Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Novel potassium binders to optimize RAASi therapy in heart failure: A systematic review and meta-analysis.

AIMS: Hyperkalemia often occurs among heart failure (HF) patients, particularly when treated with renin-angiotensin-aldosterone system inhibitors (RAASi). Even modest potassium levels variations raise the risk of mortality and prompt patients to discontinue disease-modifying treatment, as RAASi. Novel potassium binders (NPB), patiromer and sodium zirconium cyclosilicate, are effective in reducing potassium levels and are approved for the treatment of hyperkalemia in HF, but whether their use results in a real optimization of HF treatment remains to be seen. The aim of the present meta-analysis was to assess the efficacy of NPB on the optimization of RAASi therapy in HF patients. METHODS AND RESULTS: PubMed, Web of Science and Clinicaltrial.gov were searched without restrictions from inception to 06 August 2022 to identify valuable articles. The studies that met the inclusion criteria were analyzed. The prespecified primary outcome was the optimization of RAASi therapy in HF patients, defined as the proportion of patients on RAASi at the end of follow-up. Secondary outcomes were hyperkalemia events, reduction in potassium levels, and adverse drugs reactions. Six studies with a total of 1390 patients were included. NPB improved RAASi therapy optimization in HF by 14% (95% CI: 4-26%), decreased hyperkalemia events by 29% (95% CI: 55-92%), and reduced potassium levels by 0.31 mEq/L (95% CI: 0.18-0.44) compared to placebo, maintaining a good safety profile. CONCLUSION: NPB are effective in allowing RAASi therapy optimization in patients affected by HF, in reducing hyperkalemia events and potassium levels. SYSTEMATIC REVIEW REGISTRATION: CRD42022351811 URL: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=351811.

Structural characterization, biofunctionality, and environmental factors impacting rheological properties of exopolysaccharide produced by probiotic Lactococcus lactis C15.

Exopolysaccharides (EPSs) possess distinctive rheological and physicochemical properties and innovative functionality. This study aimed to investigate the physicochemical, bioactive, and rheological properties of an EPS secreted by Lactococcus lactis subsp. lactis C15. EPS-C15 was found to have an average molecular weight of 8.8 × 105 Da and was identified as a hetero-EPS composed of arabinose, xylose, mannose, and glucose with a molar ratio of 2.0:2.7:1.0:21.3, respectively. The particle size and zeta potential represented 311.2 nm and - 12.44 mV, respectively. FITR exhibited that EPS-C15 possessed a typical polysaccharide structure. NMR displayed that EPS-C15 structure is → 3)α-d-Glcvi (1 → 3)α-d-Xylv (1 → 6)α-d-Glciv(1 → 4)α-d-Glc(1 → 3)ß-d-Man(1 → 2)α-d-Glci(1 → . EPS-C15 scavenged DPPH and ABTS free radicals with 50.3% and 46.4% capacities, respectively. Results show that the antiproliferative activities of EPS-C15 revealed inhibitions of 49.7% and 88.1% against MCF-7 and Caco-2 cells, respectively. EPS-C15 has antibacterial properties that inhibited Staphylococcus aureus (29.45%), Salmonella typhimurium (29.83%), Listeria monocytogenes (30.33%), and E. coli O157:H7 (33.57%). The viscosity of EPS-C15 decreased as the shear rate increased. The rheological properties of the EPS-C15 were affected by changes in pH levels and the addition of salts. EPS-C15 is a promising biomaterial that has potential applications in various industries, such as food, pharmaceuticals, and healthcare.

Physicochemical, rheological, and bioactive properties of exopolysaccharide produced by a potential probiotic Enterococcus faecalis 84B.

