The aim of this study was to explore the relationship between follow-up imaging characteristics and overall survival (OS) in advanced hepatocellular carcinoma (HCC) patients under sorafenib treatment.Associations between OS and objective response (OR) by mRECIST or early tumor shrinkage  were analyzed in HCC patients treated with sorafenib within a multicenter phase II trial (SORAMIC). 115 patients were included in this substudy. The relationship between survival and OR or ETS were explored. Landmark analyses were performed according to OR at fixed time points. Cox proportional hazards models with OR and ETS as a time-dependent covariate were used to compare survival with factors known to influence OS.p < 0.001), and longer progression-free survival . Patients with ETS ≥ 20% had longer OS  and PFS  than patients with ETS < 20%. Besides OR and ETS, male gender, lower bilirubin and ALBI grade were associated with improved OS in univariate analysis. Separate models of multivariable analysis confirmed OR and ETS as independent predictors of OS.The OR rate was 29.5%. Responders had significantly better OS than non-responders (median 30.3 vs. 11.4 months; HR, 0.38 , OR according to mRECIST and ETS in patients receiving sorafenib treatment are independent prognostic factors for OS. These parameters can be used for assessment of treatment benefit and optimal treatment sequencing in patients with advanced HCC.The online version contains supplementary material available at 10.1186/s40644-021-00439-x. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the third leading cause of cancer-related death . ApproxiSorafenib is a multitarget tyrosine kinase inhibitor that interrupts tumor proliferation and angiogenesis, and was shown in the phase III SHARP trial and the Asia-Pacific trial to improve overall survival (OS) in HCC , 3. AlthAlthough patients continue to receive their assigned treatment unless disease progression is encountered at follow-up imaging, not all patients with disease control benefit equally from treatment. A retrospective analysis of HCC patients receiving sorafenib revealed that patients who had stable disease (SD) for more than three months had similar OS to patients who had an OR, while patients with SD for a shorter duration had worse outcomes, similar to patients with progressive disease (PD) .In clinical practice, early identification of patients benefitting from sorafenib treatment is crucial to avoid overtreatment, which may lead to toxicities or suboptimal treatment sequencing, especially in light of alternative treatments –15. EarlThis study aimed to evaluate the prognostic role of OR and ETS in patients receiving sorafenib therapy for the treatment of advanced HCC in a Western cohort.SORAMIC was a prospective, randomized, controlled, phase II trial comparing the effects of sorafenib monotherapy and a combination of selective internal radiation therapy (Yttrium-90 radioembolization) and sorafenib, performed in 38 centers in 12 countries in Europe and Turkey. The inclusion criteria for SORAMIC have been described previously . The maiThe present study represents a post hoc analysis of patients in the sorafenib-only arm of the palliative trial cohort. The study cohort comprised 208 patients randomized to receive sorafenib monotherapy. The study protocol was approved by the institutional review board of participating centers. Written, informed consent was obtained from all patients. The primary aim of the analysis was to explore the radiological response rate to sorafenib treatment and its correlation with OS. Criteria were OR, ETS, and depth of response (DpR).Follow-up imaging every three months was recommended, but was not a mandatory part of the SORAMIC trial. The imaging modality – computed tomography (CT) or magnetic resonance imaging (MRI) – was chosen by the local investigator. Centralized image assessment was not included in the main study. Imaging follow-up of a total of 136 patients was available for review. The following inclusion criteria were applied for this study: (1) at least one follow-up before 6 months or 6 months follow-up other than PD, (2) follow-up imaging until death or PD (last imaging within 6 months) or disease control  at 12 months after randomization, (3) minimum follow-up duration of 6 months unless PD was encountered before. A total of 115 patients were included in this study  or toxicity which required discontinuation.All patients underwent CT and MRI at baseline for study inclusion according to previously published protocols . Follow-In addition to routine response analysis according to mRECIST, progression-free survival , time to response (from randomization to first objective response), DpR , ETS (≥20% reduction in enhancing tumor diameter at the first follow-up imaging), and time to DpR (from randomization to DpR) was evaluated. For DpR and ETS assessments, only hepatic target lesions were evaluated and diameter measurements were taken according to mRECIST. In patients with disease progression, the first progression site was noted . In order to evaluate whether further subgrouping of patients translated to better survival prediction, patients with the best response of PR were dichotomized according to median DpR.http://www.r-project.org). Numerical data are presented as means with standard deviations. For categorical data, results are given as absolute numbers with percentages. For comparison of categorical data between responders and non-responders, chi-square tests were applied; for continuous data, T-tests or Mann-Whitney U tests were used for testing the homogeneity of independent samples. OS in responders and non-responders was estimated by the unweighted Kaplan-Meier method. The same analysis was repeated for patients with and without ETS. The Mantel-Byar test was used to assess statistical significance. Landmark analysis of OS by the objective response was conducted at 6 and 12 months after randomization. The log-rank test was used for the inference associated to the landmark analysis. For all variables, univariate Cox proportional hazard regression was performed as time-fixed covariates, while objective response and ETS were analyzed as a time-dependent variable. Statistically significant variables in the univariate analyses (including objective response as a time-dependent covariate) were analyzed in a multivariate Cox regression model to explore prognostic factors for OS. A separate multivariate Cox regression model was used to explore the prognostic value of ETS.All statistical analyses were performed using SAS version 9.4 for Windows  and R statistical and computing software, version 3.5.0  patients had died, with a median OS of 14.3 months. Out of 115 patients, 34 (29.5%) were responders and the remaining 81 (70.4%) were non-responders according to mRECIST. Best response during follow-up was CR in 6 (5.2%) patients, PR in 28 (24.3%) patients, SD in 50 (43.4%) patients, and PD in 31 (26.9%) patients. Median time to response was 3.8 (range 1.3–8.1) months. The baseline and clinical characteristics of patients with and without OR are shown in Table p < 0.001) . Median DpR of patients with PR was 54.4% . There was no significant OS difference between PR patients with a DpR greater than or lower than the median  in responders and 11.4 months  in non-responders . Although the difference is not significant, in none of the responders was the first progression extrahepatic .Responders also had longer PFS than non-responders  was associated with better OS, while bilirubin ≥17 μmol/L  and albumin-bilirubin (ALBI) grade 2–3  were associated with poorer OS. Multivariate Cox regression analysis revealed that OR assessed by mRECIST was an independent prognostic factor . Male gender  and ALBI grade  were also independent prognostic factors for OS . Median time from randomization to the imaging used for ETS evaluation was 2.2  months. In patients with OR, the median time to ETS was significantly shorter than the time to response . Patients with ETS ≥20% had a longer OS  and PFS  than patients with ETS < 20% . The multivariate analysis with a second model  as an independent prognostic factor, besides gender (p = 0.004) and ALBI grade (p = 0.022).Among the 115 patients, ETS ≥20% was achieved in 32 27.8%) patients. The baseline clinical characteristics of patients with ETS ≥20% compared with ETS < 20% are shown in Table .8% patieThis analysis of data from the SORAMIC trial showed that OR by mRECIST is an independent predictor of OS and PFS in patients receiving sorafenib treatment. The OR rate was 29.5%, and responders had significantly longer median OS than non-responders (30.3 vs. 11.4 months). Besides OR assessment, ETS was an independent predictive factor in our study, identifying responders to treatment earlier than assessment by mRECIST.Following the demonstration of clinical efficacy in the SHARP trial and Asia-Pacific trial, sorafenib has been the primary treatment of advanced HCC in recent years , 3. Respp < 0.001) in patients with OR after sorafenib treatment [p = 0.005), and this finding is in agreement with previous reports [Five retrospective studies used mRECIST for response assessment in patients receiving sorafenib. While four of these studies showed that OR successfully predicts OS –8, the oreatment . In this reports , 25. A s reports , 12. HowETS has been shown to predict treatment outcome in various tumor types, but, to date, only one study has evaluated ETS in patients with HCC. Takahashi et al. showed that patients with ETS ≥10% had better OS and PFS than patients with ETS < 10% after lenvatinib treatment . In thatThis study has some limitations. First, follow-up imaging was not conducted according to a standardized protocol, since follow-up imaging was at the discretion of the local investigator. However, baseline imaging of all patients was done in a standardized fashion within the diagnostic arm of the SORAMIC trial, and the images used for follow-up assessment were high-quality state-of-the-art triphasic images. Second, follow-up imaging was not available in 30.9% of the patients who received sorafenib treatment in the SORAMIC trial. There is an inherent risk of selection of responders and patients with longer survival. Patients with poorer performance status or liver function might not have undergone cross-sectional follow-up imaging due to earlier deterioration in their clinical situation. The OS of the patients included in this study was a little longer than the OS of the sorafenib arm in the SORAMIC study (14.3 vs. 11.4 months). Nevertheless, this study was conducted on patients using prospectively collected data from the phase II SORAMIC trial, and it showed, for the first time in a Western population, a correlation between OR by mRECIST and OS in HCC patients receiving sorafenib, and also, for the first time, a correlation between ETS and OS in the same cohort.OR assessments by mRECIST and ETS in HCC patients receiving sorafenib monotherapy are associated with treatment outcome and survival. Both assessments can be used to identify patients who do not benefit from sorafenib and to guide the decision-making process in the era of effective second-line therapies. However, the use of ETS needs further validation.Additional file 1: Supplementary Fig. 1. Consort diagram.Additional file 2: Supplementary Fig. 2. Examples for image analysis.Additional file 3: Supplementary Fig. 3. Overall survival of patients with partial response according to depth of response (DpR) more or less than the median DpR. CI, confidence interval; HR, hazard ratio.Additional file 4: Supplementary Fig. 4. Progression-free survival of patients with ETS ≥ 20% vs. ETS < 20%. CI, confidence interval; ETS, early tumor shrinkage; HR, hazard ratio; PFS, progression-free survival.Additional file 5: Supplementary Fig. 5. Overall survival of patients according to best response. BR, best response; CI, confidence interval; CR, complete response; HR, hazard ratio; PR, partial response; PD, progressive disease; SD, stable disease.Additional file 6: Supplementary Fig. 6. Landmark Kaplan-Meier curve as function of tumor response at 6 months. OR, objective response.Additional file 7: Supplementary Fig. 7. Landmark Kaplan-Meier curve as function of tumor response at 12 months. OR, objective response.


