Lipid network and moiety analysis for revealing enzymatic dysregulation and mechanistic alterations from lipidomics data.

Lipidomics is of growing importance for clinical and biomedical research due to many associations between lipid metabolism and diseases. The discovery of these associations is facilitated by improved lipid identification and quantification. Sophisticated computational methods are advantageous for interpreting such large-scale data for understanding metabolic processes and their underlying (patho)mechanisms. To generate hypothesis about these mechanisms, the combination of metabolic networks and graph algorithms is a powerful option to pinpoint molecular disease drivers and their interactions. Here we present lipid network explorer (LINEX$^2$), a lipid network analysis framework that fuels biological interpretation of alterations in lipid compositions. By integrating lipid-metabolic reactions from public databases, we generate dataset-specific lipid interaction networks. To aid interpretation of these networks, we present an enrichment graph algorithm that infers changes in enzymatic activity in the context of their multispecificity from lipidomics data. Our inference method successfully recovered the MBOAT7 enzyme from knock-out data. Furthermore, we mechanistically interpret lipidomic alterations of adipocytes in obesity by leveraging network enrichment and lipid moieties. We address the general lack of lipidomics data mining options to elucidate potential disease mechanisms and make lipidomics more clinically relevant.

MoSBi: Automated signature mining for molecular stratification and subtyping.

The improving access to increasing amounts of biomedical data provides completely new chances for advanced patient stratification and disease subtyping strategies. This requires computational tools that produce uniformly robust results across highly heterogeneous molecular data. Unsupervised machine learning methodologies are able to discover de novo patterns in such data. Biclustering is especially suited by simultaneously identifying sample groups and corresponding feature sets across heterogeneous omics data. The performance of available biclustering algorithms heavily depends on individual parameterization and varies with their application. Here, we developed MoSBi (molecular signature identification using biclustering), an automated multialgorithm ensemble approach that integrates results utilizing an error model-supported similarity network. We systematically evaluated the performance of 11 available and established biclustering algorithms together with MoSBi. For this, we used transcriptomics, proteomics, and metabolomics data, as well as synthetic datasets covering various data properties. Profiting from multialgorithm integration, MoSBi identified robust group and disease-specific signatures across all scenarios, overcoming single algorithm specificities. Furthermore, we developed a scalable network-based visualization of bicluster communities that supports biological hypothesis generation. MoSBi is available as an R package and web service to make automated biclustering analysis accessible for application in molecular sample stratification.

Kupffer cells are protective in alcoholic steatosis.

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases.

The lack of retropatellar resurfacing at index surgery is significantly associated with failure in patients following patellofemoral inlay arthroplasty: a multi-center study of more than 260 patients.

PURPOSE: To evaluate the clinical outcomes of patients with a minimum 2-year follow-up following contemporary patellofemoral inlay arthroplasty (PFIA) and to identify potential risk factors for failure in a multi-center study. METHODS: All patients who underwent implantation of PFIA between 09/2009 and 11/2016 at 11 specialized orthopedic referral centers were enrolled in the study and were evaluated retrospectively at a minimum 2-year follow-up. Clinical outcomes included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Tegner Scale, the visual analogue scale (VAS) for pain, and subjective patient satisfaction. Pre- and perioperative risk factors were compared among failures and non-failures to determine potential risk factors. RESULTS: A total of 263 patients (85% follow-up rate) could be enrolled. The mean age at the time of index surgery was 49 ± 12 years with a mean postoperative follow-up of 45 ± 18 months. The overall failure rate was 11% (28 patients), of which 18% (5 patients) were patients with patella resurfacing at index surgery and 82% (23 patients) were patients without initial patella resurfacing. At final follow-up, 93% of the patients who did not fail were satisfied with the procedure with a mean transformed WOMAC Score of 84.5 ± 14.5 points, a mean KOOS Score of 73.3 ± 17.1 points, a mean Tegner Score of 3.4 ± 1.4 points and a mean VAS pain of 2.4 ± 2.0 points. An increased BMI was significantly correlated with a worse postoperative outcome. Concomitant procedures addressing patellofemoral instability or malalignment, the lack of patellofemoral resurfacing at the index surgery and a high BMI were significantly correlated with failure in our patient cohort. CONCLUSION: Patellofemoral inlay arthroplasty shows high patient satisfaction with good functional outcomes at short-term follow-up and thus can be considered a viable treatment option in young patients suffering from isolated patellofemoral arthritis. Patellar resurfacing at index surgery is recommended to decrease the risk of failure. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Investigating Global Lipidome Alterations with the Lipid Network Explorer.

