Cerebrospinal fluid findings in patients with myelin oligodendrocyte glycoprotein (MOG) antibodies. Part 2: Results from 108 lumbar punctures in 80 pediatric patients.

BACKGROUND: New-generation, cell-based assays have demonstrated a robust association of serum autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis, and brainstem encephalitis, as well as with neuromyelitis optica (NMO)-like or acute-disseminated encephalomyelitis (ADEM)-like presentations. However, only limited data are yet available on cerebrospinal fluid (CSF) findings in MOG-IgG-associated encephalomyelitis (MOG-EM; also termed MOG antibody-associated disease, MOGAD). OBJECTIVE: To describe systematically the CSF profile in children with MOG-EM. MATERIAL AND METHODS: Cytological and biochemical findings (including white cell counts [WCC] and differentiation; frequency and patterns of oligoclonal bands; IgG/IgM/IgA and albumin concentrations and CSF/serum ratios; intrathecal IgG/IgM/IgA fractions; locally produced IgG/IgM/IgA concentrations; immunoglobulin class patterns; IgG/IgA/IgM reibergrams; Link index; measles/rubella/zoster [MRZ] reaction; other anti-viral and anti-bacterial antibody indices; CSF total protein; CSF L-lactate) from 108 lumbar punctures in 80 pediatric patients of mainly Caucasian descent with MOG-EM were analyzed retrospectively. RESULTS: Most strikingly, CSF-restricted oligoclonal IgG bands, a hallmark of multiple sclerosis (MS), were absent in 89% of samples (N = 96), and the MRZ reaction, the most specific laboratory marker of MS known so far, in 100% (N = 29). If present at all, intrathecal IgG synthesis was low, often transient and mostly restricted to acute attacks. Intrathecal IgM synthesis was present in 21% and exclusively detectable during acute attacks. CSF WCC were elevated in 54% of samples (median 40 cells/µl; range 6-256; mostly lymphocytes and monocytes; > 100/µl in 11%). Neutrophils were present in 71% of samples; eosinophils, activated lymphocytes, and plasma cells were seen only rarely (all < 7%). Blood-CSF barrier dysfunction (as indicated by an elevated albumin CSF/serum ratio) was present in 46% of all samples (N = 79) and at least once in 48% of all patients (N = 67) tested. CSF alterations were significantly more frequent and/or more pronounced in patients with acute spinal cord or brain disease than in patients with acute ON and varied strongly depending on attack severity. CSF L-lactate levels correlated significantly with the spinal cord lesions load (measured in vertebral segments) in patients with acute myelitis (p = 0.0099). An analysis of pooled data from the pediatric and the adult cohort showed a significant relationship of QAlb (p < 0.0005), CST TP (p < 0.0001), and CSF L-lactate (p < 0.0003) during acute attacks with age. CONCLUSION: MOG-IgG-associated EM in children is characterized by CSF features that are distinct from those in MS. With regard to most parameters, no marked differences between the pediatric cohort and the adult cohort analyzed in Part 1 were noted. Our findings are important for the differential diagnosis of pediatric MS and MOG-EM and add to the understanding of the immunopathogenesis of this newly described autoimmune disease.

The role of lipoxygenases in epidermis.

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Long-term results and prognostic factors after resection of hepatic and pulmonary metastases of colorectal cancer.

BACKGROUND: Resection of colorectal liver or lung metastases is an established therapeutical concept at present. However, an affection of both these organs is frequently still regarded as incurable. METHODS: All cancer patients are documented in our prospective cancer registry since 1995. Data of patients who underwent liver and lung resection for colorectal metastases were extracted and analysed. RESULTS: Sixty-five patients underwent surgery for liver and lung metastases. In 33 cases, the first distant metastasis was diagnosed synchronously to the primary tumour. For the remaining patients, median time interval between primary tumour and first distant metastasis was 18 months (5-69 months). Complete resection was achieved in 51 patients (79 %) and was less likely in patients with synchronous disease (p = 0.017). Negative margins (p = 0.002), the absence of pulmonary involvement in synchronous metastases (p = 0.0003) and single metastases in both organs (p = 0.036) were associated with a better prognosis. Five- and 10-year survival rates for all patients are 57 and 15 % from diagnosis of the primary tumour, 37 and 14 % from resection of the first metastasis and 20 and 15 % from resection of the second metastasis. After complete resection, 5- and 10-year survival rates increased to 61 and 18 %, 43 and 17 % as well as 25 and 19 %, respectively. Long-term survivors (≥10 years) were seen only after complete resection of both metastases. CONCLUSIONS: Patients with resectable liver and lung metastases of the colorectal primary should be considered for surgery after multidisciplinary evaluation regardless of the number or size of the metastases or the disease-free intervals. Clear resection margins are the strongest prognostic parameter.

