Functional recovery of injured cavernous nerves achieved through endogenous nerve growth factor-containing bioactive fibrous membrane.

Radical prostatectomy is a highly successful treatment for prostate cancer, among the most prevalent manifestations of the illness. Damage of the cavernous nerve (CN) during prostatectomy is the main cause of postoperative erectile dysfunction (ED). In this study, the capability of a personalized bioactive fibrous membrane to regenerate injured CN was investigated. The fibrous membrane bioactivity is conferred by the selectively bound nerve growth factor (NGF) present in the rat urine. In a rat model of bilateral CN crush, the implanted bioactive fibrous membrane induces CN regeneration and restoration of erectile function, showing a significantly increased number of smooth muscle cells and content of endothelial and neuronal nitric oxide synthases (eNOS; nNOS). In addition, the bioactive fibrous membrane promotes nerve regeneration by increasing the number of myelinated axons and nNOS-positive cells, therefore reversing the CN fibrosis found in untreated rats or rats treated with a bare fibrous membrane. Therefore, this personalized regenerative strategy could overcome the recognized drawbacks of currently available treatments for CN injuries. It may constitute an effective treatment for prostate cancer patients suffering from ED after being subject to radical prostatectomy. STATEMENT OF SIGNIFICANCE: The present work introduces a unique strategy to address post-surgical ED resulting from CN injury during pelvic surgery (e.g., radical prostatectomy, radical cystoprostatectomy, abdominoperineal resection). It comprises a bioactive and cell-free fibrous implant, customized to enhance CN recovery. Pre-clinical results in a rat model of bilateral CN crush demonstrated that the bioactive fibrous implant can effectively heal injured CN, and restore penile structure and function. This implant selectively binds NGF from patient fluids (i.e. urine) due to its functionalized surface and high surface area. Moreover, its local implantation reduces adverse side effects. This tailored regenerative approach has the potential to revolutionize the treatment of ED in prostate cancer patients following radical prostatectomy, overcoming current treatment limitations.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Development of bilayered porous silk scaffolds for thymus bioengineering.

The thymus coordinates the development and selection of T cells. It is structured into two main compartments: the cortex and the medulla. The replication of such complex 3D environment has been challenged by bioengineering approaches. Nevertheless, the effect of the scaffold microstructure on thymic epithelial cell (TEC) cultures has not been deeply investigated. Here, we developed bilayered porous silk fibroin scaffolds and tested their effect on TEC co-cultures. The small and large pore scaffolds presented a mean pore size of 84.33 ± 21.51 µm and 194.90 ± 61.38 µm, respectively. The highly porous bilayered scaffolds presented a high water absorption and water content (> 94 %), together with mechanical properties in the range of the native tissue. TEC (i.e., medullary (mTEC) and cortical (cTEC) cell lines) proliferation is increased in scaffolds with larger pores. The co-culture of both TEC lines in the bilayered porous silk scaffolds presents enhanced cell proliferation and metabolic activity when compared with mTEC in single culture. Also, when the co-culture occurred with cTEC in the small pores layer and mTEC in the large pores layer, a 9.2- and 18.9-fold increase in Foxn1 and Icam1 gene expression in cTEC is evident. These results suggest that scaffold microstructure and the co-culture influence TEC's behaviour. Bilayered silk scaffolds with adjusted microstructure are a valid alternative for TEC culture, having possible applications in advanced thymus bioengineering strategies.

Laminin-2 immobilized on a 3D fibrous structure impacts cortical thymic epithelial cells behaviour and their interaction with thymocytes.

The tri-dimensionality of the thymic extracellular matrix (ECM) supports the crosstalk between thymocytes and thymic epithelial cells (TECs). The thymic ECM component laminin-2 is involved in the regulation of thymocytes and their interaction with cortical TECs (cTECs). Most in vitro studies use planar surfaces to study the interaction between ECM components and thymic cells. Herein, we developed a novel biofunctionalized culture system by immobilizing laminin-2 at the surface of porous and fibrous electrospun meshes. We aimed to study the interaction of cTECs with thymocytes in the presence of laminin-2 presented through this system. The results indicated that the presence of laminin-2, not its density, has a positive effect on the cell viability and proliferation of cTECs. qPCR results demonstrated that laminin-2 density influenced the expression of cTECs genes. An increased percentage of adherent CD4-CD8- thymocytes and a decreased percentage of CD4+CD8+ thymocytes were evident in higher laminin-2 concentrations. Higher concentrations decreased the expression of Il7 and Ccl25 in cTECs after thymocyte adhesion. Altogether, these results indicate that the interaction of thymocytes with the thymic cortical compartment is affected by laminin-2 density and supports the need for immobilized ECM proteins in porous and fibrous substrates for the study of thymus biology.