Exopolysaccharides (EPS) have attracted a great interest due to their potential health-promoting properties and industrial applications. This study aimed to investigate the physicochemical, rheological, and biological properties of an EPS produced by a potential probiotic strain Enterococcus faecalis 84B. The results show that the extracted EPS, designated EPS-84B, had an average molecular weight of 604.8 kDa, particles size diameter of 322.0 nm, and mainly composed of arabinose and glucose with a molar ratio of 1:2. Furthermore, EPS-84B exhibited a shear-thinning behavior and had a high melting point. The rheological properties of EPS-84B were strongly influenced by the type of salt than by the pH value. EPS-84B displayed ideal viscoelastic properties, with both viscous and storage moduli increasing with frequency. The antioxidant activity of EPS-84B at a concentration of 5 mg/mL was 81.1 % against DPPH and 35.2 % against ABTS. At 5 mg/mL, the antitumor activity of EPS-84B against Caco-2 and MCF-7 cell lines was 74.6 and 38.6 %, respectively. In addition, the antidiabetic activity of EPS-84B towards α-amylase and α-glucosidase was 89.6 and 90.0 %, respectively at 100 µg/mL. The inhibition of foodborne pathogens by EPS-84B was up to 32.6 %. Overall, EPS-84B has promising properties that could be utilized in food and pharmaceutical industries.

Satisfaction with Telemedicine for Cancer Pain Management: A Model of Care and Cross-Sectional Patient Satisfaction Study.

Background: Since cancer pain requires complex modalities of care, the proper strategy for addressing its telemedicine-based management should be better defined. This study aimed to trace a pathway for a progressive implementation of the telemedicine process for the treatment of pain in the setting of cancer patients. Methods: The features of the model were investigated to dissect the dropout from the telemedicine pathway. A cross-sectional patient satisfaction study was conducted. The degree of satisfaction was evaluated through a developed 22-item questionnaire (Likert scale 0−7). Results: A total of 375 video consultations for 164 patients (mean age 62.9 years) were performed through remote consultations for cancer pain management between March 2021 and February 2022. After the exclusion of 72 patients, 92 (56.1%) were included in the analysis. The dropout ratio was 8.7%. The number of visits and pharmacological therapies for neuropathic pain correlated with the risk for readmission (p < 0.05). Overall, the satisfaction was very high (mean > 5.5 for all items). Conclusion: Feedback from patients reflected high satisfaction rates with the care provided. A methodological approach based on the degree of satisfaction combined with the analysis of the pathways can help to implement the quality of a service provided through telemedicine. While not without limitations, our hybrid protocol can be useful for addressing cancer pain through a patient-centered approach.

Approaching Protein Aggregation and Structural Dynamics by Equilibrium and Nonequilibrium Paramagnetic Perturbation.

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.

The corona of protein-gold nanoparticle systems: the role of ionic strength.

The nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with ß2-microglobulin (ß2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a ß2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between ß2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard" interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between ß2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength.

Clinical Significance and Prognostic Value of Hemostasis Parameters in 337 Patients with Acute Infective Endocarditis.

(1) Background: The aim of this study was to assess the clinical significance and prognostic role of the main hemostasis parameters in infective endocarditis (IE): prothrombin time as international normalized ratio (PT-INR), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers, platelet count, homocysteine. (2) Methods: We studied 337 patients with IE. Clinical, hemato-chemical and echocardiography parameters were analyzed. Coagulation parameters were measured on admission. (3) Results: D-dimers levels (p = 0.012) and a prolonged PT-INR (p = 0.013) were associated with higher in-hospital mortality, while prolonged aPTT (p = 0.021) was associated with increased 1-year mortality. Staphylococcus aureus (S. aureus) infection (p = 0.003), prosthetic valve endocarditis (PVE) (p = 0.001), surgical indication (p = 0.002) and higher D-dimer levels (p = 0.005) were independent predictors of in-hospital mortality. PVE (p = 0.001), a higher Charlson Comorbidity Index (p = 0.049), surgical indication (p = 0.001) and prolonged aPTT (p = 0.012) were independent predictors of 1-year mortality. Higher levels of D-dimers (p < 0.001) and a shorter aPTT (p < 0.001) were associated with embolic complications of IE. S. aureus etiology was bound to higher D-dimers levels (p < 0.001) and a shorter aPTT (p = 0.006). (4) Conclusions: Elevated D-dimers are associated with a higher risk for in-hospital mortality in IE patients. High D-dimers and a short aPTT are associated with a higher risk for embolic events in IE. A longer aPTT is associated with 1-year mortality.

