addition to immunostaining with other antibodies , to run PRAME to gain a further indication of the benignity/malignancy of the lesions. We identified 323 melanocytic lesions, which were all diagnosed by two dermatopathologists at the time of diagnosis and double-blinded for the execution of this study (A.C and G.C.). After screening, only the cases in which the diagnosis was concordant between both dermatopathologists were considered.This study was approved by the local ethical committee.First, 5-micron thick tissue sections were cut from formalin-fixed paraffin-embedded (FFPE) blocks. The cases in question were all stained with anti-PRAME antibody Ab219650, rabbit monoclonal, in 1:250 dilution.To better correlate the PRAME expression with its nature , we categorized PRAME tumor cells’ percentage positivity and intensity of immunostaining in a cumulative score obtained by adding the quartile of positive tumor cells  to the PRAME expression intensity in tumor cells . More specifically, we used the following scores for the percentage positivity of tumor cells: 0% (score 0), 1% to 25% (score 1+), 26% to 50% (score 2+), 51% to 75% (score 3+), and 76% to 100% (score 4+). Furthermore, we used a score for intensity by measuring the nuclear immunostaining for PRAME as weak, moderate, or strong . Sebaceous glands were used as an internal control to confirm the functioning of the PRAME antibody stain. These characteristics of immunoexpression were estimated by both dermatopathologists during the review of the cases.A total of 275 melanocytic proliferations were examined, including 136 benign lesions, 12 lesions of the uncertain potential of malignancy, and 127 malignant lesions. In total, 156 (56.7%) patients were male, and 119 (43.2%) patients were female, with a mean age of 56.8 in the first group and 43.7 in the second group. Of the 136 melanocytic benign lesions, 84 (61.7%) were nevi with low-grade dysplasia (according to WHO 2018 ), and 52Of the 136 benign melanocytic lesions, divided into 84 with low-grade dysplasia and 52 with high-grade dysplasia, 81/84 (96.4%) were negative or had a score of 1+ for PRAME, while 4/52  nevi with high-grade dysplasia were positive for PRAME with an immunoscore of 1+/2+ A. RegardIn recent years, the continuous need to refine the diagnosis of MM associated with the difficulty and inter-observer variability of atypical pigmented lesions has made it possible to search for new markers that are useful in both the diagnostic and potentially therapeutic fields .Although initially discovered in tumor-reactive T-cell clones with cutaneous melanoma  and usedRegarding the pigmented melanocytic lesions, of the 145 cases reported by Lezcano et al., 86.4% were completely negative for PRAME, with 13.6% of mild and focal positivity in some nevi such as dysplastic, recurrent, etc. In our work, we found a slightly higher negative value, equal to 96.4% for nevi with low-grade dysplasia (according to WHO 2018), and we reported a positivity rate for PRAME (7.6%) in nevi with high-grade dysplasia with a score of 1+/2+. These data are particularly interesting because they seem to confirm the trend of the results reported in the paper by Gassenmaier et al.  in whichRegarding the 12 lesions with uncertain potential for malignancy comprising 4 MELTUMP, 3 SAMPUS, and 5 SPARK nevus, we also reported a certain degree of heterogeneity of immunostaining for PRAME, characterized by areas of greater positivity (score 3+) and areas of positivity equal to 2+ or also 1+. As already addressed by Raghavan et al. , the lesIn conclusion, our study also supports the evidence already present in the literature, attributing a definitely significant role to PRAME in the complex differential diagnosis between dysplastic melanocytic nevi and malignant melanomas, while remembering that there is a subgroup of lesions that can express, even quite intensely, this immunomarker. In the various histotypes of malignant melanoma, PRAME appears to be quite reliable, even considering that some particular types, including nevoid melanoma, spindle cell melanoma, and desmoplastic melanoma , tend to lose immunopositivity, and therefore, the dermatopathologist must know this eventuality. Overall, PRAME is useful but not conclusive when applied to lesions with an uncertain potential for malignancy such as MELTUMP, SAMPUS, and SPARK nevus. Future studies with more case series and other data are needed to elucidate and better understand the usefulness of PRAME in other MM subtypes and other primary skin lesions.


