Chromosome social distancing and crowd control: the dual role of Ki67.

The Ki67 protein is proposed to have two conformations; one which segregates chromosomes before anaphase, and the other which results in chromosome condensation after cell division to exclude large cytosolic components from the reforming nuclei of daughter cells.

High throughput, high resolution enzymatic lithography process: effect of crystallite size, moisture, and enzyme concentration.

By bringing enzymes into contact with predefined regions of a surface, a polymer film can be selectively degraded to form desired patterns that find a variety of applications in biotechnology and electronics. This so-called "enzymatic lithography" is an environmentally friendly process as it does not require actinic radiation or synthetic chemicals to develop the patterns. A significant challenge to using enzymatic lithography has been the need to restrict the mobility of the enzyme in order to maintain control of feature sizes. Previous approaches have resulted in low throughput and were limited to polymer films only a few nanometers thick. In this paper, we demonstrate an enzymatic lithography system based on Candida antartica lipase B (CALB) and poly(ε-caprolactone) (PCL) that can resolve fine-scale features, (<1 µm across) in thick (0.1-2.0 µm) polymer films. A Polymer Pen Lithography (PPL) tool was developed to deposit an aqueous solution of CALB onto a spin-cast PCL film. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 µm). The effect of printing conditions and relative humidity during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a "Green" approach to the fabrication of printed circuit boards.

Singlet oxygen generation on porous superhydrophobic surfaces: effect of gas flow and sensitizer wetting on trapping efficiency.

We describe physical-organic studies of singlet oxygen generation and transport into an aqueous solution supported on superhydrophobic surfaces on which silicon-phthalocyanine (Pc) particles are immobilized. Singlet oxygen ((1)O2) was trapped by a water-soluble anthracene compound and monitored in situ using a UV-vis spectrometer. When oxygen flows through the porous superhydrophobic surface, singlet oxygen generated in the plastron (i.e., the gas layer beneath the liquid) is transported into the solution within gas bubbles, thereby increasing the liquid-gas surface area over which singlet oxygen can be trapped. Higher photooxidation rates were achieved in flowing oxygen, as compared to when the gas in the plastron was static. Superhydrophobic surfaces were also synthesized so that the Pc particles were located in contact with, or isolated from, the aqueous solution to evaluate the relative effectiveness of singlet oxygen generated in solution and the gas phase, respectively; singlet oxygen generated on particles wetted by the solution was trapped more efficiently than singlet oxygen generated in the plastron, even in the presence of flowing oxygen gas. A mechanism is proposed that explains how Pc particle wetting, plastron gas composition and flow rate as well as gas saturation of the aqueous solution affect singlet oxygen trapping efficiency. These stable superhydrophobic surfaces, which can physically isolate the photosensitizer particles from the solution may be of practical importance for delivering singlet oxygen for water purification and medical devices.

Singlet oxygen generation on a superhydrophobic surface: Effect of photosensitizer coating and incident wavelength on 1O2 yields.

Photochemical generation of singlet oxygen (1O2) often relies on homogenous systems; however, a dissolved photosensitizer (PS) may be unsuitable for some applications because it is difficult to recover, expensive to replenish, and hazardous to the environment. Isolation of the PS onto a solid support can overcome these limitations, but implementation faces other challenges, including agglomeration of the solid PS, physical quenching of 1O2 by the support, photooxidation of the PS, and hypoxic environments. Here, we explore a superhydrophobic polydimethylsiloxane (SH-PDMS) support coated with the photosensitizer 5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H-porphyrin (TFPP). This approach seeks to address the challenges of a heterogeneous system by using a support that exhibits low 1O2 physical quenching rates, a fluorinated PS that is chemically resistant to photooxidation, and a superhydrophobic surface that entraps a layer of air, thus preventing hypoxia. Absorbance and fluorescence spectroscopy reveal the monomeric arrangement of TFPP on SH-PDMS surfaces, a surprising but favorable characteristic for a solid-phase PS on 1O2 yields. We also investigated the effect of incident wavelength on 1O2 yields for TFPP in aqueous solution and immobilized on SH-PDMS and found overall yields to be dependent on the absorption coefficient, while the yield per absorbed photon exhibited wavelength independence, in accordance with Kasha-Vavilov's rule.

