Adverse reactions after intravascular iodinated contrast media administration and their management.

Endovascular interventions and diagnostic examinations using iodinated contrast media (ICM) are standard of care in current vascular medicine. Although ICM use is generally considered safe, it may be associated with adverse reactions, vary from minor disturbances to rare, but severe life-threatening complications. This position paper of European Society of Vascular Medicine integrates current knowledge and summarizes the key information related to the use of intravascular ICM, serving as recommendation on prevention and management of acute, late, and very late adverse reactions. It should help the health professionals in all fields of vascular medicine to make decisions in daily practice for safe use of contrast media.

(Poly)phenol-related gut metabotypes and human health: an update.

Dietary (poly)phenols have received great interest due to their potential role in the prevention and management of non-communicable diseases. In recent years, a high inter-individual variability in the biological response to (poly)phenols has been demonstrated, which could be related to the high variability in (poly)phenol gut microbial metabolism existing within individuals. An interplay between (poly)phenols and the gut microbiota exists, with (poly)phenols being metabolised by the gut microbiota and their metabolites modulating gut microbiota diversity and composition. A number of (poly)phenol metabolising phenotypes or metabotypes have been proposed, however, potential metabotypes for most (poly)phenols have not been investigated, and the relationship between metabotypes and human health remains ambiguous. This review presents updated knowledge on the reciprocal interaction between (poly)phenols and the gut microbiome, associated gut metabotypes, and subsequent impact on human health.

Pectic-AGP is a major form of Arabidopsis AGPs.

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca2+ signaling pathway as a Ca2+-capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. This result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

Prognostic and predictive value of ultrasound-based estimated ankle brachial pressure index at early follow-up after endovascular revascularization of chronic limb-threatening ischaemia: a prospective, single-centre, service evaluation.

Background: Ankle brachial pressure index can be estimated (eABPI) using cuffless ankle Doppler ultrasound. We evaluated the prognostic value of eABPI measured during pre- and post-procedural ultrasound exams to predict the clinical outcome after endovascular revascularisations. Methods: In this prospective, single-centre, service evaluation, consecutive patients with symptomatic peripheral artery disease undergoing lower limb endovascular revascularisations between July, 26 2018 and January, 13 2022 at Surrey and Sussex Healthcare NHS Trust (Redhill, UK) were analysed. eABPI was determined using the higher acceleration index measured with angle-corrected duplex ultrasound in ankle arteries before and ≤1 month post-procedure. Clinical outcomes (mortality, major amputations, amputation-free survival [AFS], clinically driven target lesion revascularization [cdTLR], major adverse limb events [MALE; cdTLR and major amputation], wound healing) were assessed over 1 year. Findings: Of 246 patients treated, for 219 patients (median 75 [IQR 66-83] years) pre- and post-procedural eABPI (0.50 [0.33-0.59] and 0.90 [0.69-1.0], p < 0.0001) were available, respectively. In n = 199 patients with chronic limb-threatening ischaemia (CLTI) Kaplan-Meier survival analyses showed that higher post-procedural, but not pre-procedural, eABPI was associated with favourable AFS, MALE, cdTLR, and wound healing. This was confirmed in Cox regression analysis and remained significant with adjustment for pre-procedural eABPI, age, sex, co-morbidities, treated levels, wound score, and foot infection. Whereas all clinical outcomes, except for survival, were significantly better at ≥0.7 vs <0.7, wound healing (unadjusted: HR 1.7 (95% CI 1.2-2.6), adjusted: HR 2.1 (95% CI 1.3-3.1), cdTLR, and MALE (unadjusted: HR 0.41 (95% CI 0.18-0.93), adjusted: HR 0.28 (95% CI 0.11-0.74) were significantly improved at ≥0.9 vs <0.9. Interpretation: Post-procedural eABPI can provide valid, clinically important prognostic and predictive information. Our data indicate that revascularisations should target values of at least 0.9 to achieve optimal outcomes. Future studies need to confirm generalisability and cost-effectiveness in a wider context. Funding: European Partnership on Metrology, co-financed from European Union's Horizon Europe Research and Innovation Programme and UK Research and Innovation.

Permethylation as a strategy for high-molecular-weight polysaccharide structure analysis by nuclear magnetic resonance-Case study of Xylella fastidiosa extracellular polysaccharide.

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Digitization of Follow-Up Care in Orthopedic and Trauma Surgery With Video Consultations: Health Economic Evaluation Study From a Health Provider's Perspective.

