The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homologue. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Schwannoma of Nasopharynx-A Rare Case with A Complex Presentation.

Nasopharyngeal tumors are rare, and schwannomas originating from this location are extremely uncommon. Schwannomas are tumors arising from the Schwann cells of peripheral nerve sheath which are neuroectodermal in origin. These are benign, slow growing, well-encapsulated tumors and are mostly seen in the head and neck region. We present a rare case of schwannoma with nasopharynx as its epicenter in an elderly female patient who presented with complaints of hematemesis, bilateral nasal obstruction and stertorous breathing. Diagnostic nasal endoscopy revealed a smooth mucosa covered globular mass occupying bilateral choana. Contrast-enhanced computed tomography of Nose and paranasal sinuses revealed a homogenous mass occupying the entire nasopharynx extending into the nasal and oropharyngeal cavities. The patient underwent Trans-nasal Endoscopic excision under general anesthesia. Nasopharyngeal tumors have a wide range of presentation and a high index of clinical suspicion combined with imaging modalities is required for diagnosis and pre operative planning. Some tumors can cause life-threatening airway obstruction due to delayed presentation and should be managed efficiently and meticulously, with endoscopic excision being a viable approach for cases confined within the nasopharynx.

Determining the Best Thyroid Imaging Reporting and Data System: A Prospective Study Comparing the Diagnostic Performance of ACR, EU, and K TIRADS in the Evaluation of Thyroid Nodules.

Background Many different risk stratification systems have been formulated for thyroid nodules, differing in their fine-needle aspiration cytology (FNAC) indication, suggesting a lack of consensus around the world. Purpose This prospective study was conducted to find the best guideline for risk stratification, for a better malignancy yield, and with reduced rates of negative FNACs among three Thyroid Imaging, Reporting, and Data System (TIRADS) guidelines. Materials and Methods A total of 625 thyroid nodules with conclusive FNAC or histopathological diagnosis were included in the study. Various sonographic parameters were recorded. They were classified into categories as per the three guidelines and compared with FNAC diagnosis. The guidelines were evaluated in terms of sensitivity, specificity, predictive values, and diagnostic accuracy. Sensitivity and specificity were compared by McNemar's test. Results American College of Radiology (ACR) TIRADS had the highest diagnostic accuracy (56.8%), specificity (50.75%), positive predictive value (23.92%), lowest rates of negative FNACs (76.08%), and high negative predictive value (97.84 %). Korean (K) TIRADS had the maximum sensitivity (97.75%), highest negative predictive value (98.44%), and gross malignancy yield. European TIRADS was between the two other guidelines in most parameters with specificity like K TIRADS. Conclusion All the three guidelines are very good screening tools, with comparable high sensitivity. ACR TIRADS is better in terms of specificity and reduced rates of negative FNACs. Including the presence of a suspicious cervical lymph node as a criterion and more frequent follow-up might further improve the diagnostic performance of the guideline.

Modeling of Cu(II)-based protein spin labels using rotamer libraries.

The bifunctional spin label double-histidine copper-(II) capped with nitrilotriacetate [dHis-Cu(II)-NTA], used in conjunction with electron paramagnetic resonance (EPR) methods can provide high-resolution distance data for investigating protein structure and backbone conformational diversity. Quantitative utilization of this data is limited due to a lack of rapid and accurate dHis-Cu(II)-NTA modeling methods that can be used to translate experimental data into modeling restraints. Here, we develop two dHis-Cu(II)-NTA rotamer libraries using a set of recently published molecular dynamics simulations and a semi-empirical meta-dynamics-based conformational ensemble sampling tool for use with the recently developed chiLife bifunctional spin label modeling method. The accuracy of both the libraries and the modeling method are tested by comparing model predictions to experimentally determined distance distributions. We show that this method is accurate with absolute deviation between the predicted and experimental modes between 0.0-1.2 Å with an average of 0.6 Å over the test data used. In doing so, we also validate the generality of the chiLife bifunctional label modeling method. Taken together, the increased structural resolution and modeling accuracy of dHis-Cu(II)-NTA over other spin labels promise improvements in the accuracy and resolution of protein models by EPR.

