BEND3 safeguards pluripotency by repressing differentiation-associated genes.

BEN domain-containing proteins are emerging rapidly as an important class of factors involved in modulating gene expression, yet the molecular basis of how they regulate chromatin function and transcription remains to be established. BEND3 is a quadruple BEN domain-containing protein that associates with heterochromatin and functions as a transcriptional repressor. We find that BEND3 is highly expressed in pluripotent cells, and the induction of differentiation results in the down-regulation of BEND3. The removal of BEND3 from pluripotent cells results in cells exhibiting upregulation of the differentiation-inducing gene expression signature. We find that BEND3 binds to the promoters of differentiation-associated factors and key cell cycle regulators, including CDKN1A, encoding the cell cycle inhibitor p21, and represses the expression of differentiation-associated genes by enhancing H3K27me3 decoration at these promoters. Our results support a model in which transcription repression mediated by BEND3 is essential for normal development and to prevent differentiation.

A green cellulose nanofiber-based printed electrode for practical highly sensitive amoxicillin detection.

Simple electrochemical detection of the antibiotic amoxicillin (AMX) in water is crucial to mitigate health and environmental risks; however, the process requires single-use electrodes, which can increase the waste generated as well as the cost. Cellulose nanofibers (CNFs) are biodegradable materials that can be used as electrode frameworks. In this study, a sensitive single-use CNF-based printed electrode modified with polybenzimidazole (PBI)-wrapped multi-walled carbon nanotubes (MWCNTs) is developed for AMX detection. The CNF-based printed electrode achieved a detection limit of 0.3 µM and exhibited a wider detection range of 0.3-500 µM compared with electrodes developed in previous studies. In addition, the electrode reactions of AMX were electrochemically investigated and found to primarily involve the adsorbed species at low AMX concentrations and be diffusion-controlled at high AMX concentrations. Finally, the printed electrodes were used for the easy and practical determination of AMX in seawater and tap water by a soaking method. Satisfactory results were obtained, and the final concentrations of AMX were determined using simple calibration equations. Therefore, this CNF-based electrode exhibits great potential for practical real-time AMX detection in the field.

T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction.

DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.

Phosphorylation of Orc6 During Mitosis Regulates DNA Replication and Ribosome Biogenesis.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

Application of Nanocellulose Biofilter from Pineapple Peel Waste for Water Microbes Removal.

This research aimed to assess the effectiveness of nanocellulose biofilter (NCB) made from pineapple peel waste to reduce the number of microbes in water. Further processing of cellulose from nata de pina into nano size was proposed, then transformed into a filter membrane. Three types of NCB were developed: bacterial cellulose acetate membrane, bacterial cellulose acetate membrane with TiO2 treatment, and bacterial cellulose acetate membrane with TiO2 and graphite nanoplatelet treatment. These NCBs were used to filter microbes in several water sources in Padang City, West Sumatra Province. The filtering process was carried out using a filter holder where the NCB had been installed. The number of microbes contained in the water, including E. Coli, was determined before and after filtering. As a result, all NCBs reduced the total microbes in water by about 50%. Furthermore, when applied to water pollutant bacteria, E. Coli, all prepared NCBs reduced them by more than 90%. The effectiveness of all NCBs to remove microbes' contamination, especially bacteria, looks very promising with or without TiO2 and graphene reinforcement. Although the efficacy of all NBC for microbial water purification was relatively similar, further experiments to clarify the superior of TiO2 and graphite nanoplatelet on NCB need to be carried out, especially in reducing chemical contamination.

Properties of bacterial cellulose acetate nanocomposite with TiO2 nanoparticle and graphene reinforcement.

