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The integrated and indivisible nature of the SDGs is facing implementation challenges due to the silo approaches. We present the three interconnected foci (SDG interactions, modeling, and tools) at the science-policy interface to address these challenges. Accounting for them will support accelerated SDG progress, operationalizing the integration and indivisibility principles.

Abstract Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Abstract Extracellular vesicles (EVs) accumulate during packed red blood cell (PRBC) storage. To date, the involvement of EVs in transfusion-related immunomodulation (TRIM) has not been prospectively evaluated in intensive care unit (ICU) patients. This was a prospective subanalysis of a recent observational feasibility study in postoperative ICU patients after: (1) open aortic surgery (Aorta), (2) bilateral lung transplantation (LuTx), and (3) other types of surgery (Comparison). Patient plasma was collected three times each before and after leukoreduced PRBC transfusion at 30-min intervals. The total number of EVs and EVs derived from erythrocytes (EryEVs), total platelets (total PEVs), activated platelets, granulocytes (GEVs), monocytes, and myeloid cells in PRBC samples and patient plasma were analyzed by flow cytometry. Statistical analysis was performed by Spearman’s correlation test, linear mixed models and pairwise comparisons by Wilcoxon matched-pairs test. Twenty-three patients (Aorta n = 5, LuTx n = 9, Comparison n = 9) were included in the final analysis. All EV subgroups analyzed were detectable in all PRBCs samples (n = 23), but concentrations did not correlate with storage time. Moreover, all EVs analyzed were detectable in all plasma samples (n = 138), and EV counts were consistent before transfusion. Concentrations of total EVs, EryEVs, total PEVs, and GEVs increased after transfusion compared with baseline in the entire cohort but not in specific study groups. Furthermore, the change in plasma EV counts (total EVs and EryEVs) after transfusion correlated with PRBC storage time in the entire cohort. Extracellular vesicles were detectable in all PRBC and plasma samples. Individual EV subtypes increased after transfusion in the entire cohort, and in part correlated with storage duration. Future clinical studies to investigate the role of EVs in TRIM are warranted and should anticipate a larger sample size. Trial registration: Clinicaltrials.gov: NCT03782623.

Abstract Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

This article details the adaptation of the FOCUS (Fundamentals of Conceptual Understanding and Success) model (Mireles, Acee, & Gerber, 2014) implemented to improve student success in general education and developmental mathematics at a four-year rural university. The model has been demonstrated successful when implemented at a four-year large urban university in the south. The modification of the model described herein has also addressed another standard approach for improving student success used by many postsecondary institutions: drop-in Mathematics Tutoring Centers (MTCs), which provide extra learning support for students enrolled in developmental and general education mathematics courses (Gallimore & Steward, 2014). This article details the development and implementation of a modified FOCUS model to improve student success, specifically in mathematics. Students' success before and after the changes are implemented is compared. Findings indicate that the redesign via a modified FOCUS model addressed many barriers to MTC use and coincided with an increase in positive student outcomes in developmental and general education mathematics courses.

Background: The automated NavX Ensite Precision latency-map (LM) algorithm aims to identify atrial tachycardia (AT) mechanisms. However, data on a direct comparison of this algorithm with conventional mapping are scarce. Methods: Patients scheduled for AT ablation were randomized to mapping with the LM- algorithm (LM group) or to conventional mapping (conventional only group: ConvO), using entrainment and local activation mapping techniques. Several outcomes were exploratively analyzed. Primary endpoint was intraprocedural AT Termination. If AT termination with only automated 3D-Mapping failed, additional conventional methods were applied (conversion). Results: A total of 63 patients (mean 67 years, 34 % female) were enrolled. In the LM group (n = 31), the correct AT mechanism was identified in 14 patients (45 %) using the algorithm alone compared to 30 patients (94 %) with conventional methods. Time to termination of the first AT was not different between groups (LM group 34 ± 20 vs. ConvO 43.1 ± 28.3 min; p = 0.2). However, when AT termination did not occur with LM algorithm, time to termination prolonged significantly (65 ± 35 min; p = 0.01). After applying conventional methods (conversion), procedural termination rates did not differ between LM group (90 %) vs. ConvO (94 %) (p = 0.3). During a follow-up time of 20 ± 9 months, no differences were observed in clinical outcomes. Conclusion: In this small prospective, randomized study, the use of the LM algorithm alone may lead to AT termination, but less accurate than conventional methods.

