DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

DEAD box RNA helicases act as nucleotide exchange factors for casein kinase 2.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.

The Psychological and Professional Burden Experienced by Nurses Who Worked in COVID-19 Clinics during the Pandemic: A Content Analysis.

INTRODUCTION: Since the beginning of the pandemic, nurses have played a key role in providing care for COVID-19 patients. Infection risk and fear, use of personal protective equipment, and social isolation were related to high levels of stress and extreme psychological drain among front-line healthcare providers. AIM: The aim of this study was to explore how front-line nurses experienced psychological and professional burdens during the coronavirus outbreak. MATERIAL AND METHODS: The study used a qualitative research design. Semi-structured interviews were applied as the method of data collection. Ten nurses from COVID-19 wards and units of two general public hospitals participated in the study. A content analysis approach was employed to analyze the data. RESULTS: Data analysis revealed two main categories, namely: (A) front-line experience "From fear to empowerment", and (B) caring and management "From powerlessness to adaptation". Eight sub-categories were developed and included within the corresponding main categories. The study's findings demonstrated that the pandemic caused significant psychological and professional strain on front-line nurses, with feelings of fear, anxiety, and uncertainty predominating. Nonetheless, the process of adaptation and adjustment brought about sentiments of self-actualization and empowerment. CONCLUSIONS: A thorough understanding of the psychological and professional burden experienced by the front-line nurses is crucial to ensure that nurses receive appropriate support and that quality care is sustained under highly demanding healthcare conditions.

MYT1L haploinsufficiency in human neurons and mice causes autism-associated phenotypes that can be reversed by genetic and pharmacologic intervention.

MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice. Mutant mice exhibit neurodevelopmental delays with thinner cortices, behavioural phenotypes, and gene expression changes that resemble those of ASD patients. MYT1L target genes, including WNT and NOTCH, are activated upon MYT1L depletion and their chemical inhibition can rescue delayed neurogenesis in vitro. MYT1L deficiency also causes upregulation of the main cardiac sodium channel, SCN5A, and neuronal hyperactivity, which could be restored by shRNA-mediated knockdown of SCN5A or MYT1L overexpression in postmitotic neurons. Acute application of the sodium channel blocker, lamotrigine, also rescued electrophysiological defects in vitro and behaviour phenotypes in vivo. Hence, MYT1L mutation causes both developmental and postmitotic neurological defects. However, acute intervention can normalise resulting electrophysiological and behavioural phenotypes in adulthood.

Alterations in Laboratory Parameters Related to Disease Severity in Vaccinated Patients Against SARS-CoV-2.

Background: Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide with global financial and health care systems consequences. It is already well recognized that immunization against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a precondition for blocking mutations and prevent the emergence of variants. The aim of the study was to investigate the possible relationship between COVID-19 vaccines and the commonly used disease-related blood biomarkers. Methods: Adult patients with confirmed SARS-CoV-2 infection who were hospitalized from November 8, 2021, to December 31, 2021, were included. The retrospective study was conducted in Patras University Hospital, Greece. Two groups of patients were assessed, the ones who were previously vaccinated against SARS-CoV-2 (group A, n = 21), and those who were not (group B, n = 55). After analysis of peripheral blood, we calculated on admission day for each patient the total white blood cell (WBC), absolute lymphocytes count (ALC), absolute monocyte count, D-dimers, C-reactive protein (CRP) plasma levels, lactate dehydrogenase (LDH), ferritin, high-sensitive troponin, as well as the arterial oxygen partial pressure/fractional inspired oxygen (PO2/FiO2) ratio. Results: The median age of all patients was 65.3 ± 15.2 years old; 68.4% were men and 31.6% were women. Comorbidities were present in 51 patients (67.1%). Hypertension and diabetes were observed as the most common comorbidities (33.3%). About 72.4% of the patients were unvaccinated or have received the first dose of vaccine, and 27.6% were completely vaccinated. No statistical difference was found in the total WBC count and ALC between the two groups (group A vs. group B: 8,168.95 ± 7,584.4 vs. 8,521.9 ± 6,571.3, P = 0.848 and 3,052.1 ± 7,230.7 vs. 1,279.6 ± 1,218.6, P = 0.087). Monocytes count in both groups did not show statistical difference: group A vs. group B: 672.6 ± 384.7 vs. 637.9 ± 477.8 (P = 0.754). Similarly, no difference for D-dimers (1,348.5 ± 1,397.6 vs. 1,850.9 ± 3,877.5, P = 0.575), ferritin (1,082.8 ± 1,399.5 vs. 1,327.4 ± 1,307.8, P = 0.508), high-sensitive troponin (113.6 ± 318.1 vs. 157.5 ± 48.8, P = 0.252), and CRP (6.92 ± 4.9 vs. 7.4 ± 5.9, P = 0.732). For LDH plasma levels, the statistical difference was significant (274.2 ± 85.6 vs. 387.5 ± 223.4, P = 0.003), as well as for the PO2/FiO2 ratio (355.6 ± 129.7 vs. 260.5 ± 123.3, P = 0,006). Conclusions: In a mixed population hospitalized for COVID-19, only LDH plasma levels and the PaO2/FiO2 on admission day showed statistically significant difference between vaccinated and unvaccinated patients. Although unvaccinated patients are more likely to develop severe illness, they did not express significantly higher values of commonly used plasma biomarkers such as ferritin, CRP, and D-dimers which are related to disease severity.