Superhydrophobic photosensitizers. Mechanistic studies of (1)O2 generation in the plastron and solid/liquid droplet interface.

We describe here a physical-organic study of the first triphasic superhydrophobic sensitizer for photooxidations in water droplets. Control of synthetic parameters enables the mechanistic study of "borderline" two- and three-phase superhydrophobic sensitizer surfaces where (1)O2 is generated in compartments that are wetted, partially wetted, or remain dry in the plastron (i.e., air layer beneath the droplet). The superhydrophobic surface is synthesized by partially embedding silicon phthalocyanine (Pc) sensitizing particles to specific locations on polydimethylsiloxane (PDMS) posts printed in a square array (1 mm tall posts on 0.5 mm pitch). In the presence of red light and oxygen, singlet oxygen is formed on the superhydrophobic surface and reacts with 9,10-anthracene dipropionate dianion (1) within a freestanding water droplet to produce an endoperoxide in 54-72% yields. Control of the (1)O2 chemistry was achieved by the synthesis of superhydrophobic surfaces enriched with Pc particles either at the PDMS end-tips or at PDMS post bases. Much of the (1)O2 that reacts with anthracene 1 in the droplets was generated by the sensitizer "wetted" at the Pc particle/water droplet interface and gave the highest endoperoxide yields. About 20% of the (1)O2 can be introduced into the droplet from the plastron. The results indicate that the superhydrophobic sensitizer surface offers a unique system to study (1)O2 transfer routes where a balance of gas and liquid contributions of (1)O2 is tunable within the same superhydrophobic surface.

Patterned enzymatic degradation of poly(ε-caprolactone) by high-affinity microcontact printing and polymer pen lithography.

This paper reports deposition of Candida antarctica Lipase B (CALB) on relatively thick poly(ε-caprolactone) (PCL) films (300-500 nm) to create well-defined patterns using two different writing techniques: high-affinity microcontact (HA-µCL) and polymer pen (PPL) lithography. For both, an aqueous CALB ink is absorbed onto a polydimethylsiloxane (PDMS) writing implement (PDMS stamp or a PDMS pen tip), which is transferred to a spun-cast PCL film. HA-µCL experiments demonstrated the importance of applied pressure to obtain high-resolution patterns since uniform contact is needed between raised 20 µm parallel line regions of the PDMS stamp and the surface. AFM imaging shows pattern formation evolves gradually over incubation time only in areas stamped with CALB cutting through spherulites without apparent influence by grain boundaries. Strong binding of CALB to PCL is postulated as the mechanism by which lateral diffusion is limited. PPL enables formation of an arbitrary image by appropriate programming of the robot. The PDMS pen tips were coated with an aqueous CALB solution and then brought into contact with the PCL film to transfer CALB onto the surface. By repeating the ink transfer step multiple times where pen tips are brought into contact with the PCL film at a different locations, a pattern of dots is formed. After printing, patterns were developed at 37 °C and 95% RH. Over a 7-day period, CALB progressively etched the PCL down to the silicon wafer on which it was spun (350 nm) giving round holes with diameters about 10 µm. AFM images show the formation of steep PCL walls indicating CALB degraded the PCL film in areas to which it was applied. This work demonstrates that high-resolution patterns can be achieved without immobilizing the enzyme on the surface of polymeric stamps that limits the depth of features obtained as well as the throughput of the process.

Superhydrophobic Tipped Antimicrobial Photodynamic Therapy Device for the In Vivo Treatment of Periodontitis Using a Wistar Rat Model.