Site of Tracheostomy and Its Influence on The Surgical Outcome and Quality of Life After Tracheal Resection and Anastomosis in Patients with Tracheal Stenosis.

Introduction With the advances in critical care, the incidence of post intubation tracheal stenosis is increasing. Tracheal resection and anastomosis have been the gold standard for the management of grades III and IV stenosis. Scientific evidence from the literature on the determining factors and outcomes of surgery is not well described. Objective This study was aimed at determining the influence of tracheostoma site on the surgical outcomes and postoperative quality of life of patients undergoing tracheal resection anastomosis. Methods Thirteen patients who underwent tracheal resection and anastomosis during a period of 3 years were followed up prospectively for 3 months to determine the degree of improvement in their quality of life postsurgery by comparing the pre and postoperative validated Tamil/vernacular version of RAND SF-36 scores and Medical Research Council (MRC) dyspnea score. Results As per preoperative computed tomography (CT), the mean length of stenosis was found to be 1.5 cm while the mean length of trachea resected was 4.75 cm. We achieved a decannulation rate of 61.53%. There was an estimated loss of 3.20 +/- 1.90 cm of normal trachea from the lower border of the stenosis until the lower border of the stoma that was lost during resection. Analysis of SF-36 and MRC dyspnea scores revealed significant improvement in the domains of physical function postoperatively in comparison with the preoperative scores ( p < 0.05). Conclusion Diligent placement of tracheostomy in an emergency setting with respect to the stenotic segment plays a pivotal role in minimizing the length of the resected segment of normal trachea.

Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics.

Electron paramagnetic resonance (EPR) has become a powerful probe of conformational heterogeneity and dynamics of biomolecules. In this Review, we discuss different computational modeling techniques that enrich the interpretation of EPR measurements of dynamics or distance restraints. A variety of spin labels are surveyed to provide a background for the discussion of modeling tools. Molecular dynamics (MD) simulations of models containing spin labels provide dynamical properties of biomolecules and their labels. These simulations can be used to predict EPR spectra, sample stable conformations and sample rotameric preferences of label sidechains. For molecular motions longer than milliseconds, enhanced sampling strategies and de novo prediction software incorporating or validated by EPR measurements are able to efficiently refine or predict protein conformations, respectively. To sample large-amplitude conformational transition, a coarse-grained or an atomistic weighted ensemble (WE) strategy can be guided with EPR insights. Looking forward, we anticipate an integrative strategy for efficient sampling of alternate conformations by de novo predictions, followed by validations by systematic EPR measurements and MD simulations. Continuous pathways between alternate states can be further sampled by WE-MD including all intermediate states.

One More Chance to See the World: A Rare Case Report on Sphenoid Mucocele Causing Vision Loss.

Mucocele of the sphenoid sinus is one of the rare diseases which an ENT surgeon can encounter in a clinical setup. It can often present as a chronic headache. It can remain undiagnosed unless a symptom due to the compression effect of the mucocele, such as diminution of vision, ophthalmoplegia, or any intracranial complication, occurs. Early diagnosis and emergency surgical intervention are imperative to prevent complications. Although late presentation can have a risk of permanent vision loss, improvement in vision postsurgery does not necessarily depend on the duration of symptoms. Here, we present a rare case scenario where, even after the delayed presentation, the patient had a significant vision improvement postsurgery.

Utility of the Growth Differentiation Factor-15 in the Differential Diagnosis of Follicular-Patterned Lesions of the Thyroid on Cytopathologic and Histopathologic Samples.

