Evaluation of the Therapeutical Effect of Matricaria Chamomilla Extract vs. Galantamine on Animal Model Memory and Behavior Using 18F-FDG PET/MRI.

The memory-enhancing activity of Matricaria chamomilla hydroalcoholic extract (MCE) is already being investigated by behavioral and biochemical assays in scopolamine-induced amnesia rat models, while the effects of scopolamine (Sco) on cerebral glucose metabolism are examined as well. Nevertheless, the study of the metabolic profile determined by an enriched MCE has not been performed before. The present experiments compared metabolic quantification in characteristic cerebral regions and behavioral characteristics for normal, only diseased, diseased, and MCE- vs. Galantamine (Gal)-treated Wistar rats. A memory deficit was induced by four weeks of daily intraperitoneal Sco injection. Starting on the eighth day, the treatment was intraperitoneally administered 30 min after Sco injection for a period of three weeks. The memory assessment comprised three maze tests. Glucose metabolism was quantified after the 18F-FDG PET examination. The right amygdala, piriform, and entorhinal cortex showed the highest differential radiopharmaceutical uptake of the 50 regions analyzed. Rats treated with MCE show metabolic similarity with normal rats, while the Gal-treated group shows features closer to the diseased group. Behavioral assessments evidenced a less anxious status and a better locomotor activity manifested by the MCE-treated group compared to the Gal-treated group. These findings prove evident metabolic ameliorative qualities of MCE over Gal classic treatment, suggesting that the extract could be a potent neuropharmacological agent against amnesia.

Dynamics of integrin α5ß1, fibronectin, and their complex reveal sites of interaction and conformational change.

Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.

Force interacts with macromolecular structure in activation of TGF-ß.

Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-ß1 precursor (pro-TGF-ß1), integrins bind to the prodomain, apply force, and release the TGF-ß growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVß6 binds pro-TGF-ß1 in an orientation biologically relevant for force-dependent release of TGF-ß from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-ß orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin ß-subunit. Simulations of force-dependent activation of TGF-ß demonstrate evolutionary specializations for force application through the TGF-ß prodomain and through the ß- and not α-subunit of the integrin.

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation.

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF-ß family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid-cleaved GDF8 pro-complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro-complexes reveals a V-shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring-like, cross-armed conformation of latent TGF-ß1. Surprisingly, Tolloid-cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-ß1 to the α1-helix, latency lasso, and ß1-strand in the prodomain and to the ß6'- and ß7'-strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Risk Factors Associated with Retinopathy of Prematurity in Very and Extremely Preterm Infants.

Background and Objectives: Retinopathy of prematurity (ROP) is the leading cause of blindness in preterm infants. We studied the relationship between different perinatal characteristics, i.e., sex; gestational age (GA); birth weight (BW); C-reactive protein (CRP) and lactate dehydrogenase (LDH) concentrations; ventilation, continuous positive airway pressure (CPAP), and surfactant administration; and the incidence of Stage 1-3 ROP. Materials and Methods: This study included 247 preterm infants with gestational age (GA) < 32 weeks that were successfully screened for ROP. Univariate and multivariate binary analyses were performed to find the most significant risk factors for ROP (Stage 1-3), while multivariate multinomial analysis was used to find the most significant risk factors for specific ROP stages, i.e., Stage 1, 2, and 3. Results: The incidence of ROP (Stage 1-3) was 66.40% (164 infants), while that of Stage 1, 2, and 3 ROP was 15.38% (38 infants), 27.53% (68 infants), and 23.48% (58 infants), respectively. Following univariate analysis, multiple perinatal characteristics, i.e., GA; BW; and ventilation, CPAP, and surfactant administration, were found to be statistically significant risk factors for ROP (p < 0.001). However, in a multivariate model using the same characteristics, only BW and ventilation were significant ROP predictors (p < 0.001 and p < 0.05, respectively). Multivariate multinomial analysis revealed that BW was only significantly correlated with Stage 2 and 3 ROP (p < 0.05 and p < 0.001, respectively), while ventilation was only significantly correlated with Stage 2 ROP (p < 0.05). Conclusions: The results indicate that GA; BW; and the use of ventilation, CPAP, and surfactant were all significant risk factors for ROP (Stage 1-3), but only BW and ventilation were significantly correlated with ROP and specific stages of the disease, namely Stage 2 and 3 ROP and Stage 2 ROP, respectively, in multivariate models.

Molecular characterization of the ß-amyloid(4-10) epitope of plaque specific Aß antibodies by affinity-mass spectrometry using alanine site mutation.

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the ß-amyloid(1-42) (Aß) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aß-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aß antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aß(4-10) peptide has been identified as an epitope for the polyclonal anti-Aß(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aß-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aß(4-10) peptides upon affinity binding to a monoclonal anti-Aß(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aß(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aß(1-17) to the monoclonal Aß antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aß-specific antibodies with improved therapeutic efficacy.