Lipids play an important role in biological systems and have the potential to serve as biomarkers in medical applications. Advances in lipidomics allow identification of hundreds of lipid species from biological samples. However, a systems biological analysis of the lipidome, by incorporating pathway information remains challenging, leaving lipidomics behind compared to other omics disciplines. An especially uncharted territory is the integration of statistical and network-based approaches for studying global lipidome changes. Here we developed the Lipid Network Explorer (LINEX), a web-tool addressing this gap by providing a way to visualize and analyze functional lipid metabolic networks. It utilizes metabolic rules to match biochemically connected lipids on a species level and combine it with a statistical correlation and testing analysis. Researchers can customize the biochemical rules considered, to their tissue or organism specific analysis and easily share them. We demonstrate the benefits of combining network-based analyses with statistics using publicly available lipidomics data sets. LINEX facilitates a biochemical knowledge-based data analysis for lipidomics. It is availableas a web-application and as a publicly available docker container.

Nonalcoholic fatty liver disease stratification by liver lipidomics.

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic dysfunction leading to hepatic steatosis. However, NAFLD's global impact on the liver lipidome is poorly understood. Using high-resolution shotgun mass spectrometry, we quantified the molar abundance of 316 species from 22 major lipid classes in liver biopsies of 365 patients, including nonsteatotic patients with normal or excessive weight, patients diagnosed with NAFL (nonalcoholic fatty liver) or NASH (nonalcoholic steatohepatitis), and patients bearing common mutations of NAFLD-related protein factors. We confirmed the progressive accumulation of di- and triacylglycerols and cholesteryl esters in the liver of NAFL and NASH patients, while the bulk composition of glycerophospho- and sphingolipids remained unchanged. Further stratification by biclustering analysis identified sphingomyelin species comprising n24:2 fatty acid moieties as membrane lipid markers of NAFLD. Normalized relative abundance of sphingomyelins SM 43:3;2 and SM 43:1;2 containing n24:2 and n24:0 fatty acid moieties, respectively, showed opposite trends during NAFLD progression and distinguished NAFL and NASH lipidomes from the lipidome of nonsteatotic livers. Together with several glycerophospholipids containing a C22:6 fatty acid moiety, these lipids serve as markers of early and advanced stages of NAFL.

Lessons from the COVID-19 pandemic for advancing computational drug repurposing strategies.

Responding quickly to unknown pathogens is crucial to stop uncontrolled spread of diseases that lead to epidemics, such as the novel coronavirus, and to keep protective measures at a level that causes as little social and economic harm as possible. This can be achieved through computational approaches that significantly speed up drug discovery. A powerful approach is to restrict the search to existing drugs through drug repurposing, which can vastly accelerate the usually long approval process. In this Review, we examine a representative set of currently used computational approaches to identify repurposable drugs for COVID-19, as well as their underlying data resources. Furthermore, we compare drug candidates predicted by computational methods to drugs being assessed by clinical trials. Finally, we discuss lessons learned from the reviewed research efforts, including how to successfully connect computational approaches with experimental studies, and propose a unified drug repurposing strategy for better preparedness in the case of future outbreaks.

Exploring the SARS-CoV-2 virus-host-drug interactome for drug repurposing.

