Blockade of interleukin-6 receptor attenuates apoptosis and modulates the inflammatory response in Mycoplasma pneumoniae infected A549 cells.

OBJECTIVE: To investigate the effect of silencing the interleukin (IL)-6 gene on the induction of inflammation, oxidative stress and apoptosis in Mycoplasma pneumoniae (MP) infected A549 cells and its mechanism of action. METHODS: IL-6 small interfering RNA (siRNA) was synthesized and transfected into A549 cells, which were divided into a blank control group, a negative control group, and an IL-6 siRNA group. The mRNA and protein expression of IL-6 and the protein expression of CyclinD1, Cleaved caspase-3, Bax, B-cell lymphoma 2 (Bcl-2), signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p-STAT3), matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Besides, cell viability and apoptosis were determined. Additionally, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), IL-1ß, IL-8 and tumor necrosis factor (TNF)-α were measured. RESULTS: The mRNA and protein levels of IL-6 in the IL-6 siRNA group were lower than those in the blank and negative control groups (P < 0.05). The IL-6 siRNA group had higher viability but lower apoptosis rate of A549 cells at 24 h, 48 h and 72 h than the blank and negative control groups (P < 0.05). The IL-6 siRNA group had lower protein expression levels of Cleaved caspase-3 and Bax, but higher protein expression levels of CyclinD1 and Bcl-2 than the blank and negative control groups (P < 0.05). The IL-6 siRNA group had lower levels of IL-6, IL-8, TNF-α and MDA, but higher levels of SOD and GSH-PX than the blank and negative control groups (P < 0.05). CONCLUSION: Silencing the IL-6 gene can reduce the MP-induced inflammatory response and oxidative stress of A549 cells, enhance cell viability and inhibit apoptosis. Meanwhile, it was also found that STAT3 expression was inhibited after silencing IL-6 gene expression. Therefore, it is speculated that IL-6 may play a role by regulating STAT3, but its exact molecular biological mechanism still needs to be further explored.

Improving diagnosing performance for malignant parotid gland tumors using machine learning with multifeatures based on diffusion-weighted magnetic resonance imaging.

In this study, the performance of machine learning in classifying parotid gland tumors based on diffusion-related features obtained from the parotid gland tumor, the peritumor parotid gland, and the contralateral parotid gland was evaluated. Seventy-eight patients participated in this study and underwent magnetic resonance diffusion-weighted imaging. Three regions of interest, including the parotid gland tumor, the peritumor parotid gland, and the contralateral parotid gland, were manually contoured for 92 tumors, including 20 malignant tumors (MTs), 42 Warthin tumors (WTs), and 30 pleomorphic adenomas (PMAs). We recorded multiple apparent diffusion coefficient (ADC) features and applied a machine-learning method with the features to classify the three types of tumors. With only mean ADC of tumors, the area under the curve of the classification model was 0.63, 0.85, and 0.87 for MTs, WTs, and PMAs, respectively. The performance metrics were improved to 0.81, 0.89, and 0.92, respectively, with multiple features. Apart from the ADC features of parotid gland tumor, the features of the peritumor and contralateral parotid glands proved advantageous for tumor classification. Combining machine learning and multiple features provides excellent discrimination of tumor types and can be a practical tool in the clinical diagnosis of parotid gland tumors.

Preclinical Studies of OBI-999: A Novel Globo H-Targeting Antibody-Drug Conjugate.

Globo H (GH), a hexasaccharide, is expressed at low levels in normal tissues but is highly expressed in multiple cancer types, rendering it a promising target for cancer immunotherapy. OBI-999, a novel antibody-drug conjugate, is derived from a conjugation of a GH-specific mAb with a monomethyl auristatin E (MMAE) payload through a site-specific ThioBridge and a cleavable linker. OBI-999 high homogeneity with a drug-to-antibody ratio of 4 (>95%) was achieved using ThioBridge. OBI-999 displayed GH-dependent cellular internalization and trafficked to endosome and lysosome within 1 and 5 hours, respectively. Furthermore, OBI-999 showed low nanomolar cytotoxicity in the assay with high GH expression on tumor cells and exhibited a bystander killing effect on tumor cells with minimal GH expression. Tissue distribution indicated that OBI-999 and free MMAE gradually accumulated in the tumor, reaching maximum level at 168 hours after treatment, whereas OBI-999 and free MMAE decreased quickly at 4 hours after treatment in normal organs. Maximum MMAE level in the tumor was 16-fold higher than in serum, suggesting that OBI-999 is stable during circulation and MMAE is selectively released in the tumor. Excellent tumor growth inhibition of OBI-999 was demonstrated in breast, gastric, and pancreatic cancer xenograft or lung patient-derived xenograft models in a dose-dependent manner. The highest nonseverely toxic dose in cynomolgus monkeys is 10 mg/kg determined by a 3-week repeated-dose toxicology study demonstrating an acceptable safety margin. Taken together, these results support further clinical development of OBI-999, which is currently in a phase I/II clinical study in multiple solid tumors (NCT04084366). OBI-999, the first GH-targeting ADC, displayed excellent tumor inhibition in animal models across multiple cancer types, including breast, gastric, pancreatic, and lung cancers, warranting further investigation in the treatment of solid tumors.