Agricultural waste is considered a promising source for bacterial cellulose production. This study aims to observe the influence of TiO2 nanoparticles and graphene on the characteristic of bacterial cellulose acetate-based nanocomposite membranes for bacterial filtration in waters. Bacterial cellulose was produced from the pineapple peel waste using fermentation process. High-pressure homogenization process was applied to reduce bacterial nanocellulose size and esterification process was carried out to produce cellulose acetate. Nanocomposite membranes were synthesized with reinforcement of TiO2 nanoparticles 1 % and graphene nanopowder 1 %. The nanocomposite membrane was characterized using an FTIR, SEM, XRD, BET, tensile testing, and bacterial filtration effectiveness using the plate count method. The results showed that the main cellulose structure was identified at the diffraction angle 22° and the cellulose structure slightly changed at the peak of diffraction angles of 14° and 16°. In addition, the crystallinity of bacterial cellulose increased from 72.5 % to 75.9 %, and the functional group analysis showed that several peak shifts indicated a change in the functional group of membrane. Similarly, the surface morphology of membrane became rougher with the structure of mesoporous membrane. Moreover, adding TiO2 and graphene increases crystallinity and bacterial filtration effectiveness of nanocomposite membrane.

A BEN-domain protein and polycomb complex work coordinately to regulate transcription.

Transcription regulation is an important mechanism that controls pluripotency and differentiation. Transcription factors dictate cell fate decisions by functioning cooperatively with chromatin regulators. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein regulates the expression of differentiation-associated genes by modulating the chromatin architecture at promoters. We highlight the collaboration of BEND3 with the polycomb repressive complex in coordinating transcription repression and propose a model highlighting the relevance of the BEND3-PRC2 axis in gene regulation and chromatin organization.Abbreviations: BEND3, BANP, E5R and Nac1 domain; rDNA, ribosomal DNA; PRC2, Polycomb Repressive Complex 2; H3K27me3, Histone H3 Lysine 27 methylation; PcG, Polycomb group.

Rapid analysis to distinguish porcine and bovine gelatin using PANI/NiO nanoparticles modified Quartz Crystal Microbalance (QCM) sensor.

Rapid analysis to distinguish porcine and bovine gelatin using a modified Quartz Crystal Microbalance (QCM) sensor has been studied. The PANI was deposited on the sensor surface using electropolymerization, and then nickel nanoparticles were deposited by layer by layer (LbL) technique. The modified QCM sensor's performance was compared to an unmodified sensor in porcine and bovine gelatin at neutral, acidic, and alkaline conditions. The result shows that the unmodified sensor cannot distinguish between porcine and bovine gelatin, whereas the modified QCM sensor produces a different response. Porcine gelatin shows an increasing frequency response, but in contrast, bovine gelatin decreases frequency response at the alkaline condition. The time response was 2 min with a detection limit of 51.2 ppm and 8.7 ppm for porcine and bovine gelatin, respectively. Further investigation shows that the modified sensor can analyze porcine gelatin contamination in the a mixed gelatin sample.

Fluorescence spectrophotometry for COVID-19 determination in clinical swab samples.

Considering the limitations of the assays currently available for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants, a simple and rapid method using fluorescence spectrophotometry was developed to detect coronavirus disease 2019 (COVID-19). Forty clinical swab samples were collected from the nasopharyngeal and oropharyngeal cavities of COVID-19-positive and -negative. Each sample was divided into two parts. The first part of the samples was analyzed using reverse transcription-polymerase chain reaction (RT-qPCR) as the control method to identify COVID-19-positive and -negative samples. The second part of the samples was analyzed using fluorescence spectrophotometry. Fluorescence measurements were performed at excitation and emission wavelengths ranging from 200 to 800 nm. Twenty COVID-19-positive samples and twenty COVID-19-negative samples were detected based on RT-qPCR results. The fluorescence spectrum data indicated that the COVID-19-positive and -negative samples had significantly different characteristics. All positive samples could be distinguished from negative samples by fluorescence spectrophotometry. Principal component analysis showed that COVID-19-positive samples were clustered separately from COVID-19-negative samples. The specificity and accuracy of this experiment reached 100%. Limit of detection (LOD) obtained 42.20 copies/ml (Ct value of 33.65 cycles) for E gene and 63.60 copies/ml (Ct value of 31.36 cycles) for ORF1ab gene. This identification process only required 4 min. Thus, this technique offers an efficient and accurate method to identify an individual with active SARS-CoV-2 infection and can be easily adapted for the early investigation of COVID-19, in general.

