Affinity State Research Articles

Optimization of Several Parameters Towards 30% Efficiency Perovskite Based Solar Cell Using SCAPS-1D Software

A simulated perovskite solar cell based on a P3HT/MAPbI3/C60 structure is examined to achieve 30% PCE using SCAPS-1D software. Several aspects of the perovskite layer were evaluated, including the perovskite layer thickness, CB and VB effective density of state, band gap, and electron affinity. These factors show great influence on the device performance. The best device based on the best examined parameter has exhibited a PCE of 32.1% correlated with FF of 84.8%, VOC of 1.23V and JSC of 30.77 mA.cm-2. Such a result is promising towards achieving high PCE for perovskite-based solar cells by optimizing several factors, including active layer thickness, energy band gap, electron affinity, and effective state density for CB and VB. However, for the experimentally produced solar cells, the preparation condition and other factors may render this result. A low effective DOS of (1x1016m-3) is desired for both CB and VB to achieve high solar cell performance.

A Phylogenetic Model of Established and Enabled Biome Shifts.

Where each species actually lives is distinct from where it could potentially survive and persist. This suggests that it may be important to distinguish established from enabled biome affinities when considering how ancestral species moved and evolved among major habitat types. We introduce a new phylogenetic method, called RFBS, to model how anagenetic and cladogenetic events cause established and enabled biome affinities (or, more generally, other discrete realized versus fundamental niche states) to shift over evolutionary timescale. We provide practical guidelines for how to assign established and enabled biome affinity states to extant taxa, using the flowering plant clade Viburnum as a case study. Through a battery of simulation experiments, we show that RFBS performs well, even when we have realistically imperfect knowledge of enabled biome affinities for most analyzed species. We also show that RFBS reliably discerns established from enabled affinities, with similar accuracy to standard competing models that ignore the existence of enabled biome affinities. Lastly, we apply RFBS to Viburnum to infer ancestral biomes throughout the tree and to highlight instances where repeated shifts between established affinities for warm and cold temperate forest biomes were enabled by a stable and slowly-evolving enabled affinity for both temperate biomes.

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Effect of 7-ketocholesterol incorporation on substrate binding affinity and turnover rate of the organic cation transporter 2 (OCT2)

The organic cation transporter 2 (OCT2) is pivotal in the renal elimination of several positively charged molecules. OCT2 mode of transport is profoundly influenced by the level of membrane cholesterol. The aim of this study was to investigate the effect of oxidized cholesterol on OCT2 transport activity in human embryonic kidney 293 cells stably transfected with OCT2 (OCT2-HEK293) and in primary renal proximal tubular epithelial cells (RPTEC). Cholesterol was exchanged with 7-ketocholesterol, the main product of cholesterol auto-oxidation, by exposing cells to sterol-saturated methyl-β-cyclodextrin (mβcd). After a 30 min-exposure, approximately 50% of the endogenous cholesterol was replaced by 7-ketocholesterol without significant changes in total sterol level. In the presence of 7-ketocholesterol, [3H]1-methyl-4-phenylpyridinium (MPP+) uptake was significantly reduced in both cell lines. 7-ketocholesterol incorporation did not affect lipid raft integrity, nor OCT2 surface expression and spatial organization. The inhibitory effect of 7-ketocholesterol on MPP+ uptake was abolished by the presence of MPP+ in the trans-compartment. In the presence of 7-ketocholesterol, both Kt and Vmax of MPP+ influx decreased. Molecular docking using OCT2 structure in outward occluded conformation showed overlapping poses and similar binding energies between cholesterol and 7-ketocholesterol. The thermal stability of OCT2 was not changed when cholesterol was replaced with 7-ketocholesterol. We conclude that 7-ketocholesterol confers a higher rigidity to the carrier by reducing its conformational entropy, arguably as a result of changes in plasma membrane physical properties, thereby facilitating the achievement of a higher affinity state at the expense of the mobility and overall cycling rate of the transporter.