Limited options exist for treatment of periodontitis; scaling and root planing (SRP) are not sufficient to eradicate P. gingivalis and the resulting inflammatory disease. Chlorhexidine (CHX), used as an adjuvant to SRP, may reduce bacterial loads but leads to pain and staining, while evidence for its efficacy is lacking. Antibiotics are effective but can lead to drug-resistance. The rising concern of antibiotic resistance limits the future use of this treatment approach. This study evaluates the efficacy of a novel superhydrophobic (SH) antimicrobial photodynamic therapy (aPDT) device as an adjuvant to SRP for the treatment of periodontitis induced in a Wistar rat in vivo model relative to CHX. The SH-aPDT device comprises an SH silicone rubber strip coated with verteporfin photosensitizer (PS), sterilized, and secured onto a tapered plastic optical fiber tip connected to a red diode laser. The superhydrophobic polydimethylsiloxane (PDMS) strips were fabricated by using a novel soluble template method that creates a medical-grade elastomer with hierarchical surface roughness without the use of nanoparticles. Superhydrophobicity minimizes direct contact of the PS-coated surface with bacterial biofilms. Upon insertion of the device tip into the pocket and energizing the laser, the device generates singlet oxygen that effectively targets and eliminates bacteria within the periodontal pocket. SH-aPDT treatment using 125 J/cm2 of red light on three consecutive days reduced P. gingivalis significantly more than SRP-CHX controls (p < 0.05). Clinical parameters significantly improved (p < 0.05), and histology and stereometry results demonstrated SH-aPDT to be the most effective treatment for improving healing and reducing inflammation, with an increase in fibroblast cells and extracellular matrix and a reduction in vascularization, inflammatory cells, and COX-2 expression. The SH-aPDT approach resulted in complete disease clearance assessed 30 days after treatment initiation with significant reduction of the periodontal pocket and re-formation of the junctional epithelium at the enamel-cementum junction. PS isolation on a SH strip minimizes the potential for bacteria to develop resistance, where the treatment may be aided by the oxygen supply retained within the SH surface.

Generating singlet oxygen bubbles: a new mechanism for gas-liquid oxidations in water.

Laser-coupled microphotoreactors were developed to bubble singlet oxygen [(1)O(2) ((1)Δ(g))] into an aqueous solution containing an oxidizable compound. The reactors consisted of custom-modified SMA fiberoptic receptacles loaded with 150 µm silicon phthalocyanine glass sensitizer particles, where the particles were isolated from direct contact with water by a membrane adhesively bonded to the bottom of each device. A tube fed O(2) gas to the reactor chambers. In the presence of O(2), singlet oxygen was generated by illuminating the sensitizer particles with 669 nm light from an optical fiber coupled to the top of the reactor. The generated (1)O(2) was transported through the membrane by the O(2) stream and formed bubbles in solution. In solution, singlet oxygen reacted with probe compounds (9,10-anthracene dipropionate dianion, trans-2-methyl-2-pentanoate anion, N-benzoyl-D,L-methionine, or N-acetyl-D,L-methionine) to give oxidized products in two stages. The early stage was rapid and showed that (1)O(2) transfer occurred via bubbles mainly in the bulk water solution. The later stage was slow; it arose only from (1)O(2)-probe molecule contact at the gas/liquid interface. A mechanism is proposed that involves (1)O(2) mass transfer and solvation, where smaller bubbles provide better penetration of (1)O(2) into the flowing stream due to higher surface-to-volume contact between the probe molecules and (1)O(2).

Bacterial inactivation by a singlet oxygen bubbler: identifying factors controlling the toxicity of (1)O2 bubbles.