Respiratory syncytial virus (RSV) infects infants and children, predisposing them to development of asthma during adulthood. Epithelial neuroendocrine phenotypes may be associated with development of asthma. This study hopes to ascertain if RSV infection promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway. R language. An in vitro model was constructed with RSV infected human respiratory epithelial cells, and then real-time qPCR and immunofluorescence were used to detect the expression of different epithelial biomarkers and airway neuropeptides. The acute and chronic infection model of RSV infection was established by intranasal injection of RSV into guinea pigs. Immunohistochemistry and Western blot were used to detect the expression of pulmonary neuroendocrine cells markers ENO2 and neuropeptides.  The GSE6802 data set was obtained from the GEO database, and the differential genes were analyzed using the  The expression levels of ENO2, SP, CGRP, and NODAL/ACTRII were significantly higher in the RSV infection group than those of the control group, which were abrogated by siRNA-NODAL. In vivo, we found that the expression levels of ENO2, SP, and CGRP were significantly higher than that of the control group.  RSV promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway. Respiratory syncytial virus (RSV) is an enveloped RNA virus with a single-stranded negative chain genome. It is a common pathogen of lower respiratory tract, infecting infants and young children and comes with symptoms such as coughing, wheezing, and breathing difficulties. Infection with RSV in infancy promoted the incidence of asthma in adulthood significantly . Airway P (SP) increased significantly after RSV acute infection [Of recent, the relationship between neuropeptide-mediated neurogenic inflammation and asthma is attracting much attention in the scientific research. Neuropeptides are mainly produced by nonadrenergic noncholinergic (NANC) sensory nerves and pulmonary neuroendocrine cells (PNECs). The bronchial epithelium is composed of ciliated cells, goblet cells, basal cells, brush cells, Clara cells, and PNECs. The PNECs contain a variety of active amines and peptides, such as 5-hydroxytryptamine, calcitonin, calcitonin gene-related peptide (CGRP), and enkephalin . These snfection  . Our prnfection . RSV cannfection . Howeverβ family, which is mainly expressed in embryonic tissue and is generally not expressed in normal adult tissue [P. aeruginosa and A. baumannii, the expression of NODAL is inhibited, while RSV infection promotes the expression of NODAL. The upregulated NODAL may participate in the development of AHR by promoting the differentiation of Th2 and Th17 and inhibiting the differentiation of Th1 cells, thus inducing airway inflammation and AHR [NODAL is an important part of the TGF-t tissue . Recent t tissue , 11. Our and AHR . Further and AHR .Using bioinformatics to analyze RSV-infected BECs, we found that RSV infection can promote the expression levels of PNECs marker enolase-2 (ENO2) and NODAL/ACTRII in BECs. Going with our previous study, RSV infection promotes airway epithelial cell differentiation through NODAL/ACTRII. We opted to study whether RSV infection promotes epithelial differentiation into neuroendocrine phenotypes through the NODAL signaling pathway. This is imperative to clarify the epithelial differentiation induced by RSV infection and its related mechanisms, and it can also provide new clinical ideas for prevention and treatment of AHR.https://www.ncbi.nlm.nih.gov/geo/).The data set GSE6802 is the mRNA expression profile of BECs (BEAS-2B) treated by different microorganisms and the samples including 3 controls and 3 samples infected with RSV strain A2 (multiplicity of infection (MOI) = 1) for 4 h. R software  and bioconductor (http://www.bioconductor.org/) was used to process raw data. After the data set was calibrated and normalized, the limma package was used for screening .The data set GSE6802 was obtained from the Gene Expression Omnibus (GEO) database (2. RSV (A2 type) was stored by the Department of Microbiology, Central South University. 70% confluent of Hela cells was infected with 100 μL of RSV for 2 h at 37°C [μm filter. The collected virus was tested for 50% cell culture infectious dose (TCID50) and stored at -80°C until use. RSV infection was verified by indirect immunofluorescence (IFA) with RSV major surface glycoprotein G monoclonal antibody  as a primary antibody and FITC-conjucted antibody  as secondary antibody.Human BECs (BEAS-2B) and Hela were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37°C, and 5% CO at 37°C , 15, and6 cells per well and cultured to 30-50% confluence. Then, a final concentration of 20 μM siRNAs was transfected into cells for 48 h by riboFECT CP Transfection Kit (166 T), and then the medium to antibiotic-free medium BEGM was changed. Subsequently, the cells were infected with RSV at MOI of 0.2. After 2 h of RSV adsorption, the viral suspension was removed, cells washed twice, and the cells were then cultured in a fresh medium containing 2% FBS.The knockdown of NODAL in BECs was achieved by transfection of NODAL siRNA, and control siRNA was purchased from Ribobio . Seed cells were inoculated into 6-well plates at 10n = 12) and RSV group (n = 12). After anesthetizing the guinea pigs with ether, 106 pfu of RSV in 100 μl was intranasally inoculated. The guinea pigs were sacrificed on days 7 and 28 of postinfection with 6 guinea pigs from each group. RSV infection was assayed using IFA.One-month-old female guinea pigs weighing 150–200 g  were kept in a pathogen-free environment. Four healthy guinea pigs were randomly selected and executed, and their lungs were taken for follow-up studies. The remaining guinea pigs were then randomly divided into the control group , and then the cDNA was synthesized by reverse transcription and amplified using a qPCR kit  according to the manufacturer's instructions. qPCR was performed at 94°C for 3 min and 35 cycles of 94°C for 45 s, 51°C for 45 s, and 72°C for 45 s. GAPDH was used for normalization, and relative quantity of the mRNA expression was calculated by using the 2ΔΔCT− method.Primers were designed using the Primer Premier 5.0 program  and syntμm paraffin sections with HE, and observed the pathological morphology under an optical microscope.The left lung tissues taken from each animal were fixed in 10% formaldehyde solution for 24 h and then embedded in paraffin wax blocks. We stained 5 The BECs were fixed by 95% ethanol and 0.1% Triton-X100. The samples were then blocked with normal goat serum for 20 min and incubated overnight with SP , VIP , or CGRP  rabbit primary antibody at 4°C. Then, the samples were incubated with a FITC-conjugated goat anti-rabbit secondary antibody  at room temperature for 1 h. After counterstained with DAPI for 10 min, the samples were observed with a laser scanning confocal microscopy. Images were acquired with ×40 objectives using a digital camera. The integral optical density values (IOD) of the VIP, SP, and CGRP protein were measured by Image-Pro Plus 6.0.μg protein sample was obtained for 12-15% SDS-PAGE electrophoresis and transferred onto the PVDF membrane and then probed with rabbit anti-ENO2 , Rabbit anti-SP , Rabbit anti-VIP , or Rabbit anti-CGRP  primary antibodies overnight at 4×C. The secondary antibody of horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin G (IgG)  was diluted to 1 : 3,000. The signals were detected using ECL . The bands were visualized with the chemiluminescence detection system , and the protein expression levels were normalized to GAPDH levels. The integrated density of proteins was quantified using ImageJ software (NIH).Lung tissue and cellular protein were extracted using RIPA buffer  with 0.1% PMSF, lysed on ice for 30 min, and centrifuged at 12,000 r for 10 min. The protein was quantified using a BCA Kit, and a 30 2O2 at room temperature for 5-10 min to inactivate endogenous enzymes. After heating the 0.01 M sodium citrate buffer (pH 6.0) to about 95×C, brought the sections to a boil in it for 20 min, and the blocking solution was added at room temperature for 20 min. The sample was incubated overnight at 4×C with rabbit anti-ENO2 polyclonal antibody , then incubated with goat anti-rabbit IgG antibody and streptavidin-POD working solution in turn. Subsequently, the slides were stained with DAB staining solution for 15 min, counterstained with hematoxylin, and then observed under a light microscope. Images were acquired with ×10 objectives using a digital camera. The IOD of the ENO2 protein was measured by Image-Pro Plus 6.0.Following routine deparaffin and hydration, sections were treated with 3% Ht-test was used for comparison between two groups, two-way ANOVA was used for comparison among multiple groups, and LSD was used for posthoc test. p < 0.01 was considered significant.SPSS21.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation. From the data set GSE6802, we screened 295 significantly different genes, of which 224 were upregulated and 71 were downregulated , and theIn order to detect the expression of NODAL signaling pathway, we infected BECs with RSV for 48 h and confirmed the infection using IFA. Compared with uninfected cells, green fluorescence was seen in the BECs . Then, wNext, we investigated whether the differentiation of PNECs induced by RSV infection was related to NODAL signaling molecules. The results of qRT-PCR showed that when compared with the siRNA-NC group, the expression levels of ENO2 mRNA in the siRNA-NODAL group were significantly reduced . The resThen, we examined the neuropeptide expression in cells after RSV-infected BECs. The results of qRT-PCR showed that the mRNA expression of SP and CGRP increased. However, when we interfered BECs with siRNA-NODAL, prior to RSV infection for 48 h, the results showed that the mRNA of SP and CGRP was reduced, while the mRNA level of VIP increased after NODAL knockdown. This indicates that RSV can change the expression levels of intracellular neuropeptides through the NODAL signaling pathway and promote the release of excitatory neurotransmitters SP and CGRP . In addiThe effect of RSV infection in the lungs of guinea pigs was detected with IFA, and the RSV group showed significant green fluorescence compared to the control group . DiffereIn RSV-infected guinea pigs, the expression levels of NODAL and ACTRII in the lung tissue were upregulated . The mRNAsthma is a disease that confers a public health threat over years. It is accompanied by recurrent wheezing, coughing, chest tightness, shortness of breath and other symptoms, even reversible expiratory airflow limitation . Of receγ-aminobutyric acid, thereby exacerbating allergic asthma [RSV infection can cause airway mucosal damage, increased mucus secretion, decreased ciliated cells inflammatory cell infiltration, andchanges in the ability of epithelial cells to differentiate and even affect the brain function . With thc asthma . As an ic asthma  found thAt present, the molecular biological mechanism of RSV infection affecting epithelial population differentiation and resulting in pathophysiological changes of airways is still unclear. To this effect, we analyzed RSV-infected BEC samples through bioinformatics and found that RSV infection can promote the expression of PNECs markers ENO2, NODAL, and its ACTRII receptor. Further studies verified that the expression levels of ENO2 and NODAL/ACTRII were significantly higher in the RSV infection group than that of the control group, which was abrogated by siRNA-NODAL. Moreover, the neuropeptide SP and CGRP increased in the RSV group and downregulated after siRNA-NODAL treatment. This further illustrates that RSV infection may promote BECs to differentiate into neuroendocrine phenotypes and subsequent neural network remodeling through the NODAL/ACTRII signaling pathway.Next, the acute (7 d) and chronic 28 d) infection model of RSV infection was established by intranasal injection of RSV into guinea pigs  d infect. We founIn future experiments, we will continue to study the mechanism by which RSV infection may promote neural network remodeling through the NODAL/ACTRII signaling pathway. In vitro, the constructed NODAL overexpression vector will be used to study further, and the mechanism of RSV induced neuronal differentiation. In vivo, we will construct the NODAL gene knockout animal model to detect the expression changes of PNECs markers ENO2 and neuropeptide SP, CGRP, and VIP in the lungs of RSV-infected animals.In conclusion, the above studies suggested that RSV can promote the differentiation of BECs into neuroendocrine phenotype through NODAL signaling pathway and thus may contribute in the development of asthma.