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases.

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems. Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively). Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.

Metronidazole Delivery Nanosystem Able To Reduce the Pathogenicity of Bacteria in Colorectal Infection.

Metronidazole (MTZ) is a drug potentially used for the treatment of intestinal infections, namely, the ones caused by colorectal surgery. The traditional routes of administration decrease its local effectiveness and present off-site effects. To circumvent such limitations, herein a drug delivery system (DDS) based on MTZ-loaded nanoparticles (NPs) immobilized at the surface of electrospun fibrous meshes is proposed. MTZ at different concentrations (1, 2, 5, and 10 mg mL-1) was loaded into chitosan-sodium tripolyphosphate NPs. The MTZ loaded into NPs at the highest concentration showed a quick release in the first 12 h, followed by a gradual release. This DDS was not toxic to human colonic cells. When tested against different bacterial strains, a significant reduction of Escherichia coli and Staphylococcus aureus was observed, but no effect was found against Enterococcus faecalis. Therefore, this DDS offers high potential to locally prevent the occurrence of infections after colorectal anastomosis.

The intervening domain is required for DNA-binding and functional identity of plant MADS transcription factors.

The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.

Recapitulation of Thymic Function by Tissue Engineering Strategies.

The thymus is responsible for the development and selection of T lymphocytes, which in turn also participate in the maturation of thymic epithelial cells. These events occur through the close interactions between hematopoietic stem cells and developing thymocytes with the thymic stromal cells within an intricate 3D network. The complex thymic microenvironment and function, and the current therapies to induce thymic regeneration or to overcome the lack of a functional thymus are herein reviewed. The recapitulation of the thymic function using tissue engineering strategies has been explored as a way to control the body's tolerance to external grafts and to generate ex vivo T cells for transplantation. In this review, the main advances in the thymus tissue engineering field are disclosed, including both scaffold- and cell-based strategies. In light of the current gaps and limitations of the developed systems, the design of novel biomaterials for this purpose with unique features is also discussed.

Fibronectin-Functionalized Fibrous Meshes as a Substrate to Support Cultures of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

A prion-like domain in ELF3 functions as a thermosensor in Arabidopsis.

Temperature controls plant growth and development, and climate change has already altered the phenology of wild plants and crops1. However, the mechanisms by which plants sense temperature are not well understood. The evening complex is a major signalling hub and a core component of the plant circadian clock2,3. The evening complex acts as a temperature-responsive transcriptional repressor, providing rhythmicity and temperature responsiveness to growth through unknown mechanisms2,4-6. The evening complex consists of EARLY FLOWERING 3 (ELF3)4,7, a large scaffold protein and key component of temperature sensing; ELF4, a small α-helical protein; and LUX ARRYTHMO (LUX), a DNA-binding protein required to recruit the evening complex to transcriptional targets. ELF3 contains a polyglutamine (polyQ) repeat8-10, embedded within a predicted prion domain (PrD). Here we find that the length of the polyQ repeat correlates with thermal responsiveness. We show that ELF3 proteins in plants from hotter climates, with no detectable PrD, are active at high temperatures, and lack thermal responsiveness. The temperature sensitivity of ELF3 is also modulated by the levels of ELF4, indicating that ELF4 can stabilize the function of ELF3. In both Arabidopsis and a heterologous system, ELF3 fused with green fluorescent protein forms speckles within minutes in response to higher temperatures, in a PrD-dependent manner. A purified fragment encompassing the ELF3 PrD reversibly forms liquid droplets in response to increasing temperatures in vitro, indicating that these properties reflect a direct biophysical response conferred by the PrD. The ability of temperature to rapidly shift ELF3 between active and inactive states via phase transition represents a previously unknown thermosensory mechanism.

Molecular mechanisms of Evening Complex activity in Arabidopsis.

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily.

