The prevalence of peri-implantitis ranges between 7 and 38.4% depending on risk indicators such as smoking, diabetes mellitus, lack of periodontal maintenance program, and history or presence of periodontitis. Currently, the possible effect of the type of superstructure on peri-implant health is unclear. This cross-sectional study aims to investigate the influence of the superstructure on the prevalence of peri-implant mucositis, peri-implantitis and peri-implant dysbiosis.During a 32-month recruitment period dental implants were assessed to diagnose healthy peri-implant tissues, mucositis or peri-implantitis. The study included 1097 implants in 196 patients. Out of all peri-implantitis cases 20 randomly chosen submucosal biofilms from implants with fixed denture (FD) originating from 13 patients and 11 biofilms from implants with removable dentures (RD) originating from 3 patients were studied for microbiome analysis. Composition of transcriptionally active biofilms was revealed by RNAseq. Metatranscriptomic profiles were created for thirty-one peri-implant biofilms suffering from peri-implantitis and microbiome changes associated with superstructure types were identified.p =  < 0.001) and implant level (p = 0.03) between the prevalence of peri-implantitis and RD. Eight bacterial species were associated either with FD or RD by linear discriminant analysis effect size method. However, significant intergroup confounders (e.g. smoking) were present.16.41% of the implants were diagnosed with peri-implantitis, 25.00% of implants with RD and 12.68% of implants with FD, respectively. Multivariate analysis showed a significant positive association on patient (Fusobacterium nucleatum subspecies animalis and Prevotella intermedia require further investigation.Within the limitations of the present work, RDs appear to be a risk indicator for peri-implantitis and seem to facilitate expansion of specific periodontopathogens. Potential ecological and pathological consequences of shift in microbiome from RDs towards higher activity of  Dental implants are a valuable addition to the treatment options for partially and fully edentulous patients , 2. HowePeri-implant mucositis (i) is an inflammation of the peri-implant soft tissues diagnosable by bleeding on probing (BoP) with or without increased probing depth, relative to baseline measurements, without bone loss exceeding the level of physiological crestal remodeling. Peri-implantitis (ii) is diagnosed by radiographic bone loss compared to earlier radiographs with clinical signs of inflammation such as BOP or suppuration with or without increased probing depth or mucosal recession . The surPeri-implant biofilm, a sessile consortium of diverse oral microorganisms, can play a decisive role in peri-implant physiology –13. In hThe superstructure is the part of the implant restoration penetrating the protective border of the peri-implant mucosa and is permanently exposed to colonization by the oral biofilms . DiffereRammelsberg et al. prospectively evaluated 1569 implants in 630 patients and concluded that the type of superstructure has an effect on implant prognosis, e.g. a tendency toward a greater incidence of complications for implants restored with RDs than for single crowns .Accordingly, we examined implant patients to test the hypothesis, that RD is associated with higher prevalence of peri-implantitis and used metatranscriptomics to study the associated microbiota aiming to identify RD as a risk indicator for peri-implantitis.This is a cross-sectional study aligned with the STROBE statement for cross-sectional studies. The study participants were patients of the Department of Prosthetic Dentistry and Biomedical Materials Science of Hannover Medical School and were recruited between October 2016 and May 2019. Patients were approached during their regular treatment sessions and gave oral and written consent to participate in the study. The survey of the data and the inclusion of study participants took place in consideration of the ethics committee of Hannover Medical School (vote No. 3086-2016).Only patients with at least one root-shaped titanium dental implant restored and in function for at least 12 months, were included in the study. Exclusion criteria were head or neck radiation during the last 6 months, antibiotics during the last 3 months, patients with a “heart passport”, immune-compromised patients, patients with uncontrolled diabetes, expecting or lactating mothers. The patient recruitment process and clinical assessment was performed by one examiner following a standardized protocol.The collection of data was subject to special data protection measures, each patient was assigned a sequential number. All digital data were stored on the internal, secure and password-protected drives of Hannover Medical School. The examinations were carried out by an experienced practitioner to achieve standardization of the examination procedures, the probing pressure, the radiographic evaluation, the visual assessment and the subsequent diagnosis.All implants were examined clinically and, when indicated, radiographically. Healthy implants without clinical signs of inflammation (BOP) were not examined radiographically. However, fistula, suppuration, bleeding on probing with an increase of PD or recession over time in relation to former examinations and pain were indications for radiographs. All patients that were diagnosed with peri-implant mucositis received radiographic examination to eventually diagnose peri-implantitis. The following parameters were collected:i. age and gender, ii. bleeding on probing, iii. probing depth, iv. presence of periodontitis, v. history of periodontitis vi. smoking status, vii. residual teeth, viii. type of superstructure (fixed or removable), ix. full-mouth plaque index x. number of implants xi. implant age xii. type of implant. Additionally, submucosal plaque samples were collected. Clinical examination was performed with the help of a periodontal probe . Four aspects of each implant were assessed.To evaluate the influence of the superstructure on peri-implant health, the exposure variable superstructure was categorized into (a) removable (RD) and (b) fixed dentures (FD). Based on the available information, one of the following diagnoses was made for each implant following the definition for peri-implant mucositis and peri-implantitis of group 4 of the World Workshop on Periodontal and Peri-implant disease : i. healSuperstructures were defined as fixed dentures when they were cemented or screwed retained. Bar-supported, locator-supported, and telescope-prostheses were defined as removable. No distinction was made between overdentures and telescoping bridges.Probing depth (PD) was measured using a periodontal probe . Measurements were taken at four implant sites . Peri-implant mucositis was evaluated utilizing the bleeding on probing index (BOP). If BOP occurred in one aspect, the whole implant was positive. Presence of periodontitis was defined at CPI  Code ≥ 3 . Full-moSubmucosal plaque samples and samples of the peri-implant crevicular fluids were collected at six implant sites  after drying the area. Samples were collected with the help of paper points  inserted in the peri-implant pocket for 30s and a titanium curette. Paper points were pooled for each implant and stored in RNA-protect at − 80 °C  RNA was isolated based on previously described protocol . BrieflyThe data obtained were transferred from the analogue diagnostic results to an Excel  spreadsheet. The Excel spreadsheet was imported into SPSS, version 26  and then a (1) descriptive analysis of the data was made, (2) the data were checked for normal distribution with the Kolmogorov–Smirnov (KS) test and histogram graphs (3) a multifactorial regression analysis was performed in Excel. A KS test and visual evaluation of histogram for 1097 samples confirmed a normal distribution of the data.Contingency table analysis was applied to study the relationship between superstructure and diagnosis groups. Pearson Chi-Square statistics and adjusted residual values were calculated to identify associations.Mean values of diagnoses  were compared, using a two-sample t-test. Based on a univariate correlation analysis, possible cofactors were identified and considered for the multivariate adjusted model. In the next step, an adjusted sensitivity analysis was carried out, in which the parameters previously identified as cofactors were incorporated in the model. An adjusted regression analysis was performed.PRIMER and PERMANOVA+, the suite of univariate, graphical and multivariate routines were used to analyze the microbiome data. Transcriptionally active microbiome profiles were screened for species associated with denture superstructures. We used blank samples and the correlation analysis to identify and remove the contaminating reads . BrieflyA total of 196 patients were examined. The recruitment process is depicted in Fig. Descriptive analysis revealed that 1097 implants were included in the cross-sectional study, of which 372 were diagnosed as healthy and 725 as diseased (Table Peri-implant mucositis occurred in both types of dentures at a rate of about 50%. Peri-implantitis occurred roughly twice as often (25%) with RDs than with FDs (12.68%).df = 2, p =  < 0.001, minimum expected count = 54). Low or high adjusted residuals values (< − 2 or > 2) for health and peri-implantitis but not for mucositis  indicated that there were fewer health cases and more peri-implantitis cases than expected for RD group. [Contingency table analysis was performed using a Crosstabs function in SPSS on implant cases weighted by diagnosis frequencies and revealed that restoration type was associated with diagnosis  was found. In the group of RD, a mean value of 0.96 with approximately the same variance was found. The t-statistic was − 4.48, indicating that peri-implant health was significantly better in the group of fixed dentures.t test, assuming equal variances  between the type of superstructure (removable / fixed) and the diagnosis . Possible covariates that were not associated with our primary outcome in the univariate analysis were not included in the multivariate analysis. Consequently, the variables, which were found to be significant were used in the adjusted sensitivity analysis of the primary objective.A two-way correlation analysis showed that there is a significant association (p = 0.0304). Using the patient as the statistical unit (Table p < 0.001). The multiple linear regression on patient-level based on a binominal regression (peri-implantitis vs. no peri-implantitis) showed consistent results (Table p = 0.001) and oral hygiene (p < 0.001) were significantly associated with peri-implantitis.Using the implant as the statistical unit Table , the mults Table . AdditioSecondary outcomes are significant associations between higher mean values of diagnosis and insufficient oral hygiene, higher implant age on patient-level as well as higher implant age, history and presence of periodontitis, edentulism, insufficient oral hygiene, and higher patient age on implant-level.RNAseq-based biofilm profiles obtained with the small patient subgroup suggest that RDs favor expansion of specific periodontopathogens.Clinical characteristics of samples selected for biofilm RNA-seq-based analysis is shown in Table Porphyromonas sp., 4 Fusobacterium nucleatum subsp., 6 Prevotella or closely related Alloprevotella species. 