Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

p14ARF forms meso-scale assemblies upon phase separation with NPM1.

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14ARF). Oncogenic stress induces p14ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14ARF by NPM1 are unclear because the structural features of the p14ARF-NPM1 complex remain elusive. Here we show that NPM1 sequesters p14ARF within phase-separated condensates, facilitating the assembly of p14ARF into a gel-like meso-scale network. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and ß-strands within a partially folded N-terminal domain of p14ARF. Those hydrophobic interactions promote phase separation with NPM1, enhance nucleolar partitioning of p14ARF, restrict p14ARF and NPM1 diffusion within condensates and in nucleoli, and reduce cell viability. Our structural model provides novel insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

NMR Signatures and Electronic Structure of Ti Sites in Titanosilicalite-1 from Solid-State 47/49Ti NMR Spectroscopy.

Although titanosilicalite-1 (TS-1) is among the most successful oxidation catalysts used in industry, its active site structure is still debated. Recent efforts have mostly focused on understanding the role of defect sites and extraframework Ti. Here, we report the 47/49Ti signature of TS-1 and molecular analogues [Ti(OTBOS)4] and [Ti(OTBOS)3(OiPr)] using novel MAS CryoProbe to enhance the sensitivity. While the dehydrated TS-1 displays chemical shifts similar to those of molecular homologues, confirming the tetrahedral environment of Ti consistent with X-ray absorption spectroscopy, it is associated with a distribution of larger quadrupolar coupling constants, indicating an asymmetric environment. Detailed computational studies on cluster models highlights the high sensitivity of the NMR signatures (chemical shift and quadrupolar coupling constant) to small local structural changes. These calculations show that, while it will be difficult to distinguish mono- vs dinuclear sites, the sensitivity of the 47/49Ti NMR signature should enable distinguishing the Ti location among specific T site positions.

Lumbar Puncture and Meningitis in Infants with Proven Early- or Late-Onset Sepsis: An Italian Prospective Multicenter Observational Study.

Background: To evaluate the rates of lumbar puncture (LP) in infants with culture-proven sepsis. Study design: We prospectively enrolled 400 infants with early- or late-onset sepsis due to Group B streptococcus (GBS) or Eschericha coli, diagnosed within 90 days of life. Rates of LP and potential variables associated with LP performance were evaluated. Moreover, cerebrospinal fluid (CSF) characteristics and results of the molecular analysis were investigated. Results: LP was performed in 228/400 (57.0%) infants; 123/228 LPs (53.9%) were performed after antibiotic initiation, hampering the ability to identify the pathogen in the CSF culture. However, polymerase chain reaction increased the probability of positive results of CSF analysis compared to microbiological culture (28/79, 35.4% vs. 14/79, 17.7%, p = 0.001). Severe clinical presentation and GBS infection were associated with higher LP rates. The rate of meningitis was 28.5% (65/228). Conclusions: Rates of LP are low in culture-proven neonatal sepsis and antibiotics are frequently given before LP is carried out. Thus meningitis may be underestimated, and the chances of giving an effective therapy to the newborn are reduced. LP should be performed before the start of antibiotics when there is a clinical suspicion of infection.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Timing of Symptoms of Early-Onset Sepsis after Intrapartum Antibiotic Prophylaxis: Can It Inform the Neonatal Management?

The effectiveness of "inadequate" intrapartum antibiotic prophylaxis (IAP administered < 4 h prior to delivery) in preventing early-onset sepsis (EOS) is debated. Italian prospective surveillance cohort data (2003-2022) were used to study the type and duration of IAP according to the timing of symptoms onset of group B streptococcus (GBS) and E. coli culture-confirmed EOS cases. IAP was defined "active" when the pathogen yielded in cultures was susceptible. We identified 263 EOS cases (GBS = 191; E. coli = 72). Among GBS EOS, 25% had received IAP (always active when beta-lactams were administered). Most IAP-exposed neonates with GBS were symptomatic at birth (67%) or remained asymptomatic (25%), regardless of IAP duration. Among E. coli EOS, 60% were IAP-exposed. However, IAP was active in only 8% of cases, and these newborns remained asymptomatic or presented with symptoms prior to 6 h of life. In contrast, most newborns exposed to an "inactive" IAP (52%) developed symptoms from 1 to >48 h of life. The key element to define IAP "adequate" seems the pathogen's antimicrobial susceptibility rather than its duration. Newborns exposed to an active antimicrobial (as frequently occurs with GBS infections), who remain asymptomatic in the first 6 h of life, are likely uninfected. Because E. coli isolates are often unsusceptible to beta-lactam antibiotics, IAP-exposed neonates frequently develop symptoms of EOS after birth, up to 48 h of life and beyond.