Up to a third of the world’s population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Abstract Background Dietary fiber is an integral part of a healthy diet, but questions remain about the mechanisms that underlie effects and the causal contributions of the gut microbiota. Here, we performed a 6-week exploratory trial in adults with excess weight (BMI: 25–35 kg/m2) to compare the effects of a high-dose (females: 25 g/day; males: 35 g/day) supplement of fermentable corn bran arabinoxylan (AX; n = 15) with that of microbiota-non-accessible microcrystalline cellulose (MCC; n = 16). Obesity-related surrogate endpoints and biomarkers of host-microbiome interactions implicated in the pathophysiology of obesity (trimethylamine N-oxide, gut hormones, cytokines, and measures of intestinal barrier integrity) were assessed. We then determined whether clinical outcomes could be predicted by fecal microbiota features or mechanistic biomarkers. Results AX enhanced satiety after a meal and decreased homeostatic model assessment of insulin resistance (HOMA-IR), while MCC reduced tumor necrosis factor-α and fecal calprotectin. Machine learning models determined that effects on satiety could be predicted by fecal bacterial taxa that utilized AX, as identified by bioorthogonal non-canonical amino acid tagging. Reductions in HOMA-IR and calprotectin were associated with shifts in fecal bile acids, but correlations were negative, suggesting that the benefits of fiber may not be mediated by their effects on bile acid pools. Biomarkers of host-microbiome interactions often linked to bacterial metabolites derived from fiber fermentation (short-chain fatty acids) were not affected by AX supplementation when compared to non-accessible MCC. Conclusion This study demonstrates the efficacy of purified dietary fibers when used as supplements and suggests that satietogenic effects of AX may be linked to bacterial taxa that ferment the fiber or utilize breakdown products. Other effects are likely microbiome independent. The findings provide a basis for fiber-type specific therapeutic applications and their personalization. Trial registration Clinicaltrials.gov, NCT02322112 , registered on July 3, 2015. Video Abstract

Abstract Background Cold urticaria (ColdU) is characterized by pruritic wheals following exposure of the skin to cold. Many patients show insufficient response to antihistamines, the first line treatment. Based on the high efficacy of interleukin‐1(IL‐1)‐inhibition in cold‐induced urticarial autoinflammatory diseases, we assessed the effects of rilonacept, an IL‐1 inhibitor, in ColdU patients unresponsive to standard treatment. Methods In this randomized, double‐blind, placebo‐controlled two‐center study, we included 20 patients with ColdU. In the first part, patients received 320 mg rilonacept or placebo (1:1) followed by weekly doses of 160 mg rilonacept or placebo for 6 weeks. In the second part, all patients received weekly 160 mg or 320 mg rilonacept for 6 weeks, open‐label. The primary endpoint was change in critical temperature threshold (CTT). Secondary endpoints included changes in quality of life impairment (Dermatology Life Quality Index, DLQI), differences of inflammatory mediators upon cold provocation and safety assessment over the study period. Results Baseline mean CTTs were 20.2°C (placebo) and 17.3°C (rilonacept). Mean CTTs did not change significantly during the 6‐week double‐blind treatment (placebo – 0.45°C; rilonacept +0.89°C). IL‐6, IL‐18 and HSP‐70 blood levels showed interindividual variability without significant changes during hand cold water bath provocation in placebo‐ or rilonacept‐treated patients. In contrast, DLQI significantly improved in the rilonacept (mean DLQI reduction of 3.8; p = 0.002) but not in the placebo group (mean DLQI reduction of 0). Comparing baseline with the rilonacept open‐label treatment, there were no changes in CTTs or DLQI scores. Conclusion IL‐1 inhibition with rilonacept did not improve ColdU, but demonstrated a good safety profile. Clinical Trial Registration EudraCT number: 2012‐005726‐30. ClinicalTrials.gov identifier: NCT02171416.

