Multicellular Layers Research Articles

Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

Impaired breakdown of Herwig's epithelial root sheath disturbs tooth root development.

Wnt/β-catenin signaling plays a variety of roles in both the dental epithelium and mesenchyme at most stages of tooth development. In this study, we verified the roles of Hertwig's epithelial root sheath (HERS) breakdown in tooth root development. This breakdown results in formation of epithelial cell rests of Malassez (ERM). Following induction of β-catenin stabilization in the epithelium of developing tooth at the moment of HERS breakdown, HERS failed to break down for ERM formation. HERS with stabilized β-catenin was altered into a multicellular layer enveloping elongated root dentin with higher expression of junctional proteins such as Zo-1 and E-cadherin. Importantly, this impairment of HERS breakdown led to arrest of further root elongation. In addition, the portion of root dentin enveloped by the undissociated HERS remained in a hypomineralized state. The odontoblasts showed ectopically higher expression of pyrophosphate regulators including Ank and Npp1, whereas Tnap expression was unchanged. Our data suggest that Wnt/β-catenin signaling is decreased in HERS for ERM formation during root development. Furthermore, ERM formation is important for further elongation and dentin mineralization of the tooth roots. These findings may provide new insight to understand the contribution of ERM to root formation.

Histological analysis for pulp mineralisation after severe intrusive luxation of immature molars in rats.

Pulp mineralisation is a survival process that may occur in the pulp of immature teeth following trauma. However, the mechanism of this process remains unclear. The aim of this study was to evaluate the histological manifestations of pulp mineralisation after intrusion in immature molars of rats. Three-week-old male Sprague-Dawley rats were subjected to intrusive luxation of the right maxillary second molar by an impact force from a striking instrument through a metal force transfer rod. The left maxillary second molar of each rat was used as a control. The control and injured maxillae were collected at 3, 7, 10, 14, and 30 days after trauma (n=15 per time group) and evaluated using haematoxylin and eosin staining and immunohistochemistry. Independent two-tailed Student's t-test was used for statistical comparison of the immunoreactive area. Pulp atrophy and mineralisation were observed in 30%-40% of the animals, and no pulp necrosis occurred. Ten days after trauma, pulp mineralisation, with osteoid tissue rather than reparative dentin, formed around the newly vascularised areas in the coronal pulp. CD90-immunoreactive cells were observed in the sub-odontoblastic multicellular layer in control molars, whereas the number of these cells was decreased in the traumatised teeth. CD105 localised in cells around the pulp osteoid tissue of the traumatised teeth, whereas in control teeth, it was only expressed in the vascular endothelial cells of capillaries in the odontoblastic or sub-odontoblastic layers. In specimens with pulp atrophy at 3-10 days after trauma, hypoxia inducible factor expression and CD11b-immunoreactive inflammatory cells increased. Following intrusive luxation of immature teeth without crown fractures in rats, no pulp necrosis occurred. Instead, pulp atrophy and osteogenesis around neovascularisation with activated CD105-immunoreactive cells were observed in the coronal pulp microenvironment characterised by hypoxia and inflammation.

Effect of paclitaxel priming on doxorubicin penetration in a multicellular layer model of human colorectal cancer cells

Tumor priming is considered a promising strategy for improving drug distribution in malignant tissues. Multicellular layers (MCLs) of human cancer cells are potentially useful models for evaluating tumor-priming agents. We evaluated the priming effects of paclitaxel (PTX) on doxorubicin (DOX) penetration using MCLs of the human colorectal cancer cell lines including DLD-1, HCT-116, and HT-29. The penetration of DOX treated at 50 μM for 3 h was highly limited in all three MCLs. The penetration of the priming agent PTX into MCLs was determined using rhodamine-labeled PTX and appeared to be cell line-dependent: full penetration was observed in HCT-116 and HT-29 MCLs, whereas only limited penetration occurred in DLD-1 MCLs. PTX pretreatment at 20 μM for 24 or 48 h induced a tumor-priming effect in DOX distribution, with a 3 to 5.6-fold-increase in HCT-116 and HT-29 MCLs but a less than two-fold increase in DLD-1 MCLs. PTX treatment decreased fibronectin expression in HCT-116 and HT-29 MCLs but not in DLD-1, suggesting that the prominent priming effect of PTX in HCT-116 and HT-29 MCLs may be associated with the downregulation of fibronectin expression. Our study demonstrated that MCLs of human cancer cells are a useful model not only for the study of drug penetration into tumor tissues but also for screening and evaluating tumor-priming agents.

