Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity.

BACKGROUND: Cancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms. RESULTS: In sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms. CONCLUSION: This work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Noncanonical Functions of the Polycomb Group Protein EZH2 in Breast Cancer.

Enhancer of Zeste Homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that is critical for determining cell identity. An epigenetic writer, EZH2 has a well-defined role in transcriptional repression by depositing trimethyl marks on lysine 27 of histone H3. However, there is mounting evidence that histone methyltransferases like EZH2 exert histone methyltransferase-independent functions. The relevance of these functions to breast cancer progression and their regulatory mechanisms are only beginning to become understood. Here, we review the current understanding of EZH2 H3K27me3-independent, noncanonical, functions and their regulation in breast cancer.

The importance of the bacterial cell wall in uranium(VI) biosorption.

The bacterial cell envelope, in particular the cell wall, is considered the main controlling factor in the biosorption of aqueous uranium(vi) by microorganisms. However, the specific roles of the cell wall, associated biomolecules, and other components of the cell envelope are not well defined. Here we report findings on the biosorption of uranium by isolated cell envelope components and associated biomolecules, with P. putida 33015 and B. subtilis 168 investigated as representative strains for the differences in Gram-negative and Gram-positive cell envelope architecture, respectively. The cell wall and cell surface membrane were isolated from intact cells and characterised by X-ray Photoelectron Spectroscopy (XPS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FT-IR) spectroscopy; revealing variations in the abundance of functional moieties and biomolecules associated with components of the cell envelope. Uranium biosorption was investigated as a function of cell envelope component and pH, comparing with intact cells. The isolated cell wall from both strains exhibited the greatest uranium biosorption capacity. Deprotonation of favourable functional groups on the biomass as the pH increased from 3 to 5.5 increased their uranium biosorption capacity by approximately 3 fold. The results from ATR-FT-IR indicated that uranium(vi) biosorption was mediated by phosphate and carboxyl groups associated with proteins and phosphorylated biopolymers of the cell envelope. This includes outer membrane phospholipids and LPS of Gram-negative bacteria and teichoic acids, surface proteins and peptidoglycan from Gram-positive bacteria. As a result, the biosorption process of uranium(vi) to microorganisms is controlled by surface interactions, resulting in higher accumulation of uranium in the cell envelope. This demonstrates the importance of bacterial cell wall as the key mediator of uranium biosorption with microorganisms.

Integrating untargeted metabolomics, genetically informed causal inference, and pathway enrichment to define the obesity metabolome.

BACKGROUND: Obesity and its associated diseases are major health problems characterized by extensive metabolic disturbances. Understanding the causal connections between these phenotypes and variation in metabolite levels can uncover relevant biology and inform novel intervention strategies. Recent studies have combined metabolite profiling with genetic instrumental variable (IV) analysis (Mendelian randomization) to infer the direction of causality between metabolites and obesity, but often omitted a large portion of untargeted profiling data consisting of unknown, unidentified metabolite signals. METHODS: We expanded upon previous research by identifying body mass index (BMI)-associated metabolites in multiple untargeted metabolomics datasets, and then performing bidirectional IV analysis to classify metabolites based on their inferred causal relationships with BMI. Meta-analysis and pathway analysis of both known and unknown metabolites across datasets were enabled by our recently developed bioinformatics suite, PAIRUP-MS. RESULTS: We identified ten known metabolites that are more likely to be causes (e.g., alpha-hydroxybutyrate) or effects (e.g., valine) of BMI, or may have more complex bidirectional cause-effect relationships with BMI (e.g., glycine). Importantly, we also identified about five times more unknown than known metabolites in each of these three categories. Pathway analysis incorporating both known and unknown metabolites prioritized 40 enriched (p < 0.05) metabolite sets for the cause versus effect groups, providing further support that these two metabolite groups are linked to obesity via distinct biological mechanisms. CONCLUSIONS: These findings demonstrate the potential utility of our approach to uncover causal connections with obesity from untargeted metabolomics datasets. Combining genetically informed causal inference with the ability to map unknown metabolites across datasets provides a path to jointly analyze many untargeted datasets with obesity or other phenotypes. This approach, applied to larger datasets with genotype and untargeted metabolite data, should generate sufficient power for robust discovery and replication of causal biological connections between metabolites and various human diseases.