Classification of parotid gland tumors by using multimodal MRI and deep learning.

Various MRI sequences have shown their potential to discriminate parotid gland tumors, including but not limited to T2 -weighted, postcontrast T1 -weighted, and diffusion-weighted images. In this study, we present a fully automatic system for the diagnosis of parotid gland tumors by using deep learning methods trained on multimodal MRI images. We used a two-dimensional convolution neural network, U-Net, to segment and classify parotid gland tumors. The U-Net model was trained with transfer learning, and a specific design of the batch distribution optimized the model accuracy. We also selected five combinations of MRI contrasts as the input data of the neural network and compared the classification accuracy of parotid gland tumors. The results indicated that the deep learning model with diffusion-related parameters performed better than those with structural MR images. The performance results (n = 85) of the diffusion-based model were as follows: accuracy of 0.81, 0.76, and 0.71, sensitivity of 0.83, 0.63, and 0.33, and specificity of 0.80, 0.84, and 0.87 for Warthin tumors, pleomorphic adenomas, and malignant tumors, respectively. Combining diffusion-weighted and contrast-enhanced T1 -weighted images did not improve the prediction accuracy. In summary, the proposed deep learning model could classify Warthin tumor and pleomorphic adenoma tumor but not malignant tumor.

Two-channel autofluorescence analysis for oral cancer.

We created a two-channel autofluorescence test to detect oral cancer. The wavelengths 375 and 460 nm, with filters of 479 and 525 nm, were designed to excite and detect reduced-form nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Patients with oral cancer or with precancerous lesions, and a control group with healthy oral mucosae, were enrolled. The lesion in the autofluorescent image was the region of interest. The average intensity and heterogeneity of the NADH and FAD were calculated. The redox ratio [(NADH)/(NADH + FAD)] was also computed. A quadratic discriminant analysis (QDA) was used to compute boundaries based on sensitivity and specificity. We analyzed 49 oral cancer lesions, 34 precancerous lesions, and 77 healthy oral mucosae. A boundary (sensitivity: 0.974 and specificity: 0.898) between the oral cancer lesions and healthy oral mucosae was validated. Oral cancer and precancerous lesions were also differentiated from healthy oral mucosae (sensitivity: 0.919 and specificity: 0.755). The two-channel autofluorescence detection device and analyses of the intensity and heterogeneity of NADH, and of FAD, and the redox ratio combined with a QDA classifier can differentiate oral cancer and precancerous lesions from healthy oral mucosae.

miR-590 promotes the proliferation of HUMSCs and induces ECM synthesis by targeting Smad7.

MicroRNA (miR)-590 has been established to be a promoter of cell proliferation, migration and invasion, and an inhibitor of apoptosis in numerous cancer cell lines. However, its effects on non-cancer cells remain to be elucidated. miR-590 was transfected into human umbilical cord mesenchymal stem cells (HUMSCs), and the cell proliferation rate was determined using a Cell-Light 5-ethynyl-20-deoxyuridine Apollo 567 kit and the presence of extracellular matrix (ECM) proteins were detected using western blot analysis and immunofluorescence microscopy. Using bioinformatic analysis and dual-luciferase assays, the novel target miR-590 was identified. In addition, the effects of miR-590 on cell proliferation and ECM enhancement were also evaluated. The results of the present study demonstrated that miR-590 interacts directly with the 3'-untranslated region of Mothers Against Decapentaplegic Homolog 7 (Smad7), which is an important factor in transforming growth factor-ß signaling pathway. Overexpression of miR-590 downregulated Smad7 expression at the mRNA and protein level, and subsequently resulted in cell proliferation and ECM accumulation. Additionally, the transfection of small interfering RNA targeting Smad7 in HUMSCs produced similar effects on cell proliferation and ECM to the overexpression of miR-590. The results of the present study indicated that miR-590 affects HUMSC proliferation by directly targeting Smad7.

