Periodontal ligament-associated protein-1 knockout mice regulate the differentiation of osteoclasts and osteoblasts through TGF-ß1/Smad signaling pathway.

It has been known that periodontal ligament-associated protein-1 (PLAP-1/Asporin) not only inhibits cartilage formation in osteoarthritis, but it also influences the healing of skull defect. However, the effect and mechanism of PLAP-1/Asporin on the mutual regulation of osteoclasts and osteoblasts in periodontitis are not clear. In this study, we utilized a PLAP-1/Asporin gene knockout (KO) mouse model to research this unknown issue. We cultured mouse bone marrow mesenchymal stem cells with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) for osteogenic induction in vitro. The molecular mechanism of PLAP-1/Asporin in the regulation of osteoblasts was detected by immunoprecipitation, immunofluorescence, and inhibitors of signaling pathways. The results showed that the KO of PLAP-1/Asporin promoted osteogenic differentiation through transforming growth factor beta 1 (TGF-ß1)/Smad3 in inflammatory environments. We further found the KO of PLAP-1/Asporin inhibited osteoclast differentiation and promoted osteogenic differentiation through the TGF-ß1/Smad signaling pathway in an inflammatory coculture system. The experimental periodontitis model was established by silk ligation and the alveolar bone formation in PLAP-1/Asporin KO mice was promoted through TGF-ß1/Smad3 signaling pathway. The subcutaneous osteogenesis model in nude mice also confirmed that the KO of PLAP-1/Asporin promoted bone formation by the histochemical staining. In conclusion, PLAP-1/Asporin regulated the differentiation of osteoclasts and osteoblasts through TGF-ß1/Smad signaling pathway. The results of this study lay a theoretical foundation for the further study of the pathological mechanism underlying alveolar bone resorption, and the prevention and treatment of periodontitis.

HDAC inhibitor regulates the tumor immune microenvironment via pyroptosis in triple negative breast cancer.

Pyroptosis, an inflammatory form of cell death, promotes the release of immunogenic substances and stimulates immune cell recruitment, a process, which could turn cold tumors into hot ones. Thus, instigating pyroptosis in triple-negative breast cancer (TNBC) serves as a viable method for restoring antitumor immunity. We analyzed the effects of Histone Deacetylase Inhibitors (HDACi) on TNBC cells using the Cell Counting Kit-8 and colony formation assay. Apoptosis and lactate dehydrogenase (LDH) release assays were utilized to determine the form of cell death. The pyroptotic executor was validated by quantitative real-time polymerase chain reaction and western blot. Transcriptome was analyzed to investigate pyroptosis-inducing mechanisms. A subcutaneously transplanted tumor model was generated in BALB/c mice to evaluate infiltration of immune cells. HDACi significantly diminished cell proliferation, and pyroptotic "balloon"-like cells became apparent. HDACi led to an intra and extracellular material exchange, signified by the release of LDH and the uptake of propidium iodide. Among the gasdermin family, TNBC cells expressed maximum quantities of GSDME, and expression of GSDMA, GSDMB, and GSDME were augmented post HDACi treatment. Pyroptosis was instigated via the activation of the caspase 3-GSDME pathway with the potential mechanisms being cell cycle arrest and altered intracellular REDOX balance due to aberrant glutathione metabolism. In vivo experiments demonstrated that HDACi can activate pyroptosis, limit tumor growth, and escalate CD8+ lymphocyte and CD11b+ cell infiltration along with an increased presence of granzyme B in tumors. HDACi can instigate pyroptosis in TNBC, promoting infiltration of immune cells and consequently intensifying the efficacy of anticancer immunity.

Targeting the inward rectifier potassium channel 5.1 in thyroid cancer: artificial intelligence-facilitated molecular docking for drug discovery.

