Cholesterol-Bearing Polysaccharide-Based Nanogels for Development of Novel Immunotherapy and Regenerative Medicine.

Novel functional biomaterials are expected to bring about breakthroughs in developing immunotherapy and regenerative medicine through their application as drug delivery systems and scaffolds. Nanogels are defined as nanoparticles with a particle size of 100 nm or less and as having a gel structure. Nanogels have a three-dimensional network structure of cross-linked polymer chains, which have a high water content, a volume phase transition much faster than that of a macrogel, and a quick response to external stimuli. As it is possible to transmit substances according to the three-dimensional mesh size of the gel, a major feature is that relatively large substances, such as proteins and nucleic acids, can be taken into the gel. Furthermore, by organizing nanogels as a building block, they can be applied as a scaffold material for tissue regeneration. This review provides a brief overview of the current developments in nanogels in general, especially drug delivery, therapeutic applications, and tissue engineering. In particular, polysaccharide-based nanogels are interesting because they have excellent complexation properties and are highly biocompatible.

Ammonia Toxicity and Associated Protein Oxidation: A Single-Cell Surface Enhanced Raman Spectroscopy Study.

Ammonia (NH3) is a commonly used industrial chemical to which exposure at high concentrations can result in severe skin damage. Moreover, high levels of ammonia in the human body can lead to hyperammonemia conditions and enhanced cancer metabolism. In this work, the toxicity mechanism of NH3 has been studied against human dermal fibroblast (HDF) cells using surface-enhanced Raman spectroscopy (SERS). For this purpose, gold nanoparticles of size 50 nm have been prepared and used as probes for Raman signal enhancement, after being internalized inside HDF cells. Following the exposure to ammonia, HDF cells showed a significant variation in the protein ternary structure's signals, demonstrating their denaturation and oxidation process, together with early signs of apoptosis. Meaningful changes were observed especially in the Raman vibrations of sulfur-containing amino acids (cysteine and methionine) together with aromatic residues. Fluorescence microscopy revealed the formation of reactive oxygen and nitrogen species in cells, which confirmed their stressed condition and to whom the causes of protein degradation can be attributed. These findings can provide new insights into the mechanism of ammonia toxicity and protein oxidation at a single-cell level, demonstrating the high potential of the SERS technique in investigating the cellular response to toxic compounds.

A Natural Organic Compound "Decursin" Has Both Antitumor and Renal Protective Effects: Treatment for Osteosarcoma.

Osteosarcoma is a rare malignant tumor that commonly occurs in children. Anticancer drugs, for example, cisplatin, aid in postsurgery recovery but induce side effects such as renal damage, affecting the life prognosis of patients. Decursin which is one of the bioactive components has been reported for its anti-inflammatory, antioxidant, and antitumor effects, but the effect on osteosarcoma is unexplained. In this study, the research theme was to examine the sensitizing effect of decursin and its influence on cisplatin-induced nephrotoxicity. The cell viability and half maximal inhibitory concentration (IC50), apoptosis induction, and effect on cell cycle and Akt pathways were examined. In vivo, we examine the effects of decursin on tumors and mice bodies. Additionally, the effects of the cisplatin-decursin combination were evaluated in vitro and in vivo. Decursin suppressed cell viability and induced apoptosis via the cell cycle. Decursin also inhibited the Akt pathway by suppressing the phosphorylation of Akt. It enhanced apoptosis induction and lowered cell viability in combination with cisplatin. The increasing tumor volume was suppressed in the decursin-administrated group with further suppression in combination with cisplatin compared to sole cisplatin administration. The decrease in renal function and renal epithelial cell damage caused by cisplatin was improved by the combinatorial treatment with decursin. Therefore, decursin demonstrated an antitumor effect on the osteosarcoma cells and a renal protective effect in combination with cisplatin. Therefore, decursin is a prospective therapeutic agent against osteosarcoma.

Raman Multi-Omic Snapshot and Statistical Validation of Structural Differences between Herpes Simplex Type I and Epstein-Barr Viruses.

