Signature construction and molecular subtype identification based on liver-specific genes for prediction of prognosis, immune activity, and anti-cancer drug sensitivity in hepatocellular carcinoma.

BACKGROUND: Liver specific genes (LSGs) are crucial for hepatocyte differentiation and maintaining normal liver function. A deep understanding of LSGs and their heterogeneity in hepatocellular carcinoma (HCC) is necessary to provide clues for HCC diagnosis, prognosis, and treatment. METHODS: The bulk and single-cell RNA-seq data of HCC were downloaded from TCGA, ICGC, and GEO databases. Through unsupervised cluster analysis, LSGs-based HCC subtypes were identified in TCGA-HCC samples. The prognostic effects of the subtypes were investigated with survival analyses. With GSVA and Wilcoxon test, the LSGs score, stemness score, aging score, immune score and stromal score of the samples were estimated and compared. The HCC subtype-specific genes were identified. The subtypes and their differences were validated in ICGC-HCC samples. LASSO regression analysis was used for key gene selection and risk model construction for HCC overall survival. The model performance was estimated and validated. The key genes were validated for their heterogeneities in HCC cell lines with quantitative real-time PCR and at single-cell level. Their dysregulations were investigated at protein level. Their correlations with HCC response to anti-cancer drugs were estimated in HCC cell lines. RESULTS: We identified three LSGs-based HCC subtypes with different prognosis, tumor stemness, and aging level. The C1 subtype with low LSGs score and high immune score presented a poor survival, while the C2 subtype with high LSGs score and immune score indicated an enduring survival. Although no significant survival difference between C2 and C3 HCCs was shown, the C2 HCCs presented higher immune score and stroma score. The HCC subtypes and their differences were confirmed in ICGC-HCC dataset. A five-gene prognostic signature for HCC survival was constructed. Its good performance was shown in both the training and validation datasets. The five genes presented significant heterogeneities in different HCC cell lines and hepatocyte subclusters. Their dysregulations were confirmed at protein level. Furthermore, their significant associations with HCC sensitivities to anti-cancer drugs were shown. CONCLUSIONS: LSGs-based HCC subtype classification and the five-gene risk model might provide useful clues not only for HCC stratification and risk prediction, but also for the development of more personalized therapies for effective HCC treatment.

Antibody and transcription landscape in peripheral blood mononuclear cells of elderly adults over 70 years of age with third dose of COVID-19 BBIBP-CorV and ZF2001 booster vaccine.

BACKGROUND: In the context of the COVID-19 pandemic and extensive vaccination, it is important to explore the immune response of elderly adults to homologous and heterologous booster vaccines of COVID-19. At this point, we detected serum IgG antibodies and PBMC sample transcriptome profiles in 46 participants under 70 years old and 25 participants over 70 years old who received the third dose of the BBIBP-CorV and ZF2001 vaccines. RESULTS: On day 7, the antibody levels of people over 70 years old after the third dose of booster vaccine were lower than those of young people, and the transcriptional responses of innate and adaptive immunity were also weak. The age of the participants showed a significant negative correlation with functions related to T-cell differentiation and costimulation. Nevertheless, 28 days after the third dose, the IgG antibodies of elderly adults reached equivalence to those of younger adults, and immune-related transcriptional regulation was significantly improved. The age showed a significant positive correlation with functions related to "chemokine receptor binding", "chemokine activity", and "chemokine-mediated signaling pathway". CONCLUSIONS: Our results document that the response of elderly adults to the third dose of the vaccine was delayed, but still able to achieve comparable immune effects compared to younger adults, in regard to antibody responses as well as at the transcript level.

