The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP

Transmorphic phage-guided systemic delivery of TNFα gene for the treatment of human pediatric medulloblastoma

Medulloblastoma is the most common childhood brain tumor with an unfavorable prognosis and limited options of harmful treatments that are associated with devastating long-term side effects. Therefore, the development of safe, noninvasive, and effective therapeutic approaches is required to save the quality of life of young medulloblastoma survivors. We postulated that therapeutic targeting is a solution. Thus, we used a recently designed tumor-targeted bacteriophage (phage)-derived particle, named transmorphic phage/AAV, TPA, to deliver a transgene expressing the tumor necrosis factor-alpha (TNFα) for targeted systemic therapy of medulloblastoma. This vector was engineered to display the double-cyclic RGD4C ligand to selectively target tumors after intravenous administration. Furthermore, the lack of native phage tropism in mammalian cells warrants safe and selective systemic delivery to the tumor microenvironment. In vitro RGD4C.TPA.TNFα treatment of human medulloblastoma cells generated efficient and selective TNFα expression, subsequently triggering cell death. Combination with the chemotherapeutic drug cisplatin used clinically against medulloblastoma resulted in augmented effect through the enhancement of TNFα gene expression. Systemic administration of RGD4C.TPA.TNFα to mice-bearing subcutaneous medulloblastoma xenografts resulted in selective tumor homing of these particles and consequently, targeted tumor expression of TNFα, apoptosis, and destruction of the tumor vasculature. Thus, our RGD4C.TPA.TNFα particle provides selective and efficient systemic delivery of TNFα to medulloblastoma, yielding a potential TNFα anti-medulloblastoma therapy while sparing healthy tissues from the systemic toxicity of this cytokine

Targeting human osteoarthritic chondrocytes with ligand directed bacteriophage-based particles

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs

Bio-electrospraying 3-D Organotypic Human Skin Cultures

Organotypic 3D tissue models have greatly contributed to understand a wide range of molecular and cellular characteristics within a functional or diseased tissue. Human skin reconstructs which act as models are most useful for a wide range of investigations, ranging from tissue engineering and regenerative medicine, drug development, screening, and discovery to name a few. There are many approaches for reconstructing 3D skin tissue models, however, to date there have been very few that are able to generate organotypic 3D constructs with a single technology having minimal processing steps to finally scalability. The many manifestations of 3D bioprinting have contributed to this endeavor, having said that, the technology's limitations have tempered those reconstructed models, as they are known to contain low cell numbers/concentrations to those having damaged/dead molecules/cells within the reconstructed tissue, which are not desirable, for exploring as tissues models. Contrary to 3D bioprinting approaches, bio-electrosprays have been demonstrated to possess the ability to handle large concentrations of cells and molecules to whole fertilized embryos without damaging them from a molecular level upwards. Consequently, this article demonstrates, for the first time, bio-electrospray's capacity to reconstruct skin-like structures in vitro and its potential in reconstructing full-thickness 3D organotypic human skin tissues

Factors affecting compressive strength and expansion due to alkali-silica reaction of fly ash-based alkaline activated mortar

Data availability: Data will be made available on request.Copyright © 2023 The Author(s). The development of environmentally friendly alkaline-activated materials (AAMs) holds promise, as AAMs can be derived from waste materials. This study aims to investigate the factors influencing (i) compressive strength and (ii) expansion due to alkali-silica reaction (ASR) in AAMs. These factors include alkaline concentration, heat curing conditions, fineness of fly ash, and the liquid alkaline-to-binder (L/B) ratio. The findings indicate that the higher concentrations of NaOH solution led to an increase in AAM compressive strength due to the enhanced dissolution and polymerization rates in a more alkaline environment. Heat curing stimulated chemical reactions and structure formation, while the reduced water content resulted in lower porosity and higher compressive strength in the hardened cement. Finer fly ash yielded greater compressive strength than coarser ash, as its smaller spherical particles contributed to denser and firmer structures. The presence of calcium minerals, from both Ordinary Portland Cement (OPC) and high-calcium fly ash, bolstered the strength of hardened products. Moreover, calcium minerals like CaO, Ca(OH)2, and CaSO4 were found to induce ASR expansion by promoting gel formation, leading to later-stage expansion in the hardened cement or concrete. However, finely milled fly ash as a precursor significantly reduced ASR expansion in AAMs, by approximately 40% compared to ordinary Portland cement. This study provides valuable insights for civil engineers for better understanding of AAM behavior and makes contributions to the safety and sustainability of cement and concrete systems.Thailand Science Research and Innovation (TSRI) under Fundamental Fund 2023 (Project: Advanced Construction Towards Thailand 4.0); Department of Civil Engineering, Faculty of Engineering, Chiang Mai University (CMU)

Validation of the Thai Osteoporosis Foundation and Royal College of Orthopaedic Surgeons of Thailand Clinical Practice Guideline for bone mineral density measurement in postmenopausal women