Exploring Nurses' Working Experiences during the First Wave of COVID-19 Outbreak.

During the COVID-19 outbreak, nurses employed in the clinical sector faced a number of difficulties associated with excessive workload, increased stress, and role ambiguity, which impacted nurses themselves and patient care. The aim of the present study was to investigate how Greek hospital nurses working in non-COVID units experienced the virus outbreak during the first wave of the pandemic. A descriptive qualitative research design was applied using a content analysis approach. To recruit the study participants a purposive sampling strategy was used. Ten nurses participated in the study. Data collection was conducted through semi-structured interviews. Content analysis revealed three themes namely, (a) emotional burden, (b) professional commitment, and (c) abrupt changes. Six subthemes were formulated and assimilated under each main theme respectively. Organizational changes, emotional burdens and feelings of fear and uncertainty, appeared to have a crucial effect on nurses and patient care. However, the professional commitment and the nurses' effort to provide excellent nursing care remained high. Nurses demonstrated that despite the burdens caused by the COVID-19 outbreak, the pandemic era created opportunities for thoroughness and accuracy in nursing care.

Cracking chromatin with proteomics: From chromatome to histone modifications.

Chromatin is the assembly of genomic DNA and proteins packaged in the nucleus of eukaryotic cells, which together are crucial in regulating a plethora of cellular processes. Histones may be the best known class of protein constituents in chromatin, which are decorated by a range of post-translational modifications to recruit accessory proteins and protein complexes to execute specific functions, ranging from DNA compaction, repair, transcription, and duplication, all in a dynamic fashion and depending on the cellular state. The key role of chromatin in cellular fitness is emphasized by the deregulation of chromatin determinants predisposing to different diseases, including cancer. For this reason, deep investigation of chromatin composition is fundamental to better understand cellular physiology. Proteomic approaches have played a crucial role to understand critical aspects of this complex interplay, benefiting from the ability to identify and quantify proteins and their modifications in an unbiased manner. This review gives an overview of the proteomic approaches that have been developed by combining mass spectrometry-based with tailored biochemical and genetic methods to examine overall protein make-up of chromatin, to characterize chromatin domains, to determine protein interactions, and to decipher the broad spectrum of histone modifications that represent the quintessence of chromatin function.

Floating marine litter detection algorithms and techniques using optical remote sensing data: A review.

Floating Marine Litter (FML) are mainly plastics or synthetic polymers that float on the sea surface after being deliberately discarded or unintentionally lost along beaches, rivers or marine environments. In recent years, much focus has been placed on locating, tracking and removing plastic items in both coastal areas and in the open ocean. The use of high-resolution multispectral satellite images for such purpose is very promising, since satellite images can systematically monitor much larger areas in comparison to the traditional in situ observations. This paper contains a literature review of the published research regarding the optical remote detection of floating marine debris and the proposed associated methodologies. The main aim of this review is to compile all available information on detection methodologies, providing at the same time valuable insights into the different approaches used for floating marine litter monitoring. First, a brief introduction into the theoretical basis of a spaceborne floating marine litter detection system is given. Next, published articles, or relevant research work have been compartmentalised, for analysing the proposed procedures and assisting in a further assessment of their methodological frameworks. Lastly, conclusions and bottlenecks of the existing knowledge on marine litter detection from space are derived. Although the remote detection of floating marine litter is currently limited by inherent restrictions of the available satellite sensors specifications, we highlight how the methodological processing chain can significantly affect the future accuracy of plastic detection from space.

