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Experimental and Molecular Pathology Jun 2024Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study...
OBJECTIVE
Little information is available concerning protein expression of the free fatty acid receptor 2 (FFAR2), especially in tumours. Therefore, the aim of the present study was to comprehensively characterise the expression profile of FFAR2 in a large series of human normal and neoplastic tissues using immunohistochemistry thus providing a basis for further in-depth investigations into its potential diagnostic or therapeutic importance.
METHODS
We developed a novel rabbit polyclonal anti-FFAR2 antibody, 0524, directed against the C-terminal region of human FFAR2. Antibody specificity was confirmed via Western blot analyses and immunocytochemistry using the FFAR2-expressing cell line BON-1 and FFAR2-specific small interfering RNA as well as native and FFAR2-transfected HEK-293 cells. The antibody was then used for immunohistochemical analyses of various formalin-fixed, paraffin-embedded specimens of normal and neoplastic human tissues.
RESULTS
In normal tissues, FFAR2 was mainly present in distinct cell populations of the cerebral cortex, follicular cells and C cells of the thyroid, cardiomyocytes of the heart, bronchial epithelia and glands, hepatocytes and bile duct epithelia of the liver, gall bladder epithelium, exocrine and β-cells of the endocrine pancreas, glomerular mesangial cells and podocytes as well as collecting ducts of the kidney, intestinal mucosa (particularly enteroendocrine cells), prostate epithelium, seminiferous tubules of the testicles, and placental syncytiotrophoblasts. In neoplastic tissues, FFAR2 was particularly prevalent in papillary thyroid carcinomas, parathyroid adenomas, and gastric, colon, pancreatic, hepatocellular, cholangiocellular, urinary bladder, breast, cervical, and ovarian carcinomas.
CONCLUSIONS
We generated and characterised a novel rabbit polyclonal anti-human FFAR2 antibody that is well-suited for visualising FFAR2 expression in human routine pathology tissues. This antibody is also suitable for Western blot and immunocytochemistry experiments. To our knowledge, this antibody enabled the first broad FFAR2 protein expression profile in various normal and neoplastic human tissues.
Topics: Humans; Neoplasms; HEK293 Cells; Animals; Rabbits; Immunohistochemistry; Receptors, G-Protein-Coupled; Female; Male
PubMed: 38788249
DOI: 10.1016/j.yexmp.2024.104902 -
MSystems Jun 2024In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical...
In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. and were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of and spp. in 50% (7/14) of stripped milk samples and growth of spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.
Topics: Animals; Cattle; Milk; Microbiota; Female; DNA, Bacterial; Bacteria; Mammary Glands, Animal; Specimen Handling; RNA, Ribosomal, 16S
PubMed: 38785438
DOI: 10.1128/msystems.01158-23 -
The International Journal of... May 2024The development of skin appendages, including hair follicles, teeth and mammary glands is initiated through the formation of the placode, a local thickening of the...
The development of skin appendages, including hair follicles, teeth and mammary glands is initiated through the formation of the placode, a local thickening of the epithelium. The Wnt/β-catenin signaling cascade is an evolutionary conserved pathway with an essential role in placode morphogenesis, but its downstream targets and their exact functions remain ill defined. In this study, we identify () as a novel target of the Wnt/β-catenin pathway and demonstrate its expression pattern in the signaling centers of developing hair follicles and teeth. Ascl transcription factors belong to the superfamily of basic helix-loop-helix transcriptional regulators involved in cell fate determination in many tissues. However, their specific role in the developing skin remains largely unknown. We report that null mice have no overt phenotype. Absence of Ascl4 did not impair hair follicle morphogenesis or hair shaft formation suggesting that it is non-essential for hair follicle development. No tooth or mammary gland abnormalities were detected either. We suggest that other transcription factors may functionally compensate for the absence of Ascl4, but further research is warranted to assess this possibility.
PubMed: 38770834
DOI: 10.1387/ijdb.240007vp -
The EMBO Journal Jun 2024How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from...
How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis.
Topics: Animals; Mice; Mammary Glands, Animal; Female; Cell Lineage; Mesenchymal Stem Cells; Epithelial Cells; Cell Differentiation; Multipotent Stem Cells; Fibroblast Growth Factor 10; Morphogenesis; Single-Cell Analysis; Mesoderm
PubMed: 38760574
DOI: 10.1038/s44318-024-00115-3 -
Current Ophthalmology Reports Jun 2024This study is to highlight the incidence of corneal pseudomicrocysts in FDA-approved antibody-drug conjugates (ADCs), and success of preventive therapies for...
