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Motivation: Finding genes that are preferentially expressed in a particular tissue or condition is a problem that cannot be solved by standard statistical testing procedures. A relatively unknown procedure that can be used is the intersection–union test (IUT). However, two disadvantages of the IUT are that it is conservative and it conveys only the information of the least differing target tissue–other tissue pair. [ABSTRACT FROM PUBLISHER]

Abstract Background The 3' untranslated regions (UTRs) of transcripts are not well characterized for many genes and often extend beyond the annotated regions. Since Affymetrix 3' expression arrays were designed based on expressed sequence tags, many probesets map to intergenic regions downstream of genes. We used expression information from these probesets to predict transcript extension beyond currently known boundaries. Results Based on our dataset encompassing expression in 22 different murine tissues, we identified 845 genes with predicted 3'UTR extensions. These extensions have a similar conservation as known 3'UTRs, which is distinctly higher than intergenic regions. We verified 8 of the predictions by PCR and found all of the predicted regions to be expressed. The method can be extended to other 3' expression microarray platforms as we demonstrate with human data. Additional confirming evidence was obtained from public paired end read data. Conclusions We show that many genes have 3'UTR regions extending beyond currently known gene regions and provide a method to identify such regions based on microarray expression data. Since 3' UTR contain microRNA binding sites and other stability determining regions, identification of the full length 3' UTR is important to elucidate posttranscriptional regulation.

The fusion index is a key indicator for quantifying the differentiation of a myoblast population, which is often calculated manually. In addition to being time-consuming, manual quantification is also error prone and subjective. Several software tools have been proposed for addressing these limitations but suffer from various drawbacks, including unintuitive interfaces and limited performance. In this study, we describe MyoFInDer, a Python-based program for the automated computation of the fusion index of skeletal muscle. At the core of MyoFInDer is a powerful artificial intelligence-based image segmentation model. MyoFInDer also determines the total nuclei count and the percentage of stained area and allows for manual verification and correction. MyoFInDer can reliably determine the fusion index, with a high correlation to manual counting. Compared with other tools, MyoFInDer stands out as it minimizes the interoperator variability, minimizes process time and displays the best correlation to manual counting. Therefore, it is a suitable choice for calculating fusion index in an automated way, and gives researchers access to the high performance and flexibility of a modern artificial intelligence model. As a free and open-source project, MyoFInDer can be modified or extended to meet specific needs.

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC -p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl -/- and PWS-IC -p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular parallels between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Failure to close the abdomen after intestinal or multivisceral transplantation (Tx) remains a frequently occurring problem. Two attractive reconstruction methods, especially in large abdominal wall defects, are full-thickness abdominal wall vascularized composite allograft (AW-VCA) and nonvascularized rectus fascia (NVRF) Tx. This review compares surgical technique, immunology, integration, clinical experience, and indications of both techniques. In AW-VCA Tx, vascular anastomosis is required and the graft undergoes hypotrophy post-Tx. Furthermore, it has immunologic benefits and good clinical outcome. NVRF Tx is an easy technique without the need for vascular anastomosis. Moreover, a rapid integration and neovascularization occurs with excellent clinical outcome., Competing Interests: Disclosure L.J. Ceulemans and L. Thorrez were granted an FTBO grant from KU Leuven; L.J. Ceulemans is appointed as senior clinical investigator for Research Foundation Flanders (FWO)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Failure to close the abdominal wall after intestinal transplantation (ITx) or multivisceral Tx remains a surgical challenge. An attractive method is the use of nonvascularized rectus fascia (NVRF) in which both layers of the donor abdominal rectus fascia are used as an inlay patch without vascular anastomosis. How this graft integrates over time remains unknown. The study aims to provide a multilevel analysis of the neovascularization and integration process of the NVRF., Methods: Three NVRF-Tx were performed after ITx. Clinical, radiological, histological, and immunological data were analyzed to get insights into the neovascularization and integration process of the NVRF. Moreover, cryogenic contrast-enhanced microfocus computed tomography (microCT) analysis was used for detailed reconstruction of the vasculature in and around the NVRF (3-dimensional histology)., Results: Two men (31- and 51-y-old) and 1 woman (49-y-old) underwent 2 multivisceral Tx and 1 combined liver-ITx, respectively. A CT scan showed contrast enhancement around the fascia graft at 5 days post-Tx. At 6 weeks, newly formed blood vessels were visualized around the graft with Doppler ultrasound. Biopsies at 2 weeks post-Tx revealed inflammation around the NVRF and early fibrosis. At 6 months, classical 2-dimensional histological analysis of a biopsy confirmed integration of the fascia graft with strong fibrotic reaction without signs of rejection. A cryogenic contrast-enhanced microCT scan of the same biopsy revealed the presence of microvasculature, enveloping and penetrating the donor fascia., Conclusions: We showed clinical, histological, and microCT evidence of the neovascularization and integration process of the NVRF after Tx., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Engineered myogenic microtissues derived from human skeletal myoblasts offer unique opportunities for varying skeletal muscle tissue engineering applications, such as in vitro drug-testing and disease modelling. However, more complex models require the incorporation of vascular structures, which remains to be challenging. In this study, myogenic spheroids were generated using a high-throughput, non-adhesive micropatterned surface. Since monoculture spheroids containing human skeletal myoblasts were unable to remain their integrity, co-culture spheroids combining human skeletal myoblasts and human adipose-derived stem cells were created. When using the optimal ratio, uniform and viable spheroids with enhanced myogenic properties were achieved. Applying a pre-vascularization strategy, through addition of endothelial cells, resulted in the formation of spheroids containing capillary-like networks, lumina and collagen in the extracellular matrix, whilst retaining myogenicity. Moreover, sprouting of endothelial cells from the spheroids when encapsulated in fibrin was allowed. The possibility of spheroids, from different maturation stages, to assemble into a more large construct was proven by doublet fusion experiments. The relevance of using three-dimensional microtissues with tissue-specific microarchitecture and increased complexity, together with the high-throughput generation approach, makes the generated spheroids a suitable tool for in vitro drug-testing and human disease modeling., (Creative Commons Attribution license.)