Efficient analysis of pharmaceutical drug substances and products using a solid-state NMR CryoProbe.

Solid-state nuclear magnetic resonance (ssNMR) is a high-resolution and versatile spectroscopic tool for characterizing pharmaceutical solids. However, the inherent low sensitivity of NMR remains a significant challenge in the analysis of natural abundance drug substances and products. Here, we report, for the first time, the application of a CPMAS CryoProbe™ to improve the sensitivity of 13C and 15N detection by approximately 5 to 6 times for solid-state analysis of a commercial pharmaceutical drug posaconazole (POSA). The sensitivity enhancement enables two-dimensional (2D) 13C-13C and 1H-15N correlation experiments, which are otherwise time-prohibitive using regular MAS probes, for resonance assignment and structural elucidation. These polarization transfer and correlation experiments reveal drug-drug and drug-polymer interactions in amorphous POSA and its amorphous solid dispersion formulation. Our results demonstrated that the CPMAS CryoProbe™ can be widely applied for routine pharmaceutical analysis and advanced structural investigations with significantly enhanced efficiency and throughput.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Understanding Factors in Group B Streptococcus Late-Onset Disease.

Group B streptococcus (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis in infants. Rates of GBS early onset disease have declined following the widcespread use of intrapartum antibiotic prophylaxis; hence, late-onset infections (LOGBS) are currently a common presentation of neonatal GBS dicsease. The pathogenesis, mode of transmission, and risk factors associated with LOGBS are unclear, which interfere with effective prevention efforts. GBS may be transmitted from the mother to the infant at the time of delivery or during the postpartum period via contaminated breast milk, or as nosocomial or community-acquired infection. Maternal GBS colonization, prematurity, young maternal age, HIV exposure, and ethnicity (Black) are identified as risk factors for LOGBS disease; however, further studies are necessary to confirm additional risk factors, if any, for the implementation of effective prevention strategies. This narrative review discusses current and previous studies that have reported LOGBS. Few well-designed studies have described this condition; therefore, reliable assessment of maternal GBS colonization, breastfeeding, and twin delivery as risk factors for LOGBS remains limited.

A global point prevalence survey of antimicrobial use in neonatal intensive care units: The no-more-antibiotics and resistance (NO-MAS-R) study.

BACKGROUND: Global assessment of antimicrobial agents prescribed to infants in the neonatal intensive care unit (NICU) may inform antimicrobial stewardship efforts. METHODS: We conducted a one-day global point prevalence study of all antimicrobials provided to NICU infants. Demographic, clinical, and microbiologic data were obtained including NICU level, census, birth weight, gestational/chronologic age, diagnoses, antimicrobial therapy (reason for use; length of therapy), antimicrobial stewardship program (ASP), and 30-day in-hospital mortality. FINDINGS: On July 1, 2019, 26% of infants (580/2,265; range, 0-100%; median gestational age, 33 weeks; median birth weight, 1800 g) in 84 NICUs (51, high-income; 33, low-to-middle income) from 29 countries (14, high-income; 15, low-to-middle income) in five continents received ≥1 antimicrobial agent (92%, antibacterial; 19%, antifungal; 4%, antiviral). The most common reasons for antibiotic therapy were "rule-out" sepsis (32%) and "culture-negative" sepsis (16%) with ampicillin (40%), gentamicin (35%), amikacin (19%), vancomycin (15%), and meropenem (9%) used most frequently. For definitive treatment of presumed/confirmed infection, vancomycin (26%), amikacin (20%), and meropenem (16%) were the most prescribed agents. Length of therapy for culture-positive and "culture-negative" infections was 12 days (median; IQR, 8-14) and 7 days (median; IQR, 5-10), respectively. Mortality was 6% (42%, infection-related). An NICU ASP was associated with lower rate of antibiotic utilization (p = 0·02). INTERPRETATION: Global NICU antibiotic use was frequent and prolonged regardless of culture results. NICU-specific ASPs were associated with lower antibiotic utilization rates, suggesting the need for their implementation worldwide. FUNDING: Merck & Co.; The Ohio State University College of Medicine Barnes Medical Student Research Scholarship.

