Constricted State Research Articles

Dysregulated DnaB unwinding induces replisome decoupling and daughter strand gaps that are countered by RecA polymerization.

The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress. This surplus ssDNA can stimulate RecA recruitment to initiate recombinational repair, restart, or activation of the transcriptional SOS response. To better understand the consequences of dysregulated unwinding, we combined targeted genomic dnaB mutations with an inducible RecA filament inhibition strategy to examine the dependencies on RecA in mitigating replisome decoupling phenotypes. Without RecA filamentation, dnaB:mut strains had reduced growth rates, decreased mutagenesis, but a greater burden from endogenous damage. Interestingly, disruption of RecA filamentation in these dnaB:mut strains also reduced cellular filamentation but increased markers of double strand breaks and ssDNA gaps as detected by in situ fluorescence microscopy and FACS assays, TUNEL and PLUG, respectively. Overall, RecA plays a critical role in strain survival by protecting and processing ssDNA gaps caused by dysregulated helicase activity in vivo.

Recount Restriction and Identity in Margaret Atwood’s Surfacing

Margaret Atwood’s Surfacing crams the effects of the demarcation of identity revels in Canadian history where the question of Canadian national identity was sprouting, becoming a pointer that was more clearly defined and more intentionally sought out by Canadian artists and citizens. Atwood’s Surfacing can be considered in light of these historical developments, in sketching the establishment of recount restriction and not an avowal of the positive effects with such identifiers can bring. As a Canadian writer and a woman in particular, the narrator’s communal groupings stipulate her victimhood, a notion that explores in relation to Atwood’s major work of Canadian literary works. The narrator’s initial state of close proximity total psychological constriction is pinpointing the peril of social borders, and it is only in evading the identifiers that define her in peculiar of national, gender, and epistemological establish that she comes to a sort of completeness. This breaks out takes the form of a turn towards the chaotic world as a truly borderless space where it is doable to mendan inconsistent self.

Experimental investigation on “glow flow” phenomenon during current-zero period between transverse magnetic contacts

The inter-electrode state of the transverse magnetic field contact during the current-zero period is determined by the evolution of the arcing process. It has an important effect on whether the vacuum switch can be successfully broken. In this paper, a series of contacts were subjected to arcing and fixed gap distance experiments. The results show that a special inter-electrode phenomenon (glow flow) appears during the current-zero period when the current exceeds the threshold for the transition from diffused to constricted state of the vacuum arc. The mechanism behind the formation of this phenomenon was elucidated through an analysis of multiple sets of experimental results. It is found that the inter-electrode phenomenon arises from the disruption of the equilibrium process between the pressure from the arc column plasma and the anode vapor flux on the near-anode side at the current-zero. The copper vapor falls and is ionized by collisions with heat-emitting electrons. The experimental results and theoretical analysis also show that this phenomenon has a time interval with the reignition phenomenon. When this phenomenon is intense to a certain degree, the reignition will occur with high probability. The observation and theoretical analysis results of this paper are relatively consistent with the research results of other scholars, which can help to improve the vacuum arc characteristics and interrupting mechanism. This can provide a basis for improving the arc extinction performance of the vacuum switch.

Effects of low-dose x-ray radiation at varying dose rates on human aortic smooth muscle in vitro.

Radiation has a variety of uses in the medical field such as diagnostic imaging and cancer treatment. However, we still know very little about radiation's effect on cells in the body particularly at low doses such as those used in x-ray and CT imaging. Our group has designed studies to determine the effects of low doses of x-rays on vascular cells. We helped to design a novel x-ray fluorescent experimental setup to irradiate human aortic smooth muscle cells with dosing rates varying from at the lower end of the medical imaging range. This novel setup allows us to expose the cells inside a standard incubator with x-rays that have very precisely controlled energy, total dose, and dose rate. Three specific protocols were conducted in 24 hour intervals over several days, ultimately showing clear and statistically significant differences between dose rates even when the total dose was kept constant. As dose rate increased, the total number of cells and the number of cells in a constricted state increased, while no significant increase in cell death was observed. We have shown, for the first time, that x-rays, at doses similar to what is given to patients during standard x-ray imaging, can cause changes in vascular cells in culture. Furthermore, RT-PCR was conducted at 12 hour intervals, showing along with the assays that radiation quantitatively changes the amount of collagen in vascular cells, thus supporting our previous assays. Future experiments will include determining a specific relationship between and mechanisms behind varying dose rate and the number of cells, as well as determining whether changing other characteristics of radiation has an impact on the cells being irradiated.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

