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The growing demand for sustainable and alternative protein sources has spurred interest in insect-based and plant-based proteins. Protaetia brevitarsis (PB) larvae and isolated soy protein (ISP) are notable in this regard, offering potential health benefits and nutritional enhancements. We assessed the feasibility of PB larvae and ISP mixtures as alternative food ingredients. Methods included the optimized purification and freeze-drying of PB larvae, extraction and refinement of legume proteins, physicochemical and antioxidant capacity evaluations, DPPH radical scavenging activity measurement, total phenolic and flavonoids content quantification, general component analysis, amino acid profiling using HPLC, fatty acid profiling through gas chromatography, and mineral content analysis using inductively coupled plasma spectrometry. The study found that certain PB:ISP ratios, particularly a 7:3 ratio, significantly improved the blend’s antioxidant capacity, as evidenced by DPPH scavenging activity. This ratio also impacted the nutritional profile by altering the mixture’s general components, with a notable increase in moisture, crude protein, and fiber and a decrease in crude fat and ash. Amino acid analysis revealed a balanced presence of essential and non-essential amino acids. The fatty acid profile was rich in unsaturated fatty acids, especially in certain ratios. Mineral analysis showed a complex interplay between PB larvae and ISP, with some minerals decreasing and others increasing in the blend. PB larvae and ISP mixtures have significant potential as alternative protein sources, offering a diversified nutritional profile and enhanced antioxidant properties. The 7:3 ratio of PB larvae to ISP has been shown to be particularly effective, suggesting that this ratio may offer an optimal balance for enhancing the overall nutritional quality of the mixture. This study sets the stage for future research to further explore and optimize the potential of these mixtures for human consumption while considering the challenges of consumer acceptance and long-term safety.

Glioma accounts for 80% of all malignant brain tumours and is the most common adult primary brain tumour. Age is an important factor affecting the development of cancer, as somatic mutations accumulate with age. Here, we aimed to analyse the significance of age-dependent non-silent somatic mutations in glioma prognosis. Histological tumour grade depends on age at diagnosis in patients with IDH1, TP53, ATRX, and EGFR mutations. Age of patients with wild-type IDH1 and EGFR increased with increase in tumour grade, while the age of patients with IDH1 or EGFR mutation remained constant. However, the age of patients with EGFR mutation was higher than that of patients with IDH1 mutation. The hierarchical clustering of patients was dominantly separated by IDH1 and EGFR mutations. Furthermore, patients with IDH1 mutation were dominantly separated by TP53 and ATRX double mutation and its double wild-type counterpart. The age of patients with ATRX and TP53 mutation was lower than that of patients with wild-type ATRX and TP53. Patients with the double mutation showed poorer prognosis than those with the double wild type genotype. Unlike IDH1 mutant, IDH1 wild-type showed upregulation of expression of epithelial mesenchymal transition associated genes.

Cancer: Tipping the signaling balance Reducing the levels of the multifunctional protein transforming growth factor (TGF)-β in cancer cells prevents tumor growth in mice. Previous studies have shown that high levels of TGF-β in cancerous tissue are associated with accelerated disease progression. Hye Jin Choi and Jae J Song at Yonsei University in Seoul, South Korea, and colleagues infected cancer cells with genetically modified viruses that reduced the expression of the gene encoding TGF-β. The resulting decrease in TGF-β protein led to cell death by stimulating the production of reactive oxygen species and signaling through the apoptosis signal-regulating kinase 1 (ASK1) pathway. When tumor-bearing mice were infected with these modified viruses, their overall survival was improved. Further understanding the mechanisms through which TGF-β regulates cancer cell survival will contribute to the development of new approaches in cancer treatment.

Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques., (© 2024. Springer Nature Limited.)