Gut bacteria are essential for normal cuticle development in herbivorous turtle ants.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15N isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

Sensitivity boosts by the CPMAS CryoProbe for challenging biological assemblies.

Despite breakthroughs in MAS NMR hardware and experimental methodologies, sensitivity remains a major challenge for large and complex biological systems. Here, we report that 3-4 fold higher sensitivities can be obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments that are otherwise time-prohibitive, such as 2D 15N-15N proton-driven spin diffusion and 15N-13C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins - demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of applications, especially for biological systems suffering from low intrinsic sensitivity and at physiological temperatures.

Control over the fibrillization yield by varying the oligomeric nucleation propensities of self-assembling peptides.

Self-assembling peptides are an exemplary class of supramolecular biomaterials of broad biomedical utility. Mechanistic studies on the peptide self-assembly demonstrated the importance of the oligomeric intermediates towards the properties of the supramolecular biomaterials being formed. In this study, we demonstrate how the overall yield of the supramolecular assemblies are moderated through subtle molecular changes in the peptide monomers. This strategy is exemplified with a set of surfactant-like peptides (SLPs) with different ß-sheet propensities and charged residues flanking the aggregation domains. By integrating different techniques, we show that these molecular changes can alter both the nucleation propensity of the oligomeric intermediates and the thermodynamic stability of the fibril structures. We demonstrate that the amount of assembled nanofibers are critically defined by the oligomeric nucleation propensities. Our findings offer guidance on designing self-assembling peptides for different biomedical applications, as well as insights into the role of protein gatekeeper sequences in preventing amyloidosis.

NPM1 exhibits structural and dynamic heterogeneity upon phase separation with the p14ARF tumor suppressor.

Nucleophosmin (NPM1) is an abundant nucleolar protein that aids in the maturation of pre-ribosomal particles and participates in oncogenic stress responses through its interaction with the Alternative Reading Frame tumor suppressor (p14ARF). NPM1 mediates multiple mechanisms of phase separation which contribute to the liquid-like properties of nucleoli. However, the effects of phase separation on the structure and dynamics of NPM1 are poorly understood. Here we show that NPM1 undergoes phase separation with p14ARF in vitro, forming condensates that immobilize both proteins. We probed the structure and dynamics of NPM1 within the condensed phase using solid-state NMR spectroscopy. Our results demonstrate that within the condensed phase, the NPM1 oligomerization domain forms an immobile scaffold, while the central intrinsically disordered region and the C-terminal nucleic acid binding domain exhibit relative mobility.

Italian Recommendations for Placental Transfusion Strategies.

At delivery, if the cord is not clamped, blood continues to pass from the placenta to the newborn during the first minutes of life, allowing the transfer of 25-35 ml/kg of placental blood to the newborn, depending on gestational age, the timing of cord clamping, the position of the infant at birth, the onset of respiration, and administration of uterotonics to the mother. However, deriving benefits from delayed cord clamping (DCC) are not merely related to placental-to-fetal blood transfusion; establishing spontaneous ventilation before cutting the cord improves venous return to the right heart and pulmonary blood flow, protecting the newborn from the transient low cardiac output, and systemic arterial pressure fluctuations. Recent meta-analyses showed that delayed cord clamping reduces mortality and red blood cell transfusions in preterm newborns and increases iron stores in term newborns. Various authors suggested umbilical cord milking (UCM) as a safe alternative when delayed cord clamping is not feasible. Many scientific societies recommend waiting 30-60 s before clamping the cord for both term and preterm newborns not requiring resuscitation. To improve the uptake of placental transfusion strategies, in 2016 an Italian Task Force for the Management of Umbilical Cord Clamping drafted national recommendations for the management of cord clamping in term and preterm deliveries. The task force performed a detailed review of the literature using the GRADE methodological approach. The document analyzed all clinical scenarios that operators could deal with in the delivery room, including cord blood gas analysis during delayed cord clamping and time to cord clamping in the case of umbilical cord blood banking. The panel intended to promote a more physiological and individualized approach to cord clamping, specifically for the most preterm newborn. A feasible option to implement delayed cord clamping in very preterm deliveries is to move the neonatologist to the mother's bedside to assess the newborn's clinical condition at birth. This option could safely guarantee the first steps of stabilization before clamping the cord and allow DCC in the first 30 s of life, without delaying resuscitation. Contra-indications to placental transfusion strategies are clinical situations that may endanger mother 's health and those that may delay immediate newborn's resuscitation when required.

Recording 13C-15N HMQC 2D sparse spectra in solids in 30 s.

We propose a dipolar HMQC Hadamard-encoded (D-HMQC-Hn) experiment for fast 2D correlations of abundant nuclei in solids. The main limitation of the Hadamard methods resides in the length of the encoding pulses, which results from a compromise between the selectivity and the sensitivity due to losses. For this reason, these methods should mainly be used with sparse spectra, and they profit from the increased separation of the resonances at high magnetic fields. In the case of the D-HMQC-Hn experiments, we give a simple rule that allows directly setting the optimum length of the selective pulses, versus the minimum separation of the resonances in the indirect dimension. The demonstration has been performed on a fully 13C,15N labelled f-MLF sample, and it allowed recording the build-up curves of the 13C-15N cross-peaks within 10 min. However, the method could also be used in the case of less sensitive samples, but with more accumulations.

Current practice of iron prophylaxis in preterm and low birth weight neonates: A survey among Italian Neonatal Units.

BACKGROUND: Preterm babies are at high risk of iron deficiency. METHODS: We investigated current practices regarding iron prophylaxis in preterm and low birth weight newborns among Local Neonatal Units (LNUs, n = 74) and Neonatal Intensive Care Units (NICUs, n = 20) of three Italian Regions (Piemonte, Marche and Lazio). RESULTS: Birth weight is considered an indicative parameter in only 64% of LNUs and 71% of NICUs, with a significant difference between LNUs in the three regions (86%, 20% and 62%, respectively; p < 0.001). Iron is recommended to infants with a birth weight between 2000 and 2500 g in only 25% of LNUs and 21% of NICUs, and to late-preterm (gestational age between 34 and 37 weeks) in a minority of Units (26% of LNUs, 7% of NICUs). CONCLUSIONS: Our pilot survey documents a great variability and the urgent need to standardize practices according to literature recommendations.

Placental Transfusion Strategies in Italy: A Nationwide Survey of Tertiary-Care Delivery Wards.

Objective Delayed umbilical cord clamping and cord milking are placental transfusion strategies that produce higher iron stores and better hemodynamic conditions in a newborn. We aimed to evaluate their current practice in tertiary-care delivery wards in Italy. Study Design A multiple-choice questionnaire was e-mailed to all the 101 Italian tertiary-care delivery wards. The comparative analysis between categorical variables was performed by the χ2 test. Result We obtained an 85% (86/101) response rate. Where placental transfusion strategies were applied, in 61% of cases they were performed in less than half of deliveries. Obstetric-neonatal guidelines were available in 21% of the centers. Where they were available, application of delayed clamping and milking was significantly more frequent and clamping time was longer. Conclusion This first Italian nationwide survey on placental transfusion strategies showed low application rate and variability in knowledge and execution. Availability of obstetric-neonatal guidelines, knowledge of benefits, and cooperation within the delivery team were associated with enhanced implementation.

Interactions between silica nanoparticles and phospholipid membranes.

UNLABELLED: Silica nanoparticles (SNPs) are widely used for biomedical applications. However, their parenteral administration may induce hemolysis. Molecular mechanisms leading to this effect are still controversially discussed. We therefore used a combination of biophysical techniques to investigate the interaction of hemolytic and non-hemolytic SNPs with model phospholipid membranes. METHODS: Interaction of SNPs with membranes was studied using a dye-leakage assay, dynamic light scattering (DLS), isothermal titration calorimetry, and solid state nuclear magnetic resonance. RESULTS AND DISCUSSION: The dye leakage assay revealed that only hemolytic, negatively charged SNPs, but not non-hemolytic positively charged SNPs, destabilized POPC based phospholipid bilayers. Interaction of SNPs with lipid vesicles leading to particle agglomeration was demonstrated by DLS. Isothermal titration calorimetry confirmed the interaction between negatively charged SNPs and phospholipids, which is characterized by an exothermic reaction enthalpy ΔH(0)SNP of -0.04cal/g at 25°C. Calorimetric titrations at different temperatures revealed a molar heat capacity change of zero. This finding excluded a contribution of electrostatic interactions. Mechanistic insight was provided by solid state phosphorus-31 NMR and deuterium NMR measurements. CONCLUSIONS: Our results demonstrate that electrostatic interaction between hemolytic SNPs and model phospholipid membranes is negligible. SNPs induce membrane destabilization and adsorptive processes induced by agglomeration of phospholipid vesicles. The interaction is driven by van der Waals forces at the level of the hydration layer on the vesicles surface.