Photodynamic Therapy Minimally Affects HEMA-DMAEMA Hydrogel Viscoelasticity.

Soft matter implants are a rapidly growing field in medicine for reconstructive surgery, aesthetic treatments, and regenerative medicine. Though these procedures are efficacious, all implants carry risks associated with microbial infection which are often aggressive. Preventative and responsive measures exist but are limited in applicability to soft materials. Photodynamic therapy (PDT) presents a means to perform safe and effective antimicrobial treatments in proximity to soft implants. HEMA-DMAEMA hydrogels are prepared with the photosensitizer methylene blue included at 10 and 100 µM in solution used for swelling over 2 or 4 days. Thirty minutes or 5 h of LED illumination at 9.20 m W c m 2 $9.20\frac{{mW}}{{c{m}^2}}$ is then used for PDT-induced generation of reactive oxygen species in direct contact with hydrogels to test viable limits of treatment. Frequency sweep rheological measurements reveal minimal overall changes in terms of loss modulus and loss factor but a statistically significant drop in storage modulus for some PDT doses, though within the range of controls and biological variation. These mild impacts suggest the feasibility of PDT application for infection clearing in proximity to soft implants. Future investigation with additional hydrogel varieties and current implant models will further detail the safety of PDT in implant applications.

Gene targeting as a therapeutic avenue in diseases mediated by the complement alternative pathway.

The complement alternative pathway (AP) is implicated in numerous diseases affecting many organs, ranging from the rare hematological disease paroxysmal nocturnal hemoglobinuria (PNH), to the common blinding disease age-related macular degeneration (AMD). Critically, the AP amplifies any activating trigger driving a downstream inflammatory response; thus, components of the pathway have become targets for drugs of varying modality. Recent validation from clinical trials using drug modalities such as inhibitory antibodies has paved the path for gene targeting of the AP or downstream effectors. Gene targeting in the complement field currently focuses on supplementation or suppression of complement regulators in AMD and PNH, largely because the eye and liver are highly amenable to drug delivery through local (eye) or systemic (liver) routes. Targeting the liver could facilitate treatment of numerous diseases as this organ generates most of the systemic complement pool. This review explains key concepts of RNA and DNA targeting and discusses assets in clinical development for the treatment of diseases driven by the alternative pathway, including the RNA-targeting therapeutics ALN-CC5, ARO-C3, and IONIS-FB-LRX, and the gene therapies GT005 and HMR59. These therapies are but the spearhead of potential drug candidates that might revolutionize the field in coming years.

Early onset of senescence and imbalanced epidermal homeostasis across the decades in photoexposed human skin: Fingerprints of inflammaging.

Inflammaging is a theory of ageing which purports that low-level chronic inflammation leads to cellular dysfunction and premature ageing of surrounding tissue. Skin is susceptible to inflammaging because it is the first line of defence from the environment, particularly solar radiation. To better understand the impact of ageing and photoexposure on epidermal biology, we performed a system biology-based analysis of photoexposed face and arm, and photoprotected buttock sites, from women between the ages of 20s to 70s. Biopsies were analysed by histology, transcriptomics, and proteomics and skin surface biomarkers collected from tape strips. We identified morphological changes with age of epidermal thinning, rete ridge pathlength loss and stratum corneum thickening. The SASP biomarkers IL-8 and IL-1RA/IL1-α were consistently elevated in face across age and cis/trans-urocanic acid were elevated in arms and face with age. In older arms, the DNA damage response biomarker 53BP1 showed higher puncti numbers in basal layers and epigenetic ageing were accelerated. Genes associated with differentiation and senescence showed increasing expression in the 30s whereas genes associated with hypoxia and glycolysis increased in the 50's. Proteomics comparing 60's vs 20's confirmed elevated levels of differentiation and glycolytic-related proteins. Representative immunostaining for proteins of differentiation, senescence and oxygen sensing/hypoxia showed similar relationships. This system biology-based analysis provides a body of evidence that young photoexposed skin is undergoing inflammaging. We propose the presence of chronic inflammation in young skin contributes to an imbalance of epidermal homeostasis that leads to a prematurely aged appearance during later life.

Trophoblast lineage-specific differentiation and associated alterations in preeclampsia and fetal growth restriction.