AbstractObjectiveThe primary objective of this study was to determine the sensitivity, specificity, and predictive values of the Thai Osteoporosis Foundation (TOPF) and Royal College of Orthopaedic Surgeons of Thailand (RCOST) Clinical Practice Guideline for bone mineral density (BMD) measurement for the detection of postmenopausal osteoporosis. Its secondary objective was to find better indicators to detect postmenopausal osteoporosis.MethodsPostmenopausal women were enrolled in this study between June and December 2014. The clinical risk factors following TOPF and RCOST Clinical Practice Guideline for BMD measurement were collected. Bone mineral density was measured using dual energy X-ray absorptiometry.ResultsFour hundred postmenopausal women were enrolled in the study. The mean age of the studied population was 66.16 ± 6.04 years. Twenty-seven percent of the participants had either osteoporosis of the lumbar spine, femoral neck, or total hip, of which 13.3% had osteoporosis at the lumbar spine, 21.3% had osteoporosis at the femoral neck, and 2.5% had osteoporosis of the total hip. The sensitivity and specificity for detecting osteoporosis of the whole TOPF and RCOST guideline were 96.2% and 16.7%, 98.8% and 18.7%, 90.0% and 15.1%, and 97.2% and 19.5% at the lumbar spine, femoral neck, total hip, and any sites, respectively. Multiple logistic regression analysis revealed that only OSTA ≤−1, osteopenia on X-ray and low trauma fracture after age of 40 years were significant clinical risk factors in the detection of postmenopausal osteoporosis. The Receiver Operating Characteristics (ROC) curve was used to obtain the optimum probability value of osteoporosis at any sites which revealed that the probability value of 0.2222236 would have a sensitivity of 67% and specificity of 62% as the optimal cut point to detect osteoporosis. A simple flow diagram of “OSTA ≤−1”, “Osteopenia on X-ray” and “A history of low trauma fracture after age of 40 years” was developed as a better trade-off guideline for BMD measurement.ConclusionsThis study revealed that the TOPF and RCOST guideline for BMD measurement provided a high true positive rate of disease detection but with an expense of high false positive rate. The simple flow diagram was proposed as a more appropriate guideline for BMD measurement in postmenopausal women

Salivary gland proteome during adult development and after blood feeding of female anopheles dissidens mosquitoes (Diptera Culicidae)

Understanding changes in mosquito salivary proteins during the time that sporozoite maturation occurs and after blood feeding may give information regarding the roles of salivary proteins during the malarial transmission. Anopheles dissidens (formerly Anopheles barbirostris species A1) is a potential vector of Plasmodium vivax in Thailand. In this study, analyses of the proteomic profiles of female An. dissidens salivary glands during adult development and after blood feeding were carried out using two-dimensional gel electrophoresis coupled with nano-liquid chromatography-mass spectrometry. Results showed at least 17 major salivary gland proteins present from day one to day 21 post emergence at 8 different time points sampled. Although there was variation observed, the patterns of protein expression could be placed into one of four groups. Fifteen protein spots showed significant depletion after blood feeding with the percentages of the amount of depletion ranging from 8.5% to 68.11%. The overall results identified various proteins, including a putative mucin-like protein, an anti-platelet protein, a long form D7 salivary protein, a putative gVAG protein precursor, a D7-related 3.2 protein, gSG7 salivary proteins, and a gSG6 protein. These results allow better understanding of the changes of the salivary proteins during the adult mosquito development. They also provide candidate proteins to investigate any possible link or not between sporozoite maturation, or survival of skin stage sporozoites, and salivary proteins

Doxorubicin improves cancer cell targeting by filamentous phage gene delivery vectors.

Merging targeted systemic gene delivery and systemic chemotherapy against cancer, chemovirotherapy, has the potential to improve chemotherapy and gene therapy treatments and overcome cancer resistance. We introduced a bacteriophage (phage) vector, named human adeno-associated virus (AAV)/phage or AAVP, for the systemic targeting of therapeutic genes to cancer. The vector was designed as a hybrid between a recombinant adeno-associated virus genome (rAAV) and a filamentous phage capsid. To achieve tumor targeting, we displayed on the phage capsid the double-cyclic CDCRGDCFC (RGD4C) ligand that binds the alpha-V/beta-3 (αvβ3) integrin receptor. Here, we investigated a combination of doxorubicin chemotherapeutic drug and targeted gene delivery by the RGD4C/AAVP vector. Firstly, we showed that doxorubicin boosts transgene expression from the RGD4C/AAVP in two-dimensional (2D) cell cultures and three-dimensional (3D) tumor spheres established from human and murine cancer cells, while preserving selective gene delivery by RGD4C/AAVP. Next, we confirmed that doxorubicin does not increase vector attachment to cancer cells nor vector cell entry. In contrast, doxorubicin may alter the intracellular trafficking of the vector by facilitating nuclear accumulation of the RGD4C/AAVP genome through destabilization of the nuclear membrane. Finally, a combination of doxorubicin and RGD4C/AAVP-targeted suicide gene therapy exerts a synergistic effect to destroy human and murine tumor cells in 2D and 3D tumor sphere settings