The RNA-binding protein KSRP aggravates malignant progression of clear cell renal cell carcinoma through transcriptional inhibition and post-transcriptional destabilization of the NEDD4L ubiquitin ligase.

BACKGROUND: KH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood. METHODS: KSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC. RESULTS: Our results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3'UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses. CONCLUSION: The current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

The Immunological Microenvironment and the Emerging Role of Stem Cells Therapy in Peyronie's Disease: A Systematic Narrative Review.

Current literature has indicated that Peyronie's disease (PD) could be initiated by microtrauma and the subsequent inflammation episodes that follow. PD could be sorted into acute or chronic status, and it can differ when selecting the clinical therapeutics. PD would cause pain and penile deformity to diseased men and impair their erectile function. Occasionally, surgical revision of the penis might be needed to correct the penile curvature. We find that there are limited effective options of intra-lesion injections for the PD plaques. By searching the databases and screening the literature with the PRISMA 2020 guideline, we observed that several preclinical studies that applied stem cell therapy in treating PD were fruitful in the acute phase. Although in the chronic phase of PD, erectile parameters were not significantly improved, and therefore, future studies might be better elevated in certain aspects, such as the sites selected for harvesting stem cells or changing the centrifugation forces. In this review, we concluded the contemporary understanding of inflammatory microenvironments in PD, the stem cell therapy in PD, and our perspectives on future studies. We concluded that there may be great potential in stem cell therapy for treating both acute and chronic phases PD.

Proteomics-based identification of TMED9 is linked to vascular invasion and poor prognoses in patients with hepatocellular carcinoma.

BACKGROUND: Due to the difficulties in early diagnosing and treating hepatocellular carcinoma (HCC), prognoses for patients remained poor in the past decade. In this study, we established a screening model to discover novel prognostic biomarkers in HCC patients. METHODS: Candidate biomarkers were screened by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of five HCC normal (N)/tumor (T) paired tissues and preliminarily verified them through several in silico database analyses. Expression levels and functional roles of candidate biomarkers were respectively evaluated by immunohistochemical staining in N/T paired tissue (n = 120) and MTS, colony formation, and transwell migration/invasion assays in HCC cell lines. Associations of clinicopathological features and prognoses with candidate biomarkers in HCC patients were analyzed from GEO and TCGA datasets and our recruited cohort. RESULTS: We found that the transmembrane P24 trafficking protein 9 (TMED9) protein was elevated in HCC tissues according to a global proteomic analysis. Higher messenger (m)RNA and protein levels of TMED9 were observed in HCC tissues compared to normal liver tissues or pre-neoplastic lesions. The TMED9 mRNA expression level was significantly associated with an advanced stage and a poor prognosis of overall survival (OS, p = 0.00084) in HCC patients. Moreover, the TMED9 protein expression level was positively correlated with vascular invasion (p = 0.026), OS (p = 0.044), and disease-free survival (p = 0.015) in our recruited Taiwanese cohort. In vitro, manipulation of TMED9 expression in HCC cells significantly affected cell migratory, invasive, proliferative, and colony-forming abilities. CONCLUSIONS: Ours is the first work to identify an oncogenic role of TMED9 in HCC cells and may provide insights into the application of TMED9 as a novel predictor of clinical outcomes and a potential therapeutic target in patients with HCC.

Dopamine receptor D2 genetic variations is associated with the risk and clinicopathological variables of urothelial cell carcinoma in a Taiwanese population.

Dopamine receptor D2 (DRD2) is overexpressed in several kinds of cancers and was correlated with the prognosis of these cancers. Polymorphisms within the DRD2 gene were shown to be associated with lung and colon cancers. The purpose of this study was to explore effects of DRD2 gene polymorphisms on the susceptibility to and clinicopathological characteristics of urothelial cell carcinoma (UCC). In total, 369 patients diagnosed with UCC and 738 healthy controls were enrolled to analyze DRD2 genotypes at four loci (rs1799732, -141C>del; rs1079597, TaqIB; rs6277, 957C>T; and rs1800497, TaqIA) using a TaqMan-based real-time polymerase chain reaction (PCR). We found a significantly lower risk for UCC in individuals with the DRD2 rs6277 CT genotype compared to those with the wild-type CC genotype (adjusted odds ratio (AOR)=0.405, 95% confidence interval (CI): 0.196~0.837, p=0.015). In 124 younger patients (aged < 65 years) of the recruited UCC cohort, patients who carried at least one T allele of DRD2 rs1800497 were at higher risk (AOR=2.270, 95% CI: 1.060~4.860, p=0.033) of developing an invasive stage (pT2~pT4). In 128 female patients of the recruited UCC cohort, patients who carried at least one deletion allele of DRD2 rs1799732 showed a higher incidence of having an invasive stage (AOR=2.585, 95% CI: 1.066~6.264, p=0.032) and a large tumor (AOR=2.778, 95% CI: 1.146~6.735, p=0.021). Further analyses of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets revealed correlations of the expression of DRD2 with an invasive tumor, tumor metastasis, and the lower survival rate in patients with UCC. Our findings suggest that DRD2 levels might affect the progression of UCC, and the polymorphisms rs6277, rs1800497, and rs1799732 of DRD2 are probably associated with the susceptibility and clinicopathologic development of UCC in a Taiwanese population.

