IER5, a DNA damage response gene, is required for Notch-mediated induction of squamous cell differentiation.

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A, a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.

Stromal vascular fraction cells as biologic coating of mesh for hernia repair.

BACKGROUND: The interest in non-manipulated cells originating from adipose tissue has raised tremendously in the field of tissue engineering and regenerative medicine. The resulting stromal vascular fraction (SVF) cells have been successfully used in numerous clinical applications. The aim of this experimental work is, first to combine a macroporous synthetic mesh with SVF isolated using a mechanical disruption process, and to assess the effect of those cells on the early healing phase of hernia. METHODS: Human SVF cells combined with fibrin were used to coat commercial titanized polypropylene meshes. In vitro, viability and growth of the SVF cells were assessed using live/dead staining and scanning electron microscopy. The influence of SVF cells on abdominal wall hernia healing was conducted on immunodeficient rats, with a focus on short-term vascularization and fibrogenesis. RESULTS: Macroporous meshes were easily coated with SVF using a fibrin gel as temporary carrier. The in vitro experiments showed that the whole process including the isolation of human SVF cells and their coating on PP meshes did not impact on the SVF cells' viability and on their capacity to attach and to proliferate. In vivo, the SVF cells were well tolerated by the animals, and coating mesh with SVF resulted in a decrease degree of vascularity compared to control group at day 21. CONCLUSIONS: The utilization of SVF-coated mesh influences the level of angiogenesis during the early onset of tissue healing. Further long-term animal experiments are needed to confirm that this effect correlates with a more robust mesh integration compared to non-SVF-coated mesh.

Efficacy of antimicrobial agents delivered to hernia meshes using an adaptable thermo-responsive hyaluronic acid-based coating.

PURPOSE: Mesh-related infection is a critical outcome for patients with hernia defect stabilized with synthetic or biological meshes. Even though bioactive meshes loaded with antibiotics or antiseptics are slowly emerging in the market, the available solutions still lack versatility. Here, we proposed a polymer solution, i.e., hyaluronic acid-poly(N-isopropylacrylamide) (HApN), which forms a hydrogel to be used as coating for meshes only when it reaches body temperature. METHODS: We assessed how the gelation of HApN was influenced by the incorporation of different antibiotic and antiseptic formulations, and how this gel can be used to coat several mesh types. The impact of the coating on the elastic behavior of a macroporous mesh was tested under cyclic elongation condition. Finally, we selected two different coating formulations, one based on antibiotics (gentamicin + rifampicin) and one based on antiseptic (chlorhexidine) and tested in vitro their antimicrobial efficacies. RESULTS: HApN can be used as carrier for different antimicrobial agents, without having a strong influence on its gelation behavior. Porous or dense meshes can be coated with this polymer, even though the stability was not optimal on macroporous meshes such as Optilene when pores are too large. HApN loaded with drugs inhibited in vitro the growth of several Gram-positive and Gram-negative bacteria. CONCLUSION: Compared to the available technologies developed to endow meshes with antibacterial activity, the proposed HApN offers further versatility with potential to prevent mesh-related infection in hernioplasty.

A critical review of the in vitro and in vivo models for the evaluation of anti-infective meshes.

BACKGROUND: Infectious complications following mesh implantation for abdominal wall repair appear in 0.7 up to 26.6% of hernia repairs and can have a detrimental impact for the patient. To prevent or to treat mesh-related infection, the scientific community is currently developing a veritable arsenal of antibacterial meshes. The numerous and increasing reports published every year describing new technologies indicate a clear clinical need, and an academic interest in solving this problem. Nevertheless, to really appreciate, to challenge, to compare and to optimize the antibacterial properties of next generation meshes, it is important to know which models are available and to understand them. PURPOSE: We proposed for the first time, a complete overview focusing only on the in vitro and in vivo models which have been employed specifically in the field of antibacterial meshes for hernia repair. RESULTS AND CONCLUSION: From this investigation, it is clear that there has been vast progress and breadth in new technologies and models to test them. However, it also shows that standardization or adoption of a more restricted number of models would improve comparability and be a benefit to the field of study.