Mycobacterium leprae, can significantly improve our understanding of the disease’s complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period.Hansen’s disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, M. leprae genomes to further investigate M. leprae’s genetic variation in Europe, with a dedicated focus on bacterial genomes from previously unstudied regions , from multiple sites in a single region , and from two Iberian leprosaria. Overall, our data confirm the existence of similar phylogeographic patterns across Europe, including high diversity in leprosaria. Further, we identified a new genotype in Belarus. By doubling the number of complete ancient M. leprae genomes, our results improve our knowledge of the past phylogeography of M. leprae and reveal a particularly high M. leprae diversity in European medieval leprosaria.Here, we reconstructed 19 ancient M. leprae, we gained first insights into the disease’s global history in relation to major historic events such as the Roman expansion or the beginning of the regular transatlantic long distance trade. In summary, our findings highlight how studying ancient M. leprae genomes worldwide improves our understanding of leprosy’s global history and can contribute to current models of M. leprae’s worldwide dissemination, including interspecies transmissions.Our findings allow us to detect similar patterns of strain diversity across Europe with branch 3 as the most common branch and the leprosaria as centers for high diversity. The higher resolution of our phylogeny tree also refined our understanding of the interspecies transfer between red squirrels and humans pointing to a late antique/early medieval transmission. Furthermore, with our new estimates on the past population diversity of The online version contains supplementary material available at 10.1186/s12915-021-01120-2. Mycobacterium leprae or Mycobacterium lepromatosis, is one of the oldest recorded diseases known to humankind. Its notoriety relates both to its potential to cause extreme physical manifestations of infection, which include damage to the peripheral nervous system, mucosal membranes, skin, and ultimately the extremities [Hansen’s disease (leprosy), caused by infection with remities , and itsremities , 3. The remities . More reremities  from theremities . This geremities , althougremities . It was remities .Geospatial analyses of archeological skeletons of individuals with Hansen’s disease have done little to change this narrative. Until recently, the earliest individuals with evidence for infection have been dated to the 2nd millennium BCE in India , fourth Archeological and historical sources all demonstrate that Hansen’s disease was widespread in Europe by the Middle Ages, being increasingly identified from the Roman and early Medieval periods (200 BCE–600 CE). The period from 1000 to 1300 CE saw the increased foundation of leprosaria across the continent , 22. TheFor as yet unclear reasons, the disease prevalence began to decline in Europe from the fourteenth century, although pockets of infection remained until the nineteenth century, e.g., in Scandinavia  or even M. leprae’s evolutionary history benefits from the genetic investigation of archeological human remains (skeletons or preserved bodies such as mummies). Initially, PCR-based analyses identified four major SNP (single-nucleotide polymorphism) types from 1 to 4, which allowed an assessment of the phylogeographic distribution of archeological evidence within a framework of modern strain distribution [M. leprae, the distribution of SNP types correlated with geographic location and could largely be explained by major population movements [M. leprae from archeological human samples were reconstructed in 2013 [M. leprae genomes into five major branches (0-4) [M. leprae genomes [M. leprae studies [The understanding of ribution . Later, ribution . At thisovements , 43. Priovements . Furtherovements . Due to  in 2013 . Investies (0-4) . Later s genomes , 46 impr genomes –47. BothM. leprae DNA strains made it clear that there is no longer a simple correlation between the diverse bacterial genomes and their geographic origins. Hence, questions concerning the origin of leprosy are still unresolved. Ancient DNA studies have uncovered a high genetic diversity of M. leprae strains in medieval Europe [M. leprae strains had spread worldwide from the European continent. For example, it is assumed that Europeans spread branch 3 genomes to the Americas in the sixteenth century and later, through contact via the slave trade [M. leprae seen today, including the most basal (branch 0) currently associated with modern East Asian samples [M. leprae genome outside Europe, does not further refine the localization of leprosy’s origin [M. leprae in modern red squirrels in Britain [Genome-wide analyses of ancient and modern l Europe , 45, revve trade , where tve trade . More in samples , were prs origin . Further Britain , genetic Britain . In addiM. leprae genomes which were also suitable for phylogenetic analysis, doubling the number of published ancient genomes, and to identify a new SNP subtype, named 3Q, in Belarus. The analysis of these new genomes supports the previous findings of high genetic diversity in medieval leprosy strains across Europe and indicates that this was also the case in at least one other medieval leprosarium site, the Hospital of Sant Llàtzer (Barcelona).Here, we address these gaps by examining 41 individuals including 39 with osteological or historical contextual evidence of Hansen’s disease, and two were exclusively identified as positive for leprosy by genetic examination. These individuals originated from 20 archeological sites across Europe dating from the sixth to the twentieth centuries CE including areas for which previously no genome-wide data existed: Belarus, Iberia, Russia, and Scotland. To further assess intra-regional strain diversity, we investigated skeletons from multiple sites of the same region  and the remains of people buried in two leprosaria in Portugal and Spain. We were able to reconstruct 19 Samples from a total of 41 individuals were investigated in this study  individuals from ten archeological sites, spanning the period of the sixth–seventh centuries CE to the twentieth century  and Denmark (Jorgen507) presented in a previous study [We combined the 19 new genomes with 177 published modern and ancient  genomes , 116–121hylogeny . Four Ibhylogeny –111. Thius study . None ofus study  and Bodyus study , are divus study –111, excIn total, 12 of our 19 (63.2%) genomes position on branch 3  and branch off basally to strains isolated from modern British red squirrels (Table The Bergen sample forms a sister branch to the previously published Jorgen625 (Denmark) strain . Two M. M. leprae genomes exclusively  and UF800  are placed in branch 2F , Bergen (n = 2), CHRY023 (n = 3), CHRY044 (n = 6), EDI006 (n = 1), JDS097 (n = 2), Kirk Hill (n = 5), PAVd’09_I.5 (n = 2), COR_XVIII (n = 5), R7546-671 (n = 4), UF101 (n = 8), UF21 (n = 1), UF25 (n = 6), UF700 (n = 5), UF703 (n = 2), UF800 (n = 7), UF803 (n = 1), and UF8 (n = 3); details of these unique SNPs can be found in Additional file mce) operons, secretion system, and cell surface components  was acquired by all modern strains . Indel analysis was not performed due to limited genome quality.Using SnpEff , a rangergen n = , CHRY023I006 n = , JDS097 M. leprae strains isolated from red squirrels [M. leprae genomes reconstructed from two medieval Barcelona individuals (UF101 and UF700). Therefore, we investigated further into details of SNP differences and similarities of these M. leprae genomes. The modern M. leprae genomes from red squirrels differ from each other by zero to three nucleotides (0–0.11%). The two medieval M. leprae genomes from the individuals UF101 and UF700 from Barcelona differ in 23 nucleotides (0.82%). When comparing either the M. leprae genome from UF101 or UF700 to the M. leprae genomes from red squirrels, they are different at 41–45 positions (1.47 to 1.61%). Furthermore, the two medieval M. leprae genomes isolated human individuals and the five modern M. leprae strains from red squirrels share three specific SNPs: a synonymous SNP at position 1348383, and two non-synonymous SNPs at the positions 2271752 and 2495453. The T-to-G SNP at position 2271752 is coding for “rpoC” gene and calling glycine instead of valine, the G-to-T SNP at position is coding for the gene “mntH” and here the amino acid serine is called instead of alanine.Finally, we analyzed the specific SNPs to branch 3—the branch in which most of our genomes fall—in comparison to the other branches. We found 16 SNPs including 50% of missense mutations  starting around 250 CE (± 250 years) and 1600 CE (± ca. 100 years), which potentially tie in with major changes in human connectivity  that circulate within single locations, in this case medieval leprosaria , where three different strains were found in individuals from a similar time frame . TherefoAs not all individuals with Hansen’s disease were buried in leprosarium contexts, and to assess circulating strains in one location over a long period of time, we also investigated the region of Cambridge. Here, we have the highest number of genomes from one geographical area from non-leprosarium contexts; two early Anglo-Saxon individuals that date prior to the widespread foundation of leprosaria in England , and two from the tenth–twelfth century  and one of thirteenth century date  when leprosaria are known to exist in this region of England . InteresAlthough it is necessary to study a greater number of individuals from non-leprosarium contexts from the same location, the high strain diversity found in the leprosarium at Barcelona raises interesting questions about the nature of leprosarium sites and whether individuals entering some of these institutions originated from diverse locations. It is thought that most leprosaria were founded to serve local people but some possibly also admitted the “wandering leprous,” meaning individuals with no accommodation alternatives , 127, whQuestions surrounding the importance of pilgrimage in the spread of the disease have already been raised through the identification of the “Pilgrim Burial” at Winchester and the mobility of people with the disease might have been underestimated in the past . BarceloM. leprae genome  which sits in branch 4 basal to the Body188 genome from the Czech Republic , might raledonia ) and mosaledonia –45, 49. 1th CE whaledonia , 38. ThiM. leprae strain presented here. Historic reports confirm the prevalence of Hansen’s disease in Russia in the late nineteenth to early twentieth century [Interestingly, we observe a close phylogenetic relationship between the nineteenth and twentieth century strain from Russia to those in modern Ethiopia   in Jena, and the University of Tartu. All laboratories fulfil the requirements for ancient DNA research , 150. ToIn this study, we produced double-stranded non-UDG and UDG-treated, as well as single-stranded DNA libraries , or the Institute of Genomics Core Facility at the University of Tartu (UTIG).M. leprae reads using the EAGER pipeline [M. leprae genome, the complete ancient genome was reconstructed using software integrated into the EAGER pipeline [For a detailed description of the downstream analysis, see Additional file pipeline . For thopipeline   through time (Fig. Ne is not an estimate of the actual census size of the population, it should well reflect the relative changes in the number of transmitted bacteria.Only the high-quality samples with a minimum 3-fold coverage of at least 60% of the genomic sites were used for Bayesian time-aware phylogeny and past population dynamics inference with BEAST  (AdditioAdditional file 1: Supplementary Information Supplementary Note 1 Archaeological information for the sites and samples. Radiocarbon dates. Supplementary Note 2 Radiocarbon Dating Supplemetary Note 3 Applied Methods. Fig. S1. Sample Processing and Genome-wide analyses Fig. S2. Combined damage profiles. Fig. S3. Phylogenetic trees of 1-fold covered genomes. Fig. S4. Phylogenetic trees of 3-fold covered genomes. Fig. S5. Bayesian Maximum Clade Credibility time-aware tree for the leprosy genomes. Fig. S6. Date Randomization Test for the M. leprae dataset. Fig. S7. TempEst analysis for the M. leprae dataset. Table S1. Sample information. Table S2. Eager Report. Table S3. SNP subtyping. Table S6. Unique SNPs and virulence factors.Additional file 2: Table S4. Result table of the SNP effect analysis.Additional file 3: Table S5. SNP distance matrix.