Components of the Effectiveness of Endodontic Treatment Based on Long-term X-ray Criteria

The objective of the research was to determine the effectiveness of obturation of the root canals and crown part of the teeth, and to identify the most important factors influencing its results, based on the X-ray examination.&#x0D; Materials and methods: The study was conducted at the Dental Center (University Clinic, Ivano-Frankivsk National Medical University). Evaluation of 140 orthopantomograms, computed tomograms and digital intraoral images of patient, aged 18-68 years with endodontically treated teeth, was made with the aim to determine the obturation state of root canals and the state of periodontal tissues. The radiological picture of 579 teeth and 1120 roots in particular was evaluated.&#x0D; Results: the state of apical constriction, the state of obturation of root canals, sealing conditions of coronal part of the tooth, the state of periodontal tissues were analyzed, compared and discussed.&#x0D; Conclusions: According to the results obtained in this study, the relationships between the presence of periapical changes and state of apical constructions, obturation level of filling material in roots canals, coronal leakage, and periodontal tissue changes were found.&#x0D; Keywords: periapical changes, endodontic treatment, coronal leakage, x-ray examination

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

Spatiotemporally resolved characteristics of arc plasma in non-thermal multi-arc generator at atmospheric pressure

The spatiotemporally resolved characteristics of arc plasma in a non-thermal multi-arc generator with three high-voltage electrodes and a common grounded one were investigated by means of arc current signal, discharge image signal, and image analysis. Experimental results show that the spatial distribution of multi-arc plasma can be adjusted by the gas flow rate, and multi-arc plasma tends to distribute in the center of the arc chamber with the decrease in the gas flow rate. Once the confluence of three independent arc columns occurs, a common arc root will be shared by three arcs. The common arc-root attachment varies between constricted state and diffuse state, and the variation duration was in a cycle of about 1 ms, which is associated with beat vibration caused by the small deviation among the frequencies of three arc currents. This phenomenon suggests that the size of the arc root attachment can be adjusted by means of the frequency of the driving power supply.

Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness.

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.

CryoEM structure of the super-constricted two-start dynamin 1 filament

Dynamin belongs to the large GTPase superfamily, and mediates the fission of vesicles during endocytosis. Dynamin molecules are recruited to the neck of budding vesicles to assemble into a helical collar and to constrict the underlying membrane. Two helical forms were observed: the one-start helix in the constricted state and the two-start helix in the super-constricted state. Here we report the cryoEM structure of a super-constricted two-start dynamin 1 filament at 3.74 Å resolution. The two strands are joined by the conserved GTPase dimeric interface. In comparison with the one-start structure, a rotation around Hinge 1 is observed, essential for communicating the chemical power of the GTPase domain and the mechanical force of the Stalk and PH domain onto the underlying membrane. The Stalk interfaces are well conserved and serve as fulcrums for adapting to changing curvatures. Relative to one-start, small rotations per interface accumulate to bring a drastic change in the helical pitch. Elasticity theory rationalizes the diversity of dynamin helical symmetries and suggests corresponding functional significance.