Bradyarrhythmia treatment is often not timely enough, posing a potential threat to health. It is necessary to find a strategy with stable curative effects and high safety. Mahuang-Fuzi-Xixin (MFX) decoction and Shenmai injection (SMI) are compound Chinese Patent Medicines. Recent evidence has shown that the combined use of these two drugs can effectively treat bradyarrhythmia.  To evaluate the effect of MFX decoction combined with SMI on bradyarrhythmia.  PRISMA was followed as the guideline for this systematic review. RevMan 5.4 software was applied for meta-analysis. P < 0.01); after treatment, MFX combined with SMI treatment and SMI alone had no significant difference in heart rate. In the subgroup of patients with an age less than 60, the effect of MFX combined with SMI treatment on the 24-hour mean heart rate was better than that of SMI alone , P < 0.01), as did the 24-hour minimal heart rate , P < 0.01). In addition, the effect of MFX combined with SMI treatment on the Traditional Chinese Medicine syndrome score (TCMSS) was significantly better than that of SMI alone , P < 0.01). In terms of safety, two adverse events were reported in the SMI combined with MFX group compared to 12 in the SMI alone group.  Eight studies were included in the analysis, with a total of 340 patients in the intervention and control groups. The results of the meta-analysis showed that the effective rate of MFX combined with SMI treatment was higher than that of SMI alone ,  MFX combined with SMI treatment is effective in treating bradyarrhythmia. However, the results were heterogeneous. The safety of MFX combined with SMI treatment should be verified and treated with caution. Bradyarrhythmia is a common cardiovascular disease (CVD), and every year one million people have pacemakers installed worldwide , 2. Bradred ginseng and Ophiopogon japonicus, which have been used clinically to treat bradyarrhythmia [ephedra, asarum, and aconite [Traditional Chinese medicine injection (TCMI) is a proprietary Chinese medicine with a long history of development . People rhythmia , 11. Mah aconite . It is u aconite . MFX dec aconite . For exa aconite . Other s aconite . MFX comThis study included clinical studies in recent years, with the effective rate, 24-hour mean heart rate, 24-hour minimal heart, rate and TCM syndrome score (TCMSS) as outcome indicators, aiming to evaluate the effectiveness of the combination of MFX with SMI for treating bradyarrhythmia.This research method strictly adhered to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). As of November 2021, we searched the following 6 databases: the Chinese Biomedical Database (CBM), Web of Science, EMBASE, PubMed, the China National Knowledge Infrastructure Database (CNKI), and the Cochrane Library. The language of the references was limited to Chinese and English. Search terms included “Shenmai injection,” “Mahuang-Fuzi-Xixin decoction,” “bradyarrhythmia,” “bradyarrhythmias,” “bradycardia,” “Bradycardias,” “slow arrhythmia,” and “arrhythmia.” A search methodology that combined subject terms and free word lines was utilized. In addition, to avoid omissions, related references were manually searched.Endnote X9 was applied to import the search results, followed by removing duplicate literature. The inclusion criteria met the following criteria: (1) compared the clinical trials of bradyarrhythmia between the SMI alone treatment group and the MFX decoction combined with SMI treatment group, (2) the control group received SMI alone, (3) the intervention group received MFX decoction combined with SMI treatment, and (4) at least one of the following results were reported: (1) efficacy, (2) 24-hour mean heart rate, (3) 24-hour minimal heart rate, and (4) Traditional Chinese Medicine Syndrome Score (TCMSS). Exclusion criteria met the following criteria: (1) incomplete data, (2) different baseline levels of patients, and (3) animal experiments, meeting reports, review comments, repeated published studies, and other nonconforming studies.Two experienced researchers searched and selected studies based on inclusion and exclusion criteria. If there was a disagreement in the data extraction step, the third researcher discussed and resolved the disagreement. These data were extracted: the name of the first author, the year of publication, the sample size, gender distribution, intervention measures, duration of treatment, comorbidity, outcome, and adverse events.The methodological quality of the included studies was evaluated according to the Cochrane Collaboration's tool. The risk of bias assessment mainly included five aspects: (1) the bias generated by the randomization process; (2) the deviation caused by the deviation from the expected intervention; (3) the deviation caused by the missing result data; (4) the deviation of the measurement result; and (5) the bias in the selection of the reported result. Finally, a judgment of the overall risk of bias was generated. The risk of bias was categorized into three levels: “low,” “high,” and “unclear.”χ2 test and I2 statistic. If there was heterogeneity (I2 > 50% or P < 0.05), the random effects model was used for data analysis; otherwise, a fixed effects model was adopted. At the same time, sensitivity analysis or subgroup analysis was used to determine the source of heterogeneity. Our study did not perform a funnel plot analysis because only 8 (less than 10) studies were included. RevMan 5.4 software was employed for statistical analysis.For binary variables, the odds ratio (OR) was used to assess the effect; for continuous variables, the mean difference (MD) was used. For the above variables, the 95% confidence interval (CI) was calculated. The heterogeneity among different studies was assessed by the We retrieved a total of 488 studies from 6 databases. First, 120 duplicate studies were removed. Then, the title and abstract were read by experienced researchers, and 282 studies were removed because they were case reports, conference papers, or other nonconforming study types. Next, the full text was carefully checked, and 78 studies were not included in this step because their interventions or results did not meet the inclusion criteria. In the final stage, 8 studies were included , 16–22. Eight studies were included in the analysis, with a total of 340 patients in the intervention and control groups (170 in the intervention group and 170 in the control group). The detailed characteristics of these studies are shown in All studies