PURPOSE OF REVIEW
This study is to highlight the incidence of corneal pseudomicrocysts in FDA-approved antibody-drug conjugates (ADCs), and success of preventive therapies for pseudomicrocysts and related ocular surface adverse events (AEs).
RECENT FINDINGS
ADCs are an emerging class of selective cancer therapies that consist of a potent cytotoxin connected to a monoclonal antibody (mAb) that targets antigens expressed on malignant cells. Currently, there are 11 FDA-approved ADCs with over 164 in clinical trials. Various AEs have been attributed to ADCs, including ocular surface AEs (keratitis/keratopathy, dry eye, conjunctivitis, blurred vision, corneal pseudomicrocysts). While the severity and prevalence of ADC-induced ocular surface AEs are well reported, the reporting of corneal pseudomicrocysts is limited, complicating the development of therapies to prevent or treat ADC-related ocular surface toxicity.
SUMMARY
Three of 11 FDA-approved ADCs have been implicated with corneal pseudomicrocysts, with incidence ranging from 41 to 100% of patients. Of the six ADCs that reported ocular surface AEs, only three had ocular substudies to investigate the benefit of preventive therapies including topical steroids, vasoconstrictors, and preservative-free lubricants. Current preventive therapies demonstrate limited efficacy at mitigating pseudomicrocysts and other ocular surface AEs.
PubMed: 38756824
DOI: 10.1007/s40135-024-00322-5 -
Translational Breast Cancer Research :... 2023Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer. However, if left untreated, about 50% of DCIS progress. Preventing such a...
BACKGROUND
Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer. However, if left untreated, about 50% of DCIS progress. Preventing such a progression is of paramount importance. Cumulative evidence indicated that the mevalonate cascade, the core of cholesterol biosynthesis, contributes to the regulation of the Hippo signaling pathway providing the isoprenoids required for GTPase activation, the nuclear accumulation of the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) coactivator, and the subsequent gene transcription and that the disruption of this cooperation associated with tumor progression.
METHODS
In this study, we investigated whether such a disruption occurred already during the transformation of the normal mammary epithelium into DCIS. To this aim, we interrogated a publicly available dataset, and we explored the interrelationship of the genes involved in the cholesterol biosynthesis and the association with those coding for the core components of the Hippo signaling pathway in a set of patient-matched samples of DCIS and corresponding histologically normal (HN) epithelium.
RESULTS
Most genes involved in cholesterol biosynthesis were more expressed in DCIS than in the corresponding HN epithelium. This differential expression was associated with a substantial change in their correlation profile. In particular, 3-hydroxy-3-methylglutaryl coenzyme-A reductase () and lost the positive association shown in the HN epithelium, and their negative association with switched to a positive one. Also, , which plays a crucial role in isoprenoids production, significantly changed its correlation profile. The positive association between and or disappeared, whereas the positive association with , which drives the irreversible commitment to cholesterol, switched to a negative one in DCIS.
CONCLUSIONS
Present findings corroborated the hypothesis that a dysfunctional mevalonate pathway possibly concurs with DCIS development by leading to abnormal production of isoprenoids, which in turn activate GTPases and promote YAP/TAZ nuclear translocation, and suggested the safe and low-cost treatment with statins as the possible winning strategy to contrast this metabolic dysfunction.
PubMed: 38751474
DOI: 10.21037/tbcr-23-42 -
BioRxiv : the Preprint Server For... May 2024Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation...
Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from or mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from or mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of , LOH of or , and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.
PubMed: 38746396
DOI: 10.1101/2024.05.01.591587 -
Cancer Science May 2024Metaplastic breast cancer is a rare, aggressive, and chemotherapy-resistant subtype of breast cancers, accounting for less than 1% of invasive breast cancers,... (Review)
Review
Metaplastic breast cancer is a rare, aggressive, and chemotherapy-resistant subtype of breast cancers, accounting for less than 1% of invasive breast cancers, characterized by adenocarcinoma with spindle cells, squamous epithelium, and/or mesenchymal tissue differentiation. The majority of metaplastic breast cancers exhibit the characteristics of triple-negative breast cancer and have unfavorable prognoses with a lower survival rate. This subtype often displays gene alterations in the PI3K/AKT pathway, Wnt/β-catenin pathway, and cell cycle dysregulation and demonstrates epithelial-mesenchymal transition, immune response changes, TP53 mutation, EGFR amplification, and so on. Currently, the optimal treatment of metaplastic breast cancer remains uncertain. This article provides a comprehensive review on the clinical features, molecular characteristics, invasion and metastasis patterns, and prognosis of metaplastic breast cancer, as well as recent advancements in treatment strategies.