Profiling transcriptional heterogeneity of epithelium, fibroblasts, and immune cells in esophageal squamous cell carcinoma by single-cell RNA sequencing.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies with complex tumor microenvironment (TME) which has been proven to be associated with therapeutic failure or resistance. A deeper understanding of the complex TME and cellular heterogeneity is urgently needed in ESCC. Here, we generated single-cell RNA sequencing (scRNA-seq) of 25 796 immune and 8197 non-immune cells from three primary tumor and paired normal samples in ESCC patients. The results revealed intratumoral and intertumoral epithelium heterogeneity and tremendously differences in tumor and normal epithelium. The infiltration of myofibroblasts, one subtype of fibroblasts, might play important roles in the progression of ESCC. We also found that some differentially expressed genes and markers in epithelium and fibroblast subtypes showed prognostic values for ESCC. Diverse cell subtypes of T cells and myeloid cells were identified, including tumor-enriched HAVCR2+ CD4+ T cells with significantly exhausted signature. The epithelium and myeloid cells had more frequent cell-cell communication compared with epithelium and T cells. Taken together, this study provided in-depth insights into the cellular heterogeneity of TME in ESCC and highlighted potential therapeutic targets including for immunotherapy.

CD5L-associated gene analyses highlight the dysregulations, prognostic effects, immune associations, and drug-sensitivity predicative potentials of LCAT and CDC20 in hepatocellular carcinoma.

BACKGROUND: The dysregulation of CD5L has been reported in hepatocellular carcinoma (HCC). However, its functions in HCC were controversial. In this study, we aimed to identify CD5L-associated pathways and markers and explore their values in HCC diagnosis, prognosis and treatment. METHODS: HCC datasets with gene expression profiles and clinical data in TCGA and ICGC were downloaded. The immune/stroma cell infiltrations were estimated with xCell. CD5L-associated pathways and CD5L-associated genes (CD5L-AGs) were identified with gene expression comparisons and gene set enrichment analysis (GSEA). Cox regression, Kaplan-Meier survival analysis, and least absolute shrinkage and selection operator (LASSO) regression analysis were performed. The correlations of the key genes with immune/stroma infiltrations, immunoregulators, and anti-cancer drug sensitivities in HCC were investigated. At protein level, the key genes dysregulations, their correlations and prognostic values were validated in clinical proteomic tumor analysis consortium (CPTAC) database. Serum CD5L and LCAT activity in 50 HCC and 30 normal samples were evaluated and compared. The correlations of serum LCAT activity with alpha-fetoprotein (AFP), albumin (ALB) and high-density lipoprotein (HDL) in HCC were also investigated. RESULTS: Through systemic analyses, 14 CD5L-associated biological pathways, 256 CD5L-AGs and 28 CD5L-associated prognostic and diagnostic genes (CD5L-APDGs) were identified. A risk model consisting of LCAT and CDC20 was constructed for HCC overall survival (OS), which could discriminate HCC OS status effectively in both the training and the validation sets. CD5L, LCAT and CDC20 were shown to be significantly correlated with immune/stroma cell infiltrations, immunoregulators and 31 anti-cancer drug sensitivities in HCC. At protein level, the dysregulations of CD5L, LCAT and CDC20 were confirmed. LCAT and CDC20 were shown to be significantly correlated with proliferation marker MKI67. In serum, no significance of CD5L was shown. However, the lower activity of LCAT in HCC serum was obvious, as well as its significant positive correlations ALB and HDL concentrations. CONCLUSIONS: CD5L, LCAT and CDC20 were dysregulated in HCC both at mRNA and protein levels. The LCAT-CDC20 signature might be new predicator for HCC OS. The associations of the three genes with HCC microenvironment and anti-cancer drug sensitivities would provide new clues for HCC immunotherapy and chemotherapy.

Lattice vibration characteristics in layered InSe films and the electronic behavior of field-effect transistors.