BACKGROUND: Recommendations for health care digitization as issued with the Riyadh Declaration led to an uptake in telemedicine to cope with the COVID-19 pandemic. Evaluations based on clinical data are needed to support stakeholders' decision-making on the long-term implementation of digital health. OBJECTIVE: This health economic evaluation aims to provide the first German analysis of the suitability of video consultations in the follow-up care of patients in orthopedic and trauma surgery, investigate the financial impact on hospital operations and personnel costs, and provide a basis for decisions on digitizing outpatient care. METHODS: We conducted a randomized controlled trial that evaluated video consultations versus face-to-face consultations in the follow-up care of patients in orthopedic and trauma surgery at a German university hospital. We recruited 60 patients who had previously been treated conservatively or surgically for various knee or shoulder injuries. A digital health app and a browser-based software were used to conduct video consultations. The suitability of telemedicine was assessed using the Telemedicine Satisfaction Questionnaire and the EQ-5D-5L questionnaire. Economic analyses included average time spent by physician per consultation, associated personnel costs and capacities for additional treatable patients, and the break-even point for video consultation software fees. RESULTS: After 4 withdrawals in each arm, data from a total of 52 patients (telemedicine group: n=26; control group: n=26) were used for our analyses. In the telemedicine group, 77% (20/26) of all patients agreed that telemedicine provided for their health care needs, and 69% (18/26) found telemedicine an acceptable way to receive health care services. In addition, no significant difference was found in the change of patient utility between groups after 3 months (mean 0.02, SD 0.06 vs mean 0.07, SD 0.17; P=.35). Treatment duration was significantly shorter in the intervention group (mean 8.23, SD 4.45 minutes vs mean 10.92, SD 5.58 minutes; P=.02). The use of telemedicine saved 25% (€2.14 [US $2.35]/€8.67 [US $9.53]) in personnel costs and increased the number of treatable patients by 172 annually, assuming 2 hours of video consultations per week. Sensitivity analysis for scaling up video consultations to 10% of the hospital's outpatient cases resulted in personnel cost savings of €73,056 (US $ 80,275.39) for a senior physician. A total of 23 video consultations per month were required to recoup the software fees of telemedicine through reduced personnel costs (break-even point ranging from 12-38 in the sensitivity analysis). CONCLUSIONS: Our study supports stakeholders' decision-making on the long-term implementation of digital health by demonstrating that video consultations in the follow-up care of patients in orthopedic and trauma surgery result in cost savings and productivity gains for clinics with no negative impact on patient utility. TRIAL REGISTRATION: German Clinical Trials Register DRKS00023445; https://drks.de/search/en/trial/DRKS00023445.

A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate.

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novel wzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4- linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higher Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties.

Comparison of Nanocrystalline Hydroxyapatite Bone Graft with Empty Defects in Long Bone Fractures: A Retrospective Case-Control Study.

BACKGROUND The regeneration of bone defects is indicated to restore lost tissue mass and functionality. Ostim®, an absorbable nanocrystalline hydroxyapatite (NCHA) paste, is indicated to enhance bone regeneration in bone defects due to trauma or surgery. This retrospective study of 110 patients with long-bone fracture defects presenting at a single trauma center between 2010 and 2012 aimed to compare outcomes with and without the use of Ostim® absorbable nanocrystalline hydroxyapatite paste. MATERIAL AND METHODS The study encompassed fractures in 110 patients - 55 patients received any defect augmentation (ED) and 55 patients were treated with NCHA augmentation. Fractures were located at the distal radius (66.4%, n=73), proximal humerus (5.5%, n=6), and proximal tibia (28.2%, n=31). Evaluating the clinical follow-up, the study encompassed post-surgery complications (eg, non-unions, infection). Bone healing was evaluated by conventional radiographs. RESULTS Postoperative complications occurred in 45.5% of patients regardless of the treatment (P=1.0). The non-union rate in both groups was 5.5% (n=8, P=1.0), and the risk for infection was lower in the NCHA group (3.6%, ED: n=3, NCHA: n=1, p=0.62). Patients suffered open fractures were treated in the NCHA group (100%, n=7, P=0.003). Radiological assessment demonstrated comparable healing of the fracture border, fracture gap, and articular surface (P>0.05). CONCLUSIONS The findings from this retrospective study support previous studies that have shown Ostim® absorbable nanocrystalline hydroxyapatite paste enhances outcomes and reduces the risk of complications when used to repair bone defects in long-bone fractures in trauma patients. NCHA paste augmentation is suitable for use in traumatic long-bone fractures.

