FRET-FISH probes chromatin compaction at individual genomic loci in single cells

The density or compaction of chromatin throughout the cell nucleus is a key biophysical property that influences DNA replication, transcription, and repair. Chromatin accessibility is often used as a proxy for chromatin compaction or density, however it is not clear how these two properties relate to each other, given the lack of tools for directly probing compaction at defined genomic loci. To fill in this gap, here we developed FRET-FISH, a microscopy-based method combining fluorescence resonance energy transference (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at selected loci in single cells. We optimized FRET-FISH by testing different probe designs in situ in fixed cells, readily detecting FRET generated by DNA FISH probes. To validate FRET-FISH, we compared it with ATAC-seq and Hi-C, demonstrating that local chromatin compaction and accessibility are strongly correlated and that the frequency of intra-genic contacts measured by Hi-C may be an even better proxy for local chromatin density. To further validate FRET-FISH, we showed that it can detect expected differences in chromatin compaction along the nuclear radius, with peripheral loci being more compacted and central ones less compacted. Lastly, we assessed the sensitivity of FRET-FISH, demonstrating its ability to reproducibly detect differences in chromatin density (i) upon treatment of cells with drugs that perturb global chromatin condensation; (ii) during prolonged cell culture; and (iii) in different phases of the cell cycle. We conclude that FRET-FISH is a robust tool for probing chromatin compaction at selected loci in single cells and for studying inter-allelic and cell-to-cell variability in chromatin density.

Touchable cell biophysics property recognition platforms enable multifunctional blood smart health care

As a crucial biophysical property, red blood cell (RBC) deformability is pathologically altered in numerous disease states, and biochemical and structural changes occur over time in stored samples of otherwise normal RBCs. However, there is still a gap in applying it further to point-of-care blood devices due to the large external equipment (high-resolution microscope and microfluidic pump), associated operational difficulties, and professional analysis. Herein, we revolutionarily propose a smart optofluidic system to provide a differential diagnosis for blood testing via precise cell biophysics property recognition both mechanically and morphologically. Deformation of the RBC population is caused by pressing the hydrogel via an integrated mechanical transfer device. The biophysical properties of the cell population are obtained by the designed smartphone algorithm. Artificial intelligence-based modeling of cell biophysics properties related to blood diseases and quality was developed for online testing. We currently achieve 100% diagnostic accuracy for five typical clinical blood diseases (90 megaloblastic anemia, 78 myelofibrosis, 84 iron deficiency anemia, 48 thrombotic thrombocytopenic purpura, and 48 thalassemias) via real-world prospective implementation; furthermore, personalized blood quality (for transfusion in cardiac surgery) monitoring is achieved with an accuracy of 96.9%. This work suggests a potential basis for next-generation blood smart health care devices.

The emergence of a collective sensory response threshold in ant colonies

The sensory response threshold is a fundamental biophysical property of biological systems that underlies many physiological and computational functions, and its systematic study has played a pivotal role in uncovering the principles of neural computation. Here, we show that ant colonies, which perform computational tasks at the group level, have emergent collective sensory response thresholds. Colonies respond collectively to step changes in temperature and evacuate the nest during severe perturbations. This response is characterized by a group-size dependent threshold, and the underlying dynamics are dominated by social feedback between the ants. Using a binary network model, we demonstrate that a balance between short-range excitatory and long-range inhibitory interactions can explain the emergence of the collective response threshold and its size dependency. Our findings illustrate how simple social dynamics allow insect colonies to integrate information about the external environment and their internal state to produce adaptive collective responses.

Abundance Imparts Evolutionary Constraints of Similar Magnitude on the Buried, Surface, and Disordered Regions of Proteins

An understanding of the forces shaping protein conservation is key, both for the fundamental knowledge it represents and to allow for optimal use of evolutionary information in practical applications. Sequence conservation is typically examined at one of two levels. The first is a residue-level, where intra-protein differences are analyzed and the second is a protein-level, where inter-protein differences are studied. At a residue level, we know that solvent-accessibility is a prime determinant of conservation. By inverting this logic, we inferred that disordered regions are slightly more solvent-accessible on average than the most exposed surface residues in domains. By integrating abundance information with evolutionary data within and across proteins, we confirmed a previously reported strong surface-core association in the evolution of structured regions, but we found a comparatively weak association between disordered and structured regions. The facts that disordered and structured regions experience different structural constraints and evolve independently provide a unique setup to examine an outstanding question: why is a protein’s abundance the main determinant of its sequence conservation? Indeed, any structural or biophysical property linked to the abundance-conservation relationship should increase the relative conservation of regions concerned with that property (e.g., disordered residues with mis-interactions, domain residues with misfolding). Surprisingly, however, we found the conservation of disordered and structured regions to increase in equal proportion with abundance. This observation implies that either abundance-related constraints are structure-independent, or multiple constraints apply to different regions and perfectly balance each other.