Generation and reactivity of gas-phase phenyloxenium ion

Phenyloxenium ion (POI) is an important intermediate in organic reactions, however, its generation and reactivity have not been extensively investigated. In this study, POI is produced in the gas phase by electrospray ionization tandem mass spectrometry (ESI-MS/MS), isolated for gas phase ion-molecule reactions, and used theoretical calculations to reveal mechanisms to those reactions. It forms adduct ions with H2O, methanol, ethanol, benzene, and toluene, although the structures of the adduct ions are not well-resolved. In contrast, ortho-cyclization with nitriles has been verified by the reaction with acetonitrile and theoretical calculation. Further, Hydride transfer reaction during the reaction with tetrahydrofuran in the gas phase led to experimental values of the hydride affinity and electronic state of POI. Electron transfer studies revealed that the ionization potential of phenoxy radical locates in the range of 8.01–9.19 eV, in line with the calculated value of 8.60 eV for the singlet state POI.

Multi-area state estimation for active distribution networks under multiple uncertainties: An affine approach

An enormous challenge for the state estimation (SE) process in large-scale distribution networks lies in how to deal with multiple uncertainties and the increasing computational tasks simultaneously. This paper aims to address the issue and propose a multi-area architecture for perceiving system status with uncertain inputs. The first step involves the introduction of a novel Louvain-flow-based partition structure, which effectively divides large-scale unbalanced distribution networks into several sub-areas. Subsequently, an affine state estimation (ASE) model, employing the Neumann series method, is formulated for each sub-area, taking into account uncertainties in system measurements and line parameters. Finally, the interior-point filter line-search algorithm combined with consensus theory is utilized to solve all local ASE models, and the interaction of boundary state between adjacent sub-areas is focused and completed, to output the global state results of the distribution network precisely. Case studies are presented to verify that the proposed distributed ASE model yields obvious advantages in tracking system uncertainties by contrast with the existing SE model.

Junctional Adhesion Molecule a (JAM-A) Associates with CD36 upon Platelet Activation and Cytoskeleton in a Phosphorylation-Dependent Manner to Promote Platelet-Platelet Contacts

Background: Platelets play a fundamental role inmaintaining hemostasis. Stability of hemostatic plug is crucial for arresting blood loss during vascular breach. JAM-A is a member of the immunoglobulin superfamily that restricts α IIbβ 3 to its low affinity state by recruiting CSK to the α IIbβ 3-c-Src complex in resting platelets. Agonist-induced inside-out signaling causes JAM-A to dissociate from the α IIbβ 3-Src complex, thus facilitating activation of α IIbβ 3 and outside-in signaling. However, the function of JAM-A in the process of hemostasis is poorly understood. Aim: To evaluate the role of JAM-A in activated platelets and on hemostasis. Methods: Washed human platelets were used in these studies. Full-length cytoplasmic domain of JAM-A (cyto JAM-A) and JAM-A cytoplasmic domain lacking C-terminal PDZ-domain-binding motif (ΔPDZ cyto JAM-A) were expressed as GST-fusion proteins in bacteria using pGEX4T-1 vector. GST-fusion proteins were purified using glutathione beads. Pull-down assays were performed by incubating glutathione GST-fusion proteins beads with lysates of washed human platelets (4×10 8/mL). GST beads alone without the fusion proteins were used as a control. Immunoprecipitation studies were performed using washed human platelets (4x10 8/mL) lysed with CHAPS lysis buffer and precleared by using Protein G Sepharose beads. Specific primary antibodies and isotype specific IgGs (control) were used for immunoprecipitation followed by western blotting. To identify protein complexes two-dimensional blue-native polyacrylamide gel electrophoresis was performed in which protein complexes were first separated based on their size (1 st dimension) and individual proteins in each complex were separated using SDS PAGE (2 nd dimension). JAM-A S 284 phosphorylation was detected using anti-Phospho-S 284. Rap1 activation assay was performed by pull-down GTP-bound Rap1 following the manufacturer's instructions. Results: CD9 and α IIbβ 3 co-immunoprecipitated with JAM-A from resting platelet lysates suggesting that CD9 is a part of the JAM-A/α IIbβ 3 complex. In a pull-down assay recombinant full-length JAM-A cytoplasmic tail fused with GST, but not JAM-A tail lacking the PDZ domain motif fused with GST pull-down CD9 and α IIbβ 3 from resting platelet lysates suggesting that PDZ-domain binding motif of JAM-A is responsible for its interaction with CD9/α IIbβ 3 complex. Interestingly, platelet activation resulted in the dissociation of JAM-A from the CD9/α IIbβ 3 complex as seen by loss of interaction between JAM-A and CD9/α IIbβ 3 in co-immunoprecipitation and blue-native PAGE assays. It is reported that platelet activation results in rapid phosphorylation of JAM-A on S 284 in a PKC-dependent manner. We found that S 284 phosphorylated JAM-A associates with CD36 and this complex moves to the Triton X-100 insoluble cytoskeletal fraction upon platelet activation suggesting that phosphorylation of JAM-A may assist its localization to cytoskeleton. Interestingly, we found that induction of conformational change in α IIbβ 3 using Mn 2+ (10μM) activates JAM-A S 284 phosphorylation. Furthermore, Mn 2+ also induced a robust activation of Rap1, a key signaling molecule involved in junctional assembly. Both JAM-A phosphorylation and Rap1 activation were significantly ( P<0.05) inhibited by a pan PKC inhibitor, bisindolylmaleimide (10μM) suggesting that S 284 phosphorylated JAM-A/CD36 complex may support junctional assembly within the platelet plug. Conclusion: JAM-A, through its cytoplasmic domain, associates with α IIbβ 3 and CD9 in resting platelets. Upon platelet activation JAM-A rapidly dissociates from this complex and is phosphorylated on S 284. Phosphorylated JAM-A then associates with CD36 and cytoskeleton leading to activation of Rap1, a protein important in junctional assembly between platelets within a hemostatic plug.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