Background Follicular-patterned lesions are a major gray zone in thyroid cytopathology. The recently introduced 2022 World Health Organization (WHO) classification emphasizes the importance of genetic alterations in thyroid neoplasms with the introduction of certain newer terminologies that are expected to cause remarkable changes in cytopathologic and histopathologic reporting. Although molecular assays such as the Afirma gene expression classifier and the ThyroSeq are already in use, there has been an ongoing search for further reliable molecular markers. The growth differentiation factor-15 (GDF-15) is one among them. This study aimed to determine the diagnostic utility of GDF-15 mRNA expression in frozen tissue and fine-needle aspiration (FNA) samples from follicular-patterned thyroid lesions and neoplasms. Methodology The real-time quantitative polymerase chain reaction was performed on 75 frozen tissue and FNA samples each from 19 cases of follicular thyroid hyperplasia (FTH), 10 nodular goiters (NGs), 17 follicular thyroid adenomas (FTAs), eight follicular thyroid carcinomas (FTCs), 12 follicular variant of papillary thyroid carcinomas (FVPTCs), and nine classic papillary thyroid carcinomas (CPTCs) that were diagnosed according to the 2017 WHO classification of thyroid neoplasms. The GDF-15 mRNA expression in all these cases was assessed and compared with the control thyroid tissue samples. One-way analysis of variance and the Kruskal-Wallis test were performed using GraphPad Prism 8 software to determine the significance of differences in the GDF-15 mRNA levels among various thyroid lesions. Results A higher GDF-15 mRNA expression was noted in the malignant thyroid neoplasms including FTC, FVPTC, and CPTC in comparison to FTA, with a fold change between the malignant and benign groups being more than 244.18 times. A difference in the fold change was noted between FTH and FTA with an increase in GDF-15 mRNA level in the latter, which was statistically not significant. Conclusions The fact that GDF-15 mRNA was studied both on fine-needle aspiration cytologic and the frozen tissue material and that the majority of the lesions studied were follicular-patterned establishes the GDF-15 as a potential marker not only for diagnosing malignant thyroid neoplasms of the follicular epithelium but also in distinguishing benign and malignant follicular-patterned neoplasms of the thyroid.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (NTA) Motif.

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu2+ (NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron-electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu2+ (NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+ (NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu2+ (NTA) motif in PDS experiments.

Double Quantum Coherence ESR at Q-Band Enhances the Sensitivity of Distance Measurements at Submicromolar Concentrations.

Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts.

Endoscope-Assisted Removal of Post-Traumatic Orbital Epidermoid Inclusion Cyst: A Useful Adjunct.

Orbital epidermoid cysts are uncommon lesions within the bony orbit with varied symptomatology related to both the eye and the sino-nasal system. They are often slow-growing cystic masses which may cause facial asymmetry and visual loss due to pressure symptoms. Cross-sectional imaging such as computed tomography and magnetic resonance imaging are contributory and useful for assessment of the size and actual extent and should be mandatory before planning any surgical intervention. Open approaches and needle aspiration have been traditionally described; however, the use of the rigid nasal endoscope in the intraorbital compartment is a useful adjunct for exploration of the extent of the lesion and for complete surgical clearance. In this report, a 69 years old female with an old post- traumatic orbital epidermoid cyst which was removed completely using an endoscope via transorbital route was presented with the review of literature.

Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation.

The catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1-1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare-events sampling strategy to gain mechanistic information on protein function at the atomic level.

Kaposiform Haemangioendothelioma of the Sublingual Gland in an Adult.

Kaposiform haemangioendotheliomas (KHE) are extremely rare, borderline malignant, locally aggressive vascular tumours. KHE is usually found in the retroperitoneum, over the extremities, the soft tissues of the trunk, mediastinum and the head and neck regions. We present a very rare case of KHE of the sublingual gland in an adult, which was not associated with Kasabach-Merritt phenomenon and was resected completely without any recurrence. To the best of our knowledge, this is the first reported case in the literature of a KHE arising from the salivary glands in an adult.

"Store-bought is fine": Sensitivity considerations using shaped pulses for DEER measurements on Cu(II) labels.