2 Tannerella sp., Treponema denticola, Selenomonas sputigena, and Anaeroglobus geminatus. All these species have been associated with periodontal and/or peri-implant pathologies. We observed two separate clusters that grouped “RD” samples from three patients  in this cohort of university clinic outpatients. RD was identified as an independent risk indicator in the adjusted model. Scientific data on peri-implant health in direct comparison between RDs and FDs is scarce . In lineFusobacterium nucleatum subsp. animalis, Porphyromonas gingivalis, Prevotella nigrescens, Tannerella forsythia and Fusobacterium nucleatum subsp vincentii [. In some cases, biofilms were dominated by less studied species, often representing Prevotella genus, e.g., P. pleuritidis, P. maculosa, or P. denticola but the ecological role and pathogenic potential of this species is poorly explored [Cardiobacterium hominis, a species that can cause infective endocarditis [RNAseq profiling of submucosal biofilms in peri-implantitis revealed active microbial communities usually dominated by well-known oral opportunistic pathogens, e.g., incentii . In someexplored . We alsocarditis . Thus, mFusobacterium nucleatum and Prevotella intermedia are both associated with oral and extraoral pathologies, are equipped with numerous virulence factors, and have big impact on microbiome ecology [Fusobacterium nucleatum ss animalis and not the other subspecies were associated with removable dentures. This provokes a question about genetic determinants that drive superstructure specificity.Hence, profiling reveals the active community members and can capture dynamic changes in biofilms. Two important periodontopathogens expended in microbiomes from implants with RD.  ecology , 20, 44.Fusobacterium species and subspecies were shown to differentially affect the composition and architecture of supra- and subgingival biofilms models, suggesting high functional diversity in Fusobacterium genus. [Prevotella intermedia, the other potential biomarker, commonly produces beta-lactamases and can resist higher concentrations of few antibiotics [Prevotella intermedia sensu lato) or subspecies (the Fusobacterium nucleatum subspecies) can be challenging and studies addressing genomic diversity of these taxa could improve the genomic reference for microbiome studies. Organoid and animal models can be used to establish a casual effect of biofilm shift (towards specific periodontopathogens) on peri-implant tissue in context of superstructure type or implant material [Interestingly, m genus.  Prevotelibiotics –48. A vaibiotics . Distingmaterial .Meijer et al. (2014) summarized two prospective studies and found a 10-year incidence of peri-implant mucositis and peri-implantitis in edentulous patients with overdentures in 57% and 29.7%, respectively . These nIn a systematic review and meta-analysis, both current periodontitis  and histIt may be assumed that the study cohort is representative for a university population. It would be advisable to perform a follow-on study that includes i. multiple centers, ii. private practice and focus on different populations in terms of e.g. geography, socioeconomic status and oral health behavior.By including more than one implant of the same patient, pseudo replication may occur. The presented statistical evaluation considers this putative bias as we did patient-level analysis with one random implant for each patient and found consistent results. Furthermore, the clinical and microbiological status may strongly differ for implants in the same individual warranting implant-level analysis. A mixed-multi-level-model as described in  would beThe prevalence of peri-implant mucositis (50%) and peri-implantitis (16%) both on implant- and patient-level observed in the present patient population correspond to the numbers published in recent reviews and the results on the patient level with only one implant per patient included in the analysis. Consequently, generalizability of the study results may be assumed, however subject to the limitations.For microbiome analysis we analyzed only a small number of biofilm samples as a first pilot to investigate whether further research in this direction is promising. The metadata of the randomly selected patients shows that the implants in the RDs group are from (i) smokers and (ii) from only three individuals. This limits the conclusions that can be drawn from our findings as the effect may also be due to these cofactors. In future, it is necessary to validate the findings of the present microbial analysis in a more representative cohort in more individuals carrying RDs in a matched-case analysis.Conclusively, microbiome analysis gave first insights into superstructure-specific effects on biofilms but bigger number of patients needs to be profiled to confirm our observations.For future research the incidence of peri-implant disease in association with the type of superstructure for determining possible preventive aspects or the determination of success / or loss rates seems highly interesting. Consequently, the previously examined patients would have to be re-examined after a period of 5–10 years to elucidate the predictive value of peri-implant mucositis and the type of superstructure on the progression and initiation of peri-implantitis as the positive predictive value of peri-implant mucositis for the development of peri-implantitis has not yet been clarified until today .Possible future approaches to investigate causality of the relationship between prevalence of peri-implantitis and RDs are quantitative and qualitative analyzes of the peri-implant bacterial biofilm and its changes over time.F. nucleatum ss animalis and Prevotella intermedia and its therapeutic consequences require further investigation.Peri-implant mucositis occurs at a rate of about 50%, regardless of the type of denture, while peri-implantitis is twice as likely (25.00%) for RDs than for FDs (12.68%). The hypothesis that the type of superstructure has an impact on peri-implant health must be accepted in this patient population as RD could be identified as an independent risk indicator for peri-implantitis in a model adjusted for co-factors. Potential role of shift in microbiome from RDs towards higher activity of


Walnuts contain nutrients that are associated with improved cognitive health. To our knowledge, no review has systematically examined the effects of walnuts on cognitive function and risk for cognitive decline.To conduct a systematic review and meta-analysis evaluating the effects of walnut intake on cognition-related outcomes and risk-factors for cognitive decline in adults.®, Commonwealth Agricultural Bureau, and Cochrane Central Register of Controlled Trials were searched for randomized controlled trials (RCTs) and observational studies published until April 2020 on walnut intake, cognition , and selected risk factors for cognitive decline (e.g. glucose homeostasis and inflammation). Risk-of-bias and strength-of-evidence assessments were conducted using standard validated tools. Random-effects meta-analyses were conducted when ≥3 studies reported quantitative data for each outcome.Medline32 RCT and 7 observational study publications were included. Meta-analysis of cognition-related outcomes could not be conducted due to heterogeneity of tests. None of the 5 cognition RCTs found significant effects of walnuts on overall cognition, although 3 studies found improvements on subdomains and/or subgroups. All 7 observational studies found significant associations and a dose-response relationship between walnut intake and cognition-related outcomes. Meta-analyses of 27 RCTs reporting glucose homeostasis and inflammation outcomes, selected risk factors for cognitive decline, did not show significant effects of walnut intake.This is a systematic review and meta-analysis of 5 randomized clinical trials and 7 observational study articles of the impact of walnut intake on cognition decline and 27 randomized clinical trials of the effect of walnut intake on risk factors for cognitive decline including glucose homeostasis and inflammation.The non-uniformity of tests performed to measure cognitive function in the various studies did not allow for a meta-analysis of these studies. A definitive conclusion could therefore not be reached regarding the effect of walnut intake on cognitive decline.The evidence available does not show an association between walnut intake and glucose homeostasis or inflammation.Due to the non-uniformity of tests for cognition-related outcomes, definitive conclusions regarding the effect of walnut consumption on cognition could not be reached. Additionally, evidence does not show associations between walnut intake and glucose homeostasis or inflammation, cognitive decline risk-factors. High-quality studies with standardized measures are needed to clarify the role of walnuts in cognitive health. The prevalence of age-related cognitive decline is expected to grow with increases in life expectancy ,2. AdditWalnuts contain nutrients that may intervene in the development of cognitive decline, in part by targeting cardiometabolic risk factors . These nWhile a role for walnuts in promoting cognitive health has been previously reviewed ,17, to oThis systematic review was conducted following standard methodology outlined in the Cochrane Handbook  and the ®, Commonwealth Agricultural Bureau (CAB), and Cochrane Central Register of Controlled Trials from 1946 to April 2020. The search strategy included terms for walnut interventions of interest and outcomes of interest (described later) and was limited to human studies and the English language. The complete search strategy is shown in Supplemental Table S1. The reference lists of prior relevant systematic reviews were screened to identify additional eligible studies not captured by the database searches.We conducted electronic searches on MedlineWe included all interventional and observational studies in adults (≥18 years) who were healthy or at increased risk for cognitive decline. Walnut interventions of interest included whole walnuts, walnut oil, or walnut extract. The primary outcomes of interest were cognition-related outcomes . Secondary outcomes included risk factors for cognitive decline. With input from key informants, we identified major risk factors for cognitive-decline as alterations