Impaired Brain Growth in Myelin Oligodendrocyte Glycoprotein Antibody-Associated Acute Disseminated Encephalomyelitis.

BACKGROUND AND OBJECTIVES: Acute disseminated encephalomyelitis (ADEM) is the most common phenotype in pediatric myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease. A previous study demonstrated impaired brain growth in ADEM. However, the effect of MOG antibodies on brain growth remains unknown. Here, we performed brain volume analyses in MOG-positive and MOG-negative ADEM at onset and over time. METHODS: In this observational cohort study, we included a total of 62 MRI scans from 24 patients with ADEM (54.2% female; median age 5 years), of which 16 (66.7%) were MOG positive. Patients were compared with healthy controls from the NIH pediatric MRI data repository and a matched local cohort. Mixed-effect models were applied to assess group differences and other relevant factors, including relapses. RESULTS: At baseline and before any steroid treatment, patients with ADEM, irrespective of MOG antibody status, showed reduced brain volume compared with matched controls (median [interquartile range] 1,741.9 cm3 [1,645.1-1,805.2] vs 1,810.4 cm3 [1,786.5-1,836.2]). Longitudinal analysis revealed reduced brain growth for both MOG-positive and MOG-negative patients with ADEM. However, MOG-negative patients showed a stronger reduction (-138.3 cm3 [95% CI -193.6 to -82.9]) than MOG-positive patients (-50.0 cm3 [-126.5 to -5.2]), independent of age, sex, and treatment. Relapsing patients (all MOG positive) showed additional brain volume loss (-15.8 cm3 [-68.9 to 37.3]). DISCUSSION: Patients with ADEM exhibit brain volume loss and failure of age-expected brain growth. Importantly, MOG-negative status was associated with a more pronounced brain volume loss compared with MOG-positive patients.

12R-lipoxygenase deficiency disrupts epidermal barrier function.

12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX-deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX-eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.

High Velocity Suspension Flame Spraying (HVSFS) of Metal Suspensions.

Thermal spraying of metal materials is one of the key applications of this technology in industry for over a hundred years. The variety of metal-based feedstocks (powders and wires) used for thermal spray is incredibly large and utilization covers abrasion and corrosion protection, as well as tribological and electrical applications. Spraying metals using suspension- or precursor-based thermal spray methods is a relatively new and unusual approach. This publication deals with three metal types, a NiCr 80/20, copper (Cu), and silver (Ag), sprayed as fine-grained powders dispersed in aqueous solvent. Suspensions were sprayed by means of high-velocity suspension spraying (HVSFS) employing a modified TopGun system. The aim was to prepare thin and dense metal coatings (10-70 µm) and to evaluate the process limits regarding the oxygen content of the coatings. In case of Cu and Ag, possible applications demand high purity with low oxidation of the coating to achieve for instance a high electrical conductivity or catalytic activity. For NiCr however, it was found that coatings with a fine dispersion of oxides can be usable for applications where a tunable resistivity is in demand. The paper describes the suspension preparation and presents results of spray experiments performed on metal substrates. Results are evaluated with respect to the phase composition and the achieved coating morphology. It turns out that the oxidation content and spray efficiency is strongly controlled by the oxygen fuel ratio and spray distance.

Thin Degradable Coatings for Optimization of Osteointegration Associated with Simultaneous Infection Prophylaxis.

One of the most common causes of implant failure is aseptic prosthesis loosening. Another frequent complication after prosthesis implant is the microbial colonization of the prosthesis surface, which often leads to a replacement of the prosthesis. One approach to reduce these complications is the application of bioactive substances to implant surfaces. Both an antibiotic prophylaxis and a faster osteointegration can be obtained by incorporation of bactericidal active metals in degradable calcium phosphate (CaP) coatings. In this study, thin degradable calcium phosphate ceramic coatings doped with silver (Ag), copper (Cu), and bismuth (Bi) on a titanium substrate were prepared with the aid of the high-velocity suspension flame spraying (HVSFS) coating process. To characterize the samples surface roughness, brightfield microscopy of the coatings, X-ray diffraction (XRD)-analysis for definition of the phase composition of the layers, Raman spectroscopy for determination of the phase composition of the contained metals, element-mapping for Cu-content verification, release kinetics for detection of metal ions and ceramic components of the coatings were carried out. The aim of this study was to evaluate in vitro biocompatibility and antimicrobial activity of the coatings. For biocompatibility testing, growth experiments were performed using the cell culture line MG-63. Cell viability was investigated by Giemsa staining and live/dead assay. The WST-1 kit was used to quantify cell proliferation and vitality in vitro and the lactate dehydrogenase (LDH) kit to quantify cytotoxicity. The formation of hydroxyapatite crystals in simulated body fluid was investigated to predict bioactivity in vivo. The Safe Airborne Antibacterial Assay with Staphylococcus aureus (S. aureus) was used for antimicrobial testing. The results showed good biocompatibility of all the metal doped CaP coatings, furthermore Cu and Ag doped layers showed significant antibacterial effects against S. aureus.

