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.

Evaluation of the Nutritional Quality of Chinese Processed Meat Products: Comparison of Two Nutrient Profile Models.

Processed meat products are one of the most consumed pre-packaged foods in China. They are also group-1 carcinogens, whose consumption has proved to be positively associated with the risk of noncommunicable diseases (NCDs). The purpose of this study is to analyze the nutrient content on the food label of processed meat products based on the China Standardized Database for the Composition of Pre-packaged Food and the National Open Database of the UK and France. The Chilean front-of-pack warning label (FOPWL) and the Chinese Healthier Choice Logo were used to compare the nutrient content of processed meat products from the three countries. It was found that cured meat products have the highest median energy (483 kcal/100 g), total fat content (38.7 g/100 g), and sodium content (2076 mg/100 g) and dried meat products have the highest median protein content (30.2 g/100 g) and carbohydrate content (38.2 g/100 g). In addition, there were significant differences in energy content and contents of total fat, protein, and carbohydrate across different products of the three countries (p < 0.001). A large number of processed meat products currently collected did not meet the criteria of the Chilean FOPWL and the Chinese Healthier Choice Logo. This study provided information on the healthiness of Chinese processed meat products and provided data for improving food formulations for different categories of processed meat products.

Modifying the Oxidative Potentials of Imines in a Dye Loaded Capsule for Photocatalytic Cyclization with Hydrogen Evolution.

Modifying redox potential of substrates and intermediates to balance pairs of redox steps are important stages for multistep photosynthesis but faced marked challenges. Through co-clathration of iridium photosensitizer and imine substrate within one packet of a metal-organic capsule to shift the redox potentials of substrate, herein, we reported a multiphoton enzymatic strategy for the generation of heterocycles by intramolecular C-X hydrogen evolution cross-couplings. The cage facilitated a pre-equilibrium substrate-involving clathrate that cathodic shifts the oxidation potential of the substrate-dye-host ternary complex and configuration inversion of substrate via spatial constraints in the confined space. The new two photon excitation strategy enabled the precise control of the multistep electron transfer between each pair (photosensitizer, substrate and the capsule), endowing the catalytic system proceeding smoothly with an enzymatic fashion. Three kinds of 2-subsituted (-OH, -NH2 , and -SH) imines and N-aryl enamines all give the corresponding cyclization products efficiently under visible light irradiation, demonstrating the promising of the microenvironment driven thermodynamic activation in the host-dye-substrate ternary for synergistic combination of multistep photocatalytic transformations.

Sodium Content in Pre-Packaged Foods in China: A Food Label Analysis.

Sodium intake from pre-packaged foods is increasing in China and is well above the WHO recommendation of 5 g per day. The purpose of this study is to analyze the sodium content of pre-packaged foods collected by the National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention (NINH, China CDC) in 20 provinces of China from 2017 to 2022. The proportion of pre-packaged foods that meet or exceed the low-sodium, medium-sodium, and high-sodium classifications were analyzed. The proportion of pre-packaged foods that meet and do not meet the WHO global sodium benchmarks and the difference in sodium content between these foods was also calculated. High-sodium foods include sauces, dips, and dressings (3896 mg/100 g), convenience foods (1578 mg/100 g), processed fish products (1470 mg/100 g), processed meat products (1323 mg/100 g), processed poultry products (1240 mg/100 g), snack foods (750 mg/100 g), processed egg products (741 mg/100 g), and fine dried noodles (602 mg/100 g). A large number of pre-packaged foods currently collected in China have a sodium content above sodium benchmarks. This study provided data to support the assessment of sodium intake from pre-packaged foods in the Chinese population and the implementation of comprehensive salt reduction strategies.

Mixed-ligand metal-organic frameworks as an effective photocatalyst for selective oxidation reaction.

By incorporating tetrakis(4-carboxyphenyl)porphyrin and bis(3,5-dicarboxyphenyl)pyridine into one single metal-organic framework (MOF), a multifunctional mixed-ligand Zn-MIX with large pores was obtained. Under visible-light irradiation, Zn-MIX exhibits high photocatalytic activity for the oxidation of amines and sulfides.

Beyond TORCH: A narrative review of the impact of antenatal and perinatal infections on the risk of disability.