randomly grouped patients but did not report the allocation concealment method and blinding. Two studies did not report predetermined results, nor did they report other biases. The Cochrane risk of bias results is shown in After evaluating the bias risk of included RCTs, the risk of random sequence generation and allocation concealment was found to be low. However, the blinding of participants and blinded reporting of outcome assessments remained unclear in these studies. But it was found that there were three studies with biased reporting of primary outcome and incomplete outcome data , 18, 20,2 = 0). These studies had no differences in patient conditions, clinical methodology, or other characteristics. Our results indicated that MFX decoction combined with SMI was effective in the treatment of bradyarrhythmia , P < 0.00001)  .2 = 99%) (2 = 0). Although the heterogeneity of the aged >60 subgroup was reduced, it still had high heterogeneity (I2 = 53%)  . Therefo2 = 53%) . It was 2 = 98%)  (P < 0.00001) in patients aged less than 60 years.Heart rate, a measure of bradyarrhythmia, is often measured with a 24-hour mean heart rate and a 24-hour minimal heart rate to assess the extent of improvement of disease. Four studies , 21, 22 2 = 98%) . Therefo2 = 21%) . This inP < 0.00001)  . HoweverThree studies , 19, 21 Through our analysis, it was found that, compared with SMI treatment alone, MFX decoction combined with SMI treatment could further improve the effective rate, 24-hour mean heart rate, and 24-hour minimal heart rate in patients less than 60 years of age and reduce the TCMSS. In terms of safety, our results showed that there were no serious adverse reactions in the included studies, and the adverse reaction rate of the intervention group was lower than that of the control group. Among the included studies, 6 studies reported treatment duration (4 weeks or 1 month), and the variety in treatment duration was small. The electrocardiogram (ECG) is an important way to continuously record heart rhythm . HoweverBradyarrhythmia is a common disease  with symβ receptors, increase heart rate, and increase myocardial contractility. Its active ingredient, ephedra fruit polysaccharide, can improve the blood rheology of model rats and resist the formation of blood stasis [β-AR) and electrophysiological properties and ionic cardiomyocyte membrane channels [Blood rheology is the main factor affecting the development of arrhythmia. The increase in blood viscosity and vascular resistance in patients will cause ischemia and hypoxia in local tissues and organs . Relevand stasis . There hd stasis . This wad stasis . It has d stasis . The mold stasis . Red gind stasis . The mecchannels . These aOur research had certain limitations. First, from the included studies, both the intervention and control groups showed high safety. However, only 3 studies reported the occurrence of adverse reactions, and the evidence was not very reliable. Therefore, we believe that the safety of the intervention cannot be fully shown. In the future, more studies need to be included to prove this hypothesis. Second, these studies had small sample sizes, and no detailed randomization or blocking strategy was provided, leading to a poor evaluation methodology. Third, the mechanism of the combination therapy was not fully revealed. Nevertheless, this is the first study to provide some evidence for the adjuvant treatment of bradyarrhythmia using combined TCMs.


Escherichia coli that forms biofilms poorly, with aromatic and heteroaromatic moieties leading to the best performing polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expression of the adhesin curli. In the presence of selected polymers, MC4100 can reach levels of biomass production and curli expression similar or higher than PHL644, demonstrating that synthetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.The intrinsic resilience of biofilms to environmental conditions makes them an attractive platform for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiology and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using a combination of controlled radical polymerization and dynamic covalent chemistry, we prepare a set of synthetic polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties. We then demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in MC4100, a strain of  Here, we show how synthetic polymers, prepared by a combination of controlled radical polymerization and in-situ screening, can be used to address a biological challenge – how to improve biofilm formation – to provide solutions to a field – biocatalysis – of industrial and societal relevance. E. coli, a workhorse in biotechnology. To the best of our knowledge, currently there are no methods that provide this simplicity and versatility when promoting biofilms for beneficial bacteria. Traits for biofilms formation are often introduced in bacteria through gene-editing and experimental evolution, which are costly and time consuming, and require expertise in molecular biology and/or microbiology. We believe that the presented methodology demonstrates an innovative use of synthetic materials. Rather than trying to inhibit formation of biofilms, we have used synthetic polymers to induce the formation of these communities of bacteria, and exploited these polymer-induced biofilms for a beneficial application, in this case biocatalysis.We present in the attached manuscript a new materials-based methodology to induce biofilms in beneficial bacteria. Our solution relies on synthetic polymers, that work as simple additives during microbial culture, to promote biofilm formation. We have also demonstrated that these polymer-induced biofilms can increase the biocatalytic activity of 1.1–7 Biofilms are far more resistant than planktonic bacteria to environmental conditions, including extreme pH and temperatures, or to the presence of detrimental chemicals and metabolites, including antibiotic treatment.2,4,8,9 As a consequence, bacterial biofilms are often seen as problematic and difficult to eradicate, and have been associated for example with biofouling10 or hospital-acquired infections.11 This awareness of problematic biofilms means that the role of beneficial biofilms and microbial communities are often ignored and overlooked. For instance, biofilms of beneficial microorganisms are critical in maintaining a healthy microbiota, which plays a critical role in human, animal and plant health.12–14 As such, strategies