Aging is a natural process that compromises the immune system's functionality increasing the risk of infectious, tumors, and autoimmune diseases. The thymus involution is an age-dependent process characterized by decreased cellularity, peripheral lymphocyte infiltration into the perivascular space, and expansion of adipose tissue. All those modifications hamper the functionality of the organ and lead to a decline of naïve T-cell production with a shrinking of the T-cell repertoire. Thymus atrophy is described in several disorders including autoimmune diseases. The quantification of T-cell receptor excision circles (TRECs) in recent thymus emigrants is a standard procedure to investigate the thymic function. In this chapter, we discuss the methodology used to quantify this molecule in peripheral blood mononuclear cells and isolated CD4+ and CD8+ T cells., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chondrosarcoma (CS) is a malignant cartilage-forming bone tumor that is inherently resistant to chemotherapy and radiotherapy, leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvβ3 and αvβ5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human CS cells (SW1353), but not in primary normal chondrocytes. The RGD4C.PDP- Luc particle carrying a luciferase reporter gene, Luc , effectively and selectively mediated gene delivery to SW1353 cells, but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP -sTRAIL encoding a human sTRAIL, resulted in the expression of TRAIL and subsequent cell death without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP- sTRAIL to mice with established human CS resulted in a decrease in tumor size and tumor viability. Altogether, RGD4C.PDP- sTRAIL can be used to target systemic treatment of CS with the sTRAIL., Competing Interests: A.H. is inventor on patents 10799542 and 11603540 describing the vector constructs reported here. S.W., K.S., and A.H. are inventors on a patent application 20200239535 related to this work. They will be entitled to royalties if licensing or commercialization occur., (© 2024 The Author(s).)

Objective: Soluble CD27 is a promising cerebrospinal fluid inflammatory biomarker in multiple sclerosis. In this study, we investigate relevant immune and neuro-pathological features of soluble CD27 in multiple sclerosis., Methods: Protein levels of soluble CD27 were correlated to inflammatory cell subpopulations and inflammatory cytokines and chemokines detected in cerebrospinal fluid of 137 patients with multiple sclerosis and 47 patients with inflammatory and non-inflammatory neurological disease from three independent cohorts. Production of soluble CD27 was investigated in cell cultures of activated T and B cells and CD27-knockout T cells. In a study including matched cerebrospinal fluid and post-mortem brain tissues of patients with multiple sclerosis and control cases, levels of soluble CD27 were correlated with perivascular and meningeal infiltrates and with neuropathological features., Results: We demonstrate that soluble CD27 favours the differentiation of interferon-γ-producing T cells and is released through a secretory mechanism activated by TCR engagement and regulated by neutral sphingomyelinase. We also show that the levels of soluble CD27 correlate with the representation of inflammatory T cell subsets in the CSF of patients with relapsing-remitting multiple sclerosis and with the magnitude of perivascular and meningeal CD27 + CD4 + and CD8 + T cell infiltrates in post-mortem central nervous system tissue, defining a subgroup of patients with extensive active inflammatory lesions., Interpretation: Our results demonstrate that soluble CD27 is a biomarker of disease activity, potentially informative for personalized treatment and monitoring of treatment outcomes., (© 2024. The Author(s).)

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats ( ITRs ) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to α v β 3 and α v β 5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express α v β 5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA- tmTRAIL ) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

The prevalence of type 2 diabetes (T2DM) is associated with diet. While consumption of plant-based foods may reduce blood sugar levels, the impact of consuming plant-based foods on fasting blood sugar levels has not been well defined. This cross-sectional study was conducted at the Health-Promoting Hospital in Pak Phun Municipality, Thailand. It included 61 patients with T2DM and 74 controls matched for age and gender. Dietary intake levels among T2DM and controls were assessed by a validated food-frequency questionnaire from which plant-based-food scores were calculated. This study found significant differences between specific plant foods and fasting blood sugar levels in patients with T2DM. Adherence to a plant-based diet appeared to influence fasting blood sugar levels. Patients who consumed higher amounts of certain vegetables and fruits showed lower fasting blood sugar levels. Diabetic patients consumed more legumes than controls, but the consumption of cereals and nuts/seeds in the two groups were similar. Consumption of nuts and seeds was also associated with a 76.3% reduction in the risk of a T2DM diagnosis. These findings suggest the potential efficacy of glycemic control in T2DM patients. More work is required to explore strategies for preventing and treating metabolic disorders through dietary modification.

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., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNF α ) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

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.