BACKGROUND: Recurrent and metastatic thyroid cancer is more invasive and can transform to dedifferentiated thyroid cancer, thus leading to a severe decline in the 10-year survival. The thyroid-stimulating hormone receptor (TSHR) plays an important role in differentiation process. We aim to find a therapeutic target in redifferentiation strategies for thyroid cancer. METHODS: Our study integrated the differentially expressed genes acquired from the Gene Expression Omnibus database by comparing TSHR expression levels in the Cancer Genome Atlas database. We conducted functional enrichment analysis and verified the expression of these genes by RT-PCR in 68 pairs of thyroid tumor and paratumor tissues. Artificial intelligence-enabled virtual screening was combined with the VirtualFlow platform for deep docking. RESULTS: We identified five genes (KCNJ16, SLC26A4, TG, TPO, and SYT1) as potential cancer treatment targets. TSHR and KCNJ16 were downregulated in the thyroid tumor tissues, compared with paired normal tissues. In addition, KCNJ16 was lower in the vascular/capsular invasion group. Enrichment analyses revealed that KCNJ16 may play a significant role in cell growth and differentiation. The inward rectifier potassium channel 5.1 (Kir5.1, encoded by KCNJ16) emerged as an interesting target in thyroid cancer. Artificial intelligence-facilitated molecular docking identified Z2087256678_2, Z2211139111_1, Z2211139111_2, and PV-000592319198_1 (-7.3 kcal/mol) as the most potent commercially available molecular targeting Kir5.1. CONCLUSION: This study may provide greater insights into the differentiation features associated with TSHR expression in thyroid cancer, and Kir5.1 may be a potential therapeutic target in the redifferentiation strategies for recurrent and metastatic thyroid cancer.

Quantification of Intracellular Proteins in Single Cells Based on Engineered Picoliter Droplets.

Unlike conventional bulk measurements, single-cell protein analysis permits quantification of protein expression in individual cells. This has shed light on the cell-to-cell variation in heterogeneous biological systems, such as solid tumors, brain tissues, and developing embryos. Herein, a microfluidic method is developed to profile protein expression in individual cells by performing single-cell intracellular protein immunoassay in picoliter paired droplets. The high sensitivity of single-cell protein analysis on a chip is achieved by the confined reaction volume of picoliter droplets, efficient kinetic characteristics of the immunoassay through active mixing, and minimum single-cell protein loss by integrated operations. The abundance of an intracellular prostate specific antigen at the single-cell level is measured, and then the platform is applied to identify cell types and investigate heterogeneity within cell populations. Overall, a paired chip for single-cell immunoassay establishes a foundation for parallel, sensitive, and integrated protein quantification at the single-cell level and will find wide applications in the field of single-cell proteomics.

A multicenter, prospective study to observe the initial management of patients with differentiated thyroid cancer in China (DTCC study).

BACKGROUND: To assess the gaps between the initial management of patients with differentiated thyroid cancer (DTC) in real clinical practice and the recommendations of the 2012 Chinese DTC guidelines. METHODS: This multicenter, prospective study was conducted at nine tertiary hospitals across China. Eligible patients were those having intermediate or high-risk DTC after first-time thyroidectomy. During 1 year of follow-up, comprehensive medical records were collected and summarized using descriptive statistics. RESULTS: Of 2013 patients, 1874 (93.1%) underwent standard surgery according to the guidelines (including total lobectomy plus isthmusectomy and total/near total thyroidectomy), and 1993 (99.0%) underwent lymph node dissection; only 56 (2.8%) had postoperative complications. Overall, 982/2013 patients (48.8%) received radioactive iodine (RAI) therapy after thyroidectomy. Of all enrolled patients, 61.4% achieved the target serum thyroid-stimulating hormone level, with a median time to target of 234.0 days (95% CI: 222.0-252.0). At 1 year of follow-up, proportions of patients with excellent response, incomplete structural response, biochemical incomplete response, and indeterminate response were 34.6, 11.2, 6.6, and 47.5%, respectively; recurrence or metastasis occurred in 27 patients (1.3%). During the overall study period, 209 patients (10.4%) had at least one adverse event: 65.1% of cases were mild, 24.9% moderate, and 10.1% severe. CONCLUSIONS: This was the first large-scale prospective study of how patients with DTC in China are treated in actual practice. Initial DTC management is generally safe and adheres to the 2012 Chinese guidelines but could be improved, and the level of guideline adherence did not produce the anticipated treatment response at 1 year of follow-up.