Mass cytometry (MC) is a powerful method for mapping complex cellular systems at single-cell levels, based on the detection of cellular proteins. Numerous studies have been performed using human blood, but there is a lack of protocols describing the processing and labeling of bronchoalveolar lavage fluid (BALF) and nasal polyps (NP) for acquisition by MC. These specimens are essential in the investigation of immune cell characteristics in airway diseases such as asthma and chronic rhinosinusitis with NP (CRSwNP). Here we optimized a workflow for processing, labeling, and acquisition of BALF and NP cells by MC. Among three methods tested for NP digestion, combined enzymatic/mechanical processing yielded maximum cell recovery, viability and labeling patterns compared to the other methods. Treatment with DNAse improved sample acquisition by MC. In a final step, we performed a comparison of blood, BALF and NP cell composition using a 31-marker MC antibody panel, revealing expected differences between the different tissue but also heterogeneity among the BALF and NP samples. We here introduce an optimized workflow for the MC analysis of human NP and BALF, which enables comparative analysis of different samples in larger cohorts. A deeper understanding of immune cell characteristics in these samples may guide future researchers and clinicians to a better disease management.

Category: Trauma; Ankle; Sports Introduction/Purpose: Lower extremity fractures are relatively common among winter sports participants. The purpose of this study is to evaluate the demographics, injury mechanisms and environmental features among patients evaluated for lower leg fractures at a high-volume Colorado ski resort during five consecutive ski seasons. Methods: We conducted a retrospective descriptive analysis of patients with lower leg fractures at the Denver Health Winter Park Medical Center during the 2012/13 to 2016/17 ski seasons. We included all fractures below the knee (excluding tibial plateau and isolated fibular head fractures) sustained by skiing or snowboarding. Chart review was performed on the patient cohort, confirming diagnosis and evaluating factors associated with these fractures. Results: There were 346 lower leg, ankle, and/or foot fractures (5.4% of all clinic visits) during the study period. The average age was 33.1 years (range 4-74) with the majority being male (60.7%) and mainly as a result of skiing (84.7%). The majority of fractures were sustained among skiers and snowboarders who self-identified as beginner or intermediate skill level (64.8%), and on easy (green) or intermediate (blue) runs (64.9%). The most common fractures sustained were complete tibia-fibula fractures (30.9%) and lateral malleolus fractures (27.5%). More than half (57.0%) of complete tibia-fibula fractures were classified and boot top (mid- shaft just above the ski boot) fractures. The most common type of lateral malleolus fractures was Danis-Weber B (72.0%) with only 5.4% being Weber C fractures. Foot fractures were rare with only 12 total cases (3.5%). Conclusion: Skiers are much more likely to sustain lower leg fractures compared to snowboarders. Men were much more likely than women to sustain lower leg fractures. Most fractures occurred in mild or moderately difficult ski runs relative to advanced runs. Fractures appear to be more common with advancing age. Complete tibia-fibula fractures were the most common fractures sustained in all riders with lateral malleolus fractures being the second most common. Foot fractures are very rare in this population. Given the frequency of these injuries, additional education around prevention may be warranted.

PurposeTo investigate the efficacy and safety of buparlisib, an oral pan-PI3K inhibitor, in relapsed or refractory thymomas.MethodsThis was a single center, single arm, open label phase II trial of buparlisib in patients with recurrent thymoma who have progressed after at least one prior line of treatment. The primary endpoint was objective response rate (complete response [CR] + partial response [PR]). Secondary endpoints included toxicity; progression free survival (PFS); overall survival (OS); disease control rate (DCR), i.e., the percentage of patients who achieve either PR or CR or stable disease [SD] for at least 4 months.ResultsBetween 10/13/2014 and 1/18/2017, 14 patients with stage IV disease were enrolled. Median age was 58y (23–74). 71% were females and 71% white. All patients had WHO B2 (29%) or B3 (71%) thymoma. Patients received buparlisib for a median of 4.5m (2–33). At a median follow up of 16.6m (2.4–31.3), onr patients (7%) achieved a PR. DCR was 50%. Median PFS was 11.1m (95% CI 2.9 – 18.8). Median OS, updated as of March, 2021 was 22.5m (10.7–31.3). Most common grade 3-4 adverse events related to buparlisib were dyspnea (21%), rash (14%), elevated transaminases (14%), cough (7%), pneumonitis (7%), anxiety (7%), fatigue (7%) and hyperglycemia (7%). Reasons for treatment discontinuation included progression of disease (n= 5), rash (n=4), pulmonary toxicity (n=3), sinusitis (n=1), and disseminated toxoplasmosis plus autoimmune cholangitis (n=1). As of 3/2021, 8 patients have died, 7 due to disease progression and 1 due to central nervous system toxoplasmosis and autoimmune cholangitis.ConclusionBuparlisib showed modest activity in patients with relapsed or refractory thymomas. Further investigation of PI3K pathway targeted therapy in thymoma is warranted. (clinicaltrials.gov ID: NCT02220855).Clinical trial registrationclinicaltrials.gov, identifier (NCT02220855)

