Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

Coronaviruses (CoVs) are enveloped single-stranded RNA viruses that predominantly attack the human respiratory system. In recent decades, several deadly human CoVs, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have brought great impact on public health and economics. However, their high infectivity and the demand for high biosafety level facilities restrict the pathogenesis research of CoV infection. Exacerbated inflammatory cell infiltration is associated with poor prognosis in CoV-associated diseases. In this study, we used human CoV 229E (HCoV-229E), a CoV associated with relatively fewer biohazards, to investigate the pathogenesis of CoV infection and the regulation of neutrophil functions by CoV-infected lung cells. Induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells (iAECIIs) exhibiting specific biomarkers and phenotypes were employed as an experimental model for CoV infection. After infection, the detection of dsRNA, S, and N proteins validated the infection of iAECIIs with HCoV-229E. The culture medium conditioned by the infected iAECIIs promoted the migration of neutrophils as well as their adhesion to the infected iAECIIs. Cytokine array revealed the elevated secretion of cytokines associated with chemotaxis and adhesion into the conditioned media from the infected iAECIIs. The importance of IL-8 secretion and ICAM-1 expression for neutrophil migration and adhesion, respectively, was demonstrated by using neutralizing antibodies. Moreover, next-generation sequencing analysis of the transcriptome revealed the upregulation of genes associated with cytokine signaling. To summarize, we established an in vitro model of CoV infection that can be applied for the study of the immune system perturbations during severe coronaviral disease.

Detection of Interleukin-1 ß (IL-1ß) in Single Human Blastocyst-Conditioned Medium Using Ultrasensitive Bead-Based Digital Microfluidic Chip and Its Relationship with Embryonic Implantation Potential.

The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of cytokines produced by a single embryo in successful implantation is largely unknown. This study aimed to investigate the role of IL-1ß expressed in a single-embryo-conditioned medium (ECM) in embryo implantation. Seventy samples of single ECM were analyzed by a specially designed magnetic-beads-based microfluidic chip from 15 women. We discovered that IL-1ß level increased as the embryo developed, and the difference was significant. In addition, receiver operator characteristic (ROC) curves analysis showed a higher chance of pregnancy when the IL-1ß level on day 5 ECM was below 79.37 pg/mL and the difference between day 5 and day 3 was below 24.90 pg/mL. Our study discovered a possible association between embryonic proteomic expression and successful implantation, which might facilitate single-embryo transfer in the future by helping clinicians identify the embryo with the greatest implantation potential.

Dysregulation of the circRNA_0087207/miR-548c-3p/PLSR1-TGFB2 axis in Leber hereditary optic neuropathy in vitro.

BACKGROUND: Leber hereditary optic neuropathy (LHON) is mainly the degeneration of retinal ganglion cells (RGCs) associated with high apoptosis and reactive oxygen species (ROS) levels, which is accepted to be caused by the mutations in the subunits of complex I of the mitochondrial electron transport chain. The treatment is still infant while efforts of correcting genes or using antioxidants do not bring good and consistent results. Unaffected carrier carries LHON mutation but shows normal phenotype, suggesting that the disease's pathogenesis is complex, in which secondary factors exist and cooperate with the primary complex I dysfunction. METHODS: Using LHON patient-specific induced pluripotent stem cells (iPSCs) as the in vitro disease model, we previously demonstrated that circRNA_0087207 had the most significantly higher expression level in the LHON patient-iPSC-derived RGCs compared with the unaffected carrier-iPSC-derived RGCs. To elaborate the underlying pathologies regulated by circRNA_008720 mechanistically, bioinformatics analysis was conducted and elucidated that circRNA_0087207 could act as a sponge of miR-548c-3p and modulate PLSCR1/TGFB2 levels in ND4 mutation-carrying LHON patient-iPSC-derived RGCs. RESULTS: Using LHON iPSC-derived RGCs as the disease-based platform, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on targeted mRNA of miR-548c-3p showed the connection with apoptosis, suggesting downregulation of miR548c-3p contributes to the apoptosis of LHON patient RGCs. CONCLUSION: We showed that the downregulation of miR548c-3p plays a critical role in modulating cellular dysfunction and the apoptotic program of RGCs in LHON.

Overcoming the challenges of scalable iPSC generation in translation medicine.

BACKGROUND: The potential of induced pluripotent stem cells (iPSCs) in revolutionizing regenerative medicine cannot be overstated. iPSCs offer a profound opportunity for therapies involving cell replacement, disease modeling, and cell transplantation. However, the widespread application of iPSC cellular therapy faces hurdles, including the imperative to regulate iPSC differentiation rigorously and the inherent genetic disparities among individuals. To address these challenges, the concept of iPSC super donors emerges, holding exceptional genetic attributes and advantageous traits. These super donors serve as a wellspring of standardized, high-quality cell sources, mitigating inter-individual variations and augmenting the efficacy of therapy. METHODS: In pursuit of this goal, our study embarked on the establishment of iPSC cell lines specifically sourced from donors possessing the HLA type (A33:03-B58:01-DRB1*03:01). The reprogramming process was meticulously executed, resulting in the successful generation of iPSC lines from these carefully selected donors. Subsequently, an extensive characterization was conducted to comprehensively understand the features and attributes of these iPSC lines. RESULTS: The outcomes of our research were highly promising. The reprogramming efforts culminated in the generation of iPSC lines from donors with the specified HLA type. These iPSC lines displayed a range of distinctive characteristics that were thoroughly examined and documented. This successful generation of iPSC lines from super donors possessing advantageous genetic traits represents a significant stride towards the realization of their potential in therapeutic applications. CONCLUSION: In summary, our study marks a crucial milestone in the realm of regenerative medicine. The establishment of iPSC lines from super donors with specific HLA types signifies a paradigm shift in addressing challenges related to iPSC cellular therapy. The standardized and high-quality cell sources derived from these super donors hold immense potential for various therapeutic applications. As we move forward, these findings provide a solid foundation for further research and development, ultimately propelling the field of regenerative medicine toward new horizons of efficacy and accessibility.

Modifications of lipid pathways restrict SARS-CoV-2 propagation in human induced pluripotent stem cell-derived 3D airway organoids.

BACKGROUND: Modifications of lipid metabolism were closely associated with the manifestations and prognosis of coronavirus disease of 2019 (COVID-19). Pre-existing metabolic conditions exacerbated the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection while modulations of aberrant lipid metabolisms alleviated the manifestations. To elucidate the underlying mechanisms, an experimental platform that reproduces human respiratory physiology is required. METHODS: Here we generated induced pluripotent stem cell-derived airway organoids (iPSC-AOs) that resemble the human native airway. Single-cell sequencing (ScRNAseq) and microscopic examination verified the cellular heterogeneity and microstructures of iPSC-AOs, respectively. We subjected iPSC-AOs to SARS-CoV-2 infection and investigated the treatment effect of lipid modifiers statin drugs on viral pathogenesis, gene expression, and the intracellular trafficking of the SARS-CoV-2 entry receptor angiotensin-converting enzyme-2 (ACE-2). RESULTS: In SARS-CoV-2-infected iPSC-AOs, immunofluorescence staining detected the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins and bioinformatics analysis further showed the aberrant enrichment of lipid-associated pathways. In addition, SARS-CoV-2 hijacked the host RNA replication machinery and generated the new isoforms of a high-density lipoprotein constituent apolipoprotein A1 (APOA1) and the virus-scavenging protein deleted in malignant brain tumors 1 (DMBT1). Manipulating lipid homeostasis using cholesterol-lowering drugs (e.g. Statins) relocated the viral entry receptor angiotensin-converting enzyme-2 (ACE-2) and decreased N protein expression, leading to the reduction of SARS-CoV-2 entry and replication. The same lipid modifications suppressed the entry of luciferase-expressing SARS-CoV-2 pseudoviruses containing the S proteins derived from different SARS-CoV-2 variants, i.e. wild-type, alpha, delta, and omicron. CONCLUSIONS: Together, our data demonstrated that modifications of lipid pathways restrict SARS-CoV-2 propagation in the iPSC-AOs, which the inhibition is speculated through the translocation of ACE2 from the cell membrane to the cytosol. Considering the highly frequent mutation and generation of SARS-CoV-2 variants, targeting host metabolisms of cholesterol or other lipids may represent an alternative approach against SARS-CoV-2 infection.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

Nature-Inspired Surface Structures Design for Antimicrobial Applications.