in blood lipids, glucose metabolism, blood pressure, endothelial function, inflammation, and oxidative stress ,10. SincWe excluded studies that compared interventions in which the effects of walnuts could not be isolated , or did not correlate walnut consumptions with any one of the outcomes of interest. Detailed study eligibility criteria are listed in http://abstrackr.cebm.brown.edu/). For all abstracts deemed potentially relevant by both screeners, full-text articles were retrieved and independently screened by two investigators based on the study eligibility criteria. All abstract and full-text screening conflicts or disagreements were resolved through discussions between the two investigators or through the consensus of the entire research team. Excluded articles and reasons for exclusion are listed in Supplemental Table S3.Titles and abstracts identified from the literature searches were independently screened by two investigators to exclude irrelevant citations using the open-source online software Abstrackr (We used a two-stage approach for data extractions. During the scoping stage (stage 1) of the systematic review, we extracted information from all included studies about study design, types of exposure/interventions, and outcomes reported in each included study. Due to the large number of inflammation and glucose homeostasis measures investigated across studies, we set aside (i.e. not performing the next stage of data extraction) the measures if they were reported in 2 or fewer studies.For the second-stage data extraction, we designed customized Excel data extraction forms for interventional and observational studies. The items extracted included the following: funding sources, location, study population characteristics, enrolled and analysed sample sizes, study design features, walnut intake doses , relevant outcomes assessed and methods used, confounders and effect modifiers used in statistical analysis, and results. For interventional studies, we additionally extracted information on walnut interventions and comparators, adherence, and adverse effects. For studies with multiple walnut-free comparator arms, the intervention that was most similar to the walnut group was chosen as the comparator for our analyses. Quantitative results that were needed for meta-analysis were extracted wherever provided, otherwise, qualitative results were extracted . For all studies, results from the most adjusted statistical model were extracted in preference over crude or age-adjusted measures. The extraction forms were piloted on several studies and revised according to group discussions. Data from each study were extracted by one investigator and confirmed by ≥1 other investigator. Data discrepancies were identified and resolved through group discussions.via group discussion. For interventional studies, we used the Cochrane risk of bias (ROB 2.0) tool [For each included study, two independent investigators conducted risk of bias (ROB) assessments and discrepancies were resolved .0) tool  to evalu. The maximum number of stars was 9 for prospective cohort studies and 10 for cross-sectional studies. Following the criteria used in previous reviews, 0–4 stars was considered high risk of bias, 5–7 was some concerns, and 8–9 (prospective cohort) or 8–10  was considered low risk of bias [The ROB of prospective cohort studies was assessed using the Newcastle Ottawa Scale (NOS) for cohort studies . The NOS of bias ,25.All of the included studies were summarized in narrative form and in the summary tables that tabulated key features of the study populations, design, intervention, outcomes, and results. Summary tables were organized by study type  and by outcomes of interest (cognition-related outcomes and outcomes related to risk-factors for cognitive decline).diff = Base+ SDfinal − 2 x Corr x SDBase x SDFinal), where SDBase is the SD at baseline and SDfinal is the SD at the end of the intervention. We assumed a correlation coefficient (Corr) value of 0.50 to impute the missing SD of the mean within-group change. Sensitivity analyses that used Corr values of 0.20 and 0.80 were conducted to evaluate the impacts of the correlation assumptions on the meta-analysis results, and none showed appreciable impacts on the pooled results. Studies were excluded from meta-analyses if the required information for the aforementioned calculations was not reported, not standardisable, or otherwise implausible for any given outcome. We used both the Q statistic (considered significant when p < .10) and the I2 index to quantify the extent of statistical heterogeneity [2 values below 25%, between 25 and 70%, and above 70%, respectively.We did not perform meta-analyses to quantitatively synthesize the results from observational studies because these studies used a wide variety of outcome measures and no outcome measure of interest was reported in more than two studies. For RCTs, we performed random-effects meta-analyses where there were ≥ 3 unique studies reporting the same outcomes in light of clinical heterogeneity . We used the reported or calculated net change (difference of the 2 within-group changes from baseline) between the walnut and comparison groups as the effect size measure in the meta-analysis. If the standard deviations (SDs) of the within-group changes were not reported, the SD of the mean within-group change was estimated by using the following formula: SDogeneity . Low, mop values ≤ .05 were considered significant. The analytic data sets can be found under Supplemental File 2. The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary materials.All calculations and meta-analyses were conducted using Stata SE 13 (StataCorp). Two-tailed We followed the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) approach to determine strength of evidence (SoE) for each outcome . BrieflyThe results of the search strategy for overall inclusion in this systematic review and meta-analysis can be found in Seven observational studies . Risk of bias for this study was rated as some concerns, due to missing data .In healthy young adults consuming 60 g/d walnuts or placebo for 8 weeks in a cross-over design, Pribis et al.  found nop = .016) and perception (p = .005) composite scores, in that the walnut group had less decline in performance than did the control group over the two-year intervention period. No significant differences were observed between groups in subjects from the Loma Linda site. The authors noted that Barcelona participants had significantly lower education and baseline dietary and red blood cell ALA and ALA intake than their Loma Linda counterparts.In the Walnut and Healthy Aging Study (WAHA), comprised of older adults (mean age 69.2) with mixed baseline health, Sala-Vila et al. examined the effects of consuming 30–60 g/d walnuts on cognitive function using composite scores for memory , language , perception , frontal function , and global cognition   Table 2Table 2. In addition to neuropsychological testing, Sala-Vila et al. conducted structural and functional MRIs to examine brain structure, resting state connectivity, blood flow, and the expression of functional brain networks during cognitive demands (N-back test) in the Barcelona subset . No signFour observational studies . Additionally, the effect size and significance levels were greater for the highest tertile of walnut consumption than for the middle and lower tertiles, demonstrating a dose-response effect. Similar findings were reported for the WWON group. However, risk of bias for this study was rated as some concerns, due to participant selection (non-random sampling and no description of compatibility between responders and non-responders) and exposure/outcome ascertainment , after adjustment for covariates. Overall risk of bias for this study was rated as some concerns, due to participant selection (non-random sampling and no description of compatibility between responders and non-responders) and incomplete reporting of results (significant findings only)  .p < .001). However, latent growth models found no significant association between walnut consumption and change in global cognitive status (TICS scores) over four years, after adjustment for covariates. Risk of bias for this study was rated as low . The authors noted that these findings should be interpreted with caution, as the small sample size in the highest walnut consumption frequency category was not significantly associated with improved cognitive function. Furthermore, there was no overall trend of increasing walnut intake with improved cognitive performance . Additionally, there were no significant differences between walnut intake groups in rates of cognitive decline over the six years of follow-up on any of the composite scores . Overall risk of bias was rated as some concerns, due to participant selection , no description of subjects lost to follow-up, and selective reporting  compared to men , possibly due to increased likelihood of reporting depression symptoms. Risk of bias for this study was rated as some concerns, due to participant selection, lack of adjustment for important covariates (e.g. education), and exposure/outcome ascertainment .Ten randomized   .Six randomized . Findings from the two studies that could not be meta-analysed were consistent with the meta-analysis as no significant difference in HOMA-IR was found between walnut and control interventions [Supplemental File 2).Findings within these six individual RCTs were consistent; none found a significant difference in HOMA-IR between walnut and control interventions. Random-effects meta-analysis of the four RCTs reporting quantitative HOMA-IR data  did not ventions ,60  .Findings were consistent; ten studies did not observe significant effects of walnut on hsCRP, and one study reported a significant reduction in hsCRP with walnut consumption . Random- = .251) . In the Ten randomized cross-over trials reported sICAM-1 as an outcome ,58,63,652 = 93.8%; p < 0.0001). Findings from the three studies that could not be meta-analysed [Supplemental File 2).Findings were relatively consistent across studies; nine of the ten RCTs found no significant differences in sICAM-1 between walnut and control interventions. Only one study found that the walnut intervention significantly reduced sICAM-1 compared to control.  