An Overview on the Potential Roles of EGCG in the Treatment of COVID-19 Infection.

Coronavirus disease-19 (COVID-19) pandemic is currently ongoing worldwide and causes a lot of deaths in many countries. Although different vaccines for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have been developed and are now available, there are no effective antiviral drugs to treat the disease, except for Remdesivir authorized by the US FDA to counteract the emergency. Thus, it can be useful to find alternative therapies based on the employment of natural compounds, with antiviral features, to circumvent SARS-CoV-2 infection. Pre-clinical studies highlighted the antiviral activities of epigallocatechin-3-gallate (EGCG), a catechin primarily found in green tea, against various viruses, including SARS-CoV-2. In this review, we summarize this experimental evidence and highlight the potential use of EGCG as an alternative therapeutic choice for the treatment of SARS-CoV-2 infection.

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19.

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

COVID-19 Vaccine mRNABNT162b2 Elicits Human Antibody Response in Milk of Breastfeeding Women.

OBJECTIVE: The objective of this research is to demonstrate the release of SARS-CoV-2 Spike (S) antibodies in human milk samples obtained by patients who have been vaccinated with mRNABNT162b2 vaccine. METHODS: Milk and serum samples were collected in 10 volunteers 20 days after the first dose and 7 seven days after the second dose of the mRNABNT162b2 vaccine. Anti-SARS-CoV-2 S antibodies were measured by the Elecsys® Anti-SARS-CoV-2 S ECLIA assay (Roche Diagnostics AG, Rotkreuz, Switzerland), a quantitative electrochemiluminescence immunometric method. RESULTS: At first sample, anti-SARS-CoV-2 S antibodies were detected in all serum samples (103.9 ± 54.9 U/mL) and only in two (40%) milk samples with a low concentration (1.2 ± 0.3 U/mL). At the second sample, collected 7 days after the second dose, anti-SARS-CoV-2 S antibodies were detected in all serum samples (3875.7 ± 3504.6 UI/mL) and in all milk samples (41.5 ± 47.5 UI/mL). No correlation was found between the level of serum and milk antibodies; the milk antibodies/serum antibodies ratio was on average 2% (range: 0.2-8.4%). CONCLUSION: We demonstrated a release of anti-SARS-CoV-2 S antibodies in the breast milk of women vaccinated with mRNABNT162b2. Vaccinating breastfeeding women could be a strategy to protect their infants from COVID-19 infection.

Multidimensional Statistical Technique for Interpreting the Spontaneous Breakthrough Cancer Pain Phenomenon. A Secondary Analysis from the IOPS-MS Study.

Breakthrough cancer pain (BTcP) is a temporary exacerbation of pain that "breaks through" a phase of adequate pain control by an opioid-based therapy. The non-predictable BTcP (NP-BTcP) is a subtype of BTcP that occurs in the absence of any specific activity. Since NP-BTcP has an important clinical impact, this analysis is aimed at characterizing the NP-BTcP phenomenon through a multidimensional statistical technique. This is a secondary analysis based on the Italian Oncologic Pain multiSetting-Multicentric Survey (IOPS-MS). A correlation analysis was performed to characterize the NP-BTcP profile about its intensity, number of episodes per day, and type. The multiple correspondence analysis (MCA) determined the identification of four groups (phenotypes). A univariate analysis was performed to assess differences between the four phenotypes and selected covariates. The four phenotypes represent the hierarchical classification according to the status of NP-BTcP: from the best (phenotype 1) to the worst (phenotype 4). The univariate analysis found a significant association between the onset time >10 min in the phenotype 1 (37.3%)' vs. the onset > 10 min in phenotype 4 (25.8%) (p < 0.001). Phenotype 1 was characterized by the gastrointestinal type of cancer (26.4%) with respect to phenotype 4, where the most frequent cancer affected the lung (28.8%) (p < 0.001). Phenotype 4 was mainly managed with rapid-onset opioids, while in phenotype 1, many patients were treated with oral, subcutaneous, or intravenous morphine (56.4% and 44.4%, respectively; p = 0.008). The ability to characterize NP-BTcP can offer enormous benefits for the management of this serious aspect of cancer pain. Although requiring validation, this strategy can provide many indications for identifying the diagnostic and therapeutic gaps in NP-BTcP management.