Raman spectroscopy was applied to study the structural differences between herpes simplex virus Type I (HSV-1) and Epstein-Barr virus (EBV). Raman spectra were first collected with statistical validity on clusters of the respective virions and analyzed according to principal component analysis (PCA). Then, average spectra were computed and a machine-learning approach applied to deconvolute them into sub-band components in order to perform comparative analyses. The Raman results revealed marked structural differences between the two viral strains, which could mainly be traced back to the massive presence of carbohydrates in the glycoproteins of EBV virions. Clear differences could also be recorded for selected tyrosine and tryptophan Raman bands sensitive to pH at the virion/environment interface. According to the observed spectral differences, Raman signatures of known biomolecules were interpreted to link structural differences with the viral functions of the two strains. The present study confirms the unique ability of Raman spectroscopy for answering structural questions at the molecular level in virology and, despite the structural complexity of viral structures, its capacity to readily and reliably differentiate between different virus types and strains.

TGF-ß inhibitor treatment of H2O2-induced cystitis models provides biochemical mechanism for elucidating interstitial cystitis/painful bladder syndrome patients.

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disease for which no effective treatment is available. Transforming growth factor-ß (TGF-ß) is thought to be involved in the pathogenesis of IC/PBS, and previous studies have suggested that administrations of a TGF-ß inhibitor significantly ameliorated IC/PBS in a mouse model. However, the molecular mechanisms underlying the therapeutic effect of a TGF-b inhibitor on IC/PBS has not been comprehensively analyzed. TGF-ß has a variety of actions, such as regulation of immune cells and fibrosis. In our study, we induced IC/PBS-like disease in mice by an intravesical administration of hydrogen peroxide (H2O2) and examined the effects of three TGF-ß inhibitors, Repsox, SB431542, and SB505124, on the urinary functions as well as histological and gene expression profiles in the bladder. TGF-ß inhibitor treatment improved urinary function and histological changes in the IC/PBS mouse model, and SB431542 was most effective among the TGF-ß inhibitors. In our present study, TGF-ß inhibitor treatment improved abnormal enhancement of nociceptive mechanisms, immunity and inflammation, fibrosis, and dysfunction of bladder urothelium. These results show that multiple mechanisms are involved in the improvement of urinary function by TGF-ß inhibitor.

Raman Fingerprints of SARS-CoV-2 Omicron Subvariants: Molecular Roots of Virological Characteristics and Evolutionary Directions.

The latest RNA genomic mutation of SARS-CoV-2 virus, termed the Omicron variant, has generated a stream of highly contagious and antibody-resistant strains, which in turn led to classifying Omicron as a variant of concern. We systematically collected Raman spectra from six Omicron subvariants available in Japan (i.e., BA.1.18, BA.2, BA.4, BA.5, XE, and BA.2.75) and applied machine-learning algorithms to decrypt their structural characteristics at the molecular scale. Unique Raman fingerprints of sulfur-containing amino acid rotamers, RNA purines and pyrimidines, tyrosine phenol ring configurations, and secondary protein structures clearly differentiated the six Omicron subvariants. These spectral characteristics, which were linked to infectiousness, transmissibility, and propensity for immune evasion, revealed evolutionary motifs to be compared with the outputs of genomic studies. The availability of a Raman "metabolomic snapshot", which was then translated into a barcode to enable a prompt subvariant identification, opened the way to rationalize in real-time SARS-CoV-2 activity and variability. As a proof of concept, we applied the Raman barcode procedure to a nasal swab sample retrieved from a SARS-CoV-2 patient and identified its Omicron subvariant by coupling a commercially available magnetic bead technology with our newly developed Raman analyses.

Effects of tea, catechins and catechin derivatives on Omicron subvariants of SARS-CoV-2.