The narrow excitation bandwidth of monochromic pulses is a sensitivity limitation for pulsed dipolar spectroscopy on Cu(II)-based measurements. In response, frequency-swept pulses with large excitation bandwidths have been adopted to probe a greater range of the EPR spectrum. However, much of the work utilizing frequency-swept pulses in Cu(II) distance measurements has been carried out on home-built spectrometers and equipment. Herein, we carry out systematic Cu(II) based distance measurements to demonstrate the capability of chirp pulses on commercial instrumentation. More importantly we delineate sensitivity considerations under acquisition schemes that are necessary for robust distance measurements using Cu(II) labels for proteins. We show that a 200 MHz sweeping bandwidth chirp pulse can improve the sensitivity of long-range distance measurements by factors of three to four. The sensitivity of short-range distances only increases slightly due to special considerations for the chirp pulse duration relative to the period length of the modulated dipolar signal. Enhancements in sensitivity also dramatically reduce measurement collection times enabling rapid collection of orientationally averaged Cu(II) distance measurements in under two hours.

Efficient sampling of molecular orientations for Cu(II)-based DEER on protein labels.

Combining rigid Cu(II) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron-nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu(II)-labeled proteins to evaluate pulse excitation in the context of double electron-electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment and the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations.

Veillonella atypica causing retropharyngeal abscess: A rare case presentation.

Veillonella species are obligate anaerobes which are part of the human oral, gut and vaginal microbiota. The genus Veillonella consists of 16 characterized species. Very few infections due to Veillonella atypica have been reported till date. Here we present a case of retropharyngeal abscess due to this organism in a 55-year-old lady.

A new 13C trityl-based spin label enables the use of DEER for distance measurements.

Triarylmethyl (TAM)-based labels, while still underutilized, are a powerful class of labels for pulsed-Electron Spin Resonance (ESR) distance measurements. They feature slow relaxation rates for long-lasting signals, high stability for cellular experiments, and narrow spectral features for efficient excitation of the spins. However, the typical narrow line shape limits the available distance measurements to only single-frequency experiments, such as Double Quantum Coherence (DQC) and Relaxation Induced Dipolar Modulation Enhancement (RIDME), which can be complicated to perform or hard to process. Therefore, widespread usage of TAM labels can be enhanced by the use of Double Electron-Electron Resonance (DEER) distance measurements. In this work, we developed a new spin label, 13C1-mOX063-d24, with a 13C isotope as the radical center. Due to the resolved hyperfine splitting, the spectrum is sufficiently broadened to permit DEER-based experiments at Q-band spectrometers. Additionally, this new label can be incorporated orthogonally with Cu(II)-based protein label. The orthogonal labeling scheme enables DEER distance measurement at X-band frequencies. Overall, the new trityl label allows for DEER-based distance measurements that complement existing TAM-label DQC and RIDME experiments.

Plasma Cell Granuloma of the Maxillary Sinus-A Case Report and Review.

Plasma cell granuloma is a rare, benign, space occupying lesions occurring after recurrent infections. It most commonly involves young adults with lungs being the most common site. They are usually rounded masses with bony expansion and destruction without any life threatening complications and surgery being the best choice of treatment. Here we reported a 33 year-old female with plasma cell granuloma of the maxillary sinus treated with surgery and no recurrence has been noted.

Synthesis of Square Planar Cu4 Clusters.

Template-assisted synthesis of well-defined polynuclear clusters remains a challenge for [M4 ] square planar topologies. Herein, we present a tetraamine scaffold R L(NH2 )4 , where L is a rigidified resorcin[4]arene, to direct the formation of C4 -symmetric R L(NH)4 Cu4 clusters with Cu-Cu distances around 2.7 Å, suggesting metal-metal direct interactions are operative since the sum of copper's van der Waals radii is 2.8 Å. DFT calculations display HOMO to HOMO-3 residing all within a 0.1 eV gap. These four orbitals display significant electron density contribution from the Cu centers suggesting a delocalized electronic structure. The one-electron oxidized [Cu4 ]+ species was probed by variable temperature X-band continuous wave-electron paramagnetic resonance (CW-EPR), which displays a multiline spectrum at room temperature. This work presents a novel synthetic strategy for [M4 ] clusters and a new platform to investigate activation of small molecules.