Next-generation sequencing identifies recurrent copy number variations in invasive breast carcinomas from Ghana.

African and African-American (AA) women have higher incidence of triple-negative breast cancers (TNBC) with high histological grade and aggressive clinical behavior, but the reasons are not fully understood. We recently found that the oncogenic protein EZH2 is overexpressed in Ghanaian breast cancer patients, with 16% of the tumors expressing cytoplasmic EZH2. Understanding the molecular underpinnings of these aggressive tumors may lead to the identification of potential targetable oncogenic drivers. We characterized the copy number variations of 11 Ghanaian breast tumor patients by targeted multiplexed PCR-based DNA next-generation sequencing (NGS) over 130 cancer-relevant genes. While the DNA quality was not optimal for mutation analysis, 90% of the tumors had frequent recurrent copy number alterations (CNAs) of 17 genes: SDHC, RECQL4, TFE3, BCL11A, BCL2L1, PDGFRA, DEK, SMUG1, AKT3, SMARCA4, VHL, KLF6, CCNE1, G6PD, FGF3, ABL1, and CCND1, with the top oncogenic functions being mitotic G1-G1/S-phase regulation, gene transcription, apoptosis, and PI3K/AKT pathway. The most common recurrent high-level CNAs were gains of RECQL4 and SDHC, in 50% and 60% of cases, respectively. Network analyses revealed a significant predicted interaction among 12 of the 17 (70.6%) genes with high-level CNAs (p = 5.7E-07), which was highly correlated with EZH2 expression (r = 0.4-0.75). By immunohistochemistry, RECQL4 and SDHC proteins were upregulated in 53 of 86 (61.6%) and 48 of 86 (56%) of Ghanaian invasive carcinoma tissue samples. In conclusion, our data show that invasive carcinomas from Ghana exhibit recurrent CNAs in 17 genes, with functions in oncogenic pathways, including PI3K/AKT and G1-G1/S regulation, which may have implications for the biology and treatment of invasive carcinomas in African and AA women.

Fluorescence Spectroscopy Analysis of the Bacteria-Mineral Interface: Adsorption of Lipopolysaccharides to Silica and Alumina.

We present here a quantification of the sorption process and molecular conformation involved in the attachment of bacterial cell wall lipopolysaccharides (LPSs), extracted from Escherichia coli, to silica (SiO2) and alumina (Al2O3) particles. We propose that interfacial forces govern the physicochemical interactions of the bacterial cell wall with minerals in the natural environment, and the molecular conformation of LPS cell wall components depends on both the local charge at the point of binding and hydrogen bonding potential. This has an effect on bacterial adaptation to the host environment through adhesion, growth, function, and ability to form biofilms. Photophysical techniques were used to investigate adsorption of fluorescently labeled LPS onto mineral surfaces as model systems for bacterial attachment. Adsorption of macromolecules in dilute solutions was studied as a function of pH and ionic strength in the presence of alumina and silica via fluorescence, potentiometric, and mass spectrometry techniques. The effect of silica and alumina particles on bacterial growth as a function of pH was also investigated using spectrophotometry. The alumina and silica particles were used to mimic active sites on the surface of clay and soil particles, which serve as a point of attachment of bacteria in natural systems. It was found that LPS had a high adsorption affinity for Al2O3 while adsorbing weakly to SiO2 surfaces. Strong adsorption was observed at low pH for both minerals and varied with both pH and mineral concentration, likely in part due to conformational rearrangement of the LPS macromolecules. Bacterial growth was also enhanced in the presence of the particles at low pH values. This demonstrates that at a molecular level, bacterial cell wall components are able to adapt their conformation, depending on the solution pH, in order to maximize attachment to substrates and guarantee community survival.