Coronavirus Disease-2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Various studies exist about the molecular mechanisms of viral infection. However, such information is spread across many publications and it is very time-consuming to integrate, and exploit. We develop CoVex, an interactive online platform for SARS-CoV-2 host interactome exploration and drug (target) identification. CoVex integrates virus-human protein interactions, human protein-protein interactions, and drug-target interactions. It allows visual exploration of the virus-host interactome and implements systems medicine algorithms for network-based prediction of drug candidates. Thus, CoVex is a resource to understand molecular mechanisms of pathogenicity and to prioritize candidate therapeutics. We investigate recent hypotheses on a systems biology level to explore mechanistic virus life cycle drivers, and to extract drug repurposing candidates. CoVex renders COVID-19 drug research systems-medicine-ready by giving the scientific community direct access to network medicine algorithms. It is available at https://exbio.wzw.tum.de/covex/.

Synthesis and µ-Opioid Activity of the Primary Metabolites of Carfentanil.

Carfentanil is a synthetic opioid significantly more potent than clinically prescribed fentanyl. The primary metabolites of carfentanil, generated from human liver microsomes, were structurally confirmed through chemical synthesis. The synthesized compounds were evaluated for µ-opioid receptor (MOR) functional activity. Of the six metabolites assayed, a major metabolite showed comparable activity to the parent opioid. Three other metabolites showed significant MOR functional activity. The availability of the metabolites could aid improvements in the analysis of biomedical samples obtained from suspected human exposures to carfentanil and development of treatment protocols.

The evolutionary convergence of mid-Mesozoic lacewings and Cenozoic butterflies.

Mid-Mesozoic kalligrammatid lacewings (Neuroptera) entered the fossil record 165 million years ago (Ma) and disappeared 45 Ma later. Extant papilionoid butterflies (Lepidoptera) probably originated 80-70 Ma, long after kalligrammatids became extinct. Although poor preservation of kalligrammatid fossils previously prevented their detailed morphological and ecological characterization, we examine new, well-preserved, kalligrammatid fossils from Middle Jurassic and Early Cretaceous sites in northeastern China to unravel a surprising array of similar morphological and ecological features in these two, unrelated clades. We used polarized light and epifluorescence photography, SEM imaging, energy dispersive spectrometry and time-of-flight secondary ion mass spectrometry to examine kalligrammatid fossils and their environment. We mapped the evolution of specific traits onto a kalligrammatid phylogeny and discovered that these extinct lacewings convergently evolved wing eyespots that possibly contained melanin, and wing scales, elongate tubular proboscides, similar feeding styles, and seed-plant associations, similar to butterflies. Long-proboscid kalligrammatid lacewings lived in ecosystems with gymnosperm-insect relationships and likely accessed bennettitalean pollination drops and pollen. This system later was replaced by mid-Cretaceous angiosperms and their insect pollinators.

Hemoglobin-derived porphyrins preserved in a Middle Eocene blood-engorged mosquito.

Although hematophagy is found in ~14,000 species of extant insects, the fossil record of blood-feeding insects is extremely poor and largely confined to specimens identified as hematophagic based on their taxonomic affinities with extant hematophagic insects; direct evidence of hematophagy is limited to four insect fossils in which trypanosomes and the malarial protozoan Plasmodium have been found. Here, we describe a blood-engorged mosquito from the Middle Eocene Kishenehn Formation in Montana. This unique specimen provided the opportunity to ask whether or not hemoglobin, or biomolecules derived from hemoglobin, were preserved in the fossilized blood meal. The abdomen of the fossil mosquito was shown to contain very high levels of iron, and mass spectrometry data provided a convincing identification of porphyrin molecules derived from the oxygen-carrying heme moiety of hemoglobin. These data confirm the existence of taphonomic conditions conducive to the preservation of biomolecules through deep time and support previous reports of the existence of heme-derived porphyrins in terrestrial fossils.

Do we need synthetic osteotomy augmentation materials for opening-wedge high tibial osteotomy.

