Antibody-free profiling of transcription factor occupancy during early embryogenesis by FitCUT&RUN

Key transcription factors (TFs) play critical roles in zygotic genome activation (ZGA) during early embryogenesis, while genome-wide occupancies of only a few factors have been profiled during ZGA due to the limitation of cell numbers or the lack of high-quality antibodies. Here, we present FitCUT&RUN, a modified CUT&RUN method, in which an Fc fragment of immunoglobulin G is used for tagging, to profile TF occupancy in an antibody-free manner and demonstrate its reliability and robustness using as few as five thousand K562 cells. We applied FitCUT&RUN to zebrafish undergoing embryogenesis to generate reliable occupancy profiles of three known activators of zebrafish ZGA: Nanog, Pou5f3 and Sox19b. By profiling the time-series occupancy of Nanog during zebrafish ZGA, we observed a clear trend toward a gradual increase in Nanog occupancy and found that Nanog occupancy prior to the major phase of ZGA is critical for the activation of a significant proportion of early transcribed genes. Our results further suggested that the sequential binding of Nanog may be controlled by replication timing and the presence of Nanog motifs.

Coupling of Ca2+-triggered unclamping and membrane fusion during neurotransmitter release

Neurotransmitter (NT) release is accomplished by a machinery that unclamps fusion in response to calcium and then fuses the synaptic vesicle and plasma membranes. These are often thought of as distinct tasks assigned to non-overlapping components. Vesicle release rates have a power law dependence on [Ca2+] with an exponent of 3-5, long taken to indicate that 3-5 Ca2+ ions bind the calcium sensor Synaptotagmin to trigger release. However, dependencies at low [Ca2+] are inconsistent with simple sequential binding to a single Ca2+ sensor followed by a final fusion step. Here we developed coarse-grained molecular dynamics simulations of the NT release machinery accounting for Synaptotagmin-mediated unclamping and SNARE-mediated fusion. Calcium-triggered unclamping and SNARE-mediated fusion emerged from simulations as contemporaneous, coupled processes. Increasing cytosolic [Ca2+], the instantaneous fusion rate increased as SNAREpins were progressively and reversibly released by dissociation of Synaptotagmin-SNAREpin complexes. Simulations reproduced the observed dependence of release rates on [Ca2+], but the power law was unrelated to the number of Ca2+ ions required. Action potential-evoked vesicle release probabilities depended on the number of transiently unclamped SNAREpins, explaining experimental dependencies of release probabilities on both unclamping and membrane-fusing machinery components. These results describe a highly cooperative NT release machinery with intrinsically inseparable unclamping and membrane-fusing functionalities.

Correlation in Domain Fluctuations Navigates Target Search of a Viral Peptide along RNA

Biological macromolecules often exhibit correlation in fluctuations involving distinct domains. This study decodes their functional implications in RNA-protein recognition and target-specific binding. The target search of a peptide along RNA in viral TAR-Tat complex is closely monitored using atomistic simulations, steered molecular dynamics simulations, free energy calculations, and a machine-learning-based clustering technique. An anti-correlated domain fluctuation is identified between the tetraloop and the bulge region in the apo form of TAR RNA that sets a hierarchy in the domain-specific fluctuations at each binding event and that directs succeeding binding footsteps. Thus, at each binding footstep, the dynamic partner selects an RNA location for binding where it senses higher fluctuation, which is conventionally reduced upon binding. This event stimulates an alternate domain- fluctuation which then dictates sequential binding footstep/s and thus, the search progresses. Our cross-correlation maps show that the fluctuations relay from one domain to another specific domain till the anti-correlation between that inter-domain fluctuations sustains. Artificial attenuation of that hierarchical domain fluctuation inhibits specific RNA binding. The binding is completed with the arrival of a few long-lived water molecules that mediate slightly distant RNA-protein sites and finally stabilizes the overall complex. The study underscores the functional importance of naturally designed fluctuating RNA motifs (bulge, tetraloop) and their interplay in dictating the directionality of the search in a highly dynamic environment.