PAIRUP-MS: Pathway analysis and imputation to relate unknowns in profiles from mass spectrometry-based metabolite data.

Metabolomics is a powerful approach for discovering biomarkers and for characterizing the biochemical consequences of genetic variation. While untargeted metabolite profiling can measure thousands of signals in a single experiment, many biologically meaningful signals cannot be readily identified as known metabolites nor compared across datasets, making it difficult to infer biology and to conduct well-powered meta-analyses across studies. To overcome these challenges, we developed a suite of computational methods, PAIRUP-MS, to match metabolite signals across mass spectrometry-based profiling datasets and to generate metabolic pathway annotations for these signals. To pair up signals measured in different datasets, where retention times (RT) are often not comparable or even available, we implemented an imputation-based approach that only requires mass-to-charge ratios (m/z). As validation, we treated each shared known metabolite as an unmatched signal and showed that PAIRUP-MS correctly matched 70-88% of these metabolites from among thousands of signals, equaling or outperforming a standard m/z- and RT-based approach. We performed further validation using genetic data: the most stringent set of matched signals and shared knowns showed comparable consistency of genetic associations across datasets. Next, we developed a pathway reconstitution method to annotate unknown signals using curated metabolic pathways containing known metabolites. We performed genetic validation for the generated annotations, showing that annotated signals associated with gene variants were more likely to be enriched for pathways functionally related to the genes compared to random expectation. Finally, we applied PAIRUP-MS to study associations between metabolites and genetic variants or body mass index (BMI) across multiple datasets, identifying up to ~6 times more significant signals and many more BMI-associated pathways compared to the standard practice of only analyzing known metabolites. These results demonstrate that PAIRUP-MS enables analysis of unknown signals in a robust, biologically meaningful manner and provides a path to more comprehensive, well-powered studies of untargeted metabolomics data.

Influence of saprophytic fungi and inorganic additives on enzyme activities and chemical properties of the biodegradation process of wheat straw for the production of organo-mineral amendments.

Cellulose and lignin as main components of crop residues have a significant influence on composting operations and composition of the final products. Both are strongly associated, and lignin can be considered an important barrier during the biodegradation process of lignocellulosic materials. Saprophytic fungi are efficient lignin degraders due to their complex enzymatic system. Therefore, the influence of the inoculation of saprophytic fungi (Coriolopsis rigida, Pleurotus ostreatus, Trichoderma harzianum and Trametes versicolor) and the supply of inorganic additives (Al2O3, Fe2O3 and allophanic soil) that promote the stabilization of carbon (C), were analyzed in the biodegradation of wheat straw (WS). The activity of Laccase (LAC), manganese peroxidase (MnP) and ß-glucosidase and changes in temperature, pH and E4/E6 ratio were analyzed in a biodegradation process of 126 days. The activity of LAC, MnP and the E4/E6 ratio were significantly influenced and increased (enzymes) by fungi species, inorganic additives, and time of inorganic material addition, as well as their interactions (p < 0.05). The WS inoculated with T. versicolor showed the highest average activities for LAC, MnP and ß-glucosidase (2000, 220 UL-1 and 400 µmol pNP g-1 h-1 respectively). Furthermore, the addition of Al2O3 and Fe2O3 increased all the activities regarded to the decomposition of WS and influenced the changes associated with the stabilization of OM in composted WS. In conclusion, the inoculation of WS with T. versicolor in combination with metal oxides improved the enzyme related to the biodegradation process of WS favorizing its stabilization in the medium time, which is of importance in the composting of residues with high C/N ratio.