The Omicron subvariants of SARS-CoV-2 have multiple mutations in the S-proteins and show high transmissibility. We previously reported that tea catechin (-)-epigallocatechin gallate (EGCG) and its derivatives including theaflavin-3,3'-di-O-digallate (TFDG) strongly inactivated the conventional SARS-CoV-2 by binding to the receptor binding domain (RBD) of the S-protein. Here we show that Omicron subvariants were effectively inactivated by green tea, Matcha, and black tea. EGCG and TFDG strongly suppressed infectivity of BA.1 and XE subvariants, while effect on BA.2.75 was weaker. Neutralization assay showed that EGCG and TFDG inhibited interaction between BA.1 RBD and ACE2. In silico analyses suggested that N460K, G446S and F490S mutations in RBDs crucially influenced the binding of EGCG/TFDG to the RBDs. Healthy volunteers consumed a candy containing green tea or black tea, and saliva collected from them immediately after the candy consumption significantly decreased BA.1 virus infectivity in vitro. These results indicate specific amino acid substitutions in RBDs that crucially influence the binding of EGCG/TFDG to the RBDs and different susceptibility of each Omicron subvariant to EGCG/TFDG. The study may suggest molecular basis for potential usefulness of these compounds in suppression of mutant viruses that could emerge in the future and cause next pandemic.

Size control of induced pluripotent stem cells colonies in two-dimensional culture for differentiation into functional monocyte-like cells.

BACKGROUND AIMS: Monocytes, derived from hematopoietic stem cells (HSCs), play a pivotal role in the immune response to cancer. Although they are an attractive source of cell therapy for cancer, a method for ex vivo expansion has not yet been established. Monocytes differentiated from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs), can be an alternative source of HSC-derived monocytes because of their self-renewal and pluripotency. To develop a standardized method for the generation of iPSC-derived monocytes for future clinical applications, we aim to control the size of the iPSC colony. METHODS: To this end, we developed a plate with multiple dots containing a chemical substrate for the iPSC scaffold. iPSCs placed in the plate expanded only on the dots and created colonies of the same size. The cells were then differentiated into monocytes by adding cytokines to the colonies. RESULTS: The dot plate substantially reduced variability in monocyte-like cell generation when compared with cultivating cells on a plate with the substrate covering the entire surface area. Furthermore, more monocyte-like cells were obtained by adjusting the dot size and the distance between the dots. The iPSC-derived monocyte-like cells phagocytosed cancer cells and secreted proinflammatory cytokines. The cells also expressed Fc receptors and exerted immunoglobulin G-mediated killing of cancer cells with the corresponding antibodies. CONCLUSIONS: The dot plate enabled the control of iPSC colony size in two-dimensional culture, which resulted in a reduction in the generation-variation of functional monocyte-like cells. This standardized method for generating iPSC-derived monocyte-like cells using the dot plate could also facilitate the development of an automated closed system on a large scale for clinical applications.

In Situ Raman Study of Neurodegenerated Human Neuroblastoma Cells Exposed to Outer-Membrane Vesicles Isolated from Porphyromonas gingivalis.

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer's disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells' Raman profiles were then discussed in view of key factors including the formation of amyloid ß (Aß) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies.

Milk Casein Inhibits Effect of Black Tea Galloylated Theaflavins to Inactivate SARS-CoV-2 In Vitro.

Continuing caution is required against the potential emergence of SARS-CoV-2 novel mutants that could pose the next global health and socioeconomical threats. If virus in saliva can be inactivated by a beverage, such a beverage may be useful because the saliva of infected persons is the major origin of droplets and aerosols that mediate human-to-human viral transmission. We previously reported that SARS-CoV-2 was significantly inactivated by treatment in vitro with tea including green tea and black tea. Catechins and its derived compounds galloylated theaflavins (gTFs) bound to the receptor-binding domain (RBD) of the S-protein and blocked interaction between RBD and ACE2. Black tea is often consumed with sugar, milk, lemon juice, etc., and it remains unclarified whether these ingredients may influence the anti-SARS-CoV-2 effect of black tea. Here, we examined the effect of black tea on Omicron subvariants in the presence of these ingredients. The infectivity of Omicron subvariants was decreased to 1/100 or lower after treatment with black tea for 10 s. One or two teaspoons of milk (4~8 mL) completely blocked the anti-viral effect of a cup of tea (125 mL), whereas an addition of sugar or lemon juice failed to do so. The suppressive effect was dose-dependently exerted by milk casein but not whey proteins. gTFs were coprecipitated with casein after acidification of milk-supplemented black tea, strongly suggesting the binding of gTFs to casein. The present study demonstrates for the first time that an addition of milk cancelled the anti-SARS-CoV-2 effect of black tea due to binding of casein to gTFs.

Instantaneous Inactivation of Herpes Simplex Virus by Silicon Nitride Bioceramics.

Hydrolytic reactions taking place at the surface of a silicon nitride (Si3N4) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si3N4 is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si3N4 showed that ammonia and its nitrogen radical byproducts, produced upon Si3N4 hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO2 particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si3N4's antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si3N4 particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.

Hypoxia with or without Treadmill Exercises Affects Slow-Twitch Muscle Atrophy and Joint Destruction in a Rat Model of Rheumatoid Arthritis.

The effects of treadmill running under hypoxic conditions on joints and muscles of collagen-induced arthritis (CIA) rats were investigated. CIA rats were divided into normoxia no-exercise, hypoxia no-exercise (Hypo-no), and hypoxia exercise (Hypo-ex) groups. Changes were examined on days 2 and 44 of hypoxia with or without treadmill exercises. In the early stage of hypoxia, the expression of hypoxia-inducible factor (HIF)-1α increased in the Hypo-no and Hypo-ex groups. The expression of the egl-9 family hypoxia-inducible factor 1 (EGLN1) and vascular endothelial growth factor (VEGF) in the Hypo-ex group also increased. Under sustained hypoxia, the Hypo-no and Hypo-ex groups did not show increased expression of HIF-1α or VEGF, but p70S6K levels were elevated. Histologically, joint destruction was alleviated in the Hypo-no group, the loss of muscle weight in slow-twitch muscles was prevented, and muscle fibrosis was suppressed. In the Hypo-ex group, the preventive effect of a reduction in the slow-twitch muscle cross-sectional area was enhanced. Thus, chronic hypoxia in an animal model of rheumatoid arthritis controlled arthritis and joint destruction and prevented slow-twitch muscle atrophy and fibrosis. The combination of hypoxia with treadmill running further enhanced the preventive effects on slow-twitch muscle atrophy.

Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.

BACKGROUND/AIM: Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-ß) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. MATERIALS AND METHODS: We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. RESULTS: The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. CONCLUSION: Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.

Anti-Inflammatory Effects of ß-Cryptoxanthin on 5-Fluorouracil-Induced Cytokine Expression in Human Oral Mucosal Keratinocytes.

Oral mucositis is a typical adverse effect of chemotherapy, causing oral pain that significantly reduces the patient's quality of life. ß-cryptoxanthin (ß-cry) is a carotenoid abundant in citrus fruits with antioxidant and anti-inflammatory effects. However, the ß-cry effect on oral mucositis remains unclear. We investigated the effects of 5-fluorouracil (5-FU) and ß-cry on human normal oral mucosal keratinocytes (hOMK). hOMK was seeded on a culture plate and cultured with 5-FU and ß-cry. The cell number, mRNA expression of inflammatory cytokines and matrix metalloproteinases (MMPs), and production of inflammatory cytokines in hOMK were evaluated. Additionally, the cell count and inflammatory cytokine production were analyzed when hOMK was co-stimulated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in addition to 5-FU. The numbers of hOMK significantly reduced with 5-FU stimulation, whereas it increased with ß-cry treatment. mRNA expression of interleukin (IL)-6, IL-8, metalloproteinase (MMP)-2, and MMP-9 and protein production of IL-6 and IL-8 in hOMK were augmented on 5-FU stimulation. Simultaneously, ß-cry treatment significantly suppressed IL-8 and MMP-9 mRNA expression, and IL-8 production was induced on 5-FU stimulation. Co-stimulation with P. gingivalis LPS and 5-FU enhanced IL-6 and IL-8 production in hOMK. ß-cry could enhance cell proliferation and suppress 5-FU-induced expression of inflammatory cytokines and MMP in hOMK. Thus, ß-cry can alleviate the symptoms of chemotherapy-induced oral mucositis, and its combination with oral care is effective in managing oral mucositis.