A microphotoreactor device was developed to generate bubbles (1.4 mm diameter, 90 µL) containing singlet oxygen at levels toxic to bacteria and fungus. As singlet oxygen decays rapidly to triplet oxygen, the bubbles leave behind no waste or byproducts other than O(2). From a comparative study in deaerated, air saturated, and oxygenated solutions, it was reasoned that the singlet oxygen bubbles inactivate Escherichia coli and Aspergillus fumigatus, mainly by an oxygen gradient inside and outside of the bubble such that singlet oxygen is solvated and diffuses through the aqueous solution until it reacts with the target organism. Thus, singlet oxygen bubble toxicity was inversely proportional to the amount of dissolved oxygen in solution. In a second mechanism, singlet oxygen interacts directly with E. coli that accumulate at the gas-liquid interface although this mechanism operates at a rate approximately 10 times slower. Due to encapsulation in the gaseous core of the bubble and a 0.98 ms lifetime, the bubbles can traverse relatively long 0.39 mm distances carrying (1)O(2) far into the solution; by comparison the diffusion distance of (1)O(2) fully solvated in H(2)O is much shorter (~150 nm). Bubbles that reached the outer air-water interface contained no (1)O(2). The mechanism by which (1)O(2) deactivated organisms was explored through the addition of detergent molecules and Ca(2+) ions. Results indicate that the preferential accumulation of E. coli at the air-water interface of the bubble leads to enhanced toxicity of bubbles containing (1)O(2). The singlet oxygen device offers intriguing possibilities for creating new types of disinfection strategies based on photodynamic ((1)O(2)) bubble carriers.

Evaluation of photosensitizer-containing superhydrophobic surfaces for the antibacterial treatment of periodontal biofilms.

Antimicrobial photodynamic therapy (aPDT) is a promising approach to control biofilms involved in periodontal diseases. However, certain challenges, such as staining of teeth, preferential interaction of photosensitizer (PS) with Gram-positive versus Gram-negative bacteria, and insufficient oxygen in hypoxic periodontal pockets have presented barriers to its use in the clinic. To overcome these challenges, a novel superhydrophobic (SH) film that generates airborne singlet oxygen has been developed. The SH-aPDT approach isolates the PS onto a topologically rough solid SH film on which channels allow air to diffuse to the PS surface, thus ensuring sufficient oxygen supply. Upon illumination, gas phase singlet oxygen (1O2) is produced and diffuses from the SH surface to the underlying biofilm. The killing efficacy was assessed as a function of transmitted fluence (17.9-89.5 J/cm2) and chorin e6 loading (96-1110 nmol/cm2) by counting of colony forming units, biofilm metabolism by XTT and confocal microscopy. The decrease in viability of both Gram-positive and Gram-negative bacteria in a multi-species biofilm was found to be linearly dependent on the fluence as well as the loading of the PS up to 71.6 J/cm2 when 1110 nmols/cm2 of chlorin e6 was used. A > 4.6 log bacterial reduction was observed under these conditions (p < 0.05). This novel SH-aPDT approach shows promise as an effective method to disinfect multi-species bacterial biofilms associated with periodontal disease and will be evaluated in animal models in future studies.

Ratchetlike slip angle anisotropy on printed superhydrophobic surfaces.

The fabrication and properties of superhydrophobic surfaces that exhibit ratchet-like anisotropic slip angle behavior is described. The surface is composed of arrays of poly(dimethylsiloxane) (PDMS) posts fabricated by a type of 3D printing. By controlling the dispense parameters, regular arrays of asymmetric posts were deposited such that the slope of the posts was varied from 0 to 50 relative to the surface normal. Advancing and receding contact angles as well as slip angles were measured as a function of the post slope and droplet volume. Ratchetlike slip angle anisotropy was observed on surfaces composed of sloped features. The maximum slip angle difference (for a 180° tilt angle variation) was 32° for 20 µL droplets on surfaces with posts fabricated with a slope of 50°. This slip angle anisotropy is attributed to an increase in the triple contact line (TCL) length as the droplet is tilted in a direction against the post slope whereas the TCL decreases continuously when the drop travels in a direction parallel to the post slope. The increasing length of the TCL creates an increased energy barrier that accounts for the higher slip angles in this direction.

Polymers from fatty acids: poly(ω-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies.