Mutations in Recessive Congenital Ichthyoses Illuminate the Origin and Functions of the Corneocyte Lipid Envelope.

The corneocyte lipid envelope (CLE), a monolayer of ω-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue. We provide multiple lines of evidence that the CLE originates as lamellar body-limiting membranes fuse with the plasma membrane: (i) ABCA12 patients and Abca12-/- mice display normal CLEs; (ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; (iii) CLEs are absent in VSP33B-negative patients; (iv) limiting membranes of lamellar bodies are defective in lipid-synthetic autosomal recessive congenital ichthyoses; and (v) lipoxygenases, lipase activity, and LIPN co-localize within putative lamellar bodies.

Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation.

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.

3D Powder Printed Bioglass and ß-Tricalcium Phosphate Bone Scaffolds.

The use of both bioglass (BG) and ß tricalcium phosphate (ß-TCP) for bone replacement applications has been studied extensively due to the materials' high biocompatibility and ability to resorb when implanted in the body. 3D printing has been explored as a fast and versatile technique for the fabrication of porous bone scaffolds. This project investigates the effects of using different combinations of a composite BG and ß-TCP powder for 3D printing of porous bone scaffolds. Porous 3D powder printed bone scaffolds of BG, ß-TCP, 50/50 BG/ß-TCP and 70/30 BG/ß-TCP compositions were subject to a variety of characterization and biocompatibility tests. The porosity characteristics, surface roughness, mechanical strength, viability for cell proliferation, material cytotoxicity and in vitro bioactivity were assessed. The results show that the scaffolds can support osteoblast-like MG-63 cells growth both on the surface of and within the scaffold material and do not show alarming cytotoxicity; the porosity and surface characteristics of the scaffolds are appropriate. Of the two tested composite materials, the 70/30 BG/ß-TCP scaffold proved to be superior in terms of biocompatibility and mechanical strength. The mechanical strength of the scaffolds makes them unsuitable for load bearing applications. However, they can be useful for other applications such as bone fillers.

Modulation of epidermal tumor development caused by targeted overexpression of epidermis-type 12S-lipoxygenase.

In contrast to other 12S-lipoxygenase (LOX) isoforms expressed in the skin of mice, epidermis-type (e) 12S-LOX was found to be transcriptionally down-regulated in the course of epidermal tumor development in NMRI mice. This may indicate that this enzyme is related to antitumorigenic rather than protumorigenic effects. To test this hypothesis, two transgenic mouse lines were generated that differentially expressed e12S-LOX under the control of the bovine keratin 6 promoter known to be constitutively up-regulated in mouse skin tumors. As compared with the wild-type, low transgene expression correlated with a decreased skin tumor response paralleled by an up-regulation of leukocyte-type 12S-LOX and an accumulation of the linoleic acid derivative 13S-hydroxyoctadecadienoic acid. In contrast, high transgene expression coincided with an increased tumor response paralleled by a strong keratin 6 promoter-driven up-regulation of the transgenic e12S-LOX and an accumulation of the arachidonic acid derivative 12S-hydroxyeicosatetraenoic acid as the predominant LOX product. These results indicate a complex interaction between different LOX isoforms and an opposite role of arachidonic acid and linoleic acid products in the modulation of skin carcinogenesis.

Mutation spectrum and functional analysis of epidermis-type lipoxygenases in patients with autosomal recessive congenital ichthyosis.

Autosomal-recessive congenital ichthyosis (ARCI) is a clinically and genetically heterogeneous group of severe hereditary keratinization disorders characterized by intense scaling of the whole integument, and differences in color and shape. It is often associated with erythema. To date, six loci for ARCI have been mapped. Mutations in ALOXE3 and ALOX12B on chromosome 17p13, which code for two different epidermal lipoxygenases, were recently found in patients with ichthyosiform erythroderma from Turkey, France, and North Africa. Here we describe molecular and clinical findings in 17 families with ARCI originating from Central Europe, Turkey, and the Indian subcontinent, with mutations in ALOXE3 or ALOX12B. We identified 11 novel point mutations in ALOX12B (one nonsense mutation and 10 missense mutations) and four different inactivating mutations in ALOXE3. The gene products of ALOX12B and ALOXE3, the epidermal lipoxygenases 12R-LOX and eLOX3, respectively, are preferentially synthesized in the skin. They act in sequence to convert arachidonic acid via 12(R)-HPETE to the corresponding epoxyalcohol, 8(R)-hydroxy-11(R),12(R)-epoxyeicosatrienoic acid. To assess the impairment of enzyme activity, we expressed the mutated genes in vitro and determined the activity of the recombinant proteins toward their genuine substrates. All but one of the recombinant mutants were enzymatically inactive. The characterization of disease-causing mutations in ALOXE3 and ALOX12B and the resulting ARCI phenotypes did not result in clear diagnostic criteria; however, we found a first correlation between the genetic findings and the clinical presentation of ichthyosis.

