Tracking single-cell evolution using clock-like chromatin accessibility loci.

Single-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.

The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples.

Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.

ACAT1 promotes proliferation and metastasis of bladder cancer via AKT/GSK3ß/c-Myc signaling pathway.

Acetyl-CoA acetyltransferase 1 (ACAT1) plays a significant role in the regulation of gene expression and tumorigenesis. However, the biological role of ACAT1 in bladder cancer (BLCA) has yet to be elucidated. This research aimed to elucidate the bioinformatics features and biological functions of ACAT1 in BLCA. Here, we demonstrate that ACAT1 is elevated in BLCA tissues and is correlated with specific clinicopathological features and an unfavorable prognosis for survival in BLCA patients. ACAT1 was identified as an independent risk factor in BLCA. Phenotypically, both in vitro and in vivo, ACAT1 knockdown suppressed BLCA cell proliferation and migration, while ACAT1 overexpression had the opposite effect. Mechanistic assays revealed that ACAT1 enhances BLCA cell proliferation and metastasis through the AKT/GSK3ß/c-Myc signaling pathway by modulating the cell cycle and EMT. Taken together, the results of our study reveal that ACAT1 is an oncogenic driver in BLCA that enhances tumor proliferation and metastasis, indicating its potential as a diagnostic and therapeutic target for this disease.

Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

Evaluation of DNA extracted from residual blood clots after serological testing.

DNA quality is of paramount importance for molecular biology research. This study aimed to assess the DNA extracted from residual blood clots after serological testing, focusing on the impact of blood clot segments, extraction kits, temporary storage durations (TSDs), and thawing methods on DNA quality. We divided the residual blood clot column (BCC) from healthy donors into three segments and utilized two different extraction kits. The BCCs were subjected to four TSDs at 4°C (7 days, 10 days, 1 month, and 2 months) and three thawing methods (4°C, room temperature, and 37°C). We found that the TIANamp Blood Clot DNA Kit yielded consistently high-quality DNA from each segment with stable A260/280 and A260/230 ratios. The DNA yield showed a strong positive correlation with leukocyte concentration, and a satisfactory median DNA yield of 28.79 µg/g BCC was obtained across all segments. DNA integrity, as measured by the DNA integrity number and DNA fragment peak size, decreased with increasing TSD at 4°C, with a notable decrease after 10 days of storage. Thawing at 37°C resulted in the lowest DNA fragment peak size. In conclusion, BCC could be an ideal DNA source with satisfactory yield and purity. A prolonged TSD at 4°C leads to an obvious decrease in DNA integrity, and thawing the frozen BCC at 37°C decreases DNA fragment sizes. To maintain DNA integrity, BCCs should be cryopreserved as soon as possible after short TSDs at 4°C and thawed at 4°C.

Causal association of smoking, blood lipids, and bladder cancer: Insights from a multivariable and mediation mendelian randomization investigation.

We used Mendelian randomization (MR) to examine the relationship between smoking, various categories of blood lipids, and bladder cancer (BLCA). Data for this study were drawn from the genome-wide association studies of the GSCAN consortium (~1.2 million participants), a subset of the UK Biobank (~120,000 participants), and the FinnGen consortium (2,072 cases and 307,082 controls). Initially, we utilized inverse variance weighted (IVW), complementary and sensitivity analyses, multivariable MR, and meta-analysis to confirm the association between blood lipids and BLCA. We then performed mediation MR to elucidate the relationship between smoking, blood lipids, and BLCA. Our analysis identified five lipids, including triglycerides in very large HDL, cholesterol in small VLDL, free cholesterol in very large HDL, total free cholesterol, and apolipoprotein B, as having strong and inverse associations with BLCA. These lipids demonstrated no heterogeneity or pleiotropy and exhibited consistent direction and magnitude across IVW, weighted median, and MR-Egger analyses. Our mediation MR further revealed that triglycerides in very large HDL and cholesterol in small VLDL could reduce the impact of smoking on BLCA, mediating -4.3% and -4.5% of the effect, respectively. In conclusion, our study identified five lipids exhibiting a robust inverse relationship with BLCA, two of which can buffer the impact of smoking on BLCA.

UPP1 enhances bladder cancer progression and gemcitabine resistance through AKT.