High tibial osteotomy (HTO) is an increasing popular method to treat unicompartimental osteoarthritis of the knee in younger, active patients. In so doing one tries to delay the need for total or unicompartimental joint replacement. The augmentation of HTO opening gaps with supporting material is discussed controversially, especially after the introduction of locking plates, which contribute to the decline of the non-union rate. Currently, we do not recommend synthetic augmentation, when using locking plates in HTO with opening angles less than 10 degrees . In our recent randomized study we could histologically and radiologically demonstrate the complete rebuilding of lamelliform bone in patients without synthetic augmentation, whilst bony ingrowth into the hydroxyapatite/tricalcium phosphate (HA/TCP) wedge of augmented osteotomies just slowly progressed. In contrast to unaugmented osteotomies, there was no advantage in using HA/TCP wedges or the combination of HA/TCP wedges and platelet rich plasma (PRP) as supporting material after 12 months. In osteotomies where an opening angle bigger than 7.5 degrees is chosen, rigid locking plates should be used. In our opinion, autologous iliac crest graft should be used in the high-risk patients (obese, smoker, opening angle bigger than 10 degrees ). Whether synthetic augmentation combined with PRP is equal or even superior to autologous iliac crest graft in openings bigger than 10 degrees has not been proven yet.

The reverse shoulder prosthesis for primary and secondary treatment of proximal humeral fractures: a case report.

Humeral head fractures are very common among elderly people, often requiring shoulder arthroplasty. One requirement for good function after shoulder arthroplasty is an intact or at least reparable rotator cuff. In patients with multifractured and or osteoporotic tuberosities, refixation leads to the potential risk of redislocation and resorption of the tuberosities or coexisting irreparable rotator cuff tears lead to a high failure rate after implantation of traditional fracture prosthesis, whereas the reverse shoulder arthroplasty may provide better outcome. Here we report of a 79-year-old woman, who fractured both humeral heads at different times. Her right side was treated with a fracture prosthesis, which had to be converted after 2 years to a reverse shoulder prosthesis. Because of coexisting irreversible rotator cuff tear accompanying the second humeral head fracture on her left side, this patient was primarily treated with a reverse shoulder prosthesis. During the most recent follow up, 33 months after reverse shoulder arthroplasty on the left side and 39 months on the right side, the age- and gender-adapted constant score was 88 compared to 59 on the right side. The primary or secondary implantation of the reverse shoulder prosthesis in proximal humeral fractures has to be planned carefully, since long-term results are still lacking and treatment options after failed reverse shoulder arthroplasty are few. Generally, primary implantation of traditional fracture prosthesis is indicated in most cases of humeral head fractures; but in carefully selected cases primary reverse shoulder arthroplasty may be superior and lead to better outcome. Therefore, future research should be conducted to find criteria where the reverse shoulder arthroplasty is indicated as first line treatment of proximal humeral head fractures in elderly patients.

Prospective randomized clinical comparison of femoral transfixation versus bioscrew fixation in hamstring tendon ACL reconstruction--a preliminary report.

PURPOSE: The purpose of this study is to clinically evaluate hamstring tendon anterior cruciate ligament (ACL)-reconstruction using femoral fixation with bioresorbable interference screws and with a bioresorbable transfixation device. HYPOTHESIS: The ACL-reconstruction using the transfixation device at the femoral side leads to less knee laxity and therefore to a better clinical outcome for the patient. TYPE OF STUDY: Prospective randomized clinical outcome study. METHODS: From February 2002 to December 2002, a total of 68 patients with hamstring ACL reconstruction using a femoral fixation once with TransFix (n=38; m:22 and f:16; median age=28.5 range 15-47) and the second with bioscrew (BS) (n=30; m:20, f:10; median age=25.5 range 13-61) completed the follow-up period. Patients in each group got a clinical assessment at 3, 6, and 12 months after surgery. The measurement of anterior translation of the tibia has been performed using the Rolimeter device. RESULTS: No significant differences in the knee laxity testing using the Rolimeter device were seen between both groups and over time within these groups. Ninety percent of all patients had functionally normal or near normal International Knee Documentation Committee (IKDC) knee ligament ratings. The TF-group included 17 grade A, 19 grade B, and 2 grade C knees, and the BS-group had 12 grade A, 13 grade B, and 5 grade C knees. The IKDC rating, the OAK-score, the Tegner-activity-score, and the Lysholm-score did not show significant differences between the TF-group and the BS-group. CONCLUSION: We disproved our hypothesis that the transfixation technique leads to less laxity and therefore to a better clinical outcome when compared to the use of BS. The clinical results in this study clarified that this technique is an effective and safe method for femoral hamstring fixation in ACL-reconstruction. However, this technique revealed no advantage compared to the bioscrew fixation technique within the short-term follow-up.

Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells.

OBJECTIVE: Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle exhibit long-time proliferation, high self-renewal, and multipotent differentiation. This study was undertaken to investigate the ability of MDSCs that were retrovirally transduced to express bone morphogenetic protein 4 (BMP-4) to differentiate into chondrocytes in vitro and in vivo and enhance articular cartilage repair. METHODS: Using monolayer and micromass pellet culture systems, we evaluated the in vitro chondrogenic differentiation of LacZ- and BMP-4-transduced MDSCs with or without transforming growth factor beta1 (TGFbeta1) stimulation. We used a nude rat model of a full-thickness articular cartilage defect to assess the duration of LacZ transgene expression and evaluate the ability of transplanted cells to acquire a chondrocytic phenotype. We evaluated cartilage repair macroscopically and histologically 4, 8, 12, and 24 weeks after surgery, and performed histologic grading of the repaired tissues. RESULTS: BMP-4-expressing MDSCs acquired a chondrocytic phenotype in vitro more effectively than did MDSCs expressing only LacZ; the addition of TGFbeta1 did not alter chondrogenic differentiation of the BMP-4-transduced MDSCs. LacZ expression within the repaired tissue continued for up to 12 weeks. Four weeks after surgery, we detected donor cells that coexpressed beta-galactosidase and type II collagen. Histologic scoring of the defect sites 24 weeks after transplantation revealed significantly better cartilage repair in animals that received BMP-4-transduced MDSCs than in those that received MDSCs expressing only LacZ. CONCLUSION: Local delivery of BMP-4 by genetically engineered MDSCs enhanced chondrogenesis and significantly improved articular cartilage repair in rats.

The autologous osteochondral transplantation of the knee: clinical results, radiographic findings and histological aspects.

INTRODUCTION: The osteochondral transplantation (OCT) is a well accepted treatment option for focal cartilage lesions in the knee joint, whereas the fate of the transplanted cartilage is still unclear and the clinical outcome is variable. The purpose of this study was to evaluate the histological character of autologous transplanted cartilage and to correlate technical aspects and the patients' history with the clinical outcome. MATERIAL AND METHODS: The OCT was performed in 27 patients (median age of 32 (22-43) years) with a focal chondral lesion at the medial femoral condyle. We investigated the clinical outcome after a median follow-up of 13.5 (5-28) months using the Lysholm-score and the integration of the transplanted plugs using an MRI-scoring system. Biopsy specimens from representative patients (n = 8) were evaluated with histological staining and immunohistochemistry. RESULTS: The median Lysholm-score was 80 (range 45-98). The wide range of the Lysholm-score in clinical outcome did not show significant differences in: follow-up, concomitant injuries, defect size or genesis. The MRI analysis revealed in all cases a regular osseous integration of the subchondral bone, but a failed chondral integration. The congruency of the plugs to the joint surface was often incorrect, however a correlation between the MRI-score and the clinical outcome could not be shown. Histology of the transplanted cartilage revealed small changes in immunohistochemistry after a relatively short-term follow-up, whereas the cartilage has still the typical hyaline character. Often, the surrounding cartilage consists of fibrous and granulation tissue. CONCLUSION: The congruency of the joint surface can not be restored to the original status, particularly in larger defects with irregular shapes. However, we did not find any aspects which affected the function of the knee joint following OCT. It can be assumed that remaining lesions at the surrounding cartilage could maintain the inflammatory process and therefore maintain the pain and a low knee function. Further investigations are needed to specify the effects of the OCT on the transplanted cartilage and its influence on the later clinical outcome.