to establish and maintain healthy microbiotas will have an impact in agriculture15 and consumer health,16,17 and have been at the heart of traditional manufacturing of foods such as cheese, vinegar or fortified wines.18–20 Moreover, the intrinsic resilience of biofilms can be exploited to underpin other beneficial applications, including bioremediation21,22 or biocatalysis.4,23–26The vast majority of bacteria live in biofilms, microbial communities where cells stick to each other, and are protected by an extracellular matrix of biopolymers.27,28 or non-pathogenic strains of Escherichia coli,29 are not necessarily good at forming biofilms. Strains that form biofilms efficiently can appear as a result of evolution (e.g. the E. coli PHL644 strain used in this work),30 a process that often requires lengthy incubation times and multiple rounds of culture, something that limits its practicality. Alternatively, relevant genes for adhesion and biofilm formation can be introduced via genetic modification or synthetic biology approaches, although this process is often reserved to introduce genes needed for biocatalytic activity and bioprocessing. Moreover, for some applications such as bioremediation, food manufacture, or agriculture, the release of genetically modified organisms into the environment is a concern.One of the challenges in exploiting beneficial biofilms for biotechnology or health is that some of the candidate microorganisms, such as probiotics2 Often, adhesion to surfaces and hosts can trigger this transition to a biofilm phenotype.2 We postulate here that a similar transition should be observed following the binding of microorganisms to synthetic polymers. We had already identified that this was the case for human pathogen Vibrio cholerae, that upon interaction with cationic polymers, would adopt a non-virulent sessile lifestyle, characterized by an increase in biofilm formation and a decrease in toxin production.31,32Interestingly, the switch between planktonic and biofilm lifestyles is often dictated by environmental factors such as the presence or absence of certain nutrients, pH, or temperature.E. coli, resulting in increased biocatalytic activity in a model biotransformation , both key biofilm biomarkers in this organism. Our results indicate that hydrophobic polymers outperform mildly cationic polymers, with increasingly hydrophobic materials often resulting in higher levels of biofilm formation. Throughout the manuscript we demonstrate that, in the presence of these synthetic polymers, E. coli MC4100, a poor biofilm former, can reach levels of biomass production, and curli expression similar or higher than PHL644, a good biofilm former with a single point mutation in the OmpR regulator which increases curli expression. Finally, we show that these polymers can be used to increase the biocatalytic activity of engineered E. coli biofilms in the transformation of serine and 5-fluoroindole into 5-fluorotryptophan. All together, our results demonstrate that the use of these synthetic polymers as additives in microbial culture can be a cheap alternative to engineering microbial biofilms using gene editing or experimental evolution.In this manuscript, we show that synthetic polymers induce the nucleation of biofilms in biotechnology relevant ormation . First, 2.2.1.V. cholerae,31,32 for which we had seen an increase in biofilm formation upon incubation with cationic polymers, we postulate here that this polymer-induced biofilm formation can be exploited in other microorganisms for the production of biofilms for biotechnology. As mentioned above, the relevance of microbial biofilms for biotechnology is slowly becoming apparent, as the protective environment offered by biofilms can be exploited for food production,18–20 bioremediation21,22 or biocatalysis.4,23–26 In our case, we focused on biocatalysis, where biofilms can protect microbial cells from harsh conditions such as extreme pH or temperature, or from the presence of detrimental chemicals such as organic solvents. In our previous work, we had exploited cationic polymers to induce clustering in a range of bacteria, including Vibrio harveyi,33–36V. cholerae,31,32Pseudomonas aeruginosa,34,35Staphylococcus aureus35 and E. coli,34,35 and, as just mentioned, we observed increased biofilm formation for V. cholerae. However, we also observed that cationic polymers were toxic to some of the E. coli strains employed.34 Thus, we decided to explore mildly cationic polymers that would be partially protonated in the culture media. Since both electrostatic and hydrophobic interactions are involved in the initial stages of adhesion by bacteria to surfaces and hosts,37 we decided to also investigate hydrophobic polymers. To have quick access to this broad chemical space, we employed a post-polymerization modification strategy developed in our team  P1 with aldehydes under aqueous conditions, and the in situ evaluation of activity of the formed functional polymers. The post-polymerization modification is often done under mildly acidic conditions (here 100 mM acetic acid at pH 3), giving us versatility when exploring a broad chemical space, and access to functional polymers simply by choosing the required aldehydes. To date this in situ screening methodology has only been applied to the delivery of nucleic acids,39–41 and we wanted to demonstrate here its versatility to develop other functional polymers with biological relevance.Following our previous work with our team .38 This E. coli using isovaleraldehyde , benzaldehyde  and 1H-indole-3-carbaldehyde  as representative hydrophobic aldehydes  and 2-amino-3-formylpyridine  as representative mild cationic aldehydes (cpKaH 3.3) should be partially protonated at pH 3, it was also investigated. E. coli K-12 strain PHL644 was used as a model strain,30 as it has already been investigated as a platform for biocatalysis using biofilms.42–44 Cationic polymers were prepared by mixing stock solutions of P1 and aldehyde in 100 mM acetic acid to give a final 125 mM concentration of aldehyde and a 1 : 1 ratio between aldehyde and hydrazide moieties. Hydrophobic polymers were prepared following the same protocol but using 95% DMSO-d6/5% 100 mM acetic acid as the solvent. Following incubation of P1 and the aldehydes over 24 h at 60 °C, aldehyde loading on the polymer was analysed by NMR as reported.38,39 In all cases this loading was consistent with our previous results, with most aldehydes giving approximately 70% loading . The formed polymers were then added without further purification to suspensions of E. coli PHL644 bacteria in a 100 mM aqueous solution of NaCl, to ensure that the polymers remained protonated after their preparation at pH 3. Following incubation for 24 h  were the aldehydes that resulted in higher levels of crystal violet staining, we decided to investigate 2-naphthaldehyde (2Nph), anthracene-9-carbaldehyde (9AntA) and pyrene-1-carbaldehyde (1PyrA) as additional aromatic aldehydes  and 1ldehydes  and 5 daldehydes . E. coliy MC4100 . Overallmod-4ImA , suggestmod-2NphA, P1-mod-9AntA and P1-mod-1PyrA, which after 24 h helped MC4100 reach significantly higher levels of biomass production than PHL644  than MC4100 . Interes2.3.31–36 Here, we wanted to evaluate if a similar aggregation was being induced by hydrophobic polymers. To this end, aggregation of bacteria was first evaluated monitoring changes in the optical density for E. coli MC4100 cultures in the absence and presence of these functional polymers , as a result of bacterial clusters scattering more light than individual bacteria. These aggregates will gradually sediment, leading to a decrease in optical density. On the other hand, fast sedimentation can already happen at early timepoints in the experiments, when aggregation of bacteria is very fast.33,46 The timing of these two phenomena is highly dependent on small changes in the concentration of bacteria and polymer and, as such, there is a lot of batch-to-batch variability. In our case, all functional polymers induced changes in the optical density of the culture when compared to untreated bacteria. These changes were more obvious when the difference between the maximum and minimum optical density was plotted  was used to measure csgB promoter activity.48 The csgB promoter region was fused to gfp, the gene encoding green fluorescent protein (GFP) to monitor expression of curli through time using a simple fluorescence readout. Curli expression is maximal at temperatures of around 30 °C and in conditions of low osmolarity.47 Under these conditions, and in the absence of polymers, it took E. coli MC4100 more than 20 h to produce significant levels of GFP fluorescence, reaching maximum GFP expression approximately after 38 h of incubation ,51 was incubated in the absence and presence of the functional polymers as described above, and then treated with a buffer containing both starting materials for this biotransformation. The supernatants were then collected and the amount of 5-fluoroindole depleted  related to this work.MH-009-D2MH00607C-s001


The present review provides an overview of the literature from the more than 20 and 10 years, respectively, that these two tracers have been used for the study of atherosclerosis, with emphasis on what new information they have provided in relation to diabetes and which questions remain insufficiently elucidated.Arteriosclerosis and its sequelae are the most common cause of death in diabetic patients and one of the reasons why diabetes has entered the top 10 causes of death worldwide, fatalities having doubled since 2000. The literature in the field claims almost unanimously that arteriosclerosis is more frequent or develops more rapidly in diabetic than non-diabetic subjects, and that the disease is caused by arterial inflammation, the control of which should therefore be the goal of therapeutic efforts. These views are mostly based on indirect methodologies, including studies of artery wall thickness or stiffness, or on conventional CT-based imaging used to demonstrate tissue changes occurring late in the disease process. In contrast, imaging with positron emission tomography and computed tomography (PET/CT) applying the tracers  Atherosclerosis is a significant co-morbidity in diabetes mellitus, and its sequalae, acute myocardial infarction, stroke, and peripheral artery disease, are major reasons why diabetes has recently entered the top ten causes of death worldwide . However18F-fluorodeoxyglucose (FDG) and 18F-sodium fluoride (NaF), which enable the detection and measurement of inflammation and active microcalcification, respectively, in early-stage atherosclerosis, long before CT-detectable calcification is formed [This review focuses on the use of molecular imaging with positron emission tomography (PET) in the form of PET/CT to assess atherosclerosis in diabetes. Even though a multitude of PET tracers have been applied, only two have been studied sufficiently to be of interest in this context, i.e., s formed . Multipls formed ,6,7 and s formed ,10 and cs formed ,12,13, rs formed ,15,16, ds formed ,18,19, tWe take a molecular perspective in focusing on PET/CT, because the superior sensitivity of PET enables detection of molecular changes long before tissue changes become apparent using CT. The strength of CT is its much higher spatial resolution, which can show more precisely where in the body the molecular changes are taking place ,21. In dOur systematic literature search see  yielded Inflammation mechanisms and targeting  ,27,28,29Early detection and prevalence of arterial FDG or NaF uptake ,33,34,35Cardiovascular risk in DM ,37,38,Disease progression ,40,Therapy ,47,48,49Below and in  the liteSeven studies dealt with the carotids ,31,33,3718F-flouromethylcholine and FDG in many organs in the same mice models with similar methods, and found that whole aortic FDG uptake was higher than aortic 18F-flouromethylcholine uptake in both diabetic (4.7 vs. 2.9) and nondiabetic mice (5.0 vs. 1.5) [Three studies were animal experiments from the PET Centre in Turku, Finland. Silvola et al. investigated the effects of age, duration of a high-fat diet, and type 2 diabetes mellitus (T2DM) on atherosclerotic plaque development and the uptake of FDG. Atherosclerotic low-density lipoprotein receptor deficient mice were compared to atherosclerotic and T2DM mice that overexpressed insulin-like growth factor II, and C57BL/6N mice on normal chow. From ages four to six months and twelve months and older, plaque size increased, and the macrophage density decreased. Compared with controls, PET showed increased aortic FDG uptake at four and six months, but not at twelve months and older. Autoradiography demonstrated focal FDG uptake in plaques at all time points with highest uptake in plaques with high macrophage density. There were no differences in plaque size, macrophage density, or uptake of FDG between the two types of mice at any time point . Hellbervs. 1.5) . Finallyvs. 1.5) .Among the six human studies, Kim et al. compared carotid FDG uptake and intima-media thickness in three age- and sex-matched samples, each of 30 subjects: one with T2DM, another with impaired glucose tolerance, and a third with normal glucose tolerance. This study also examined associations with the Framingham Risk Score, high sensitivity C-reactive protein (hsCRP), adiponectin, and anti-inflammatory adipokine. They measured the maximal standardized uptake value (SUVmax) and the target-to-background ratio (TBRmax), i.e., the SUVmax divided by blood pool radioactivity, and found that both parameters were slightly higher in the sample with impaired glucose tolerance, and clearly higher in T2DM patients than in normal glucose tolerance subjects. Meanwhile, there were no significant differences with regard to carotid intima-media thickness. TBRmax values had the strongest positive correlation with hsCRP levels among cardiovascular risk factors, but other inflammatory markers were not associated with TBRmax values. Their conclusion was that vascular inflammation measured by FDG PET was increased in healthy individuals without hyperlipidemia, but with elevated hsCRP, even if the increases of SUVmean and TBRmean  in the impaired glucose tolerance group were very modest compared to the increases in the T2DM group  . Yang etBucerius et al.  examinedp = 0.001) and cSBP , but not with the augmentation index. The TBRmax of each separate segment was also significantly associated with PWV and cSBP. In a multiple linear regression model including age, sex, BMI, hemoglobin A1c (HbA1c), hsCRP, cholesterol, cSBP, and PWV, PWV was the strongest determinant of mean TBRmax. Seven patients had a score of 0, and seven scored 4 for calcification. The latter was only present in the abdominal aorta and iliac artery. TBRmax was lowest in the carotids, highest in the caudal parts of the aorta, and intermediate in the iliac and femoral arteries [The potential relationship between FDG uptake in major arteries and arterial stiffness assessed as central systolic blood pressure (cSBP), carotid-femoral PWV, and the augmentation index was examined by De Boer et al. in 44 patients with early T2DM (mean duration one year) without CVD and anti-diabetic medication. Coronary arteries were excluded in the study because of the large FDG uptake due to cardiac metabolism. SUVmax was normalized for blood glucose levels, as described above, and CT-detectable arterial calcification scored from 0 to 4  were endpoints. Corrected TBRmax was significantly associated with PWV  had, in general, higher visual uptake in all three arterial beds than patients without risk factors (n = 23): femoral 70% vs. 22%, iliac 54% vs. 30%, abdominal aorta 53% vs. 35% [It seems that Yun et al. were the first to report arterial FDG uptake in diabetic patients. Using visual inspection, they looked for FGD uptake in the abdominal aorta and iliac and proximal femoral arteries of a heterogeneous group of 156 patients, many of whom had various atherogenic risk factors referred to PET for various reasons. Of only 13 patients with diabetes (of unspecified type and duration), 9 (69%) had aortic and iliac artery uptake and 12 (92%) had femoral artery FDG uptake. Patients with ≥1 cardiovascular risk factor  and vascular inflammation in 275 outpatients with mean fasting blood glucose of 103.2–107.2 mg/dL and HbA1c 5.91–6.0%. They further focused on 18 patients whose AGE value was >14.2 units/mL, examining whether changes in AGE levels after treatment with oral hypoglycemia agents (OHAs) correlate with those of TBR. It did not provide much information about FDG uptake in diabetes, although carotid FDG uptake was measured in prediabetic and a few treated T2DM patients. The serum AGE level was independently associated with carotid TBRmax and pioglitazone, but not with glimepiride, reduced AGE, or HbA1c levels. TBRmax values were not compared to those of a relevant control group; nor were they given for the 18 treated patients. It was only stated that the ΔAGEs obtained by OHA treatment correlated positively with ΔTBR .Bernelot Moens et al. measured carotid FDG uptake in patients with peripheral artery disease (PAD) defined by their ankle-brachial index (<0.9 and/or decreased >0.15 after a treadmill test). The study compared 11 controls with 11 patients with PAD but no diabetes, 11 with PAD+T2DM, all on oral therapy (T2DM), and 12 with PAD+T2DM on combined oral/insulin (insulin-dependent) therapy; it was found that blood-glucose-normalized TBRmax was 32% higher in the first group, 95% higher in the second, and 122% higher in the third group, despite comparable PAD severity, BMI, and CRP in the three groups. Multivariate regression analysis showed that HbA1c and plasma insulin levels correlated with TBR, but the dose of exogenous insulin did not .Bural et al. studied 110 oncologic patients imaged with FDG PET/CT, 55 insulin dependent diabetes patients, and 56 comparable nondiabetic controls; they observed that the average blood-glucose-corrected SUVmax and SUVmean valules were significantly higher  in insulin-dependent subjects in all arterial segments . Calcification by CT was 27% higher, and both FDG uptake and calcification were most pronounced in the arch and abdominal part of the aorta and in the iliac arteries .Finally, only two studies dealt with arterial NaF uptake. In an early report, Janssen et al. noted by visual inspection the “linear” NaF uptake in the femoral arteries of 159 (39%) of 409 oncologic patients, 44 of whom had unspecified diabetes. Among these, 43 had calcification by CT, of which 28 were NaF positive, while only one was negative according to both CT and NaF PET . In contStrobl et al. analyzed 315 consecutive patients scanned with PET/CT for non-cardiovascular indications, such as malignant tumors, to study the impact of age, gender, and cardiovascular risk factors on vessel wall inflammation (FDG uptake) and the calcified plaque burden (by CT) in the common carotids, thoracic and abdominal aorta, and iliac arteries. Only 15 patients had diabetes (fasting glucose ≥ 200 mg/dL or treatment with a hypoglycemic agent). The calcified plaque score was associated with diabetes in all four vascular beds and, overall, was more closely associated with cardiovascular risk factors compared to TBR, which was not associated with diabetes in any of the four vascular beds. In the aorta, TBRmax was associated with age ≥ 65 years and male gender; in the