Nanohydroxyapatite Coating Attenuates Fibrotic and Immune Responses to Promote Keratoprosthesis Biointegration in Advanced Ocular Surface Disorders.

Keratoprosthesis (KPro) implantation is frequently the only recourse for patients with severe corneal disease. However, problems arise due to inadequate biointegration of the KPro, particularly the PMMA optical cylinder, such as tissue detachment, tissue melting, or eye-threatening infection in the interface. Here, using the AuroKPro as a model prosthesis, a surface functionalization approach─coating the optical cylinder with nanohydroxyapatite (nHAp)─was trialed in rabbit eyes with and without a proceeding chemical injury. In chemically injured eyes, which simulated total limbal epithelial stem cell deficiency, clear benefits were conferred by the coating. The total modified Hackett-McDonald score and area of tissue apposition differences 12 weeks after implantation were 5.0 and 22.5%, respectively. Mechanical push-in tests revealed that 31.8% greater work was required to detach the tissues. These differences were less marked in uninjured eyes, which showed total score and tissue apposition differences of 2.5 and 11.5%, respectively, and a work difference of 23.5%. The improved biointegration could be contributed by the attenuated expression of fibronectin (p = 0.036), collagen 3A1 (p = 0.033), and α-smooth muscle actin (p = 0.045)─proteins typically upregulated during nonadherent fibrous capsule envelopment of bioinert material─adjacent to the optical cylinders. The coating also appeared to induce a less immunogenic milieu in the ocular surface tissue, evidenced by the markedly lower expression of tear proteins associated with immune and stimulus responses. Collectively, the level of these tear proteins in eyes with coated prostheses was 1.1 ± 13.0% of naïve eyes: substantially lower than with noncoated KPros (246.5 ± 79.3% of naïve, p = 0.038). Together, our results indicated that nHAp coating may reduce the risk of prosthesis failure in severely injured eyes, which are representative of the cohort of KPro patients.

Hybrid liposome-erythrocyte drug delivery system for tumor therapy with enhanced targeting and blood circulation.

Liposome, a widely used drug delivery system (DDS), still shows several disadvantages such as dominant clearance by liver and poor target organ deposition. To overcome the drawbacks of liposomes, we developed a novel red blood cell (RBC)-liposome combined DDS to modulate the tumor accumulation and extend the blood circulation life of the existing liposomal DDS. Here, RBCs, an ideal natural carrier DDS, were utilized to carry liposomes and avoid them undergo the fast clearance in the blood. In this study, liposomes could either absorbed onto RBCs' surface or fuse with RBCs' membrane by merely altering the interaction time at 37°C, while the interaction between liposome and RBCs would not affect RBCs' characteristics. In the in vivo antitumor therapeutic efficacy study, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes attached onto RBCs' surfaces exhibited lung targeting effect (via RBC-hitchhiking approach) and reduced clearance in the liver, while DPPC liposomes fused with RBCs had prolong blood circulation up to 48 h and no enrichment in any organ. Furthermore, 20 mol% of DPPC liposomes were replaced with pH-sensitive phospholipid 1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine (DOPE) as it could respond to the low pH tumor microenvironment and then accumulate in the tumor. The DOPE attached/fusion RBCs showed partial enrichment in lung and about 5-8% tumor accumulation, which were significantly higher than (about 0.7%) the conventional liposomal DDS. Thus, RBC-liposome composite DDS is able to improve the liposomal tumor accumulation and blood circulation and shows the clinical application promises of using autologous RBCs for antitumor therapy.

Bioresorbable Polymeric Scaffold in Cardiovascular Applications.

Advances in material science and innovative medical technologies have allowed the development of less invasive interventional procedures for deploying implant devices, including scaffolds for cardiac tissue engineering. Biodegradable materials (e.g., resorbable polymers) are employed in devices that are only needed for a transient period. In the case of coronary stents, the device is only required for 6-8 months before positive remodelling takes place. Hence, biodegradable polymeric stents have been considered to promote this positive remodelling and eliminate the issue of permanent caging of the vessel. In tissue engineering, the role of the scaffold is to support favourable cell-scaffold interaction to stimulate formation of functional tissue. The ideal outcome is for the cells to produce their own extracellular matrix over time and eventually replace the implanted scaffold or tissue engineered construct. Synthetic biodegradable polymers are the favoured candidates as scaffolds, because their degradation rates can be manipulated over a broad time scale, and they may be functionalised easily. This review presents an overview of coronary heart disease, the limitations of current interventions and how biomaterials can be used to potentially circumvent these shortcomings in bioresorbable stents, vascular grafts and cardiac patches. The material specifications, type of polymers used, current progress and future challenges for each application will be discussed in this manuscript.