Infections associated with mesh repairs of abdominal wall hernias: Are antimicrobial biomaterials the longed-for solution?

The incidence of mesh-related infection after abdominal wall hernia repair is low, generally between 1 and 4%; however, worldwide, this corresponds to tens of thousands of difficult cases to treat annually. Adopting best practices in prevention is one of the keys to reduce the incidence of mesh-related infection. Once the infection is established, however, only a limited number of options are available that provides an efficient and successful treatment outcome. Over the past few years, there has been a tremendous amount of research dedicated to the functionalization of prosthetic meshes with antimicrobial properties, with some receiving regulatory approval and are currently available for clinical use. In this context, it is important to review the clinical importance of mesh infection, its risk factors, prophylaxis and pathogenicity. In addition, we give an overview of the main functionalization approaches that have been applied on meshes to confer anti-bacterial protection, the respective benefits and limitations, and finally some relevant future directions.

Drug delivery systems functionalized with bone mineral seeking agents for bone targeted therapeutics.

The systemic administration of drugs to treat bone diseases is often associated with poor uptake of the drug in the targeted tissue, potential systemic toxicity and suboptimal efficacy. In order to overcome these limitations, many micro- and nano-sized drug carriers have been developed for the treatment of bone pathologies that exhibit specific affinity for bone. Drug carriers can be functionalized with bone mineral seekers (BMS), creating a targeted drug delivery system (DDS) which is able to bind to bone and release therapeutics directly at the site of interest. This class of advanced DDS is of tremendous interest due to their strong affinity to bone, with great expectation to treat life-threatening bone disorders such as osteomyelitis, osteosarcoma or even osteoporosis. In this review, we first explain the mechanisms behind the affinity of several well-known BMS to bone, and then we present several effective approaches allowing the incorporation BMS into advanced DDS. Finally, we report the therapeutic applications of BMS based DDS under development or already established. Understanding the mechanisms behind the biological activity of recently developed BMS and their integration into advanced therapeutic delivery systems are essential prerequisites for further development of bone-targeting therapies with optimal efficacy.

Surface-enrichment with hydroxyapatite nanoparticles in stereolithography-fabricated composite polymer scaffolds promotes bone repair.

Fabrication of composite scaffolds using stereolithography (SLA) for bone tissue engineering has shown great promises. However, in order to trigger effective bone formation and implant integration, exogenous growth factors are commonly combined to scaffold materials. In this study, we fabricated biodegradable composite scaffolds using SLA and endowed them with osteopromotive properties in the absence of biologics. First we prepared photo-crosslinkable poly(trimethylene carbonate) (PTMC) resins containing 20 and 40wt% of hydroxyapatite (HA) nanoparticles and fabricated scaffolds with controlled macro-architecture. Then, we conducted experiments to investigate how the incorporation of HA in photo-crosslinked PTMC matrices improved human bone marrow stem cells osteogenic differentiation in vitro and kinetic of bone healing in vivo. We observed that bone regeneration was significantly improved using composite scaffolds containing as low as 20wt% of HA, along with difference in terms of osteogenesis and degree of implant osseointegration. Further investigations revealed that SLA process was responsible for the formation of a rich microscale layer of HA corralling scaffolds. To summarize, this work is of substantial importance as it shows how the fabrication of hierarchical biomaterials via surface-enrichment of functional HA nanoparticles in composite polymer stereolithographic structures could impact in vitro and in vivo osteogenesis. STATEMENT OF SIGNIFICANCE: This study reports for the first time the enhance osteopromotion of composite biomaterials, with controlled macro-architecture and microscale distribution of hydroxyapatite particles, manufactured by stereolithography. In this process, the hydroxyapatite particles are not only embedded into an erodible polymer matrix, as reported so far in the literature, but concentrated at the surface of the structures. This leads to robust in vivo bone formation at low concentration of hydroxyapatite. The reported 3D self-corralling composite architecture provides significant opportunities to develop functional biomaterials for bone repair and tissue engineering.