carotids, it was also associated with a BMI ≥ 30, but in the iliac arteries with age only .p < 0.001), contrary to the findings of Strobl et al. [Lee et al.  analyzedl et al. . Carotidl et al. .Takx et al. studied 68 patients with T2DM and arterial disease, defined as an ankle-brachial index of <0.9 as part of a randomized controlled trial on the effect of vitamin K2 on T2DM. They looked for potential determinants of modifiable determinants of NaF uptake in the femoral arteries, and found that higher CT calcium mass, total cholesterol, and HbA1c were associated with higher NaF TBRmax .Reijrink et al. analyzed arterial FDG and NaF uptake in 10 patients with early T2DM from the so-called RELEASE trial  to investigate—as the authors put it—the “prospective correlation” between tracers over time (five years) and whether they are “prospectively (FDG) and retrospectively (NaF) related to progression of systemic arterial disease”. Pre-scan glucose-normalized FDG and NaF TBRmax values were measured in the carotids, three parts of the thoracic aorta, and in the iliac and femoral arteries using different generation PET scanners on two separate occasions. After five years, the arterial calcified plaque score, which was assessed visually based on low dose CT scans (CPscore), had increased by 24%, whereas the measured PWV remained stable (+7.2%), as did many other parameters. The baseline mean FDG TBRmax demonstrated a strong positive correlation with five-year mean NaF TBRmax, as did the latter with the five-year CPscore. Baseline FDG uptake did not correlate with age, the baseline CPscore, the follow-up CPscore, the follow-up PWV, or the ΔCPscore. The CPscores at baseline and at follow-up were strongly correlated. Follow-up NaF uptake correlated positively with the baseline and five-year CPscores and ΔCPscores, whereas it was not associated with %change in CPscore, PWV, or ΔPWV. The authors concluded that “systemic arterial inflammation is an important pathogenetic factor in systemic arterial microcalcification development” . The datp < 0.001), that NaF-positive disease was an independent “predictor” of subsequent CCS progression , and that all subjects (15/15) with ≥2 NaF-positive coronary arteries progressed in CCS [Bellinge et al. examined 41 patients with diabetes of a duration of 8–11 years and no history of coronary artery disease; they focused on whether localized coronary artery NaF uptake “predicts” development of new CT-detectable calcifications at least two years later. The patients were T2DM, and about 2/3 received statins, and nearly 1/3 insulin; all had previously undergone coronary calcium score (CCS) screening using CT and NaF PET/CT as baseline assessments for a trial on the arterial effects of vitamin K1 and colchicine in subjects with diabetes . Rescand in CCS . p < 0.01), vs. an insignificant increase with glimepiride, from 1.35 to 1.41  and 21 to glimepiride (0.5–4 mg/d), and completed the trial with two PET/CT scans. Plaque TBRmax, uncorrected for blood glucose levels, was determined as the average of the common carotid arteries and the ascending aorta. The treatments reduced fasting plasma glucose and HbA1c values comparably, but only pioglitazone decreased atherosclerotic plaque inflammation, from 1.46 to 1.32 ( to 1.41 . Comparep = 0.033), whereas there was an insignificant increase in that of the glimepiride group . Similar changes were observed with regard to hsCRP. Neither drug affected the vascular remodeling of the coronary arteries, as assessed by vessel diameter or calcification score [Nitta et al. studied patients from the same trial, leaving out those with a visually graded myocardial FDG uptake ≥2 out of a maximal score of 3. Twenty-five pioglitazone-treated patients were compared to 22 glimepiride-treated patients. After a 16-week treatment course, fasting plasma glucose and glycosylated hemoglobin values were comparably reduced in both groups. The reduction in TBRmax from the baseline was significant for the pioglitazone group  to add-on therapy. Moreover, Δ120-min post-load plasma glucose and ΔPEDF were independent correlates of ΔLMT-TBR. Understandably, the authors concluded that 120-min post-load plasma glucose and PEDF values may be markers and potential therapeutic targets of coronary artery inflammation in T2DM [Finally, in a sub-study of the Honda trial , Tahara  in T2DM .In this section, there was a single animal FDG study. Virta et al. used a mouse model of atherosclerosis and T2DM to study the effects of the dipeptidyl peptidase-4 inhibitor linagliptin on atherosclerotic plaque and hepatic inflammation. PET imaging was not applied, but histology, radioactivity counting, and autoradiography of the excised aorta and liver demonstrated that 12 weeks of treatment with linagliptin improved glucose tolerance and reduced hepatic inflammation, but had no effect on the aortic plaque burden or atherosclerotic inflammation . In the p = 0.024), and CRP-levels were not significantly affected by resveratrol treatment (p = 0.091) [In the remaining two human studies on FDG uptake, Ripa et al. conducted an RCT of 51 patients with T2DM of 10–12 years duration vs. 51 placebo-treated patients; they found that the insulin releasing drug liraglutide, administered at 1.8 mg/d for 26 weeks, had virtually no effect on FDG uptake in the carotids and ascending aorta, albeit an exploratory analysis suggested a borderline significant effect in individuals with a history of CVD . The oth= 0.091) .Zwakenberg et al. found in a randomized control trial with 35 T2DM subjects with CVD that six months of treatment with the vitamin K analog menaquinone-7 (360 µg/d) was associated with an insignificant change in femoral artery NaF uptake, compared to placebo. Calcification progression measured with CT was not reduced by vitamin K supplementation . FinallyOnly 28 articles were found suitable for analysis, despite the fact that early reports on FDG- and NaF-PET/CT imaging of atherosclerosis appeared more than 20 and 10 years ago, respectively ,51. The Correspondingly, a large proportion of studies cite inflammation as the cause of atherosclerosis, often with reference to old papers that did not actually prove this assumption, but rather present hypotheses sustained by a statistically indicated association between markers of inflammation and increased carotid intima-media thickness, PWV, CT-detectable arterial macrocalcification, or post-mortem-identified calcification ,63,64,65Publications on this theme all