Traumatic Experiences in Selected Contemporary Poems by Patricia Smith

The thesis depicts the traumatic experiences in selected poems by the American contemporary poet Patricia Smith. It investigates the psychological impacts that affect Smith’s poetry. It examines how the language used in Smith’s poems reflects the traumatic experiences that she has been through. The thesis follows Judith Herman's encounter with trauma. According to Herman, after passing a traumatic experience, the traumatized person suffers from posttraumatic stress disorder or PTSD. PTSD folds in three main symptoms hyperarousal, intrusion, and constriction. According to Herman, the survivor starts his recovery journey after a struggling time with traumatic disorders. The recovery process consists of three main stages; establishment of safety, remembrance and mourning, and reconnection. The study demonstrates how the hyperarousal’s symptoms are noticeably mirrored in the language of Smith’s poems. Furthermore, it traces the disturbing intruders that overwhelm Smith’s mind during waking and sleeping time. The thesis determines the state of constriction that controls Smith’s soul as a result of the many racial and traumatic experiences that she passes through. The second part of the study investigates Smith’s journey in getting over her pains and restoring power over herself. The study concludes that Patricia Smith suffers from many traumatic symptoms in most of her poems. She takes deep steps in her recovery journey, but she is still surrounded by the foreshadows of the previous experiences. The study affirms the rule of literature in healing traumatic pain. Therefore, Smith uses her literary tools for retelling her traumatic experiences.

Thermoresponsive, hollow, degradable core-shell nanoparticles for intra-articular delivery of anti-inflammatory peptide

Inflammation following joint trauma contributes to cartilage degradation and progression of post traumatic osteoarthritis (PTOA). Therefore, drug delivery vehicles that deliver effective anti-inflammatory treatments have the potential to prevent PTOA. We have developed solid and hollow, thermoresponsive nanoparticles for the controlled release of our anti-inflammatory MK2-inhibiting (MK2i) peptide for intra-articular injection to halt inflammation that contributes to the advancement of PTOA. This system exploits the thermosensitive characteristic of N-isopropyl acrylamide (NIPAm) to transition phases when passing through its lower critical solution temperature (LCST). The nanoparticles (NPs) swell below the LCST and constrict above it.Non-crosslinked poly(NIPAm) (pNIPAm), held above its LCST, formed hydrophobic cores around which shells composed of NIPAm, degradable crosslinker N, N′-bis (acryloyl) cystamine (BAC), sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and acrylic acid (AAc) were polymerized. Removal of the non-crosslinked pNIPAm cores via diffusion produced thermosensitive, degradable nanoparticles with low density, or hollow, cores. The data presented here revealed low-density, termed hollow, nanoparticles (hNPs) load and release significantly more MK2i than solid nanoparticles (sNPs). Furthermore, drug loading below the LCST of NIPAm results in roughly 2.5 times more therapeutic encapsulation compared to loading particles in their constricted state. Hollow nanoparticles increase drug loading compared to solid nanoparticles, are taken up into chondrocytes within 24 h, cleared from the cells within 6 days, significantly decrease the secretion of the proinflammatory cytokine IL-6, and, via intra-articular injection, are successfully delivered into the joint space of rats. The peptide loaded nanoparticles provide a reproducible platform for intra-articular delivery of therapeutics.

Contacts and Confirmations that Regulate Hexameric Helicases at the Replication Fork