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics.

IL-23 is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin and have a similar protein scaffold to antibodies. Adnectin 2 was found to bind to IL-23 and compete with the IL-23/IL-23R interaction, posing a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between IL-23 and Adnectin 2 and to determine the correlation between the two orthogonal methods. This review summarizes the current structural knowledge about IL-23 and focuses on the applicability of hydrogen/deuterium exchange mass spectrometry to investigate the higher order structure of proteins, which plays an important role in the discovery of new and improved biotherapeutics.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

Structure and dynamic regulation of Abl kinases.

The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity.

Enhanced SH3/linker interaction overcomes Abl kinase activation by gatekeeper and myristic acid binding pocket mutations and increases sensitivity to small molecule inhibitors.

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Electron transfer control in soluble methane monooxygenase.

The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.

Tiny Lungs, Big Differences: Navigating the Varied COVID-19 Landscape in Neonates vs. Infants via Biomarkers and Lung Ultrasound.

Due to their susceptibilities, neonates and infants face unique SARS-CoV-2 challenges. This retrospective study will compare the illness course, symptoms, biomarkers, and lung damage in neonates and infants with SARS-CoV-2 infection from February 2020 to October 2023. This study was conducted at two hospitals in Timisoara, Romania, using real-time multiplex PCR to diagnose and lung ultrasonography (LUS) to assess lung involvement. Neonates had a more severe clinical presentation, an increased immune response, and greater lung involvement. Neonates had more PCR-positive tests (p = 0.0089) and longer hospital stays (p = 0.0002). In neonates, LDH, CRP, and ferritin levels were higher, indicating a stronger inflammatory response. Reduced oxygen saturation in neonates indicates respiratory dysfunction. The symptoms were varied. Infants had fever, cough, and rhinorrhea, while neonates had psychomotor agitation, acute dehydration syndrome, and candidiasis. This study emphasizes individualized care and close monitoring for neonatal SARS-CoV-2 infections. Newborn lung ultrasonography showed different variances and severity levels, emphasizing the need for targeted surveillance and therapy. Newborns have high lung ultrasound scores (LUSS), indicating significant lung involvement. Both groups had initial lung involvement, but understanding these modest differences is crucial to improving care for these vulnerable populations.

Small Scale, High Precision: Robotic Surgery in Neonatal and Pediatric Patients-A Narrative Review.

This narrative review explores the evolution and implications of robotic-assisted surgery in pediatric and neonatal cases, focusing on its advantages, drawbacks, and the specific diseases amenable to this innovative technology. Following PRISMA guidelines, 56 relevant articles from the past five years were selected, emphasizing advancements in precision, reduced trauma, and expedited recovery times for pediatric patients. Despite challenges like cost and training, ongoing research shapes pediatric robotic-assisted surgery, promising improved outcomes. The technology's benefits include enhanced precision, minimized scarring, and faster recovery, addressing the challenges in delicate pediatric procedures. Challenges encompass cost, training, and instrument design, but ongoing refinements aim to overcome these. This review underscores psychological and musculoskeletal considerations for patients and surgeons. While acknowledging limitations and preferred pathologies, this review outlines the transformative potential of robotic-assisted surgery in reshaping pediatric surgical care. This comprehensive assessment concludes that, despite challenges, ongoing advancements promise a future of enhanced precision and tailored care in pediatric surgery.

Evaluating the Role of Breast Ultrasound in Early Detection of Breast Cancer in Low- and Middle-Income Countries: A Comprehensive Narrative Review.

Breast cancer, affecting both genders, but mostly females, exhibits shifting demographic patterns, with an increasing incidence in younger age groups. Early identification through mammography, clinical examinations, and breast self-exams enhances treatment efficacy, but challenges persist in low- and medium-income countries due to limited imaging resources. This review assesses the feasibility of employing breast ultrasound as the primary breast cancer screening method, particularly in resource-constrained regions. Following the PRISMA guidelines, this study examines 52 publications from the last five years. Breast ultrasound, distinct from mammography, offers advantages like radiation-free imaging, suitability for repeated screenings, and preference for younger populations. Real-time imaging and dense breast tissue evaluation enhance sensitivity, accessibility, and cost-effectiveness. However, limitations include reduced specificity, operator dependence, and challenges in detecting microcalcifications. Automatic breast ultrasound (ABUS) addresses some issues but faces constraints like potential inaccuracies and limited microcalcification detection. The analysis underscores the need for a comprehensive approach to breast cancer screening, emphasizing international collaboration and addressing limitations, especially in resource-constrained settings. Despite advancements, notably with ABUS, the primary goal is to contribute insights for optimizing breast cancer screening globally, improving outcomes, and mitigating the impact of this debilitating disease.