Cladophora) and epiphytic diatoms (bSi). Offshore, dSi increased about 1 μM per year from 25 μM in the late 1980’s to nearly 40 μM in 2019. Nearshore dSi fluctuated dramatically annually, from near zero to concentrations similar to offshore. Both Cladophora and its epiphytes contained significant bSi, reaching up to 30% of dry mass (300 mg Si g dry mass-1) of the assemblage in summer. Microscopic analyses including localization with a Si-specific-stain and X-ray microanalysis showed bSi in epiphytic diatom cells walls, but the nature and localization of Si in macroalgae remained unclear. A simple model was developed estimating Si demand of algae using the areal macroalgal biomass, growth rates inferred from P-content, and bSi content, and comparing Si demand with dSi available in the water column. This indicated that 7–70% of the dSi in water overlying nearshore benthic algal beds could be removed per day. Key elements of the Si cycle, including which organisms sequester bSi and how rapidly Si is recycled, remain unclear. This work has implications for coastal marine waters where large macroalgal biomass accumulates but bSi content is virtually unknown.Si cycling is linked with processes from global carbon sequestration to community composition and is especially important in aquatic ecosystems. Lake Michigan has seen dramatic fluctuations in dissolved silica (dSi) over several decades, which have been examined in the context of planktonic processes (diatom blooms), but the role of benthic organisms  in Si cycling have not been explored. To assess significance of nearshore benthic algae in Si dynamics, we assembled dSi data from an offshore site sampled since the late 1980’s, and sampled off three Milwaukee beaches during 2005–19. Using colorimetric assays and alkaline digestion, we measured dSi, biogenic silica in particulate suspended material (pSi) and biogenic silica in benthic macroalgae ( The in situ growth rate of benthic algal assemblages (d-1) was calculated using the measured P content of samples using a Droop-type relationship between growth rate and P content published for Lake Huron Cladophora by Auer and Canale ([2) were derived from aerial photographs , with nearshore area constrained by the 10 m depth contour, above which Cladophora growth is most abundant [2) was broken into depth contours 0–5 m and 5–10 m, by the mean depth in each area (i.e. 2.5 m or 7.5 m) and this was multiplied by the average dSi (10 μM) over the summer 2006 period. By dividing the dSi available by the daily Si demand, we calculated the potential proportion of dSi that could be used by the benthic algal assemblages and expressed this as a percentage.To assess potential significance of benthic algae in nearshore Si cycling, the Si demand represented by the biomass was estimated with a simple model based on data collected in 2005–6. First, we estimated benthic algal Si demand. The average Cladophora-epiphyte biomass .abundant , 25. TheData were analyzed using General Linear Model procedures, typically linear regression and Analysis of Variance, using logarithmic transformations when necessary to meet assumption of normality and homoscedasticity , dSi within the upper 60 m has increased from the period 1988–91, when typical dSi was on the order of 10 μM and maximum values were 20–25 μM, to during 2007–9 and 2018–9 when dSi averages close to 30 μM and values up to 40 μm have been recorded  -1 (i.e. over 30% of dry mass) in late July—August at Atwater Beach . In the years 2005–2011, bSi values were higher in summer than other seasons (P < 0.05), but not significantly different among winter, spring and fall, and also significantly higher at the Atwater site versus the Linwood or Bradford Beach sites (P < 0.05). These differences observed in bSi among seasons and sites in 2005–11, did not hold in 2018 and 2019 (P > 0.05 in both cases). Although we have no objective measures of changing Cladophora growth or appearance on beaches over the course of this study, subjectively, beach accumulation was considerably worse in the earlier years, and this is reflected in declining frequency of Google searches by the public for information on “Cladophora” over the period  show modest average dSi concentrations of 245.1 (± 143.1) μM over the period 1980 to 2009, with no clear evidence of temporal trends, and no correlation between river discharge volume and dSi measured at nearshore sites. However, we have not adequately assessed bSi in river inputs because such measurements have rarely been taken. Riverine bSi is dominantly from terrestrial vegetation, and there is evidence that this plays a role in global silica cycling equivalent to that of oceanic diatoms [Increases in nearshore dSi are most likely driven by upwelling from deep lake waters; the frequency and magnitude appear to be consistent with patterns of measured and modelled upwelling . River i diatoms ; river bCladophora typically showed dense epiphyte loads, dominated by diatoms  present -1, while Cladophora with low epiphyte load had just 9.61 ± 1.79 mg Si (g dry mass)-1. Malkin et al. [Cladophora-epiphyte assemblages was similar to the present study: 56–224 mg Si (g dry mass)-1, they also reported cultured Cladophora (without epiphytes) had only 4 mg Si (g dry mass)-1. Combining our summer estimates of bSi in the Cladophora-epiphyte assemblage, our P content measured in benthic samples in 2006 (see above), and average C and N contents determined on Cladophora samples collected in 2005–6, we can approximate the molar C:N:P:Si stoichiometry of the assemblage as 331:21:1:62. Previous Lake Michigan Si budgets have not included these benthic algal assemblages [ diatoms . Species present . Element present , and epiilaments . PDMPO sn et al.  publisheemblages  and theyCladophora itself clearly contains some bSi, though its structural or physiological roles are unclear. Moore and Traquair [Cladophora was promoted by Si and inhibited by the silicate analog GeO2 and they speculated that electron dense areas in Cladophora cell walls might be sites of Si deposition. It is not especially surprising that Cladophora might contain bSi because it in the evolutionary lineage leading to higher plants and many angiosperms, including freshwater macrophytes, take up dSi [Elodea, Potamogeton and Myriophylum species use bSi structurally and contain 2–70 mg Si (g dry mass)-1 -1 -1 -1 , the neass m-2, . Howeveractivity , nutrienactivity ), benthiactivity . Furtheractivity . The criactivity , but theDiporeia in digesting diatom frustules, but it is also worth noting that Diporeia has significantly declined in Lake Michigan following the invasion by Dressenid mussels [In both the open lake and nearshore, recycling of bSi is a particularly critical component of Si budgets, yet our understanding is dated and fragmented. In open Lake Michigan waters, 80–100% of bSi may be recycled annually , 54, 55  mussels , 60. Alt mussels . There i mussels . Since t mussels , invasio mussels .In conclusion, we have shown that benthic macroalgae and their epiphytes constitute a significant pool of bSi in nearshore Lake Michigan with potentially significant effects on dSi, but many key elements of the nearshore silica cycle such as rates of recycling of algal bSi pools remain poorly understood. Our work also has implications for coastal marine waters where large biomasses of macroalgae accumulate but bSi pools within this biomass, or contribution to Si cycling is virtually unknown.S1 FigAverage counts of diatoms in samples collected June through August at two Lake Michigan stations at 100 m depth. 1985–8 data from Sandgren and Lehman , 2008 data from Simmons et al. .(PDF)Click here for additional data file.S2 Figtrends.google.com). Mean water level in Lake Michigan from US Army Corp of Engineers (www.lre.usace.army.mil/Missions/Great-Lakes-Information/Great-Lakes-Information-2/Water-Level-Data/).Relative popularity of Google searches for the term “Cladophora” in Wisconsin, 2004 through 2019 by month, assessed with Google Trends ((PDF)Click here for additional data file.S3 Figre.ssec.wisc.edu/?products=WICoast.100&center=43.086,-87.864&zoom=13) and matching geo-referenced charts with depth contours  for three sites near Milwaukee, Wisconsin. Regions outlined in red represent the extent of benthic Cladophora distributions selected down to the 10 m depth contour.Aerial photographs Click here for additional data file.