Intestinal Barrier Biomarker ZO1 and Endotoxin Are Increased in Blood of Patients With COVID-19-associated Pneumonia.

BACKGROUND/AIM: The present study was undertaken to investigate (i) whether hospitalized patients with COVID-19 pneumonia present intestinal barrier dysfunction with consequent translocation of endotoxin into the systemic circulation and (ii) whether intestinal barrier biomarkers have any prognostic role in terms of progression to severe respiratory failure. PATIENTS AND METHODS: In this prospective study, 22 patients with COVID-19-associated pneumonia and 19 patients with non-COVID-19-related community-acquired pneumonia (CAP group) were studied while 12 healthy persons comprised the control group. Blood samples were collected on admission and analysed for serum levels of endotoxin and zonula occludens-1 (ZO1). Clinical courses regarding progression to severe respiratory failure (SRF) requiring mechanical ventilation were recorded. RESULTS: Patients with COVID-19-associated pneumonia and patients with CAP presented significantly higher serum endotoxin and ZO1 concentrations on admission as compared to healthy controls. There was no difference in endotoxin levels between patients with COVID-19-related pneumonia and patients with CAP. In patients with COVID-19-related pneumonia, serum endotoxin concentrations were positively correlated with C-reactive protein and ferritin values. There were no significant differences in serum endotoxin and ZO1 concentrations between patients with severe and not severe COVID-19-related pneumonia, nor between patients who developed SRF and those who did not Conclusion: Patients with COVID-19-related pneumonia present intestinal barrier dysfunction leading to systemic endotoxemia. Admission values of endotoxin and ZO1 do not have any prognostic role for progression to SRF.

Clinical Analysis of Statistical Data on the Characteristics of Patients with Acute Respiratory Distress Syndrome in a Greek ICU.

Acute respiratory distress syndrome (ARDS) is a potentially life-threatening condition for a many patients. The associated mortality rate may exceed 50% in some cases. The etiopathogenesis of the syndrome remains unclear, and in Greece the exact number of people suffering or dying of ARDS each year is unknown. In this study, we sought to record the clinical characteristics and outcomes of all patients with laboratory confirmed ARDS. This is a retrospective single-center observational study, conducted in mixed, closed-type ICU with 12 beds at a Greek public health system hospital. The medical records of patients admitted to the ICU from January 1, 2015, until December 31, 2016, were retrospectively studied, and statistical analyses were performed using SPSS version 22.0 software. Patients were assessed using the Fisher exact test assuming variances. All tests were two-tailed. In total, 15 patients developed ARDS (5.4%). According to the existence of radiographic pulmonary edema image with bilateral infiltrates, 12 patients were classified in the early phase of the syndrome described by the term exudative phase and 3 patients were classified in the third stage of final fibrosis (fibrotic stage). According to the Fisher test, there is a significant correlation between age and outcome. The incidence of the syndrome among hospitalized patients in ICU is in line with the current literature, while the main predisposing factor in this study was acute pancreatitis.

Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification.

The design and fabrication of a continuous-flow µPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the µPCR device in the established PCB industry. The µPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented µPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.

EZHIP/CXorf67 mimics K27M mutated oncohistones and functions as an intrinsic inhibitor of PRC2 function in aggressive posterior fossa ependymoma.

BACKGROUND: Posterior fossa A (PFA) ependymomas are one of 9 molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis, and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of chromosome X open reading frame 67 (CXorf67) as a hallmark of PFA ependymoma and showed that CXorf67 can interact with enhancer of zeste homolog 2 (EZH2), thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. METHODS: We performed mass spectrometry and peptide modeling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot, and methyltransferase assays. RESULTS: We find that the inhibitory mechanism of CXorf67 is similar to diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain (Su(var)3-9/enhancer-of-zeste/trithorax) of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function, causing de-repression of PRC2 target genes, including genes involved in neurodevelopment. CONCLUSIONS: Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 as "EZH Inhibitory Protein" (EZHIP).

Greek nurses' knowledge on the prevention of surgical site infection: an investigation.

