Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

A Preliminary Study for an Intraoperative 3D Bioprinting Treatment of Severe Burn Injuries.

Intraoperative three-dimensional fabrication of living tissues could be the next biomedical revolution in patient treatment. APPROACH: We developed a surgery-ready robotic three-dimensional bioprinter and demonstrated that a bioprinting procedure using medical grade hydrogel could be performed using a 6-axis robotic arm in vivo for treating burn injuries. RESULTS: We conducted a pilot swine animal study on a deep third-degree severe burn model. We observed that the use of cell-laden bioink as treatment substantially affects skin regeneration, producing in situ fibroblast growth factor and vascular endothelial growth factor, necessary for tissue regeneration and re-epidermalization of the wound. CONCLUSIONS: We described an animal study of intraoperative three-dimensional bioprinting living tissue. This emerging technology brings the first proof of in vivo skin printing feasibility using a surgery-ready robotic arm-based bioprinter. Our positive outcome in skin regeneration, joined with this procedure's feasibility, allow us to envision the possibility of using this innovative approach in a human clinical trial in the near future.

From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Vascular Tissue Engineering: Challenges and Requirements for an Ideal Large Scale Blood Vessel.

Since the emergence of regenerative medicine and tissue engineering more than half a century ago, one obstacle has persisted: the in vitro creation of large-scale vascular tissue (>1 cm3) to meet the clinical needs of viable tissue grafts but also for biological research applications. Considerable advancements in biofabrication have been made since Weinberg and Bell, in 1986, created the first blood vessel from collagen, endothelial cells, smooth muscle cells and fibroblasts. The synergistic combination of advances in fabrication methods, availability of cell source, biomaterials formulation and vascular tissue development, promises new strategies for the creation of autologous blood vessels, recapitulating biological functions, structural functions, but also the mechanical functions of a native blood vessel. In this review, the main technological advancements in bio-fabrication are discussed with a particular highlights on 3D bioprinting technologies. The choice of the main biomaterials and cell sources, the use of dynamic maturation systems such as bioreactors and the associated clinical trials will be detailed. The remaining challenges in this complex engineering field will finally be discussed.

Gold nanobipyramids integrated ultrasensitive optical and electrochemical biosensor for Aflatoxin B1 detection.

This work reports the development of an electrical and optical biosensing for label-free detection of Aflatoxin B1 (AFB1) using gold (Au) nanobipyramids (NBPs). AuNBPs were synthesized through a two-step seed-mediated growth process followed by an exchange of capping agent from surfactant to lipoic acid. Pure and monodispersed AuNBPs of 70 nm base length were obtained and deposited on indium tin oxide (ITO)-coated glass substrate modified with self-assembled (3-Aminopropyl) triethoxysilane (APTES) film. The characterization of the obtained surfaces using spectroscopy, microscopy and diffractometry confirms the formation of AuNBPs, the conjugation to ITO electrode substrate and the immobilization of anti-AFB1 antibodies. AuNBPs modified ITO substrates were used for both electrochemical and Surface Plasmon Resonance biosensing studies. Localized Surface Plasmon Resonance (LSPR) local field enhancement was demonstrated. SPR based AFB1 detection was found to be linear in the 0.1-500 nM range with a limit of detection of 0.4 nM, whereas, impedimetric AFB1 detection was shown to be linear in the 0.1-25 nM range with a limit of detection of 0.1 nM. The practical utility of the impedimetric sensor was tested in spiked maize samples and 95-100% recovery percentage was found together with low relative standard deviation, proof of the robustness of this AFB1 sensor.

Integrated graphene quantum dot decorated functionalized nanosheet biosensor for mycotoxin detection.

Decoration of graphene quantum dots (GQDs) on molybdenum disulfide (MoS2) nanosheets serves as an active electrode material which enhances the electrochemical performance of the analyte detection system. Herein, ionic surfactant cetyltrimethylammonium bromide (CTAB)-exfoliated MoS2 nanosheets decorated with GQD material are used to construct an electrochemical biosensor for aflatoxin B1 (AFB1) detection. An antibody of AFB1 (aAFB1) was immobilized on the electrophoretically deposited MoS2@GQDs film on the indium tin oxide (ITO)-coated glass surface using a crosslinker for the fabrication of the biosensor. The immunosensing study investigated by the electrochemical method revealed a signal response in the range of 0.1 to 3.0 ng/mL AFB1 concentration with a detection limit of 0.09 ng/mL. Also, electrochemical parameters such as diffusion coefficient and heterogeneous electron transfer (HET) were calculated and found to be 1.67 × 10-5 cm2/s and 2 × 10-5 cm/s, respectively. The effective conjugation of MoS2@GQDs that provides abundant exposed edge sites, large surface area, improved electrical conductivity, and electrocatalytic activity has led to an excellent biosensing performance with enhanced electrochemical parameters. Validation of the fabricated immunosensor was performed in a spiked maize sample, and a good percentage of recoveries within an acceptable range were obtained (80.2 to 98.3%).Graphical abstract.