Structural Characterization of a Minimal Antibody against Human APOBEC3B.

APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.

Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing.

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Šresolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

Structural basis of superinfection exclusion by bacteriophage T4 Spackle.

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages - a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

Structural basis of host protein hijacking in human T-cell leukemia virus integration.

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.

Analytical applications of electrodes modified by gold nanoparticles: dopamine detection.

Dopamine oxidation was studied on modified gold (nano-Au) electrodes obtained by Layer-by-Layer deposition of gold nanoparticles and polyacrylic acid. A gradual loss of electrochemical activity for the dopamine oxidation reaction is observed at pH 7. Simultaneous SPR spectroscopy and cyclic voltammetry indicate the formation of an adsorbed layer on the electrode surface at this pH value. Investigations, performed through electrochemical and SPR measurements at pH 4, give evidence for a reversible process. At this pH value both dopamine oxidation and reduction current peaks show linear dependence on the dopamine concentration and may be used for analytical applications. The use of the nano-Au electrode allows resolving the peaks corresponding to ascorbic acid and to dopamine oxidation by 240 mV thus providing a high selectively for dopamine detection in the presence of ascorbic acid. The detection limit of this electrode for dopamine is below 4 microM in the presence of 1 mM ascorbic acid. The sensitivity normalized to the macroscopic electrode surface is about 10 mA cm(-2) mM(-1) at sweep rate of 10 V/s.

Corrosion Inhibition of Honeycomb Waste Extracts for 304 Stainless Steel in Sulfuric Acid Solution.

The extract of honeycomb waste was studied as a corrosion inhibitor on 304 stainless steel in H2SO4 solutions. The honeycomb waste was obtained from beekeeping at Lawang-Malang, East Java, Indonesia. Electrochemical and scanning electron microscopy methods were used to investigate the performance of the corrosion inhibition process. The inhibition efficiency of the inhibitor (2000 mg/L) reached 97.29% in 0.5 M H2SO4 and decreased with the acid concentration. Kinetic parameters were calculated to explain the effect of acid concentration on the inhibition process. The study on the adsorption behavior of the extracts followed the Frumkin isotherm model. The adsorption of the inhibitor on the 304 stainless steel surface was confirmed by the negative and lower values of Gibbs free energy. The obtained scanning electron microscopy (SEM) images were confirmed by comparing the surface of the specimens with and without inhibitor after corroding for one week. The results indicated that the extract acted as a good inhibitor for 304 stainless steel in acid corrosion.

The anti-parasitic agent suramin and several of its analogues are inhibitors of the DNA binding protein Mcm10.

Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP Ki 170 nM, SPR KD 460 nM) and cell activity (IC50 38 µM). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.

Crystal Structure of Entamoeba histolytica Cdc45 Suggests a Conformational Switch that May Regulate DNA Replication.

Cdc45 plays a critical role at the core of the eukaryotic DNA replisome, serving as an essential scaffolding component of the replicative helicase holoenzyme Cdc45-MCM-GINS (CMG) complex. A 1.66-Å-resolution crystal structure of the full-length Cdc45 protein from Entamoeba histolytica shows a protein fold similar to that observed previously for human Cdc45 in its active conformation, featuring the overall disk-like monomer shape and intimate contacts between the N- and C-terminal DHH domains. However, the E. histolytica Cdc45 structure shows several unique features, including a distinct orientation of the C-terminal DHHA1 domain, concomitant disordering of the adjacent protruding α-helical segment implicated in DNA polymerase ε interactions, and a unique conformation of the GINS/Mcm5-binding loop. These structural observations collectively suggest the possibility that Cdc45 can sample multiple conformations corresponding to different functional states. We propose that such conformational switch of Cdc45 may allow regulation of protein-protein interactions important in DNA replication.