Understanding how temperature affects the structural and electronic properties for two-dimensional (2D) semiconductors could promote the application and development of nanoelectronic devices. Here, the temperature dependence of lattice structure for indium selenide (InSe) nanosheets and the corresponding electronic properties of 3 nm indium-deposited InSe field-effect transistors (FETs) are systematically demonstrated. Analyses of Raman spectra suggest that the difference of phonon frequency (Δω) for the A[Formula: see text] mode is found to be 3.14 cm-1, which is larger than that of the E[Formula: see text] mode due to the stronger electron-phonon coupling for the A[Formula: see text] mode. The device performance based on indium-deposited InSe is systematically explained using Kelvin probe force microscopy (KPFM) and the predicted energy band structure. Furthermore, FETs based on temperature and variable thickness InSe flakes are designed as applicable devices. Our findings are of fundamental importance to explain the underlying physics in intrinsic InSe transistors and improve further applications.

Evaluation of Hg methylation in the water-level-fluctuation zone of the Three Gorges Reservoir region by using the MeHg/HgT ratio.

In the recent decade, the hydroelectric reservoir is identified as a methylmercury (MeHg) hotspot and gained much attention. The artificial water level management in the Three Gorges Reservoir (TGR) in China formed a water-level-fluctuation zone (WLFZ) undergoing flooding drying rotations annually. However, the mercury (Hg) methylation and major geochemical driving factors at different elevations in the WLFZ remain unclear. Here we use total Hg (HgT) normalized MeHg (MeHg/HgT ratio) to evaluate Hg methylation degree in a one-year field study at 155, 165 m elevations in the WLFZ and with >175 m elevation as the reference. Results demonstrate that MeHg/HgT ratio at the WLFZ could reach 4.1% in soils, and both 155 and 165 m elevations have a higher Hg methylation degree than the >175 m elevation. However, the differences in MeHg/HgT ratios both in soils and waters between 155 and 165 m elevations are not significant. This indicates the influence of different submerging periods on the MeHg/HgT at the WLFZ elevations is not observed. The significant correlation between the MeHg/HgT ratio and soil organic carbon (SOC) content implies a MeHg retention in re-exposed soils after flooding. Decoupling of MeHg/HgT ratios between submerged soil and overlying water are found at both elevations and therefore make MeHg/HgT in waters alone cannot be used to evaluate Hg methylation degree in this study. The calculation of HgT and MeHg partitioning coefficient (Kd) found an immobilization of MeHg by submerged soils at the WLFZ during the flooding period. Major geochemical factors, determined through principal component analysis (PCA), in affecting Hg methylation are the redox cycling of sulfur and the distribution of organic matters in the WLFZ.

Interaction between galectin-3 and cystinosin uncovers a pathogenic role of inflammation in kidney involvement of cystinosis.

Inflammation is involved in the pathogenesis of many disorders. However, the underlying mechanisms are often unknown. Here, we test whether cystinosin, the protein involved in cystinosis, is a critical regulator of galectin-3, a member of the ß-galactosidase binding protein family, during inflammation. Cystinosis is a lysosomal storage disorder and, despite ubiquitous expression of cystinosin, the kidney is the primary organ impacted by the disease. Cystinosin was found to enhance lysosomal localization and degradation of galectin-3. In Ctns-/- mice, a mouse model of cystinosis, galectin-3 is overexpressed in the kidney. The absence of galectin-3 in cystinotic mice ameliorates pathologic renal function and structure and decreases macrophage/monocyte infiltration in the kidney of the Ctns-/-Gal3-/- mice compared to Ctns-/- mice. These data strongly suggest that galectin-3 mediates inflammation involved in kidney disease progression in cystinosis. Furthermore, galectin-3 was found to interact with the pro-inflammatory cytokine Monocyte Chemoattractant Protein-1, which stimulates the recruitment of monocytes/macrophages, and proved to be significantly increased in the serum of Ctns-/- mice and also patients with cystinosis. Thus, our findings highlight a new role for cystinosin and galectin-3 interaction in inflammation and provide an additional mechanistic explanation for the kidney disease of cystinosis. This may lead to the identification of new drug targets to delay cystinosis progression.

Large-Scale Growth and Field-Effect Transistors Electrical Engineering of Atomic-Layer SnS2.