Mechanosignals in abdominal aortic aneurysms.

Cumulative evidence has shown that mechanical and frictional forces exert distinct effects in the multi-cellular aortic layers and play a significant role in the development of abdominal aortic aneurysms (AAA). These mechanical cues collectively trigger signaling cascades relying on mechanosensory cellular hubs that regulate vascular remodeling programs leading to the exaggerated degradation of the extracellular matrix (ECM), culminating in lethal aortic rupture. In this review, we provide an update and summarize the current understanding of the mechanotransduction networks in different cell types during AAA development. We focus on different mechanosensors and stressors that accumulate in the AAA sac and the mechanotransduction cascades that contribute to inflammation, oxidative stress, remodeling, and ECM degradation. We provide perspectives on manipulating this mechano-machinery as a new direction for future research in AAA.

Novel Ground-Up 3D Multicellular Simulators for Synthetic Biology CAD Integrating Stochastic Gillespie Simulations Benchmarked with Topologically Variable SBML Models.

The elevation of Synthetic Biology from single cells to multicellular simulations would be a significant scale-up. The spatiotemporal behavior of cellular populations has the potential to be prototyped in silico for computer assisted design through ergonomic interfaces. Such a platform would have great practical potential across medicine, industry, research, education and accessible archiving in bioinformatics. Existing Synthetic Biology CAD systems are considered limited regarding population level behavior, and this work explored the in silico challenges posed from biological and computational perspectives. Retaining the connection to Synthetic Biology CAD, an extension of the Infobiotics Workbench Suite was considered, with potential for the integration of genetic regulatory models and/or chemical reaction networks through Next Generation Stochastic Simulator (NGSS) Gillespie algorithms. These were executed using SBML models generated by in-house SBML-Constructor over numerous topologies and benchmarked in association with multicellular simulation layers. Regarding multicellularity, two ground-up multicellular solutions were developed, including the use of Unreal Engine 4 contrasted with CPU multithreading and Blender visualization, resulting in a comparison of real-time versus batch-processed simulations. In conclusion, high-performance computing and client-server architectures could be considered for future works, along with the inclusion of numerous biologically and physically informed features, whilst still pursuing ergonomic solutions.

Flame Retardant Coatings: Additives, Binders, and Fillers.

This review provides an intensive overview of flame retardant coating systems. The occurrence of flame due to thermal degradation of the polymer substrate as a result of overheating is one of the major concerns. Hence, coating is the best solution to this problem as it prevents the substrate from igniting the flame. In this review, the descriptions of several classifications of coating and their relation to thermal degradation and flammability were discussed. The details of flame retardants and flame retardant coatings in terms of principles, types, mechanisms, and properties were explained as well. This overview imparted the importance of intumescent flame retardant coatings in preventing the spread of flame via the formation of a multicellular charred layer. Thus, the intended intumescence can reduce the risk of flame from inherently flammable materials used to maintain a high standard of living.

A char stratification approach to characterization and quantitative thermal insulation performance of hydrocarbon intumescent coatings

To protect structural steel in the event of a hydrocarbon fire, epoxy-based intumescent coatings, which expand to form a multicellular char layer, are increasingly used. Consequently, to improve formulations and understand the performance of such coatings, it is of great industrial and scientific interest to establish relationships between the char properties and the critical heating times of coated steel plates. In this work, a so-called Stratification Parameter (SP) that combines the relative expansions and insulation efficiencies of individual char layers (termed the sponge-like, the macroporous, and the compact phase, respectively), is introduced to characterize the properties of the multilayered intumescent coating char. An approximate linear relationship was revealed between the SP of the char and the fire-resistance performance (i.e., critical times to 400 and 550°C) of the intumescent coating. Furthermore, owing to the mapped-out exponential relationship between the dynamic viscosity minimum and the char appearance, it was discovered that the SP of the char can be effectively used to point out the required dynamic viscosity change of an intumescent coating to form a given char. Finally, results of thermogravimetric analyses suggest that constraining the coating mass loss during char degradation (to less than 28 wt% for the coatings of the present work) may prevent the formation of the less efficient sponge-like phase. The stratification methodology developed can be used to estimate expected thermal insulation properties of non-uniform hydrocarbon intumescent coating chars.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