Effects of the Hypnotic Alkylphenol Derivative Propofol on Breast Cancer Progression. A Focus on Preclinical and Clinical Studies.

Propofol is a hypnotic alkylphenol derivative with many biological activities. It is predominantly used in anesthesia and is the most used parenteral anesthetic agent in the United States. Accumulating preclinical studies have shown that this compound may inhibit cancer recurrence and metastasis. Nevertheless, other investigations provided evidence that this compound may promote breast cancer cell progression by modulating different molecular pathways. Clinical data on this topic are scarce and derive from retrospective analyses. For this reason, we reviewed and evaluated the available data to reveal insight into this controversial issue. More preclinical and clinical investigations are necessary to determine the potential role of propofol in the proliferation of breast cancer cells.

Structure of Nanobody Nb23.

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

The Role of Anti-Nerve Growth Factor Monoclonal Antibodies in the Control of Chronic Cancer and Non-Cancer Pain.

Nerve growth factor (NGF) belongs to the neurotrophin family and plays a fundamental role in the endurance of sensory and sympathetic neurons during embryogenesis. NGF, by interacting with tropomyosin receptor kinase A receptor (TrkA), modulates the pain pathway through the enhancement of the neurotrophic and nociceptor functions. Moreover, it has been demonstrated that NGF is upregulated in patients with chronic pain syndromes, which are difficult to treat. Thus, new non-pharmacological approaches, based on the use of different species-specific monoclonal antibodies (mAbs) targeting the NGF pathway, have been tested for the treatment of chronic pain in preclinical and clinical studies. With regard to preclinical investigations, anti-NGF mAbs have been used for the management of osteoarthritis (OA) and chronic low back pain animal models, with encouraging results. Moreover, anti-NGF mAb therapy is effective in animal models of neuropathic cancer pain. As regards patients with OA, although phase II and phase III clinical trials with tanezumab led to pain reduction, the safety was not observed in all these patients. Here, we review the preclinical and clinical studies on anti-NGF mAb therapy in chronic syndromes, dissect the role of NGF in pain transduction, and highlight the use of anti-NGF mAbs in humans.

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function.

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer's disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53's transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent anticancer agent.

Entropy of Two-Molecule Correlated Translational-Rotational Motions Using the kth Nearest Neighbor Method.

The entropy associated with rotations, translations, and their coupled motions provides an important contribution to the free energy of many physicochemical processes such as association and solvation. The kth nearest neighbor method, which offers a convenient way to estimate the entropy in high-dimensional spaces, has been previously applied for translational-rotational entropy estimation. Here, we explore the possibility of extending the kth nearest neighbor method to the computation of the entropy of correlated translation-rotations of two molecules, i.e., in the product space of two translation-rotations, both referred to the same independent reference system, which is relevant for all cases in which the correlated translational-rotational motion of more than two molecules is involved. Numerical tests show that, albeit the relatively high dimensionality (12) of the space, the kth nearest neighbor approach provides an accurate estimate for the entropy of two correlated translational-rotational motions, even when computed from a limited number of samples.

Advantages and issues of concern regarding approaches to peripheral nerve block for total hip arthroplasty.

In older patients with comorbidities, hip fractures are both an important and debilitating condition. Since multimodal and multidisciplinary perioperative strategies can hasten functional recovery after surgery improving clinical outcomes, the choice of the most effective and safest pathway represents a great challenge. A key point of concern is the anesthetic approach and above all the choice of the locoregional anesthesia combined with general or neuraxial anesthesia.

Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy.

BACKGROUND: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, ß2-microglobulin (ß2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit ß2m fibril formation in vitro. METHODS: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on ß2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. RESULTS: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of ß2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. CONCLUSIONS: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.

Correction: Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra.

Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247-6259, DOI: .