2D layers of metal dichalcogenides are of considerable interest for high-performance electronic devices for their unique electronic properties and atomically thin geometry. 2D SnS2 nanosheets with a bandgap of ≈2.6 eV have been attracting intensive attention as one potential candidate for modern electrocatalysis, electronic, and/or optoelectronic fields. However, the controllable growth of large-size and high-quality SnS2 atomic layers still remains a challenge. Herein, a salt-assisted chemical vapor deposition method is provided to synthesize atomic-layer SnS2 with a large crystal size up to 410 µm and good uniformity. Particularly, the as-fabricated SnS2 nanosheet-based field-effect transistors (FETs) show high mobility (2.58 cm2 V-1 s-1 ) and high on/off ratio (≈108 ), which is superior to other reported SnS2 -based FETs. Additionally, the effects of temperature on the electrical properties are systematically investigated. It is shown that the scattering mechanism transforms from charged impurities scattering to electron-phonon scattering with the temperature. Moreover, SnS2 can serve as an ideal material for energy storage and catalyst support. The high performance together with controllable growth of SnS2 endow it with great potential for future applications in electrocatalysis, electronics, and optoelectronics.

Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A.

The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Identification of Neutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors of Neutrophil Exocytosis and Inflammation: DRUGGABILITY OF THE SMALL GTPase Rab27a.

Neutrophils constitute the first line of cellular defense in response to bacterial and fungal infections and rely on granular proteins to kill microorganisms, but uncontrolled secretion of neutrophil cargos is injurious to the host and should be closely regulated. Thus, increased plasma levels of neutrophil secretory proteins, including myeloperoxidase and elastase, are associated with tissue damage and are hallmarks of systemic inflammation. Here, we describe a novel high-throughput screening approach to identify small molecule inhibitors of the interaction between the small GTPase Rab27a and its effector JFC1, two central regulators of neutrophil exocytosis. Using this assay, we have identified small molecule inhibitors of Rab27a-JFC1 binding that were also active in cell-based neutrophil-specific exocytosis assays, demonstrating the druggability of Rab GTPases and their effectors. These compounds, named Nexinhibs (neutrophil exocytosis inhibitors), inhibit exocytosis of azurophilic granules in human neutrophils without affecting other important innate immune responses, including phagocytosis and neutrophil extracellular trap production. Furthermore, the compounds are reversible and potent inhibitors of the extracellular production of superoxide anion by preventing the up-regulation of the granule membrane-associated subunit of the NADPH oxidase at the plasma membrane. Nexinhibs also inhibit the up-regulation of activation signature molecules, including the adhesion molecules CD11b and CD66b. Importantly, by using a mouse model of endotoxin-induced systemic inflammation, we show that these inhibitors have significant activity in vivo manifested by decreased plasma levels of neutrophil secretory proteins and significantly decreased tissue infiltration by inflammatory neutrophils. Altogether, our data present the first neutrophil exocytosis-specific inhibitor with in vivo anti-inflammatory activity, supporting its potential use as an inhibitor of systemic inflammation.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.

Spectral assignments in the infrared absorption region and anomalous thermal hysteresis in the interband electronic transition of vanadium dioxide films.

The metal-insulator transition (MIT) is of key importance for understanding the fundamental electronic interaction that determines the physical properties of vanadium dioxide (VO2) film. Here, the spectral slopes of transmittance and reflectance in the infrared absorption region (about 0.62-1.63 eV) and the interband electronic transitions for VO2 films with thicknesses of 27, 40 and 63 nm have been investigated. The potential applications of the spectral slopes were presented in detail. It is found that the variation of resistivity and transmittance increases with the spectral slopes of transmittance and reflectance. It is surprising that the resistivity of the VO2 film with a thickness of 27 nm is larger than that of the VO2 film with a thickness of 40 nm in the metal state. In addition, an anomalous counterclockwise thermal hysteresis with higher energy from the interband electronic transition was also found during the MIT process for the thinnest film. It is believed that this remarkable phenomenon could be related to the correlation effects in the rutile phase, which could lead to the splitting of the a1g band into Hubbard bands. The lower Hubbard band would result in an electronic transition blue-shift with the empty e band, which can explain the origin of the counterclockwise thermal hysteresis and the abnormal resistivity in the metal state.