The Molecular Mechanisms of Plant-Derived Compounds Targeting Brain Cancer.

Glioblastoma multiforme (GBM) is one of the most aggressive and malignant forms of brain tumors. Despite recent advances in operative and postoperative treatments, it is almost impossible to perform complete resection of these tumors owing to their invasive and diffuse nature. Several natural plant-derived products, however, have been demonstrated to have promising therapeutic effects, such that they may serve as resources for anticancer drug discovery. The therapeutic effects of one such plant product, n-butylidenephthalide (BP), are wide-ranging in nature, including impacts on cancer cell apoptosis, cell cycle arrest, and cancer cell senescence. The compound also exhibits a relatively high level of penetration through the blood-brain barrier (BBB). Taken together, its actions have been shown to have anti-proliferative, anti-chemoresistance, anti-invasion, anti-migration, and anti-dissemination effects against GBM. In addition, a local drug delivery system for the subcutaneous and intracranial implantation of BP wafers that significantly reduce tumor size in xenograft models, as well as orthotopic and spontaneous brain tumors in animal models, has been developed. Isochaihulactone (ICL), another kind of plant product, possesses a broad spectrum of pharmacological activities, including impacts on cancer cell apoptosis and cell cycle arrest, as well as anti-proliferative and anti-chemoresistance effects. Furthermore, these actions have been specifically shown to have cancer-fighting effects on GBM. In short, the results of various studies reviewed herein have provided substantial evidence indicating that BP and ICH are promising novel anticancer compounds with good potential for clinical applications.

Association between allergic disease, sleep-disordered breathing, and childhood nocturnal enuresis: a population-based case-control study.

BACKGROUND: Little is known about the associations between allergic disease, sleep-disordered breathing (SDB), and childhood nocturnal enuresis (NE). We examined whether allergic disease and SDB were associated with childhood NE. METHODS: Data were assessed from the 2007-2012 Taiwan National Health Insurance Research Database. We enrolled 4308 children aged 5-18 years having NE diagnosis and age- and sex-matched 4308 children as the control group. The odds ratios of NE were calculated to determine an association with preexisting allergic disease and SDB. RESULTS: A total of 8616 children were included in the analysis. Prevalence of allergic diseases and SDB was significantly higher for the NE group than the control group (all p < 0.001). After adjusting odds ratios for potential confounding factors, except asthma, children with allergic rhinitis, atopic dermatitis, allergic conjunctivitis, and obstructive sleep apnea (OSA) had significantly higher odds of NE compared with children never diagnosed. With stratification for sex, girls with allergic rhinitis, atopic dermatitis, allergic conjunctivitis, OSA, and snoring had significantly higher odds of NE, compared with girls never diagnosed. Only boys with allergic rhinitis and OSA were associated with increased odds of NE. With stratification for age, children aged 5-12 years with allergic rhinitis, atopic dermatitis, allergic conjunctivitis, and OSA had significantly higher odds of NE compared with those never diagnosed. Odds of NE increased with the number of comorbid allergic diseases. CONCLUSIONS: Allergic diseases and SDB are associated with increased odds of childhood NE. The odds of NE increased with the number of comorbid allergic diseases present.

Genetic Variations of Melatonin Receptor Type 1A are Associated with the Clinicopathologic Development of Urothelial Cell Carcinoma.