Current Trends in Diagnosis and Treatment Approach of Diabetic Retinopathy during Pregnancy: A Narrative Review.

Diabetes mellitus during pregnancy and gestational diabetes are major concerns worldwide. These conditions may lead to the development of severe diabetic retinopathy during pregnancy or worsen pre-existing cases. Gestational diabetes also increases the risk of diabetes for both the mother and the fetus in the future. Understanding the prevalence, evaluating risk factors contributing to pathogenesis, and identifying treatment challenges related to diabetic retinopathy in expectant mothers are all of utmost importance. Pregnancy-related physiological changes, including those in metabolism, blood flow, immunity, and hormones, can contribute to the development or worsening of diabetic retinopathy. If left untreated, this condition may eventually result in irreversible vision loss. Treatment options such as laser therapy, intravitreal anti-vascular endothelial growth factor drugs, and intravitreal steroids pose challenges in managing these patients without endangering the developing baby and mother. This narrative review describes the management of diabetic retinopathy during pregnancy, highlights its risk factors, pathophysiology, and diagnostic methods, and offers recommendations based on findings from previous literature.

The Great Saphenous Vein Proximal Part: Branches, Anatomical Variations, and Their Implications for Clinical Practice and Venous Reflux Surgery.

The anatomical variations in the lower limb veins play a critical role in venous reflux surgeries. This study presents an analysis of the great saphenous vein (GSV) proximal part's anatomical peculiarities, with 257 patients included, who were operated for venous reflux. This study highlighted a progressive increase in the GSV diameter in conjunction with the complexity of the anatomical variations, ranging from no tributaries to more than five tributaries, an anterior accessory GSV, or venous aneurysms. Statistical analysis evidenced this expansion to be significantly correlated with the variations. Additionally, the progression of the chronic venous disease (CVD) stages was notably more prevalent in the complex anatomical variations, suggesting a nuanced interplay between the GSV anatomy and CVD severity. Conclusively, our research articulates the paramount importance of recognizing GSV anatomical variations in optimizing surgical outcomes for CVD patients. These insights not only pave the way for enhanced diagnostic accuracy but also support the strategic framework within which surgical and interventional treatments are devised, advocating for personalized approaches to venous reflux surgery.

Aldose Reductase as a Key Target in the Prevention and Treatment of Diabetic Retinopathy: A Comprehensive Review.

The escalating global prevalence of diabetes mellitus (DM) over the past two decades has led to a persistent high incidence of diabetic retinopathy (DR), necessitating screening for early symptoms and proper treatment. Effective management of DR aims to decrease vision impairment by controlling modifiable risk factors including hypertension, obesity, and dyslipidemia. Moreover, systemic medications and plant-based therapy show promise in advancing DR treatment. One of the key mechanisms related to DR pathogenesis is the polyol pathway, through which aldose reductase (AR) catalyzes the conversion of glucose to sorbitol within various tissues, including the retina, lens, ciliary body and iris. Elevated glucose levels activate AR, leading to osmotic stress, advanced glycation end-product formation, and oxidative damage. This further implies chronic inflammation, vascular permeability, and angiogenesis. Our comprehensive narrative review describes the therapeutic potential of aldose reductase inhibitors in treating DR, where both synthetic and natural inhibitors have been studied in recent decades. Our synthesis aims to guide future research and clinical interventions in DR management.

Stratifying Disease Severity in Pediatric COVID-19: A Correlative Study of Serum Biomarkers and Lung Ultrasound-A Retrospective Observational Dual-Center Study.

The COVID-19 pandemic, caused by SARS-CoV-2, has manifested distinct impacts on infants and children. This study delves into the intricate connection between lung ultrasound (LUS) findings and serum biomarkers in neonates and infants with COVID-19. Exploring factors contributing to the mild symptoms in this demographic, including immune responses and pre-existing immunity, the study spans 3 years and 9 months, involving 42 patients. Respiratory and gastrointestinal symptoms predominate, and LUS emerges as a vital, non-irradiating tool for evaluating pulmonary abnormalities. Serum biomarkers like CRP, procalcitonin, and cytokines provide key insights into the pathophysiology. Correlations reveal nuanced links between LUS score and clinical parameters, unveiling associations with hospitalization duration (rho = 0.49), oxygen saturation (rho = -0.88), and inflammatory markers, like ferritin (rho = 0.62), LDH (rho = 0.73), and D-dimer (rho = 0.73) with significance level (p < 0.05). The absence of large consolidations in LUS suggests unique pulmonary characteristics. The novelty of these findings lies in the comprehensive integration of LUS with serum biomarkers to assess and monitor the severity of lung involvement in neonates and infants affected by SARS-CoV-2. This approach offers valuable insights into disease severity, biomarker levels, the duration of hospitalization, and oxygen saturation, providing a multifaceted understanding of COVID-19's impact on this vulnerable population.