UPP1, a crucial pyrimidine metabolism-related enzyme, catalyzes the reversible phosphorylation of uridine to uracil and ribose-1-phosphate. However, the effects of UPP1 in bladder cancer (BLCA) have not been elucidated. AKT, which is activated mainly through dual phosphorylation (Thr308 and Ser473), promotes tumorigenesis by phosphorylating downstream substrates. This study demonstrated that UPP1 promotes BLCA cell proliferation, migration, invasion, and gemcitabine resistance by activating the AKT signaling pathway in vitro and in vivo. Additionally, UPP1 promoted AKT activation by facilitating the binding of AKT to PDK1 and PDK2 and the recruitment of phosphatidylinositol 3,4,5-triphosphate to AKT. Moreover, the beneficial effects of UPP1 on BLCA tumorigenesis were mitigated upon UPP1 mutation with Arg94 or MK2206 treatment (AKT-specific inhibitor). AKT overexpression or SC79 (AKT-specific activator) treatment restored tumor malignancy and drug resistance. Thus, this study revealed that UPP1 is a crucial oncogene and a potential therapeutic target for BLCA and that UPP1 activates the AKT signaling pathway and enhances tumorigenesis and drug resistance to gemcitabine.

Parkin inhibits proliferation and migration of bladder cancer via ubiquitinating Catalase.

PRKN is a key gene involved in mitophagy in Parkinson's disease. However, recent studies have demonstrated that it also plays a role in the development and metastasis of several types of cancers, both in a mitophagy-dependent and mitophagy-independent manner. Despite this, the potential effects and underlying mechanisms of Parkin on bladder cancer (BLCA) remain unknown. Therefore, in this study, we investigated the expression of Parkin in various BLCA cohorts derived from human. Here we show that PRKN expression was low and that PRKN acts as a tumor suppressor by inhibiting the proliferation and migration of BLCA cells in a mitophagy-independent manner. We further identified Catalase as a binding partner and substrate of Parkin, which is an important antioxidant enzyme that regulates intracellular ROS levels during cancer progression. Our data showed that knockdown of CAT led to increased intracellular ROS levels, which suppressed cell proliferation and migration. Conversely, upregulation of Catalase decreased intracellular ROS levels, promoting cell growth and migration. Importantly, we found that Parkin upregulation partially restored these effects. Moreover, we discovered that USP30, a known Parkin substrate, could deubiquitinate and stabilize Catalase. Overall, our study reveals a novel function of Parkin and identifies a potential therapeutic target in BLCA.

Acyl-coenzyme A: cholesterol acyltransferase inhibitor avasimibe suppresses tumorigenesis and induces G1-phase cell-cycle arrest by activating PPARγ signaling pathway in bladder cancer.

Reprogramming of energy metabolism is one of the most important characteristics of tumors. Bladder cancer (BLCA) cells contain higher levels of cholesterol content compared to normal cells, and acyl-coenzyme A (CoA): cholesterol acyltransferase-1 (ACAT1) plays a crucial role in the esterification of cholesterol. Avasimibe is a drug that has been used in the treatment of atherosclerosis, and it can effectively inhibit ACAT1. We observed that ACAT1 was significantly up-regulated in BLCA and positively correlated with tumor grade. By avasimibe administration, the proliferation and migration ability of BLCA cells were reduced, while the production of ROS was strongly increased, accompanied by the up-regulated expression of ROS metabolism-related proteins SOD2 and catalase. Furthermore, BLCA cell cycle was arrested at the G1 phase, accompanied by the downregulation of cell cycle-related proteins (CCNA1/2, CCND1, CDK2 and CDK4), while the PPARγ was found to be up-regulated at both transcriptional and protein levels after avasimibe treatment. Then we found that the PPARγ antagonist GW9662 could reverse the effect of avasimibe on the cell cycle. Moreover, xenograft and pulmonary metastasis models further demonstrated that avasimibe could inhibit tumor cell growth and metastasis in vivo. Taken together, our results for the first time revealed that avasimibe can inhibit BLCA progression and metastasis, and PPARγ signaling pathway may play a key role in regulation of cell cycle distribution induced by avasimibe.

USP43 stabilizes c-Myc to promote glycolysis and metastasis in bladder cancer.

A hallmark of tumor cells, including bladder cancer (BLCA) cells, is metabolic reprogramming toward aerobic glycolysis (Warburg effect). The classical oncogene MYC, which is crucial in regulating glycolysis, is amplified and activated in BLCA. However, direct targeting of the c-Myc oncoprotein, which regulates glycolytic metabolism, presents great challenges and necessitates the discovery of a more clarified regulatory mechanism to develop selective targeted therapy. In this study, a siRNA library targeting deubiquitinases identified a candidate enzyme named USP43, which may regulate glycolytic metabolism and c-Myc transcriptional activity. Further investigation using functional assays and molecular studies revealed a USP43/c-Myc positive feedback loop that contributes to the progression of BLCA. Moreover, USP43 stabilizes c-Myc by deubiquitinating c-Myc at K148 and K289 primarily through deubiquitinase activity. Additionally, upregulation of USP43 protein in BLCA increased the chance of interaction with c-Myc and interfered with FBXW7 access and degradation of c-Myc. These findings suggest that USP43 is a potential therapeutic target for indirectly targeting glycolytic metabolism and the c-Myc oncoprotein consequently enhancing the efficacy of bladder cancer treatment.