Autologous osteochondral mosaicplasty for treatment of a posttraumatic defect of the lateral tibial plateau: a case report with two-year follow-up.

Posttraumatic osteochondral defects following a tibial plateau fracture are common and a serious complication that may lead to the development of posttraumatic arthrosis. Successful reconstruction of the tibial plateau must include restoration of limb alignment, repair of bone defects, restoration of the articular cartilage, and preservation of the menisci. When osteochondral defects are present, the use of bulk bone grafts to restore the original articular surface anatomy of the tibial plateau is difficult due to incongruity between the graft and the original joint surface. Recognizing this, an autologous osteochondral transplantation utilizing the mosaic technique was performed successfully on a 32-year-old male alpine skier with a posttraumatic osteochondral defect following a tibial plateau fracture. At 2 years postsurgery, the patient had regained the capacity to perform most activities of daily living and to participate in sports. Clinical examination revealed an improvement of the Lysholm score from 48 points to 72 points.

Differences in the rehabilitation period following two methods of anterior cruciate ligament replacement: semitendinosus/gracilis tendon vs. ligamentum patellae.

This study compared patient outcome during the early rehabilitation phase following two different methods of anterior cruciate ligament (ACL) reconstruction: ligamentum patellae (LP) and semitendinosus/gracilis tendon (SG) based reconstruction. The study included 50 consecutive patients treated by each method, examined 6 weeks and 3, 6 and 12 months after surgery. Patients in the SG group showed significantly better Lysholm scores at 6 and 12 months, Tegner Activity Scale scores at 3 months, and pain profile assessments at 6 weeks and 3 months than those in the LP group. Significant advantages were observed in LP group in the Overall Knee Score at 6 weeks and in range-of-motion at 6 weeks and 3 and 6 months post-surgery. Stability tests revealed no significant differences between patients in the two groups. SG-based reconstruction of the ACL thus demonstrates advantages over LP-based reconstruction regarding pain and function, while LP-based reconstruction was associated with an earlier return of motion.

The role of cell type in bone healing mediated by ex vivo gene therapy.

BACKGROUND: The ideal cellular vehicle for use in cell-mediated gene therapy to enhance bone healing has not yet been identified. The purpose of this study was to compare the capacity of two types of cells transduced with retro-bone morphogenetic protein 4 (BMP4)-muscle-derived cells (MDCs) and unfractioned bone marrow stromal cells (BMSCs). METHOD: Primary rat MDCs and unfractioned rat BMSCs were transduced with a retrovirus to express BMP4. A 7-mm, critical-sized femur defect was created in adult rats, and 5 x 10(6) transduced cells were implanted into the femoral defect. Bone healing was monitored radiographically and histologically at 4, 8, and 12 weeks post-implantation. RESULTS: All specimens in the MDC-BMP4 group and BMSC-BMP4 group showed a bridging callus at 8 and 12 weeks. At 12 weeks post-implantation the calluses of the MDC-BMP4 femora displayed significantly higher bone photodensity than the BMSC-BMP4 femora (P<0.05). Histomorphometry revealed no difference between the two treatment groups. However, non-union between newly formed and original bone was observed in none of the MDC femora but in six femora from the BMSC-BMP4 group. CONCLUSION: Both MDCs and unfractioned BMSCs can improve healing of a critical-sized bone defect following transduction of the cells with retroBMP4. However, MDCs appear to yield superior results when compared with BMSCs in terms of improved healing of segmental defects.