Lymph Node Metastasis Prediction from Primary Breast Cancer US Images Using Deep Learning.

Background Deep learning (DL) algorithms are gaining extensive attention for their excellent performance in image recognition tasks. DL models can automatically make a quantitative assessment of complex medical image characteristics and achieve increased accuracy in diagnosis with higher efficiency. Purpose To determine the feasibility of using a DL approach to predict clinically negative axillary lymph node metastasis from US images in patients with primary breast cancer. Materials and Methods A data set of US images in patients with primary breast cancer with clinically negative axillary lymph nodes from Tongji Hospital (974 imaging studies from 2016 to 2018, 756 patients) and an independent test set from Hubei Cancer Hospital (81 imaging studies from 2018 to 2019, 78 patients) were collected. Axillary lymph node status was confirmed with pathologic examination. Three different convolutional neural networks (CNNs) of Inception V3, Inception-ResNet V2, and ResNet-101 architectures were trained on 90% of the Tongji Hospital data set and tested on the remaining 10%, as well as on the independent test set. The performance of the models was compared with that of five radiologists. The models' performance was analyzed in terms of accuracy, sensitivity, specificity, receiver operating characteristic curves, areas under the receiver operating characteristic curve (AUCs), and heat maps. Results The best-performing CNN model, Inception V3, achieved an AUC of 0.89 (95% confidence interval [CI]: 0.83, 0.95) in the prediction of the final clinical diagnosis of axillary lymph node metastasis in the independent test set. The model achieved 85% sensitivity (35 of 41 images; 95% CI: 70%, 94%) and 73% specificity (29 of 40 images; 95% CI: 56%, 85%), and the radiologists achieved 73% sensitivity (30 of 41 images; 95% CI: 57%, 85%; P = .17) and 63% specificity (25 of 40 images; 95% CI: 46%, 77%; P = .34). Conclusion Using US images from patients with primary breast cancer, deep learning models can effectively predict clinically negative axillary lymph node metastasis. Artificial intelligence may provide an early diagnostic strategy for lymph node metastasis in patients with breast cancer with clinically negative lymph nodes. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Bae in this issue.

Novel sequential treatment with palbociclib enhances the effect of cisplatin in RB-proficient triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive malignancy that lacks sensitivity to chemotherapy, endocrine therapy or targeted therapy. CDK4/6 inhibitors, combined with endocrine therapy, have been shown to be effective in postmenopausal women with HR-positive, HER2-negative advanced or metastatic breast cancer. Therefore, we investigated whether the CDK4/6 inhibitor palbociclib (PD) could enhance the effects of cisplatin (CDDP) on TNBC. METHODS: The effects of different drug regimens consisting of PD and CDDP on MDA-MB-231 and RB-knockdown MDA-MB-231 (sh-MDA-MB-231) cells were assessed in vitro and in vivo. MDA-MB-468 and RB-overexpressing MDA-MB-468 cells were used to assess the effect of the PD-CDDP regimens in vitro. Immunoblotting illustrated the role of the cyclin D1/RB/E2F axis signalling pathway. RESULTS: PD induced G1 phase cell cycle arrest in the MDA-MB-231 cell line. However, synchronous treatment with PD and CDDP for 24 h, treatment with PD for 24 h followed by CDDP and treatment with CDDP for 24 h followed by PD had no influence on MDA-MB-231 cell apoptosis. We further investigated the effect of PD or CDDP withdrawal on the effects of sequential treatment and found that PD treatment for 48 h followed by withdrawal for 48 h and subsequent CDDP treatment (PD-CDDP) significantly increased apoptosis and inhibited the cell viability and colony formation of MDA-MB-231 cells, while with other regimens, PD and CDDP had an additive or antagonistic response. The preferential use of PD increased DNA damage induced by CDDP, as measured through γH2AX immunofluorescence. These findings were not observed in sh-MDA-MB-231 cells, and experiments to assess cell function in MDA-MB-468 and RB-overexpressing MDA-MB-468 cells yielded similar results, which indicated that PD enhanced the sensitivity of TNBC cells to CDDP in an RB-dependent manner. In vivo, compared with single drug treatment, combination treatment inhibited tumour growth and Ki-67 expression in MDA-MB-231 xenograft models. Western blot analysis revealed that PD enhanced sensitivity to CDDP through the CDK4/6-cyclin D1-RB-E2F pathway. CONCLUSIONS: Pre-treatment with PD synchronized the tumour cell cycle through the CDK4/6-cyclin D1-RB-E2F pathway, which increased the antitumour effect of CDDP. Thus, PD-CDDP might be an effective treatment for RB-proficient TNBC patients.