Data analytics are an increasingly popular method for talent identification, and are used for a variety of decision making purposes, such as rotations and playing time. However, coaches often rely on their perceptions and experiences to identify talent and player attributes that are important to success. Therefore, the purposes of this study were to evaluate differences between coach perceptions of player ability against actual performances as well as to determine whether these perceptions differed as a head or assistant coach. Participants were six (two head; four assistant) college coaches who were asked to collectively identify the five most important attributes when evaluating a basketball player. Then, before the season began, all coaches were asked to independently score each of their athletes on these attributes using a 100mm Visual Analog Scale. These scores were compared to player performances during the season. Results were mixed, and while there were correlations between some player performance variables and coach perceptions, they varied wildly, and coaches’ perceptions of their athletes had little consistent correlation to their performances. Furthermore, there were few agreements between head coaches and their assistants or between assistants. Findings suggest that while coach perceptions and talent identification have their place, the use of data analytics in sports may provide additional support when making coaching decisions such as playing time. Therefore, coaches should recognize their own limitations of player talent and balance these “feelings” with statistical evidence.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell‐penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live‐cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein‐based treatment strategies. [ABSTRACT FROM AUTHOR]

(1) Background: Cell salvage is highly recommended in orthopedic surgery to avoid allogeneic transfusions. Preparational steps during cell salvage may induce extracellular vesicle (EV) formation with potential thrombogenic activity. The purpose of our study was to assess the appearance of EVs at retransfusion. (2) Methods: After ethics committee approval and informed consent, blood was withdrawn from the autotransfusion system (Xtra, Sorin, Germany) of 23 patients undergoing joint arthroplasty. EVs were assessed by flow cytometry in two times centrifugated samples. EVs were stained with specific antibodies against cellular origins from platelets (CD41), myeloid cells (CD15), monocytes (CD14), and erythrocytes (CD235a). The measured events/µL in the flow cytometer were corrected to the number of EVs in the retransfusate. (3) Results: We measured low event rates of EVs from platelets and myeloid origin (

We currently do not understand the fundamental physical processes that govern mass and energy flow through the Earth’s magnetosphere. Knowledge of these processes is critical to understanding the mass loss rate of Earth’s atmosphere, as well as for determining the role that a planetary magnetic field plays in atmospheric retention, and therefore habitability, for Earth-like planets beyond the solar system. Mass and energy flow processes are challenging to determine at Earth in part because Earth’s planetary magnetic field creates a complex “system of systems” composed of interdependent plasma populations and overlapping spatial regions that perpetually exchange mass and energy across a broad range of temporal and spatial scales. Further, the primary mass carrier in the magnetosphere is cold plasma (as cold as ∼0.1 eV), which is invisible to many space-borne instruments that operate in the inner magnetosphere. The Plasma Imaging LOcal and Tomographic experiment (PILOT) mission concept, described here, provides the transformational multi-scale observations required to answer fundamental open questions about mass and energy flow dynamics in the Earth’s magnetosphere. PILOT uses a constellation of spacecraft to make radio tomographic, remote sensing, and in-situ measurements simultaneously, fully capturing cold plasma mass dynamics and its impact on magnetospheric systems over an unprecedented range of spatial and temporal scales. This article details the scientific motivation for the PILOT mission concept as well as a potential mission implementation.

Abstract Background Extracorporeal circulation during major cardiac surgery triggers a systemic inflammatory response affecting the clinical course and outcome. Recently, extracellular vesicle (EV) research has shed light onto a novel cellular communication network during inflammation. Hemoadsorption (HA) systems have shown divergent results in modulating the systemic inflammatory response during cardiopulmonary bypass (CPB) surgery. To date, the effect of HA on circulating microvesicles (MVs) in patients undergoing CPB surgery is unknown. Methods Count and function of MVs, as part of the extracellular vesicle fraction, were assessed in a subcohort of a single-center, blinded, controlled study investigating the effect of the CytoSorb device during CPB. A total of 18 patients undergoing elective CPB surgery with (n = 9) and without (n = 9) HA device were included in the study. MV phenotyping and counting was conducted via flow cytometry and procoagulatory potential was measured by tissue factor-dependent MV assays. Results Both study groups exhibited comparable counts and post-operative kinetics in MV subsets. Tissue factor-dependent procoagulatory potential was not detectable in plasma at any timepoint. Post-operative course and laboratory parameters showed no correlation with MV counts in patients undergoing CPB surgery. Conclusion Additional artificial surfaces to the CPB-circuit introduced by the use of the HA device showed no effect on circulating MV count and function in these patients. Larger studies are needed to assess and clarify the effect of HA on circulating vesicle counts and function. Trial registration ClinicalTrials.Gov Identifier: NCT01879176; registration date: June 17, 2013; https://clinicaltrials.gov/ct2/show/NCT01879176

Extracellular vesicles (EV) act as a cellular communication tool by carrying lipids, proteins and micro RNA (miR) between cells, thereby playing a pivotal role in thromboembolic processes. The effect of P2Y12 inhibitors on pro-coagulatory, phosphatidylserine (PS)-expressing EV has been investigated previously, but only in vitro or during confounding clinical conditions, such as acute coronary syndrome. Hence, we enrolled 62 consecutive patients 12 month after percutaneous coronary intervention and stent implantation and consequent treatment with dual-antiplatelet therapy consisting of low-dose aspirin and P2Y12 inhibitors. Blood for platelet function testing and EV and miR measurements was taken on the last day of P2Y12 inhibitor intake (baseline, on-treatment) and 10, 30 and 180 days thereafter (off-treatment). We did not observe any influence of P2Y12 inhibitors on the levels of PS-EV or EV sub-population from platelets, erythrocytes, monocytes or endothelial cells, respectively. There was no relationship between platelet function and EV levels in plasma. However, the association of miR-21 and miR-150 with platelet EVs was significantly different between on- and off-treatment measurements. Hence, our study suggests no influence of P2Y12 inhibition on the count of EVs in plasma, but on the potential cargo of platelet-derived EV.

Abstract Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell‐derived extracellular vesicles (EVs). Here we demonstrate that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell‐secreted soluble factors via tangential flow‐filtration (TFF) and subtractive tandem mass‐tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein‐based flow cytometry, super‐resolution and electron microscopy verified EV identity. PLX‐EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose‐dependently inhibited T cell proliferation in vitro. Corona removal by size‐exclusion or ultracentrifugation abrogated angiogenesis. Re‐establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super‐resolution microscopy, electron microscopy and zeta‐potential shift, and served as a proof‐of‐concept. Understanding EV corona formation will improve rational EV‐inspired nano‐therapy design.

Chlamydia trachomatis (Ct) is the most common cause of genital tract infections as well as preventable blindness worldwide. Pattern recognition receptors such as toll-like receptors (TLRs) represent the initial step in recognizing pathogenic microorganisms and are crucial for the initiation of an appropriate immune response. However, our understanding of TLR-signaling in Chlamydia-infected immune cells is incomplete. For a better comprehension of pathological inflammatory responses, robust models for interrogating TLR-signaling upon chlamydial infections are needed. To analyze the TLR response, we developed and utilized a highly sensitive and selective fluorescent transcriptional cellular reporter system to measure the activity of the transcription factor NF-κB. Upon incubation of the reporter cells with different preparations of Ct, we were able to pinpoint which components of TLRs are involved in the recognition of Ct. We identified CD14 associated with unique characteristics of different serovars as the crucial factor of the TLR4/CD14/MD2 complex for Ct-mediated activation of the NF-κB pathway. Furthermore, we found the TLR4/CD14/MD2 complex to be decisive for the uptake of Ct-derived lipopolysaccharides but not for infection and replication of Ct. Imaging flow cytometry provided information about inclusion formation in myeloid- as well as lymphocytic cells and was highest for Ct L2 with at least 25% of inclusion forming cells. Ct E inclusion formation was eminent in Jurkat cells without CD14 expression (11.1%). Thus, our model enables to determine Ct uptake and signal induction by pinpointing individual components of the recognition and signaling pathways to better understand the immune response towards infectious pathogens.