Adipocyte differentiation of 3T3-L1 preadipocytes is dependent on lipoxygenase activity during the initial stages of the differentiation process.

Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARgamma (peroxisome proliferator-activated receptor gamma) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARgamma ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARgamma ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3-4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARgamma ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.

UV-A and UV-C light induced hydrophilization of dental implants.

OBJECTIVES: Wettability is increasingly considered to be an important factor determining biological responses to implant materials. In this context, the purpose of this study was to compare the dynamic wettability of dental implants made from different bulk materials and modified by different surface modifications, and to analyze the respective changes of wettability upon irradiating these implants by UV-A or UV-C light. METHODS: Four original screw-type implants were investigated: One grit-blasted/acid-etched and one anodically oxidized titanium, one zirconia and one polyetheretherketone implant. Additionally, experimental, screwless, machined titanium cylinders were included in the study. Part of that cylinders and of blasted/etched implants were further modified by a magnetron-sputtered photocatalytic anatase thin film. Scanning electron microscopy was used to investigate the surface micro- and nanostructures. Samples were treated by UV-A (382nm, 25mWcm(-2)) and UV-C (260nm, 15mWcm(-2)) for entire 40min, respectively, and their wettability was quantified by dynamic contact angle (CA) analysis from multi-loop Wilhelmy experiments. RESULTS: All implants are characterized by submicron- and nanosized surface features. Unexposed implants were hydrophobic (CA>90°). Upon UV-A, solely the implants with anatase coating became superhydrophilic (CA<5°). Upon UV-C, the blasted/etched implants turned superhydrophilic, the anodized titanium and the zirconia implants were considerably (CA=34° and 27°, respectively) and the PEEK implants slightly (CA=79°) hydrophilized. SIGNIFICANCE: The wettability of implant surfaces can be improved by UV irradiation. The efficiency of UV-A and UV-C irradiation to lower the CA by photocatalysis or photolysis, however, is strongly dependent on the specific material and surface. Thus, attempts to photofunctionalize these surfaces by irradiation is expected to result in a different pattern of bioresponses.

Arachidonate 8(S)-lipoxygenase.

The recently identified mouse 8(S)-lipoxygenase almost exclusively directs oxygen insertion into the 8(S) position of arachidonic acid and, with lower efficiency, into the 9(S) position of linoleic acid. The protein of 677 amino acids displays 78% sequence identity to human 15(S)-lipoxygenase-2 which is considered to be its human orthologue. The 8(S)-lipoxygenase gene, Alox15b, consisting of 14 exons and spanning 14.5 kb is located within a gene cluster of related epidermis-type lipoxygenases at the central region of mouse chromosome 11. 8(S)-Lipoxygenase is predominantly expressed in stratifying epithelia of mice, constitutively in the hair follicle, forestomach, and foot-sole and inducible in the back skin with strain-dependent variations. The expression is restricted to terminally differentiating keratinocytes, in particular the stratum granulosum and 8(S)-lipoxygenase activity seems to be involved in terminal differentiation of mouse epidermis. Tumor-specific up-regulation of 8(S)-lipoxygenase expression and activity indicate a critical role of this enzyme in malignant progression during tumor development in mouse skin.

A European training system in cardiothoracic surgery: is it time?

OBJECTIVE: Training in cardiothoracic surgery across Europe remains diverse and variable despite the ever closer integration of European countries at all levels and in all areas of life. Coupled with the increasing ease of movement across Europe, the need for uniform training programmes has arisen to allow for equivalent accreditation and certification. METHODS: We review the current training paradigms within the specialty across the world and in Europe and also explore the concept of competence. RESULTS: There are diverse training systems across the world and in Europe in particular. Competence-based training is the new model of training; however, competence remains difficult to define and measure. We propose a European Training Programme in Cardiothoracic Surgery that aims to standardize training across the European countries. CONCLUSIONS: The difficulties in unifying training across Europe are numerous, but it is time to implement a European Training System in Cardiothoracic Surgery that will deliver a competence-based curriculum.

Aloxe3 knockout mice reveal a function of epidermal lipoxygenase-3 as hepoxilin synthase and its pivotal role in barrier formation.

Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway.