Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration.

Silk fibroin (SF) is a promising biomaterial for tendon repair, but its relatively rigid mechanical properties and low cell affinity have limited its application in regenerative medicine. Meanwhile, gelatin-based polymers have advantages in cell attachment and tissue remodeling but have insufficient mechanical strength to regenerate tough tissue such as tendons. Taking these aspects into account, in this study, gelatin methacryloyl (GelMA) is combined with SF to create a mechanically strong and bioactive nanofibrous scaffold (SG). The mechanical properties of SG nanofibers can be flexibly modulated by varying the ratio of SF and GelMA. Compared to SF nanofibers, mesenchymal stem cells (MSCs) seeded on SG fibers with optimal composition (SG7) exhibit enhanced growth, proliferation, vascular endothelial growth factor production, and tenogenic gene expression behavior. Conditioned media from MSCs cultured on SG7 scaffolds can greatly promote the migration and proliferation of tenocytes. Histological analysis and tenogenesis-related immunofluorescence staining indicate SG7 scaffolds demonstrate enhanced in vivo tendon tissue regeneration compared to other groups. Therefore, rational combinations of SF and GelMA hybrid nanofibers may help to improve therapeutic outcomes and address the challenges of tissue-engineered scaffolds for tendon regeneration.

Test-retest reliability and validity of the Sitting Balance Measure-Korean in individuals with incomplete spinal cord injury.

STUDY DESIGN: Cross-cultural reliability and validity. OBJECTIVES: To develop and validate the Korean version of the Sitting Balance Measure (SBM-K) in Korean persons with incomplete spinal cord injury (ISCI). SETTING: Tertiary care center. METHODS: Twenty-nine persons with ISCI were evaluated using SBM-K, which was validated using the kappa coefficient and intraclass coefficient (ICC). The correlation between SBM-K individual items and total score was analyzed using Spearman's correlation, and the internal consistency of test items was measured using Cronbach's alpha. Additionally, the standard error measurement (SEM) and minimal detectable change (MDC) were measured. For the clinical validity of SBM-K, the correlation of SBM-K with the modified Sitting Balance Scale (mSBS) and the Korean-Spinal Cord Independence Measure-III (KSCIM-III) was determined via Spearman's correlation. Linear regression was performed to determine whether SBM-K could predict KSCIM-III. RESULTS: The weighted kappa score of the SBM-K individual items and ICC of SBM-K total score were 0.76-0.83 (good-very good) and 0.98 (0.95-0.99), respectively. The correlation between the SBM-K total score and individual items was notable (r = 0.78-0.98). Cronbach's alpha, SEM, and MDC of SBM-K were 0.98, 0.59, and 1.64, respectively. The clinical validity of SBM-K correlated with mSBS (r = 0.88) and KSCIM-III (r = 0.65-0.89). SBM-K accounted for 17-72% of the variance in predicting KSCIM-III. CONCLUSIONS: SBM-K showed sufficient test-retest reliability, validity, and marginal measurement errors. SBM-K can serve as an optimal clinical assessment tool for Korean ISCI patients and may provide clinicians with reliable sitting balance assessment in Korean clinical settings.

Bioengineered Multicellular Liver Microtissues for Modeling Advanced Hepatic Fibrosis Driven Through Non-Alcoholic Fatty Liver Disease.

Despite considerable efforts in modeling liver disease in vitro, it remains difficult to recapitulate the pathogenesis of the advanced phases of non-alcoholic fatty liver disease (NAFLD) with inflammation and fibrosis. Here, a liver-on-a-chip platform with bioengineered multicellular liver microtissues is developed, composed of four major types of liver cells (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) to implement a human hepatic fibrosis model driven by NAFLD: i) lipid accumulation in hepatocytes (steatosis), ii) neovascularization by endothelial cells, iii) inflammation by activated Kupffer cells (steatohepatitis), and iv) extracellular matrix deposition by activated stellate cells (fibrosis). In this model, the presence of stellate cells in the liver-on-a-chip model with fat supplementation showed elevated inflammatory responses and fibrosis marker up-regulation. Compared to transforming growth factor-beta-induced hepatic fibrosis models, this model includes the native pathological and chronological steps of NAFLD which shows i) higher fibrotic phenotypes, ii) increased expression of fibrosis markers, and iii) efficient drug transport and metabolism. Taken together, the proposed platform will enable a better understanding of the mechanisms underlying fibrosis progression in NAFLD as well as the identification of new drugs for the different stages of NAFLD.

Cancer-on-a-Chip for Modeling Immune Checkpoint Inhibitor and Tumor Interactions.