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue., (© 2024 IOP Publishing Ltd.)

Purpose: The purpose of this study was to describe the immunoarchitecture of normal extraocular muscles (EOMs) in terms of presence, distribution, and organization of various immune cells., Methods: We performed unilateral orbital exenterations in six fresh human cadavers from elderly patients, followed by dissection of the medial, lateral, superior and inferior rectus, superior and inferior oblique, and superior palpebral levator muscle in their entirety. We further cross sectioned each EOM in an anterior, central, and posterior third. After immunohistochemical staining for CD3, CD8, CD20, CD138, CD68, and podoplanin, quantitative analysis was performed., Results: We found all EOMs (rectus, oblique, and levator muscles) to harbor both T- and B-lymphocytes, with a B-lymphocyte dominance and an absence of plasma cells. The highest prevalence of immune cells was seen in the muscle bellies, with, on average, 488 ± 63 CD3+ T-lymphocytes and 44 ± 110 CD20+ B-lymphocytes per mm2, and significant differences from the anterior (T-lymphocytes) and posterior (T- and B-lymphocytes) thirds. T- and B-lymphocytes were primarily organized in hotspots in the vicinity of blood vessels. In addition, a small resident population of macrophages scattered throughout the specimens was detected. No lymphatic vessels were found in any of the EOMs., Conclusions: These findings can serve as a reference dataset in the assessment of EOM biopsies in the diagnostic process of inflammatory orbital and systemic disorders. Moreover, from a regenerative perspective, our results highlight the importance of taking into account the presence of a resident immune cell population when studying the host immune response on transplanted tissues or engineered constructs.

For tissue engineering of skeletal muscles, there is a need for biomaterials which do not only allow cell attachment, proliferation, and differentiation, but also support the physiological conditions of the tissue. Next to the chemical nature and structure of the biomaterial, its response to the application of biophysical stimuli, such as mechanical deformation or application of electrical pulses, can impact in vitro tissue culture. In this study, gelatin methacryloyl (GelMA) is modified with hydrophilic 2-acryloxyethyltrimethylammonium chloride (AETA) and 3-sulfopropyl acrylate potassium (SPA) ionic comonomers to obtain a piezoionic hydrogel. Rheology, mass swelling, gel fraction, and mechanical characteristics are determined. The piezoionic properties of the SPA and AETA-modified GelMA are confirmed by a significant increase in ionic conductivity and an electrical response as a function of mechanical stress. Murine myoblasts display a viability of >95% after 1 week on the piezoionic hydrogels, confirming their biocompatibility. The GelMA modifications do not influence the fusion capacity of the seeded myoblasts or myotube width after myotube formation. These results describe a novel functionalization providing new possibilities to exploit piezo-effects in the tissue engineering field., (© 2023 Wiley-VCH GmbH.)