In Situ Raman Analysis of Biofilm Exopolysaccharides Formed in Streptococcus mutans and Streptococcus sanguinis Commensal Cultures.

This study probed in vitro the mechanisms of competition/coexistence between Streptococcus sanguinis (known for being correlated with health in the oral cavity) and Streptococcus mutans (responsible for aciduric oral environment and formation of caries) by means of quantitative Raman spectroscopy and imaging. In situ Raman assessments of live bacterial culture/coculture focusing on biofilm exopolysaccharides supported the hypothesis that both species engaged in antagonistic interactions. Experiments of simultaneous colonization always resulted in coexistence, but they also revealed fundamental alterations of the biofilm with respect to their water-insoluble glucan structure. Raman spectra (collected at fixed time but different bacterial ratios) showed clear changes in chemical bonds in glucans, which pointed to an action by Streptococcus sanguinis to discontinue the impermeability of the biofilm constructed by Streptococcus mutans. The concurrent effects of glycosidic bond cleavage in water-insoluble α - 1,3-glucan and oxidation at various sites in glucans' molecular chains supported the hypothesis that secretion of oxygen radicals was the main "chemical weapon" used by Streptococcus sanguinis in coculture.

Prophylactic Application of Human Adipose Tissue-Derived Products to Prevent Radiation Disorders.

BACKGROUND: Radiation therapy is a mainstay treatment for malignancies, but it can induce deterministic adverse effects in surrounding healthy tissues, including atrophy, fibrosis, ischemia, and impaired wound healing. This exploratory study investigated whether prophylactic administration of products containing adipose tissue-derived stem cells immediately after radiotherapy could prevent the development of long-term functional disorders in irradiated tissues. METHODS: A total irradiation dose of 40 Gy (10 Gy, four times weekly) was delivered to the dorsal skin of nude mice. Subsequently, a prophylactic treatment with vehicle, fat tissue, stromal vascular fraction, or micronized cellular adipose matrix was injected subcutaneously into the irradiated area. Six months after these prophylactic treatments, a cutaneous punch wound was created to evaluate histologic changes and wound healing. RESULTS: Histologic assessments demonstrated dermal thickening, atrophy, and increased collagen deposits in the subcutaneous fatty layer 6 months after radiotherapy. In addition, wound healing was significantly delayed. The prophylactic treatments with three different types of human adipose tissue-derived products significantly prevented radiation-induced histologic changes and accelerated wound healing compared with the vehicle-treated irradiated group. CONCLUSIONS: This is the first study to demonstrate potential for prophylactic treatments after radiotherapy, which could prevent the progression of chronic radiation therapy disorders. The results could have a substantial impact on current anticancer radiotherapies; a next-generation radiation therapy may need to be always combined with a stem cell therapy. Such prophylactic treatments have the potential to improve wound healing of irradiated tissue and clinical outcomes of reconstructive surgery required after cancer radiotherapy. CLINICAL RELEVANCE STATEMENT: The results of this study suggest that it is clinically possible to prevent the development of radiation disorders using stem cell therapies. This study may provide a new concept of prophylactic treatment, which would be a paradigm shift in radiotherapy.

Micronized Cellular Adipose Matrix Promotes the Therapeutic Effect of an Artificial Nerve Conduit in Peripheral Nerve Gap Injury.