ASTEMIZOLE, AN INHIBITOR OF ETHER-À-GO-GO-1 POTASSIUM CHANNEL, INCREASES THE ACTIVITY OF THE TYROSINE KINASE INHIBITOR GEFITINIB IN BREAST CANCER CELLS.

BACKGROUND: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression. OBJECTIVE: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells. MATERIALS AND METHODS: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using dose-response curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy-tometry. RESULTS: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle. CONCLUSIONS: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1.

CCN6 regulates IGF2BP2 and HMGA2 signaling in metaplastic carcinomas of the breast.

PURPOSE: Metaplastic breast carcinomas are an aggressive subtype of triple-negative breast cancer (TNBC) in which part or all of the adenocarcinoma transforms into a non-glandular component (e.g., spindled, squamous, or heterologous). We discovered that mammary-specific Ccn6/Wisp3 knockout mice develop mammary carcinomas with spindle and squamous differentiation that share upregulation of the oncofetal proteins IGF2BP2 (IMP2) and HMGA2 with human metaplastic carcinomas. Here, we investigated the functional relationship between CCN6, IGF2BP2, and HMGA2 proteins in vitro and in vivo, and their expression in human tissue samples. METHODS: MMTV-cre;Ccn6fl/fl tumors and spindle TNBC cell lines were treated with recombinant CCN6 protein or vehicle. IGF2BP2 was downregulated using shRNAs in HME cells with stable CCN6 shRNA knockdown, and subjected to invasion and adhesion assays. Thirty-one human metaplastic carcinomas were arrayed in a tissue microarray (TMA) and immunostained for CCN6, IGF2BP2, and HMGA2. RESULTS: CCN6 regulates IGF2BP2 and HMGA2 protein expression in MMTV-cre;Ccn6fl/fl tumors, in MDA-MB-231 and - 468, and in HME cells. CCN6 recombinant protein reduced IGF2BP2 and HMGA2 protein expression, and decreased growth of MMTV-cre;Ccn6fl/fl tumors in vivo. IGF2BP2 shRNA knockdown was sufficient to reverse the invasive abilities conferred by CCN6 knockdown in HME cells. Analyses of the TCGA Breast Cancer Cohort (n = 1238) showed that IGF2BP2 and HMGA2 are significantly upregulated in metaplastic carcinoma compared to other breast cancer subtypes. In clinical samples, low CCN6 is frequent in tumors with high IGF2BP2/HMGA2 with spindle and squamous differentiation. CONCLUSIONS: These data shed light into the pathogenesis of metaplastic carcinoma and demonstrate a novel CCN6/IGF2BP2/HMGA2 oncogenic pathway with biomarker and therapeutic implications.

Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca2+ concentrations ([Ca2+]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca2+ oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 µg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization.

EZH2 expands breast stem cells through activation of NOTCH1 signaling.

Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation.

The role of extracellular DNA in uranium precipitation and biomineralisation.

Bacterial extra polymeric substances (EPS) have been associated with the extracellular precipitation of uranium. Here we report findings on the biomineralisation of uranium, with extracellular DNA (eDNA) used as a model biomolecule representative of EPS. The complexation and precipitation of eDNA with uranium were investigated as a function of pH, ionic strength and varying concentrations of reactants. The role of phosphate moieties in the biomineralisation mechanism was studied by enzymatically releasing phosphate (ePO4) from eDNA compared to abiotic phosphate (aPO4). The eDNA-uranium precipitates and uranium minerals obtained were characterised by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FT-IR) spectroscopy, Scanning Electron Microscopy-Energy Dispersive X-Ray analysis (SEM-EDX), X-Ray Powder Diffraction (XRD) and X-Ray Photoelectron Spectroscopy (XPS). ATR-FT-IR showed that at pH 5, the eDNA-uranium precipitation mechanism was predominantly mediated by interactions with phosphate moieties from eDNA. At pH 2, the uranium interactions with eDNA occur mainly through phosphate. The solubility equilibrium was dependent on pH with the formation of precipitate reduced as the pH increased. The XRD data confirmed the formation of a uranium phosphate precipitate when synthesised using ePO4. XPS and SEM-EDX studies showed the incorporation of carbon and nitrogen groups from the enzymatic orthophosphate hydrolysis on the obtained precipitated. These results suggested that the removal of uranium from solution occurs via two mechanisms: complexation by eDNA molecules and precipitation of a uranium phosphate mineral of the type (UO2HPO4)·xH2O by enzymatic orthophosphate hydrolysis. This demonstrated that eDNA from bacterial EPS is a key contributor to uranium biomineralisation.