Inhibition of the PI3K-Akt-mTOR signaling pathway in T lymphocytes in patients with active tuberculosis.

OBJECTIVES: To investigate PI3K-Akt-mTOR signaling pathway changes and the proliferation of FoxP3+Treg cells in patients with active tuberculosis. METHODS: We isolated PBMCs and CD4+CD25+FoxP3+Treg cells from peripheral blood collected from patients with active tuberculosis and healthy controls. We compared the proportion and MFI of PI3K-Akt-mTOR pathway components and PTEN by flow cytometry using specific cell-surface and intracellular markers. Moreover, we detected the specific secretory proteins ESAT-6 and Ag85B, cytokines IL-10, TGF-ß1 and IL-35 in serum by ELISA. RESULTS: Compared with healthy controls, the proportions of CD3+Akt+, CD3+p-Akt+, CD3+mTOR+, CD3+p-mTOR+ and CD3+PTEN+ cells, in the T lymphocyte population of patients with active tuberculosis, were decreased (p<0.05), while CD3+FoxP3+ cells were increased (p=0.013). Similarly, for CD4+CD25+FoxP3+Treg cells, the proportions of Akt+ cells, p-Akt+ cells, mTOR+ cells, p-mTOR+ cells and PTEN+ cells were decreased (p<0.05) in patients with active tuberculosis. Compared with healthy controls, the levels of ESAT-6 and Ag85B were higher in patients with active tuberculosis (p<0.001). Levels of IL-10 and TGF-ß1 were higher (p<0.001), whereas the level of IL-35 was lower (p<0.001). CONCLUSION: The PI3K-Akt-mTOR signaling pathway in T lymphocytes and CD4+CD25+FoxP3+Treg cells was inhibited, which could explain why M.tuberculosis can induce FoxP3+Treg cell to expand.

Carboxyl-functionalized graphene oxide composites as SPR biosensors with enhanced sensitivity for immunoaffinity detection.

This work demonstrates the excellent potential of carboxyl-functionalized graphene oxide (GO-COOH) composites to form biocompatible surfaces on sensing films for use in surface plasmon resonance (SPR)-based immunoaffinity biosensors. Carboxyl-functionalization of graphene carbon can modulate its visible spectrum, and can therefore be used to improve and control the plasmonic coupling mechanism. The binding properties of the molecules between a sensing film and a protein were elucidated at various flow rates of those molecules. The bio-specific binding interaction among the molecules was investigated by performing an antigen and antibody affinity immunoassay. The results thus obtained revealed that the overall affinity binding value, KA, of the Au/GO-COOH chip can be significantly enhanced by up to ∼5.15 times that of the Au/GO chip. With respect to the shifts of the SPR angles of the chips, the affinity immunoassay interaction at a BSA concentration of 1µg/ml for an Au/GO-COOH chip, an Au/GO chip and a traditional SPR chip are 35.5m°, 9.128m° and 8.816m°, respectively. The enhancement of the antigen-antibody interaction of the Au/GO-COOH chip cause this chip to become four times as sensitive to the SPR angle shift and to have the lowest antibody detection limit of 0.01pg/ml. These results indicate the potential of the chip in detecting specific proteins, and the development of real-time in vivo blood analysis and diagnosis based on cancer tumor markers.

A potential risk of overestimating apparent diffusion coefficient in parotid glands.

OBJECTIVES: To investigate transient signal loss on diffusion weighted images (DWI) and overestimation of apparent diffusion coefficient (ADC) in parotid glands using single shot echoplanar DWI (EPDWI). MATERIALS AND METHODS: This study enrolled 6 healthy subjects and 7 patients receiving radiotherapy. All participants received dynamic EPDWI with a total of 8 repetitions. Imaging quality of DWI was evaluated. Probability of severe overestimation of ADC (soADC), defined by an ADC ratio more than 1.2, was calculated. Error on T2WI, DWI, and ADC was computed. Statistical analysis included paired Student t testing and Mann-Whitney U test. A P value less than 0.05 was considered statistically significant. RESULTS: Transient signal loss was visually detected on some excitations of DWI but not on T2WI or mean DWI. soADC occurred randomly among 8 excitations and 3 directions of diffusion encoding gradients. Probability of soADC was significantly higher in radiotherapy group (42.86%) than in healthy group (24.39%). The mean error percentage decreased as the number of excitations increased on all images, and, it was smallest on T2WI, followed by DWI and ADC in an increasing order. CONCLUSIONS: Transient signal loss on DWI was successfully detected by dynamic EPDWI. The signal loss on DWI and overestimation of ADC could be partially remedied by increasing the number of excitations.