A label-free ultrasensitive microfluidic surface Plasmon resonance biosensor for Aflatoxin B1 detection using nanoparticles integrated gold chip.

The Surface Plasmon resonance (SPR) based label-free detection of small targeted molecules is a great challenge and require substantial signal amplification for the accurate and precise quantification. The incorporation of noble metal nanoparticles (NPs) like gold (Au) NPs for the fabrication of SPR biosensor has shown remarkable impact both for anchoring the signal amplification and generate plasmonic resonant coupling between NPs and chip surface. In this work, we present comparative studies related to the fabrication of self-assembled monolayer (SAM) and the influence of AuNPs on Au chip for Aflatoxin B1 (AFB1) detection using SPRi apparatus. The SAM Au chip was sequentially modified by EDC-NHS crosslinkers, grafting of protein-A and finally interaction with anti-AFB1 antibodies. Similar multilayer chip surface was prepared using functionalized lipoic acid AuNPs deposited on SAM Au chips followed by in situ activation of functional groups using EDC-NHS crosslinkers, grafting of protein-A and immobilization of anti-AFB1 antibodies. This multilayer functionalized AuNPs modified Au chip was successfully utilized for AFB1 detection ranging from 0.01 to 50 nM with a limit of detection of 0.003 nM. When compared to bare self-assembled Au chip which was shown to exhibit a limit of detection of 0.19 nM and a linear detection ranging from 1 to 50 nM, the AuNPs modified Au chip was proven to clearly be a better analytical tool. Finally, validation of the proposed biosensor was evaluated by spiked wheat samples and average recoveries (93 and 90.1%) were found to be acceptable.

SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring.

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (<3%) and a greater analytical range (0.03-20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID's one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

Bioinspired Multi-Activities 4D Printing Objects: A New Approach Toward Complex Tissue Engineering.

4D printing is an innovative approach which might in a near future lead to the achievement of highly complex smart materials. The authors describe a new strategy for the achievement of 4D printed objects with multiple biological activities. These activities are generated through the entrapment, during 3D printing, of two distinct enzymes (alkaline phosphatase and thrombin). These two enzymes give then the ability to the 4D printed object to generate bioactivities useful for in vitro tissue engineering. Indeed, it is shown that the entrapped alkaline phosphatase enables the localized and pre-programmed calcification of some 3D object parts while the diffusion of thrombin from the object permits the formation of fibrin biofilm (including living cells) directly at the surface of 3D object. Both activities and enzyme behavior within the 4D printed hydrogel are characterized through enzymatic measurements, microscopy, magnetic resonance imaging (MRI), and cell seeding.

Massively parallel and multiplex blood group genotyping using next-generation-sequencing.

OBJECTIVES: Thirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run. DESIGN AND METHOD: After an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina). RESULTS: In a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%. CONCLUSION: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure.

Human Skin 3D Bioprinting Using Scaffold-Free Approach.

Organ in vitro synthesis is one of the last bottlenecks between tissue engineering and transplantation of synthetic organs. Bioprinting has proven its capacity to produce 3D objects composed of living cells but highly organized tissues such as full thickness skin (dermis + epidermis) are rarely attained. The focus of the present study is to demonstrate the capability of a newly developed ink formulation and the use of an open source printer, for the production of a really complete skin model. Proofs are given through immunostaining and electronic microscopy that the bioprinted skin presents all characteristics of human skin, both at the molecular and macromolecular level. Finally, the printability of large skin objects is demonstrated with the printing of an adult-size ear.

Multiplex cell microarrays for high-throughput screening.

Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cell microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. It is usual to functionalize the surface of a substrate to design cell microarrays. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA spotted on the surface of a substrate is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technology, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices or printed directly on bio-ink material. We are providing herein an overview of the latest developments in optical cell microarrays allowing high-throughput and high-content analysis.

Adding Biomolecular Recognition Capability to 3D Printed Objects.

Three-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems.

Cancer-Cells on Chip for Label-Free Detection of Secreted Molecules.

In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24 h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and ß-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip.

Paper electrodes for bioelectrochemistry: Biosensors and biofuel cells.

Paper-based analytical devices (PAD) emerge in the scientific community since 2007 as low-cost, wearable and disposable devices for point-of-care diagnostic due to the widespread availability, long-time knowledge and easy manufacturing of cellulose. Rapidly, electrodes were introduced in PAD for electrochemical measurements. Together with biological components, a new generation of electrochemical biosensors was born. This review aims to take an inventory of existing electrochemical paper-based biosensors and biofuel cells and to identify, at the light of newly acquired data, suitable methodologies and crucial parameters in this field. Paper selection, electrode material, hydrophobization of cellulose, dedicated electrochemical devices and electrode configuration in biosensors and biofuel cells will be discussed.

Cells on chip for multiplex screening.

Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cells microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA, spotted on the surface of a substrate, is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technologies, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices, or printed directly in bio-ink material. We are providing herein an overview of the last developments in optical cell microarrays allowing high-throughput and high-content analysis.

Development and Validation of a Fully Automated Platform for Extended Blood Group Genotyping.

Thirty-five blood group systems, containing >300 antigens, are listed by the International Society of Blood Transfusion. Most of these antigens result from a single nucleotide polymorphism. Blood group typing is conventionally performed by serology. However, this technique has some limitations and cannot respond to the growing demand of blood products typed for a large number of antigens. The knowledge of the molecular basis of these red blood cell systems allowed the implementation of molecular biology methods in immunohematology laboratories. Here, we describe a blood group genotyping assay based on the use of TKL immobilization support and microarray-based HIFI technology that takes approximately 4 hours and 30 minutes from whole-blood samples to results analysis. Targets amplified by multiplex PCR were hybridized on the chip, and a revelation step allowed the simultaneous identification of up to 24 blood group antigens, leading to the determination of extended genotypes. Two panels of multiplex PCR were developed: Panel 1 (KEL1/2, KEL3/4; JK1/2; FY1/2; MNS1/2, MNS3/4, FY*Fy et FY*X) and Panel 2 (YT1/2; CO1/2; DO1/2, HY+, Jo(a+); LU1/2; DI1/2). We present the results of the evaluation of our platform on a panel of 583 and 190 blood donor samples for Panel 1 and 2, respectively. Good correlations (99% to 100%) with reference were obtained.

Microarrays in blood group genotyping.

Thirty-five blood group systems, containing more than 300 antigens, are listed by the International Society of Blood Transfusion (ISBT). Most of these antigens result from a single-nucleotide polymorphism (SNP). Blood group typing is conventionally carried out by serology. However, this technique has certain limitations and cannot respond to the growing demand for blood products typed for a large number of antigens. Here we describe a blood group genotyping assay, from genomic DNA extraction from whole-blood samples to results. After DNA extraction, the on-chip test is based on the hybridization of targets beforehand amplified by multiplex polymerase chain reaction, followed by a revelation step allowing the simultaneous identification of up to 24 blood group antigens and leading to the determination of extended genotypes.

Magnetically-assisted impedimetric detection of bacteria using phage-modified carbon microarrays.

This study presents an investigation on the possibility of improving the detection limit of bacteria with an inexpensive electrochemical, impedimetric sensor platform, by integrating the sensor with magnetic manipulation. The approach uses T4 bacteriophage coated Dynabeads to selectively capture and concentrate E. coli K12 cells from samples, to increase the sensitivity of detection at the surface of functionalized screen-printed carbon microarrays. Fluorescence and flow cytometry measurements indicate that the surface modification of the magnetic beads, with phages, and binding with the bacteria, were successful. Integration of the screen-printed carbon-based impedimetric sensor, with a magnetic manipulation system, was found to improve the sensitivity of the device, decreasing the limit of detection of E. coli K12 from 10(4) to 10(3) cfu/mL. We have also demonstrated that this approach provides for more specific detection of bacteria, enabling the operator to account for non-specific adsorption, and detection of bacteria in more complex (real) samples (milk).