Infection with the rabies virus (RABV) results in a 100% lethal neurological disease once symptoms develop. Post-exposure prophylaxis (PEP) consists of a combination of vaccination and anti-rabies immunoglobulins (RIGs); it is 100% effective if administered early after exposure. Because of its limited availability, alternatives for RIGs are needed. To that end, we evaluated a panel of 33 different lectins for their effect on RABV infection in cell culture. Several lectins, with either mannose or GlcNAc specificity, elicited anti-RABV activity, of which the GlcNAc-specific Urtica dioica agglutinin (UDA) was selected for further studies. UDA was found to prevent the entry of the virus into the host cell. To further assess the potential of UDA, a physiologically relevant RABV infection muscle explant model was developed. Strips of dissected swine skeletal muscle that were kept in a culture medium could be productively infected with the RABV. When the infection of the muscle strips was carried out in the presence of UDA, RABV replication was completely prevented. Thus, we developed a physiologically relevant RABV muscle infection model. UDA (i) may serve as a reference for further studies and (ii) holds promise as a cheap and simple-to-produce alternative for RIGs in PEP.

Competing Interests: None declared.

Background: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points., Methods: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems., Results: We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/β-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs., Conclusions: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms., (© 2023. The Author(s).)

Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wüst, Terrie, Müntefering, Ruck and Thorrez.)

Skeletal muscle tissue engineering (SMTE) aims at the in vitro generation of 3D skeletal muscle engineered constructs which mimic the native muscle structure and function. Although native skeletal muscle is a highly dynamic tissue, most research approaches still focus on static cell culture methods, while research on stimulation protocols indicates a positive effect, especially on myogenesis. A more mature muscle construct may be needed especially for the potential applications for regenerative medicine purposes, disease or drug disposition models. Most efforts towards dynamic cell or tissue culture methods have been geared towards mechanical or electrical stimulation or a combination of those. In the context of dynamic methods, pulsed electromagnetic field (PEMF) stimulation has been extensively used in bone tissue engineering, but the impact of PEMF on skeletal muscle development is poorly explored. Here, we evaluated the effects of PEMF stimulation on human skeletal muscle cells both in 2D and 3D experiments. First, PEMF was applied on 2D cultures of human myoblasts during differentiation. In 2D, enhanced myogenesis was observed, as evidenced by an increased myotube diameter and fusion index. Second, 2D results were translated towards 3D bioartificial muscles (BAMs). BAMs were subjected to PEMF for varying exposure times, where a 2-h daily stimulation was found to be effective in enhancing 3D myotube formation. Third, applying this protocol for the entire 16-days culture period was compared to a stimulation starting at day 8, once the myotubes were formed. The latter was found to result in significantly higher myotube diameter, fusion index, and increased myosin heavy chain 1 expression. This work shows the potential of electromagnetic stimulation for enhancing myotube formation both in 2D and 3D, warranting its further consideration in dynamic culturing techniques., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Terrie, Burattini, Van Vlierberghe, Fassina and Thorrez.)

A wide range of synthetic and natural biomaterials is available for skeletal muscle tissue engineering. One class of natural biomaterials consists of the extracellular matrix (ECM) from donor skeletal muscle. To obtain this ECM, the cellular compartment must be completely removed while retaining the native composition and ultrastructure of the tissue as much as possible. In this review, the progress and challenges in the field of skeletal muscle decellularization are discussed by reviewing the different decellularization methods available and by highlighting the different applications of the scaffolds. Decellularized skeletal muscle has mainly been studied in the context of regeneration with a focus on its tissue-specific morphological features as well as biochemical cues to stimulate muscle regeneration. However, in this review, the potential applications of decellularized skeletal muscle are expanded beyond the regenerative setting to demonstrate its versatility as a biomaterial. Acellular matrices are discussed as a platform to study cell-matrix interactions and drug screening. Decellularized skeletal muscle ECM can also be further processed to re-engineer its structure. An overview is presented of materials processed from decellularized skeletal muscle, ranging from injectable hydrogels, bioinks for 3D bioprinting, electrospun nanofibers to coatings for cell culture., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Different types of proteins diverge at vastly different rates. Moreover, the same type of protein has been observed to evolve with different rates in different phylogenetic lineages. In the present study we measured the rates of protein evolution in Eutheria (placental mammals) and Metatheria (marsupials) on a genome-wide basis and we propose that the gene position in the genome landscape has an important influence on the rate of protein divergence., Results: We analyzed a protein-encoding gene set (n = 15,727) common to 16 mammals (12 Eutheria and 4 Metatheria). Using sliding windows that averaged regional effects of protein divergence we constructed landscapes in which strong and lineage-specific regional effects were seen on the molecular clock rate of protein divergence. Within each lineage, the relatively high rates were preferentially found in subtelomeric chromosomal regions. Such regions were observed to contain important and well-studied loci for fetal growth, uterine function and the generation of diversity in the adaptive repertoire of immunoglobulins., Conclusions: A genome landscape approach visualizes lineage-specific regional differences between Eutherian and Metatherian rates of protein evolution. This phenomenon of chromosomal position is a new element that explains at least part of the lineage-specific effects and differences between proteins on the molecular clock rates., (© 2021. The Author(s).)