Quantitative assessment of angioplasty-induced vascular inflammation with 19F cardiovascular magnetic resonance imaging.

BACKGROUND: Macrophages play a pivotal role in vascular inflammation and predict cardiovascular complications. Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorocarbon allows a background-free direct quantification of macrophage abundance in experimental vascular disease models in mice. Recently, perfluorooctyl bromide-nanoemulsion (PFOB-NE) was applied to effectively image macrophage infiltration in a pig model of myocardial infarction using clinical MRI scanners. In the present proof-of-concept approach, we aimed to non-invasively image monocyte/macrophage infiltration in response to carotid artery angioplasty in pigs using 19F MRI to assess early inflammatory response to mechanical injury. METHODS: In eight minipigs, two different types of vascular injury were conducted: a mild injury employing balloon oversize angioplasty only (BA, n = 4) and a severe injury provoked by BA in combination with endothelial denudation (BA + ECDN, n = 4). PFOB-NE was administered intravenously three days after injury followed by 1H and 19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using X-ray angiography, intravascular ultrasound and immunohistology in at least 10 segments per carotid artery. RESULTS: Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and concomitant infiltration of monocytes/macrophages. No severe adverse reactions were observed following PFOB-NE administration. In vivo 19F signals were only detected in the four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8. Ex vivo analysis revealed a linear correlation of 19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was estimated at approximately 410 cells/mm2. CONCLUSIONS: In this proof-of-concept study, 19F MRI enabled quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This may provide the opportunity to non-invasively monitor vascular inflammation with MRI in patients after angioplasty or even in atherosclerotic plaques.

Mesoporous Bioactive Glass-Incorporated Injectable Strontium-Containing Calcium Phosphate Cement Enhanced Osteoconductivity in a Critical-Sized Metaphyseal Defect in Osteoporotic Rats.

In this study, the in vitro and in vivo bone formation behavior of mesoporous bioactive glass (MBG) particles incorporated in a pasty strontium-containing calcium phosphate bone cement (pS100G10) was studied in a metaphyseal fracture-defect model in ovariectomized rats and compared to a plain pasty strontium-containing calcium phosphate bone cement (pS100) and control (empty defect) group, respectively. In vitro testing showed good cytocompatibility on human preosteoblasts and ongoing dissolution of the MBG component. Neither the released strontium nor the BMG particles from the pS100G10 had a negative influence on cell viability. Forty-five female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) pS100 (n = 15), (2) pS100G10 (n = 15), and (3) empty defect (n = 15). Twelve weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur in all animals. The originated fracture-defect was substituted with pS100 or pS100G10 or left empty. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the pS100G10 group compared to the pS100 (p = 0.03) and empty defect groups (p = 0.0001), indicating enhanced osteoconductivity with the incorporation of MBG. Immunohistochemistry revealed a significant decrease in the RANKL/OPG ratio for pS100 (p = 0.004) and pS100G10 (p = 0.003) compared to the empty defect group. pS100G10 showed a statistically higher expression of BMP-2. In addition, a statistically significant higher gene expression of alkaline phosphatase, osteoprotegerin, collagen1a1, collagen10a1 with a simultaneous decrease in RANKL, and carbonic anhydrase was seen in the pS100 and pS100G10 groups compared to the empty defect group. Mass spectrometric imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) showed the release of Sr2+ ions from both pS100 and pS100G10, with a gradient into the interface region. ToF-SIMS imaging also revealed that resorption of the MBG particles allowed for new bone formation in cement pores. In summary, the current work shows better bone formation of the injectable pasty strontium-containing calcium phosphate bone cement with incorporated mesoporous bioactive glass compared to the bioactive-free bone cement and empty defects and can be considered for clinical application for osteopenic fracture defects in the future.

Traumatic Fracture Treatment: Calcium Phosphate Bone Substitute Case-Control Study in Humerus, Radius, Tibia Fractures-Assessing Efficacy and Recovery Outcomes.

To date, insufficient investigation has been carried out on the biocompatibility of synthetic bioactive bone substitute materials after traumatically induced bone fractures in clinical conditions. This study encompasses the safety, resorption, healing process, and complications of surgical treatment. Our current hypothesis posits that calcium phosphate-based bone substitutes could improve bone healing. In this retrospective case-control study, over 290 patients who underwent surgical treatment for acute fractures were examined. Bone defects were augmented with calcium phosphate-based bone substitute material (CP) in comparison to with empty defect treatment (ED) between 2011 and 2018. A novel scoring system for fracture healing was introduced to assess bone healing in up to six radiological follow-up examinations. Furthermore, demographic data, concomitant diseases, and complications were subjected to analysis. Data analysis disclosed significantly fewer postoperative complications in the CP group relative to the ED group (p < 0.001). The CP group revealed decreased risks of experiencing complications (p < 0.001), arthrosis (p = 0.01), and neurological diseases (p < 0.001). The fracture edge, the fracture gap, and the articular surface were definably enhanced. Osteosynthesis and general bone density demonstrated similarity (p > 0.05). Subgroup analysis focusing on patients aged 64 years and older revealed a diminished complication incidence within the CP group (p = 0.025). Notably, the application of CP bone substitute materials showed discernible benefits in geriatric patients, evident by decreased rates of pseudarthrosis (p = 0.059). Intermediate follow-up evaluations disclosed marked enhancements in fracture gap, edge, and articular surface conditions through the utilization of CP-based substitutes (p < 0.05). In conclusion, calcium phosphate-based bone substitute materials assert their clinical integrity by demonstrating safety in clinical applications. They substantiate an accelerated early osseous healing trajectory while concurrently decreasing the severity of complications within the bone substitute cohort. In vivo advantages were demonstrated for CP bone graft substitutes.

Analysis of endovascular therapy for peripheral arterial disease in all German hospitals.

Background: The quality of vascular care has significantly improved in part by the expansion of endovascular techniques for the treatment of symptomatic peripheral artery disease (PAD) in recent years. In Germany these are primarily provided by the three disciplines of vascular surgery, angiology, and interventional radiology (IR). However, the relative contribute of angiologists to the total number of cases performed is unknown. Patients and methods: In the present study, we analysed the respective contribution of vascular surgery, angiology, and IR to the delivery of endovascular revascularisations in symptomatic PAD in Germany based on the legally mandatory quality reports representative for the reporting year 2018. Results: Vascular surgery is the most common speciality reporting procedures in German hospitals (n=579; 25.1%), followed by IR (n=264; 11.5%), angiology (n=189; 8.2%) and cardiology (n=17; 0.7%). The combination of vascular surgery and IR was reported in 202 (8.8%), vascular surgery and angiology in 167 (7.2%) and angiology and IR in 65 (2.8%) hospitals, and 63 (2.7%) hospitals reported the combination of all three disciplines. Not every department performed catheter interventions. The analysis of procedures per centre revealed that angiology centres provided the highest numbers for both basic procedures and more complex techniques such as atherectomy, rotational thrombectomy, lithoplasty, selective thrombolysis or the use of re-entry devices. In total, angiology centres provided 24.4% of the total procedures or 23.9% of the so-called basic procedures as a surrogate for patient numbers. Conclusions: While each of the disciplines contribute significantly to the endovascular procedures, angiology centres perform more procedures per centre and more complex procedures than the other disciplines highlighting the important quantitative and qualitative contribution of angiology specialists to the care of vascular patients. The inpatient catheter interventional care of patients with PAD is still too rarely carried out in a multi-disciplinary manner in Germany.

Xylella fastidiosa modulates exopolysaccharide polymer length and the dynamics of biofilm development with a ß-1,4-endoglucanase.

IMPORTANCE: It is well established that exopolysaccharide (EPS) is an integral structural component of bacterial biofilms necessary for assembly and maintenance of the three-dimensional architecture of the biofilm. However, the process and role of EPS turnover within a developing biofilm is not fully understood. Here, we demonstrated that Xylella fastidiosa uses a self-produced endoglucanase to enzymatically process its own EPS to modulate EPS polymer length. This enzymatic processing of EPS dictates the early stages of X. fastidiosa's biofilm development, which, in turn, affects its behavior in planta. A deletion mutant that cannot produce the endoglucanase was hypervirulent, thereby linking enzymatic processing of EPS to attenuation of virulence in symptomatic hosts, which may be a vestige of X. fastidiosa's commensal behavior in many of its other non-symptomatic hosts.