DLGAP5 triggers proliferation and metastasis of bladder cancer by stabilizing E2F1 via USP11.

Bladder cancer (BLCA) is one of the most widespread malignancies worldwide, and displays significant tumor heterogeneity. Understanding the molecular mechanisms exploitable for treating aggressive BLCA represents a crucial objective. Despite the involvement of DLGAP5 in tumors, its precise molecular role in BLCA remains unclear. BLCA tissues exhibit a substantial increase in DLGAP5 expression compared with normal bladder tissues. This heightened DLGAP5 expression positively correlated with the tumor's clinical stage and significantly affected prognosis negatively. Additionally, experiments conducted in vitro and in vivo revealed that alterations in DLGAP5 expression notably influence cell proliferation and migration. Mechanistically, the findings demonstrated that DLGAP5 was a direct binding partner of E2F1 and that DLGAP5 stabilized E2F1 by preventing the ubiquitination of E2F1 through USP11. Furthermore, as a pivotal transcription factor, E2F1 fosters the transcription of DLGAP5, establishing a positive feedback loop between DLGAP5 and E2F1 that accelerates BLCA development. In summary, this study identified DLGAP5 as an oncogene in BLCA. Our research unveils a novel oncogenic mechanism in BLCA and offers a potential target for both diagnosing and treating BLCA.

TRAIP suppresses bladder cancer progression by catalyzing K48-linked polyubiquitination of MYC.

TRAF-interacting protein (TRAIP), an E3 ligase containing a RING domain, has emerged as a significant contributor to maintaining genome integrity and is closely associated with cancer. Our study reveals that TRAIP shows reduced expression in bladder cancer (BLCA), which correlates with an unfavorable prognosis. In vitro and in vivo, TRAIP inhibits proliferation and migration of BLCA cells. MYC has been identified as a novel target for TRAIP, wherein direct interaction promotes K48-linked polyubiquitination at neighboring K428 and K430 residues, ultimately resulting in proteasome-dependent degradation and downregulation of MYC transcriptional activity. This mechanism effectively impedes the progression of BLCA. Restoring MYC expression reverses suppressed proliferation and migration of BLCA cells induced by TRAIP. Moreover, our results suggest that MYC may bind to the transcriptional start region of TRAIP, thereby exerting regulatory control over TRAIP transcription. Consequently, this interaction establishes a negative feedback loop that regulates MYC expression, preventing excessive levels. Taken together, this study reveals a mechanism that TRAIP inhibits proliferation and migration of BLCA by promoting ubiquitin-mediated degradation of MYC.

Pectolinarigenin inhibits bladder urothelial carcinoma cell proliferation by regulating DNA damage/autophagy pathways.

Pectolinarigenin (PEC), an active compound isolated from traditional herbal medicine, has shown potential anti-tumor properties against various types of cancer cells. However, its mechanism of action in bladder cancer (BLCA), which is one of the fatal human carcinomas, remains unexplored. In this study, we first revealed that PEC, as a potential DNA topoisomerase II alpha (TOP2A) poison, can target TOP2A and cause significant DNA damage. PEC induced G2/M phase cell cycle arrest via p53 pathway. Simultaneously, PEC can perform its unique function by inhibiting the late autophagic flux. The blocking of autophagy caused proliferation inhibition of BLCA and further enhanced the DNA damage effect of PEC. In addition, we proved that PEC could intensify the cytotoxic effect of gemcitabine (GEM) on BLCA cells in vivo and in vitro. Summarily, we first systematically revealed that PEC had great potential as a novel TOP2A poison and an inhibitor of late autophagic flux in treating BLCA.

Aspartoacylase suppresses prostate cancer progression by blocking LYN activation.