LFA-1 Activation in T-Cell Migration and Immunological Synapse Formation.

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation.

More of the same? Voxelotor spawns a successor, but on what success does it build?

More of the same? Voxelotor spawns a successor, but on what success does it build?

Design, synthesis, and evaluation of substituted alkylindoles that activate G protein-coupled receptors distinct from the cannabinoid CB1 and CB2 receptors

The alkylindole (AI), WIN55212-2, modulates the activity of several proteins, including cannabinoid receptors 1 and 2 (CB1R, CB2R), and at least additional G protein-coupled receptor (GPCR) that remains uncharacterized with respect to its molecular identity and pharmacological profile. Evidence suggests that such AI-sensitive GPCRs are expressed by the human kidney cell line HEK293. We synthesized fourteen novel AI analogues and evaluated their activities at AI-sensitive GPCRs using [35S]GTPγS and [3H]WIN55212-2 binding in HEK293 cell membranes, and performed in silico pharmacophore modeling to identify characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R. Compounds 10 and 12 stimulated [35S]GTPγS binding (EC50s = 3.5 and 1.1 nM, respectively), and this response was pertussis toxin-sensitive, indicating that AI-sensitive GPCRs couple to Gi/o proteins. Five AI analogues reliably distinguished two binding sites that correspond to the high and low affinity state of AI-sensitive GPCRs coupled or not to G proteins. In silico pharmacophore modeling suggest 3 characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R: 1) an s-cis orientation of the two aromatic rings in AI analogues, 2) a narrow dihedral angle between the carbonyl group and the indole ring plane [i.e., O-C(carbonyl)-C3-C2] and 3) the presence of a carbonyl oxygen. The substituted alkylindoles reported here represent novel chemical tools to study AI-sensitive GPCRs.

Vacuum Spin LED: First Step towards Vacuum Semiconductor Spintronics.

Improving the efficiency of spin generation, injection, and detection remains a key challenge for semiconductor spintronics. Electrical injection and optical orientation are two methods of creating spin polarization in semiconductors, which traditionally require specially tailored p-n junctions, tunnel or Schottky barriers. Alternatively, we introduce here a novel concept for spin-polarized electron emission/injection combining the optocoupler principle based on vacuum spin-polarized light-emitting diode (spin VLED) making it possible to measure the free electron beam polarization injected into the III-V heterostructure with quantum wells (QWs) based on the detection of polarized cathodoluminescence (CL). To study the spin-dependent emission/injection, we developed spin VLEDs, which consist of a compact proximity-focused vacuum tube with a spin-polarized electron source (p-GaAs(Cs,O) or Na2KSb) and the spin detector (III-V heterostructure), both activated to a negative electron affinity (NEA) state. The coupling between the photon helicity and the spin angular momentum of the electrons in the photoemission and injection/detection processes is realized without using either magnetic material or a magnetic field. Spin-current detection efficiency in spin VLED is found to be 27% at room temperature. The created vacuum spin LED paves the way for optical generation and spin manipulation in the developing vacuum semiconductor spintronics.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain

AbstractType 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain’s thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.

