The effects of different impeller combinations in the Sphingan WL gum fermentation process.

The fermentation of the high-viscosity polysaccharide WL gum has always been associated with poor mass transfer. Appropriate impeller configurations are key factors in maintaining homogeneity and sufficient mass transfer conditions. Therefore, a flat-folded disc turbine impeller (FFDT) taking into account both the reduced cavitation effect and the increased contact area was designed. Besides, a curved cross impeller (CC) and a fishbone-shaped impeller (FS) generating axial flow were also designed. The energy consumption and efficiency of the designed impellers and eight reported impellers were evaluated through fermentation and principal component analysis (PCA). Compared to the commonly-used six-blade flat-blade disc turbine (FBDT), the ungassed power number of FFDT was reduced by 50 %. Combinations of six-blade Brumajin impeller (BM) + FFDT and CC + FFDT produced high WL gum production and viscosity (34.0 g/L, 35.50 g/L, and 62.64 Pa·s, 61.68 Pa·s, respectively) and were suitable impellers for WL biosynthesis. WL gum from BM + FFDT showed higher viscosity, viscoelasticity, and molecular weight than that from FBDT + FBDT. In addition, fewer amino acids and pyruvic acid intermediates were formed using BM + FFDT, indicating a greater metabolic flux towards WL gum synthesis. This work provided an important reference for the design of impellers in high-viscosity fermentation systems.

Chronotoxici-Plate Containing Droplet-Engineered Rhythmic Liver Organoids for Drug Toxicity Evaluation.

The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi-timepoint drug administration strategies across a wide range of drugs. Here, droplet-engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici-plate as an in vitro high-throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time-dependent variations are demonstrated in toxicity on the Chronotoxici-plate, highlighting the importance of considering time-dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing.

Single-cell RNA sequencing reveals aberrant sphingolipid metabolism in non-small cell lung cancer impacts tumor-associated macrophages and stimulates angiogenesis via macrophage inhibitory factor signaling.

BACKGROUND: Sphingolipids not only serve as structural components for maintaining cell membrane fluidity but also function as bioactive molecules involved in cell signaling and the regulation of various biological processes. Their pivotal role in cancer cell development, encompassing cancer cell proliferation, migration, angiogenesis, and metastasis, has been a focal point for decades. However, the contribution of sphingolipids to the complexity of tumor microenvironment promoting cancer progression has been rarely investigated. METHODS: Through the integration of publicly available bulk RNA-seq and single-cell RNA-seq data, we conducted a comprehensive analysis to compare the transcriptomic features between tumors and adjacent normal tissues, thus elucidating the intricacies of the tumor microenvironment (TME). RESULTS: Disparities in sphingolipid metabolism (SLM)-associated genes were observed between normal and cancerous tissues, with the TME characterized by the enrichment of sphingolipid signaling in macrophages. Cellular interaction analysis revealed robust communication between macrophages and cancer cells exhibiting low SLM, identifying the crucial ligand-receptor pair, macrophage inhibitory factor (MIF)-CD74. Pseudo-time analysis unveiled the involvement of SLM in modulating macrophage polarization towards either M1 or M2 phenotypes. Categorizing macrophages into six subclusters based on gene expression patterns and function, the SPP1+ cluster, RGS1+ cluster, and CXCL10+ cluster were likely implicated in sphingolipid-induced M2 macrophage polarization. Additionally, the CXCL10+, AGER+, and FABP4+ clusters were likely to be involved in angiogenesis through their interaction with endothelial cells. CONCLUSION: Based on multiple scRNA-seq datasets, we propose that a MIF-targeted strategy could potentially impede the polarization from M1 to M2 and impair tumor angiogenesis in low-SLM non-small cell lung cancer (NSCLC), demonstrating its potent antitumor efficacy.

Adsorption and desorption behavior of Zn2+ in a flow-through electrosorption reactor.

As heavy metal industrial wastewater increases in volume and complexity, we need more efficient, cheaper, and renewable technologies to curb its environmental impact. Compared to advection electrosorption, through-flow electrosorption is a hotspot technique that makes more efficient use of the adsorption capacity of activated carbon fiber mats. A cascade flow-through electrosorption assembly based on activated carbon fiber was used to obtain the best adsorption of Zn2+ in water at a voltage of 2 V, pH value of 8, plate spacing of 3 mm, and temperature of 15°C. The process is more closely fitted to the secondary adsorption kinetic equation and the Langmuir equation. The adsorption capacity of the module decreases at a progressively slower rate with the number of cycles and will eventually retain 75% of its peak value with significant regenerability. The study of this module can provide technical support for treating heavy metal wastewater.

Protocol to encapsulate cerebral organoids with alginate hydrogel shell to induce volumetric compression.

In vitro organoids, including cerebral organoids, are usually developed without mechanical compression, which may contribute to a delay in maturation. Here, we present a protocol for encapsulating cerebral organoids with a thin shell of low-concentration alginate hydrogel. We describe steps for organoid generation, microfluidic chip culture, Matrigel coating, expansion culture, and alginate encapsulation. We then detail procedures for maturation culture and organoid characterization. The moderate compressive stimulation that the shell provides promotes cell proliferation and neuronal maturation. For complete details on the use and execution of this protocol, please refer to Tang et al.1.

Microglial Reactivity Correlates with Presynaptic Loss Independent of ß-Amyloid and Tau.

OBJECTIVE: Triggering receptor expressed on myeloid cells-2 (TREM2) and progranulin (PGRN) are critical regulators of microglia activation and can be detected in cerebrospinal fluid (CSF). However, whether microglial reactivity is detrimental or neuroprotective for Alzheimer disease (AD) is still debatable. METHODS: We identified 663 participants with baseline ß-amyloid (Aß) positron emission tomography (PET) and CSF biomarker data, including phosphorylated tau181 (p-Tau181), soluble TREM2 (sTREM2), PGRN, and growth-associated protein-43 (GAP-43). Among them, 254 participants had concurrent longitudinal CSF biomarkers. We used multivariate regression analysis to study the associations of CSF microglial biomarkers with Aß PET, CSF p-Tau181, and CSF GAP-43 cross-sectionally and longitudinally. A Chinese aging cohort's independent CSF samples (n = 65) were analyzed as a validation. RESULTS: Higher baseline levels of CSF microglial biomarkers were related to faster rates of CSF sTREM2 increase and CSF PGRN decrease. Elevated CSF p-Tau181 was associated with higher levels of CSF microglial biomarkers and faster rates of CSF sTREM2 increase and CSF PGRN decrease. In both cohorts, higher Aß burden was associated with attenuated CSF p-Tau181 effects on CSF microglial biomarker increases. Independent of Aß PET and CSF p-Tau181 pathologies, higher levels of CSF sTREM2 but not CSF PGRN were related to elevated CSF GAP-43 levels and faster rates of CSF GAP-43 increase. INTERPRETATION: These findings suggest that higher Aß burden may attenuate the p-Tau-associated microglial responses, and TREM2-related microglial reactivity may independently correlate with GAP-43-related presynaptic loss. This study highlights the two-edged role of microglial reactivity in AD and other neurodegenerative diseases. ANN NEUROL 2024;95:917-928.

A novel PDPN antagonist peptide CY12-RP2 inhibits melanoma growth via Wnt/ß-catenin and modulates the immune cells.

BACKGROUND: Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. METHODS: PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. RESULTS: PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/ß-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1ß, TNF-α, and IFN-γ. CONCLUSIONS: This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/ß-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/ß-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.

Rubusoside mitigates neuroinflammation and cellular apoptosis in Parkinson's disease, and alters gut microbiota and metabolite composition.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative condition characterized by the progressive loss of dopaminergic neurons within the substantia nigra. Neuroinflammation plays a pivotal role in the pathogenesis of PD, involving the activation of microglia cells, heightened production of proinflammatory cytokines, and perturbations in the composition of the gut microbiota. Rubusoside (Ru), the principal steviol bisglucoside present in Rubus chingii var. suavissimus (S.K.Lee) L.T.Lu (Rosaceae), has been documented for its anti-inflammatory properties in diverse disease models. Nonetheless, there is an imperative need to comprehensively assess and elucidate the protective and anti-inflammatory attributes of Ru concerning PD, as well as to uncover the underlying mechanism involved. OBJECTIVE: The aim of this study is to evaluate the neuroprotective and anti-inflammatory effects of Ru on PD and investigate its potential mechanisms associated with microbes. RESEARCH DESIGN AND METHODS: We pre-treated mice and cell lines with Ru in order to simulate the progression of PD and the neuroinflammatory state. The mouse model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), SN4741 cells were induced by 1-methyl-4-phenylpyridine (mpp+), and BV-2 cells were induced by lipopolysaccharide (LPS). We assessed the impact of Ru on motor function, neuroinflammation, neuron apoptosis, the composition of gut microbes, and their metabolites. RESULTS: Ru treatment reduces the release of pro-inflammatory mediators by inhibiting microglia activation. It also prevents neuronal apoptosis, thereby safeguarding dopaminergic neurons and ameliorating motor dysfunction. Furthermore, it induces alterations in the fecal microbiota composition and metabolites profile in PD mice. In vitro experiments have demonstrated that Ru inhibits neuronal apoptosis in SN4741 cells induced by mpp+, suppresses the production of pro-inflammatory mediators, and activates the c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (p38 MAPK), and nuclear factor kappa-B (NF-κB) signaling pathways. CONCLUSION: Ru exhibits inhibitory effects on the MPTP-induced PD model by mitigating neuroinflammation and neuronal apoptosis while also inducing changes in the gut microbiota and metabolite composition.

Stress regulates Alzheimer's disease progression via selective enrichment of CD8+ T cells.

This study investigates stress's impact on Alzheimer's disease (AD) using male APP/PS1 transgenic mice. Negative stressors (chronic social defeat, restraint) and positive hedonia (environmental enrichment, EE) were applied. Stress worsens AD pathology, while EE slows progression. Brain RNA sequencing reveals interleukin-6 (IL-6) and IL-10 as key stress-related AD regulators. Flow cytometry shows that the CD8+/CD4+ T cell ratio shifts in response to stress exposure and EE. Stress exposure increases CD8+/CD4+ ratio, opposite to EE. Depletion and enrichment of CD8+ T cells both accelerate AD, indicating immune intervention's negative impact. Stress management and balanced immunity may aid AD therapy, highlighting novel potential treatment.

Volumetric compression by heterogeneous scaffold embedding promotes cerebral organoid maturation and does not impede growth.

Although biochemical regulation has been extensively studied in organoid modeling protocols, the role of mechanoregulation in directing stem cell fate and organoid development has been relatively unexplored. To accurately replicate the dynamic organoid development observed in nature, in this study, we present a method of heterogeneous embedding using an alginate-shell-Matrigel-core system. This approach allows for cell-Matrigel remodeling by the inner layer and provides short-term moderate-normal compression through the soft alginate outer layer. Our results show that the time-limited confinement contributes to increased expression of neuronal markers such as neurofilament (NF) and microtubule-associated protein 2 (MAP2). Compared with non-alginate embedding and alginate compression groups, volume growth remains unimpeded. Our findings demonstrate the temporary mechanical regulation of cerebral organoid growth, which exhibits a regular growth profile with enhanced maturation. These results highlight the importance and potential practical applications of mechanoregulation in the establishment of brain organoids. A record of this paper's transparent peer review process is included in the supplemental information.

Attachment culture of cortical organoids at the microwell air-liquid interface.

The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.

Pure-Phase, Large-Grained Wide-Band-Gap Perovskite Films for High-Efficiency, Four-Terminal Perovskite/Silicon Tandem Solar Cells.

High-quality, stable perovskite films with a wide band gap between 1.65 and 1.80 eV are highly suitable for efficient and cost-competitive silicon-based tandem solar cells. Herein, we demonstrate that the combined strategies of the Pb(SCN)2 additive and air annealing can enable the Cs0.22FA0.78Pb(I0.85Br0.15)3 films with a wide band gap of 1.65 eV and favored properties including pure composition, high crystallinity, micro-sized grains, and reduced defects. With these desired films, the average efficiencies of semitransparent perovskite solar cells (PSCs) are boosted from (18.13 ± 0.31) to (20.35 ± 0.28)%. Further, the semitransparent PSC is used to assemble the four-terminal perovskite/TOPCon tandem solar cell. Benefiting from its excellent performance and preferred optical properties, the obtained tandem solar cell yields a milestone efficiency of 30.32%.

Integrative analysis of DNA methylomes reveals novel cell-free biomarkers in lung adenocarcinoma.

Lung cancer is a leading cause of cancer-related deaths worldwide, with a low 5-year survival rate due in part to a lack of clinically useful biomarkers. Recent studies have identified DNA methylation changes as potential cancer biomarkers. The present study identified cancer-specific CpG methylation changes by comparing genome-wide methylation data of cfDNA from lung adenocarcinomas (LUAD) patients and healthy donors in the discovery cohort. A total of 725 cell-free CpGs associated with LUAD risk were identified. Then XGBoost algorithm was performed to identify seven CpGs associated with LUAD risk. In the training phase, the 7-CpGs methylation panel was established to classify two different prognostic subgroups and showed a significant association with overall survival (OS) in LUAD patients. We found that the methylation of cg02261780 was negatively correlated with the expression of its representing gene GNA11. The methylation and expression of GNA11 were significantly associated with LAUD prognosis. Based on bisulfite PCR, the methylation levels of five CpGs (cg02261780, cg09595050, cg20193802, cg15309457, and cg05726109) were further validated in tumor tissues and matched non-malignant tissues from 20 LUAD patients. Finally, validation of the seven CpGs with RRBS data of cfDNA methylation was conducted and further proved the reliability of the 7-CpGs methylation panel. In conclusion, our study identified seven novel methylation markers from cfDNA methylation data which may contribute to better prognosis for LUAD patients.

TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation.

BACKGROUND: Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Its role in LUAD has gained attention, but the detailed function and mechanism have not been clarified completely at present. METHODS: Tumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, we detected the influence of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis. RESULTS: We found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. We also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, we found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. We further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. CONCLUSIONS: Our study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

Protocol for generating in vitro glioma models using human-induced pluripotent- or embryonic-stem-cell-derived cerebral organoids.

In glioma modeling, existing organoid protocols lack the ability to replicate glioma cell invasion and interaction with normal brain tissue. Here, we present a protocol for generating in vitro brain disease models using human-induced pluripotent- or embryonic-stem-cell-derived cerebral organoids (COs). We describe steps for forming glioma organoids by co-culturing forebrain organoids with U-87 MG cells. We also detail vibratome sectioning of COs to prevent cell death and enhance contact between U-87 MG cells and cerebral tissues.

A specific RAGE-binding peptide inhibits triple negative breast cancer growth through blocking of Erk1/2/NF-κB pathway.

Triple-negative breast cancer (TNBC) is an aggressive cancer that poses a significant threat to women's health. Unfortunately, the lack of clinical targets leads the poor clinical outcomes in TNBC. Many cancers demonstrate overexpression of receptor for advanced glycation end products (RAGE), which can contribute to cancer progression. Despite the potential therapeutic value of blocking RAGE for TNBC treatment, effective peptide drugs have yet to be developed. In our study, we observed that RAGE was highly expressed in TNBC and was associated with poor disease progression. We subsequently investigated the antitumor effects and underlying mechanisms of the RAGE antagonist peptide RP7 in both in vitro and in vivo models of TNBC. Our study revealed that RP7 selectively binds to RAGE-overexpressing TNBC cell lines, including MDA-MB-231 and BT549, and significantly inhibits cell viability, migration, and invasion in both cell lines. Furthermore, RP7-treatment suppressed tumor growth in TNBC xenograft mouse models without inducing detectable toxicity in normal tissues. Mechanistically, RP7 was found to inhibit the phosphorylation of ERK1/2, IKKα/ß, IKBα, and p65 to block the NF-κB pathway, prevent the entry of p65 into the nucleus, decrease the protein expression of Bcl-2 and HMGB1, and promote the release of cytochrome C from the mitochondria into the cytoplasm. These effects were observed to activate apoptosis and inhibit epithelial-mesenchymal transition (EMT) in TNBC cells. This study highlights RAGE as a candidate therapeutic target for TNBC treatment and suggests that the RAGE antagonist peptide RP7 is a promising anticancer drug for TNBC.

FANCI is Associated with Poor Prognosis and Immune Infiltration in Liver Hepatocellular Carcinoma.

Objective: This study aimed to validate FANCI as a potential marker for both prognosis and therapy in liver hepatocellular carcinoma. Method: FANCI expression data were acquired from GEPIA, HPA, TCGA, and GEO databases. The impact of clinicopathological features was analyzed by UALCAN. The prognosis of Liver Hepatocellular Carcinoma (LIHC) patients with highly expressed FANCI was constructed utilizing Kaplan-Meier Plotter. GEO2R was employed to identify differentially expressed genes (DEGs). Metascape was used to analyze functional pathways correlations. Protein-Protein interaction (PPI) networks were generated by Cytoscape. Furthermore, molecular complex detection (MCODE) was utilized to recognize Hub genes, which were selected to establish a prognostic model. Lastly, the relationship between FANCI and immune cell infiltration in LIHC was examined. Results: Compared to adjacent tissues, FANCI expression levels were significantly higher in LIHC tissues and were positively correlated to the cancer grade, stage, and prior hepatitis B virus (HBV) infection. High expression of FANCI was found to be associated with poor prognosis in LIHC (HR=1.89, p<0.001). DEGs that were positively correlated with FANCI were involved in various processes, including the cell cycle, VEGF pathway, immune system processes, and biogenesis of ribonucleoproteins. MCM10, TPX2, PRC1, and KIF11 were identified as key genes closely related to FANCI and poor prognosis. A reliable five-variable prognostic model was constructed with strong predictive capability. Lastly, a positive correlation was observed between FANCI expression and tumor-infiltration levels of CD8+ T cells, B cells, regulatory T (Tregs), CD4+ T helper 2 (Th2), and macrophage M2 cells. Conclusion: FANCI may hold promise as a potential biomarker for predicting prognostic outcomes, and a valuable therapeutic target for LIHC patients, with a focus on anti-proliferation, anti-chemoresistance, and combination with immunotherapy.

Role of MAIT cells in gastrointestinal tract bacterial infections in humans: More than a gut feeling.

Mucosa-associated invariant T (MAIT) cells are the largest population of unconventional T cells in humans. These antimicrobial T cells are poised with rapid effector responses following recognition of the cognate riboflavin (vitamin B2)-like metabolite antigens derived from microbial riboflavin biosynthetic pathway. Presentation of this unique class of small molecule metabolite antigens is mediated by the highly evolutionarily conserved major histocompatibility complex class I-related protein. In humans, MAIT cells are widely found along the upper and lower gastrointestinal tracts owing to their high expression of chemokine receptors and homing molecules directing them to these tissue sites. In this review, we discuss recent findings regarding the roles MAIT cells play in various gastrointestinal bacterial infections, and how their roles appear to differ depending on the etiological agents and the anatomical location. We further discuss the potential mechanisms by which MAIT cells contribute to pathogen control, orchestrate adaptive immunity, as well as their potential contribution to inflammation and tissue damage during gastrointestinal bacterial infections, and the ensuing tissue repair following resolution. Finally, we propose and discuss the use of the emerging three-dimensional organoid technology to test different hypotheses regarding the role of MAIT cells in gastrointestinal bacterial infections, inflammation, and immunity.