We examined the relationships between pattern electroretinogram and optical coherence tomography derived optic nerve head measurements, after controlling for disc area.  Thirty-two eyes from 20 subjects with preperimetric glaucoma underwent pattern electroretinogram and optical coherence tomography. Pattern electroretinogram parameters  and optic nerve head measurements  were analyzed after controlling for disc area. r ≥ 0.503, p ≤ 0.004). All pattern electroretinogram parameters significantly correlated with Bruch's membrane opening-minimum rim width sectors—temporal superior and nasal inferior —and retinal nerve fiber layer sectors—superior, nasal superior, and inferior . Magnitude and MagnitudeD explained an additional 26.8% and 25.2% of variance in rim area , p=0.003, and B = 0.160 , p=0.004), respectively. MagnitudeD and MagnitudeD/Magnitude ratio explained an additional 13.4% and 12.8% of the variance in Bruch's membrane opening-minimum rim width global , p=0.035), respectively. All Bruch's membrane opening-minimum rim width sectors and retinal nerve fiber layer sectors  were significantly correlated with rim area .  Magnitude and MagnitudeD were significantly associated with rim area ( PERG abnormalities can predict rim area loss in preperimetric glaucoma after controlling for disc area. We recommend controlling for disc area to increase diagnostic accuracy in early glaucoma. Glaucoma, a disease of progressive optic neuropathy, features optic disc cupping and consequent visual field (VF) deficits . Due to Structural change in the optic nerve precedes VF change,  and at lA morphologic target in diagnosis of glaucoma, the neuroretinal rim (NRR) area is an intrapapillary reflection of the amount of optic nerve fibers . The corFollowing the above discussion on the importance of ONH morphology in glaucoma diagnosis and the impact of taking disc size into consideration when assessing ONH morphology, we aimed to examine the relationships between PERG parameters and ONH morphologic measures, after accounting for disc area variability in PPG participants.® NOVA PERG , and OCT testing using Cirrus  and SPECTRALIS® OCT . In this study, only scans with a signal strength greater than 7 were used, as per Cirrus HD-OCT recommendations  [Twenty PPG subjects (32 untreated eyes) with normal Humphrey 24-2 VF tests and suspicious ONH were consecutively recruited at Manhattan Eye, Ear and Throat Hospital. In this cross-sectional study, participants underwent a complete comprehensive ophthalmologic examination, PERG tests using Diopsysngth ≥6) . OCT scaD, and cylinder correction within 3.0 D were included. Using HFA 24-2 SITA-standard test, only participants with stage 0 (no VF losses) based on the Glaucoma Staging System (GSS 2) were enrolled in this study [PPG participants were recruited according to the following criteria: the presence of a glaucomatous ONH appearance  and a normal Humphrey Field Analyzer (HFA) 24-2 Swedish Interactive Thresholding Algorithm (SITA) standard test at the baseline visit. Participants within 18–80 years of age with best corrected visual acuity better than or equal to 20/40, spherical refraction within ±6.0 ® NOVA-ss-PERG Contrast Sensitivity module. The test was performed in a dark room to standardize environment luminance—free of visual and audible distractions. The patient's seat height was adjusted so the tested eye stayed in a horizontal plane with the center of the monitor. The forehead skin was cleaned using NuPerp® Skin Prep Gel  and the lower eyelids, using OCuSOFT® Lid Scrub Original  to ensure good and stable electrical activity. Disposable hypoallergenic skin sensors Silver/Silver Chloride ink (Diopsys® proprietary skin sensor) were applied on the lower lids of both eyes, close to the lid margins while avoiding eyelashes. One ground sensor (Diopsys® EEG electrode) was applied in the central forehead area with a small amount of conductive paste . Then, cables from the Diopsys® NOVA ss-PERG device were connected to the electrodes. A total of 3 electrodes were used per test per patient (two active/reference and one ground electrodes). Subjects were fitted with the appropriate correction for a viewing distance of 24 inches and were instructed to fixate on a target at the center of the monitor in front of them. An occluding lens was inserted into the trial lens to cover the eye that was not being tested. No pupil dilation was needed. Subjects were asked to blink freely. However, if more than 4 artifacts were recorded over one 25-second test period, subjects were subsequently instructed to reduce blinking frequency and eye lubricants were offered when needed.The steady state (ss-PERG) was recorded using a commercially available system, Diopsys2 and the luminance for black was 20.5 cd/m2 and 52.5 cd/m2 yielding a mean luminance of 112.3 cd/m2 and 128.2 cd/m2, respectively. All recorded signals underwent band filtration (0.5–100 Hz) and amplification  and averaged at least 150 frames. The signal was sampled at 1920 samples per second by an analog to digital (A/D) converter. The voltage range of the (A/D) converter was programmed between −5 V and +5 V. Sweeps contaminated by eye blinks or gross eye saccades were rejected automatically over a threshold voltage of 50 μV, and these sections were identified as “artifacts” in the report. Synchronized single-channel electroretinograms (ERGs) were recorded, generating a time series of 384 data points per analysis frame (200 ms). An automatic fast Fourier transformation (FFT) was applied to the PERG waveforms to isolate the desired component at 15 rps. Other frequencies, such as those originating from eye muscles, were rejected. The ss-PERG test results were saved in a Structured Query Language (SQL) database and presented in a report form to be used for analysis. For every subject, four preprogrammed full “contrast sensitivity protocols” were performed one after another. A “contrast sensitivity protocol” consisted of two 25-second recordings for each eye: first with high contrast (85%) diffuse retinal stimulation and then with low contrast (75%) pattern stimulation. The device collects 5 frames of data per second, totaling 125 frames of data, and the first 10 frames (2 seconds) of data are discarded. A result was categorized as nonreliable if there were more than 4 artifacts.The stimulus was presented on a gamma corrected Acer V176BM 17-inch monitor, having a refresh rate of 75 frames/second. Luminance output over time was verified using a luminance meter MAVO-SPOT 2 USB . The pattern stimulus consisted of black/white alternating square bars, reversing at 15 reversals/second (rps) with a duration of 25 seconds for high contrast (HC 85%) and 25 seconds for low contrast (LC 75%) for a total of 50 seconds per eye. The stimulus field subtends a visual angle of 1439.90 arc minutes. Each bar will subtend 22.49 arc minutes, for a total of 64 bars. A red target subtending 50.79 arc minutes was used as a fixation target and was centered on the stimulus field. The luminance of the white bars for 85% and 75% contrast was 204 cd/mµV) represents the amplitude of the signal strength at the specific reversal rate of 15 Hz in the frequency domain, while MagD (µV) represents an adjusted amplitude of the PERG signal impacted by phase variability throughout the waveform recording. A recording where the phase of the response is consistent will produce a MagD value close to that of Mag, whereas a recording where the phase of the response varies will produce a MagD value lower than that of Mag. This is due to the fact that averaging responses which are out-of-phase with each other will cause some degree of cancellation. The MagD/Mag ratio is a ratio that is a within-subject representation of the phase consistency of ss-PERG. The SNR represents the level of electrical noise compared with the level of the PERG signal at 15 Hz.For each eye, three PERG measurements (Magnitude (Mag), MagnitudeD (MagD), and MagD/Mag ratio) were collected and calculated, as well as the number of artifacts and signal to noise ratio (SNR). Mag (The Glaucoma Module Premium Edition (GMPE) software was used for the SPECTRALIS® Spectral Domain (SD) OCT . It measures the minimum distance between the internal limiting membrane and Bruch's membrane opening around the optic nerve head, also known as BMO-MRW, which increases accuracy of the anatomic disc margin and the NRR measurements. This software uses the center of the BMO-MRW and of the fovea as fixed landmarks in creation of an anatomic map of the patient's eye, allowing higher sensitivity for structural change . Cirrus®Descriptive statistics were used to evaluate continuous and demographic data. Mean and standard deviations values were determined for each ss-PERG , HFA SITA-Standard (24-2) tests, and all ONH OCT-based measurements.A Pearson correlation analysis was conducted between PERG parameters  and ONH measurements . Partial correlation analysis was conducted after controlling for disc area. In the prediction of the rim area change, a hierarchical linear regression was used, where the disc area was entered in step 1 of the model, and Mag was entered in step 2. An identical model was used by replacing Mag with MagD and subsequently with MagD/Mag ratio in step 2.In the prediction of BMO-MRW global, an identical hierarchical model was used, after controlling for disc area (step 1). PERG parameters were entered one by one in step 2.Statistical analyses were performed with commercially available software .Thirty-two eyes (20 patients) with PPG were initially recruited. All 32 eyes had testing with PERG and Cirrus OCT. 28 eyes additionally had testing with SPECTRALIS® OCT . The characteristics of the study population are summarized in Tables r ≥ 0.508, p=0.003), rim area , and average C/D ratio . No significant correlations were found between PERG parameters and vertical C/D ratio as well as cup volume. After controlling for disc area, Mag and MagD remained significantly correlated with rim area . No significant correlation was found between PERG parameters and average C/D ratio, vertical C/D ratio, and cup volume , p=0.003). When Mag was replaced by MagD in the same model, MagD (step 2) explained an additional 25.2% of variance in rim area , p=0.004)  and disc area (p volume . In two p=0.004) . MagD/Mar ≥ 0.431, p ≤ 0.025) and temporal superior (TS) . Mag and MagD were significantly correlated with the temporal (T) sector . MagD was significantly correlated with BMO-MRW global , NI , T , and TS  sectors. MagD/Mag ratio was significantly correlated with BMO-MRW global , NI , nasal superior (NS) sectors , and TS  , p=0.031). After replacing MagD by MagD/Mag ratio, the ratio (step 2) explained an additional 12.8% of variance , p=0.035)  (p=0.034) . In the p=0.035) .r ≥ 0.408, p ≤ 0.031). Mag and MagD were significantly associated with RNFL sectors NI and TS . MagD and MagD/Mag ratio were significantly associated with RNFL sector temporal inferior (TI) . After controlling for disc area, all PERG parameters remained significant with RNFL sectors I, S, and NS . Mag and MagD remained significantly correlated with NI and were correlated with global . Only MagD/Mag ratio remained significantly associated with N  , superior (S), nasal (N), and NS (p=0.028) .r ≥ 0.497, p ≤ 0.008)    and with≤ 0.045) .R2 = 0.268, p=0.003)     and for p=0.004) . No signp=0.119) .2) besides its shape and pallor [Assessment of RGC damage is necessary in management of glaucoma. Measures like perimetry and OCT allow for clinical decision-making, but we have yet to establish a diagnostic tool which allows for the quantification of remaining or lost RGCs and their axons , 23. Thed pallor .The optic disc itself is a representation of nerve fiber health as the size of the optic disc is positively correlated to the number of nerve fibers present , 25–27. In the current study, our measurements were taken using both SPECTRALIS® and Cirrus OCT devices. Mwanza et al. showed that Cirrus HD-OCT ONH parameters, particularly vertical rim thickness, rim area, and vertical C/D ratio, have excellent ability to discriminate between normal eyes and those with even mild glaucoma . Thus, wIn addition to wide optic disc variability and myopia disrupting disc area measurement, further factors contribute to underestimation or overestimation of this variable. While SD-OCT devices like Cirrus OCT are known to have advancements such as enhanced resolution, reduced acquisition time, and less operator dependence, limitations exist which can affect accuracy of measurements such as disc area . For exaFor at least two decades, PERG has been known to detect RGC dysfunction in patients with OHT and normal VF or glaucoma suspects –35. StudMost of these studies involved transient state PERG (ts-PERG) devices with reversal rate of the checkerboard pattern visual stimulus <15 reversals/second . The ss-PERG differs from ts-PERG in that it operates at higher temporal frequencies, above 10 reversals/s, causing overlap of the successive waveforms. Mag (amplitude) reflects the strength of the electrical response and corresponds to the number of living RGCs. MagD (latency) indicates the presence of RGCs in distress, and it can be thought of as the timing of the RGC response. MagD/Mag ratio is the ratio between the two, and the closer MagD values are to Mag values, the better the RGC function is. In this study, we used ss-PERG technology with the reversal frequency of 15 reversals/second, providing better diagnostic capabilities in early glaucoma , 41. ss-In this study, after controlling for disc size, Mag and MagD were significantly correlated with rim area , suggestBruch's membrane opening-minimum rim width consists of the minimum distance between the BMO, considered to be the outer border of the neural tissues at the optic nerve head, and the internal limiting membrane , 55–60. 