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. The hexameric helicase is at the leading front of the replisome and is a nexus of control for both the initiation and elongation phases of DNA replication. These proteins are comprised of a N‐terminal DNA binding domain and a C‐terminal motor domain that assemble into a toroidal hexameric structure. Two main superfamilies of hexameric helicases exist: the RecA family, which contain helicases from phage and bacteria, and the AAA+ family, which contain helicases from archaea and eukaryotes. Interestingly, the measured translocation polarity of 5′‐3′ or 3′‐5′, respectively, dictates the orientation of these helicases at the replication fork.Using site specific DNA cleavage experiments and presteady‐state FRET, we have unequivocally shown the then N‐terminal domain of the archaeal MCM helicase leads the way for unwinding (N‐first). The MCM hexamer itself can bind model DNA substrates in multiple conformations; however, the correct ‘N‐first’ conformation is dictated by 1) the presence of an excluded strand, 2) the ssDNA polarity (3′‐5′), and 3) the C‐terminal winged‐helix (WH). This orientation is opposite to that of the bacterial DnaB helicase which proceeds C‐first in the 5′‐3′ direction.To determine the cellular impact of the excluded strand interaction in the steric exclusion and unwinding model (SEW), we created and tested several CRISPR/Cas9 genetic mutations of E. coli dnaB that correlate with mutations on the exterior surface. These DnaB mutations alter the conformation of the hexamer creating a spectrum of constricted states that all result in faster DNA unwinding. Genetic dnaB mutations lead to decoupling within the replisome, buildup of ssDNA, activation of the SOS response, and loss of genomic integrity. Therefore, the SEW interaction and the central channel diameter is adaptive and dynamic to properly maintain the DNA unwinding within the confines of the replisome to regulate speed of DNA replication and prevent chromosomal instability.Support or Funding InformationNational Science Foundation Division of Molecular and Cellular Biosciences (NSF1613534)

Selectivity filter modalities and rapid inactivation of the hERG1 channel

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.

Novel, vessel anatomy adjusting drug-coated balloon-Preclinical evaluation in peripheral porcine arteries.

The diameter of balloons or stents is selected according to the estimated reference vessel diameter and do not adapt to the vessel anatomy. The aim of the present preclinical studies was to investigate a novel, vessel anatomy adjusting hypercompliant drug-coated balloon catheter (HCDCB). Hypercompliant balloon membranes were coated in a constricted state with high drug density. Drug adherence was investigated in vitro, transfer to the porcine peripheral arteries and longitudinal distribution in vivo. In young domestic swine, neointimal proliferation was induced by vessel overstretch and continuous irritation by permanent stents. Uncoated hypercompliant balloons (HCB), and standard uncoated balloons and drug-coated balloons (DCB) served as controls. Efficacy was assessed by angiography, optical coherence tomography (OCT), and histomorphometry. HCDCB lost 18.0 ± 3.9% of dose during in vitro simulated delivery to the lesion. Drug transfer to the vessel wall was 13.9 ± 6.4% and drug concentration was 1,044 ± 529 ng/mg tissue. Four weeks after treatment, the histomorphometric neointimal area was smaller with HCDCB versus uncoated HCB (2.39 ± 0.55 mm2 vs. 3.26 ± 0.72 mm2 , p = .038) and area stenosis (OCT) was less (11.6 ± 6.9% vs. 24.7 ± 9.7%, p = .022). No premature death occurred and no in-life clinical symptoms or treatment-associated thrombi were observed. HCDCB were found to inhibit excessive neointimal proliferation. Balloon adaption to different vessel diameters and shapes may provide drug-delivery in irregular lumen and facilitate balloon selection.

Instabilities of a constricted gas discharge with respect to two-dimensional wave perturbations

Modelling of the positive column in the glow discharge of neon was performed under discharge conditions covering the areas of diffuse and constricted states, as well as the transition region between them. An analysis of the stability of the stationary solution for diffuse and constricted regimes of the discharge was made. The spectral problem was solved in order to find the eigenmodes and eigenvalues corresponding to the growth increment of the perturbations of the electron number density and electrical potential. It was shown that the discharge in the diffusion mode is stable with respect to the perturbations under consideration, and the appearance and propagation of essentially two-dimensional spatially periodic perturbations is possible in the constricted mode of the discharge. A two-dimensional structure (topology) of plasma parameters was calculated and illustrated. The results of the theory were compared to the experimental data.

Radial structure of the constricted positive column: Modeling and experiment.