Кашель у детей: современный подход к рациональной фармакотерапии

Cough in children is one of the most common symptoms both in outpatient and hospital practices. It is the main reason why parents of these patients (especially of younger kids) seek for medical care. The article highlights the current view on the cough in pediatric practice. It elucidates cough classification, etiology, and therapy in children. The authors describe characteristics of the respiratory epithelium, a multicellular layer, that extends from the nasopharynx to the small airways and acts as a respiratory barrier coordinating immune response in the early childhood, undergoing the process of development, maturation, and regeneration to adapt to the changing environment. The article presents findings of the clinical studies assessing the medications of choice that were used for the treatment of coughs in children. The selection of right drugs should be based on individual patient’s needs and depend on the extent of cough impact on the patient condition. Also, it is necessary to consider whether the cough is intense, productive, for how long it lasts and to analyse bronchial mucus characteristics. KEYWORDS: cough, children, respiratory epithelium, immune response, immunity, antitussives, butamirate. FOR CITATION: Nikolaeva S.V., Kanner E.V., Kanner I.D. et al. Cough in children: the current approach to rational pharmacotherapy. Russian Medical Inquiry. 2022;6(11):650–655 (in Russ.). DOI: 10.32364/2587-6821-2022-6-11-650-655.

Radiosensitisation of SCCVII tumours and normal tissues in mice by the DNA-dependent protein kinase inhibitor AZD7648

Background and purposeInhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. Materials and methodsRadiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2′-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. ResultsAZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. ConclusionAZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates forany diffusion limitations of the prodrug in tumour tissue. Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively. NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

The Orchid Velamen: A Model System for Studying Patterned Secondary Cell Wall Development?

Understanding the mechanisms through which plants generate secondary cell walls is of more than academic interest: the physical properties of plant-derived materials, including timber and textiles, all depend upon secondary wall cellulose organization. Processes controlling cellulose in the secondary cell wall and their reliance on microtubules have been documented in recent decades, but this understanding is complicated, as secondary walls normally form in the plant’s interior where live cell imaging is more difficult. We investigated secondary wall formation in the orchid velamen, a multicellular epidermal layer found around orchid roots that consists of dead cells with lignified secondary cell walls. The patterns of cell wall ridges that form within the velamen vary between different orchid species, but immunolabelling demonstrated that wall deposition is controlled by microtubules. As these patterning events occur at the outer surface of the root, and as orchids are adaptable for tissue culture and genetic manipulation, we conclude that the orchid root velamen may indeed be a suitable model system for studying the organization of the plant cell wall. Notably, roots of the commonly grown orchid Laelia anceps appear ideally suited for developing this research.

Morphological features of rye grain with low pentosan content

Background. The grain coat plays a major role in the development of rye cultivars with low levels of water-soluble pentosans (WSP). Grain coat thinness is a diagnostic trait for low WSP (arabinoxylans) content. To improve the technology of low-pentosan rye breeding, it becomes important to study the effect of changes in the anatomy of low-pentosan grains on morphological characteristics.Materials and methods. Grains with thin coats (transparent) were identified with the LFS-1 diaphanoscope in the populations of 7 commercial winter rye cultivars grown in Russian Federation. Anatomical and morphological features of seed coats were studied on thick- and thin-coated grains identified in the rye cultivars ‘Era’ and ‘Vyatka’. The thickness of grain coats and the aleurone layer were assessed according to L. N. Lyubarsky.Results and conclusions. In thin-coated rye grains, we found a reduction in the thickness of the pericarp and seed coats by 50–70% and in the aleurone layer by 32.1–39.6%, compared to thick-coated grains. With a constant cell size in “transparent” grains, a decrease in the thickness of the aleurone layer and coat occurs at the expense of the reduction in not only the cell walls, but also the intercellular space. The pericarp in the tissues of the second multicellular layer contains the bulk of the protective biologically passive WSP. It was proved that there were no significant changes in grain parameters, such as length, width and relative volume, depending on coat thinness. Significant differences were found in the 1000 grain weight. In “transparent” grains the index increased by 5.2–19.7%, compared with “nontransparent” ones.

A New Mathematical Model for Controlling Tumor Growth Based on Microenvironment Acidity and Oxygen Concentration.

Hypoxia and the pH level of the tumor microenvironment have a great impact on the treatment of tumors. Here, the tumor growth is controlled by regulating the oxygen concentration and the acidity of the tumor microenvironment by introducing a two-dimensional multiscale cellular automata model of avascular tumor growth. The spatiotemporal evolution of tumor growth and metabolic variations is modeled based on biological assumptions, physical structure, states of cells, and transition rules. Each cell is allocated to one of the following states: proliferating cancer, nonproliferating cancer, necrotic, and normal cells. According to the response of the microenvironmental conditions, each cell consumes/produces metabolic factors and updates its state based on some stochastic rules. The input parameters are compatible with cancer biology using experimental data. The effect of neighborhoods during mitosis and simulating spatial heterogeneity is studied by considering multicellular layer structure of tumor. A simple Darwinist mutation is considered by introducing a critical parameter (Nmm) that affects division probability of the proliferative tumor cells based on the microenvironmental conditions and cancer hallmarks. The results show that Nmm regulation has a significant influence on the dynamics of tumor growth, the growth fraction, necrotic fraction, and the concentration levels of the metabolic factors. The model not only is able to simulate the in vivo tumor growth quantitatively and qualitatively but also can simulate the concentration of metabolic factors, oxygen, and acidity graphically. The results show the spatial heterogeneity effects on the proliferation of cancer cells and the rest of the system. By increasing Nmm, tumor shrinkage and significant increasing in the oxygen concentration and the pH value of the tumor microenvironment are observed. The results demonstrate the model's ability, providing an essential tool for simulating different tumor evolution scenarios of a patient and reliable prediction of spatiotemporal progression of tumors for utilizing in personalized therapy.

Urinary bladder reconstruction using autologous collagenous connective tissue membrane "Biosheet®" induced by in-body tissue architecture: A pilot study.

IntroductionIn-body tissue architecture (iBTA) technology, based on cell-free tissue engineering, can produces collagenous tissues for implantation by subcutaneous embedding a designed mold. The aim of this study was to evaluate the biocompatibility of iBTA-induced “Biosheet®” collagenous sheets, as scaffold materials for bladder reconstruction.MethodsCanine Biosheet® implants were prepared by embedding molds into subcutaneous pouches in beagles for 8 weeks. A part of canine bladder wall was excised (2 × 2 cm) and repaired by patching the same sized autologous Biosheet®. The Biosheet® implants were harvested 4 weeks (n = 1) and 12 weeks (n = 3) after the implantation and evaluated histologically.ResultsNo disruption of the patched Biosheet® implants or urinary leakage into the peritoneal cavity was observed during the entire observation periods. There were no signs of chronic inflammation or Biosheet® rejection. The urine-contacting surface of luminal surface of the Biosheet® was covered with a multicellular layer of urothelium cells 4 weeks after implantation. α-SMA-positive muscle cells were observed at the margin of the Biosheet® implants at 12 weeks after the implantation. In addition, in the center of the Biosheet® implants, the formation of microvessels stained as α-SMA-positive was observed.ConclusionBiosheet® implants have biocompatibility as a scaffold for bladder reconstruction, indicating that they may be applicable for full-thickness bladder wall substitution. Further studies are required for definitive evaluation as a scaffold for bladder reconstruction.

Intra-tumoral drug concentration mapping within solid tumor micro-milieu using in-vitro model and doxorubicin as a model drug