used FDG and showed—despite differences in purpose and methodology—that arterial inflammation, assessed with FDG-PET, is a frequent occurrence, most pronounced in the lower parts of the aorta and present to a lesser extent upwards and downwards ,34, albeWith regard to prevalence, the present FDG studies suggest that arterial inflammation probably does occur more frequently in subjects with prediabetes and T2DM ,33,34, wThis reflects an ongoing lack of understanding regarding the link between ongoing inflammation, mirrored by increased FDG uptake, and active microcalcification, reflected by NaF accumulation. This is also illustrated also by the fact that multiple PET studies have demonstrated limited co-localization of arterial FDG and NaF uptake to be the norm rather than the exception : see thed) for 18F-NaF binding to areas of calcification ex vivo is 0.6 pM, thus indicating that the ultra-high affinity [In contrast to the uncertainty of FDG measurements of inflammation, NaF uptake into tissue and the binding of cells is phenomenally solid. The dissociation constant  in 315 patients scanned for non-CVD reasons, 15 of whom had unspecified diabetes, and found that increased blood-glucose-corrected FDG uptake was not statistically associated with diabetes in any of the four vascular beds, whereas CT-detectable calcification was associated with all four of them . In contThe studies on progression could not explain a possible association between increased arterial FDG and increased NaF uptake. They suggested that the presence of microcalcification by NaF-PET is a precursor or predictor of upcoming cardiovascular events attributable to later-onset CT-detectable calcification, but whether this is actually the case is impossible to judge from the existing literature. However, it seems rather clear from PET studies of atherosclerosis in patients without diabetes ,53,54, aWhat remains to be discussed are aspects of therapy; these are important for our understanding because a positive therapeutic effect may bridge gaps of ignorance and sometimes point to unexpected disease mechanisms. This is what the three careful studies from the same Japanese institution did. Dealing with FDG uptake in the carotids, the ascending aorta, and the left main coronary artery, two of them demonstrated an FDG-reducing effect of about 10% following only four months of treatment with the insulin-sensitivity improving drug pioglitazone, whereas glimepiride, which stimulates pancreatic insulin production, had no effect ,42. As sp = 0.052) in treated patients with a history of CVD [Of other FDG studies of therapy, the Finnish study of atherosclerotic mice on a high-fat diet showed that added linagliptin improved glucose tolerance and reduced hepatic inflammation, but had no effect on the plaque burden or atherosclerotic inflammation, according to ex vivo examinations of the thoracic aorta and liver . Among ty of CVD . In a suy of CVD .Finally, among three NaF studies, Zwakenberg et al. found that, in T2DM patients with CVD, vitamin K supplementation for six months had no effect on femoral artery NaF uptake . SimilarHence, it remains to be seen whether pioglitazone or another insulin sensitizer given for longer than four months can inhibit arterial inflammation in diabetics by more than the 10% shown in the Japanese studies; it is also unclear whether this type of drug can also reduce arterial wall NaF uptake and the later development of CT-detectable calcification. Elucidating these matters would significantly improve our understanding of atherosclerosis, and have a major impact on the treatment of atherosclerosis and its serious derivative diseases.Unfortunately, studies on PET imaging of atherosclerosis in DM are hampered by many of the same shortcomings as PET studies of atherosclerosis in general . That isWhen it comes to studies on diabetes, a particular variant was that 5 of the 20 studies reporting FDG uptake ,37,45,47Many studies were retrospective, typically based on trials conducted for other purposes in patients with primary diseases other than diabetes, or were reanalyses of samples of other materials. There is low demand for prospective studies designed to study atherosclerosis in diabetes, and longitudinal studies with a follow-up period of more than a couple of years, which attempt to map the natural history of atherosclerosis in diabetes, are nonexistent. Hence, it is impossible to decipher the actual meaning or impact of the many reported significant “associations”, typically in the shape of a linear correlation, which was statistically significant at the 5% level, but often with a wide scatter, precluding solid assumptions about the underlying relationships. Some authors called associations of this kind ‘predictive’ of, for instance, future CV events, even though nothing can be stated for the individual subject with a reasonably high likelihood of, say, 85% or more. Therefore, the somewhat tedious conclusion is that a number of interesting observations have been made, but also that the interpretation of these findings is difficult and calls for new, more highly targeted, and better designed studies, the primary purpose of which is to illuminate the molecular mechanisms of diabetes.Presumably, the same atherosclerosis mechanisms are at play in non-diabetic and diabetic patients, but NaF studies about this are lacking, as almost all the information comes from FDG studies.Arterial inflammation is very often present in diabetics, but the nature and frequency of the potential transition to microcalcification is not known, nor are the nature and frequency of the transition from microcalcification to CT-detectable calcification, although an increasing body of literature sustains the notion that NaF is a precursor for CT-detectable calcification.NaF uptake signals ongoing microcalcification, is associated with known cardiovascular risk factors, and appears to herald later cardiovascular events .Arterial inflammation in diabetics can be reduced by short-term treatment with the insulin sensitizer pioglitazone. However, it is unknown whether glitazone drugs can also reduce arterial NaF uptake, and this question deserves investigation in prospective longitudinal trials.With these reservations, the most striking findings in prediabetic and T2DM patients can be summarized as follows see also :PresumabUsing the principles of a systematic review including the Patient, Intervention, Comparison, Outcome Study (PICOS) approach , we sear