Calcium Activation of the Androgen Receptor in Prostate Cells.

Background: Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. Methods: The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. Results: To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. Conclusions: The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Neutron spectrometry of D2O-moderated 252Cf with Bonner sphere spectrometer.

For neutron spectrometry of the D2O-moderated 252Cf source with a Bonner sphere spectrometer (BSS), it is difficult to use the large and heavy shadow cone to correct the neutron scattering effect. To overcome this problem, Monte Carlo (MC) simulation method was applied to calculate the neutron scattering ratio and to establish the BSS response functions. The simulated response functions were verified by experimental measurements in reference mono-energetic neutron fields. MC simulation based scattering-correction was validated by measurement of 252Cf neutron field. The measured and simulated values of the neutron scattering ratio were very close with relative errors within ±6%. Finally, the neutron spectrum and the spectrum averaged conversion coefficients of the D2O-moderated 252Cf were measured using BSS after scattering-correction by MC simulation, and the results agreed with the values recommended by ISO 8529-1:2021. It shows that the MC simulation can be a useful substitute to shadow cones method for neutron scattering-correction.

Constructing biomimetic liver models through biomaterials and vasculature engineering.

The occurrence of various liver diseases can lead to organ failure of the liver, which is one of the leading causes of mortality worldwide. Liver tissue engineering see the potential for replacing liver transplantation and drug toxicity studies facing donor shortages. The basic elements in liver tissue engineering are cells and biomaterials. Both mature hepatocytes and differentiated stem cells can be used as the main source of cells to construct spheroids and organoids, achieving improved cell function. To mimic the extracellular matrix (ECM) environment, biomaterials need to be biocompatible and bioactive, which also help support cell proliferation and differentiation and allow ECM deposition and vascularized structures formation. In addition, advanced manufacturing approaches are required to construct the extracellular microenvironment, and it has been proved that the structured three-dimensional culture system can help to improve the activity of hepatocytes and the characterization of specific proteins. In summary, we review biomaterials for liver tissue engineering, including natural hydrogels and synthetic polymers, and advanced processing techniques for building vascularized microenvironments, including bioassembly, bioprinting and microfluidic methods. We then summarize the application fields including transplant and regeneration, disease models and drug cytotoxicity analysis. In the end, we put the challenges and prospects of vascularized liver tissue engineering.

Regulable Supporting Baths for Embedded Printing of Soft Biomaterials with Variable Stiffness.

Three-dimensional (3D) embedded printing is emerging as a potential solution for the fabrication of complex biological structures and with ultrasoft biomaterials. For the supporting medium, bulk gels can support a wide range of bioinks with higher printing resolution as well as better finishing surfaces than granular microgel baths. However, the difficulties of regulating the physical properties of existing bulk gel supporting baths limit the further development of this method. This work has developed a bulk gel supporting bath with easily regulable physical properties to facilitate soft-material fabrication. The proposed bath is composed based on the hydrophobic association between a hydrophobically modified hydroxypropylmethyl cellulose (H-HPMC) and Pluronic F-127 (PF-127). Its rheological properties can be easily regulated; in the preprinting stage by varying the relative concentration of components, during printing by changing the temperature, and postprinting by adding additives with strong hydrophobicity or hydrophilicity. This has made the supporting bath not only available for various bioinks with a range of printing windows but also easy to be removed. Also, the removal strategy is independent of printing conditions like temperature and ions, which empowers the bath to hold great potential for the embedded printing of commonly used biomaterials. The adjustable rheological properties of the bath were leveraged to characterize the embedded printing quantitatively, involving the disturbance during the printing, filament cross-sectional shape, printing resolution, continuity, and the coalescence between adjacent filaments. The match between the bioink and the bath was also explored. Furthermore, low-viscosity bioinks (with 0.008-2.4 Pa s viscosity) were patterned into various 3D complex delicate soft structures (with a 0.5-5 kPa compressive modulus). It is believed that such an easily regulable assembled bath could serve as an available tool to support the complex biological structure fabrication and open unique prospects for personalized medicine.

A versatile embedding medium for freeform bioprinting with multi-crosslinking methods.

Embedded freeform writing addresses the contradiction between the material printability and biocompatibility for conventional extrusion-based bioprinting. However, the existing embedding mediums have limitations concerning the restricted printing temperature window, compatibility with bioinks or crosslinkers, and difficulties on medium removal. This work demonstrates a new embedding medium to meet the above demands, which composes of hydrophobically modified hydroxypropylmethyl cellulose and Pluronic F-127. The adjustable hydrophobic and hydrophilic associations between the components permit tunable thermoresponsive rheological properties, providing a programmable printing window. These associations are hardly compromised by additives without strong hydrophilic groups, which means it is compatible with the majority of bioink choices. We use polyethylene glycol 400, a strong hydrophilic polymer, to facilitate easy medium removal. The proposed medium enables freeform writing of the millimetric complex tubular structures with great shape fidelity and cell viability. Moreover, five bioinks with up to five different crosslinking methods are patterned into arbitrary geometries in one single medium, demonstrating its potential in heterogeneous tissue regeneration. Utilizing the rheological properties of the medium, an enhanced adhesion writing method is developed to optimize the structure's strand-to-strand adhesion. In summary, this versatile embedding medium provides excellent compatibility with multi-crosslinking methods and a tunable printing window, opening new opportunities for heterogeneous tissue regeneration.