Which Is Stronger? A Continuing Battle Between Cry Toxins and Insects

In this article, we review the latest works on the insecticidal mechanisms of Bacillus thuringiensis Cry toxins and the resistance mechanisms of insects against Cry toxins. Currently, there are two models of insecticidal mechanisms for Cry toxins, namely, the sequential binding model and the signaling pathway model. In the sequential binding model, Cry toxins are activated to bind to their cognate receptors in the mid-intestinal epithelial cell membrane, such as the glycophosphatidylinositol (GPI)-anchored aminopeptidases-N (APNs), alkaline phosphatases (ALPs), cadherins, and ABC transporters, to form pores that elicit cell lysis, while in the signaling pathway model, the activated Cry toxins first bind to the cadherin receptor, triggering an extensive cell signaling cascade to induce cell apoptosis. However, these two models cannot seem to fully describe the complexity of the insecticidal process of Cry toxins, and new models are required. Regarding the resistance mechanism against Cry toxins, the main method insects employed is to reduce the effective binding of Cry toxins to their cognate cell membrane receptors by gene mutations, or to reduce the expression levels of the corresponding receptors by trans-regulation. Moreover, the epigenetic mechanisms, host intestinal microbiota, and detoxification enzymes also play significant roles in the insects’ resistance against Cry toxins. Today, high-throughput sequencing technologies like transcriptomics, proteomics, and metagenomics are powerful weapons for studying the insecticidal mechanisms of Cry toxins and the resistance mechanisms of insects. We believe that this review shall shed some light on the interactions between Cry toxins and insects, which can further facilitate the development and utilization of Cry toxins.

The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1)

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro. In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo. Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

Hemoglobin Non-equilibrium Oxygen Dissociation Curve

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve (ODC), which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the ODC of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of RBCs through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium ODC, and show that under these conditions Hb cooperativity has two apparent components in the Adair, Perutz, and MWC models of Hb. The first component, which we call sequential cooperativity, accounts for ∼70% of Hb cooperativity, and emerges from the constraint of sequential binding that is shared by the three models. The second component, which we call conformational cooperativity, accounts for ∼30% of Hb cooperativity, and is due either to a conformational equilibrium between low affinity and high affinity tetramers (as in the MWC model), or to a conformational change from low to high affinity once two of the tetramer sites are occupied (Perutz model).

Sequential Detection of Palladium and Chromium Oxyanion by a Fluorescein Based Chemosensor in Mixed Aqueous Media

A new highly selective chemosensor, based on fluorescein-allyloxy benzene conjugate 1, was developed for the sequential detection of palladium and chromium oxyanions in a mixed aqueous media, and was studied by UV-visible and fluorescence spectroscopy. The sensing of palladium ions produces a chemodosimetric and ratiometric change in the emission band of 1 from 450 to 525 nm, followed by the sensing of chromate ions by 2 that quenches the emission band at 525 nm in a buffered H2O: DMF solution (9:1, pH = 7.4). The rate constants of palladium and chromate ions were found to be 8.6 × 105 M−1, 2.1 × 105 M−1, and 5.4 × 104 M−1 respectively. The chemosensor 1 has a palladium detection limit of 49 ppb while the sequential detection limit of chromate ions (CrO42− and Cr2O72−) were 127 and 259 ppb. The ratiometric change in the emission is produced due to the deallylation of 1 by palladium to produce 2 that restores the ESIPT (excited state intramolecular proton transfer) of the phenolic ring and enhances the electron transfer (ET) phenomenon from the phenolic group to fluorescein. The sequential binding of chromate ions to 2 inhibits the ESIPT and causes chelation enhanced quenching (CHEQ) of the fluorescence.

Architecture of Allosteric Structure. Equation of State Containing Three Unknown Quantities for Fractional Saturation of whole Human Blood with O2: Formatting the Adair Equation to Accommodate Ordered Equivalent Sequences of O2-Binding Reactions and a Single Conformation Change Not Concerted with O2 Binding

<p>O₂-Equilibrium binding data of hemoglobin in whole blood under standard conditions (Kernohan JC. & Roughton FJW (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status, ed Rorth and Astrup, Copenhagen, Munksgaard, pp 65-72; Severinghaus JW in <i>ibid</i> pp. xx-xx) was fitted to an equation of state comprised of three unknown quantities: <i>K</i>α, the equilibrium constant for binding O₂ by equivalent low affinity α-chains;<i> K_ΔC</i>, a dimensionless equilibrium constant describing the conformation change between low- and high-affinity conformations of hemoglobin, ^Tstate and ^Rstate; <i>K</i>_β, the equilibrium constant for binding O₂ by equivalent high affinity β-chains, the Perutz/Adair Equation. Values of the unknown quantities at pH 7.4 and 37^oC are: <i>K</i>_α= 15,090 L/mol; <i>K_ΔC</i> = 0.0260; <i>K</i>_β = 393,900 L/mol. </p> <p> </p> <p>A graph of predicted <i>versus</i> observed values of fractional saturation, <i>F</i>, is linear: <i>F</i>_PRE = 0.9998 <i>F</i>_OBS – 0.0005, r² =0.9997. The Perutz/Adair equation of state is defined as such insofar as all aspects of the stereochemical model (Perutz MF (1970) Nature London 228, 726-739) are imposed on the earlier sequential binding model of Adair (1925) JBC 63, 493-545.</p> <br>