DNA Nanolithography Enables a Highly Ordered Recognition Interface in a Microfluidic Chip for the Efficient Capture and Release of Circulating Tumor Cells.

Microfluidic chips with nano-scale structures have shown great potential, but the fabrication and cost issues restrict their application. Herein, we propose a conceptually new "DNA nanolithography in a microfluidic chip" by using sub-10 nm three-dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN-Chip). The nano-scale framework ensures that the aptamer is in a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN-Chip was enhanced nearly 60 % due to the highly precise dimension and rigid framework of TDNs. In addition, the scaffolds make DNase I more accessible to the aptamer with up to 83 % release efficiency and 91 % cell viability, which is fully compatible with downstream molecular analysis. Overall, this strategy provides a novel perspective on engineering nano-scaffolds to achieve a more ordered nano-topography of microfluidic chips.

Stable Colloidosomes Formed by Self-Assembly of Colloidal Surfactant for Highly Robust Digital PCR.

As the third-generation nucleic acid amplification technology, digital polymerase chain reaction (PCR) has been widely adopted in the analysis of nucleic acids. However, further application of this powerful technology is hindered due to the limitation of surfactants. Here, for the first time, we propose the use of colloidosomes self-assembled from fluorinated silica nanoparticle for digital PCR to address this limitation. A one-step fluorinated silica nanoparticle synthesis method is proposed, which is much more convenient and reproducible compared with the synthesis of conventional fluorine-based surfactants. Fluorinated silica nanoparticles facilitate the formation of colloidosomes with excellent stability capable of enduring the rapid temperature changes associated with the PCR and avoiding material exchange (cross-talk) between droplets for high-fidelity analysis. The colloidosome digital PCR method was developed using these colloidosomes as highly parallel reactors for single-copy nucleic acid amplification and rare mutant detection. The method is robust and accurate, and it offers possibilities for a great variety of applications, such as gene expression studies, single cell analysis, and circulating tumor DNA detection.

Centrifugal-Driven Droplet Generation Method with Minimal Waste for Single-Cell Whole Genome Amplification.

High-quality whole-genome amplification (WGA) of individual cells is the primary step for characterizing the genetic information on single cells in biology and medicine. As the most popular single-cell WGA method, multiple displacement amplification (MDA) is often plagued by the nonuniform amplification. The droplet MDA has been an innovative tool to solve this dilemma by mitigating the amplification bias and increasing the genomic coverage. Despite these advantages, the time-consuming droplet generation process, the waste of small volume samples and the difficulty of parallel operation for multiple single-cell samples remain major obstacles. Herein, we introduce a centrifugal-driven droplet generation method for rapid and convenient generation of uniform droplets from a relatively small volume sample (5 µL) in 60s with more than 98% sample utilization. We have performed quantitative digital droplet PCR using this method, demonstrating its capability of amplifying nucleic acids at the single-molecule level. Single-cell centrifugal-driven droplet MDA (cd-MDA) has also been conducted for single-cell sequencing, achieving uniform amplification and broad genomic coverage. With the single-molecule sensitivity, minimum sample waste, high genomic coverage, and excellent sequencing evenness, this centrifugal-driven droplet generation method is promising for convenient and scalable use in digital PCR and single-cell whole-genome research.

