Polymer architecture as key to unprecedented high-resolution 3D-printing performance : the case of biodegradable hexa-functional telechelic urethane-based poly-ε-caprolactone

Two-photon polymerization (2PP) is a high-resolution 3D-printing technology with a very rapidly expanding field of applications, including tissue engineering (TE). In this field, 2PP offers unprecedented possibilities for systematic studies of both cell–cell and cell–material interactions in 3D. For TE applications, the reliable production of biodegradable micro-scaffolds in porous, complex architectures is essential. However, the number of biodegradable materials that support the required level of spatial resolution is very limited, being a major bottleneck for the use of 2PP in the TE field. Herein, we introduce a hexa-functional urethane-based biodegradable precursor that overcomes the limitations associated with the high-resolution printing of current biodegradable precursors. The precursor is a telechelic urethane-based poly-ε-caprolactone (PCL) possessing three acrylate functionalities at each polymer end group which enables the reliable production of complex architectures owing to its superior physical properties as compared to the traditional di-acrylate terminated analogs. The newly developed hexa-functional telechelic urethane-based PCL reveals enhanced crosslinking kinetics and one order of magnitude higher Young’s modulus compared to the di-functional precursor (57.8 versus 6.3 MPa), providing an efficient and solvent-free 2PP processing at fast scanning speeds of up to 100 mm s−1 with unprecedented feature resolutions (143 ± 18 nm at 100 mm s−1 scanning speed). The crosslinked hexa-functional polymer combines strength and flexibility owing to the segregation between its hard polyacrylate and soft PCL segments, which makes it suitable for biological systems in contrast to the highly crosslinked and rigid structures typically manufactured by 2PP. Furthermore, it revealed lower degradation rate compared to its di-functional analog, which can be considered as an advantage in terms of biocompatibility due to the slower formation of acidic degradation products. Extracts of the developed polymers did not show a cytotoxic effect on the L929 fibroblasts as confirmed via ISO 10993-5 standard protocol. The presented precursor design constitutes a simple and effective approach that can be easily translated towards other biodegradable polymers for the manufacturing of biodegradable constructs with nano-scale precision, offering for the first time to use the true capabilities of 2PP for TE applications with the use of synthetic biodegradable polymers

Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes

Cancer immunotherapy can induce durable antitumor responses. However, many patients poorly respond to such therapies. Here we describe a generic antitumor therapy that is based on the intratumor delivery of mRNA that codes for the necroptosis executioner mixed lineage kinase domain-like (MLKL) protein. This intervention stalls primary tumor growth and protects against distal and disseminated tumor formation in syngeneic mouse melanoma and colon carcinoma models. Moreover, MLKL-mRNA treatment combined with immune checkpoint blockade further improves the antitumor activity. MLKL-mRNA treatment rapidly induces T cell responses directed against tumor neo-antigens and requires CD4(+) and CD8(+) T cells to prevent tumor growth. Type I interferon signaling and Batf3-dependent dendritic cells are essential for this mRNA treatment to elicit tumor antigen-specific T cell responses. Moreover, MLKL-mRNA treatment blunts the growth of human lymphoma in mice with a reconstituted human adaptive immune system. MLKL-based treatment can thus be exploited as an effective antitumor immunotherapy

Intelligent hydrogel design: Towards more performing hydrogel processing

Despite their highly attractive properties, 3D printing of hydrogel materials can be rather challenging. Herein, we present a novel hydrogel material that can be easily processed into three-dimensional scaffolds using different 3D printing technologies. An acrylate-terminated, urethane-based PEG was prepared by reacting PEG 2000 with isophorone diisocyanate (IPDI) and monoacrylated PEG (336 g/mol) in a 1:2:2 molar ratio (WO 2017/005613 A1). For melt 3D-printing, pure polymer was used (Tm 38°C). For bioprinting, a 50 wt% solution with 3 wt% Laponite was used. Please click Additional Files below to see the full abstract

Removal of the N-glycosylation sequon at position N116 located in p27 of the respiratory syncytial virus fusion protein elicits enhanced antibody responses after DNA immunization

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates

Inhibition of caspase-1 prolongs survival of mice infected with rabies virus

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response

High resolution synchrotron imaging of wheat root hairs growing in soil and image based modelling of phosphate uptake

Root hairs are known to be highly important for uptake of sparingly soluble nutrients, particularly in nutrient deficient soils. Development of increasingly sophisticated mathematical models has allowed uptake characteristics to be quantified. However, modelling has been constrained by a lack of methods for imaging live root hairs growing in real soils.We developed a plant growth protocol and used Synchrotron Radiation X-ray Tomographic Microscopy (SRXTM) to uncover the 3D interactions of root hairs in real soil. We developed a model of phosphate uptake by root hairs based directly on the geometry of hairs and associated soil pores as revealed by imaging.Previous modelling studies found that root hairs dominate phosphate uptake. By contrast, our study suggests that hairs and roots contribute equally. We show that uptake by hairs is more localised than by roots and strongly dependent on root hair and aggregate orientation.The ability to image hair-soil interactions enables a step change in modelling approaches, allowing a more realistic treatment of processes at the scale of individual root hairs in soil pores

Worldwide absence of canonical benzimidazole resistance-associated mutations within β-tubulin genes from Ascaris

Background: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with β-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the β-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the β-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. Methods: To further determine the putative absence of key β-tubulin polymorphisms, we screened two β-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris β-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. Results: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of β-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for β-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of β-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. Conclusions: Our work suggests an absence of canonical β-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy. Graphical Abstract

Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms