Foraging for the Optimal Dressing Scaffold to Carry Adipose-Derived Stromal/Progenitor Cells for Cell Therapy.

In our daily plastic surgery practice, we have seen many chronic wounds that need new biotechnology to help and improve wound healing. Stem cells play a crucial role in regenerative medicine. Many pre-clinical researches had reported the beneficial paracrine effects of stem cell therapy for chronic wounds. Cell-friendly scaffolds may provide the protection and three-dimensional space required for adherence of stem cells, thus allowing these stem cells to proliferate and differentiate for treatment purpose. A successful scaffold may enhance the effects of stem cell therapy. In this presented series, the authors attempted to identify the most suitable scaffolds from several commercially available wound dressings that could sustain adipose-derived stromal/progenitor cells (ADSCs) survival. Therefore, we isolated ADSCs containing the green fluorescent protein (GFP) from GFP transgenic rats. The GFP (+) ADSCs and their progenies could be easily observed using a fluorescence microscope. Moreover, we analyzed the cytokines secreted in condition medium (CM) to understand the activities of ADSCs in various dressings. Our results showed that the foam dressings, hydrofiber, chitosan, and alginate plus carboxymethylcellulose were identified as the most suitable dressing materials. Higher concentrations of transforming growth factor beta (TGF-ß) and vascular endothelial growth factor (VEGF) were observed 48 h after loading them with GFP (+) ADSCs. Therefore, multiple topical cell therapy using ADSCs can be performed by applying suitable dressing scaffolds without repeated needle injections to deliver the stem cells into the wound bed. Based on their fluorescence property, the GFP (+) ADSCs can also possibly be used for testing biocompatibility of medical materials in the future.

Reconstruction of the orbital floor using supercritical CO2 decellularized porcine bone graft.

Orbital floor fractures subsequently lead to consequences such as diplopia and enophthalmos. The graft materials used in orbital floor fractures varied from autografts to alloplastic grafts, which possess certain limitations. In the present study, a novel porcine bone matrix decellularized by supercritical CO2 (scCO2), ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework. The study was approved by the institutional review board (IRB) of Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUH). Ten cases underwent orbital floor reconstruction in KMUH in 2019. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. Nine out of ten cases used 1 piece of customized ABCcolla® Collagen Bone Graft in each defect. The other case used 2 pieces of customized ABCcolla® Collagen Bone Graft in one defect area due to the curved outline of the defect. In the outpatient clinic, all 10 cases showed improvement of enophthalmos on CT (computerized tomography) at week 8 follow-up. No replacement of implants was needed during follow-ups. To conclude, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate and low complication rate.

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro.

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.

A Novel Non-contact Self-Injection-Locked Radar for Vital Sign Sensing and Body Movement Monitoring in COVID-19 Isolation Ward.

BACKGROUND: The outbreak of Coronavirus disease (COVID-19) pandemic has become the most serious global health issue. Isolation policy in hospitals is one of the most crucial protocols to prevent nosocomial infection of COVID-19. It is important to monitor and assess the physical conditions of the patients in isolation. METHODS: Our institution has installed the novel non-contact wireless sensor for vital sign sensing and body movement monitoring for patients in COVID-19 isolation ward. RESULTS: We have collected and compared data between the radar record with the nurse's handover record of two patients, one recorded for 13 days and the other recorded for 5 days. The P value by Fisher's exact test were 0.139 (temperature, P > 0.05) and 0.292 (heart beat rate, P > 0.05) respectively. CONCLUSIONS: This is the first report about the application experience of this equipment. Therefore we attempted to share the experience and try to apply this equipment in COVID-19 patients in future to offer the more reliable and safe policy.

Correction to: A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

In the online version of the article [ 1 ], Figure S1 was mistakenly replaced with Figure 1.

A genetically encoded near-infrared fluorescent calcium ion indicator.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.

A Green BODIPY-Based, Super-Fluorogenic, Protein-Specific Labelling Agent.

We report the development of YC23, a novel green BODIPY-based dimaleimide derivative that undergoes a fluorogenic addition reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578.

BACKGROUND: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. RESULTS: Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. CONCLUSION: K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity.

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.

Ring Substituent Effects on the Thiol Addition and Hydrolysis Reactions of N-Arylmaleimides.

Maleimide groups are used extensively in bioconjugation reactions, but limited kinetic information is available regarding their thiol addition and hydrolysis reactions. We prepared a series of fluorogenic coumarin maleimide derivatives that differ by the substituent on their maleimide C═C bond. Fluorescence-based kinetic studies of the reaction with ß-mercaptoethanol (BME) yielded the second-order rate constants (k2), while pH-rate studies from pH 7 to 9 gave base-catalyzed hydrolysis rate constants (kOH). Linear free-energy relationships were studied through the correlation of log k2 and log kOH to both electronic (σ(+)) and steric (Es(norm)) parameters of the C═C substituent. These correlations revealed the thiol addition reaction is primarily sensitive to the electronic effects, while steric effects dominate the hydrolysis reaction. These mechanistic studies provide the basis for the design of novel bioconjugation reactants or fluorogenic labeling agents.

Coumarin-based fluorogenic probes for no-wash protein labeling.

A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.

Antihelminthic niclosamide modulates dendritic cells activation and function.

Dendritic cells (DCs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. Accordingly, DCs are considered to be a major target in the development of immunomodulating compounds. In this study, the effect of niclosamide, a Food and Drug Administration-approved antihelminthic drug, on the activation of lipopolysaccharide (LPS)-stimulated murine bone marrow-derived DCs was examined. Our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of LPS-activated DCs. In addition, niclosamide also affected the expression of MHC and costimulatory molecules and influenced the ability of the cells to take up antigens. Therefore, in mixed cell cultures composed of syngeneic OVA-specific T cells and DCs, niclosamide-treated DCs showed a decreased ability to stimulate T cell proliferation and IFN-γ production. Furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (CHS) in mice during sensitization with 2,4-dinitro-1-fluorobenzene. Blocking the LPS-induced activation of MAPK-ERK, JNK and NF-κB may contribute to the inhibitory effect of niclosamide on DC activation. Collectively, our findings suggest that niclosamide can manipulate the function of DCs. These results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or DC-mediated autoimmune diseases.