Characterization of a novel positive allosteric modulator of the α1A-Adrenergic receptor

α1-Adrenergic Receptors (ARs) are G-protein Coupled Receptors (GPCRs) that regulate the sympathetic nervous system via the binding and activation of norepinephrine (NE) and epinephrine (Epi). α1-ARs control various aspects of neurotransmission, cognition, cardiovascular functions as well as other organ systems. However, therapeutic drug development for these receptors, particularly agonists, has been stagnant due to unwanted effects on blood pressure regulation. We report the synthesis and characterization of the first positive allosteric modulator (PAM) for the α1-AR based upon the derivation of the α1A-AR selective imidazoline agonist, cirazoline. Compound 3 (Cmpd-3) binds the α1A-AR with high and low affinity sites (0.13pM; 54 ​nM) typical of GPCR agonists, and reverts to a single low affinity site of 100 ​nM upon the addition of GTP. Comparison of Cmpd-3 versus other orthosteric α1A-AR-selective imidazoline ligands reveal unique properties that are consistent with a type I PAM. Cmpd-3 is both conformationally and ligand-selective for the α1A-AR subtype. In competition binding studies, Cmpd-3 potentiates NE-binding at the α1A-AR only on the high affinity state of NE with no effect on the Epi-bound α1A-AR. Moreover, Cmpd-3 demonstrates signaling-bias and potentiates the NE-mediated cAMP response of the α1A-AR at nM concentrations with no effects on the NE-mediated inositol phosphate response. There are no effects of Cmpd-3 on the signaling at the α1B- or α1D-AR subtypes. Cmpd-3 displays characteristics of a pure PAM with no intrinsic agonist properties. Specific derivation of Cmpd-3 at the R1 ortho-position recapitulated PAM characteristics. Our results characterize the first PAM for the α1-AR and holds promise for a first-in-class therapeutic to treat various diseases without the side effect of increasing blood pressure intrinsic to classical orthosteric agonists.

Conformational dynamics of the Hsp70 chaperone throughout key steps of its ATPase cycle

The 70 kDa heat shock proteins (Hsp70s) are highly versatile molecular chaperones that assist in a wide variety of protein-folding processes. They exert their functions by continuously cycling between states of low and high affinity for client polypeptides, driven by ATP-binding and hydrolysis. This cycling is tuned by cochaperones and clients. Although structures for the high and low client affinity conformations of Hsp70 and Hsp70 domains in complex with various cochaperones and peptide clients are available, it is unclear how structural rearrangements in the presence of cochaperones and clients are orchestrated in space and time. Here, we report insights into the conformational dynamics of the prokaryotic model Hsp70 DnaK throughout its adenosine-5'-triphosphate hydrolysis (ATPase) cycle using proximity-induced fluorescence quenching. Our data suggest that ATP and cochaperone-induced structural rearrangements in DnaK occur in a sequential manner and resolve hitherto unpredicted cochaperone and client-induced structural rearrangements. Peptides induce large conformational changes in DnaK·ATP prior to ATP hydrolysis, whereas a protein client induces significantly smaller changes but is much more effective in stimulating ATP hydrolysis. Analysis of the enthalpies of activation for the ATP-induced opening of the DnaK lid in the presence of clients indicates that the lid does not exert an enthalpic pulling force onto bound clients, suggesting entropic pulling as a major mechanism for client unfolding. Our data reveal important insights into the mechanics, allostery, and dynamics of Hsp70 chaperones. We established a methodology for understanding the link between dynamics and function, Hsp70 diversity, and activity modulation.