Melatonin counteracts tumor occurrence and tumor cell progression in several cancer types in vitro and in vivo. It acts predominantly through its melatonin receptor type 1A (MTNR1A), and genetic variations of MTNR1A affect the susceptibility several diseases and cancer. The purpose of this study was to explore the effect of MTNR1A gene polymorphisms on the susceptibility to and clinicopathological characteristics of urothelial cell carcinoma (UCC). We recruited 272 patients with UCC and 272 normal controls to analyze three common single-nucleotide polymorphisms (SNPs) (rs2119882, rs13140012, and rs6553010) of MTNR1A related to cancer risk and clinicopathological relevance according to a TaqMan-based real-time polymerase chain reaction (PCR). We found that these three SNPs of MTNR1A were not associated with UCC susceptibility. However, patients with UCC who had at least one G allele of MTNR1A rs6553010 (in intron 1) were at higher risk (1.768-fold, 95% confidence interval: 1.068~1.849) of developing an invasive stage (p < 0.026), compared to those patients with AA homozygotes. In conclusion, polymorphic genotypes of rs6553010 of MTNR1A might contribute to the ability to predict aggressive phenotypes of UCC. This is the first study to provide insights into risk factors associated with intronic MTNR1A variants in the clinicopathologic development of UCC in Taiwan.

Effects of everolimus on tuberous sclerosis complex-associated renal angiomyolipoma: A preliminary report.

AIM: Tuberous sclerosis complex (TSC) presents with multisystem benign neoplasm induced by dysregulation of the mammalian target of rapamycin pathway. This study aimed to examine the effects of oral everolimus at either 2.5 or 5.0 mg daily on the treatment of TSC-associated renal angiomyolipoma (AML). METHODS: Between July 2012 and August 2015, patients with TSC-associated renal AML were selected for everolimus therapy protocol. An oral everolimus starting dose at 2.5 mg was administered daily, and was gradually increased to 5.0 mg daily. All patients were evaluated using magnetic resonance imaging or computed tomography scanning at baseline, 12, 24, and 36 months after the start of treatment for measuring the changes of renal AML mass volume. RESULTS: Eight patients were finally enrolled for analysis in this study. Everolimus treatment had a statistically significant effect on the renal AML volume reduction during follow-up (P < 0.05). Renal AML mass volume reduction rates were 10.5-45.3% in four patients with everolimus 2.5 mg and 40.7-73.1% in four patients with everolimus 5.0 mg daily; the difference was statistically significant between the two groups (P < 0.05). Longitudinal follow-up for response to everolimus showed volume reduction rates to be around 10.5-73.1% in the initial 6-24 months after everolimus treatment, which remained stable during follow-up up to 36 months. CONCLUSION: The results suggest that an oral everolimus is effective and provides a non-invasive way to treat TSC-associated renal AML, and patients are likely to require maintenance therapy to continue to derive benefit.

Combined effects of GSTO1 and SULT1A1 polymorphisms and cigarette smoking on urothelial carcinoma risk in a Taiwanese population.

BACKGROUND/PURPOSE: Cigarette smoking is the main risk factor for urothelial carcinoma of the bladder (UCB). Glutathione S-transferase omega 1 (GSTO1) and sulfotransferase 1A1 (SULT1A1) have been reported to be associated with the metabolism of polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. The aim of the present study was to investigate the combined effects of polymorphisms in GSTO1 and SULT1A1 genes and cigarette smoking on UCB risk in a Taiwanese population. METHODS: A total of 300 patients with histopathologically confirmed UCB and 233 cancer-free controls were recruited from the Department of Urology of Tung's Taichung Metro Harbor Hospital and Taipei Medical University Hospital. A comprehensive interview was conducted to collect personal information, including demographic characteristics and cigarette smoking status. A multivariate-adjusted logistic regression was performed to estimate the risk of UCB. RESULTS: A significantly increased risk of UCB was observed in ever smokers [odds ratio (OR) = 2.3]. The Ala/Ala genotype of the GSTO1 gene and the Arg/Arg genotype of the SULT1A1 gene were associated with a significantly increased risk of UCB, with ORs of 1.8 [95% confidence interval (CI) = 1.2-2.6] and 2.1 (95% CI = 1.6-4.5), respectively. Significantly increased UCB risks were found in heavy smokers with the Ala/Ala genotype of the GSTO1 gene (OR = 4.2) and the Arg/Arg genotype of the SULT1A1 gene (OR = 6.8). Furthermore, a significant synergistic effect in an additive model (OR = 3.5) between the GSTO1 Ala/Ala genotype and the SULT1A1 Arg/Arg genotype on UCB risk was observed. CONCLUSION: The present study provided epidemiological evidence for a significantly increased risk of UCB in ever smokers with the Ala/Ala genotype of the GSTO1 gene and the Arg/Arg genotype of the SULT1A1 gene.