Infections and inflammation during pregnancy or early life can alter child neurodevelopment and increase the risk for structural brain abnormalities and mental health disorders. There is strong evidence that TORCH infections (i.e., Treponema pallidum, Toxoplasma gondii, rubella virus, cytomegalovirus, herpes virus) alter fetal neurodevelopment across multiple developmental domains and contribute to motor and cognitive disabilities. However, the impact of a broader range of viral and bacterial infections on fetal development and disability is less well understood. We performed a literature review of human studies to identify gaps in the link between maternal infections, inflammation, and several neurodevelopmental domains. We found strong and moderate evidence respectively for a higher risk of motor and cognitive delays and disabilities in offspring exposed to a range of non-TORCH pathogens during fetal life. In contrast, there is little evidence for an increased risk of language and sensory disabilities. While guidelines for TORCH infection prevention during pregnancy are common, further consideration for prevention of non-TORCH infections during pregnancy for fetal neuroprotection may be warranted.

Does the agricultural co-agglomeration help reduce livestock and poultry pollution? From the perspective of planting and breeding combination in China.

Given the problem of considerable livestock and poultry pollution and the differentiation of the regional agricultural layout in China, the combination of planting and breeding (CPB) forms an agricultural co-agglomeration to recycle manure waste into croplands to reduce livestock and poultry pollution. This study aims to evaluate CPB co-agglomeration and empirically examine its effects on livestock and poultry pollution. Based on provincial data from 1997 to 2020 in China, this study constructed three indicators to evaluate CPB co-agglomeration, summarized its temporal and spatial characteristics, and conducted a spatial analysis using the Spatial Lag Model (SLM) to empirically investigate its effect on livestock and poultry pollution. The results showed that: first, from 1997 to 2020, the overall level of CPB co-agglomeration in China declined and the region with higher CPB co-agglomeration level transferred from the central provinces to the west provinces. Second, livestock and poultry pollution in most provinces had significantly positive spatial correlations with adjacent regions. The co-agglomeration of CPB had a significantly positive effect on reducing livestock and poultry pollution; however, the effect had no significant spatial spillover. Third, the breeding industry agglomeration and the moderate expansion of breeding industry scale significantly reduced pollution. These findings provide a reference for reducing livestock and poultry pollution by promoting CPB co-agglomeration to establish a waste recycling system. Optimizing the layout of the planting and breeding industry helps achieve the goal of long-term sustainable development of the breeding industry.

Visible-light-driven organic oxidation over CdS-doped metal-organic frameworks.

In this research, we synthesized CdS nanoparticle-doped metal-organic frameworks (CdS@DUT-52) with different contents using a solvothermal method. CdS@DUT-52 possesses strong light absorption ability and effective electron-hole separation properties, which was proved by performing UV-vis spectroscopy and photoelectrochemical testing. It was used to trigger the photooxidation of amines, sulfides, and alcohols to produce the corresponding imines, sulfoxides, and aldehydes in the presence of air or oxygen, exhibiting considerable yields of products under visible light irradiation compared with a single component.

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.

Regulation of antitumor miR-205 targets oncogenes: Direct regulation of lymphoid specific helicase and its clinical significance.

HEADING AIMS: Breast cancer is one of the most common malignant tumors with a high incidence and leading cancer-related death in women worldwide. MiR-205 plays a crucial role in breast cancer initiation and progression. Here, we identified the relationship between miR-205 and lymphoid specific helicase and confirmed the significance of the miR-205/lymphoid specific helicase (miR-205/HELLS) axis. MATERIALS AND METHODS: Data from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were analyzed to investigate the expression level of miR-205 and HELLS in breast cancer. The TargetScan, Starbase and miRWalk databases were used to predict the candidate target genes of miR-205. Proliferation and migration abilities were examined using cell counting kit-8 assay, colony formation assays, transwell assay and wound-healing assay. Dual-luciferase reporter assay was utilized to confirm the binding of miR-205 and HELLS. Quantitative RT-PCR, western blot assays or immunohistochemistry were conducted to detect the expression level of genes in breast cancer cells or tissues. Mice xenograft models were constructed to explore the function of miR-205 and HELLS in vivo. KEY FINDINGS: Overexpressed miR-205 alleviated cancer cell proliferation and migration and influenced patients' prognosis by negatively regulating the HELLS gene. Consistently, animal experiments revealed that both overexpressing miR-205 and knocking down HELLS exhibited significant tumor growth inhibition in vivo. SIGNIFICANCE: Our study demonstrated that miR-205 targets HELLS to regulate tumor progression. MiR-205 and HELLS could be considered a novel diagnosis and therapeutic molecular marker of breast cancer.