BACKGROUND: Globally, despite prostate cancer (PCa) representing second most prevalent malignancy in male, the precise molecular mechanisms implicated in its pathogenesis remain unclear. Consequently, elucidating the key molecular regulators that govern disease progression could substantially contribute to the establishment of novel therapeutic strategies, ultimately advancing the management of PCa. METHODS: A total of 49 PCa tissues and 43 adjacent normal tissues were collected from January 2017 to December 2021 at Zhongnan Hospital of Wuhan University. The advanced transcriptomic methodologies were employed to identify differentially expressed mRNAs in PCa. The expression of aspartoacylase (ASPA) in PCa was thoroughly evaluated using quantitative real-time PCR and Western blotting techniques. To elucidate the inhibitory role of ASPA in PCa cell proliferation and metastasis, a comprehensive set of in vitro and in vivo assays were conducted, including orthotopic and tumor-bearing mouse models (n = 8 for each group). A combination of experimental approaches, such as Western blotting, luciferase assays, immunoprecipitation assays, mass spectrometry, glutathione S-transferase pull-down experiments, and rescue studies, were employed to investigate the underlying molecular mechanisms of ASPA's action in PCa. The Student's t-test was employed to assess the statistical significance between two distinct groups, while one-way analysis of variance was utilized for comparisons involving more than two groups. A two-sided P value of less than 0.05 was deemed to indicate statistical significance. RESULTS: ASPA was identified as a novel inhibitor of PCa progression. The expression of ASPA was found to be significantly down-regulated in PCa tissue samples, and its decreased expression was independently associated with patients' prognosis (HR = 0.60, 95% CI 0.40-0.92, P = 0.018). Our experiments demonstrated that modulation of ASPA activity, either through gain- or loss-of-function, led to the suppression or enhancement of PCa cell proliferation, migration, and invasion, respectively. The inhibitory role of ASPA in PCa was further confirmed using orthotopic and tumor-bearing mouse models. Mechanistically, ASPA was shown to directly interact with the LYN and inhibit the phosphorylation of LYN as well as its downstream targets, JNK1/2 and C-Jun, in both PCa cells and mouse models, in an enzyme-independent manner. Importantly, the inhibition of LYN activation by bafetinib abrogated the promoting effect of ASPA knockdown on PCa progression in both in vitro and in vivo models. Moreover, we observed an inverse relationship between ASPA expression and LYN activity in clinical PCa samples, suggesting a potential regulatory role of ASPA in modulating LYN signaling. CONCLUSION: Our findings provide novel insights into the tumor-suppressive function of ASPA in PCa and highlight its potential as a prognostic biomarker and therapeutic target for the management of this malignancy.

DNA polymerase POLD1 promotes proliferation and metastasis of bladder cancer by stabilizing MYC.

To date, most studies on the DNA polymerase, POLD1, have focused on the effect of POLD1 inactivation mutations in tumors. However, the implications of high POLD1 expression in tumorigenesis remains elusive. Here, we determine that POLD1 has a pro-carcinogenic role in bladder cancer (BLCA) and is associated to the malignancy and prognosis of BLCA. Our studies demonstrate that POLD1 promotes the proliferation and metastasis of BLCA via MYC. Mechanistically, POLD1 stabilizes MYC in a manner independent of its' DNA polymerase activity. Instead, POLD1 attenuates FBXW7-mediated ubiquitination degradation of MYC by directly binding to the MYC homology box 1 domain competitively with FBXW7. Moreover, we find that POLD1 forms a complex with MYC to promote the transcriptional activity of MYC. In turn, MYC increases expression of POLD1, forming a POLD1-MYC positive feedback loop to enhance the pro-carcinogenic effect of POLD1-MYC on BLCA. Overall, our study identifies POLD1 as a promotor of BCLA via a MYC driven mechanism and suggest its potential as biomarker for BLCA.

Melatonin inhibits bladder tumorigenesis by suppressing PPARγ/ENO1-mediated glycolysis.

Melatonin is a well-known natural hormone, which shows a potential anticancer effect in many human cancers. Bladder cancer (BLCA) is one of the most malignant human cancers in the world. Chemoresistance is an increasingly prominent phenomenon that presents an obstacle to the clinical treatment of BLCA. There is an urgent need to investigate novel drugs to improve the current clinical status. In our study, we comprehensively explored the inhibitory effect of melatonin on BLCA and found that it could suppress glycolysis process. Moreover, we discovered that ENO1, a glycolytic enzyme involved in the ninth step of glycolysis, was the downstream effector of melatonin and could be a predictive biomarker of BLCA. We also proved that enhanced glycolysis simulated by adding exogenous pyruvate could induce gemcitabine resistance, and melatonin treatment or silencing of ENO1 could intensify the cytotoxic effect of gemcitabine on BLCA cells. Excessive accumulation of reactive oxygen species (ROS) mediated the inhibitory effect of melatonin on BLCA cells. Additionally, we uncovered that PPARγ was a novel upstream regulator of ENO1, which mediated the downregulation of ENO1 caused by melatonin. Our study offers a fresh perspective on the anticancer effect of melatonin and encourages further studies on clinical chemoresistance.