We present a detailed self-consistent model of a positive column in argon glow discharge at moderate pressures and currents. This model describes the discharge transition between diffuse and constricted states. The model includes an extensive set of plasma chemical reactions and equation for inhomogeneous gas heating. The nonequilibrium behavior of an electron distribution function is also considered. One of the main features of the model is an accurate treatment of radiation trapping by solving the Holstein-Biberman equation directly. Influence of the radiation trapping on macroscopic parameters of the constricted positive column is studied. We propose a method for solving a boundary-value problem, including particle and energy balance equations for electrons, ground state atoms, atomic and molecular ions, and excited species. Unlike traditional solution approaches for similar systems, the method provides continuous Z- and S-shaped characteristics of discharge parameters, describing hysteresis in transition between diffuse and constricted discharge regimes. Performed experiments include measurements of volt-ampere characteristics and spectroscopic study of radial density profiles of excited atoms by measuring line emission and absorption, and electrons by measuring bremsstrahlung intensity. The role of resonance radiation trapping in spatial redistribution of 1s and 2pstates of argon is demonstrated. Results of modeling are compared to the experimental data.

Sequential Latent Structure Analysis of Segregated System Material in the Doll Play of Maltreated Children.

A range of assessment methodologies pertaining to attachment in children have been developed and validated during the past decades. The present study examined the validity of a sequential, profile-centered approach to the assessment of attachment disorganization using a doll play methodology developed by George and Solomon (1990). The sample was composed of sixty-two children between the age of 5 and 12 who had been removed from their family of origin due to abuse or neglect. Latent Markov Models performed on markers of defensive dysregulation (Frightening, Chaos, Constriction) yielded three distinct states, characterized respectively by themes of fear or chaos, presence of at least partial constriction, and absence of segregated system markers across doll play stories. The states generally showed a high degree of stability across scenarios. The Frightening-Chaotic state was positively associated with sexual abuse, placement in a specialized setting and caregiver-reported externalizing problems whereas the Constricted state was associated with placement in a specialized setting and self-reported hyperactivity and attention problems. These results provide support for the validity and clinical utility of this approach to assessing attachment disorganization on the doll play.

Probing the Effects of Gating on the Ion Occupancy of the K+ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy.

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.

Dynamic Sampling of Multiple Conformations in FimH Modulates Bacterial Adhesion

Gram-negative bacteria express hundreds of diverse pili assembled by the chaperone-usher pathway (CUP) for surface colonization and biofilm formation. CUP pili are multi-subunit surface appendages tipped with two-domain adhesins that recognize distinct ligands through a receptor-binding domain while anchored to an adaptor subunit through a pilin domain. Uropathogenic E. coli utilize the type 1 pilus adhesin FimH to bind mannose, which decorates the bladder epithelium. This interaction is critical in urinary tract infection (UTI) pathogenesis. Bioinformatic studies have demonstrated that the composition of three positively selected residues (PSRs) in FimH impact its role in pathogenesis. However, the mechanism by which residues that lie outside the mannose-binding pocket alter the binding and virulence of FimH remains poorly understood. Crystallographic studies indicate that FimH adopts elongated and compact conformations. We postulated that PSRs in FimH allosterically modulate a dynamic equilibrium of multiple conformations in solution, which impacts function. To address this hypothesis, we first show by small-angle X-ray scattering that FimH alleles bound to FimG peptide, which reflects a tip-like setting, vary drastically in their average conformation. PSRs that promote extension of FimH conformation exhibit high affinity for mannose. Further, collision cross-section distributions measured by ion mobility mass spectrometry indicate that FimH alleles adopt altered equilibria of two major conformations. Congruently, preliminary analyses of molecular dynamics simulations suggest that PSRs modulate flux across a conformational landscape characterized by a constricted tense state and multiple, rapidly exchanging relaxed states. Currently, we are investigating the biophysical implications of multiple relaxed states with predicted functionality on bacterial attachment. Altogether, by integrating biophysical and computational methods to map the FimH conformer-function relationship, this work begins to unravel how a conformational phase switch regulates bacterial adhesion and how evolutionary processes fine-tune protein dynamics to guide host colonization.