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic β-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. β-cell-specific Furin knockout (β Fur KO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on β Fur KO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). β Fur KO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in β-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β-cells lacking Furin , causing β-cell dysfunction., (© 2020 by the American Diabetes Association.)

Body weight loss, mostly due to the wasting of skeletal muscle and adipose tissue, is the hallmark of the so-called cachexia syndrome. Cachexia is associated with several acute and chronic disease states such as cancer, chronic obstructive pulmonary disease (COPD), heart and kidney failure, and acquired and autoimmune diseases and also pharmacological treatments such as chemotherapy. The clinical relevance of cachexia and its impact on patients' quality of life has been neglected for decades. Only recently did the international community agree upon a definition of the term cachexia, and we are still awaiting the standardization of markers and tests for the diagnosis and staging of cancer-related cachexia. In this review, we discuss cachexia, considering the evolving use of the term for diagnostic purposes and the implications it has for clinical biomarkers, to provide a comprehensive overview of its biology and clinical management. Advances and tools developed so far for the in vitro testing of cachexia and drug screening will be described. We will also evaluate the nomenclature of different forms of muscle wasting and degeneration and discuss features that distinguish cachexia from other forms of muscle wasting in the context of different conditions.

Background: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype., Methods: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status., Results: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFN γ -induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences., Conclusions: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology., (Copyright © 2020 by the American Society of Nephrology.)

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-β1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.

Cultured meat aspires to be biologically equivalent to traditional meat. If cultured meat is to be consumed, sensorial (texture, color, flavor) and nutritional characteristics are of utmost importance. This paper compares cultured meat to traditional meat from a tissue engineering and meat technological point of view, focusing on several molecular, technological and sensorial attributes. We outline the challenges and future steps to be taken for cultured meat to mimic traditional meat as closely as possible., (Copyright © 2020 Fraeye, Kratka, Vandenburgh and Thorrez.)

Introduction: Bone implant related infection is still one of the biggest challenges in bone and joint surgery. Antibiotic impregnated bone grafts seem to be promising in both treatment and prevention of these infections. However, great variance in methodology predominates this field of research. This paper gives an overview of the published literature. Methods: The PRISMA-flowchart was used as protocol for article selection. Medline was searched and articles were selected in accordance with predetermined exclusion criteria. Results: Forty-eight articles were included in the synthesis. Topics including bone graft type, manipulations of the graft, elution profile, bacterial inhibition, osteotoxicity, incorporation, special impregnation methods, clinical use and storage were investigated. Therapeutically, high initial levels seem appropriate for biofilm eradication. A single stage procedure in the treatment of bone implant related infection seems feasible. Prophylactically, the literature indicates a reduction of postoperative infections when using antibiotic impregnated bone grafts. Conclusion: Bone grafts are a suitable carrier for local antibiotic application both therapeutically and prophylactically., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca +2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.

Background: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available., Methods: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661., Findings: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection., Interpretation: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection., Funding: The Seventh Framework Programme (FP7) of the European Commission., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenitor cells are cocultured with human umbilical vein endothelial cells (HUVECs) in a fibrin hydrogel. The cell-gel mix is cast into silicone molds with end attachment sites and cultured in endothelial growth medium (EGM-2) for 1 week. The passive forces generated in the contracted hydrogel align the myogenic cells parallel to the long axis of the contracted gel such that they fuse into aligned multinucleated myofibers. This results in the formation of a 2 cm long and ~1.5 mm tick human BAM construct with endothelial networks.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)