Refining the Identity and Role of Kv4 Channels in Mouse Substantia Nigra Dopaminergic Neurons

ABSTRACTSubstantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized in vitro by a spontaneous tonic regular activity (pacemaking activity), a broad action potential and a biphasic post-inhibitory response. Several studies in rodents have underlined the central role played by the transient A-type current (IA) in the control of pacemaking activity and post-inhibitory rebound properties, thereby influencing both DA release and the physiological response of SNc neurons to incoming inhibitory inputs. Kv4.3 potassium channels were considered to be fully responsible for IA in these neurons, their density being tightly related to pacemaking frequency. In spite of this crucial electrophysiological role, we show that Kv4.3-/- transgenic mice exhibit minor alterations in locomotion and motor learning, although no compensation by functionally overlapping ion channels is observed in Kv4.3-/- SNc DA neurons. Using antigen retrieval immunohistochemistry, we further demonstrate that Kv4.2 potassium channels are also expressed in SNc DA neurons, even though their contribution to IA appears significant only in a minority of neurons (~5-10%). Using correlative analysis on recorded electrophysiological parameters and multi-compartment modeling, we then demonstrate that, rather than its conductance level, IA gating kinetics (inactivation time constant) appear as the main biophysical property defining post-inhibitory rebound delay and pacemaking frequency. Moreover, we show that the hyperpolarization-activated current (IH) has an opposing and complementary influence on the same firing features, and that the biophysical properties of IA and IH are likely coregulated in mouse SNc DA neurons.SIGNIFICANCE STATEMENTSubstantia nigra pars compacta (SNc) dopaminergic (DA) neurons are characterized by pacemaking activity, a broad action potential and biphasic post-inhibitory response. The A-type transient potassium current (IA) plays a central role in both pacemaking activity and post-inhibitory response. While it was thought so far that Kv4.3 ion channels were fully responsible for IA, using a Kv4.3-/- transgenic mouse and antigen retrieval immunohistochemistry we demonstrate that Kv4.2 channels are also expressed in SNc DA neurons, although their contribution is significant in a minority of neurons only. Using electrophysiological recordings and computational modeling, we then demonstrate that IA gating kinetics and its functional complementarity with the hyperpolarization-activated current are major determinants of both pacemaking activity and post-inhibitory response in SNc DA neurons.

Graph Signal Processing on protein residue networks helps in studying its biophysical properties

Understanding the physical and chemical properties of proteins is vital, and many efforts have been made to study the emergent properties of the macro-molecules as a combination of long chains of amino acids. Here, we present a graph signal processing based approach to model the biophysical property of proteins. For each protein inter-residue proximity-based network is used as basis graph and the respective amino acid properties are used as node-signals. Signals on node are decomposed on network’s Laplacian eigenbasis using graph Fourier transformations. We found that the intensity in low-frequency components of graph signals of residue features could be used to model few biophysical properties of proteins. Specifically, using our approach, we could model protein folding-rate, globularity and fraction of alpha-helices and beta-sheets. Our approach also allows amalgamation of different types of chemical and graph theoretic properties of residue to be used together in a multi-variable regression model to predict biophysical properties.

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis

Shelterin is a six-proteins complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

Unravelling the Complex Duplication History of Deuterostome Glycerol Transporters

Transmembrane glycerol transport is an ancient biophysical property that evolved in selected subfamilies of water channel (aquaporin) proteins. Here, we conducted broad level genome (>550) and transcriptome (>300) analyses to unravel the duplication history of the glycerol-transporting channels (glps) in Deuterostomia. We found that tandem duplication (TD) was the major mechanism of gene expansion in echinoderms and hemichordates, which, together with whole genome duplications (WGD) in the chordate lineage, continued to shape the genomic repertoires in craniates. Molecular phylogenies indicated that aqp3-like and aqp13-like channels were the probable stem subfamilies in craniates, with WGD generating aqp9 and aqp10 in gnathostomes but aqp7 arising through TD in Osteichthyes. We uncovered separate examples of gene translocations, gene conversion, and concerted evolution in humans, teleosts, and starfishes, with DNA transposons the likely drivers of gene rearrangements in paleotetraploid salmonids. Currently, gene copy numbers and BLAST are poor predictors of orthologous relationships due to asymmetric glp gene evolution in the different lineages. Such asymmetries can impact estimations of divergence times by millions of years. Experimental investigations of the salmonid channels demonstrated that approximately half of the 20 ancestral paralogs are functional, with neofunctionalization occurring at the transcriptional level rather than the protein transport properties. The combined findings resolve the origins and diversification of glps over >800 million years old and thus form the novel basis for proposing a pandeuterostome glp gene nomenclature.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance

ABSTRACTAirway mucociliary clearance (MCC) is required for host defense and often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B levels using genetic approaches. Here, we show that BPIFB1 is required for normal mucociliary clearance in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in sodium or chloride ion transport or reduced ciliary beat frequency. BPIFB1 loss resulted in airway mucus flakes with significantly increased complex viscosity, a key biophysical property of mucus known to impact MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and in the protein mesh of secreted mucus in human airway cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.