Background: Reprogrammed cellular metabolism is a cancer hallmark. In addition to increased glycolysis, the oxidation of acetate in the citric acid cycle is another common metabolic phenotype. We have recently developed a novel fluorine-18-labelled trimethylacetate-based radiotracer, [ 18 F]fluoro-pivalic acid ([ 18 F]FPIA), for imaging the transcellular flux of short-chain fatty acids, and investigated whether this radiotracer can be used for the detection of glioma growth., Methods: We evaluated the potential of [ 18 F]FPIA PET to monitor tumor growth in orthotopic patient-derived (HSJD-GBM-001) and cell line-derived (U87, LN229) glioma xenografts, and also included [ 18 F]FDG PET for comparison. We assessed proliferation (Ki-67) and the expression of lipid metabolism and transport proteins (CPT1, SLC22A2, SLC22A5, SLC25A20) by immunohistochemistry, along with etomoxir treatment to provide insights into [ 18 F]FPIA uptake., Results: Longitudinal PET imaging showed gradual increase in [ 18 F]FPIA uptake in orthotopic glioma models with disease progression ( p < 0.0001), and high tumor-to-brain contrast compared to [ 18 F]FDG ( p < 0.0001). [ 18 F]FPIA uptake correlated positively with Ki-67 ( p < 0.01), SLC22A5 ( p < 0.001) and SLC25A20 ( p = 0.001), and negatively with CPT1 ( p < 0.01) and SLC22A2 ( p < 0.01). Etomoxir reduced [ 18 F]FPIA uptake, which correlated with decreased Ki-67 ( p < 0.05)., Conclusions: Our findings support the use of [ 18 F]FPIA PET for the detection and longitudinal monitoring of glioma, showing a positive correlation with tumor proliferation, and suggest transcellular flux-mediated radiotracer uptake.

Chondrosarcoma is a cartilage-forming bone tumor, well known for intrinsic resistance to chemotherapy and radiotherapy. We have designed a targeted chondrosarcoma gene therapy using a bacteriophage (phage) particle to deliver therapeutic genes. Phage has no tropism for mammalian cells, allowing engineered phage to be targeted to specific cell surface receptors in cancer. We modified the phage capsid to display the RGD4C ligand on the pIII minor coat proteins to specifically bind to αvβ3 or αvβ5 integrin receptors. The endosomal escape peptide, H5WYG, was also displayed on recombinant pVIII major coat proteins to enhance gene delivery. Finally, a human tumor necrosis factor alpha (TNFα) therapeutic transgene expression cassette was incorporated into the phage genome. First, we found that human chondrosarcoma cells (SW1353) have high expression of αvβ3, αvβ5 integrin receptors, and both TNFα receptors. Targeted particle encoding a luciferase reporter gene efficiently and selectively mediated gene delivery to these cells. When SW1353 cells were treated with the targeted particle encoding a TNFα transgene, significant cell killing was evident and was associated with high expression of TNFα and apoptosis-related genes. In vivo, mice with established human chondrosarcoma showed suppression of tumors upon repetitive intravenous administrations of the targeted phage. These data show that our phage-based particle is a promising, selective, and efficient tool for targeted chondrosarcoma therapy., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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 (α v β 3 ) 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.

p53 gene ( TP53 ) replacement therapy has shown promising results in cancer gene therapy. However, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) can knock out the mutated TP53 ( mutTP53 ), but due to its large size, many viral vectors are not suitable or require implemented strategies that lower the therapeutic efficiency. Here, we introduced a bacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the feasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells. First, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette. Next, we investigated any negative impact on vector titers via quantitative polymerase chain reaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and immunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted bacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence without any negative impact on the active or total number of bacteriophage particles. Then, we detected expression of the Cas9 in human lung adenocarcinoma cells in a targeted and efficient manner. Finally, we proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9 phage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage for TP53 replacement therapy in lung cancer.