Role of hormones in the pregnancy and sex-specific outcomes to infections with respiratory viruses.

Pregnant women infected with pathogenic respiratory viruses, such as influenza A viruses (IAV) and coronaviruses, are at higher risk for mortality, hospitalization, preterm birth, and stillbirth. Several factors are likely to contribute to the susceptibility of pregnant individuals to severe lung disease including changes in pulmonary physiology, immune defenses, and effector functions of some immune cells. Pregnancy is also a physiologic state characterized by higher levels of multiple hormones that may impact the effector functions of immune cells, such as progesterone, estrogen, human chorionic gonadotropin, prolactin, and relaxin. Each of these hormones acts to support a tolerogenic immune state of pregnancy, which helps prevent fetal rejection, but may also contribute to an impaired antiviral response. In this review, we address the unique role of adaptive and innate immune cells in the control of pathogenic respiratory viruses and how pregnancy and specific hormones can impact their effector actions. We highlight viruses with sex-specific differences in infection outcomes and why pregnancy hormones may contribute to fetal protection but aid the virus at the expense of the mother's health.

MiR-205-5p/GGCT Attenuates Growth and Metastasis of Papillary Thyroid Cancer by Regulating CD44.

Papillary thyroid cancer (PTC) remains the most common endocrine malignancy, despite marked achieves in recent decades, and the mechanisms underlying the pathogenesis and progression for PTC are incompletely elucidated. Accumulating evidence show that γ-glutamylcyclotransferase (GGCT), an enzyme participating in glutathione homeostasis and is elevated in multiple types of tumors, represents an attractive therapeutic target. Using bioinformatics, immunohistochemistry, qRT-PCR, and Western blot assays, we found that GGCT expression was upregulated in PTC and correlated with more aggressive clinicopathological characteristics and worse prognosis. GGCT knockdown inhibited the growth and metastasis ability of PTC cells both in vitro and in vivo and reduced the expression of mesenchymal markers (N-cadherin, CD44, MMP2, and MMP9) while increasing epithelial marker (E-cadherin) in PTC cells. We confirmed binding of microRNA-205-5p (miR-205-5p) on the 3'-UTR regions of GGCT by dual-luciferase reporter assay and RNA-RNA pull-down assay. Delivery of miR-205-5p reversed the pro-malignant capacity of GGCT both in vitro and in vivo. Lastly, we found that GGCT interacted with and stabilized CD44 in PTC cells by co-immunoprecipitation and immunohistochemistry assays. Our findings illustrate a novel signaling pathway, miR-205-5p/GGCT/CD44, that involves in the carcinogenesis and progression of PTC. Development of miR-205-mimics or GGCT inhibitors as potential therapeutics for PTC may have remarkable applications.

Dye-loaded metal-organic helical capsules applied to the combination of photocatalytic H2S splitting and nitroaromatic hydrogenation.

Two dye-loaded metal-organic capsules constructed with different spatial sizes and functional groups simulated the enzymatic substrate activation for hydrogenation of nitroarenes with the kinetics obeying the Michaelis-Menten mechanism.

CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer.

BACKGROUND: Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in hematological cancers but have not yet translated in treating solid tumors. The significant hurdles limiting CAR-T therapy were from a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present TSH receptor (TSHR) as a putative target for CAR-T therapy of differentiated thyroid cancer (DTC). METHODS: We undertook a large-scale screen on thyroid cancer tissues and multiple internal organs through bioinformatical analysis and immunohistochemistry to date TSHR expression. Using 3 previously described monoclonal antibodies, we generated 3 third-generation CAR-Ts. We tested anti-TSHR CAR-T in vitro activity by T-cell function and killing assay. Then we tested preclinical therapeutical efficacy in a xenograft mouse model of DTC and analyzed mice's physical conditions and histological abnormalities to evaluate anti-TSHR CAR-T's safety. RESULTS: TSHR is highly and homogeneously expressed on 90.8% (138/152) of papillary thyroid cancer, 89.2% (33/37) of follicular thyroid cancer, 78.2% (18/23) of cervical lymph node metastases, and 86.7% of radioactive iodine resistance diseases. We developed 3 novel anti-TSHR CAR-Ts from monoclonal antibodies M22, K1-18, and K1-70; all 3 CAR-Ts mediate significant antitumor activity in vitro. Among these, we demonstrate that K1-70 CAR-T can have therapeutical efficacy in vivo, and no apparent toxicity has been observed. CONCLUSION: TSHR is a latent target antigen of CAR-T therapy for DTC. Anti-TSHR CAR-T could represent a therapeutic option for patients with locoregional relapsed or distant metastases of thyroid cancer and should be tested in carefully designed clinical trials.