OBJECTIVE: The aim of the study was to investigate nurses' knowledge regarding the prevention of surgical site infection (SSI), and to examine the relationship between nurses' demographic characteristics and educational level and their level of knowledge in prevention of SSIs. A further aim was to examine the differences in nurses' knowledge with respect to selected variables and to identify the most significant predictors of nurses' knowledge regarding the prevention of SSIs, to support the provision of high-quality nursing care. METHOD: A prospective, observational study of a convenience sample of nurses and assistant nurses working in surgical departments, in a public general hospital for adults in Attica, during May to August 2016. For data collection, an anonymous self-completion questionnaire was developed and tested for comprehension and acceptability. RESULTS: Data was collected from 148 nurses and assistant nurses, 121 (81.8%) were female, 73.6% were aged 36-50 years and 43.9% had 11-20 years of experience. With regards to educational level, 66.2% had a degree from a technological educational institute and 10.1% had a Master's degree. Furthermore, 18.2% had a surgical specialty and 59.5% had received special training on surgical infections. The majority of respondents did not chose the correct definition of the time of occurrence of SSIs. Several statistically significant correlations were observed between knowledge on safer hair removal and respndent age (p=0.037), educational level (p=0.003), professional experience (p=0.048), and training in SSIs (p=0.009). CONCLUSION: The results of this study revealed that the majority respondents had a high level of knowledge regarding the prevention of SSIs, which contrasted with a low level of knowledge regarding their full definition of the time of occurrence.

Frailty in elderly ICU patients in Greece: a prospective, observational study.

BACKGROUND: Of particular interest is the study of frailty syndrome in older patients in recent years. This syndrome is characterized by weight loss and muscle mass, a change in eating habits, movement and endurance, and a decline in cognitive function. The purpose of the study was the prevalence of frailty syndrome in subjects aged 65 years who were hospitalized in an intensive care unit (ICU) in Greece. METHODS: This is a prospective, observational study. The study sample consisted of families or carers of 36 patients over 65 years of age. The study was conducted in a General Hospital in Greece, over a period of 1 year. The Clinical Frailty Scale (CFS) and the 5 criterions frailty phenotype were used. In addition, the severity of the patients with the APACHE II, SAPS II and SOFA scales was assessed. For the analysis of the data, the SPSS 19 for windows statistical package was used and more specifically descriptive statistics and correlations with parametric methods were performed. RESULTS: Thirty-six severely ill patients aged 65 years and over (22 women, 14 men) were studied. 25% of the patients had a positive frailty phenotype, 44% were at an early stage, while 30.6% had a frailty negative phenotype. A significant correlation between CFS and APACHE (P=0.041), age (P=0.033), sex (P=0.049) and ADL mobility index (P=0.001) was found to be significant. Concerning mortality, 36.1% of patients died in ICU, 11.1% died within the next 6 months and 38.9% were alive. CONCLUSIONS: The findings of the study highlighted the onset of frailty syndrome in ICU patients. The objective assessment of the frailty syndrome of the seriously ill patient as well as the prognostic markers provides a clearer picture of its out-of-hospital condition and contributes to the collection of information on the outcomes of the in-hospital treatment.

Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

ΟBJECTIVE: To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag. RESULTS: We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus (TEV) protease cleavage site, together with a mammalian codon-optimized BirA biotin ligase fused to green fluorescent protein. We also describe platform vectors for the N- or C-terminal AVI-TEV-FLAG tagging of any complementary DNA of choice. These vectors offer versatility and efficiency in the application of metabolic biotinylation tandem affinity tagging of nuclear proteins in mammalian cells.

Therapeutic Hypothermia in the ICU: The Nursing Aspect.

Clinical studies and experimental research have described therapeutic hypothermia for patients suffering from traumatic brain injury (TBI), cardiac arrest, and neonatal hypoxic ischemic encephalopathy. This procedure is implemented by intensive care unit (ICU)-trained nurses. The aim of the present study was to compare cold compresses/ ice packs, cooling blankets, and heat-exchange systems via intravascular catheters used in the ICU for therapeutic hypothermia from a nursing perspective with respect to ease of application, additional workload, ease of temperature monitoring, and effectiveness. A questionnaire was completed by ICU nurses to evaluate these techniques for therapeutic hypothermia. The results were calculated and a score of 1 to 5 was obtained, where 1 = very bad, 2 = bad, 3 = moderate, 4 = good, and 5 = very good. Overall, heat exchange via intravascular catheters had the best score for implementation of therapeutic hypothermia. Regarding ease of the application, cold compresses/ice packs had the best score. Regarding additional workload, cold compresses/ice packs had the worst score, whereas the heat-exchange system via intravascular catheters scored the best. Regarding ease of temperature monitoring, the heat-exchange system via intravascular catheters had the best score and, regarding effectiveness, cold compresses/ice packs scored the best.

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions.

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal ß-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis.

The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.

NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.

We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.