Tyrosine kinase discoidin domain receptors DDR1 and DDR2 are coordinately deregulated in triple-negative breast cancer.

Receptor kinases Discoidin Domain Receptors (DDRs) 1 and 2 are emerging as new therapeutic targets in breast cancer (BC). However, the expression of DDR proteins during BC progression and their association with BC subtypes remain poorly defined. Herein we report the first comprehensive immunohistochemical analyses of DDR protein expression in a wide range of breast tissues. DDR1 and DDR2 expression was investigated by immunohistochemistry in 218 samples of normal breast (n = 10), ductal carcinoma in situ (DCIS, n = 10), and invasive carcinomas (n = 198), arrayed in tissue microarrays with comprehensive clinical and follow-up information. Staining was evaluated for cell type, subcellular localization, percentage and intensity (scores 1-4), and association with disease subtype and outcome. In normal epithelium and DCIS, DDR1 was highly expressed, while DDR2 was negative in normal epithelium, and in DCIS it localized to cells at the epithelial-stromal interface. Of the 198 invasive carcinomas, DDR1 was high in 87 (44 %) and low in 103 (52 %), and DDR2 was high in 110 (56 %) and low in 87 (44 %). High DDR2 was associated with high tumor grade (P = 0.002), triple-negative subtype (TNBC) (P < 0.0001), and worse survival (P = 0.037). We discovered a novel concordant deregulation of DDR expression, with a DDR1(Low)/DDR2(High) profile significantly associated with TNBC, compared to luminal tumors (P = 0.012), and with worse overall survival. In conclusion, DDR2 upregulation occurs in DCIS, before stromal invasion, and may reflect epithelial-stromal cross-talk. A DDR1(Low)/DDR2(High) protein profile is associated with TNBC and may identify invasive carcinomas with worse prognosis.

Human sperm degradation of zona pellucida proteins contributes to fertilization.

BACKGROUND: The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. METHODS: Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. RESULTS: The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. CONCLUSIONS: This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Adsorption of poly acrylic acid onto the surface of calcite: an experimental and simulation study.

Macromolecular binding to minerals is of great importance in the formation of biofilms, and carboxylate functional groups have been found to play a pivotal role in the functioning of these macromolecules. Here we present both fluorescence time-resolved anisotropy measurements and simulation data on the conformational behaviour and binding of a poly acrylic acid polymer. In solution the polymer exhibits a pH dependent behaviour, with a coiled conformation at a low pH and extended conformation at higher pH values. The polymer is readily adsorbed on the surface of calcite, preferring to bind in an extended conformation, with the strength of the adsorption dependent on the pH and presence of counter ions. We discuss the reasons why the calculated adsorption free energy differs from that obtained from a Langmuir isotherm analysis, showing that they refer to different quantities. The enhanced binding of the extended conformations shows the importance of flexibility in the binding of macromolecules.

Regulation of EZH2 protein stability: new mechanisms, roles in tumorigenesis, and roads to the clinic.