Cancer immunotherapies, including immune checkpoint inhibitor (ICI)-based therapies, have revolutionized cancer treatment. However, patient response to ICIs is highly variable, necessitating the development of methods to quickly assess efficacy. In this study, an array of miniaturized bioreactors has been developed to model tumor-immune interactions. This immunotherapeutic high-throughput observation chamber (iHOC) is designed to test the effect of anti-PD-1 antibodies on cancer spheroid (MDA-MB-231, PD-L1+) and T cell (Jurkat) interactions. This system facilitates facile monitoring of T cell inhibition and reactivation using metrics such as tumor infiltration and interleukin-2 (IL-2) secretion. Status of the tumor-immune interactions can be easily captured within the iHOC by measuring IL-2 concentration using a micropillar array where sensitive, quantitative detection is allowed after antibody coating on the surface of array. The iHOC is a platform that can be used to model and monitor cancer-immune interactions in response to immunotherapy in a high-throughput manner.

Micro and Nanoscale Technologies for Diagnosis of Viral Infections.

Viral infection is one of the leading causes of mortality worldwide. The growth of globalization significantly increases the risk of virus spreading, making it a global threat to future public health. In particular, the ongoing coronavirus disease 2019 (COVID-19) pandemic outbreak emphasizes the importance of devices and methods for rapid, sensitive, and cost-effective diagnosis of viral infections in the early stages by which their quick and global spread can be controlled. Micro and nanoscale technologies have attracted tremendous attention in recent years for a variety of medical and biological applications, especially in developing diagnostic platforms for rapid and accurate detection of viral diseases. This review addresses advances of microneedles, microchip-based integrated platforms, and nano- and microparticles for sampling, sample processing, enrichment, amplification, and detection of viral particles and antigens related to the diagnosis of viral diseases. Additionally, methods for the fabrication of microchip-based devices and commercially used devices are described. Finally, challenges and prospects on the development of micro and nanotechnologies for the early diagnosis of viral diseases are highlighted.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

Gelatin methacryloyl-based tactile sensors for medical wearables.

Gelatin methacryloyl (GelMA) is a widely used hydrogel with skin-derived gelatin acting as the main constituent. However, GelMA has not been used in the development of wearable biosensors, which are emerging devices that enable personalized healthcare monitoring. This work highlights the potential of GelMA for wearable biosensing applications by demonstrating a fully solution-processable and transparent capacitive tactile sensor with microstructured GelMA as the core dielectric layer. A robust chemical bonding and a reliable encapsulation approach are introduced to overcome detachment and water-evaporation issues in hydrogel biosensors. The resultant GelMA tactile sensor shows a high-pressure sensitivity of 0.19 kPa-1 and one order of magnitude lower limit of detection (0.1 Pa) compared to previous hydrogel pressure sensors owing to its excellent mechanical and electrical properties (dielectric constant). Furthermore, it shows durability up to 3000 test cycles because of tough chemical bonding, and long-term stability of 3 days due to the inclusion of an encapsulation layer, which prevents water evaporation (80% water content). Successful monitoring of various human physiological and motion signals demonstrates the potential of these GelMA tactile sensors for wearable biosensing applications.

A Patch of Detachable Hybrid Microneedle Depot for Localized Delivery of Mesenchymal Stem Cells in Regeneration Therapy.

Mesenchymal stem cells (MSCs) have been widely used for regenerative therapy. In most current clinical applications, MSCs are delivered by injection but face significant issues with cell viability and penetration into the target tissue due to a limited migration capacity. Some therapies have attempted to improve MSC stability by their encapsulation within biomaterials; however, these treatments still require an enormous number of cells to achieve therapeutic efficacy due to low efficiency. Additionally, while local injection allows for targeted delivery, injections with conventional syringes are highly invasive. Due to the challenges associated with stem cell delivery, a local and minimally invasive approach with high efficiency and improved cell viability is highly desired. In this study, we present a detachable hybrid microneedle depot (d-HMND) for cell delivery. Our system consists of an array of microneedles with an outer poly(lactic-co-glycolic) acid (PLGA) shell and an internal gelatin methacryloyl (GelMA)-MSC mixture (GMM). The GMM was characterized and optimized for cell viability and mechanical strength of the d-HMND required to penetrate mouse skin tissue was also determined. MSC viability and function within the d-HMND was characterized in vitro and the regenerative efficacy of the d-HMND was demonstrated in vivo using a mouse skin wound model.

Mechanical Cues Regulating Proangiogenic Potential of Human Mesenchymal Stem Cells through YAP-Mediated Mechanosensing.