Retrospective Analysis of Bone Substitute Material for Traumatic Long Bone Fractures: Sex-Specific Outcomes.

Male patients often experience increased bone and muscle loss after traumatic fractures. This study aims to compare the treatment outcomes of male and female patients with large bone defects. A total of 345 trauma patients underwent surgery, with participants divided into two groups: one receiving bone substitute material (BSM) for augmented defects (n = 192) and the other without augmentation (empty defects = ED, n = 153). Outcome parameters were assessed among female (n = 184) and male (n = 161) patients. Descriptive statistics revealed no significant differences between male and female patients. Approximately one-half of the fractures resulted from high-energy trauma (n = 187). The BSM group experienced fewer complications (p = 0.004), including pseudarthrosis (BSM: n = 1, ED: n = 7; p = 0.02). Among female patients over 65, the incidence of pseudarthrosis was lower in the BSM group (p = 0.01), while younger females showed no significant differences (p = 0.4). Radiologically, we observed premature bone healing with subsequent harmonization. Post hoc power analysis demonstrated a power of 0.99. Augmenting bone defects, especially with bone substitute material, may reduce complications, including pseudarthrosis, in female patients. Additionally, this material accelerates bone healing. Further prospective studies are necessary for confirmation.

Duckweed pectic-arabinogalactan-proteins can crosslink through borate diester bonds.

Material containing pectin and arabinogalactan-protein (AGP) was released and purified from Spirodela alcohol insoluble residues. Results of carbohydrate analyses and two-dimensional NMR spectroscopy suggest that this material is composed of apiogalacturonan and rhamnogalacturonan-I covalently attached to AGPs. 11B NMR spectroscopy indicated that some of the glycoses in this complex exist as their boric acid monoesters. Borate diesters were formed when the pectic-AGPs were allowed to react at pH above 6.2 with the boron-depleted pectic-AGPs, suggesting that in vitro two pectic-AGP molecules can crosslink to one another through borate. Borate diesters also formed when the pectic-AGPs were incubated with monomeric rhamnogalacturonan-II in the presence of Pb2+ ion at pH 9.2. This data presents evidence of the first wall polymer after rhamnogalacturonan-II to crosslink through borate diesters. We suggest that the formation of these borate-crosslinks may help Spirodela respond to high-pH condition.

Cocoa flavanols improve peakVO2 and exercise capacity in a randomized double blinded clinical trial in healthy elderly people.

Background: Loss of functional capacity is one of the hallmarks in cardiovascular aging. Cocoa flavanols (CF) exert favorable effects on endothelial function, blood pressure, and inflammation. These cardiovascular health markers worsen with increasing age and limit functional exercise capacity. Aim: To investigate the effect of CF on cardiorespiratory-fitness in healthy elderly people. Methods: In a randomized, double-masked, placebo-controlled, parallel-group dietary intervention trial, 68 healthy elderly people (55-79 years, 28 female) received either 500 mg of CF or a nutrient-matched control capsule twice a day for 30 days. Primary endpoint was defined as peak oxygen consumption (VO2) in a cardiopulmonary exercise test (CPET). Secondary endpoints were oxygen pulse (VO2 per heart rate (HR)), resting blood pressure (BP), and resting vascular function. Results: After 30 days of CF intake peakVO2 increased by 190 ml min-1 (95% CI 1-371 ml min-1) and peakVO2 per kg by 2.5 ml (min kg)-1 (95% CI 0.30-4.2 ml (min kg)-1). O2-pulse increased by 1.7 ml (95% CI 0.29-3.2 ml) and max exercise capacity by 9.6 W (95% CI 2.1-17.7 W). CF decreased resting systolic and diastolic BP by 5.4 mmHg (95% CI -10.7 to -0.1 mmHg) and 2.9 mmHg (95% CI -5.5 to -0.4 mmHg), respectively. Flow-mediated vasodilation (FMD) increased by an absolute 1.3% (95% CI 0.76-1.79%) in the CF group. Indexes of pulmonary function were not affected. No changes for primary and secondary endpoints were detected in control. Conclusion: CF substantially improve markers of cardiorespiratory fitness in healthy elderly humans highlighting their potential to preserve cardiovascular health with increasing age.

Genetic Characterization of the Acidic and Neutral Glycosphingolipid Biosynthetic Pathways in Neurospora crassa.

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.

Characterization of the need for galactofuranose during the Neurospora crassa life cycle.

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases.

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides.

Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.