S100A1 is a Potential Biomarker for Papillary Thyroid Carcinoma Diagnosis and Prognosis.

S100 calcium binding protein A1 (S100A1) is an important member of the S100 family and known to express in a variety of cancers. However, the biological functions of S100A1 in thyroid carcinoma have not been thoroughly studied. In this report, bioinformatics analyses and immunohistochemistry assays were applied to assess the expression profile of S100A1 as well as its relationship with the pathological features and prognosis of papillary thyroid carcinoma (PTC). Meanwhile, functions of S100A1 in PTC cells were analyzed with either in vitro or in vivo experiments. S100A1 was significantly up-regulated in PTC tissues compared with adjacent non-cancerous tissues. S100A1 protein expression was significantly associated with tumor size (p=0.0032) or lymph node metastasis (p=0.0331). More importantly, an elevated S100A1 expression was significantly correlated with a worse recurrence-free survival (RFS) (HR=2.26, p=0.042). Further, knockdown of S100A1 dramatically inhibited cell proliferation and migration as well as increased apoptosis of PTC cells. S100A1 knockdown inhibited tumor progression as seen in in vivo experiments. In terms of mechanism, down-regulation of S100A1 induced yes associated protein (YAP) phosphorylation in the cytoplasm and diminished Hippo/YAP pathway activation. Therefore, S100A1 may serve as a novel oncogene and a promising biomarker for PTC diagnosis and prognosis.

Triarylamine-based porous coordination polymers performing both hydrogen atom transfer and photoredox catalysis for regioselective α-amino C(sp3)-H arylation.

Direct functionalization of C(sp3)-H bonds in a predictable, selective and recyclable manner has become a central challenge in modern organic chemistry. Through incorporating different triarylamine-containing ligands into one coordination polymer, we present herein a heterogeneous approach to the combination of hydrogen atom transfer (HAT) and photoredox catalysis for regioselective C-H arylation of benzylamines. The different molecular sizes and coordination modes of the ligands, tricarboxytriphenylamine (H3 TCA) and tris(4-(pyridinyl)phenyl)amine (NPy3), in one coordination polymer consolidate the triarylamine (Ar3N) moiety into a special structural intermediate, which enhances the chemical and thermal stability of the polymers and diminishes structural relaxation during the catalytic process. The inherent redox potentials of Ar3N moieties prohibit the in situ formed Ar3N˙+ to earn an electron from C(sp3)-H nucleophiles, but allow the abstraction of a hydrogen atom from C(sp3)-H nucleophiles, enabling the formation of the C(sp3)˙ radical and the cross-coupling reaction to proceed at the most electron-rich sites with excellent regioselectivity. The new heterogeneous photoredox HAT approach skips several interactions between transient species during the typical synergistic SET/HAT cycles, demonstrating a promising redox-economical and reagent-economical heterogeneous platform that has not been reported for α-amino C-H arylation to form benzylamine derivatives. Control experiments based on monoligand coordination polymers suggested that the mixed-ligand approach improved the photochemical and photophysical properties, providing important insight into rational design and optimization of recyclable photocatalysts for rapid access to complex bioactive molecules and late-stage functionalized pharmaceuticals.

NCAPG upregulation mediated by four microRNAs combined with activation of the p53 signaling pathway is a predictor of poor prognosis in patients with breast cancer.