Stem cells secrete trophic factors that induce angiogenesis. These soluble factors are promising candidates for stem cell-based therapies, especially for cardiovascular diseases. Mechanical stimuli and biophysical factors presented in the stem cell microenvironment play important roles in guiding their behaviors. However, the complex interplay and precise role of these cues in directing pro-angiogenic signaling remain unclear. Here, a platform is designed using gelatin methacryloyl hydrogels with tunable rigidity and a dynamic mechanical compression bioreactor to evaluate the influence of matrix rigidity and mechanical stimuli on the secretion of pro-angiogenic factors from human mesenchymal stem cells (hMSCs). Cells cultured in matrices mimicking mechanical elasticity of bone tissues in vivo show elevated secretion of vascular endothelial growth factor (VEGF), one of representative signaling proteins promoting angiogenesis, as well as increased vascularization of human umbilical vein endothelial cells (HUVECs) with a supplement of conditioned media from hMSCs cultured across different conditions. When hMSCs are cultured in matrices stimulated with a range of cyclic compressions, increased VEGF secretion is observed with increasing mechanical strains, which is also in line with the enhanced tubulogenesis of HUVECs. Moreover, it is demonstrated that matrix stiffness and cyclic compression modulate secretion of pro-angiogenic molecules from hMSCs through yes-associated protein activity.

Gelatin Methacryloyl Microneedle Patches for Minimally Invasive Extraction of Skin Interstitial Fluid.

The extraction of interstitial fluid (ISF) from skin using microneedles (MNs) has attracted growing interest in recent years due to its potential for minimally invasive diagnostics and biosensors. ISF collection by absorption into a hydrogel MN patch is a promising way that requires the materials to have outstanding swelling ability. Here, a gelatin methacryloyl (GelMA) patch is developed with an 11 × 11 array of MNs for minimally invasive sampling of ISF. The properties of the patch can be tuned by altering the concentration of the GelMA prepolymer and the crosslinking time; patches are created with swelling ratios between 293% and 423% and compressive moduli between 3.34 MPa and 7.23 MPa. The optimized GelMA MN patch demonstrates efficient extraction of ISF. Furthermore, it efficiently and quantitatively detects glucose and vancomycin in ISF in an in vivo study. This minimally invasive approach of extracting ISF with a GelMA MN patch has the potential to complement blood sampling for the monitoring of target molecules from patients.

Thrombolytic Agents: Nanocarriers in Controlled Release.

Thrombosis is a life-threatening pathological condition in which blood clots form in blood vessels, obstructing or interfering with blood flow. Thrombolytic agents (TAs) are enzymes that can catalyze the conversion of plasminogen to plasmin to dissolve blood clots. The plasmin formed by TAs breaks down fibrin clots into soluble fibrin that finally dissolves thrombi. Several TAs have been developed to treat various thromboembolic diseases, such as pulmonary embolisms, acute myocardial infarction, deep vein thrombosis, and extensive coronary emboli. However, systemic TA administration can trigger non-specific activation that can increase the incidence of bleeding. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses. To overcome these limitations, various types of nanocarriers have been introduced that enhance the pharmacokinetic effects by protecting the TA from the biological environment and targeting the release into coagulation. The nanocarriers show increasing half-life, reducing side effects, and improving overall TA efficacy. In this work, the recent advances in various types of TAs and nanocarriers are thoroughly reviewed. Various types of nanocarriers, including lipid-based, polymer-based, and metal-based nanoparticles are described, for the targeted delivery of TAs. This work also provides insights into issues related to the future of TA development and successful clinical translation.

A Microfabricated Sandwiching Assay for Nanoliter and High-Throughput Biomarker Screening.

Cells secrete substances that are essential to the understanding of numerous immunological phenomena and are extensively used in clinical diagnoses. Countless techniques for screening of biomarker secretion in living cells have generated valuable information on cell function and physiology, but low volume and real-time analysis is a bottleneck for a range of approaches. Here, a simple, highly sensitive assay using a high-throughput micropillar and microwell array chip (MIMIC) platform is presented for monitoring of biomarkers secreted by cancer cells. The sensing element is a micropillar array that uses the enzyme-linked immunosorbent assay (ELISA) mechanism to detect captured biomolecules. When integrated with a microwell array where few cells are localized, interleukin 8 (IL-8) secretion can be monitored with nanoliter volume using multiple micropillar arrays. The trend of cell secretions measured using MIMICs matches the results from conventional ELISA well while it requires orders of magnitude less cells and volumes. Moreover, the proposed MIMIC is examined to be used as a drug screening platform by delivering drugs using micropillar arrays in combination with a microfluidic system and then detecting biomolecules from cells as exposed to drugs.

Interfacial geometry dictates cancer cell tumorigenicity.