Inclusion of Cross-Linked Elastin in Gelatin/PEG Hydrogels Favourably Influences Fibroblast Phenotype.

The capacity of a biomaterial to innately modulate cell behavior while meeting the mechanical property requirements of the implant is a much sought-after goal within bioengineering. Here we covalently incorporate soluble elastin into a gelatin-poly (ethylene glycol) (PEG) hydrogel for three-dimensional (3D) cell encapsulation to achieve these properties. The inclusion of elastin into a previously optimized gelatin-PEG hydrogel was then evaluated for effects on entrapped fibroblasts, with the aim to assess the hydrogel as an extracellular matrix (ECM)-mimicking 3D microenvironment for cellular guidance. Soluble elastin was incorporated both physically and covalently into novel gelatin/elastin hybrid PEG hydrogels with the aim to harness the cellular interactivity and mechanical tunability of both elastin and gelatin. This design allowed us to assess the benefits of elastin-containing hydrogels in guiding fibroblast activity for evaluation as a potential dermal replacement. It was found that a gelatin-PEG hydrogel with covalently conjugated elastin, supported neonatal fibroblast viability, promoted their proliferation from 7.3% to 13.5% and guided their behavior. The expression of collagen alpha-1(COL1A1) and elastin in gelatin/elastin hybrid gels increased 16-fold and 6-fold compared to control sample at day 9, respectively. Moreover, cells can be loaded into the hydrogel precursor solution, deposited, and the matrix cross-linked without affecting the incorporated cells adversely, thus enabling a potential injectable system for dermal wound healing.

Robust Fabrication of Composite 3D Scaffolds with Tissue-Specific Bioactivity: A Proof-of-Concept Study.

The basic requirement of any engineered scaffold is to mimic the native tissue extracellular matrix (ECM). Despite substantial strides in understanding the ECM, scaffold fabrication processes of sufficient product robustness and bioactivity require further investigation, owing to the complexity of the natural ECM. A promising bioacive platform for cardiac tissue engineering is that of decellularized porcine cardiac ECM (pcECM, used here as a soft tissue representative model). However, this platform's complexity and batch-to-batch variability serve as processing limitations in attaining a robust and tunable cardiac tissue-specific bioactive scaffold. To address these issues, we fabricated 3D composite scaffolds (3DCSs) that demonstrate comparable physical and biochemical properties to the natural pcECM using wet electrospinning and functionalization with a pcECM hydrogel. The fabricated 3DCSs are non-immunogenic in vitro and support human mesenchymal stem cells' proliferation. Most importantly, the 3DCSs demonstrate tissue-specific bioactivity in inducing spontaneous cardiac lineage differentiation in human induced pluripotent stem cells (hiPSC) and further support the viability, functionality, and maturation of hiPSC-derived cardiomyocytes. Overall, this work illustrates the technology to fabricate robust yet tunable 3D scaffolds of tissue-specific bioactivity (with a proof of concept provided for cardiac tissues) as a platform for basic materials science studies and possible future R&D application in regenerative medicine.

Microneedle-Assisted Topical Delivery of Photodynamically Active Mesoporous Formulation for Combination Therapy of Deep-Seated Melanoma.