This Article describes the synthesis and physicomechanical properties of bioplastics prepared from methyl ω-hydroxytetradecanoic acid (Me-ω-OHC14), a new monomer available by a fermentation process using an engineered Candida tropicalis strain. Melt-condensation experiments were conducted using titanium tetraisopropoxide (Ti[OiPr](4)) as a catalyst in a two-stage polymerization (2 h at 200 °C under N(2), 4 h at 220 °C under 0.1 mmHg). Poly(ω-hydroxytetradecanoate), P(ω-OHC14), M(w), determined by SEC-MALLS, increased from 53K to 110K as the Ti(OiPr)(4) concentration increased from 50 to 300 ppm. By varying the polymerization conditions (catalyst concentration, reaction time, second-stage reaction temperature) a series of P(ω-OHC14) samples were prepared with M(w) values from 53K to 140K. The synthesized polyesters with M(w) ranging from 53K to 140K were subjected to characterization by DSC, TGA, DMTA, and tensile testing. Influences of P(ω-OHC14) molecular weight, melting point, and enthalpies of melting/crystallization on material tensile properties were explored. Cold-drawing tensile tests at room temperature for P(ω-OHC14) with M(w) 53K-78K showed a brittle-to-ductile transition. In contrast, P(ω-OHC14) with M(w) 53K undergoes brittle fracture. Increasing P(ω-OHC14) M(w) above 78K resulted in a strain-hardening phenomena and tough properties with elongation at break ~700% and true tensile strength of ~50 MPa. Comparisons between high density polyethylene and P(ω-OHC14) mechanical and thermal properties as a function of their respective molecular weights are discussed.

Antimicrobial Photodynamic Inactivation Using Topical and Superhydrophobic Sensitizer Techniques: A Perspective from Diffusion in Biofilms.

This review describes nanoparticle and dye diffusion in bacterial biofilms in the context of antimicrobial photodynamic inactivation (aPDI). aPDI requires the diffusion of a photosensitizer (Sens) into the biofilm and subsequent photoactivation of oxygen for the generation of reactive oxygen species (ROS) that inactivate microbes. Molecular diffusion in biofilms has been long investigated, whereas this review is intended to draw a logical link between diffusion in biofilms and ROS, a combination that leads to the current state of aPDI and superhydrophobic aPDI (SH-aPDI). This review should be of interest to photochemists, photobiologists and researchers in material and antimicrobial sciences as is ties together conventional aPDI with the emerging subject of SH-aPDI.

Superhydrophobic Surfaces as a Source of Airborne Singlet Oxygen through Free Space for Photodynamic Therapy.

A superhydrophobic (SH) sandwich system has been developed to enable "contact-free" airborne singlet oxygen (1O2) delivery to a water droplet. The contact-free feature means that the sensitizer is physically separated from the droplet, which presents opportunities for photodynamic therapy (PDT). Trapping of airborne 1O2 in a H2O droplet residing on a lower SH surface was monitored with 9,10-anthracene dipropionate dianion by varying distances to an upper 1O2-generating surface. Short distances of 20 µm efficiently delivered airborne 1O2 to the droplet in single-digit picomolar steady-state concentrations. Delivery decreases linearly with distance, but 50% of the 1O2 steady-state concentration is trapped at a distance of 300 µm from the generating surface. The 1270 nm luminescence intensity was measured within the SH sandwich system, confirming the presence of airborne 1O2. Physical quenching of 1O2 to ground-state 3O2 by the water droplet itself and both physical and chemical quenching of 1O2 by the water droplet containing the trap 9,10-anthracene dipropionate dianion are observed. Unlike a majority of work in the field of PDT with dissolved sensitizers, where 1O2 diffuses short (hundreds of nanometers) distances, we show the delivery of airborne 1O2 via a superhydrophobic surface is effective through air in tenths of millimeters distances to oxidize an organic compound in water. Our results provide not only potential relevance to PDT but also surface bacterial inactivation processes.

Resistance to c-KIT kinase inhibitors conferred by V654A mutation.

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT.

Absence of Tumor Necrosis Factor Supports Alternative Activation of Macrophages in the Liver after Infection with Leishmania major.

The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF-/-) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF-/- mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.

Inducible IFN-γ Expression for MHC-I Upregulation in Devil Facial Tumor Cells.