Surface contamination by microorganisms such as viruses and bacteria may simultaneously aggravate the biofouling of surfaces and infection of wounds and promote cross-species transmission and the rapid evolution of microbes in emerging diseases. In addition, natural surface structures with unique anti-biofouling properties may be used as guide templates for the development of functional antimicrobial surfaces. Further, these structure-related antimicrobial surfaces can be categorized into microbicidal and anti-biofouling surfaces. This review introduces the recent advances in the development of microbicidal and anti-biofouling surfaces inspired by natural structures and discusses the related antimicrobial mechanisms, surface topography design, material application, manufacturing techniques, and antimicrobial efficiencies.

Nanoparticles-mediated CRISPR-Cas9 gene therapy in inherited retinal diseases: applications, challenges, and emerging opportunities.

Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.

Superrepellent Doubly Reentrant Geometry Promotes Antibiofouling and Prevention of Coronavirus Contamination.

The fomite transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has drawn attention because of its highly contagious nature. Therefore, surfaces that can prevent coronavirus contamination are an urgent and unmet need during the coronavirus disease 2019 (COVID-19) pandemic. Conventional surfaces are usually based on superhydrophobic or antiviral coatings. However, these coatings may be dysfunctional because of biofouling, which is the undesired adhesion of biomolecules. A superhydrophobic surface independent of the material content and coating agents may serve the purpose of antibiofouling and preventing viral transmission. Doubly reentrant topology (DRT) is a unique structure that can meet the need. This study demonstrates that the DRT surfaces possess a striking antibiofouling effect that can prevent viral contamination. This effect still exists even if the DRT surface is made of a hydrophilic material such as silicon oxide and copper. To the best of our knowledge, this work first demonstrates that fomite transmission of viruses may be prevented by minimizing the contact area between pathogens and surfaces even made of hydrophilic materials. Furthermore, the DRT geometry per se features excellent antibiofouling ability, which may shed light on the applications of pathogen elimination in alleviating the COVID-19 pandemic.

Single-type reporter multiplexing with A single droplet through bead-based digital microfluidics.

With the limited sample volume, the droplet-based microfluidic becomes attractive in biomedical diagnosis, especially for measuring multiple analytes. Usually, for multiplexing by parallel processing, a larger sample volume is required. In our previous study, simultaneously detecting two analytes from a single droplet was first achieved by measuring different fluorescence wavelengths for different analytes. However, the number of detectable analytes could be limited by the spectral resolution of fluorescence. Here a different approach is proposed for multiplexing by sharing a single droplet in multiple sub-assays. Therefore, only a single-type reporter, i.e., the fluorescence with the same wavelength, is needed for detection of different analytes from a single sample droplet, called single-type reporter multiplexing (STRM). The standard curves of two analytes, human IL-1ß and human TNF-α, are demonstrated. The required sample volume for one measurement is only 520 nL; the total duration of the on-chip process is less than 50 min. The limits of detection (LOD) of human IL-1ß and human TNF-α are about 1.14 and 0.97 pg/mL, respectively. It is shown that the proposed bead-based digital microfluidic immunoassay can achieve multiple analytes detection with low LOD from a single sample droplet using the single-type reporter, which has never been achieved before.

Bead Number Effect in a Magnetic-Beads-Based Digital Microfluidic Immunoassay.