Scanning electron microscope imaging by selective e-beaming using photoelectron beams from semiconductor photocathodes

Pulsed electron beams from a photocathode using an InGaN semiconductor have brought selectively scanning technology to scanning electron microscopes, where the electron beam irradiation intensity and area can be arbitrarily selected within the field of view in SEM images. The p-type InGaN semiconductor crystals grown in the metalorganic chemical vapor deposition equipment were used as the photocathode material for the electron beam source after the surface was activated to a negative electron affinity state in the electron gun under ultrahigh vacuum. The InGaN semiconductor photocathode produced a pulsed electron beam with a rise and fall time of 3 ns, consistent with the time structure of the irradiated pulsed laser used for the optical excitation of electrons. The InGaN photocathode-based electron gun achieved a total beam operation time of 1300 h at 15 μA beam current with a downtime rate of 4% and a current stability of 0.033% after 23 cycles of surface activation and continuous beam operation. The InGaN photocathode-based electron gun has been installed in the conventional scanning electron microscope by replacing the original field emission gun. SEM imaging was performed by selective electron beaming, in which the scanning signal of the SEM system was synchronized with the laser for photocathode excitation to irradiate arbitrary regions in the SEM image at arbitrary intensity. The accuracy of the selection of regions in the SEM image by the selective electron beam was pixel by pixel at the TV scan speed (80 ns/pix, 25 frame/s) of the SEM.

Carbon Monoxide Signaling: Examining Its Engagement with Various Molecular Targets in the Context of Binding Affinity, Concentration, and Biologic Response.

Carbon monoxide (CO) has been firmly established as an endogenous signaling molecule with a variety of pathophysiological and pharmacological functions, including immunomodulation, organ protection, and circadian clock regulation, among many others. In terms of its molecular mechanism(s) of action, CO is known to bind to a large number of hemoproteins with at least 25 identified targets, including hemoglobin, myoglobin, neuroglobin, cytochrome c oxidase, cytochrome P450, soluble guanylyl cyclase, myeloperoxidase, and some ion channels with dissociation constant values spanning the range of sub-nM to high μM. Although CO's binding affinity with a large number of targets has been extensively studied and firmly established, there is a pressing need to incorporate such binding information into the analysis of CO's biologic response in the context of affinity and dosage. Especially important is to understand the reservoir role of hemoglobin in CO storage, transport, distribution, and transfer. We critically review the literature and inject a sense of quantitative assessment into our analyses of the various relationships among binding affinity, CO concentration, target occupancy level, and anticipated pharmacological actions. We hope that this review presents a picture of the overall landscape of CO's engagement with various targets, stimulates additional research, and helps to move the CO field in the direction of examining individual targets in the context of all of the targets and the concentration of available CO. We believe that such work will help the further understanding of the relationship of CO concentration and its pathophysiological functions and the eventual development of CO-based therapeutics. SIGNIFICANCE STATEMENT: The further development of carbon monoxide (CO) as a therapeutic agent will significantly rely on the understanding of CO's engagement with therapeutically relevant targets of varying affinity. This review critically examines the literature by quantitatively analyzing the intricate relationships among targets, target affinity for CO, CO level, and the affinity state of carboxyhemoglobin and provide a holistic approach to examining the molecular mechanism(s) of action for CO.

S136: CONSTITUTIVE VLA-4 ACTIVATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE OTHER SIDE OF BCR AUTONOMOUS SIGNALING.