The EM structure of the TRAPPIII complex leads to the identification of a requirement for COPII vesicles on the macroautophagy pathway.

The transport protein particle (TRAPP) III complex, comprising the TRAPPI complex and additional subunit Trs85, is an autophagy-specific guanine nucleotide exchange factor for the Rab GTPase Ypt1 that is recruited to the phagophore assembly site when macroautophagy is induced. We present the single-particle electron microscopy structure of TRAPPIII, which reveals that the dome-shaped Trs85 subunit associates primarily with the Trs20 subunit of TRAPPI. We further demonstrate that TRAPPIII binds the coat protein complex (COP) II coat subunit Sec23. The COPII coat facilitates the budding and targeting of ER-derived vesicles with their acceptor compartment. We provide evidence that COPII-coated vesicles and the ER-Golgi fusion machinery are needed for macroautophagy. Our results imply that TRAPPIII binds to COPII vesicles at the phagophore assembly site and that COPII vesicles may provide one of the membrane sources used in autophagosome formation. These events are conserved in yeast to mammals.

Mechanisms of CPB Modified Zeolite on Mercury Adsorption in Simulated Wastewater.

A systematic study was carried out to analyze the effects of mercury(II) adsorption by surface modified zeolite (SMZ) and adsorption mechanism. Cetylpyridinium bromide (CPB) was used to prepare SMZ. The characterization methods by means of powder X-ray diffraction (XRD), Fourier transform infrared (FT-IR), scanning electron microscope (SEM) showed that both the surface and internal zeolite were covered with CPB molecules, but the main binding sites were surface. Results showed that the organic carbon and cation exchange capacity of the SMZ were 7.76 times and 4.22 times higher than those of natural zeolite (NZ), respectively. Zeta potentials before and after modification were measured at -7.80 mV and -30.27 mV, respectively. Moreover, the saturation adsorptive capacity of SMZ was 16.35 times higher than NZ in mercury-containing wastewater. The possible mechanisms of mercury elimination were surface adsorption, hydrophobic interaction, ion exchange, electricity neutralization. The adsorption process was affected little by competitive ions.

Spatiotemporal detection and analysis of exocytosis reveal fusion "hotspots" organized by the cytoskeleton in endocrine cells.

Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.

Strain and temperature dependent absorption spectra studies for identifying the phase structure and band gap of EuTiO3 perovskite films.

Post-annealing has been approved to effectively relax the out-of-plane strain in thin films. Epitaxial EuTiO3 (ETO) thin films, with and without strain, have been fabricated on (001) LaAlO3 substrates by pulsed laser deposition. The absorption and electronic transitions of the ETO thin films are investigated by means of temperature dependent transmittance spectra. The antiferrodistortive phase transition can be found at about 260-280 K. The first-principles calculations indicate there are two interband electronic transitions in ETO films. Remarkably, the direct optical band gap and higher interband transition for ETO films show variation in trends with different strains and temperatures. The strain leads to a band gap shrinkage of about 240 meV while the higher interband transition an expansion of about 140 meV. The hardening of the interband transition energies in ETO films with increasing temperature can be attributed to the Fröhlich electron-phonon interaction. The behavior can be linked to the strain and low temperature modified valence electronic structure, which is associated with rotations of the TiO6 octahedra.

Mapracorat, a selective glucocorticoid receptor agonist, upregulates RelB, an anti-inflammatory nuclear factor-kappaB protein, in human ocular cells.

Selective glucocorticoid receptor agonists (SEGRAs) are a new class of compounds under clinical evaluation for treatment of ocular inflammation. Widely prescribed therapeutics, such as glucocorticoids, are effective at reducing ocular inflammation, but their long term use predisposes to undesirable side effects. The purpose of this study was to investigate a novel SEGRA, mapracorat (BOL-303242-X), and the differences in mapracorat's mechanism of action compared with traditional steroids (i.e. dexamethasone). Keratocytes from three different humans were cultured and treated with mapracorat or dexamethasone, with and without a strong provoking agent, interleukin (IL)-1ß. The effects of mapracorat compared to dexamethasone were determined by measuring protein levels (Western blotting) and DNA binding (ELISA) for two nuclear factor-kappaB (NF-κB) family members, RelA and RelB. Cytokine production (i.e. IL-6, IL-8, prostaglandin E2 (PGE2)) was characterized by immunoassay. Our findings reveal mechanistic differences between mapracorat and traditional steroid therapies. Mapracorat showed partial attenuation of the classical NF-κB pathway, consistent with traditional steroids. However, mapracorat uniquely potentiated a novel anti-inflammatory mechanism through rapid upregulation of RelB, an anti-inflammatory member of the NF-κB alternative pathway. Mapracorat potently inhibits ocular inflammation in vitro and is a promising new treatment for ocular inflammatory disease. Mapracorat acts, in part, by a novel mechanism via upregulation of RelB in the NF-κB alternative pathway.

Injectable extracellular matrix-mimetic hydrogel based on electrospun Janus fibers.

To date, the reported injectable hydrogels have failed to mimic the fibrous architecture of the extracellular matrix (ECM), limiting their biological effects on cell growth and phenotype. Additionally, they lack the micro-sized pores present within the ECM, which is unfavorable for the facile transport of nutrients and waste. Herein, an injectable ECM-mimetic hydrogel (IEMH) was fabricated by shortening and dispersing Janus fibers capable of self-curling at body temperature into pH 7.4 phosphate buffer solution. The IEMH could be massively prepared through a side-by-side electrospinning process combined with ultraviolet irradiation. The IEMHs with only 5 wt% fibers could undergo sol-gel transition at body temperature to become solid gels with desirable stability, sturdiness, and elasticity and self-healing ability. In addition, they possessed notable pseudoplasticity, which is beneficial to injection at room temperature. The results obtained from characterization analysis via scanning electron microscopy, total internal reflection fluorescence microscopy, nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy indicate that their sol-gel transition under physiological conditions stems from the synergistic action of the tight entanglements between thermally-induced self-curling fibers and the hydrophobic interaction between the fibers. An MTT assay using C2C12 myoblast cells was performed to examine the in vitro cytotoxicity of IEMHs for biomedical applications, and the cell viability was found to be more than 95%.

Robot-assisted system for non-invasive wide-range flexible eye positioning and tracking in particle radiotherapy.

Particle (proton, carbon ion, or others) radiotherapy for ocular tumors is highly dependent on precise dose distribution, and any misalignment can result in severe complications. The proposed eye positioning and tracking system (EPTS) was designed to non-invasively position eyeballs and is reproducible enough to ensure accurate dose distribution by guiding gaze direction and tracking eye motion. Eye positioning was performed by guiding the gaze direction with separately controlled light sources. Eye tracking was performed by a robotic arm with cameras and a mirror. The cameras attached to its end received images through mirror reflection. To maintain a light weight, certain materials, such as carbon fiber, were utilized where possible. The robotic arm was controlled by a robot operating system. The robotic arm, turntables, and light source were actively and remotely controlled in real time. The videos captured by the cameras could be annotated, saved, and loaded into software. The available range of gaze guidance is 360° (azimuth). Weighing a total of 18.55 kg, the EPTS could be installed or uninstalled in 10 s. The structure, motion, and electromagnetic compatibility were verified via experiments. The EPTS shows some potential due to its non-invasive wide-range flexible eye positioning and tracking, light weight, non-collision with other equipment, and compatibility with CT imaging and dose delivery. The EPTS can also be remotely controlled in real time and offers sufficient reproducibility. This system is expected to have a positive impact on ocular particle radiotherapy.