The incidence of paediatric fractures among winter sport athletes is not adequately studied. Our objective was to categorize fractures that occurred in paediatric skiers and snowboarders at a single ski resort. X-rays of 756 skiers/snowboarders aged 3–17 diagnosed with a fracture were categorized using the Salter-Harris (SH) classification. SH fractures were seen in 158 (21%) patients, with 123 (77%) being Type II. There were no significant differences between patients with a SH fracture and patients with a non-SH fracture for age, sex, snowboarding or skiing, mechanism of injury, terrain or the resort conditions on the day of injury. The most common mechanism of injury was falling onto snow while collisions resulted in more severe injuries. Compared to fractures without growth plate involvement, a higher proportion of SH fractures were seen in the humerus, radius, fibula and thumb; a lower proportion of SH fractures were observed at the tibia and clavicle. [ABSTRACT FROM AUTHOR]

The Dunkin Hartley is the most common guinea pig strain used in biomedical research, particularly for studies of asthma, allergy, infectious disease, reproduction, and osteoarthritis. Minimally invasive blood tests, such as complete blood counts and serum biochemistry profiles, are often collected for diagnostics and laboratory analyses. However, reference intervals for these assays have not yet been well-documented in this strain. The purpose of this study was to establish reference intervals for hematologic and biochemical parameters of Dunkin Hartley guinea pigs and determine age- and sex-related differences. Hematologic and biochemical parameters were retrospectively obtained from 145 male and 68 female guinea pigs between 2 and 15 months of age. All blood parameters were analyzed by a veterinary clinical pathology laboratory. Reference intervals were established according to the American Society for Veterinary Clinical Pathology guidelines. Age- and sex-related differences were determined using unpaired t-tests or nonparametric Mann-Whitney tests. Hematocrit, red blood cell distribution width, mean platelet volume, white blood cell count, heterophils, monocytes, eosinophils, glucose, blood urea nitrogen, creatinine, calcium, magnesium, total protein, albumin, globulin, cholesterol, aspartate aminotransferase, gamma glutamyl transferase, and bicarbonate increased with age. Mean corpuscular hemoglobin concentration, cellular hemoglobin concentration mean, platelets, lymphocytes, phosphorus, albumin/globulin ratio, alkaline phosphatase, anion gap, and calculated osmolality decreased with age. Males had higher hemoglobin, hematocrit, red blood cell count, mean corpuscular hemoglobin concentration, white blood cell count, heterophils, Foa-Kurloff cells, alanine aminotransferase, and bicarbonate and lower mean corpuscular volume, red blood cell distribution width, platelets, mean platelet volume, eosinophils, total protein, albumin, globulin, cholesterol, potassium, anion gap, calculated osmolality, and iron compared to females. Establishing age and sex differences in hematologic and biochemical parameters of Dunkin Hartley guinea pigs provides valuable insight into their physiology to better evaluate diagnostics and experimental results.

The gut microbiota plays a pivotal role in the conversion of dietary flavonoids, which can affect their bioavailability and bioactivity and thereby their health-promoting properties. The ability of flavonoids to metabolically-activate the microbiota has, however, not been systematically evaluated. In the present study, we used a fluorescence-based single-cell activity measure [biorthogonal non-canonical ammino acid-tagging (BONCAT)] combined with fluorescence activated cell sorting (FACS) to determine which microorganisms are metabolically-active after amendment of the flavonoid rutin. We performed anaerobic incubations of human fecal microbiota amended with rutin and in the presence of the cellular activity marker L-azidohomoalanine (AHA) to detect metabolically-active cells. We found that 7.3% of cells in the gut microbiota were active after a 6 h incubation and 26.9% after 24 h. We then sorted BONCAT-positive cells and observed an enrichment of Lachnospiraceae (Lachnoclostridium and Eisenbergiella), Enterobacteriaceae, Tannerellaceae, and Erysipelotrichaceae species in the rutin-responsive fraction of the microbiota. There was marked inter-individual variability in the appearance of rutin conversion products after incubation with rutin. Consistent with this, there was substantial variability in the abundance of rutin-responsive microbiota among different individuals. Specifically, we observed that Enterobacteriaceae were associated with conversion of rutin into quercetin-3-glucoside (Q-glc) and Lachnospiraceae were associated with quercetin (Q) production. This suggests that individual microbiotas differ in their ability to metabolize rutin and utilize different conversion pathways.