In contrast to plasma pharmacokinetics, intratumoral pharmacokinetics of doxorubicin (DOX) determines its spatial anti-tumoral activity. Three-dimensional multicellular layers (MCL) model for solid tumors present optimum experimental platform for studying the intratumoral pharmacokinetics of DOX. This might imply new insights for understanding intratumoral pharmacokinetic parameters with realistic clinical implications. Herein, we are presenting simplified method for the spatial in-situ concentration assessment of DOX within the avascular simulating MCL solid tumor model of DLD-1 and HT-29 cell lines. DLD-1 and HT-29 formed viable well-structured MCL model abundant in extracellular matrix component (fibronectin). DOX (100 µM) showed stronger anti-proliferative effect against MCL of DLD-1 compared to HT-29 MCL (38.8% and 27.9%, respectively). The differential potencies of DOX closely correlate to the intratumoral pharmacokinetics within MCL’s of both cell lines. DOX penetrated faster and washed out slower through the MCL of DLD-1 compared to HT-29 MCL. Distribution of DOX within MCL of DLD-1 was more homogenous compared to HT-29 MCL. Tissue concentration of DOX within MCL of DLD-1 was significantly higher than HT-29 MCL’s after 96 h exposure (0.7 and 0.4 µmole/gm tissue, respectively). Concentration of DOX within MCL of both cell lines exceeded the IC50 under monolayer conditions (2.3 ± 0.6 µM and 0.6 ± 0.1 µM, respectively). In addition, DOX was extensively metabolized to less active metabolites (doxorubicinol and doxorubicinone) through the thickness of both MCL’s. In conclusion, Intratumoral pharmacokinetic barriers to DOX might be key determinant in drug resistance on the tissue level, despite cellular and molecular events.

E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications.

The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates.Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4.Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach.Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

Difference of PDT induced apoptotic gene expression in 2D and 3D nasopharyngeal carcinoma cell models

Abstract Background Nasopharyngeal Carcinoma (NPC) is one of the top 10 cancers in Hong Kong. Photodynamic Therapy (PDT) is an alternative treatment approach to NPC. We previously reported that PDT induced both apoptosis and necrosis in 2D and 3D NPC models but in different manners, yet the underline biological mechanism is still unknown. In this pilot study we measured 3 apoptotic related gene expression, including c-Myc, p53 and Notch-1 genes using 2D and 3D EBV+ c666-1 cell models at pre- and post- FosPeg (FP) PDT treatment. Methods Total 5 x 105 cells were used for 2D and 3D multicellular layer cell models. Cells were activated with laser at 20J/cm2 after 24 hours FP incubation. Total RNA were obtained using high pure RNA isolation kit (Roche) and were converted to cDNA. 20ng cDNA were used for qPCR (Light Cycler 480). Results Results revealed that 2D and 3D models expressed different mRNA levels of c-Myc, p53 and Notch-1 gene, in both control cells and PDT treated cells. Results indicated that there were 0.3 to 1.2 fold difference in mRNA expression between 2D and 3D cell models among the tested apoptotic genes. 2D model was found more susceptible to PDT inducing mRNA expression than 3D model. The difference could be explained by: 1) light and drug delivery variation; 2) intracellular oxygen difference; and 3) cell to cell communication difference in 2D and 3D cell models. Conclusion 2D and 3D cell models behave differently at both molecular and cellular levels. These differences may due to different PDT responses in 2D and 3D models. 3D cell models were found to be more suitable for PDT pharmacokinetic studies. Further investigation is needed on PDT mediated molecular changes (protein expression) in 3D models. Acknowledgement The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (project no.:2015-00-74-RGC150201).

Reliable Integrated Satellite Terrestrial Communications using MIMO for Mitigation of Microwave Absorption by Earths Oxygen

Microwaves are used to communicate with satellite and terrestrial communication networks. But as microwaves pass through the Earth’s atmosphere, the oxygen gas absorbs microwave. In this 5G era, when the whole world is moving towards high data-rates and reliable communications, this absorption affects the data transmission in Integrated Satellite/Terrestrial Communication (ISTC) systems, which leads to degradation of the system performance. The multiple-input-multiple-output (MIMO) technology has become a boon for modern wireless communication systems to achieve the necessities of higher data-rates and communication reliability. The paper analyses the MIMO effect on block error rate (BLER), error vector magnitude (EVM) and throughput performance of the data transmission with different MIMO configurations. The paper establishes that better data-rates as well as reliable data communication is achieved with higher order MIMO configurations. MIMO 8×1 provides 5, 20 and 42.5 times improved performance to BLER; 5.26%, 25% and 81.82% in throughput; and 10.34%, 23.07% and 28% in EVM calculations as comparable to MIMO 4×1, MIMO 2×1 and SISO 1×1, respectively at 15 dB signal-to-noise ratio (SNR). The authors also give a new concept of multi-cellular layers based mobile communication network, useful for future smart cities.