Random-eanalysed ,58,63 weNine randomized cross-over trials reported sVCAM-1 as an outcome ,58,63,652 = 73.2%; p = .005). Findings from three of the four studies that could not be meta-analysed were consistent with the meta-analysis; no significant difference in sVCAM-1 was found between walnut and control groups [p = .045) [Findings were inconsistent; two RCTs found that the walnut intervention significantly reduced sVCAM-1 relative to control ,65, whill groups ,55,58. H = .045)  . Findings from the one study that could not be meta-analysed was consistent with the results of the meta-analysis; no significant difference in IL-6 was found between the walnut and control intervention [Supplemental File 2).None of the six RCTs found a significant difference in IL-6 between walnut and control interventions. Random-effects meta-analysis of the five RCTs reporting plausible IL-6 data  did not rvention   .Overall, findings were consistent; four studies did not observe significant effects of walnut on TNFα ,52,54,57 = .961) . One stuFive randomized cross-over trials reporting E-selectin as an outcome were included ,54,55,652 = 68.6%; p = .023) (p = .033) .Random-effects meta-analysis of four RCTs reporting plausible data did not find significant effects on E-selectin  with moderate statistical heterogeneity (I = .023) . Only onOnly two RCTs (cross-over) ,66 reporlow was assigned for the effect of walnut consumption on cognitive function and mood in RCTs (i.e. further research is highly likely to have an important impact on our confidence in the estimate of association and is likely to change the estimate) . An evidence level of moderate was also assigned for no effect of walnut on inflammation outcomes.An evidence level of stimate) . An evidThe aim of this review was to systematically evaluate existing research on walnuts as a dietary strategy to promote cognitive health and reduce risk for cognitive decline. Previous reviews have described the beneficial effects of walnuts on cognitive function in animal studies, which include improvements in learning, memory, and anxiety behaviours in aged animals  and an aIn the majority of the included RCTs, there was no significant overall effect of walnut intake on cognition-related outcomes (cognitive function and mood), although there were a few exceptions. In the two RCTs reporting cognitive function outcomes, significant effects of 30–60 g/day of walnuts were found on subdomains of cognition and/or subgroups of study populations, although not on total cognitive scores. In the WAHA study , particiIn contrast to results from RCTs, the majority of the seven included observational studies found significant associations and a dose-response relationship between walnut intake and cognition-related outcomes. Most of these studies were of cross-sectional design but used large pre-existing datasets. In analyses of both the NHS and HPFS cohorts, higher walnut intake was related to lower risk of stroke ,33, and While these observational studies show more promise for a role of dietary walnut in maintenance of cognitive health than results from RCTs, traditionally, observational studies, and in particular, cross-sectional studies, are thought to provide lower strength-of-evidence than do the gold-standard of RCTs . AdditioIn addition to cognitive function and related outcomes, we aimed to evaluate the effect of walnut on risk factors for cognitive decline, including alterations in blood lipids, glucose metabolism, blood pressure, endothelial function, inflammation, and oxidative stress ,10. SincPrevious systematic reviews and meta-analyses did not show an effect of dietary walnut intake on blood pressure ,74, or oOverall, the available evidence reviewed in this report is not sufficient to draw a firm conclusion regarding the effect of walnut intake on either cognitive decline or on risk factors for cognitive decline in adults. The limitations identified in this review impel us to suggest some directions for future research.First, the heterogeneity in cognitive function measures, and minimal reporting of stroke and mood outcomes ruled out meta-analyses for these outcomes. Future RCTs involving cognitive outcomes should specify a minimal set of tests to be included, and these tests should be sufficiently sensitive to detect small changes in cognition-related outcomes. Second, the included studies showed variability in duration and dosage of interventions, background diet and study population characteristics, which hinders comparability of findings. This heterogeneity precludes firm conclusions on the walnut intake regimen required for cognitive benefit, and which populations are likely to experience these beneficial effects. Future studies should specify a set of standard dosages and durations of the intervention and follow-up. Additionally, measures of risk factors for cognitive decline should be incorporated into studies examining the effects of walnut on cognitive function, to elucidate possible biological mechanisms. Third, trials should focus on “at-risk” individuals (such as those with dyslipidemia), since healthy individuals consuming healthy diets are less likely to show cognitive improvement upon dietary intervention. Similarly, trials on mood outcomes should focus on subjects reporting symptoms at screening. Lastly, wherever realistic, biomarkers of nutrient intake should be used since recall of food intake has long been acknowledged to be a limitation in studies involving self-reported dietary assessments. Self-reported food intake, and the resulting risk of recall bias is of particular concern in studies on cognitive function and decline, as even mild cognitive impairment has been shown to reduce the validity of FFQs . BiomarkIn conclusion, the existing evidence, although with low level of confidence, suggests that walnut intake may have a beneficial effect on cognition-related outcomes, including cognitive function, mood, and stroke. Limitations in study design and comparability render the available evidence insufficient to draw a firm conclusion regarding the effects of walnut on cognition in adults. High quality studies and standardized interventions and measurement tools are necessary to determine the role of walnut intake in cognitive health.Click here for additional data file.


Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins. Large-scale discovery of small proteins , or “microproteins”) has relied primarily on computational analysis of genome sequences , and genMass spectrometry-based proteomics (MS) provides a direct method to detect and quantify small proteins on a global or targeted scale  Bottom up. Methods in which protein samples are enzymatically or chemically cleaved into peptides prior to MS and MS/MS analysis. Typically uses liquid chromatography (LC) to separate digested peptides prior to MS and MS/MS analysis. Typically involves fragmentation in the mass spectrometer (MS/MS) to sequence peptides.(ii) Top down. Methods in which protein samples are analyzed directly in the mass spectrometer without digestion. Can involve direct infusion or LC to separate proteins prior to MS and MS/MS analysis. Typically involves fragmentation in the mass spectrometer (MS/MS) to sequence and identify the proteins. Can require specialized instrumentation or custom setup of existing instruments.Mass spectrometry instrumentation and acquisition.(i) LC-MS. Liquid chromatography-coupled mass spectrometry and tandem mass spectrometry (LC-MS/MS).(ii) MS/MS. Typical arrangement for most MS analyses, also termed “tandem mass spectrometry” Separated proteins and/or peptides are measured in the instrument and then isolated and fragmented to record tandem mass spectra. Can be used for “bottom-up” and “top-down” approaches. Typically uses electrospray ionization (ESI) to generate ions.(iii) MALDI/ESI. Ionization methods for MS. Matrix-assisted laser desorption ionization (MALDI) is rarely attached to LC systems. ESI is typically coupled to LC systems and is the dominant ionization mode for proteomics.(iv) DDA. Data-dependent acquisition mode. Most commonly-used approach for proteomics studies. Candidate ions are selected based on peak intensity and resolution (= data dependent), isolated, and fragmented to provide MS/MS spectra.(v) DIA. Data-independent acquisition mode. A hybrid approach where LC-MS/MS data are acquired without isolating specific ions for tandem analysis. Yields complex MS/MS spectra from multiple precursors. Since all ions are fragmented for MS/MS, no information is lost in the process, and typically data quality for quantitation is higher. Requires libraries of empirical or derived data from samples to extract identification and abundance from acquired data. Existing files can be reanalyzed with new information, as no ions are discarded.Data analysis terms.(i) PRM. Parallel reaction monitoring. Typical targeted acquisition mode for specific detection and robust quantification. Can involve specialized instrumentation.De novo.(ii)  Direct sequencing of polypeptides from fragment spectra via comparison of mass differences to amino acid residues.(iii) Database search. Peptide and protein identification from fragment spectra by comparison of spectral patterns and fragment ions to databases of theoretical spectra/fragment masses. Redundant peptides measured from this approach are called a peptide spectrum match (PSM).Bottom-up approaches also face the so-called “protein inference issue”: protein identifications are inferred based on the detected peptides, which can be ambiguous and match to more than one protein or isoform . ProteinHere, we describe a selection of MS-based approaches to identify and characterize small proteins in both exploratory and targeted studies. We focus on (i) approaches for sample preparation and MS data collection for small proteins, (ii) analysis of MS data sets for small protein discovery, (iii) validation of putative small proteins, and (iv) bioinformatic and MS-based prioritization of putative small proteins. While we cannot cover all approaches due to space constraints, we provide a repertoire of approaches that can be modified for a specific research question and that optimize the output for small proteins. These methods were developed by many different groups and now help to level the playing field for small proteins.Small protein detection by MS is most frequently applied to discover small proteins from complex samples such as whole-cell lysates ; Table 1An important consideration in method choice is the use of bottom-up or top-down proteomics . The vasBelow, we outline general considerations for each step of an MS-based analysis of a sample for small proteins. The experimental workflow can be divided up in four parts , describDepending on sample complexity and the type of MS analysis, different preparation steps are required. In general, bottom-up analyses focus on efficient denaturation and digestion, while top-down approaches mainly utilize size exclusion steps to enrich small proteins . We outl(i) Sample preparation for bottom-up proteomics. Sample preparation for bottom-up proteomics typically includes denaturation, reduction, and alkylation steps to ensure optimal access of the proteases to unfolded proteins, and to prevent thiol oxidation and disulfide bridge formation. Per se, these steps do not impede small protein detection; however, most protocols were designed for protein molecular weights >10 kDa, and include efficient removal steps for small molecular