Visual Quantitative Detection of Circulating Tumor Cells with Single-Cell Sensitivity Using a Portable Microfluidic Device.

Immunocytological technologies, molecular technologies, and functional assays are widely used for detecting circulating tumor cells (CTCs) after enrichment from patients' blood sample. Unfortunately, accessibility to these technologies is limited due to the need for sophisticated instrumentation and skilled operators. Portable microfluidic devices have become attractive tools for expanding the access and efficiency of detection beyond hospitals to sites near the patient. Herein, a volumetric bar chart chip (V-Chip) is developed as a portable platform for CTC detection. The target CTCs are labeled with aptamer-conjugated nanoparticles (ACNPs) and analyzed by V-Chip through quantifying the byproduct (oxygen) of the catalytic reaction between ACNPs and hydrogen peroxide, which results in the movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Thus, the CTC number is decoded into visually quantifiable information and a linear correlation can be found between the distance moved by the ink and number of cells in the sample. This method is sensitive enough that a single cell can be detected. Furthermore, the clinical capabilities of this system are demonstrated for quantitative CTC detection in the presence of a high leukocyte background. This portable detection method shows great potential for quantification of rare cells with single-cell sensitivity for various applications.

Golgi Membrane Protein 1 (GOLM1) Promotes Growth and Metastasis of Breast Cancer Cells via Regulating Matrix Metalloproteinase-13 (MMP13).

BACKGROUND Breast cancer (BC) is the leading cause of death in women worldwide. Golgi membrane protein 1 (GOLM1) has been identified as novel regulator in carcinogenesis, but its function in BC is unclear. MATERIAL AND METHODS The expression of GOLM1 in BC tissues and cell lines was detected by using qRT-PCR assay. CCK-8 and colony-formation assays were used to evaluate BC cell growth in vivo. Wound-healing and Transwell assays were used to detect cell migration and invasion. To investigate GOLM1 functions in vivo, we established a xenograft mice model and a lung metastasis model. The level of epithelial-to-mesenchymal transition (EMT)-related markers was analyzed by immunofluorescent staining. RESULTS GOLM1 was overexpressed in BC cell lines and tissues. Overexpression of GOLM1 induced EMT and promoted proliferation, migration, and invasion of BC cells. Furthermore, overexpressing of GOLM1 markedly promoted the tumorigenicity and metastasis of BC cells in vivo, whereas knock-down of GOLM1 caused the opposite outcomes. Furthermore, we proved that GOLM1 promoted BC cell aggressiveness by regulating matrix metalloproteinase-13 (MMP13). CONCLUSIONS Our results prove that GOLM1 facilitates the growth and metastasis of breast cancer cells.

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 µm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

Comparative metabolomic analysis of Crypthecodinium cohnii in response to different dissolved oxygen levels during docosahexaenoic acid fermentation.

Oxygen supply is an important factor during Crypthecodinium cohnii fermentation for docosahexaenoic acid (DHA) production. However, few studies about the intrinsic correlation between dissolved oxygen (DO) and cellular metabolism have been reported. In this study, the responses of C. cohnii to different DO levels were evaluated. The results showed the growth and glucose consumption rates of C. cohnii were much higher under high oxygen supply condition. Furthermore, GC-MS based comparative metabolomic analysis was employed to discriminate the responsive metabolites associated with varying DO levels. The results showed the intermediates involved in glycolytic pathway and TCA cycle were up-regulated under high DO levels at exponential phase. At stationary phase, under high DO levels, metabolites involved in triacylglycerol metabolism were up-regulated, while the OPP pathway intermediate product ribose 5-phosphate was down-regulated. Together, these results provide useful insights into the functional metabolic relationship between DO levels and DHA production in C. cohnii.

Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening.

A prognostic model based on CLEC6A predicts clinical outcome of breast cancer patients.

CLEC6A, (C-type lectin domain family 6, member A), plays a prominent role in regulating innate immunity and adaptive immunity. CLEC6A has shown great potential as a target for cancer immunotherapy. This study aims to explore the prognostic value of CLEC6A, and analyze the relationship associated with the common hematological parameters in breast cancer patients. We performed a retrospective analysis on 183 breast cancer patients data in hospital information system from January 2013 to December 2015. The expression of CLEC6A was recorded via semiquantitative immunohistochemistry in breast cancer. The association between expression of CLEC6A and relative parameters were performed by Chi-square test and Fisher's exact test. Kaplan-Meier assay and Log-rank test were performed to evaluate the survival time. The Cox proportional hazards regression analysis was applied to identify prognostic factors. Nomograms were conducted to predict 1-, 3-, and 5-year disease free survival (DFS) and overall survival (OS) for breast cancer, which could be a good reference in clinical practice. The nomogram model was estimated by calibration curve analysis for its function of discrimination. The accuracy and benefit of the nomogram model were appraised by comparing it to only CLEC6A via decision curve analysis (DCA). The prediction accuracy of CLEC6A was also determined by time-dependent receiver operating characteristics (TDROC) curves, and the area under the curve (AUC) for different survival time. There were 94 cases in the CLEC6A low-expression group and 89 cases in CLEC6A high-expression group. Compared to CLEC6A low-expression group, the CLEC6A high-expression group had better survival (DFS: 56.95 vs. 70.81 months, P = 0.0078 and OS: 67.98 vs. 79.05 months, P = 0.0089). The CLEC6A was a potential prognostic factor in multivariate analysis (DFS: P = 0.023, hazard ratio (HR): 0.454, 95 % confidence interval (CI): 0.229-0.898; OS: P = 0.020, HR: 0.504, 95 %CI: 0.284-0.897). The nomogram in accordance with these potential prognostic factors was constructed to predict survival and the calibration curve analysis had indicated that the predicted line was well-matched with reference line in 1-, 3-, and 5-year DFS and OS category. The 1-, 3-, and 5-year DCA curves have revealed that nomogram model yielded larger net benefits than CLEC6A alone. Finally, the TDROC curve indicated that CLEC6A could better predict 1-year DFS and OS than others. Furthermore, we combined these potential independent prognostic factors to analyze the relationship among these hematologic index and oxidative stress indicators, and indicated that higher CLEC6A level, higher CO2 level or low CHOL level or high HDL-CHO level would have survived longer and better prognosis. In breast cancer, high expression of CLEC6A can independently predict better survival. Our nomogram consisted of CLEC6A and other indicators has good predictive performance and can facilitate clinical decision-making.

A coupling, stabilizing, and shaping strategy for breast ultrasound computed tomography (USCT) with a ring array transducer.

Breast ultrasound computed tomography (USCT) has been gradually promoted to clinical application after years of rapid development. Compared with the traditional handheld ultrasound scanning method, the scanning plane of USCT is fixed at the coronal plane, and the scanning path is designed in advance; the acoustic window is not in direct contact with the breast, a lot of coupling medium (usually degassed water is used to fill the gaps between the probe and breast. The clinical application of breast USTC faces challenges: (1) the processes of water degassing, heating, filling, draining, and cleaning prolong the entire scan cycle and reduce patient throughput. (2) The breast is not stabilized and slight movements of the breast may cause motion artifacts in the USCT images. (3) The non-normal incidence of ultrasound into the breast causes reflected and transmitted signals received with a low signal-to-noise ratio (SNR) or even unable to be detected. This article proposes a coupling, stabilizing, and shaping strategy for the clinical application of USCT with a ring array transducer. The solid gel coupling agent (SGCA) is applied for coupling, and a set of SGCA moldings is designed to stabilize and shape the breast during scanning, the breast shape and size which vary from person to person are simplified into several models. The preparation time is reduced to less than 1 min by replacing disposable moldings. The results show that the breast after shaping is close to round in the coronal plane, and slopes of the breast skin are limited in the sagittal and transverse planes, the breast subcutaneous tissue (fat and glands) has a better contrast-to-noise ratio (CNR) and can be better distinguished in the reflection images than that of the breast without shaping. The mean value of the raw beamformed data which represents the reflection signal amplitude of breast subcutaneous tissue after shaping shows 1.5 times that of the breast without shaping, the signal-to-noise ratio (SNR) of the raw transmission signal data after breast shaping is overall higher than that of the breast without shaping. The application of SGCA moldings for breast coupling, stabilizing, and shaping also benefits establishing a standardized scanning process, the standardized diagnosis of the breast lesion, and the localization of breast lesions.