2) as a global measure for NRR by means of Zeiss Cirrus OCT device, while SPECTRALIS® OCT and GMPE software provided sectorial measurements of the NRR in forms of BMO-MRW thicknesses. After controlling for disc area, significant relationships were found between all BMO-MRW sectors and rim area (In the present study, we used rim area (mmrim area . These fFurthermore, we found a significant association between RNFL thickness sectors and rim area in the I, NI, and NS sectors . These fThis study had many advantages. First, every participant had all his/her tests completed within the same day. Additionally, sufficient time was given to participants to recover between tests. Second, we used two SD-OCT devices to generate NRR data, where the rim area was used as a global measure of NRR, while BMO-MRW provided us with sectorial measurements by means of different software and algorithms. Third, we used the ss-PERG over ts-PERG, and it has been demonstrated that, in the latter, both P50 and N95 signals interact with signals from adjacent cells and neuronal generators, which complicate the interpretation of results. The ss-PERG modality, on the other hand, was less ambiguous. It increases the metabolic demand within the RGCs and leads to functional habituation . Mag andLimitations of our study include a relatively small sample size. More longitudinal studies are needed to investigate the relationships between PERG and ONH morphology after adjusting for disc size and including lamina cribrosa imaging that could shed some light on the mechanism of axonal damage. More OCT-angiography studies are needed to better understand the vascular abnormalities around the macula and the ONH and the effects of disc size on the capillary density and flow measures.In this study, we report significant associations between PERG parameters and ONH morphology measurements such as rim area and BMO-MRW sectors. After controlling for disc size in our analysis, this relationship became even more significant despite using modern OCT algorithms that incorporated the confounding effects of disc size. Furthermore, Mag and MagD were strong predictors in rim area and BMO-MRW thickness variances. The ss-PERG provides objective, functional, quantitative, and qualitative information about RGC function. When examining PPG patients, we recommend use of ss-PERG with OCT derived ONH morphology measures while controlling for disc area to increase diagnostic accuracy of the devices and to circumvent underestimation or overestimation of the NRR.


Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in the Western world, and it’s likely to parallel the increasing prevalence of type 2 diabetes, obesity, and other components of metabolic syndrome. However, optimal treatment for NAFLD has not been established yet. Therefore, this study investigated the hepatoprotective effect of silymarin on fructose-induced nonalcoholic fatty liver disease in rats.Thirty male Wistar rats were randomly divided into five groups; normal control group that consumed tap water, silymarin control group that consumed tap water and silymarin (400 mg/kg/day), fructose control group that consumed 20% fructose solution, treatment group that consumed 20% fructose solution and silymarin (200 mg/kg/day), and another treatment group that consumed 20% fructose solution and silymarin (400 mg/kg/day). Hepatic triglyceride, serum lipid profile, lipid peroxidation, antioxidant level, morphological features, and histopathological changes were investigated. The data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. Statistical significance was determined at p < 0.05.This study showed that the fructose control group had a significantly high value in the stage of steatosis grade, hepatic triglyceride, serum triglyceride, total cholesterol, low-density lipoprotein cholesterol, alanine aminotransferase, aspartate aminotransferase, and hepatic malondialdehyde concentration as compared to the normal control. However, significantly low values of reduced glutathione and plasma total antioxidant capacity were found. The altered parameters due to fructose drastic effect were ameliorated by silymarin treatment.The fructose control group developed dyslipidemia, oxidative stress, and mild steatosis that are the characteristics features of NAFLD. However, silymarin-treated groups showed amelioration in oxidative stress, dyslipidemia, and steatosis. Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in the Western world, and its prevalence is likely to parallel the increasing prevalence of diabetes, obesity, and other components of metabolic syndrome . NAFLD hFatty liver disease (FLD) is a growing public health problem worldwide. The global prevalence of NAFLD based on a meta-analysis study showed about 25.24% with the highest prevalence in the Middle East and South America and lowest in Africa. Metabolic comorbidities associated with NAFLD included obesity (51.34%), type 2 diabetes (22.51%), hyperlipidemia (69.16%), hypertension (39.34%) and metabolic syndrome (42.54%) [There is growing evidence in both animal models and human studies suggesting that high dietary intake of fructose is an important nutritional factor in the development of the metabolic syndrome and its associated complications. It was shown that fructose overconsumption in humans’ leads to dyslipidemia and ectopic lipid deposition; along with increased hepatic insulin resistance (IR) , 7. CaloSilybum marianum, which is commonly known as milk thistle. Silymarin belongs to the Aster family (Asteraceae or Compositae). The mature plant has large brilliant purple flowers and abundant thorns. The plant grows in places with sufficient sun exposure [25H22O10. Its active constituents are collectively known as silymarin [Silymarin is a natural compound that is present in species derived from exposure . Silymarilymarin .Silybum marianum group as compared to the vitamin E group was significant. In general Silybum marianum and vitamin E treatment appeared to be significantly effective in the biochemical improvement and decreasing the ALT and AST levels in patients with NAFLD [A study was done to assess the effects of metformin, pioglitazone, and silymarin on the treatment of NAFLD showed that all drugs are beneficial in improving biochemical indices in patients with NAFLD. However, changes in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in silymarin group showed more improvement than the other groups and the average difference between changes were significant between silymarin and metformin group . A studyth NAFLD , 14.Furthermore, several groups of drugs have been suggested according to the pathomechanisms of liver injury in NASH; including antioxidants, carnitine, and insulin sensitizers. However, while some agents showed modest improvements in liver function tests (LFTs) and even histologic parameters, the agents mentioned above are generally used to modify risk factors than as primary therapy for NASH . TherefoThe study was conducted at Black Lion Collage of Health and Medical Sciences, School of Medicine, Department of Biochemistry Master of Science laboratory, Department of Pharmaceutics and Social study laboratory, and Department of Microbiology and Immunology laboratory from November 2018 to June 2019.The experiment was performed after the protocol was approved by Addis Ababa University, College of Health and Medical Science, Department of Biochemistry Research and Ethical Review Committee meeting number DRERC: 06/18 with a protocol number of 9/18 following the code of ethics of animal experiments which comply with national and international scientific and ethical guidelines.Thirty male Wistar Albino rats weighing (151–170 g) were obtained from the animal experiment center of the Ethiopian Public Health Institute. Animals were maintained at 23 ± 1 °C room temperature and 12/12 h dark/light rhythm. The rats were acclimatized for two weeks before the experiment started. Female rats were excluded from the study because of their cyclic hormonal variations.Normal control group (NC) - a group that took standard chow & water only.- a group that took 20% fructose solution and standard chow.Fructose control group (FC) Silymarin control group (SC) - treatment control group that took standard chow, water and 400 mg/kg silymarin orally as a treatment.– a group that took standard chow, 20% fructose solution and 200 mg/kg silymarin orally as a treatment.Fructose + Silymarin (200 mg/Kg) group (FTH) ) – a group that took standard chow, 20% fructose solution and 400 mg/kg silymarin orally as a treatment.Fructose + Silymarin (400 mg/Kg) group . Most of the reagents were purchased from the Research Lab Fine Chem Industries . The kits for total antioxidant capacity, reduced glutathione, and lipid peroxidation parameters were purchased from HiMedia . All the chemicals used in the experiments were of analytical grade.The fructose solution was prepared according to Mamikutty et al  by dissoAt the end of the experiment day (after eight weeks), the rats were fasted overnight, anesthetized using light diethyl ether, and the blood was collected through cardiac puncture . After tSerum lipid profiles were measured with conventional laboratory methods using an auto-analyzer .To prepare lipid extracts from liver tissues, 0.5 g (wet weight) each of rat liver tissues was homogenized with 10 ml of the chloroform/methyl alcohol mixture (2/1 by volume), and then centrifuged at 2000 rpm for 20 min according to Folch et al . BrieflyAfter total lipid extraction, simple enzymatic determination of tissue triglyceride was done according to Danno et al . BrieflyLipid peroxide content was estimated according to the method of Ohkawa et al . BrieflyA direct measurement method for total antioxidant capacity using a new generation, more stable 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical cation was used to determine the total antioxidant capacity (TAC) as previously described by another study . In prinThe free sulfhydryl group was estimated by the method of Ellman  as modifAt the end of the experimental period, the rats were anesthetized using diethyl ether and blood was drawn from the heart by cardiac puncture and collected into serum separator test tubes  for serum and ethylenediaminetetraacetic acid tubes (EDTA tube)  for plasma. Serum activities of ALT and AST were measured with routine laboratory methods using an auto-analyzer .Liver tissues were cut and fixed with 4% paraformaldehyde. The tissue slices were embedded in paraffin. Tissue sections of 5 μm were stained with hematoxylin and eosin  and histp-values < 0.05 were considered statistically significant.Data were analyzed using Statistical Package for Social Science (SPSS) software (V-16.00). Data were compared using one-way ANOVA followed by posthoc Tukey’s test to determine significant difference between groups. Frequency data (pathologic grading of the fatty liver) were analyzed. A The fructose control group consumed less standard chow (67.69 g/day) than the normal control group (114.36 g/day) and the silymarin control group (108.05 g/day) (p < 0.001). Silymarin either 200 mg/kg (70.07 g/day) or 400 mg/kg (70.52 g/day) treated group showed a slightly higher chow consumption as compared to the fructose control group  in the fructose control group as compared to the normal control group . However, their high-density lipoprotein-cholesterol (HDL-C) was lower and not statistically significant. In silymarin-treated groups: TC, LDL-C, and HDL-C levels showed improvement (Table p < 0.05)  and low-density lipoprotein-cholesterol (LDL-C) as compared to the normal control group (nt Table . The Hep5) Table .Table 2p < 0.001). Both silymarin-treated groups were significantly prevented from lipid peroxidation (Table p < 0.01). The 400 mg/kg silymarin-treated group showed a significantly higher H-GSH as compared to the fructose control group (p < 0.01). However, the 200 mg/kg silymarin-treated group did not show a statistical difference (Table p < 0.01). Both silymarin-treated groups displayed a significantly higher P-TAC (p < 0.05)  as compared to the normal control group (on Table . The fruce Table . The fru5) Table .Table 3p < 0.05). Both silymarin-treated groups demonstrated lower values of ALT and AST as compared to the fructose control group  had better efficacy than a lower dose (200 mg/kg) on treating NAFLD.


The diagnostic value of platelet indices has been evaluated in various infectious diseases but not in infected nonunion. The purpose of this study was to assess the usefulness of platelet indices for diagnosis of infected nonunion after open reduction and internal fixation.This retrospective study was performed in patients who underwent primary fracture nonunion revision surgeries from January 2016 to December 2021. A total of 297 patients were included in the study: 96 with infected nonunion (group A) and 201 with aseptic nonunion (group B). Receiver operator characteristic (ROC) curve analysis was performed to evaluate diagnostic value of each index. Area under the curve (AUC), sensitivity, specificity, and positive and negative predictive values were calculated and compared.P < 0.05). ROC analysis showed PC/MPV and plasma fibrinogen to have better diagnostic value than the other coagulation indicators . The combination of ESR, plasma fibrinogen, and PC/MPV had good sensitivity and specificity for diagnosis of infected nonunion. PC/MPV had better diagnostic value than ESR and plasma fibrinogen in the subgroup of patients with coagulation-related comorbidities.Demographic characteristics were comparable between the two groups. White blood cell (WBC) count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), plasma fibrinogen, plasma D-dimer, platelet count (PC), plateletcrit, and ratio of platelet count to mean platelet volume (PC/MPV) were significantly higher, and MPV and platelet distribution width (PDW) significantly lower, in group A than in group B (Plasma fibrinogen and PC/MPV ratio might be useful parameters for early diagnosis of infected nonunion. Open reduction and internal fixation (ORIF) for fractures has been one of the most successful surgeries during the last century. Although ORIF provides rapid relief of pain and functional recovery, infected nonunion is a disruptive complication that results in higher hospitalization costs, longer treatment course, and higher morbidity and mortality rates than the primary procedure , 2. AccuMany studies have indicated a close association between coagulation and inflammation –11. CoagThe aim of this study was to evaluate the accuracy of the established inflammatory biomarkers  and the easily tested coagulation-related parameters  in the diagnosis of infected nonunion after ORIF, and to identify the coagulation-related inflammatory marker with the best diagnostic value.This study was approved by the ethical committee of our institution. All experimental procedures were conducted according to the Declaration of Helsinki.n = 16) because, in these patients, the source of pathogens is complex and duration of infection uncertain; (2) patients without complete blood workup (n = 9); and (3) patients who had received antibiotics during the 2 weeks preceding surgery . Among the remaining 351 patients, there were 54 patients with coagulation-related comorbidities , and blood disorders [n = 3]) and patients with infection at a site other than the fracture (n = 12); these patients were analyzed separately. Ultimately, 297 patients were included in the main analysis  patients who had undergone refixation surgeries (n = 96) and an aseptic nonunion group . Differentiation of infected from aseptic nonunion was based on the 2018 Fracture-Related Infection Consensus Definition ) and analyzed by the Mann–Whitney U test. Differences were considered significant at P < 0.05. Receiver operating characteristic (ROC) curves were used to evaluate the value of each biomarker for predicting infected nonunion before revision surgeries. The area under the curve (AUC), with the 95% confidence interval [CI]), and the sensitivity and specificity of different markers, were determined using the working subject curve. The Youden index (J = [sensitivity + specificity] − 1) was used to identify the optimal predictive cutoffs for the tested markers. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each test were calculated. AUC > 0.9 was considered excellent, AUC 0.8–0.9 was considered good, AUC 0.7–0.8 was considered fair, AUC 0.6–0.7 was considered poor, and AUC < 0.6 was considered to indicate no discriminatory capacity.Statistical analysis was performed using STATA 18.0 . Categorical variables were expressed as frequencies and percentages, and analyzed by Pearson Chi-square test or Fisher exact test. The Kolmogorov–Smirnov test was used to identify the normality of distribution of continuous data. Normally distributed variables were presented as mean ± standard deviation and analyzed by the Student’s Staphylococcus aureus was the most common infecting microorganism (n = 29), followed by Staphylococcus epidermidis (n = 20), Escherichia coli (n = 17), Enterobacter cloacae (13), Enterococcus faecalis (n = 8), and Streptococcus mutans (n = 4).Table P < 0.05). Table WBC count, CRP, ESR, plasma fibrinogen, plasma D-dimer, PC, plateletcrit, and PC/MPV were significantly higher, and MPV and PDW significantly lower, in group A than in group B  for PC/MPV > 31.7, plasma fibrinogen > 3.35 g/L, and ESR > 17.5 mm/h. The highest specificity (99%) was seen with the combination of PC/MPV > 31.7, plasma fibrinogen > 3.35 g/L, and ESR > 17.5 mm/h Table .Table 4DAmong the 54 patients with coagulation-related comorbidities, there were 31 patients with infected nonunion and 23 patients with aseptic nonunion. Table A review of literature confirmed that our study is the first to examine the use of coagulation-related indices  to differentiate aseptic nonunion and infected nonunion. In this study, we also evaluated the diagnostic value of the classic inflammatory markers . We found elevated plasma fibrinogen, PC/MPV, and ESR to be associated with infected nonunion. Some previous studies have also shown that elevated plasma fibrinogen and ESR are useful for diagnosing infected nonunion . OverallInflammation and infection are important regulators of coagulation and fibrinolytic system activity , 23. BecPlatelets, generated from megakaryocytes, are a part of the natural immune system and are rapidly activated during the inflammation process . In bactIn the presence of inflammation and infection, platelet production intensifies and the mean platelet volume drops, resulting in increase of the PC/MPV ratio . Paziuk We evaluated patients with coagulation-related comorbidities separately and found that PC/MPV was useful for diagnosing infected nonunion in this subgroup of patients also, with sensitivity of 77.41% and specificity of 91.3%; PC/MPV was superior to ESR (sensitivity of 64.52% and specificity of 73.91%) and plasma fibrinogen (sensitivity of 48.39% and specificity of 52.17%).There are several limitations in our study. First, due to its retrospective nature, selection bias and information bias cannot be ruled out. Second, although we attempted to identify and exclude patients who had used antibiotics in the 2 weeks preceding revision surgery, information for antibiotic use was not recorded for some patients; this might have affected our results. Third, among patients with coagulation-related comorbidities, there were very small numbers with some types of comorbidities ; thus, some subgroups were too small for meaningful statistical analysis. Large-scale prospective studies, with subgroup analyses, are needed to further investigate the value of these biomarkers for early diagnosis of infected nonunion.PC/MPV, ESR, and plasma fibrinogen level are significantly higher in patients with infected nonunion than in patients with aseptic nonunion. PC/MPV has high specificity for the diagnosis of infected nonunion. PC/MPV may be a practical and cost-efficient biomarker for detecting infected nonunion after ORIF.


Self-expanding metallic stents (SEMS) as bridge to surgery have been questioned due to the fear of perforation and tumour spread. This study aimed to compare SEMS and stoma as bridge to surgery in acute malignant large bowel obstruction in the Swedish population.Medical records of patients identified via the Swedish Colorectal Cancer Register 2007–2009 were collected and scrutinized. The inclusion criterion was decompression intended as bridge to surgery due to acute malignant large bowel obstruction. Patients who underwent decompression for other causes or had bowel perforation were excluded. Primary endpoints were 5-year overall survival and 3-year disease-free survival. Secondary endpoints were 30-day morbidity and mortality rates.P = 0.260. Likewise, 3-year disease-free survival did not differ statistically significant, SEMS 73 per cent (43 of 59 patients), stoma 65 per cent (62 of 95 patients), P = 0.32. In the SEMS group, 1.4 per cent (one patient) did not fulfil resection surgery compared to 8.8 per cent (11 patients) in the stoma group (P = 0.040). Postoperative complication and 30-day postoperative mortality rates did not differ, whereas the duration of hospital stay and proportion of permanent stoma were lower in the SEMS group.A total of 196 patients fulfilled the inclusion criterion . There was no significant difference in sex, age, ASA score, TNM stage and adjuvant chemotherapy between the SEMS and stoma groups. No patient was treated with biological agents. Five-year overall survival was comparable in SEMS, 56 per cent (40 patients), and stoma groups, 48 per cent (60 patients), This nationwide registry-based study showed that long-term survival in patients with either SEMS or stoma as bridge to surgery in acute malignant large bowel obstruction were comparable. SEMS were associated with a lower rate of permanent stoma, higher rate of resection surgery and shorter duration of hospital stay. This is a nationwide registry-based study on acute malignant large bowel obstruction comprising 196 cases in which all medical files were scrutinized. The results show that SEMS as bridge to surgery were associated with comparable long-term survival and recurrence rate compared to decompression by stoma, higher rate of fulfilled resection surgery and lower permanent stoma rate. Primary endpoints were 5-year overall survival and 3-year disease-free survival. Secondary endpoints were 30-day morbidity and mortality rates, recurrence rate, duration of stay in hospital and stoma rate at 3 years.In this retrospective nationwide population-based study, the patients were identified via the Swedish Colorectal Cancer Register (SCRCR); the registry had a coverage of 94–99 per centThe inclusion criterion was decompression with bridge to surgery intention due to malignant large bowel obstruction. Patients with prophylactic, palliative or other decompression intentions were excluded. Patients with bowel perforation at diagnosis were also excluded.The following data were retrieved: age, sex, ASA score, preoperative staging, tumour localization, pathology reports including TNM stage, types of complications, readmission, stoma at 3 years, recurrence and death of any cause. The date of death was obtained from the Swedish Population Register and the follow-up duration was 5 years for each patient. The patients were divided into two groups (SEMS and stoma), and analyses were performed per protocol.Ethical approval for this study was obtained from the Regional Ethics Committee in Lund, dnr 2010/260.The primary endpoints were 5-year overall survival (OS) and 3-year disease-free survival. OS and disease-free survival were calculated from the date of resection surgery until recurrent disease, death or end of follow-up in all cases that underwent subsequent resection. In cases that did not undergo subsequent resection OS and disease-free survival were calculated from the date of diagnosis. In the analysis of disease-free survival, all patients not radically operated  were excluded. Disease-free survival was compared between groups by calculating the risk of recurrent disease.Secondary endpoints were 30-day morbidity and mortality rates, recurrence rate , duration of stay in hospital and stoma rate at 3 years.The Clavien–Dindo classification (CD)Recurrence rate analyses were, as in disease-free survival, based on radical operations only.Finally, a subgroup analysis was conducted focused on left-sided tumours only.2 test was used to compare categorical variables, and the Mann–Whitney U test was used to compare continuous variables. In the 30-day mortality rate, Fisher’s exact test was used. Survival analysis data were measured from the day of resection surgery to death of any cause. In patients with a bridge-to-surgery intention in whom resection surgery was never performed, the day of decompression was used. The Kaplan–Meier method was used in the survival analyses. As the Kaplan–Meier curves showed that the assumption of proportional hazards was not fulfilled, survival was first analysed using logistic regression analyses. The follow-up duration was thus divided into two periods, enabling performance of Cox regression analyses. Multivariable analyses adjusting for potential confounders were performed in the two groups. Missing values were coded as a separate category.The Pearson χ® version 23, Armonk, NY, USA. Differences were considered statistically significant at P < 0.050.Analyses were performed using IBM SPSSFig. 1).A total of 542 patients in 52 different hospitals were registered as undergoing preoperative decompression with a temporary intention, of which it was possible to retrieve medical files for 519 cases. A total of 196 patients met the inclusion criteria . All patients who lacked verification of acute malignant obstruction were excluded (Table 1), although a borderline significant difference towards higher age was noted in the SEMS group (median 72 (range 41–91) years versus 66 (range 36–91) years, P = 0.060). Significantly more rectal tumours had a stoma (35 per cent) than SEMS (13 per cent). No difference was found in the proportion of patients referred to adjuvant treatment (34 per cent in the SEMS group versus 40 per cent in the stoma group) (Table 1). For adjuvant treatment, 5-fluorouracil intravenously or orally with or without concomitant oxaliplatin were used. No patient was treated with biological agents including Bevacizumab.Of the 52 hospitals, 15 did not use bridge to surgery at all, 18 centres used only stoma for decompression and 19 used both SEMS and stoma. Of these 19 centres only three were able to use SEMS off-office time. In the centres using SEMS, a total of 136 cases were treated, of which 71 were treated with SEMS and 65 with stoma, whereas the remaining 60 cases with stoma were treated in centres only using stoma. Statistically the SEMS and stoma groups did not differ significantly in sex, age, ASA score and TNM stage (P = 0.040). In the SEMS group, a patient who received neoadjuvant treatment developed bowel ischaemia after 3 months. In the stoma group, three patients did not undergo resection surgery due to complications after the initial stoma procedure , and eight patients were converted to palliative treatment due to tumour progression (five of which were during neoadjuvant treatment).One patient (1 per cent) in the SEMS group did not undergo resection surgery compared to 11 (8.8 per cent) patients in the stoma group (P = 0.260). Moreover, no statistical difference in the 3-year disease-free survival was noted ; stoma, 65 per cent (62 of 95), P = 0.320). Kaplan–Meier curves are shown in Fig. 2a. After adjusting for age, stage, sex and ASA score in the multivariable analyses, no significant difference was noted in the 5-year survival by logistic regression analyses (Table 2). In the Cox regression analyses however, a significantly better survival rate was noted in the SEMS group in the second half of the study period (2.5–5 years).The 5-year OS did not differ significantly between groups: 56 per cent (40 of 71 patients) in the SEMS group compared with 48 per cent (60 of 125) in the stoma group (As shown in Table 3).The total recurrence rate did not differ between groups: 27 per cent (16 of 59 patients) in the SEMS group and 35 per cent (33 of 95) in the stoma group. Moreover, no difference in locoregional and distant recurrences was found between groups , and stoma, 0.8 per cent (1 of 125).Table 3). The total complication rates during resection surgery were 34 and 29 per cent in the SEMS and stoma groups, respectively. A borderline difference towards more severe complications during resection surgery was noted in the SEMS group (21 per cent) compared to the stoma group (11 per cent) (P = 0.070). The combined complication rates (decompression and resection) did not differ between the groups.There was no significant difference in complication rate during decompression between the SEMS and stoma groups. The total complication rate during decompression (CD 2–5) and severe complication rate (CD 3b–5) were 24 and 14 per cent in the SEMS group and 36 and 17 per cent in the stoma group, respectively (P = 0.740).Stent perforations occurred in six of 71 patients (8 per cent), of which four were in-hospital and two were late perforations. None of the patients were treated with chemotherapy or biological agents before or at the time of perforation. No mortality directly related to perforation was noted, and there was no difference in 5-year OS (50 per cent (3 of 6 patients) in SEMS with perforation and 57 per cent (37 of 65 patients) in SEMS with no perforation; P < 0.010).The duration of hospital stay for decompression procedure was significantly lower in the SEMS group with a median of 4 days compared with 9 days in the stoma group. No difference was found for resection surgery with a median of 8 days in each group. The aggregated median duration of stay for both procedures was 12 (i.q.r. 9–18) days in the SEMS group and 17.5 (i.q.r. 14–23) days in the stoma group (P = 0.050). A borderline difference towards a lower readmission rate at 365 days was noted in the SEMS group  compared to that in the stoma group  (P = 0.070).The 30-day readmission rate was lower in the SEMS group  compared to that in the stoma group  (P = 0.030).The permanent stoma rate was lower in the SEMS group  than in the stoma group   were treated by SEMS and 54 per cent (69 patients) were operated with a stoma. Tumour and patient characteristics did not differ between groups and were similar to the main study cohort.P = 0.230), nor did 3-year disease-free survival: SEMS 78 per cent (40 of 51 patients); stoma 64 per cent (38 of 59), P = 0.090. Kaplan–Meier curves are shown in P = 0.090) and 2.62 (95 per cent c.i. 0.94 to 7.32) in 3-year disease-free survival (P = 0.070). As in the total population, the multivariable Cox regression analyses  showed no statistically significant difference in OS the first 2.5 years, hazard ratio (HR) 1.18 (95 per cent c.i. 0.56 to 2.48) but did in the 2.5–5 year period, HR 5.74 (95 per cent c.i. 1.41 to 23.38). A hazard ratio of 2.19 (95 per cent c.i. 0.98 to 4.93) in 3-year disease-free survival was noted (P = 0.060) (Appendix S1).Five-year OS did not differ statistically significantly between the groups: 64 per cent (37 of 58 patients) in the SEMS group compared to 52 per cent (36 of 69) in the stoma group . Thus, the allocation of patients could depend predominately on availability for SEMS. Besides availability, technical aspects are most likely to be the key factor in the choice of method but are less likely to influence mortality. One problem is that the number of cases where SEMS were intended but failed to be placed was not available. Thus, it was not possible to draw any firm conclusions on success rate or complete procedure-related complications for SEMS deployment, and as such, the study is to be regarded as a per-protocol analysis.On the other hand, the main strength is the long-term follow-up of the population-based, nationwide, well defined material solely constituting cases of acute malignant large bowel obstruction managed with a bridge-to-surgery approach.This study documented at least equivalent long-term survival and similar local and distant recurrence rates with the use of SEMS compared with stoma as a bridge to surgery in acute malignant large bowel obstruction. Moreover, SEMS was associated with shorter hospital stay, fewer permanent stomas and a higher rate of fulfilled resection surgery. Taken together, these data should encourage a change in the predominant approach and increase the use of SEMS as a bridge to surgery in malignant large bowel obstruction.This study was supported by Skåne County Counciĺs Research and Development Foundation grants, ALF-agreement (2018-Project 0264) between the Swedish government and the county councils, and local grant .Disclosure. The authors declare no conflict of interest.BJS Open online.zrab018_Supplementary_DataClick here for additional data file.