Lactoferrin as a therapeutic agent for attenuating hepatic stellate cell activation in thioacetamide-induced liver fibrosis.

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF's protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF's mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-ß1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF's ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1ß and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-ß1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis.

Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening lung disease with high mortality rates. The limited availability of effective drugs for IPF treatment, coupled with concerns regarding adverse effects and restricted responsiveness, underscores the need for alternative approaches. Kefir peptides (KPs) have demonstrated antioxidative, anti-inflammatory, and antifibrotic properties, along with the capability to modulate gut microbiota. This study aims to investigate the impact of KPs on bleomycin-induced pulmonary fibrosis. METHODS: Mice were treated with KPs for four days, followed by intratracheal injection of bleomycin for 21 days. Comprehensive assessments included pulmonary functional tests, micro-computed tomography (µ-CT), in vivo image analysis using MMPsense750, evaluation of inflammation- and fibrosis-related gene expression in lung tissue, and histopathological examinations. Furthermore, a detailed investigation of the gut microbiota community was performed using full-length 16 S rRNA sequencing in control mice, bleomycin-induced fibrotic mice, and KPs-pretreated fibrotic mice. RESULTS: In KPs-pretreated bleomycin-induced lung fibrotic mice, notable outcomes included the absence of significant bodyweight loss, enhanced pulmonary functions, restored lung tissue architecture, and diminished thickening of inter-alveolar septa, as elucidated by morphological and histopathological analyses. Concurrently, a reduction in the expression levels of oxidative biomarkers, inflammatory factors, and fibrotic indicators was observed. Moreover, 16 S rRNA sequencing demonstrated that KPs pretreatment induced alterations in the relative abundances of gut microbiota, notably affecting Barnesiella_intestinihominis, Kineothrix_alysoides, and Clostridium_viride. CONCLUSIONS: Kefir peptides exerted preventive effects, protecting mice against bleomycin-induced lung oxidative stress, inflammation, and fibrosis. These effects are likely linked to modifications in the gut microbiota community. The findings highlight the therapeutic potential of KPs in mitigating pulmonary fibrosis and advocate for additional exploration in clinical settings.

Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

The matrix metalloprotease A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1) was reported to be involved in tumor progression in several cancer types, but its contributions appear discrepant. At present, the role of ADAMTS1 in oral squamous cell carcinoma (SCC; OSCC) remains unclear. Herein, The Cancer Genome Atlas (TCGA) database showed that ADAMTS1 transcripts were downregulated in head and neck SCC (HNSCC) tissues compared to normal tissues, but ADAMTS1 levels were correlated with poorer prognoses of HNSCC patients. In vitro, we observed that ADAMTS1 expression levels were correlated with the invasive abilities of four OSCC cell lines, HSC-3, SCC9, HSC-3M, and SAS. Knockdown of ADAMTS1 in OSCC cells led to a decrease and its overexpression led to an increase in cell-invasive abilities in vitro as well as tumor growth and lymph node (LN) metastasis in OSCC xenografts. Mechanistic investigations showed that the cyclic increase in ADAMTS1-L1 cell adhesion molecule (L1CAM) axis-mediated epidermal growth factor receptor (EGFR) activation led to exacerbation of the invasive abilities of OSCC cells via inducing epithelial-mesenchymal transition (EMT) progression. Clinical analyses revealed that ADAMTS1, L1CAM, and EGFR levels were all correlated with worse prognoses of HNSCC patients, and patients with ADAMTS1high/L1CAMhigh or EGFRhigh tumors had the shortest overall and disease-specific survival times. As to therapeutic aspects, we discovered that an edible plant-derived flavonoid, apigenin (API), drastically inhibited expression of the ADAMTS1-L1CAM-EGFR axis and reduced the ADAMTS1-triggered invasion and LN metastasis of OSCC cells in vitro and in vivo. Most importantly, API treatment significantly prolonged survival rates of xenograft mice with OSCC. In summary, ADAMTS1 may be a useful biomarker for predicting OSCC progression, and API potentially retarded OSCC progression by targeting the ADAMTS1-L1CAM-EGFR signaling pathway.