The short chain dehydrogenase/reductase superfamily (SDR) is a large family of NAD(P)H-dependent enzymes found in all kingdoms of life. SDRs are particularly well-represented in plants, playing diverse roles in both primary and secondary metabolism. In addition, some plant SDRs are also able to catalyse a reductive cyclisation reaction critical for the biosynthesis of the iridoid backbone that contains a fused 5 and 6-membered ring scaffold. Mining the EST database of Plantago major, a medicinal plant that makes iridoids, we identified a putative 5ß-progesterone reductase gene, PmMOR (P. major multisubstrate oxido-reductase), that is 60% identical to the iridoid synthase gene from Catharanthus roseus. The PmMOR protein was recombinantly expressed and its enzymatic activity assayed against three putative substrates, 8-oxogeranial, citral and progesterone. The enzyme demonstrated promiscuous enzymatic activity and was able to not only reduce progesterone and citral, but also to catalyse the reductive cyclisation of 8-oxogeranial. The crystal structures of PmMOR wild type and PmMOR mutants in complex with NADP+ or NAD+ and either 8-oxogeranial, citral or progesterone help to reveal the substrate specificity determinants and catalytic machinery of the protein. Site-directed mutagenesis studies were performed and provide a foundation for understanding the promiscuous activity of the enzyme.

Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis.

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.

Transthyretin neuroprotection in Alzheimer's disease is dependent on proteolysis.

The deposition of amyloid ß peptide (Aß) in the hippocampus is one of the major hallmarks of Alzheimer's disease, a neurodegenerative disorder characterized by memory loss and cognitive impairment. The modulation of Aß levels in the brain results from an equilibrium between its production from the amyloid precursor protein and removal by amyloid clearance proteins, which might occur via enzymatic (Aß-degrading enzymes) or nonenzymatic (binding/transport proteins) reactions. Transthyretin (TTR) is one of the major Aß-binding proteins acting as a neuroprotector in AD. In addition, TTR cleaves Aß peptide in vitro. In this work, we show that proteolytically active TTR, and not the inactive form of the protein, impacts on Aß fibrillogenesis, degrades neuronal-secreted Aß, and reduces Aß-induced toxicity in hippocampal neurons. Our data demonstrate that TTR proteolytic activity is required for the neuroprotective effect of the protein constituting a putative novel therapeutic target for AD.

Merging of synchrotron serial crystallographic data by a genetic algorithm.

Recent advances in macromolecular crystallography have made it practical to rapidly collect hundreds of sub-data sets consisting of small oscillations of incomplete data. This approach, generally referred to as serial crystallography, has many uses, including an increased effective dose per data set, the collection of data from crystals without harvesting (in situ data collection) and studies of dynamic events such as catalytic reactions. However, selecting which data sets from this type of experiment should be merged can be challenging and new methods are required. Here, it is shown that a genetic algorithm can be used for this purpose, and five case studies are presented in which the merging statistics are significantly improved compared with conventional merging of all data.

The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization.

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock. Lux mutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein-DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using the MeshAndCollect workflow available at the ESRF.

Highly efficient baculovirus-mediated multigene delivery in primary cells.

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.

Evolution of the Plant Reproduction Master Regulators LFY and the MADS Transcription Factors: The Role of Protein Structure in the Evolutionary Development of the Flower.

Understanding the evolutionary leap from non-flowering (gymnosperms) to flowering (angiosperms) plants and the origin and vast diversification of the floral form has been one of the focuses of plant evolutionary developmental biology. The evolving diversity and increasing complexity of organisms is often due to relatively small changes in genes that direct development. These "developmental control genes" and the transcription factors (TFs) they encode, are at the origin of most morphological changes. TFs such as LEAFY (LFY) and the MADS-domain TFs act as central regulators in key developmental processes of plant reproduction including the floral transition in angiosperms and the specification of the male and female organs in both gymnosperms and angiosperms. In addition to advances in genome wide profiling and forward and reverse genetic screening, structural techniques are becoming important tools in unraveling TF function by providing atomic and molecular level information that was lacking in purely genetic approaches. Here, we summarize previous structural work and present additional biophysical and biochemical studies of the key master regulators of plant reproduction - LEAFY and the MADS-domain TFs SEPALLATA3 and AGAMOUS. We discuss the impact of structural biology on our understanding of the complex evolutionary process leading to the development of the bisexual flower.

Structural basis for the oligomerization of the MADS domain transcription factor SEPALLATA3 in Arabidopsis.

In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.

Endo-ß-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

The crystal structure of wild-type endo-ß-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (ß/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.