Within the heterogeneous architecture of tumour tissue there exists an elusive population of stem-like cells that are implicated in both recurrence and metastasis. Here, by using engineered extracellular matrices, we show that geometric features at the perimeter of tumour tissue will prime a population of cells with a stem-cell-like phenotype. These cells show characteristics of cancer stem cells in vitro, as well as enhanced tumorigenicity in murine models of primary tumour growth and pulmonary metastases. We also show that interfacial geometry modulates cell shape, adhesion through integrin α5ß1, MAPK and STAT activity, and initiation of pluripotency signalling. Our results for several human cancer cell lines suggest that interfacial geometry triggers a general mechanism for the regulation of cancer-cell state. Similar to how a growing tumour can co-opt normal soluble signalling pathways, our findings demonstrate how cancer can also exploit geometry to orchestrate oncogenesis.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Hydrogel design to overcome thermal resistance and ROS detoxification in photothermal and photodynamic therapy of cancer.

Responsive heat resistance (by heat shock protein upregulation) and spontaneous reactive oxygen species (ROS) detoxification have been regarded as the major obstacles for photothermal/photodynamic therapy of cancer. To overcome the thermal resistance and improve ROS susceptibility in breast cancer therapy, Au ion-crosslinked hydrogels including indocyanine green (ICG) and polyphenol are devised. Au ion has been introduced for gel crosslinking (by catechol-Au3+ coordination), cellular glutathione depletion, and O2 production from cellular H2O2. ICG can generate singlet oxygen from O2 (for photodynamic therapy) and induce hyperthermia (for photothermal therapy) under the near-infrared laser exposure. (-)-Epigallocatechin gallate downregulates heat shock protein to overcome heat resistance during hyperthermia and exerts multiple anticancer functions in spite of its ironical antioxidant features. Those molecules are concinnously engaged in the hydrogel structure to offer fast gel transformation, syringe injection, self-restoration, and rheological tuning for augmented photo/chemotherapy of cancer. Intratumoral injection of multifunctional hydrogel efficiently suppressed the growth of primary breast cancer and completely eliminated the residual tumor mass. Proposed hydrogel system can be applied to tumor size reduction prior to surgery of breast cancer and the complete remission after its surgery.

Gelatin methacryloyl granular scaffolds for localized mRNA delivery.

mRNA therapy is the intracellular delivery of messenger RNA (mRNA) to produce desired therapeutic proteins. Developing strategies for local mRNA delivery is still required where direct intra-articular injections are inappropriate for targeting a specific tissue. The mRNA delivery efficiency depends on protecting nucleic acids against nuclease-mediated degradation and safe site-specific intracellular delivery. Herein, we report novel mRNA-releasing matrices based on RGD-moiety-rich gelatin methacryloyl (GelMA) microporous annealed particle (MAP) scaffolds. GelMA concentration in aerogel-based microgels (µgels) produced through a microfluidic process, MAP stiffnesses, and microporosity are crucial parameters for cell adhesion, spreading, and proliferation. After being loaded with mRNA complexes, MAP scaffolds composed of 10 % GelMA µgels display excellent cell viability with increasing cell infiltration, adhesion, proliferation, and gene transfer. The intracellular delivery is achieved by the sustained release of mRNA complexes from MAP scaffolds and cell adhesion on mRNA-releasing scaffolds. These findings highlight that hybrid systems can achieve efficient protein expression by delivering mRNA complexes, making them promising mRNA-releasing biomaterials for tissue engineering.

Injectable Nanoengineered Adhesive Hydrogel for Treating Enterocutaneous Fistulas.

Enterocutaneous fistula (ECF) is a severe medical condition where an abnormal connection forms between the gastrointestinal tract and skin. ECFs are, in most cases, a result of surgical complications such as missed enterotomies or anastomotic leaks. The constant leakage of enteric and fecal contents from the fistula site leads to skin breakdown and increases the risk of infection. Despite advances in surgical techniques and postoperative management, ECF accounts for significant mortality rates, estimated between 15-20%, and causes debilitating morbidity. Therefore, there is a critical need for a simple and effective method to seal and heal ECF. Injectable hydrogels with combined properties of robust mechanical properties and cell infiltration/proliferation have the potential to block and heal ECF. Herein, we report the development of an injectable nanoengineered adhesive hydrogel (INAH) composed of a synthetic nanosilicate (Laponite®) and a gelatin-dopamine conjugate for treating ECF. The hydrogel undergoes fast cross-linking using a co-injection method, resulting in a matrix with improved mechanical and adhesive properties. INAH demonstrates appreciable blood clotting abilities and is cytocompatible with fibroblasts. The adhesive properties of the hydrogel are demonstrated in ex vivo adhesion models with skin and arteries, where the volume stability in the hydrated internal environment facilitates maintaining strong adhesion. In vivo assessments reveal that the INAH is biocompatible, supporting cell infiltration and extracellular matrix deposition while not forming fibrotic tissue. These findings suggest that this INAH holds promising translational potential for sealing and healing ECF.

Production of 2'-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.