Robot-Assisted Radical Prostatectomy by the Hugo Robotic-Assisted Surgery (RAS) System and the da Vinci System: A Comparison between the Two Platforms.

OBJECTIVE: In a previous study, we proved that an experienced urologist is more likely to adapt to the Hugo RAS system. Based on this, we further examine various parameters in this study. Parameters included in this study consisted of console time, functional outcomes, and oncological outcomes. MATERIALS AND METHODS: A total of 60 patients who underwent robot-assisted radical prostatectomy (RARP) performed by a single surgeon using the da Vinci (DV) system (n = 30) or the Hugo RAS system (n = 30) between March 2023 and August 2023 were included in the analysis. The intraoperative operative time was categorized into vesicourethral anastomosis time and overall console time. Functional and oncological outcomes were documented at the 1st and 3rd postoperative months. Parametric and non-parametric methods were adopted after checking skewness and kurtosis, and an α value of 5% was used to determine the significance. RESULTS: The vesicourethral anastomosis time was significantly lengthened (Hedge's g: 0.87; 95% confidence interval (CI): 0.34-1.39; J factor = 0.987). However, the overall console time was not affected. The functional (postoperative 3rd month: p = 0.130) and oncological outcomes (postoperative 3rd month: p = 0.103) were not significantly different. We also found that the adverse effect on surgical specimens and positive surgical margins was not affected (p = 0.552). CONCLUSION: During the process of adaptation, although intricate motions (such as the vesicourethral anastomosis time) would be lengthened, the overall console time would not change remarkably. In this process, the functional and oncological outcomes would not be compromised. This encourages urologists to adopt the Hugo RAS system in RARP if they have previous experiences of using the DV system, since their trifecta advantage would not be compromised.

Evaluating Urine Cytology Slide Digitization Efficiency: A Comparative Study Using an Artificial Intelligence-Based Heuristic Scanning Simulation and Multiple Z-Plane Scanning.

INTRODUCTION: Digitizing cytology slides presents challenges because of their three-dimensional features and uneven cell distribution. While multi-Z-plane scan is a prevalent solution, its adoption in clinical digital cytopathology is hindered by prolonged scanning times, increased image file sizes, and the requirement for cytopathologists to review multiple Z-plane images. METHODS: This study presents heuristic scan as a novel solution, using an artificial intelligence (AI)-based approach specifically designed for cytology slide scanning as an alternative to the multi-Z-plane scan. Both the 21 Z-plane scan and the heuristic scan simulation methods were used on 52 urine cytology slides from three distinct cytopreparations (Cytospin, ThinPrep, and BD CytoRich™ [SurePath]), generating whole-slide images (WSIs) via the Leica Aperio AT2 digital scanner. The AI algorithm inferred the WSI from 21 Z-planes to quantitate the total number of suspicious for high-grade urothelial carcinoma or more severe cells (SHGUC+) cells. The heuristic scan simulation calculated the total number of SHGUC+ cells from the 21 Z-plane scan data. Performance metrics including SHGUC+ cell coverage rates (calculated by dividing the number of SHGUC+ cells identified in multiple Z-planes or heuristic scan simulation by the total SHGUC+ cells in the 21 Z-planes for each WSI), scanning time, and file size were analyzed to compare the performance of each scanning method. The heuristic scan's metrics were linearly estimated from the 21 Z-plane scan data. Additionally, AI-aided interpretations of WSIs with scant SHGUC+ cells followed The Paris System guidelines and were compared with original diagnoses. RESULTS: The heuristic scan achieved median SHGUC+ cell coverage rates similar to 5 Z-plane scans across three cytopreparations (0.78-0.91 vs. 0.75-0.88, p = 0.451-0.578). Notably, it substantially reduced both scanning time (137.2-635.0 s vs. 332.6-1,278.8 s, p < 0.05) and image file size (0.51-2.10 GB vs. 1.16-3.10 GB, p < 0.05). Importantly, the heuristic scan yielded higher rates of accurate AI-aided interpretations compared to the single Z-plane scan (62.5% vs. 37.5%). CONCLUSION: We demonstrated that the heuristic scan offers a cost-effective alternative to the conventional multi-Z-plane scan in digital cytopathology. It achieves comparable SHGUC+ cell capture rates while reducing both scanning time and image file size, promising to aid digital urine cytology interpretations with a higher accuracy rate compared to the conventional single (optimal) plane scan. Further studies are needed to assess the integration of this new technology into compatible digital scanners for practical cytology slide scanning.