Correction of geometric distortion in Propeller echo planar imaging using a modified reversed gradient approach.

OBJECTIVE: This study investigates the application of a modified reversed gradient algorithm to the Propeller-EPI imaging method (periodically rotated overlapping parallel lines with enhanced reconstruction based on echo-planar imaging readout) for corrections of geometric distortions due to the EPI readout. MATERIALS AND METHODS: Propeller-EPI acquisition was executed with 360-degree rotational coverage of the k-space, from which the image pairs with opposite phase-encoding gradient polarities were extracted for reversed gradient geometric and intensity corrections. The spatial displacements obtained on a pixel-by-pixel basis were fitted using a two-dimensional polynomial followed by low-pass filtering to assure correction reliability in low-signal regions. Single-shot EPI images were obtained on a phantom, whereas high spatial resolution T2-weighted and diffusion tensor Propeller-EPI data were acquired in vivo from healthy subjects at 3.0 Tesla, to demonstrate the effectiveness of the proposed algorithm. RESULTS: Phantom images show success of the smoothed displacement map concept in providing improvements of the geometric corrections at low-signal regions. Human brain images demonstrate prominently superior reconstruction quality of Propeller-EPI images with modified reversed gradient corrections as compared with those obtained without corrections, as evidenced from verification against the distortion-free fast spin-echo images at the same level. CONCLUSIONS: The modified reversed gradient method is an effective approach to obtain high-resolution Propeller-EPI images with substantially reduced artifacts.

Gas-phase chemistry of multiply charged bioions in analytical mass spectrometry.

Ion chemistry has long played an important role in molecular mass spectrometry (MS), as it is central to the use of MS as a structural characterization tool. With the advent of ionization methods capable of producing gaseous ions from large biomolecules, the chemistry of gaseous bioions has become a highly active area of research. Gas-phase biomolecule-ion reactions are usually driven by interactions with neutral molecules, photons, electrons, ions, or surfaces. Ion dissociation or transformation into different ion types can be achieved. The types of reaction products observed depend on the characteristics of the ions, the transformation methods, and the time frame of observation. This review focuses on the gas-phase chemistries of ions derived from the electrospray ionization of peptides, proteins, and oligonucleotides, with particular emphasis on their utility in bioanalysis. Various ion-transformation strategies, which further facilitate structural interrogation by converting ions from one type to another, are also summarized.

Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation.

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74-95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA(Phe); rather, the mass was consistent with tRNA(Phe) bearing an incomplete 3'-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5'- and 3'-termini of the molecule could be used to identify the major component as tRNA(Phe)-3'adenosine (without 3'-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA(Phe) variant and the intact tRNA(Phe) was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA(Phe)-3'adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.

Electron-transfer reagent anion formation via electrospray ionization and collision-induced dissociation.

A strategy is described and demonstrated for the formation of reagent anions via electrospray ionization (ESI) for electron-transfer dissociation (ETD). To circumvent difficulties associated with formation of high mass-to-charge ratio (m/z) reagent anions, it is desirable to form ETD reagents via means other than those that require reagent molecule vaporization. ESI is a candidate method, but anions that are generally generated efficiently by ESI tend to react with multiply protonated polypeptides via proton transfer. The strategy described herein involves the use of a precursor reagent molecule that ionizes efficiently via electrospray ionization and that can subsequently be converted to an ETD reagent via gas-phase dissociation. The approach is demonstrated with arenecarboxylic acids that yield strong signals associated with the deprotonated molecule and that subsequently undergo collision-induced dissociation (CID) by loss of CO(2). In the present work, triply protonated KGAILKGAILR served as a test substrate for the CID product ions to give rise to ETD. Several precursor molecules were shown to be capable of generating ETD reagents via ESI followed by CID. These included 9-anthracenecarboxylic acid, 2-fluoro-5-iodobenzoic acid, and 2-(fluoranthene-8-carbonyl)benzoic acid. The latter molecule has the most attractive set of characteristics as a precursor for a relatively high m/z ratio ETD reagent.