The human placenta is a poorly-understood organ, but one that is critical for proper development and growth of the fetus in-utero. The epithelial cell type that contributes to primary placental functions is called "trophoblast," including two main subtypes, villous and extravillous trophoblast. Cytotrophoblast and syncytiotrophoblast comprise the villous compartment and contribute to gas and nutrient exchange, while extravillous trophoblast invade and remodel the uterine wall and vessels, in order to supply maternal blood to the growing fetus. Abnormal differentiation of trophoblast contributes to placental dysfunction and is associated with complications of pregnancy, including preeclampsia (PE) and fetal growth restriction (FGR). This review describes what is known about the cellular organization of the placenta during both normal development and in the setting of PE/FGR. It also explains known trophoblast lineage-specific markers and pathways regulating their differentiation, and how these are altered in the setting of PE/FGR, focusing on studies which have used human placental tissues. Finally, it also highlights remaining questions and needed resources to advance this field.

The characterization of Mediator 12 and 13 as conditional positive gene regulators in Arabidopsis.

Mediator 12 (MED12) and MED13 are components of the Mediator multi-protein complex, that facilitates the initial steps of gene transcription. Here, in an Arabidopsis mutant screen, we identify MED12 and MED13 as positive gene regulators, both of which contribute broadly to morc1 de-repressed gene expression. Both MED12 and MED13 are preferentially required for the expression of genes depleted in active chromatin marks, a chromatin signature shared with morc1 re-activated loci. We further discover that MED12 tends to interact with genes that are responsive to environmental stimuli, including light and radiation. We demonstrate that light-induced transient gene expression depends on MED12, and is accompanied by a concomitant increase in MED12 enrichment during induction. In contrast, the steady-state expression level of these genes show little dependence on MED12, suggesting that MED12 is primarily required to aid the expression of genes in transition from less-active to more active states.

Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action.

Bioactive natural products have evolved to inhibit specific cellular targets and have served as lead molecules for health and agricultural applications for the past century1-3. The post-genomics era has brought a renaissance in the discovery of natural products using synthetic-biology tools4-6. However, compared to traditional bioactivity-guided approaches, genome mining of natural products with specific and potent biological activities remains challenging4. Here we present the discovery and validation of a potent herbicide that targets a critical metabolic enzyme that is required for plant survival. Our approach is based on the co-clustering of a self-resistance gene in the natural-product biosynthesis gene cluster7-9, which provides insight into the potential biological activity of the encoded compound. We targeted dihydroxy-acid dehydratase in the branched-chain amino acid biosynthetic pathway in plants; the last step in this pathway is often targeted for herbicide development10. We show that the fungal sesquiterpenoid aspterric acid, which was discovered using the method described above, is a sub-micromolar inhibitor of dihydroxy-acid dehydratase that is effective as a herbicide in spray applications. The self-resistance gene astD was validated to be insensitive to aspterric acid and was deployed as a transgene in the establishment of plants that are resistant to aspterric acid. This herbicide-resistance gene combination complements the urgent ongoing efforts to overcome weed resistance11. Our discovery demonstrates the potential of using a resistance-gene-directed approach in the discovery of bioactive natural products.

Fibromodulin-deficiency alters temporospatial expression patterns of transforming growth factor-ß ligands and receptors during adult mouse skin wound healing.

Fibromodulin (FMOD) is a small leucine-rich proteoglycan required for scarless fetal cutaneous wound repair. Interestingly, increased FMOD levels have been correlated with decreased transforming growth factor (TGF)-ß1 expression in multiple fetal and adult rodent models. Our previous studies demonstrated that FMOD-deficiency in adult animals results in delayed wound closure and increased scar size accompanied by loose package collagen fiber networks with increased fibril diameter. In addition, we found that FMOD modulates in vitro expression and activities of TGF-ß ligands in an isoform-specific manner. In this study, temporospatial expression profiles of TGF-ß ligands and receptors in FMOD-null and wild-type (WT) mice were compared by immunohistochemical staining and quantitative reverse transcriptase-polymerase chain reaction using a full-thickness, primary intention wound closure model. During the inflammatory stage, elevated inflammatory infiltration accompanied by increased type I TGF-ß receptor levels in individual inflammatory cells was observed in FMOD-null wounds. This increased inflammation was correlated with accelerated epithelial migration during the proliferative stage. On the other hand, significantly more robust expression of TGF-ß3 and TGF-ß receptors in FMOD-null wounds during the proliferative stage was associated with delayed dermal cell migration and proliferation, which led to postponed granulation tissue formation and wound closure and increased scar size. Compared with WT controls, expression of TGF-ß ligands and receptors by FMOD-null dermal cells was markedly reduced during the remodeling stage, which may have contributed to the declined collagen synthesis capability and unordinary collagen architecture. Taken together, this study demonstrates that a single missing gene, FMOD, leads to conspicuous alternations in TGF-ß ligand and receptor expression at all stages of wound repair in various cell types. Therefore, FMOD critically coordinates temporospatial distribution of TGF-ß ligands and receptors in vivo, suggesting that FMOD modulates TGF-ß bioactivity in a complex way beyond simple physical binding to promote proper wound healing.