Vascularizing the brain in vitro.

The brain is arguably the most fascinating and complex organ in the human body. Recreating the brain in vitro is an ambition restricted by our limited understanding of its structure and interacting elements. One of these interacting parts, the brain microvasculature, is distinguished by a highly selective barrier known as the blood-brain barrier (BBB), limiting the transport of substances between the blood and the nervous system. Numerous in vitro models have been used to mimic the BBB and constructed by implementing a variety of microfabrication and microfluidic techniques. However, currently available models still cannot accurately imitate the in vivo characteristics of BBB. In this article, we review recent BBB models by analyzing each parameter affecting the accuracy of these models. Furthermore, we propose an investigation of the synergy between BBB models and neuronal tissue biofabrication, which results in more advanced models, including neurovascular unit microfluidic models and vascularized brain organoid-based models.

Replication Kinetics for a Reporter Merkel Cell Polyomavirus.

Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, ß-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.

A Systematic Thermal Analysis for Accurately Predicting the Extrusion Printability of Alginate-Gelatin-Based Hydrogel Bioinks.

Three-dimensional (3D) bioprinting has significant potential for addressing the global problem of organ shortages. Extrusion printing is a versatile 3D bioprinting technique, but its low accuracy currently limits the solution. This lack of precision is attributed largely to the complex thermal and dynamic properties of bioinks and makes it difficult to provide accurate estimations of the printed results. It is necessary to understand the relationship between printing temperature and materials' printability to address this issue. This paper proposes a quantitative thermal model incorporating a system's printing temperatures (syringe, ambient, and bioink) to facilitate accurate estimations of the printing outcomes. A physical model was established to reveal the relationship between temperature, pressure, and velocity in guiding the printing of sodium alginate-gelatin composite hydrogel (a popular bioink) to optimize its extrusion-based printability. The model considered the phenomenon of bioink die swells after extrusion. A series of extrusion experiments confirmed that the proposed model offers enhanced printing outcome estimations compared with conventional models. Two types of nozzles (32- and 23-gauge) were used to print several sets of lines with a linewidth step of 50 mm by regulating the extrudate's temperature, pressure, and velocity separately. The study confirmed the potential for establishing a reasonable, accurate open-loop linewidth control based on the proposed optimization method to expand the application of extrusion-based bioprinting further.

Merkel Cell Polyomavirus Encodes Circular RNAs (circRNAs) Enabling a Dynamic circRNA/microRNA/mRNA Regulatory Network.

Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.

Current Advances on 3D-Bioprinted Liver Tissue Models.

The liver, the largest gland in the human body, plays a key role in metabolism, bile production, detoxification, and water and electrolyte regulation. The toxins or drugs that the gastrointestinal system absorbs reach the liver first before entering the bloodstream. Liver disease is one of the leading causes of death worldwide. Therefore, an in vitro liver tissue model that reproduces the main functions of the liver can be a reliable platform for investigating liver diseases and developing new drugs. In addition, the limitations in traditional, planar monolayer cell cultures and animal tests for evaluating the toxicity and efficacy of drug candidates can be overcome. Currently, the newly emerging 3D bioprinting technologies have the ability to construct in vitro liver tissue models both in static scaffolds and dynamic liver-on-chip manners. This review mainly focuses on the construction and applications of liver tissue models based on 3D bioprinting. Special attention is given to 3D bioprinting strategies and bioinks for constructing liver tissue models including the cell sources and hydrogel selection. In addition, the main advantages and limitations and the major challenges and future perspectives are discussed, paving the way for the next generation of in vitro liver tissue models.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

Self-Healing Dielectric Elastomers for Damage-Tolerant Actuation and Energy Harvesting.

The actuation and energy-harvesting performance of dielectric elastomers are strongly related to their intrinsic electrical and mechanical properties. For future resilient smart transducers, a fast actuation response, efficient energy-harvesting performance, and mechanical robustness are key requirements. In this work, we demonstrate that poly(styrene-butadiene-styrene) (SBS) can be converted into a self-healing dielectric elastomer with high permittivity and low dielectric loss, which can be deformed to large mechanical strains; these are key requirements for actuation and energy-harvesting applications. Using a one-step click reaction at room temperature for 20 min, methyl-3-mercaptopropionate (M3M) was grafted to SBS and reached 95.2% of grafting ratios. The resultant M3M-SBS can be deformed to a high mechanical strain of 1000%, with a relative permittivity of εr = 7.5 and a low tan δ = 0.03. When used in a dielectric actuator, it can provide 9.2% strain at an electric field of 39.5 MV m-1 and can also generate an energy density of 11 mJ g-1 from energy harvesting. After being subjected to mechanical damage, the self-healed elastomer can recover 44% of its breakdown strength during energy harvesting. This work demonstrates a facile route to produce self-healing, high permittivity, and low dielectric loss elastomers for both actuation and energy harvesting, which is applicable to a wide range of diene elastomer systems.