PubMed: 38735837
DOI: 10.1111/cas.16208 -
BMC Biotechnology May 2024Scientists know very little about the mechanisms underlying fish skin mucus, despite the fact that it is a component of the immune system. Fish skin mucus is an...
Scientists know very little about the mechanisms underlying fish skin mucus, despite the fact that it is a component of the immune system. Fish skin mucus is an important component of defence against invasive infections. Recently, Fish skin and its mucus are gaining interest among immunologists. Characterization was done on the obtained silver nanoparticles Ag combined with Clarias gariepinus catfish epidermal mucus proteins (EMP-Ag-NPs) through UV-vis, FTIR, XRD, TEM, and SEM. Ag-NPs ranged in size from 4 to 20 nm, spherical in form and the angles were 38.10°, 44.20°, 64.40°, and 77.20°, Where wavelength change after formation of EMP-Ag-NPs as indicate of dark brown, the broad band recorded at wavelength at 391 nm. Additionally, the antimicrobial, antibiofilm and anticancer activities of EMP-Ag-NPs was assessed. The present results demonstrate high activity against unicellular fungi C. albicans, followed by E. faecalis. Antibiofilm results showed strong activity against both S. aureus and P. aeruginosa pathogens in a dose-dependent manner, without affecting planktonic cell growth. Also, cytotoxicity effect was investigated against normal cells (Vero), breast cancer cells (Mcf7) and hepatic carcinoma (HepG2) cell lines at concentrations (200-6.25 µg/mL) and current results showed highly anticancer effect of Ag-NPs at concentrations 100, 5 and 25 µg/mL exhibited rounding, shrinkage, deformation and granulation of Mcf7 and HepG2 with IC50 19.34 and 31.16 µg/mL respectively while Vero cells appeared rounded at concentration 50 µg/mL and normal shape at concentration 25, 12.5 and 6.25 µg/ml with IC50 35.85 µg/mL. This study evidence the potential efficacy of biologically generated Ag-NPs as a substitute medicinal agent against harmful microorganisms. Furthermore, it highlights their inhibitory effect on cancer cell lines.
Topics: Metal Nanoparticles; Biofilms; Silver; Animals; Humans; Catfishes; Mucus; Antineoplastic Agents; Vero Cells; Fish Proteins; Chlorocebus aethiops; Cell Line, Tumor; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Anti-Infective Agents; Staphylococcus aureus; Candida albicans; Epidermis
PubMed: 38702622
DOI: 10.1186/s12896-024-00852-7 -
Pharmaceutics Apr 2024Gemcitabine is a nucleoside analog effective against a number of cancers. However, it has an oral bioavailability of less than 10%, due to its high hydrophilicity and...
Gemcitabine is a nucleoside analog effective against a number of cancers. However, it has an oral bioavailability of less than 10%, due to its high hydrophilicity and low permeability through the intestinal epithelium. Therefore, the aim of this project was to develop a novel nanoparticulate drug delivery system for the oral delivery of gemcitabine to improve its oral bioavailability. In this study, gemcitabine-loaded β-glucan NPs were fabricated using a film-casting method followed by a freezer-milling technique. As a result, the NPs showed a small particle size of 447.6 ± 14.2 nm, and a high drug entrapment efficiency of 64.3 ± 2.1%. By encapsulating gemcitabine into β-glucan NPs, a sustained drug release profile was obtained, and the anomalous diffusion release mechanism was analyzed, indicating that the drug release was governed by diffusion through the NP matrix as well as matrix erosion. The drug-loaded NPs had a greater ex vivo drug permeation through the porcine intestinal epithelial membrane compared to the plain drug solution. Cytotoxicity studies showed a safety profile of the β-glucan polymers, and the IC of drug solution and drug-loaded β-glucan NPs were calculated as 228.8 ± 31.2 ng·mL and 306.1 ± 46.3 ng·mL, respectively. Additionally, the LD of BALB/c nude mice was determined as 204.17 mg/kg in the acute toxicity studies. Notably, pharmacokinetic studies showed that drug-loaded β-glucan NPs could achieve a 7.4-fold longer T and a 5.1-fold increase in oral bioavailability compared with plain drug solution. Finally, in vivo pharmacodynamic studies showed the promising capability of gemcitabine-loaded β-glucan NPs to inhibit the 4T1 breast tumor growth, with a 3.04- and 1.74-fold reduction compared to the untreated control and drug solution groups, respectively. In conclusion, the presented freezer-milled β-glucan NP system is a suitable drug delivery method for the oral delivery of gemcitabine and demonstrates a promising potential platform for oral chemotherapy.
PubMed: 38675207
DOI: 10.3390/pharmaceutics16040546