Background: In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator and key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. In CLL, beside the canonical antigen (Ag)-dependent mechanism, BCR signaling has been recently demonstrated to occur via an autonomous Ag-independent manner. Aims: To investigate the role of autonomous BCR signaling on constitutive VLA-4 activation state in CLL. Methods: VLA-4 activation/affinity was determined by flow cytometry (FC) using the conformation-sensitive anti-CD29 mAb HUTS21 and/or by “real-time” (FC) measuring the binding of the VLA-4 ligand LDV-FITC as reported (Tissino et al, J Exp Med, 2018) in: i) 1,984 consecutive CLL all with IGHV gene mutations/BCR features available; ii) sequential samples (0, 14, 30, 60 90 days) from CLL patients (n=26) treated in vivo with ibrutinib (IB) in real-world and from a clinical trial (NCT02827617). HUTS21 staining was also performed in the presence of: plasma depletion/replacement, soluble (s) VCAM-1, fibronectin (FN) and blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). BCR signaling were investigated by Ca++ influx assay in a 4-OH tamoxifen (4-OHT)-inducible murine TKO cell model (Dühren-von Minden M et al, Nature, 2012). Results: Out of 1,984 CLL, 1,070 (54%) expressed CD49d (cutoff 30%) and, among them, 250/1,070 (23%) were HUTS21+ (cutoff 20%), indicating an activated VLA-4 conformation. HUTS21 staining was: i) impaired by depletion of plasma from whole blood samples, and reconstituted by specific plasma components (sVCAM-1, FN); ii) impaired by pre-incubation with anti-CD49d HP1/2 blocking mAbs before addition of plasma, sVCAM-1 and FN. sVCAM-1 was higher in CD49d+ vs CD49d- CLL (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ (i.e. VLA-4 activated) cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4. CLL with mutated IGHV expressed higher levels of activated VLA-4 compared to unmutated IGHV CLL (p=0.001). Higher levels of activated VLA-4 were found in CLL using the IGHV3 and IGHV4 families, compared to cases using the IGHV1 family (p=0.043 and p=0.004). Finally, analysis of BCR stereotypy highlighted higher VLA-4 activation levels in CLL from subset#2 compared to CLL from subset#1 (p=0.02). To validate these data, murine TKO cells, expressing high VLA-4 levels, were transfected with different BCRs derived from 4 CLL with high level of constitutively activated VLA-4 (TKO-high) and 4 CLL with low level of constitutively activated VLA-4 (TKO-low). Compared to TKO-low cells, TKO-high cells showed a higher autonomous Ca++ influx (p=0.03), and consistently higher VLA-4 affinity (p=0.01). IB treatment impaired both BCR autonomous signaling and VLA-4 affinity. Notably, anti-IgM stimulation induced high Ca++ influx and high VLA-4 affinity state in both TKO-high and TKO-low, irrespective of IB treatment. According to TKO data, a decreased constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30, 60 and 90 from patients on IB, confirming an IB-dependent impairment of VLA-4 activation via BCR signal. Summary/Conclusion: The presence of a constitutively activated form of VLA-4 is observed in a fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from autonomous BCR signaling.

Kinematic Vibrational Entropy Assessment and Analysis of SARS CoV-2 Main Protease.

The three-dimensional conformations of a protein influence its function and select for the ligands it can interact with. The total free energy change during protein-ligand complex formation includes enthalphic and entropic components, which together report on the binding affinity and conformational states of the complex. However, determining the entropic contribution is computationally burdensome. Here, we apply kinematic flexibility analysis (KFA) to efficiently estimate vibrational frequencies from static protein and protein-ligand structures. The vibrational frequencies, in turn, determine the vibrational entropies of the structures and their complexes. Our estimates of the vibrational entropy change caused by ligand binding compare favorably to values obtained from a dynamic Normal Mode Analysis (NMA). Higher correlation factors can be achieved by increasing the distance cutoff in the potential energy model. Furthermore, we apply our new method to analyze the entropy changes of the SARS CoV-2 main protease when binding with different ligand inhibitors, which is relevant for the design of potential drugs.

False data injection attack detection based on interval affine state estimation

With the development and application of information communication technology, it makes the potential threat of cyber-attacks prominent increasingly that the power information network and physical network are becoming deeply integrated. In the energy management system (EMS), the state estimation module is the main target of hackers' for false data injection attacks (FDIAs) on power systems deliberately. Therefore, from the perspective of redundancy of system measurement data, it proposes a pseudo-measurement model based on a new neural network, convolutional long short-term memory (CLSTM) neural network, which is a combination of different networks. And using interval number and affine number to analyze and quantitatively describe the measurement uncertainty, this paper proposes a FDIAs detection model based on interval affine state estimation. It provides technicians with the upper and lower limit information of the systems state, which makes it easier to judge whether the measured data is attacked and thus exceeds the normal fluctuation range. This paper has tested IEEE 33-bus system and the results show that this method has obvious advantages in term of calculation speed and accuracy.