weight contaminants. In particular, filter-based methods such as filter-aided sample prep (FASP) (p (FASP)  and suspp (FASP)  efficienp (FASP) . We ther(ii) Sample preparation strategies using protein precipitation steps. The same considerations apply for sample preparation strategies using protein precipitation steps that are often applied to concentrate and purify target proteins, e.g., organic solvent precipitation using acetone, methanol, chloroform or other solvents  Enrichment using typically discarded fractions. While the above modifications reduce the likelihood of de-enriching small proteins, it is also possible to actively enrich small proteins by using only the fractions that are typically discarded in a conventional bottom-up proteomics preparation. This can be achieved by either (i) using molecular weight cut-off membranes, with large proteins being trapped using a filter-membrane with a low molecular weight cutoff, and the flow-through containing small proteins being retained for subsequent analysis, or (ii) subjecting the sample to a standard organic precipitation; the precipitated proteome is discarded, and the supernatant is digested and submitted to LC-MS/MS analysis. Both of these techniques can also be applied to top-down approaches. The small protein fractions isolated from these approaches typically coenrich for other interfering small molecules from the sample and common preparative contaminants. Hence, special care must be taken to remove these prior to MS analysis. In addition to reverse-phase desalting prior to analysis, we recommend desalting using strong cation exchange by solid-phase extraction (SCX-SPE).(iv) MS approaches. MS approaches commonly involve a prefractionation step to reduce sample complexity, with the most widely used methods being separation of the sample by (i) centrifugation, into soluble and membrane fractions; (ii) sucrose density gradients, based on hydrodynamic radius; (iii) size exclusion or ion-exchange chromatography; and (iv) SDS-PAGE. These methods can be applied to study small proteins, but separation by SDS-PAGE requires modification to prevent loss of small proteins from the sample; small proteins often stain poorly with Coomassie, or pass through the gel with minimal retention. Isolating the dye front  and a narrow range of low MWs will enrich small proteins. If no bands are detected using Coomassie staining, we suggest silver staining due to its higher sensitivity. After excision of the respective gel areas, we recommend only minimal wash steps, as small proteins may diffuse out of the gel into the wash buffer. When small proteins are not efficiently recovered with these conventional gel-based approaches, distinct MW regions can also be selected by electroeluting specific regions or by gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) and downstream processing in the liquid phase  approach , where c27–In general, we recommend fewer preparation and manipulation steps for less complex sample mixtures prior to MS analysis. For purified protein complexes, we suggest minimal desalting and purification using, for example, reversed-phase tips or cartridges, with samples then analyzed directly by MALDI-MS or -MS/MS. For complex protein mixtures and lysates, we recommend modifying S-trap protocols with minimal washing, organic extractions, or reciprocal purification using molecular weight cut-off filters.Protease digestion is the defining step of bottom-up proteomics. Digestion is often performed in-solution, in-gel, or during filter-based sample preparations. These digestion protocols use extensive washing of the sample that inherently de-enriches small proteins; due to their size, small proteins can pass through filter membranes, may not adsorb sufficiently (or may stick too tightly) to solid phases, and may be flushed during wash steps. Hence, the digest should be conducted in a way that specifically ensures inclusion of, and access to, small proteins, which can range from in-gel and in-solution digests to solid-phase supported protocols. The advantages and limitations of different digestion strategies for small proteins are detailed below.Digestion of small proteins is challenging due to the intrinsic scarcity of necessary protease cleavage sites, and the often short length of the few peptides that are generated. In general, Trypsin, mostly alone or in combination with LysC, has been the workhorse for bottom-up proteomics due to cleavage specificity (cleaves after K and R) and activity in typical preparation conditions. This excellent enzyme/combination of enzymes, in particular in consecutive denaturing conditions, has also been widely used to identify small proteins in different prokaryotes , 31 Tab. Alterna18 bed materials  and larger pore sizes (>300 Å) may offer higher recovery in bottom-up and top-down experiments. This often comes at the cost of decreased retention and recovery of hydrophilic peptides and small proteins.In bottom-up methods, proteolytic peptides are typically separated by liquid chromatography prior to acquisition of MS (ionized peptide precursors) and MS/MS data (fragmentation pattern of peptides) , 136. Thaterials  that sepFor the analysis of complex bottom-up or top-down samples, extending the length of the chromatographic gradient may offer significant improvements in data depth, as more acquisition time is available . HoweverMost proteomics labs operate electrospray ionization (ESI)-based mass spectrometers due to their direct interface with LC and high ionization efficiency. This combination allows fast acquisition of high quality data, and is equally applicable to conventional bottom-up proteomics approaches and top-down experiments . ESI-bas18  or C4/C8  tips or SCX-SPE-based desalting as outlined above. After solvent removal, these purified complexes can then be directly spotted and submitted to MALDI-MS analysis. If a MALDI-MS/MS instrument with sufficient isolation power and resolution is available, small proteins can also be identified directly from these spectra  to generate complementary data, and increase the fragment coverage of the small proteins of interest.m/z ranges are sequentially fragmented independent of precursor abundance, providing information-rich MS and MS/MS data sets that can be mined. Since no precursors are “discarded,” presumably numerous undiscovered small proteins reside in the data awaiting detection. This approach, facilitated in recent years by faster and more sensitive instruments, is expected to benefit substantially from further improvements in analytical pipelines.We anticipate that ion mobility-coupled separation of ions will improve the scope and depth of conventional Data Dependent Analysis (DDA)-based bottom-up proteomics analyses also for small proteins , 46. In de novo , plus afication –87, someication –. The morication –  control . HistoriAnother critical consideration when interpreting FDR estimates from MS data analysis is that the false positive protein identifications are distributed unevenly between large (mostly annotated) and small (mostly unannotated) proteins; custom databases will contain many more predicted small proteins than annotated proteins, such that the likelihood of a PSM matching a predicted small protein by chance is much higher than the likelihood of matching an annotated protein by chance. Moreover, there will be proportionally more PSM matches to annotated proteins than to small proteins, causing the overall FDR estimate to be skewed toward the value for annotated proteins. Consequently, reported protein-level FDRs substantially underestimate the true FDR for novel small proteins , 94, 95.ab initio gene predictions and a six-frame translation . Mo. Moab inrcolator ), and sein silico with very encouraging results for large proteomics studies and improving the number of correct PSMs  or “ProteaseGuru”  serve asst place , 109. Hopeptides , so thisproteins  to ensurproteins .B. henselae, overall almost 80% of 136 peptides implying novel sORFs, novel start sites, or expressed pseudogenes, were successfully validated by parallel reaction monitoring, with lower success rates for N-terminal extensions and in silico ORFs , determine their retention time, capture their fragmentation patterns, and compare these spectra to the experimentally observed small protein spectra. High-scoring matches add confidence to the initial MS identifications . Due to ico ORFs . Alternaapproach .A modification of this approach that is more sensitive but also more expensive, is based on synthetic, “heavy” isotope-labeled “AQUA” peptides . These hThere is increasing evidence that some, perhaps many, small proteins that can be detected by transcriptomic or MS-based approaches are nonfunctional, as defined by the lack of an effect on cell fitness , 114. HeB. henselae were predicted by LipoP , domainby LipoP  to contaby LipoP .per se (rather than a specific function) is an important prerequisite. Short ORFs immediately upstream of genes/operons may function to regulate downstream transcription or translation (Analysis of phylogenetic sequence conservation, including determining the ratio of synonymous to nonsynonymous SNPs between homologues, is highly recommended for prioritization of novel small proteins, since evidence of purifying selection supports a functional role , 93, 113nslation . Short OAn additional level of prioritization can be achieved by integrating differential gene and protein expression data, where available . RegulatEach of the approaches described above can provide evidence for small protein functions that can be used for prioritization. However, we recommend relying on multiple, independent lines of evidence, since any individual method is likely to generate a substantial number of false positives. It is also the case that the statistical power of analyses of sequence conservation and codon usage is strongly length-dependent. Hence, failure to identify evidence for small protein function using these methods should not be interpreted as a lack of function.The approaches described above are intended to provide a set of best practices for the detection and validation of small proteins using MS. We also discuss further bioinformatic analysis and prioritization strategies, which are critical for choosing the most promising candidates for subsequent physiological studies. MS approaches can also be used for these functional studies, and provide valuable information on the roles and functions of the novel small proteins. See , 142. MSIn summary, mass spectrometry-based proteomics approaches represent a powerful tool to identify and characterize small proteins. However, the standard workflows available in the field need to be adjusted depending on sample type and complexity, as outlined above. We anticipate that these strategies will provide an excellent starting point for exploratory studies of prokaryotic small proteins.