Comparative evaluation of slide scanners, scan settings, and cytopreparations for digital urine cytology.

Background: Acquiring well-focused digital images of cytology slides with scanners can be challenging due to the 3-dimensional nature of the slides. This study evaluates performances of whole-slide images (WSIs) obtained from 2 different cytopreparations by 2 distinct scanners with 3 focus modes. Methods: Fourteen urine specimens were collected from patients with urothelial carcinoma. Each specimen was equally divided into 2 portions, prepared with Cytospin and ThinPrep methods and scanned for WSIs using Leica (Aperio AT2) and Hamamatsu (NanoZoomer S360) scanners, respectively. The scan settings included 3 focus modes (default, semi-auto, and manual) for single-layer scanning, along with a manual focus mode for 21 Z-layers scanning. Performance metrics were evaluated including scanning success rate, artificial intelligence (AI) algorithm-inferred atypical cell numbers and coverage rate (atypical cell numbers in single or multiple Z-layers divided by the total atypical cell numbers in 21 Z-layers), scanning time, and image file size. Results: The default mode had scanning success rates of 85.7% or 92.9%, depending on the scanner used. The semi-auto mode increased success to 92.9% or 100%, and manual even further to 100%. However, these changes did not affect the standardized median atypical cell numbers and coverage rates. The selection of scanners, cytopreparations, and Z-stacking influenced standardized median atypical cell numbers and coverage rates, scanning times, and image file sizes. Discussion: Both scanners showed satisfactory scanning. We recommend using semi-auto or manual focus modes to achieve a scanning success rate of up to 100%. Additionally, a minimum of 9-layer Z-stacking at 1 µm intervals is required to cover 80% of atypical cells. These advanced focus methods do not impact the number of atypical cells or their coverage rate. While Z-stacking enhances the AI algorithm's inferred quantity and coverage rates of atypical cells, it simultaneously results in longer scanning times and larger image file sizes.

Knockdown of lipocalin-2 suppresses the growth and invasion of prostate cancer cells.

BACKGROUND: Lipocalin-2 (LCN2) is a member of the lipocalin superfamily, and it has an important role in the regulation of cellular oncogenesis and apoptosis. However, the role for LCN2 in prostate cancer remains unclear. METHOD: LCN2 expression has been determined by Western blotting, qRT-PCR, and immunohistochemistry in the human prostate cell lines PC3, DU145, LNCaP, and 22Rv, and in human prostate tissue array. In this study, we identified shRNA-LCN2 to determine the role of LCN2 in prostate-cancer cell proliferation, migration, and invasion. Cell proliferative ability was measured by MTT, colony-formation, and cell-cycle analysis. The role of LCN2 in prostate-cancer cell migration and invasion was analyzed by cell-migration assay and Matrigel invasion assay. The effect of LCN2 knockdown on prostate tumor growth was assessed in a subcutaneous xenograft model. RESULTS: LCN2 protein and mRNA expression are higher in PC3 and DU145 cells than in LNCaP and 22Rv cells, and prostate cancer tissue correlated significantly with tumor differentiation (P < 0.017) and Gleason's grade (P < 0.02). LCN2 knockdown in PC3 and DU145 cells decreased cell proliferation, colony formation, cell cycle arrest, migration, and invasion. Conversely, LCN2 overexpression in 22Rv cells produced the opposite effect. Subcutaneous xenografts in mice models showed decreased tumor growth in the LCN2-knockdown mice. CONCLUSIONS: Our results suggest that LCN2 might play an important role in regulation of proliferation and invasion of human prostate cancer, and that it can be a valuable marker of prostate cancer progression.

Association of epidermal growth factor receptor mutations with human papillomavirus 16/18 E6 oncoprotein expression in non-small cell lung cancer.