In a biomedical diagnosis with a limited sample volume and low concentration, droplet-based microfluidics, also called digital microfluidics, becomes a very attractive approach. Previously, our group developed a magnetic-beads-based digital microfluidic immunoassay with a bead number of around 100, requiring less than 1 µL of sample volume to achieve a pg/mL level limit of detection (LOD). However, the bead number in each measurement was not the same, causing an unstable coefficient of variation (CV) in the calibration curve. Here, we investigated whether a fixed number of beads in this bead-based digital microfluidic immunoassay could provide more stable results. First, the bead screening chips were developed to extract exactly 100, 49, and 25 magnetic beads with diameters of less than 6 µm. Then, four calibration curves were established. One calibration curve was constructed by using varying bead numbers (50-160) in the process. The other three calibration curves used a fixed number of beads, (100, 49, and 25). The results indicated that the CVs for a fixed number of beads were evidently smaller than the CVs for varying bead numbers, especially in the range of 1 pg/mL to 100 pg/mL, where the CVs for 100 beads were less than 10%. Furthermore, the calculated LOD, based on the composite calibration curves, could be reduced by three orders, from 3.0 pg/mL (for the unfixed bead number) to 0.0287 pg/mL (for 100 beads). However, when the bead numbers were too high (more than 500) or too low (25 or fewer), the bead manipulation for aggregation became more difficult in the magnetic-beads-based digital microfluidic immunoassay chip.

Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip.

The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1ß and human TNF-α, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520 nL; the total duration of the on-chip process is less than 40 min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-α change little from day 3 to day 5-6, but the concentrations of IL-1ß for some embryos might double from day 3 to day 5-6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1ß, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.

Flexible, multifunctional neural probe with liquid metal enabled, ultra-large tunable stiffness for deep-brain chemical sensing and agent delivery.

Flexible neural probes have been pursued previously to minimize the mechanical mismatch between soft neural tissues and implants and thereby improve long-term performance. However, difficulties with insertion of such probes deep into the brain severely restricts their utility. We describe a solution to this problem using gallium (Ga) in probe construction, taking advantage of the solid-to-liquid phase change of the metal at body temperature and probe shape deformation to provide temperature-dependent control of stiffness over 5 orders of magnitude. Probes in the stiff state were successfully inserted 2 cm-deep into agarose gel "brain phantoms" and into rat brains under cooled conditions where, upon Ga melting, they became ultra soft, flexible, and stretchable in all directions. The current 30 µm-thick probes incorporated multilayer, deformable microfluidic channels for chemical agent delivery, electrical interconnects through Ga wires, and high-performance electrochemical glutamate sensing. These PDMS-based microprobes of ultra-large tunable stiffness (ULTS) should serve as an attractive platform for multifunctional chronic neural implants.

Novel Passive Two-Stage Magnetic Targeting Devices for Distal Locking of Interlocking Nails.

Interlocking nailing is a common surgical operation to stabilize fractures in long bones. One of the difficult parts of the surgery is how to locate the position and direction of a screw hole on the interlocking nail, which is invisible to the naked eye after insertion of the nail into the medullary canal. Here, we propose a novel two-stage targeting process using two passive magnetic devices to locate the position and direction of the screw hole without radiation for the locking screw procedure. This involves a ring-shape positioning magnet inside the nail to generate a magnetic field for targeting. From the accuracy test results of these two-stage targeting devices, the search region can be identified in less than 20 seconds by the 1st-stage targeting device, while the total targeting time to locate the drilling position and direction takes less than 4 minutes, with 100% successful rate in 50 attempts. The drilling test further combines the two-stage targeting process and drilling process on the swine tibia, and it is shown that a 100% successful rate is achieved in all 10 attempts, where the total time needed is less than 5 minutes.

An Implantable Drug-delivery System on a Chip.

An implantable system for drug delivery provides a new strategy for drug therapy, and typically involves a microfluidic chip produced with micro or nano-technology. Implantable systems have the flexibility to conform various schemes of drug release, including zero order, pulsatile, and on demand dosing, as opposed to a standard sustained release profile. Such an implantable system is classified as allowing either controllable or uncontrollable drug release after implantation, so an active or passive delivery system respectively. The performance and related applications of these systems vary. The key points of each technology are highlighted such as performance, working principle, fabrication methods, and dimensional constrains. We here review the implantable drug-delivery system in current research with a focus on application and chip performance, and comparison for passive and active delivery system.