A. baumannii was most frequently introduced to the hospital, while the risk of colonization with VIM-encoding P. aeruginosa rose with the length of hospital stay. P. aeruginosa remains an important complication of prolonged hospital stays. The strong link to hospital-wastewater may have implications for hospital-built environments. A. baumannii can be efficiently controlled from spreading at hospital admission. OXA-encoding CEB being harder to detect in routine screening, targeted preventive measures, such as culture media selective for carbapenem-resistant bacteria, would be opportune for patients from selected regions. The CEB differences linked to ethnicity found in our study may further be supporting the tailoring of diagnostic approaches, as well as health policies upon confirmation by other studies and a better understanding of their global distribution.Antibiotic resistance represents a major global concern. The rapid spread of opportunistically pathogenic carbapenemase-encoding bacteria (CEB) requires clinicians, researchers, and policy-makers to swiftly find solutions to reduce transmission rates and the associated health burden. Epidemiological data is key to planning control measures. Our study aims to contribute by providing an analysis of 397 unique CEB isolates detected in a tertiary hospital in Germany. We propose new findings on demographic variables to support preventive sanitary precautions in routine clinical practice. Data on detected CEB was combined with patient’s demographic and clinical information for each isolate. Multiple regression techniques were applied to estimate the predictive quality of observed differences. Our findings confirm the role of age and gender in CEB colonization patterns and indicate a role for ethnicity and domicile. Also, carbapenemase-encoding  Bacteria excel at swiftly acquiring the genetic resources to thrive in environments that intend to inhibit their growth. Increasing anti-microbial resistance (AMR) continues to limit treatment options to where there may be no more cure. This growing threat to human health is a serious global concern with a significant health-economic burden. WHO considers the growing AMR issue one of the three major public health challenges of the 21st century, responsible for rising healthcare costs, extended hospital stays, treatment failures, and often death . The WorCarbapenemase-encoding bacteria (CEB) have spread worldwide . The mosEpidemiological data is of primary importance to understand the scope of the problem and to design effective control measures. We analyzed demographic and clinical data on 397 CEB detected at our University Hospital  between September 2014 and December 2019 and offer new insights on factors that may be taken into account when optimizing preventive sanitary precautions and developing safe hospital environments. The collected data included variables such as gender, age, ethnicity, residency, CEB specimen type, co-detection of CEB with other multidrug-resistant bacteria, and length of hospital stay.We analyzed retrospective data on CEB isolates that could be retrieved from the laboratory and hospital information systems of our institute, which is part of the University Hospital of Bonn, Germany (UKB). The UKB is a tertiary referral and maximum care hospital with 1,300 beds. Every year about 50,000 inpatients and 35,000 emergencies are treated, and over 350,000 outpatient procedures are provided. The UKB serves mainly German residents but also attracts patients living outside of Germany, mainly in the Arabian Peninsula. Our microbiological diagnostic unit services the University Hospital Bonn and other hospitals in the area and receives an average of 178,000 clinical samples each year. All CEB isolates from September 2014 till December 2019 were traced in the laboratory information system using species and resistance keywords by one operator. Only first isolates were selected for each pathogen–patient combination.Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE), date of hospital admission, hospitalization stay, oncological or intensive-care ward (ICU) stay, place of residency, and ethnicity. We constructed a database that was password protected and accessible by only three operators who ensured that all patient data were delinked from any results other than CEB and were fully de-identified prior to analysis after the establishment of residency and ethnicity. To infer patient ethnicity, we utilized the free version of the validated and data protection compliant software tool Onolytics . Onolytics classifies names into 189 cultural ethnic and linguistics groupings. Where available, the results were further controlled for plausibility and accuracy using additional information such as patients’ nationality.The isolate information was complemented by accessible clinical patient information including gender, age, co-colonization by methicillin-resistant All data relevant to the study are included in the article or uploaded as The ethics committee of the University Hospital Bonn confirmed that no ethics approval was required for this study.via MALDI-TOF MS  and susceptibility-tested with the VITEK 2 system . Carbapenem-resistant Enterobacterales, A. baumannii and P. aeruginosa or isolates with an unusual carbapenem-susceptibility profile (ertapenem/imipenem/meropenem) are routinely genotyped in-institute for the presence of common resistance genes, using the Allplex Entero-DR  and eazyplex SuperBug Acineto Assays .Following German guidelines, multi-resistance of gram-negative rods (MRGNs) was defined on the basis of resistance of a pathogen against three (3MRGN) or four (4MRGN) of the following antibiotic groups: acylureidopenicillins, third and fourth generation cephalosporins, carbapenems, and fluoroquinolones. At our UKB and serviced hospitals primary MRGN screening is performed on all patients who had been hospitalized abroad within the past year, on all transfers without current MRGN screening as well as on all patients that were in the same room with a patient with a 4MRGN. Screening and surveillance body sites include the anal region, the inguinal region, the throat as well as wounds if present. Surveillance after admission is ward-dependent but generally performed at least weekly. Screening and surveillance samples for CEB were routinely cultured by the laboratory on selective ESBL-media, identified e.g. age, sex, days of hospitalization, etc.) among individuals in these groups. To run the regressions we use the statistical software package Stata.We used regression techniques to assess the association between age, sex, ethnicity, place of residency, and other parameters with the occurrence and/or type of CEB colonization in patients. This allowed construction of patient and/or hospitalization driven risk profiles. First, we report the average prevalence of different types of CEB in various patient populations. Then, using multivariate regressions, we test whether the uncovered differences between groups persist when we take into account the differentials in observable characteristics  in these figures and throughout the text, refer to the significance (at the 0.05 or 0.01 level) of the point estimates obtained in the multivariate regression analysis  are shown in e.g. the prevalence of specific CEB in the patient), we also estimated the same specifications as in the multivariate analysis applying logistic models. The results of this application are shown in To test the robustness of our results, we performed some additional sensitivity analyses. Results from univariate regression analysis  of all. Among patients that were detected to carry two or more CEB, P. aeruginosa constituted only 8% (11/142) (p <.01), and Enterobacteriaceae made up more. The OXA-48- and OXA-23-encoding P. aeruginosa and OXA-23-encoding K. pneumoniae isolates that appear in our statistic all occurred in 2014 and 2015 and were not cryopreserved in a sufficiently reliable traceable manner for their validity to now be confirmed in retrospect, given their rarity; DNA contaminations can, hence, not be excluded for these six isolates.Detected species and encoded carbapenemases are displayed in Enterobacter cloacae complex ST 419 [see K. pneumoniae isolates and were detected only sporadically. Of all KPC-encoding isolates, 84% (26/31) were detected in screening and surveillance samples. Of VIM-encoding isolates, 31% (36/116) belonged to oncological and 35% (40/116) to ICU patients, making them the most frequently detected carbapenemases in both patient populations. The number of NDM carbapenemases grew steadily until 2018. ICU patients carried 26% (18/67) of the isolates, and oncological patients carried 29% (20/67). Numbers of OXA-23 carbapenemases showed only minimal variations, and only 22% (10/46) of the OXA-23-encoding bacterial strains belonged to female patients.The number of KPC, VIM, NDM, OXA-23, and OXA-48 like carbapenemases that had been detected as colonizers fluctuated each year between 2015 and 2019 (see K. pneumoniae made up 61% (85/140) of OXA-48-encoding isolates. It is known that blaOXA-48 is on an IncL-type plasmid defective for the tir gene involved in the regulation of conjugation rendering this plasmid highly conjugative. Thus it has the ability to be transferred in vivo within different members of Enterobacterales. Genetic resistance profile and MLST-type of OXA-48-encoding K. pneumoniae isolates detected between January and October 2019 are displayed in . pneumoniae isolates, encoded OXA-48-like