BACKGROUND: Stromal vascular fraction (SVF) isolated from adipose tissue has been shown to be beneficial for treating peripheral nerve injuries. Micronized cellular adipose matrix (MCAM) is an SVF-rich micronized fat tissue obtained by a series of simple mechanical processes. This study assessed the therapeutic effect of MCAM for peripheral nerve injury. METHODS: Microscopic evaluation of the cell phenotype and functions was performed to determine the adipose-derived stem cell content of the MCAM. An artificial nerve conduit (ANC) filled with MCAM was implanted into a sciatic nerve defect in immunodeficient mice. Comparisons of this treatment with an autograft, an ANC filled with SVF cells, and an ANC alone were made based on electrophysiologic characteristics, Sciatic Functional Index, and histologic analyses of regenerated nerve fiber and myelination using electron microscopy, and the preventive effect on innervated muscle atrophy. RESULTS: MCAM contained many cells with a phenotype and differentiation potency similar to those of ADSCs. The implantation experiment indicated that MCAM enhanced the efficiency of functional and structural recovery and prevented atrophy of the innervated muscle. These effects were significantly improved compared with the control group (ANC only) and comparable to those in the SVF group, whereas the improvement did not reach the same level of the autograft group. CONCLUSION: Injection of MCAM into an ANC accelerated nerve regeneration compared with use of an ANC alone, which indicates that MCAM is a promising transplant material for treatment of peripheral nerve injury and an alternative to use of SVF cells. CLINICAL RELEVANCE STATEMENT: Micronized cellular adipose matrix, which can be harvested and isolated from adipose tissue with a simple device, has been shown for the first time to be highly useful as an implantable material for new peripheral nerve regeneration.

Development of minimally invasive cancer immunotherapy using anti-disialoganglioside GD2 antibody-producing mesenchymal stem cells for a neuroblastoma mouse model.

PURPOSE: Mouse IgG anti-disialoganglioside GD2 antibody-secreting mouse mesenchymal stem cells (anti-GD2-MSCs) were developed, and their anti-tumor effects were validated in an in vivo neuroblastoma mouse model. METHODS: Anti-GD2 antibody constructs were generated, incorporating FLAG-tagged single-chain fragment variables against GD2 fused to a linker sequence, and a fragment of a stationary portion was changed from human IgG to mouse IgG and GFP protein. The construct was lentivirally introduced into mouse MSCs. A syngeneic mouse model was established through the subcutaneous transplantation of a tumor tissue fragment from a TH-MYCN transgenic mouse, and the homing effects of anti-GD2-MSCs were validated by In vivo imaging system imaging. The syngeneic model was divided into three groups according to topical injection materials: anti-GD2-MSCs with IL-2, IL-2, and PBS. The tumors were removed, and natural killer (NK) cells were counted. RESULTS: Anti-GD2-MSCs showed homing effects in syngeneic models. The growth rate of subcutaneous tumors was significantly suppressed by anti-GD2-MSCs with IL-2 (p < 0.05). Subcutaneous tumor immunostaining showed an increased NK cell infiltration in the same group (p < 0.01). CONCLUSION: Anti-GD2-MSCs using mouse IgG showed a homing effect and significant tumor growth suppression in syngeneic models. Anti-GD2-MSC-based cellular immunotherapy could be a novel therapeutic strategy for intractable neuroblastoma.

Raman Metabolomics of Candida auris Clades: Profiling and Barcode Identification.

This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls' flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α-1,3-glucan polymorph, the α-1,3-glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated ß-glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments.

Injectable Prevascularized Mature Adipose Tissues (iPAT) to Achieve Long-Term Survival in Soft Tissue Regeneration.

Soft tissue regeneration remains a challenge in reconstructive surgery. So far, both autologous fat implantations and artificial implants methods used in clinical applications lead to various disadvantages and limited lifespan. To overcome these limitations and improve the graft volume maintenance, reproducing a mature adipose tissue already including vasculature structure before implantation can be the solution. Therefore, injectable prevascularized adipose tissues (iPAT) are made from physiological collagen microfibers mixed with human mature adipocytes, adipose-derived stem cells, and human umbilical vein endothelial cells, embedded in fibrin gel. Following murine subcutaneous implantation, the iPAT show a higher cell survival (84% ± 6% viability) and volume maintenance after 3 months (up to twice heavier) when compared to non-prevascularized balls and liposuctioned fat implanted controls. This higher survival can be explained by the greater amount of blood vessels found (up to 1.6-fold increase), with balanced host anastomosis (51% ± 1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. Furthermore, with the cryopreservation possibility enabling their later reinjection, the iPAT technology has the merit to allow noninvasive soft tissue regeneration for long-term outcomes.