Histone methyltransferase SETD2: An epigenetic driver in clear cell renal cell carcinoma.

SET domain-containing 2 (SETD2) is a lysine methyltransferase that catalyzes histone H3 lysine36 trimethylation (H3K36me3) and has been revealed to play important roles in the regulation of transcriptional elongation, RNA splicing, and DNA damage repair. SETD2 mutations have been documented in several cancers, including clear cell renal cell carcinoma (ccRCC). SETD2 deficiency is associated with cancer occurrence and progression by regulating autophagy flux, general metabolic activity, and replication fork speed. Therefore, SETD2 is considered a potential epigenetic therapeutic target and is the subject of ongoing research on cancer-related diagnosis and treatment. This review presents an overview of the molecular functions of SETD2 in H3K36me3 regulation and its relationship with ccRCC, providing a theoretical basis for subsequent antitumor therapy based on SETD2 or H3K36me3 targets.

Development and validation of a prognostic nomogram for neuroendocrine prostate cancer, based on the SEER database.

Background: The tumor biology of neuroendocrine prostate cancer (NEPC) is different from that of ordinary prostate cancer, herefore, existing clinical prognosis models for prostate cancer patients are unsuitable for NEPC. The specialized individual situation assessment and clinical decision-making tools for NEPC patients are urgently needed. This study aimed to develop a valid NEPC prognostic nomogram and risk stratification model to predict risk associated with patient outcomes. Methods: We collected 340 de-novo NEPC patients from the SEER database, and randomly selected 240 of them as the training set and the remaining 100 as the validation set. Cox regression model was used to screen for risk factors affecting overall survival (OS) and cancer-specific survival (CSS) and construct a corresponding nomogram. The receiver operating characteristic (ROC) curves, calibration curves, C-indexes, and decision curve analysis (DCA) curves are used to verify and calibrate nomograms. Results: NEPC prognosis nomograms were constructed by integrating independent risk factors. The C-indexes, ROC curves, calibration curves, and DCA curves revealed excellent prediction accuracy of the prognostic nomogram. Furthermore, we demonstrated that NEPC patients in the high-risk group had significantly lower OS and CSS than those in the low-risk group with risk scores calculated from nomograms. Conclusions: The nomogram established in this research has the potential to be applied to the clinic to evaluate the prognosis of NEPC patients and support corresponding clinical decision-making.

Effects of Freezing and Rewarming Methods on RNA Quality of Blood Samples.

Background: RNA extracted from human blood has been widely applied to biological, medical, and clinical research of numerous diseases. Previous studies have demonstrated that high-quality RNA is indispensable to guarantee the reliability of downstream assays. In this study, we investigated the effects of freezing procedures, rewarming methods, and blood components on RNA quality of blood samples. Methods: Rabbit blood samples were divided into two groups: (1) whole blood (WB) and (2) blood cell components (BCC) with plasma removed. Samples were frozen using four representative freezing procedures (snap freezing in liquid nitrogen, snap freezing at -80°C, traditional slow freezing, and programmable controlled rate freezing) and rewarmed by placing at 4°C or by vortexing. RNA was extracted using the phenol-chloroform RNA extraction method and measured by an Agilent bioanalyzer. Then, human blood was used to verify the best protocol obtained from the rabbit blood experiment. Results: For the four freezing procedures, there were no differences in RNA integrity. For different rewarming methods, RNA integrity number (RIN) values of RNA extracted from frozen WB and BCC samples in the vortex group were above 9, while RNA obtained from WB showed worse quality compared with BCC in the 4°C group. For verification using human blood, RIN values of frozen human WB rewarmed by vortexing ranged from 8.0 to 9.1. Conclusions: Blood components and rewarming methods could affect the RNA quality of blood samples. For scenarios where WB samples have already been cryopreserved, the vortex rewarming method is optimal for high-quality RNA. Otherwise, we would recommend centrifuging fresh WB and cryopreserving it in the form of BCC, which showed a tendency to obtain high-quality RNA by either of the two rewarming methods.

Correction: Qian et al. Capsaicin Suppresses Cell Proliferation, Induces Cell Cycle Arrest and ROS Production in Bladder Cancer Cells through FOXO3a-Mediated Pathways. Molecules 2016, 21, 1406.

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