carbapenemases in addition to other carbapenemases. Fourteen percent (19/140) of the OXA-48-encoding isolates belonged to oncological patients and 24% (34/140) to ICU patients.A. baumannii. P. aeruginosa was more evenly found in the various specimen types, and the least efficient recovery was in anal and inguinal swabs.P. aeruginosa isolates were detected in patients below the age of 25 (n = 25). Among patients aged 25–29 years, including four oncological patients, a disproportionately high number (n=8/19) was found. While there are no gender differences in the average age, we found significant differences in the occurrence of CEB isolates in male and female patients. As the results of the multivariate regressions in Citrobacter isolates (p <.05*) were significantly more common in female patients.The total and relative frequencies with which certain carbapenemases and species were detected across different age groups are shown in Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), respectively, and five of these patients with both. There were significantly less women co-colonized with MRSA, 5/34 (14.7%) , compared to co-colonization with VRE, 22/67 (32.8%) .Thirty-four and sixty-seven patients were co-colonized with methicillin-resistant Enterobacter isolates (p <.05*) and VIM enzymes (p <.01*) were more frequent among G-residents, while KPC enzymes were solely detected in patients in this group. In contrast, A. baumannii isolates (p <.05*) and OXA-23 carbapenemases (p <.05*) were more frequent among AP-residents. The ethnicity of patients had also been retrieved, as explained in the section Materials and Methods , and by K. pneumoniae than patients of Punjabi ethnicity (p <.05*). Conversely, they were more frequently colonized by Enterobacter isolates than patients of Arabic ethnicity (p <.01*). VIM enzymes were more frequently detected among patients of German ethnicity rather than among patients of Arabic (p <.05*), Somali (p <.05*), and Turkish ethnicity (p <.05*). On the other hand, patients of German ethnicity were less frequently colonized with bacteria harboring OXA-23 carbapenemases than patients of Arabic (p <.01*) and Turkish ethnicity (p <.01*) and by bacteria harboring NDM carbapenemases than patients of Somali ethnicity (p <.01*).The human microbiome is very individual and has been correlated with factors such as ethnicity and geography . Hence, hods see Table 1.Our data suggests that residency and ethnicity play a role in terms of frequency with which certain carbapenemase-encoding species were detected in patients. Hence, we performed two further analyses. Firstly, we compared isolates of patients of German ethnicity residing in Germany with isolates of patients of Arabic, Somali, Turkish, Punjabi, or Kashmiri ethnicity residing in Germany (E. coli isolates compared to patients of Arabic ethnicity residing in the Arabian Peninsula (p <.05*). Although the substantially smaller sample size challenges the consistency of these last results, they provide further suggestive evidence that confirms the significant role of ethnicity.German ethnicity patients residing in Germany were less frequently colonized by OXA-23-encoding isolates compared to Turkish (p <.05*) and Arabic ethnicity patients residing in Germany (p <.05*), by OXA-48-encoding isolates compared to Somali ethnicity patients residing in Germany (p <.05*), and by VIM-encoding isolates compared to Kashmiri ethnicity patients. At the same time, they were associated more frequently with VIM enzymes than patients of Turkish ethnicity residing in Germany (p <.05*). Patients of Arabic ethnicity residing in Germany were more frequently colonized with carbapenemase-encoding A. baumannii isolates were more frequently detected in patients on hospital admission. Carbapenemase-encoding P. aeruginosa isolates on the other hand were more frequently detected with longer LOS.In order to differentiate CEB that are frequently introduced into the hospital from those that are more likely hospital-acquired, we studied CEB detection and the length of stay in the hospital (LOS). P. aeruginosa was detected after more than four weeks LOS. The results of the multivariate analysis confirmed the existence of a statistically significant and negative relationship between the LOS and P. aeruginosa, Proteus, Serratia, Klebsiella oxytoca, and Klebsiella aerogenes as well as with production of OXA-48-like enzymes. P. aeruginosa was the most frequently isolated CEB from oncological and ICU patients. Especially oncological patients were colonized significantly more frequently by carbapenemase-encoding P. aeruginosa than by A. baumannii (p <.01).In more than half of the cases Staphylococcus aureus (MRSA), but not vancomycin-resistant Enterococcus (VRE), more frequently colonized male patients (As in other studies on multidrug-resistant gram-negative bacteria (GNB) , in our patients . The higpatients . WhetherEnterobacterales and even by P. aeruginosa. K. pneumoniae confirmed its versatility in taking up resistance genes  in screening and surveillance samples demonstrates the importance of screening and surveillance practices  to prevece genes . That P.eriaceae .A. baumannii. The likelihood with which different CEB may be detected in various specimen types should be considered when screening patients in clinical practice.We hypothesize that several factors influence the different spectrum of CEB detected in anal swabs and stool specimen. In our experience, anal swabs are frequently erroneously collected by only swabbing the superficial area surrounding the anal sphincter. Also, only stool samples of oncological patients are routinely cultured on media on which CEB grow. Among the clinical specimens that we received, inguinal swabs were the best for detecting carbapenemase-encoding The apparent decline in occurrence of CEB after the age of 70 observed in our study is solely due to the decreasing number of people in that age group. Colonization with multidrug-resistant bacteria is generally antibiotic- and healthcare associated  and hencOur data suggests that besides the place of residence, ethnicity plays a role in terms of frequency with which certain CEB are detected in patients. Given the fact that the variability in the microbial species colonizing the human body has been shown to be linked to ethnicity as well as geography , we are A. baumannii has been documented to swiftly spread within hospital environments if effective preventive measures are inadequate (P. aeruginosa is documented to predominantly infect critically ill as well as immuno-suppressed patients (P. aeruginosa isolates detected in the siphons of the oncological ward mostly belong to the rare sequence type ST823 (adequate . We theradequate . P. aerupatients  and its pe ST823 . Future Given its frequent presence in hospital-wastewater, hotspots such as the drains, traps, sinks, faucets and toilets have undergone extensive remodeling. Nevertheless, our results and previous findings of the biofilms in the wastewater networks  further P. aeruginosa among complications of prolonged hospital stays. Continued research will further elucidate the observed gender differences in the frequency of MDR-GNB colonization/infection and the various microbial spectra linked to ethnicity and residency. The way global distribution patterns of carbapenemases are seen and studied may be substantially influenced and new screening, diagnosis, and patient care may be offered. These include more effective screening for challenging resistance enzymes linked with ethnicity/residence and the awareness of the optimal specimen for the various carbapenemase-encoding bacteria. Finally, our study has shown that even when extensive safety precautions are in place, not all hospital-acquired pathogens can be equally well contained, which prompts to continuously rethink hospital-built environments and further optimize all precautions according to risk factors and the spectrum of expected pathogens.We analyzed 397 carbapenemase-encoding isolates detected during a 5 year period with the associated demographic and clinical patient information. We found evidence of a role for ethnicity in the type of CEB colonization, and confirmed the important role of The original contributions presented in the study are included in the article/CN and MP contributed to study design, data collection, data analysis, data interpretation, the literature search, and writing of the report. CB, GN, and IH contributed to data analysis, data interpretation, and writing of the report. GB contributed to the literature search, data interpretation, and writing of the report. AH contributed to data interpretation and writing of the report. All authors contributed to the article and approved the submitted version.IH was employed by the company H.I.M.A. Consulting.