BACKGROUND: Lung cancers in women, in nonsmokers, and in patients with adenocarcinoma from Asia have more prevalent mutations in the epidermal growth factor receptor (EGFR) gene than their counterparts. However, the etiology of EGFR mutations in this population remains unclear. The authors hypothesized that the human papillomavirus (HPV) type 16/18 (HPV16/18) E6 oncoprotein may contribute to EGFR mutations in Taiwanese patients with lung cancer. METHODS: One hundred fifty-one tumors from patients with lung cancer were enrolled to determine HPV16/18 E6 and EGFR mutations using immunohistochemistry and direct sequencing, respectively. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in lung tumors and cells were evaluated using immunohistochemistry and liquid chromatography-mass spectrometry/mass spectrometry. An supF mutagenesis assay was used to determine H2 O2 -induced mutation rates of lung cancer cells with or without E6 expression. RESULTS: Patients with E6-positive tumors had a greater frequency of EGFR mutations than those with E6-negative tumors (41% vs 20%; P = .006). Levels of 8-oxo-dG were correlated with EGFR mutations (36% vs 16%; P = .012). Two stable clones of E6-overexpressing H157 and CL-3 cells were established for the supF mutagenesis assay. The data indicated that the cells with high E6 overexpression had higher H2 O2 -induced SupF gene mutation rates compared with the cells that expressed lower levels of E6 and compared with vector control cells. CONCLUSIONS: HPV16/18 E6 may contribute in part to EGFR mutations in lung cancer, at least in the Taiwanese population.

Quantitative detection and identification of Naegleria spp. in various environmental water samples using real-time quantitative PCR assay.

Naegleria spp. is a free-living amoeba that can be found in various aquatic environments. There are some Naegleria spp. that can cause fatal infections in animals and humans, and the most important source of infection is through direct water contact. In this study, a real-time quantitative PCR was developed to detect and quantify the Naegleria spp. in various environmental water samples. The water samples were taken from rivershed, water treatment plants, and thermal spring recreation areas. The total detection rate was 4.0% (7/176) for Naegleria spp. The percentages of samples containing Naegleria spp. from river water, raw drinking water, and thermal spring water were 0% (0/100), 10.7% (3/28) and 8.3% (4/48), respectively. The concentration of Naegleria spp. in detected positive raw drinking water and thermal spring water samples was in the range of 3.9-12.6 and 1.1-24.2 cells/L, respectively. The identified species included Naegleria australiensis, Naegleria lovaniensis, and Naegleria spitzbergeniensis. The presence of Naegleria spp. in various aquatic environments is considered a potential public health threat.

Real-time PCR method for the detection and quantification of Acanthamoeba species in various types of water samples.

In this study, a quantitative real-time PCR was developed to detect and quantify Acanthamoeba spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and three thermal spring recreation areas. The overall detection rate was 14.2 % (25/176) for Acanthamoeba spp. The percentages of samples containing Acanthamoeba spp. from river water, raw drinking water, and thermal spring water were 13 % (13/100), 25 % (7/28), and 10.4 % (5/48), respectively. Acanthamoeba spp. concentrations were determined according to SYBR Green quantitative real-time PCR. A plasmid-based standard curve was constructed to determine the Acanthamoeba concentration using dilution factors for achieving 1.36 × 10(9) gene copies per PCR for 18S rRNA gene in Acanthamoeba spp. The resulting concentrations varied by the type of water, in the range of 46-2.6 × 10(2) cells/l in positive raw drinking water, 2.7 × 10(2)-1.5 × 10(4) cells/l in river water, and 54-1.7 × 10(3) cells/l in thermal spring water. The presence of Acanthamoeba spp. in the raw drinking water samples was also found to have a significant difference with heterotrophic plate count. The presence of Acanthamoeba spp. in various aquatic environments may be a potential health hazard and must be further evaluated.

One-Step Robot-Assisted Complete Urinary Tract Extirpation in Man with End-Stage Renal Disease on Dialysis: The First Case Report.

Urothelial carcinoma (UC) could be observed in urinary bladder (UBUC) and upper urinary tracts (UTUC). In the National Comprehensive Cancer Network guidelines for bladder cancer, extirpative surgery is indicated in certain cases. However, some extreme cases might also need the extirpation of the majority of the urinary tract, which is called complete urinary tract extirpation (CUTE). We present a patient diagnosed with high-grade UBUC and UTUC. He underwent dialysis for end-stage renal disease (ESRD) at the same time. Considering his non-functional kidneys and removing his high-risk urothelium at the same time, we performed robot-assisted CUTE to extirpate both his upper urinary tracts, urinary bladder, and prostate. In our experience, the console time was not significantly elongated, and the perioperative course was uneventful. To our knowledge, this is the first case report adopting a robotic system in such an extreme case. We conclude that robot-assisted CUTE is worth further study regarding its oncological survival outcomes and perioperative safety in patients with ESRD on dialysis.