The importance of EZH2 as a key methyltransferase has been well documented theoretically. Practically, the first EZH2 inhibitor Tazemetostat (EPZ6438), was approved by FDA in 2020 and is used in clinic. However, for most solid tumors it is not as effective as desired and the scope of clinical indications is limited, suggesting that targeting its enzymatic activity may not be sufficient. Recent technologies focusing on the degradation of EZH2 protein have drawn attention due to their potential robust effects. This review focuses on the molecular mechanisms that regulate EZH2 protein stability via post-translational modifications (PTMs), mainly including ubiquitination, phosphorylation, and acetylation. In addition, we discuss recent advancements of multiple proteolysis targeting chimeras (PROTACs) strategies and the latest degraders that can downregulate EZH2 protein. We aim to highlight future directions to expand the application of novel EZH2 inhibitors by targeting both EZH2 enzymatic activity and protein stability.

Clinical features and predictors of delayed neurological syndrome in carbon monoxide poisoning: the AMICO study. / Caracterización clínica y factores predictivos de desarrollo de síndrome neurológico tardío en la intoxicación por monóxido de carbono: estudio AMICO.

OBJECTIVES: To identify predictors for developing delayed neurological syndrome (DNS) after an initial episode of carbon monoxide (CO) poisoning in the interest of detecting patients most likely to develop DNS so that they can be followed. MATERIAL AND METHODS: Retrospective review of cases of CO poisoning treated in the past 10 years in the emergency departments of 4 hospitals in the AMICO study (Spanish acronym for the multicenter analysis of CO poisoning). We analyzed demographic characteristics of the patients and the clinical characteristics of the initial episode. The records of the cohort of patients with available follow-up information were reviewed to find cases of DNS. Data were analyzed by multivariant analysis to determine the relationship to characteristics of the initial exposure to CO. RESULTS: A total of 240 cases were identified. The median (interquartile range) age of the patients was 36.2 years (17.6-49.6 years); 108 patients (45.0%) were men, and the poisoning was accidental in 223 cases (92.9%). The median carboxyhemoglobin concentration on presentation was 12.7% (6.2%-18.7%). Follow-up details were available for 44 patients (18.3%). Eleven of those patients (25%) developed DNS. A low initial Glasgow Coma Scale score predicted the development of DNS with an odds ratio (OR) of 0.61 (95% CI, 0.41-0.92) and an area under the receiver operating characteristic curve of 0.876 (95% CI, 0.761-0.990) (P .001). CONCLUSION: The initial Glasgow Coma Scale score seems to be a clinical predictor of DNS after CO poisoning. We consider it important to establish follow-up protocols for patients with CO poisoning treated in hospital EDs.

OBJETIVO: Identificar factores pronósticos de desarrollo de síndrome neurológico tardío (SNT) después de un episodio inicial de intoxicación por monóxido de carbono (ICO), con el fin detectar precozmente a la población más susceptible y facilitar su acceso a un seguimiento específico. METODO: Revisión retrospectiva de todos los casos de ICO que acudieron a los servicios de urgencias (SU) de 4 hospitales durante los últimos 10 años. Se analizaron datos demográficos y características clínicas en el momento del episodio. En la cohorte de pacientes con datos de seguimiento disponibles, se evaluó la aparición de SNT y su relación con diferentes variables en la exposición inicial al CO a través de técnicas de análisis multivariante. RESULTADOS: Se identificaron 240 pacientes. La mediana de edad fue de 36,2 años (17,6-49,6). De ellos 108 (45,0%) eran hombres y 223 casos (92,9%) fueron accidentales. El nivel medio de COHb fue del 12,7% (6,2-18,7). En 44 (18,3%) episodios se disponía de datos de un seguimiento específico. En esta cohorte, 11 (25%) pacientes desarrollaron SNT. Una puntuación inicial más baja en la Escala Coma de Glasgow (GCS) (OR: 0,61, IC 95%: 0,41-0,92) fue predictor independiente del desarrollo del SNT, con un ABC en la curva COR de 0,876 (IC 95%: 0,761-0,990, p 0,001). CONCLUSIONES: Una puntuación inicial baja en la GCS parece ser un predictor clínico de desarrollo de SNT en la ICO. Dada la incidencia de SNT, consideramos fundamental establecer protocolos de seguimiento específico de estos pacientes tras su asistencia inicial en los SU.