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adult-to-adult living donor liver transplantation with small partial liver grafts often requires intraoperative portal inflow modulation to prevent portal hyperperfusion and subsequent small-for-size syndrome (SFSS). However, there are concerns about the specific morbidity of these modulation techniques. This study aims to lower post-perfusion portal venous pressure and correct severe hypersplenism in a patient with end-stage liver cirrhosis by simultaneous subtotal splenectomy during auxiliary partial orthotopic liver transplantation (APOLT). A 29-year-old man was diagnosed with cryptogenic cirrhosis and severe portal hypertension suffered recurrent acute variceal bleeding, severe thrombocytopenia, and massive ascites before admission to our hospital. After the recipient's left liver was resected, we performed APOLT using his 51-year-old father's left lobe graft with a graft-to-recipient weight ratio of 0.55%. Intraoperatively, simultaneous subtotal splenectomy was performed to lower graft post-perfusion portal vein pressure below 15 mmHg and correct severe hypersplenism-related pancytopenia. The recipient's postoperative hospital course was uneventful with no occurrence of SFSS and procedure-related complications. Platelet and leukocyte counts remained in the normal ranges postoperatively. The living donor was discharged 6 days after the operation and recovered well-with no complications. After a follow-up period of 35.3 months, both the recipient and donor live with good liver function and overall condition. This is the first case report of simultaneous subtotal splenectomy during APOLT using small-for-size living-donated left liver lobes, which is demonstrated to be a viable procedure for modulating portal inflow and correcting severe hypersplenism in selected adult patients with end-stage liver cirrhosis. APOLT using a small-for-size liver graft may be a safe and feasible treatment option for selected adult patients with end-stage liver cirrhosis. Due to a critical shortage of donor organs, living donor liver transplantation (LDLT) has been increasingly used in adult recipients. In general, a graft ≥0.8% of the recipient's body weight or 30–40% of standard liver volume (SLV) is necessary to meet the recipient's functional demands, whereas procuring such a graft from a living donor may subject a healthy individual to a significantly high operative risk . To ensuAuxiliary partial orthotopic liver transplantation (APOLT), which is implanting a normal partial liver allograft while preserving part of the native liver, has emerged as an attractive alternative to liver transplantation (LT) for the treatment of selected patients , 4. The Post-transplant portal hyperperfusion is believed to be a primary contributor to the pathophysiology of SFSS. In this setting, intraoperative portal inflow modulation techniques, including splenectomy, splenic artery ligation, and portosystemic shunts, can be used to optimize the post-perfusion portal vein pressure (PVP) and thus prevent SFSS-related graft loss , 14. YosThe present case describes an adult patient diagnosed with cryptogenic cirrhosis and severe portal hypertension who underwent APOLT using a small volume living-donated left liver lobe. Simultaneous subtotal splenectomy was performed intraoperatively to lower graft PVP and correct severe hypersplenism-related pancytopenia.3 and white blood cell count 600/mm3). Serum biochemical examinations showed slightly elevated total bilirubin (2.0 mg/dl) and conjugated bilirubin (0.57 mg/dl), normal aspartate aminotransferase (26.6 IU/l) and alanine aminotransferase (6 IU/l), normal serum creatinine (67 μmol/l) and electrolytes  and low albumin level (2.91 g/dl). The blood ammonia level was 33 μmol/l, and the patient did not experience any episodes of hepatic encephalopathy previously. His international normalized ratio (INR) was slightly prolonged (1.49). Abdominal computed tomography (CT) showed atrophic liver, severely dilated gastroesophageal varices and short gastric veins, umbilical vein recanalization, splenomegaly, massive volume of free peritoneal fluid, and small volume of bilateral pleural effusion. The Child–Pugh classification was B (score 9), and the Model for End-Stage Liver Disease (MELD) score was 12. After a discussion between the patient and his parents and the multidisciplinary team, including radiologists, hepatologists, and liver transplant surgeons, the patient refused to undergo partial splenic embolization, surgical shunt or transjugular intrahepatic portosystemic shunt because the patient thought that abovementioned procedures were just temporary therapeutic options but not a cure, with the potential high risk of procedure-related complications including portal vein thrombosis, substantial infectious risk, and occurrence of hepatic encephalopathy, which may adversely influence his quality of life and the subsequent chance of LT. On the other hand, the indication for LT in this patient was relatively clear, namely decompensated liver cirrhosis, including portal hypertension, recurrent variceal hemorrhage, splenomegaly, hypersplenism, and moderate to severe ascites. In such a situation, considering the patient's own wish regarding the LT and the donor's safety being the priority during LDLT, as well as our transplant team's previous extensive experience with APOLT, and thus APOLT using a small-for-size living-donated left liver lobe was considered for this patient.A 29-year-old man was admitted to our hospital because of recurrent variceal bleeding. One year ago, he presented with sudden melena and was diagnosed with cryptogenic cirrhosis and severe portal hypertension at the local hospital. Since then, he had experienced seven episodes of acute variceal bleeding despite multiple endoscopic therapies  and regular administration of non-selective beta-blockers (carvedilol). Two months ago, he suffered from a hemorrhagic shock caused by gastrointestinal bleeding. On this admission, he had melena and his blood routine examination revealed moderate anemia (hemoglobin 75 g/l) and severe hypersplenism /SLV was 36.71%. Thus, APOLT using a left lobe liver graft was designed to decrease the morbidity of SFSS in the recipient. Given the presence of severe portal hypertension, hypersplenism, and massive splenomegaly, simultaneous subtotal splenectomy was planned, which served as a portal inflow modulation strategy and treatment for severe hypersplenism. The calculated volume of the whole spleen and the designed preserved upper pole of the spleen were 1,445 and 503 ml, respectively .The donor underwent a left hemihepatectomy with preservation of the middle hepatic vein, and the intraoperative course was uncomplicated. The actual graft weight was 405 g, GRWR was 0.55%, and GV/SLV was 31.23%. The recipient underwent APOLT with preservation of the native right lobe liver . Before Based on our and other centers' experience, vaccination against pneumococci and any other encapsulated bacteria was not performed before or after transplantation , 21. To Apart from massive ascites production, the postoperative hospital course was uneventful, and graft function recovered rapidly . It tookWith a critical shortage of donated organs and increased mortality for those on the waiting list, living donors have become an increasingly accepted potential alternative source of organs for adult patients with end-stage liver disease. Due to the assurance of donor's safety being the priority in LDLT, SFSGs have been used to minimize risks to donors in adult-to-adult LDLT. However, SFSGs are generally considered unsafe in terms of the risk of SFSS . NotablyNotably, the functional competition of the portal vein flow between the remnant native liver and the implanted graft is a significant concern of APOLT, leading to progressive growth of one-side conquering liver and chronic atrophy of the defeated liver , 25. In Previous studies have suggested that preoperative graft-to-spleen volume ratio (GSVR) at a ratio of <0.6 g/ml is a good predictor of the development of post-transplant portal hyperperfusion; therefore, it can be used to indicate whether splenectomy is required before reperfusion , 27. YaoMore recently, Zhou and Wei et al. performed simultaneous partial splenectomy during LT in patients with severe hypersplenism, which achieved a satisfactory long-term hematological response but avoided untoward complications of total splenectomy , 33. A rThis case suggests that simultaneous subtotal splenectomy during APOLT using small-for-size living-donated left liver lobes is not only an efficient intraoperative portal inflow modulation strategy but also a feasible treatment for severe hypersplenism in selected adult patients with end-stage liver cirrhosis. It could be a viable alternative to splenectomy, which preserves splenectomy's beneficial effects but avoids splenectomy-related lethal complications. Furthermore, APOLT using a small-for-size liver graft may be a safe and feasible therapeutic option for selected adult patients with end-stage liver cirrhosis, which can further expand the pool of donor grafts.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of Beijing Friendship Hospital, Capital Medical University (No. 2018-P2-127-01). The patients/participants provided their written informed consent to participate in this study and for the publication of this case report.Z-JZ and LW: conception and design of the research, analysis and interpretation of the data, and preformation of the operations. G-PZ: acquisition of data, statistical analysis, and writing of the initial draft of manuscript. WQ, Z-GZ, YL, and L-YS: clinical management and follow-up of the patient. All authors involved in the critical revision of the manuscript for important intellectual content and approved the final version of the manuscript.The present work was supported by the Capital's Funds for Health Improvement and Research (Grant/Award Number: 2020-1-2024) and Beijing Municipal Science and Technology Commission (No. Z211100002921026). The funding body had no role in the design of the study and collection, analysis, and interpretation of data, and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.