The role of non-SMC condensin I complex subunit G (NCAPG) in breast cancer remains unclear. The present study used online databases, reverse transcription-quantitative PCR, flow cytometry and western blotting to determine the expression levels, prognosis and potential molecular mechanisms underlying the role of NCAPG in breast cancer. The association between NCAPG expression and several different clinicopathological parameters in patients with breast cancer was determined, and the results revealed that NCAPG expression was negatively associated with estrogen receptor and progesterone receptor positive status, but was positively associated with HER2 positive status, Nottingham Prognostic Index score and Scarff-Bloom-Richardson grade status. Furthermore, upregulated expression levels of NCAPG resulted in a poor prognosis in patients with breast cancer. A total of 27 microRNAs (miRNAs/miRs) were predicted to target NCAPG, among which four miRNAs (miR-101-3p, miR-195-5p, miR-214-3p and miR-944) were predicted to most likely regulate NCAPG expression in breast cancer. A total of 261 co-expressed genes of NCAPG were identified, including cell division cyclin 25 homolog C (CDC25C), and pathway enrichment analysis indicated that these co-expressed genes were significantly enriched in the p53 signaling pathway. CDC25C expression was downregulated in breast cancer and was associated with a poor prognosis. These findings suggested that upregulated NCAPG expression may be a prognostic biomarker of breast cancer.

MRPL13 Promotes Tumor Cell Proliferation, Migration and EMT Process in Breast Cancer Through the PI3K-AKT-mTOR Pathway.

PURPOSE: Breast cancer (BC), with varying histopathology, biology and response to systemic treatment, is the second leading cause of cancer-related mortality. Previous studies have inferred that the expression of mitochondrial ribosomal proteins (MRPs) is possibly related to the occurrence/progression of BC. MRPL13 might be one of the potential MRP candidates that are involved in BC tumorigenesis, but its role in BC has rarely been reported. The purpose of the current study was to evaluate the prognostic significance of MRPL13, as well as to explore its potential biological functions in BC. MATERIALS AND METHODS: A series of bioinformatic and statistical methods were adopted to assess the MRPL13 expression profile, its relationship with clinicopathological characteristics, copy number variation (CNV), impact on clinical outcomes and relevant functions. All the results are analysed by 1097 BC patients collected from The Cancer Genome Atlas (TCGA) dataset and 52 clinical samples for immunohistochemistry (IHC) assay. RESULTS: The results demonstrated that the expression of MRPL13 in BC tissues was remarkably elevated than that in normal breast tissues. In addition, the Kaplan-Meier curves and Cox model indicated that patients with high MRPL13 expression were connected to a worse prognosis, heralding the independent prognostic value of this protein in BC. Moreover, an enrichment analysis showed that MRPL13 was mainly involved in cell cycle/division-related, RNA processing (degradation/splicing), MYC targets and the MTORC1 pathways. In addition, RNA interference (RNAi)-mediated MRPL13 silencing remarkedly inhibited proliferation and migration as well as the expression of EMT-related genes of BC cells in vitro. Mechanistically, attenuation of MRPL13 significantly suppressed the phosphorylation of AKT and mTOR, which could be partially abolished by 740Y-P (a PI3K agonist). CONCLUSION: Our results provide evidence for the first time that increased MRPL13 expression correlates with adverse clinicopathological variables and unfavorable clinical outcomes of BC patients. Knockdown of MRPL13 restrains the proliferation and migration potential and EMT process of BC through inhibiting PI3K/AKT/mTOR signaling pathway.

LINC00052 promotes breast cancer cell progression and metastasis by sponging miR-145-5p to modulate TGFBR2 expression.

Long non-coding RNAs (lncRNAs) may participate in biological regulatory mechanisms of tumors. The aim of the present study was to uncover the molecular mechanism of the lncRNA LINC00052 in the tumorigenesis of breast cancer (BC). LINC00052 expression in BC tissues and cell lines was detected by reverse transcription-quantitative PCR analysis. The Cell Counting Kit-8, proliferation, Transwell and wound healing assays were employed to confirm the effect of LINC00052 on cell proliferation, migration and invasion. The cell localization of LINC00052 was estimated by cytoplasmic nuclear separation assay. Finally, the potential regulatory mechanism of LINC00052 in BC was detected by western blot analysis. The expression levels of LINC00052 were found to be significantly higher in BC tissues compared with those in the adjacent normal tissues. Downregulation of LINC00052 expression in vitro significantly suppressed the proliferation, migration and invasion of BC cells. LINC00052 was mainly expressed in the cytoplasm and was considered to bind with microRNA (miR)-145-5p based on various databases. Notably, the high expression levels of LINC00052 led to the low expression levels of miR-145-5p and high expression levels of TGF-ß receptor II (TGFBR2). In conclusion, the findings of the present study demonstrated that LINC00052 may sponge miR-145-5p to upregulate TGFBR2 expression in order to promote the proliferation and metastasis of BC cells. Therefore, LINC00052 may be an effective potential target for the diagnosis and treatment of BC.