The Tasmanian devil facial tumor (DFT) disease has led to an 80% reduction in the wild Tasmanian devil (Sarcophilus harrisii) population since 1996. The limited genetic diversity of wild devils and the lack of MHC-I expression on DFT cells have been implicated in the lack of immunity against the original DFT clonal cell line (DFT1). Recently, a second transmissible tumor of independent origin (DFT2) was discovered. Surprisingly, DFT2 cells do express MHC-I, but DFT2 cells appear to be on a trajectory for reduced MHC-I expression in vivo. Thus, much of the ongoing vaccine-development efforts and conservation plans have focused on MHC-I. A major limitation in conservation efforts is the lack of species-specific tools to understand Tasmanian devil gene function and immunology. To help fill this gap, we developed an all-in-one Tet-Off vector system to regulate expression of IFN-γ in DFT cells (DFT1.Tet/IFN-γ). IFN-γ can have negative effects on cell proliferation and viability; thus, doxycycline was used to suppress IFN-γ production whilst DFT1.Tet/IFN-γ cells were expanded in cell culture. Induction of IFN-γ following removal of doxycycline led to upregulation of MHC-I but also the inhibitory checkpoint molecule PD-L1. Additionally, DFT1.Tet/IFN-γ cells were capable of stimulating MHC-I upregulation on bystander wild type DFT cells in co-culture assays in vitro. This system represents a major step forward in DFT disease immunotherapy and vaccine development efforts, and ability to understand gene function in devils. Importantly, the techniques are readily transferable for testing gene function in DFT2 cells and other non-traditional species.

Superhydrophobic Photosensitizers: Airborne 1O2 Killing of an in Vitro Oral Biofilm at the Plastron Interface.

Singlet oxygen is a potent agent for the selective killing of a wide range of harmful cells; however, current delivery methods pose significant obstacles to its widespread use as a treatment agent. Limitations include the need for photosensitizer proximity to tissue because of the short (3.5 µs) lifetime of singlet oxygen in contact with water; the strong optical absorption of the photosensitizer, which limits the penetration depth; and hypoxic environments that restrict the concentration of available oxygen. In this article, we describe a novel superhydrophobic singlet oxygen delivery device for the selective inactivation of bacterial biofilms. The device addresses the current limitations by: immobilizing photosensitizer molecules onto inert silica particles; embedding the photosensitizer-containing particles into the plastron (i.e. the fluid-free space within a superhydrophobic surface between the solid substrate and fluid layer); distributing the particles along an optically transparent substrate such that they can be uniformly illuminated; enabling the penetration of oxygen via the contiguous vapor space defined by the plastron; and stabilizing the superhydrophobic state while avoiding the direct contact of the sensitizer to biomaterials. In this way, singlet oxygen generated on the sensitizer-containing particles can diffuse across the plastron and kill bacteria even deep within the hypoxic periodontal pockets. For the first time, we demonstrate complete biofilm inactivation (>5 log killing) of Porphyromonas gingivalis, a bacterium implicated in periodontal disease using the superhydrophobic singlet oxygen delivery device. The biofilms were cultured on hydroxyapatite disks and exposed to active and control surfaces to assess the killing efficiency as monitored by colony counting and confocal microscopy. Two sensitizer particle types, a silicon phthalocyanine sol-gel and a chlorin e6 derivative covalently bound to fluorinated silica, were evaluated; the biofilm killing efficiency was found to correlate with the amount of singlet oxygen detected in separate trapping studies. Finally, we discuss the applications of such devices in the treatment of periodontitis.

Comparative Analysis of Immune Checkpoint Molecules and Their Potential Role in the Transmissible Tasmanian Devil Facial Tumor Disease.

Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil (Sarcophilus harrisii) facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology.

A dual targeted ß-defensin and exome sequencing approach to identify, validate and functionally characterise genes associated with bull fertility.

Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertility-related ß-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple ß-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine ß-defensin genes and functional analysis supports a role for ß-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle.