Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Label-Free Three-Dimensional Morphological Characterization of Cell Death Using Holographic Tomography.

This study presents a novel label-free approach for characterizing cell death states, eliminating the need for complex molecular labeling that may yield artificial or ambiguous results due to technical limitations in microscope resolution. The proposed holographic tomography technique offers a label-free avenue for capturing precise three-dimensional (3D) refractive index morphologies of cells and directly analyzing cellular parameters like area, height, volume, and nucleus/cytoplasm ratio within the 3D cellular model. We showcase holographic tomography results illustrating various cell death types and elucidate distinctive refractive index correlations with specific cell morphologies complemented by biochemical assays to verify cell death states. These findings hold promise for advancing in situ single cell state identification and diagnosis applications.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Portable particle mass spectrometer.

Particle pollutants in air have been confirmed to damage human health. The PM10 concentration is an important parameter for air quality determination. In this study, a portable quadrupole ion trap mass spectrometer (QIT-MS) was developed and used to quantitate microparticles and particulate standards. The instrument can be used to perform online analysis of various microsized particles. The instrument can be used to analyze various sizes of disperse particles with accurate mass by a histogram profile. The overall detection efficiencies of particles in the sample for polystyrene were obtained. PM10-like reference materials were used for calibration to analyze the size and mass distribution of an environmental sample. The instrument shows the potential for quantitation of different particles of an unknown sample.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Development of a focused high-energy macromolecular ion beam.

In this work, we report the development of a focused macromolecular ion beam with kinetic energy of up to 110 keV. The system consists of a quadrupole ion trap (QIT), einzel lens and linear accelerator (LINAC). Based on the combination of matrix-assisted laser desorption ionization (MALDI) and quadrupole ion trapping (QIT), ions were desorbed from the surface and trapped with an ion trap to form biomolecular ion packets. Positive- and negative-pulsed voltages were applied on each end-cap electrode of the QIT to extract the ion packets and form an ion beam that was subsequently focused via an einzel lens and accelerated by stepwise pulsed voltages. The tabletop instrument was designed and successfully demonstrated via measurements of molecular ions of insulin, cytochrome c and bovine serum albumin (BSA) with mass-to-charge ratios (m/z) ranging from ∼5.8 to 66.5 k. This is the first report of both a focused and high-kinetic-energy protein ion beam. In addition, both secondary ions and electrons were observed from the surface by hypervelocity ion beam bombardment. This focused macromolecular ion beam has demonstrated its potential in the study of interactions between large molecular ions with other molecules either in the gas phase or upon a surface.

Influence of noise-reduction techniques in sparse-data sample rotation tomographic imaging.

Data acquisition and processing is a critical issue for high-speed applications, especially in three-dimensional live cell imaging and analysis. This paper focuses on sparse-data sample rotation tomographic reconstruction and analysis with several noise-reduction techniques. For the sample rotation experiments, a live Candida rugosa sample is used and controlled by holographic optical tweezers, and the transmitted complex wavefronts of the sample are recorded with digital holographic microscopy. Three different cases of sample rotation tomography were reconstructed for dense angle with a step rotation at every 2°, and for sparse angles with step rotation at every 5° and 10°. The three cases of tomographic reconstruction performance are analyzed with consideration for data processing using four noise-reduction techniques. The experimental results demonstrate potential capability in retaining the tomographic image quality, even at the sparse angle reconstructions, with the help of noise-reduction techniques.

Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network.

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.

A portable multiple ionization source biological mass spectrometer.

In the past, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), used for large biomolecule detection, were usually installed in two separate mass spectrometers. In this study, they were equipped in the same mass spectrometer. This portable biological mass spectrometer has multiple ionization capabilities in the same mass spectrometer and shares the same mass analyzer and detector. This mass spectrometer can be operated under low vacuum (∼10-3 Torr) and can use air as the buffer gas. Therefore, the demand for pumping is reduced and rare gas feeding is no longer essential. A small scroll pump, employed to assist a miniature turbo pump, is sufficient to maintain the operational pressure. The mass spectra of biomolecules were obtained using frequency scanning instead of voltage ramping. Therefore, a wider mass-to-charge ratio (m/z) range was achieved. Furthermore, the design also couples a conversion dynode with a channeltron to enhance the mass detection range. This homemade mass spectrometer has the capability to measure charged particles with very large m/z values (m/z > 100 000). The concentrations of the studied compounds (angiotensin, insulin, cytochrome C, bovine serum albumin (BSA), immunoglobulin G, and immunoglobulin A) are from 5 femtomole to 100 picomole, and the mass resolutions are from 30 to 260. The mass range of this portable mass spectrometer was comparable with a commercial linear time-of-flight mass spectrometer owing to the use of the frequency scan.

IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression.

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.

Anti-Acne Effects of Cembrene Diterpenoids from the Cultured Soft Coral Sinularia flexibilis.

Acne is a skin disease common in adolescents and increasingly common in the adult population. The major pathologic events of acne vulgaris include increased sebum production, retention hyperkeratosis, carrying commensal skin microbiota, and inflammation. In recent years, more than 10,000 compounds have been isolated and identified from marine organisms. The aim of this study was to discover the potential anti-acne activity of fraction 9 + 10 (SF-E) of Sinularia flexibilis extract and six cembrene diterpenoids. We found that the SF-E significantly reduced Cutibacterium acnes-induced edema in Wistar rat ears. The cembrene diterpenoids including 11-dehydrosinulariolide (SC-2), 3,4:8,11-bisepoxy-7-acetoxycembra-15(17)-en-1,12-olide (SC-7), and sinularin (SC-9) reduced nitric oxide (NO) production with 50% inhibitory concentration of 5.66 ± 0.19, 15.25 ± 0.25, and 3.85 ± 0.25 µM, respectively, and inducible NO synthase expression in RAW 264.7 cells. Moreover, treatment with SC-2, SC-7, and SC-9 significantly suppressed lipopolysaccharide- and heat-killed C. acnes-induced expression of proteins involved in mitogen-activated protein kinase pathway in both RAW 264.7 and HaCaT cells. After treatment with SC-2, SC-7, and SC-9, over-proliferation of HaCaT cells was significantly terminated. In summary, SC-2, SC-7, and SC-9 showed anti-inflammatory effects in RAW 264.7 cells, suggesting that these cembrene diterpenoids obtained from S. flexibilis are natural marine products with potential anti-acne activities.

High accuracy differentiating autoimmune pancreatitis from pancreatic ductal adenocarcinoma by immunoglobulin G glycosylation.

BACKGROUND: Misdiagnosis of autoimmune pancreatitis (AIP) as pancreatic cancer (PDAC) or vice versa can cause dismal patents' outcomes. Changes in IgG glycosylation are associated with cancers and autoimmune diseases. This study investigated the IgG glycosylation profiles as diagnostic and prognostic biomarkers in PDAC and AIP. METHODS: Serum IgG-glycosylation profiles from 86 AIP patients, 115 PDAC patients, and 57 controls were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Classification and regression tree (CART) analysis was applied to build a decision tree for discriminating PDAC from AIP. The result was validated in an independent cohort. RESULTS: Compared with AIP patients and controls, PDAC patients had significantly higher agalactosylation, lower fucosylation, and sialylation of IgG1, a higher agalactosylation ratio of IgG1 and a higher agalactosylation ratio of IgG2. AIP patients had significantly higher fucosylation of IgG1 and a higher sialylation ratio of IgG subclasses 1, 2 and 4. Using the CART analysis of agalactosylation and sialylation ratios in the IgG to discriminate AIP from PDAC, the diagnostic accuracy of the glycan markers was 93.8% with 94.6% sensitivity and 92.9% specificity. There were no statistically significant difference of IgG-glycosylation profiles between diffuse type and focal type AIP. CONCLUSIONS: AIP and PDAC patients have distinct IgG-glycosylation profilings. IgG-glycosylation could different PDAC from AIP with high accuracy.

A quadrupole ion trap mass spectrometer for dry microparticle analysis.

In this work, we report a new design of a charge detection quadrupole ion trap mass spectrometer (QIT-MS) for the analysis of micro-sized dry inorganic and bioparticles including red blood cells (RBCs) and different sizes of MCF-7 breast cancer cells. The developed method is one of the fastest methods to measure the mass of micro-sized particles. This system allows the online analysis of various micro-sized particles up to 1 × 1017 Da. The calibration of the mass spectrometer has been done by using different sizes of polystyrene (PS) particles (2-15 µm). The measured masses of RBCs were around 1.8 × 1013 Da and MCF-7 cancer cells were between 1 × 1014 and 4 × 1014 Da. The calculated mass distribution profiles of the particles and cells were given as histogram profiles. The statistical data were summarized after Gaussian type fitting to the experimental histogram profiles. The new method gives very promising results for the analysis of particles and has very broad application.

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2.

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

MCP-1/MCPIP-1 Signaling Modulates the Effects of IL-1ß in Renal Cell Carcinoma through ER Stress-Mediated Apoptosis.

In renal cell carcinoma (RCC), interleukin (IL)-1ß may be a pro-metastatic cytokine. However, we have not yet noted the clinical association between tumoral expression or serum level of IL-1ß and RCC in our patient cohort. Herein, we investigate molecular mechanisms elicited by IL-1ß in RCC. We found that IL-1ß stimulates substantial monocyte chemoattractant protein (MCP)-1 production in RCC cells by activating NF-kB and AP-1. In our xenograft RCC model, intra-tumoral MCP-1 injection down-regulated Ki67 expression and reduced tumor size. Microarray analysis revealed that MCP-1 treatment altered protein-folding processes in RCC cells. MCP-1-treated RCC cells and xenograft tumors expressed MCP-1-induced protein (MCPIP) and molecules involved in endoplasmic reticulum (ER) stress-mediated apoptosis, namely C/EBP Homologous Protein (CHOP), protein kinase-like ER kinase (PERK), and calnexin (CNX). ER stress-mediated apoptosis in MCP-1-treated RCC cells was confirmed using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. Moreover, ectopic MCPIP expression increased PERK expression in Human embryonic kidney (HEK)293 cells. Our meta-analysis revealed that low MCP-1 levels reduce 1-year post-nephrectomy survival in patients with RCC. Immunohistochemistry indicated that in some RCC biopsy samples, the correlation between MCP-1 or MCPIP expression and tumor stages was inverse. Thus, MCP-1 and MCPIP potentially reduce the IL-1ß-mediated oncogenic effect in RCC; our findings suggest that ER stress is a potential RCC treatment target.

Whole-genome methylation profiling of peripheral blood mononuclear cell for acute exacerbations of chronic obstructive pulmonary disease treated with corticosteroid.

OBJECTIVE: Although association studies in the general population may be relevant for determining susceptibility to chronic obstructive pulmonary disease (COPD), they may be less applicable for pharmacogenetics research in participants who have already acquired the disease. PATIENTS AND METHODS: A genome-wide methylation profiling (generated by HumanMethylation450 BeadChips study was performed on peripheral blood mononuclear cells of 24 patients with AECOPD (acute exacerbation COPD), with good and poor responsiveness to standard corticosteroid treatment. Pyrosequencing was used to replicate the selected CpG sites in 50 patients with AECOPD with standard corticosteroid treatment. RESULTS: The results showed the patients with AECOPD with good and poor response to standard corticosteroid treatment have a distinct DNA methylation pattern. A total of 23 CpG loci located in 19 known gene regions, including gene-body and promoter, appeared to be significantly differentially methylated. Replication by pyrosequencing revealed that one CpG site in PSMD8 showed the same trend of differential methylation and reached to statistical significance as the microarray result. CONCLUSION: Our preliminary findings provide evidence for molecular heterogeneity in patients with AECOPD, which may contribute to significant differences in their response to COPD treatment.

Combining Whispering-Gallery Mode Optical Biosensors with Microfluidics for Real-Time Detection of Protein Secretion from Living Cells in Complex Media.

The noninvasive monitoring of protein secretion of cells responding to drug treatment is an effective and essential tool in latest drug development and for cytotoxicity assays. In this work, a surface functionalization method is demonstrated for specific detection of protein released from cells and a platform that integrates highly sensitive optical devices, called whispering-gallery mode biosensors, with precise microfluidics control to achieve label-free and real-time detection. Cell biomarker release is measured in real time and with nanomolar sensitivity. The surface functionalization method allows for antibodies to be immobilized on the surface for specific detection, while the microfluidics system enables detection in a continuous flow with a negligible compromise between sensitivity and flow control over stabilization and mixing. Cytochrome c detection is used to illustrate the merits of the system. Jurkat cells are treated with the toxin staurosporine to trigger cell apoptosis and cytochrome c released into the cell culture medium is monitored via the newly invented optical microfluidic platform.

Electrostatic Assist of Liquid Transfer between Flat Surfaces.

Transfer of liquid from one surface to another plays a vital role in printing processes. During liquid transfer, a liquid bridge is formed and subjected to substantial extension but incomplete liquid transfer can produce defects that are detrimental to the operation of printed electronic devices. One strategy for minimizing these defects is to apply an electric field, a technique known as electrostatic assist (ESA). However, the physical mechanisms underlying ESA remain a mystery. To better understand these mechanisms, slender-jet models are developed for both perfect dielectric and leaky dielectric axisymmetric Newtonian liquid bridges with moving contact lines. Nonlinear partial differential equations describing the evolution of the bridge radius and interfacial charge are derived and then solved using finite element methods. For perfect dielectrics, application of an electric field enhances liquid transfer to the more wettable surface over a wide range of capillary numbers. The electric field modifies the pressure differences inside the liquid bridge and, as a consequence, drives liquid toward the more wettable surface. For leaky dielectrics, charge can accumulate at the liquid-air interface. Application of an electric field can augment or oppose the influence of wettability differences, depending on the direction of the electric field and the sign of the surface charge. Flow visualization experiments reveal that when an electric field is applied, more liquid is transferred to the more wettable surface because of a modified bridge shape that causes depinning of the contact line. The measured values of the amount of liquid transferred are in good agreement with predictions of the perfect dielectric model.

Biomolecular Clusters Distribution up to Mega Dalton Region Using MALDI-Quadrupole Ion Trap Mass Spectrometer.

We present the first report on complete cluster distributions of cytochrome c (molecular weight of 12.4 kDa) and bovine serum albumin ((BSA), molecular weight of 66.4 kDa) with mass-to-charge ratio (m/z) reaching 350,000 and 1,400,000, respectively, by matrix-assisted laser desorption/ionization (MALDI). Large cluster distributions of the analytes were measured by our homemade frequency-scanned quadrupole ion trap (QIT) mass spectrometer with a charge detector. To our knowledge, we report the highest m/z clusters of these two biomolecules. The quantitative results indicate that large clusters ions of cytochrome c and BSA follow the power law (r² > 0.99) with cluster size distribution, which provides experimental evidence for the laser ablation studies of MALDI.

Quantization and Bifurcation beyond Square-Integrable Wavefunctions.

Probability interpretation is the cornerstone of standard quantum mechanics. To ensure the validity of the probability interpretation, wavefunctions have to satisfy the square-integrable (SI) condition, which gives rise to the well-known phenomenon of energy quantization in confined quantum systems. On the other hand, nonsquare-integrable (NSI) solutions to the Schrödinger equation are usually ruled out and have long been believed to be irrelevant to energy quantization. This paper proposes a quantum-trajectory approach to energy quantization by releasing the SI condition and considering both SI and NSI solutions to the Schrödinger equation. Contrary to our common belief, we find that both SI and NSI wavefunctions contribute to energy quantization. SI wavefunctions help to locate the bifurcation points at which energy has a step jump, while NSI wavefunctions form the flat parts of the stair-like distribution of the quantized energies. The consideration of NSI wavefunctions furthermore reveals a new quantum phenomenon regarding the synchronicity between the energy quantization process and the center-saddle bifurcation process.