Topical treatment using photodynamic therapy (PDT) for many types of skin cancers has largely been limited by the inability of existing photosensitizers to penetrate into the deep skin tissue. To overcome these problems, we developed a mesoporous nanovehicle with dual loading of photosensitizers and clinically relevant drugs for combination therapy, while utilizing microneedle technology to facilitate their penetration into deep skin tissue. Sub-50 nm photodynamically active mesoporous organosilica nanoparticles were synthesized with photosensitizers covalently bonded to the silica matrix, which dramatically increased the quantum yield and photostability of these photosensitizers. The mesopores of the nanoparticles were further loaded with small-molecule inhibitors, i. e., dabrafenib and trametinib, that target the hyperactive mitogen-activated protein kinase (MAPK) pathway for melanoma treatment. As-prepared empty nanovehicle was cytocompatible with normal skin cells in the dark, while NIR-irradiated drug-loaded nanovehicle showed a synergistic killing effect on skin cancer cells mainly through reactive oxygen species and caspase-activated apoptosis. The nanovehicle could significantly inhibit the proliferation of tumor cells in a 3D spheroid model in vitro. Porcine skin fluorescence imaging demonstrated that microneedles could facilitate the penetration of nanovehicle across the epidermis layer of skin to reach deep-seated melanoma sites. Tumor regression studies in a xenografted melanoma mouse model confirmed superior therapeutic efficacy of the nanovehicle through combinational PDT and targeted therapy.

Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different.

Identification and functional characterization of a novel bacterial type asparagine synthetase A: a tRNA synthetase paralog from Leishmania donovani.

Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.

Randomized, controlled trial of a sustained delivery formulation of 5-fluorouracil for the treatment of failing blebs.

PURPOSE: To determine the efficacy of a subconjunctival injection of hyaluronic acid (HA) with 5-fluorouracil (5FU) formulation as an adjunct in reviving bleb function by needling. DESIGN: Prospective, randomized, controlled trial. PARTICIPANTS: Fifty patients with previous trabeculectomy and scheduled by the managing physician for a needling intervention. METHODS: One eye of each patient was randomized to receive needling with HA-5FU mixture or needling with subconjunctival injection of 5FU solution alone. MAIN OUTCOME MEASURES: The primary outcome was the percentage of subjects with an intraocular pressure (IOP) <15 mmHg without any medications at 3 months. Secondary outcomes included the need for additional needling procedures and changes in bleb morphology. RESULTS: Forty-nine subjects (25 in the HA-5FU group and 24 in the 5FU group) completed 3 months of follow-up. At baseline, there was no significant difference between the groups in terms of demographic features, subtype of glaucoma, vertical cup-to-disc ratio, or visual field indices. The mean number of glaucoma medications at baseline was higher in the 5FU group (0.8±1.1 [mean ± standard deviation] vs. 0.2±0.6, P = 0.04). An IOP <15 mmHg without medications was reached in 48.0% of subjects in the HA-5FU group and in 33.3% of subjects in the 5FU group (P = 0.2). At 3 months, both groups demonstrated a significant decrease in IOP from baseline (HA-5FU: decrease of 5.9 mmHg [95% confidence interval, 3.4-8.4]; 5FU: decrease of 6.0 mmHg [95% confidence interval, 3.2-8.2]; P<0.001 for both). Intergroup comparisons for IOP change from baseline was not significant (P = 0.9). However, repeat needling was required more frequently in the 5FU group compared with the HA-5FU group (50.0% vs. 12.0%; P = 0.004). There were no significant differences in the number of reported adverse events, bleb vascularity, or morphology between the 2 groups. CONCLUSIONS: Subconjunctival injection of HA-5FU to revive bleb function after bleb needling is as effective as 5FU solution. Fewer repeat needlings were required after treatment with HA-5FU, suggesting that the use of a combined formulation of HA-5FU may improve the overall outcomes of bleb needlings.

Evolutionary divergence of Plasmodium falciparum: sequences, protein-protein interactions, pathways and processes.

In this article we review the organism-wide biological data available for Plasmodium falciparum (P. falciparum), a malarial parasite, in relation to the data available for other organisms. We provide comparisons at different levels such as amino acid sequences of proteins encoded in the genomes, protein-protein interaction features, metabolic and signaling pathways and processes. Our comparative analyses highlights that P. falciparum is highly diverged compared to most other eukaryotes at all these levels. Despite the extensive variation some of the physical associations between proteins, such as RNA polymerase complex and CDK-cyclin complex are expected to be conserved given their fundamental importance and ubiquitous nature. We also discuss examples of protein-protein interactions across human and P. falciparum potentially happening during pathogenesis.