Abstract Background Late antibody-mediated rejection (ABMR) triggered by donor-specific antibodies (DSA) is a cardinal cause of kidney allograft dysfunction and loss. Diagnostic criteria for this rejection type are well established, but effective treatment remains a major challenge. Recent randomized controlled trials (RCT) have failed to demonstrate the efficacy of widely used therapies, such as rituximab plus intravenous immunoglobulin or proteasome inhibition (bortezomib), reinforcing a great need for new therapeutic concepts. One promising target in this context may be interleukin-6 (IL-6), a pleiotropic cytokine known to play an important role in inflammation and adaptive immunity. Methods This investigator-driven RCT was designed to assess the safety and efficacy of clazakizumab, a genetically engineered humanized monoclonal antibody directed against IL-6. The study will include 20 DSA-positive kidney allograft recipients diagnosed with ABMR ≥ 365 days after transplantation. Participants will be recruited at two study sites in Austria and Germany (Medical University of Vienna; Charité University Medicine Berlin). First, patients will enter a three-month double-blind RCT (1,1 randomization, stratification according to ABMR phenotype and study site) and will receive either clazakizumab (subcutaneous administration of 25 mg in monthly intervals) or placebo. In a second open-label part of the trial (months 4–12), all patients will receive clazakizumab at 25 mg every month. The primary endpoint is safety and tolerability. Secondary endpoints are the pharmacokinetics and pharmacodynamics of clazakizumab, its effect on drug metabolism in the liver, DSA characteristics, morphological ABMR lesions and molecular gene expression patterns in three- and 12-month protocol biopsies, serum/urinary biomarkers of inflammation and endothelial activation/injury, Torque Teno viral load as a measure of overall immunosuppression, kidney function, urinary protein excretion, as well as transplant and patient survival. Discussion Currently, there is no treatment proven to be effective in halting the progression of late ABMR. Based on the hypothesis that antagonizing the effects of IL-6 improves the outcome of DSA-positive late ABMR by counteracting DSA-triggered inflammation and B cell/plasma cell-driven alloimmunity, we suggest that our trial has the potential to provide proof of concept of a novel treatment of this type of rejection. Trial registration ClinicalTrials.gov, NCT03444103. Registered on 23 February 2018 (retrospective registration).

Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of α2-6 sialylated structures in response to hyperoxia.

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium with a biphasic developmental cycle manifesting two distinct morphological forms: infectious elementary bodies (EBs) and replicative intracellular reticulate bodies (RBs). Current standard protocols for quantification of the isolates assess infectious particles by titering inclusion-forming units, using permissive cell lines, and analyzing via immunofluorescence. Enumeration of total particle counts is achieved by counting labeled EBs/RBs using a fluorescence microscope. Both methods are time-consuming with a high risk of observer bias. For a better assessment of C. trachomatis preparations, we developed a simple and time-saving flow cytometry-based workflow for quantifying small particles, such as EBs with a size of 300 nm. This included optimization of gain and threshold settings with the addition of a neutral density filter for small-particle discrimination. The nucleic acid dye SYBR® Green I (SGI) was used together with propidium iodide and 5(6)-carboxyfluorescein diacetate to enumerate and discriminate between live and dead bacteria. We found no significant differences between the direct particle count of SGI-stained C. trachomatis preparations measured by microscopy or flow cytometry (p > 0.05). Furthermore, we completed our results by introducing a cell culture-independent viability assay. Our measurements showed very good reproducibility and comparability to the existing state-of-the-art methods, indicating that the evaluation of C. trachomatis preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of C. trachomatis preparations for downstream applications.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols. [ABSTRACT FROM AUTHOR]