Deciphering the TCF19/miR-199a-5p/SP1/LOXL2 pathway: Implications for breast cancer metastasis and epithelial-mesenchymal transition.

Globally, breast cancer (BC) is the predominant malignancy with a significant death rate due to metastasis. The epithelial-mesenchymal transition (EMT) is a fundamental initiator for metastatic progression. Through advanced computational strategies, TCF19 was identified as a critical EMT-associated gene with diagnostic and prognostic significance in BC, based on a novel EMT score. Molecular details and the pro-EMT impact of the TCF19/miR-199a-5p/SP1/LOXL2 axis were explored in BC cell lines through in vitro validations, and the oncogenic and metastatic potential of TCF19 and LOXL2 were investigated using subcutaneous and tail-vein models. Additionally, BC-specific enrichment of TCF19 and LOXL2 was measured using a distribution landscape driven by diverse genomic analysis techniques. Molecular pathways revealed that TCF19-induced LOXL2 amplification facilitated migratory, invasive, and EMT activities of BC cells in vitro, and promoted the growth and metastatic establishment of xenografts in vivo. TCF19 decreases the expression of miR-199a-5p and alters the nuclear dynamics of SP1, modulating SP1's affinity for the LOXL2 promoter, leading to increased LOXL2 expression and more malignant characteristics in BC cells. These findings unveil a novel EMT-inducing pathway, the TCF19/miR-199a-5P/SP1/LOXL2 axis, highlighting the pivotal role of TCF19 and suggesting potential for novel therapeutic approaches for more focused BC interventions.

Directed differentiation of human embryonic stem cells into parathyroid cells and establishment of parathyroid organoids.

Differentiation of human embryonic stem cells (hESCs) into human embryonic stem cells-derived parathyroid-like cells (hESC-PT) has clinical significance in providing new therapies for congenital and acquired parathyroid insufficiency conditions. However, a highly reproducible, well-documented method for parathyroid differentiation remains unavailable. By imitating the natural process of parathyroid embryonic development, we proposed a new hypothesis about the in vitro differentiation of parathyroid-like cells. Transcriptome, differentiation marker protein detection and parathyroid hormone (PTH) secretion assays were performed after the completion of differentiation. To optimize the differentiation protocol and further improve the differentiation rate, we designed glial cells missing transcription factor 2 (GCM2) overexpression lentivirus transfection assays and constructed hESCs-derived parathyroid organoids. The new protocol enabled hESCs to differentiate into hESC-PT. HESC-PT cells expressed PTH, GCM2 and CaSR proteins, low extracellular calcium culture could stimulate hESC-PT cells to secrete PTH. hESC-PT cells overexpressing GCM2 protein secreted PTH earlier than their counterpart hESC-PT cells. Compared with the two-dimensional cell culture environment, hESCs-derived parathyroid organoids secreted more PTH. Both GCM2 lentiviral transfection and three-dimensional cultures could make hESC-PT cells functionally close to human parathyroid cells. Our study demonstrated that hESCs could differentiate into hESC-PT in vitro, which paves the road for applying the technology to treat hypoparathyroidism and introduces new approaches in the field of regenerative medicine.