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells., Competing Interests: Conflicts of interest: J.G.G., R.P., and W.A. are founders of PhageNova Bio, which has licensed intellectual property related to the AAVP technology. R.P. is the Chief Scientific Officer and a paid consultant for PhageNova Bio. J.G.G., R.P., W.A., and A.H. are inventors on issued patents and pending patent applications related to AAVP technologies and are entitled to royalties if licensing or commercialization occurs. T.Y. and A.H. are inventors on a patent application describing the vector constructs reported here and will be entitled to royalties if licensing or commercialization occurs. These arrangements are managed in accordance with established institutional conflict of interest policies of each corresponding institution., (Copyright © 2019 the Author(s). Published by PNAS.)

Glioblastoma multiforme (GBM) remains a cancer with a poor prognosis and few effective therapeutic options. Successful medical management of GBM is limited by the restricted access of drugs to the central nervous system (CNS) caused by the blood brain barrier (BBB). We previously showed that a subset of GBM are arginine auxotrophic because of transcriptional silencing of ASS1 and/or ASL and are sensitive to pegylated arginine deiminase (ADI-PEG20). However, it is unknown whether depletion of arginine in peripheral blood in vivo has therapeutic activity against intracranial disease. In the present work, we describe the efficacy of ADI-PEG20 in an intracranial model of human GBM in which tumour growth and regression are assessed in real time by measurement of luciferase activity. Animals bearing intracranial human GBM tumours of varying ASS status were treated with ADI-PEG20 alone or in combination with temozolomide and monitored for tumour growth and regression. Monotherapy ADI-PEG20 significantly reduces the intracranial growth of ASS1 negative GBM and extends survival of mice carrying ASS1 negative GBM without obvious toxicity. The combination of ADI-PEG20 with temozolomide (TMZ) demonstrates enhanced effects in both ASS1 negative and ASS1 positive backgrounds.Our data provide proof of principle for a therapeutic strategy for GBM using peripheral blood arginine depletion that does not require BBB passage of drug and is well tolerated. The ability of ADI-PEG20 to cytoreduce GBM and enhance the effects of temozolomide argues strongly for its early clinical evaluation in the treatment of GBM.

The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the &alpha; &nu; integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Cardiovascular effects (e.g., tachycardia, hypo- and/or hypertension) are often clinical outcomes of snake envenoming. Malayan krait ( Bungarus candidus ) envenoming has been reported to cause cardiovascular effects that may be related to abnormalities in parasympathetic activity. However, the exact mechanism for this effect has yet to be determined. In the present study, we investigated the in vivo and in vitro cardiovascular effects of B. candidus venoms from Southern (BC-S) and Northeastern (BC-NE) Thailand. SDS-PAGE analysis of venoms showed some differences in the protein profile of the venoms. B. candidus venoms (50 µg/kg-100 µg/kg, i.v.) caused dose-dependent hypotension in anaesthetised rats. The highest dose caused sudden hypotension (phase I) followed by a return of mean arterial pressure to baseline levels and a decrease in heart rate with transient hypertension (phase II) prior to a small decrease in blood pressure (phase III). Prior administration of monovalent antivenom significantly attenuated the hypotension induced by venoms (100 µg/kg, i.v.). The sudden hypotensive effect of BC-NE venom was abolished by prior administration of hexamethonium (10 mg/kg, i.v.) or atropine (5 mg/kg, i.v.). BC-S and BC-NE venoms (0.1 µg/kg-100 µg/ml) induced concentration-dependent relaxation (EC 50 = 8 ± 1 and 13 ± 3 µg/mL, respectively) in endothelium-intact aorta. The concentration-response curves were markedly shifted to the right by pre-incubation with L-NAME (0.2 mM), or removal of the endothelium, suggesting that endothelium-derived nitric oxide (NO) is likely to be responsible for venom-induced aortic relaxation. Our data indicate that the cardiovascular effects caused by B. candidus venoms may be due to a combination of vascular mediators (i.e., NO) and autonomic adaptation via nicotinic and muscarinic acetylcholine receptors.

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., Competing Interests: The authors have no conflicts of interest to declare.

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications.

Background: Gene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM)., Methods: We used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student's t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups., Results: We demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately., Conclusion: Our findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy.