Facile immunotargeting of nanoparticles against tumor antigens using site-specific biotinylated antibody fragments.

Nanotechnology is actively being developed for preclinical and clinical oncology applications. Nanoparticle-based immunotargeting against tumor antigens with antibodies or antibody fragments is designed to increase the nanoparticle concentration at the tumor site. However, chemical-based strategies for bioconjugating antibody fragments to nanoparticles typically result in a functionally heterogeneous population of conjugates due to alteration of amino acids within the antigen binding site. The loss of function can be prevented by expressing recombinant antibodies that contain a unique bioconjugation site, which is isolated from the antigen binding site. Biobodies are antibody fragments biosynthetically biotinylated by yeast at a specific biotin acceptor site and secreted into the culture supernatant. The high specificity and affinity between streptavidin-labeled nanoparticles and soluble biobodies allow self-assembly of immunotargeted nanoparticles directly in the yeast culture supernatant. Here, we demonstrate the versatility of biobodies for nanoparticle immunotargeting using streptavidin-labeled superparamagnetic iron oxide nanoparticles as a general, modular scaffold. Biobody-mediated targeting was performed against two antigens (mesothelin and TEM1) that are upregulated in solid tumors. The technology for biosynthetic biotinylation can be extended to proteins other than antibody fragments and adopted by fields outside of oncology for directed modification of any streptavidin-functionalized surface.

A quantitative approach to scar analysis.

Analysis of collagen architecture is essential to wound healing research. However, to date no consistent methodologies exist for quantitatively assessing dermal collagen architecture in scars. In this study, we developed a standardized approach for quantitative analysis of scar collagen morphology by confocal microscopy using fractal dimension and lacunarity analysis. Full-thickness wounds were created on adult mice, closed by primary intention, and harvested at 14 days after wounding for morphometrics and standard Fourier transform-based scar analysis as well as fractal dimension and lacunarity analysis. In addition, transmission electron microscopy was used to evaluate collagen ultrastructure. We demonstrated that fractal dimension and lacunarity analysis were superior to Fourier transform analysis in discriminating scar versus unwounded tissue in a wild-type mouse model. To fully test the robustness of this scar analysis approach, a fibromodulin-null mouse model that heals with increased scar was also used. Fractal dimension and lacunarity analysis effectively discriminated unwounded fibromodulin-null versus wild-type skin as well as healing fibromodulin-null versus wild-type wounds, whereas Fourier transform analysis failed to do so. Furthermore, fractal dimension and lacunarity data also correlated well with transmission electron microscopy collagen ultrastructure analysis, adding to their validity. These results demonstrate that fractal dimension and lacunarity are more sensitive than Fourier transform analysis for quantification of scar morphology.

Delayed wound closure in fibromodulin-deficient mice is associated with increased TGF-ß3 signaling.

Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-ß (TGF-ß) modulation. Using an adult fmod-null (fmod(-/-)) mouse model, this study further elucidates the interplay between FMOD and TGF-ß expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod(-/-) and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod(-/-) mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod(-/-) wounds exhibited early elevation (within 24 hours post-wounding) of type I and type II TGF-ß receptors as well as unexpectedly high fibroblast expression of TGF-ß3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-ß3, fmod(-/-) fibroblasts were significantly less motile than WT fibroblasts. fmod(-/-) fibroblasts were also more susceptible to migration inhibition by TGF-ß3, leading to profound delays in dermal cell migration. Increased scarring in fmod(-/-) mice indicates that TGF-ß3's antimotility effects predominate over its antifibrotic effects when high TGF-ß3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-ß bioactivity.