Decreasing incidence of late-stage breast cancer after the introduction of organized mammography screening in Italy.

BACKGROUND: After the introduction of a mammography screening program, the incidence of late-stage breast cancer is expected to decrease. The objective of the current study was to evaluate variations in the total incidence of breast cancer and in the incidence of breast cancers with a pathologic tumor (pT) classification of pT2 through pT4 after the introduction of mammography screening in 6 Italian administrative regions. METHODS: The study area included 700 municipalities, with a total population of 692,824 women ages 55 to 74 years, that were targeted by organized mammography screening between 1991 and 2005. The year screening started at the municipal level (year 1) was identified. The years of screening were numbered from 1 to 8. The ratio of the observed 2-year, age-standardized (Europe) incidence rate to the expected rate (the incidence rate ratio [IRR]) was calculated. Expected rates were estimated assuming that the incidence of breast cancer was stable and was equivalent to that in the last 3 years before year 1. RESULTS: The study was based on a total of 14,447 incident breast cancers, including 4036 pT2 through pT4 breast cancers. The total IRR was 1.35 (95% confidence interval, 1.03-1.41) in years 1 and 2, 1.16 (95% confidence interval, 1.10-1.21) in years 3 and 4, 1.14 (95% confidence interval, 1.08-1.20) in years 5 and 6, and 1.14 (95% confidence interval, 1.08-1.21) in years 7 and 8. The IRR for pT2 through pT4 breast cancers was 0.97 (95% confidence interval, 0.90-1.04) in years 1 and 2, 0.81 (95% confidence interval, 0.75-0.88) in years 3 and 4, 0.79 (95% confidence interval, 0.73-0.87) in years 5 and 6, and 0.71 (95% confidence interval, 0.64-0.79) in years 7 and 8. CONCLUSIONS: A significant and stable decrease in the incidence of late-stage breast cancer was observed from the third year of screening onward, when the IRR varied between 0.81 and 0.71.

EZH2 inhibition decreases p38 signaling and suppresses breast cancer motility and metastasis.

EZH2 is a Polycomb group protein that exerts oncogenic functions in breast cancer, where its overexpression is associated with metastatic disease. While it reportedly acts a transcriptional repressor through trimethylation of histone H3 at lysine 27, EZH2 may exhibit context-dependent activating functions. Despite associations with worse outcome and metastasis in breast cancer, a functional role of EZH2 in breast cancer metastasis in vivo has not been demonstrated. Furthermore, whether EZH2 regulates cancer cell phenotype and motility are unknown. In this study, we discovered that knockdown of EZH2 induces a phenotypic reprogramming from mesenchymal to epithelial, reduces motility, and blocks invasion in breast cancer cell lines. In vivo, EZH2 downregulation in MDA-MB-231 cells decreases spontaneous metastasis to the lungs. We uncover an unexpected role of EZH2 in inducing the p38 mitogen-activated protein kinase signaling pathway, an important regulator of breast cancer invasion and metastasis. In breast cancer cells, EZH2 binds to phosphorylated p38 (p-p38) in association with other core members of the Polycomb repressive complex 2, EED, and SUZ12, and EZH2 overexpression leads to increased levels of p-p38 and of activated, downstream pathway proteins. The effect on p-p38 was confirmed in vivo, where it correlated with decreased spontaneous metastasis. In clinical specimens of matched primary and invasive breast carcinomas, we found that EZH2 expression was upregulated in 100 % of the metastases, and that EZH2 and p-p38 were coexpressed in 63 % of cases, consistent with the functional results. Together our findings reveal a new mechanism by which EZH2 functions in breast cancer, and provide direct evidence that EZH2 inhibition reduces breast cancer metastasis in vivo.