Objective: To evaluate prevalence of overweight and obesity in second-year medical cadets and to determine risk behaviors before and after 6 months studying at Phramongkutklao College of Medicine (PCM). MATERIAL AMD METHOD: Data on self-report behavioral questionnaires was collected from 92 medical cadets before and after 6 months studying at PCM in 2008. Body weight, height, waist circumference, blood pressure and body fat were measured according to standard protocols., Results: Overall, prevalence of overweight and obese medical cadets before studying at PCM was 16.30% and 15.22% according to BMI and 27.17% and 15.22% by percent body fat. After 6 months of study, overall mean of body fat and systolic blood pressure were significantly reduced. For males, the mean of weight, BMI and waist circumference were reduced significantly. In contrast, the mean of body weight and BMI of females were significantly increased. The prevalence of overweight in total participants determined by body fat was significantly reduced from 27.17% to 15.22%. Determination by BMI, obesity was significantly reduced from 34.29% to 14.29% only in males. Behaviors that were found to be significantly increased (p < 0.001) were regular physical activity, night eating, sugar-sweet beverage consumption, amount and frequency of coffee or tea consumption. Significant decrease in time of watching TV or using a computer, and duration of sleep was also determined., Conclusion: The present study indicated that overweight, obesity, waist circumference and body fat of medical cadets were decreased after study at PCM which is possibly due to increasing exercise. Nevertheless, behavioral risks for obesity were also increasing, therefore, it might result in increased the prevalence of obesity in the future.

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Versican/proteoglycan-mesenchymal (PG-M) is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM) that is constitutively expressed in adult tissues such as dermis and blood vessels. It serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell adhesion, migration, proliferation, and differentiation. Knock-in mouse embryonic (Cspg2(Delta3/Delta3)) fibroblasts whose versican lack the A subdomain of the G1 domain exhibit low proliferation rates and acquire senescence. It was suspected that chondroitin sulfate on versican core protein would be altered when the A subdomain was disrupted, so fibroblasts were made from homozygous Cspg2(Delta3/Delta3) mouse embryos to investigate the hypothesis. Analysis of the resulting versican deposition demonstrated that the total versican deposited in the Cspg2(Delta3/Delta3) fibroblasts culture was approximately 50% of that of the wild type (WT), while the versican deposited in the ECM of Cspg2(Delta3/Delta3) fibroblasts culture was 35% of that of the WT, demonstrating the lower capacity of mutant (Cspg2(Delta3/Delta3)) versican deposited in the ECM. The analysis of CS expression in the Cspg2(Delta3/Delta3) fibroblasts culture compared with wild-type fibroblasts showed that the composition of the non-sulfate chondroitin sulfate isomer on the versican core protein increased in the cell layer but decreased in the culture medium. Interestingly, chondroitin sulfate E isomer was found in the culture medium. The amount of CS in the Cspg2(Delta3/Delta3) cell layer of fibroblasts with mutant versican was dramatically decreased, contrasted to the amount in the culture medium, which increased. It was concluded that the disruption of the A subdomain of the versican molecule leads to lowering of the amount of versican deposited in the ECM and the alteration of the composition and content of CS on the versican molecule.

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.

Objectives: To identify albuminuria prevalence and examine the association of albuminuria with plasma glucose, blood pressure and body mass index in rural Thai people aged 35 and older., Material and Method: All volunteer adults aged 35 and older at Ban Nayao, Chachoengsao Province were recruited in this cross-sectional study. Macroalbuminuria and microalbuminuria were tested in first morning urine using Combur and Micral Test strips. Fasting plasma glucose, blood pressure, weight and height were determined. Chi-square and multiple logistic regression analysis were used for analysis., Results: Of the 357 participants, 26.61% had microalbuminuria, 3.08% had macroalbuminuria, and 9.2%, 19%, 7.3% had diabetes, hypertension, and obese, respectively. The prevalence of microalbuminuria and macroalbuminuria in people with diabetes were 30.30% and 15.15%, respectively. In the obese, the prevalence was 50% and 3.8% and was 30.88% and 7.35% in the hypertensive group, respectively. Strong significant associations between plasma glucose (p = 0.013), and body mass index (p = 0.008) with the progression of albuminuria were observed. According to multiple logistic regression analysis, diabetes and obesity were independent risk factors for albuminuria statistically significant (p = 0.036 and p = 0.005, respectively, 95% CI)., Conclusion: The present study showed increased risk of albuminuria in diabetes and in obese people in a rural area of Thailand. Thus, community-controlled diabetes and weight program should be introduced to the rural community.

Background: Third-year (first preclinical year) Royal Thai Army (RTA) medical cadets were exposed to a rural community in a community medicine field activity. The poor, kind rural people gave good conditions for student's professional development and empathy., Objectives: To report a community medicine field work activity for professional development of the RTA medical school., Material and Method: Descriptive analysis on two occasions to evaluate the third-year RTA medical cadets' impressions immediately after the community medicine field activity in a rural area in central Thailand and one year later using baseline scores of cadets' impressions against our institute's objectives., Results: At the end of the field work, 90.9% of the cadets reported they achieved several institute objectives. The four most frequent mentioned were ethical development (70.6%), professionalism (84.3%), human relationship & communication skills (92.2%), and capability to work in the cmmunity (64.7%). Furthermore, RTA medical cadets rated the staff as good role models (51%)., Conclusion: The community field work course provided a good environment for the third year medical cadets in building morality and professional development to be good doctors.

Purpose/objectives: To describe patients' and their partners' perceptions of gender roles, gender power relationships, and sexuality before diagnosis of and after treatment for cervical cancer., Design: Descriptive., Setting: Southern Thailand., Sample: 97 women with cervical cancer who received cancer treatment, including radiotherapy, and their partners., Methods: Structured interview methods were used to gather information relating to gender roles, gender power relationships, and sexuality., Main Research Variables: Gender roles, gender power relationships, and sexuality., Findings: Fifty-two percent of the women (n = 50) were diagnosed with stage II cervical cancer. The percentage of women who undertook various activities specific to gender roles before their diagnoses was higher than the percentage who undertook the same activities after treatment. An increased percentage of partners undertook the women's gender-role-specific activities after the women received cancer treatment compared with the percentage who did so before diagnosis. Little change in gender power relationships was reported. A high percentage of the couples reported changes in various aspects of their sexuality after cancer treatment compared with before diagnosis., Conclusions: Gender roles, gender power relationships, and sexuality changed for women with cervical cancer and their partners after the women completed cancer treatment., Implications for Nursing: Open discussions among women with cervical cancer, their partners, and oncology nurses are necessary to identify culturally sensitive and appropriate solutions.

Aim: To determine compliance with hormone replacement therapy (HRT) over a 2-year period, reasons for discontinuing HRT and the factors associated with non-compliance., Methods: A total of 202 women attending the menopause clinic at Songklanagarind Hospital and taking HRT were included in this retrospective study. Compliance was assessed for each 6-month interval within the first 2 years. Reasons for discontinuation were requested from women who had stopped using HRT., Results: Compliance rates with HRT for the study group were 57.9% at 6 months, 42.6% at 12 months, 35.1% at 18 months and 32.7% at 24 months. The main reasons for discontinuing HRT were improvement of climacteric symptoms (20.9%), fear of cancer (16.4%) and irregular bleeding (11.9%). Logistic regression analysis revealed a significant increase in the risk of non-compliance of HRT among agriculturists or untrained workers (OR 4.7, 95% CI 1.2-18.8; reference, government employees), those with delayed onset of treatment (>1 years; OR 3.0, 95% CI 1.1-8.0; reference, 0-3 months) and those prescribed HRT for climacteric symptoms or reasons other than oophorectomy or ovarian failure (OR 18.2-41.6 depending on reasons). Agriculturists or untrained workers who delayed onset of treatment for climacteric symptoms had the highest expected non-compliance rate of 0.95%., Conclusion: Long-term compliance of HRT was not good at Songklanagarind menopause clinic. More attention has to be paid to the counseling of patients about HRT. Agricultural or untrained workers, late starting HRT, and presence of climacteric complaints were the significant factors for poor HRT compliance.