Fabrication of silk mesh with enhanced cytocompatibility: preliminary in vitro investigation toward cell-based therapy for hernia repair.

Recent studies have demonstrated that combining cells with meshes prior to implantation successfully enhanced hernia repair. The idea is to create a biologic coating surrounding the mesh with autologous cells, before transplantation into the patient. However, due to the lack of a prompt and robust cell adhesion to the meshes, extensive in vitro cultivation is required to obtain a homogenous cell layer covering the mesh. In this context, the objective of this publication is to manufacture meshes made of silk fibres and to enhance the cytoadhesion and cytocompatibility of the biomaterial by surface immobilization of a pro-adhesive wheat germ agglutinin (lectin WGA). We first investigated the affinity between the glycoprotein WGA and cells, in solution and then after covalent immobilization of WGA on silk films. Then, we manufactured meshes made of silk fibres, tailored them with WGA grafting and finally evaluated the cytocompatibility and the inflammatory response of silk and silk-lectin meshes compared to common polypropylene mesh, using fibroblasts and peripheral blood mononuclear cells, respectively. The in vitro experiments revealed that the cytocompatibility of silk can be enhanced by surface immobilization with lectin WGA without exhibiting negative response in terms of pro-inflammatory reaction. Grafting lectin to silk meshes could bring advantages to facilitate cell-coating of meshes prior to implantation, which is an imperative prerequisite for abdominal wall tissue regeneration using cell-based therapy.

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response.

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3ß complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3ß or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3ß-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.

BRCA1 is required for postreplication repair after UV-induced DNA damage.

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent but nucleotide excision repair (NER)-independent manner. More specifically, at UV-stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and postreplicative repair. These BRCA1 functions differ from those required for DSBR.

Inhibition of ALK, PI3K/MEK, and HSP90 in murine lung adenocarcinoma induced by EML4-ALK fusion oncogene.

Genetic rearrangements of the anaplastic lymphoma kinase (ALK) kinase occur in 3% to 13% of non-small cell lung cancer patients and rarely coexist with KRASor EGFR mutations. To evaluate potential treatment strategies for lung cancers driven by an activated EML4-ALK chimeric oncogene, we generated a genetically engineered mouse model that phenocopies the human disease where this rearranged gene arises. In this model, the ALK kinase inhibitor TAE684 produced greater tumor regression and improved overall survival compared with carboplatin and paclitaxel, representing clinical standard of care. 18F-FDG-PET-CT scans revealed almost complete inhibition of tumor metabolic activity within 24 hours of TAE684 exposure. In contrast, combined inhibition of the PI3K/AKT and MEK/ERK1/2 pathways did not result in significant tumor regression. We identified EML4-ALK in complex with multiple cellular chaperones including HSP90. In support of a functional reliance, treatment with geldanamycin-based HSP90 inhibitors resulted in rapid degradation of EML4-ALK in vitro and substantial, albeit transient, tumor regression in vivo. Taken together, our findings define a murine model that offers a reliable platform for the preclinical comparison of combinatorial treatment approaches for lung cancer characterized by ALK rearrangement.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration.

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Online nanoflow RP-RP-MS reveals dynamics of multicomponent Ku complex in response to DNA damage.

Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.

Does the Pyrenean salamander Euproctus asper use chemical cues for sex identification and mating behaviour?

This paper examined the potentiality of chemical cues for sex identification and mating activity in Euproctus asper. The study tested the ability of males and females to distinguish between the odour of animals of their own sex and that of animals of the opposite sex, as it was carried to them by water flowing over another animal. Their ability to distinguish between water flowing over another E. asper and water flowing over a control, where no other animal was present, was also tested. Then, the study tested their ability to distinguish between the diffusing odour of animals of their own sex, that of animals of the opposite sex, and that of a control, where no other animal was present. There was no evidence that males and females identify their mates using chemical cues. Observations of the courtship behaviour were also carried out. Mating seems to be induced by the male's display of his tail when he captures the female as she passes near him, to form an amplexus, without any obvious preliminary. On the basis of these data, the question whether the mate identification occurs during the amplexus in this species was raised.