Atomistic Characterization of Beta-2-Glycoprotein I Domain V Interaction with Anionic Membranes.

Background: Interaction of beta-2-glycoprotein I ( ß 2 GPI) with anionic membranes is crucial in antiphospholipid syndrome (APS), implicating the role of it's membrane bind-ing domain, Domain V (DV). The mechanism of DV binding to anionic lipids is not fully understood. Objectives: This study aims to elucidate the mechanism by which DV of ß 2 GPI binds to anionic membranes. Methods: We utilized molecular dynamics (MD) simulations to investigate the struc-tural basis of anionic lipid recognition by DV. To corroborate the membrane-binding mode identified in the HMMM simulations, we conducted additional simulations using a full mem-brane model. Results: The study identified critical regions in DV, namely the lysine-rich loop and the hydrophobic loop, essential for membrane association via electrostatic and hydrophobic interactions, respectively. A novel lysine pair contributing to membrane binding was also discovered, providing new insights into ß 2 GPI's membrane interaction. Simulations revealed two distinct binding modes of DV to the membrane, with mode 1 characterized by the insertion of the hydrophobic loop into the lipid bilayer, suggesting a dominant mechanism for membrane association. This interaction is pivotal for the pathogenesis of APS, as it facilitates the recognition of ß 2 GPI by antiphospholipid antibodies. Conclusion: The study advances our understanding of the molecular interactions be-tween ß 2 GPI's DV and anionic membranes, crucial for APS pathogenesis. It highlights the importance of specific regions in DV for membrane binding and reveals a predominant bind-ing mode. These findings have significant implications for APS diagnostics and therapeutics, offering a deeper insight into the molecular basis of the syndrome.

Cryo-EM structure and functional basis of prothrombin recognition by a Type-I anti-prothrombin antiphospholipid antibody.

Anti-prothrombin (anti-PT) antibodies are found in antiphospholipid patients, but how they interact with prothrombin remains elusive. Prothrombin adopts closed and open forms. We recently discovered Type-I and Type-II antibodies and proposed that Type-I recognize the open form. In this study, we report the discovery, structural and functional characterization in human plasma of a Type-I antibody, POmAb. Using surface plasmon resonance and single-molecule spectroscopy, we show that POmAb interacts with kringle-1 of prothrombin, shifting the equilibrium towards the open form. Using single-particle cryogenic electron microscopy (cryo-EM), we establish that the epitope targeted by POmAb is in kringle-1, comprising an extended binding interface centered at residues R90-Y93. The 3.2Å cryo-EM structure of the complex reveals that the epitope overlaps with the position occupied by the protease domain of prothrombin in the closed state, explaining the exclusive binding of POmAb to the open form. In human plasma, POmAb prolongs phospholipid-initiated and diluted Russel Viper Venom clotting time, which could be partly rescued by excess phospholipids, indicating POmAb is an anticoagulant but exerts a weak lupus anticoagulant effect. These studies reveal the structural basis of prothrombin recognition by a Type-I antiphospholipid antibody and uncover an exciting new strategy to achieve anticoagulation in human plasma.

From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).

Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 µM) and its potency was enhanced by 10-fold after Phe3 → ß-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 µM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.

The PALB2 DNA-binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

Forecasting the Future of Antiphospholipid Syndrome: Prospects and Challenges.

Antiphospholipid syndrome (APS) is an autoimmune condition affecting young patients, primarily women, negatively impacting their quality of life. APS is under-recognized and underdiagnosed and can have devastating results if untreated, mainly due to uncontrolled thrombosis. Research in the past decades has led to several breakthroughs with important implications for clinical practice. Here, we summarize the state of APS diagnosis, treatment, pathophysiology, and research directions that hold promise for advancing diagnosis and treatment.

Homodimeric Granzyme A opsonizes Mycobacterium tuberculosis and inhibits its intracellular growth in human monocytes via TLR4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete GzmA protective against intracellular Mtb growth. However, GzmA enzymatic activity is unnecessary for pathogen inhibition and the mechanisms of GzmA-mediated protection remain unknown. We show GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes and subsequent pathogen clearance within the phagolysosome. While monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind CD14 and TLR4. Without access to surface expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1, was associated with inhibitory activity. Further, GzmA colocalized with and was regulated by protein disulfide isomerase (PDI)A1, which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis and elimination of Mtb in human monocytes, and highlights PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Structural analyses of ß2-glycoprotein I: is there a circular conformation?

BACKGROUND: Antiphospholipid antibodies targeting ß2-glycoprotein I (ß2GPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize ß2GPI remains controversial. OBJECTIVES: This study aimed to elucidate the structure of ß2GPI and evaluate how pathogenic anti-domain I (DI) antibodies recognize it in human plasma. METHODS: ß2GPI was made recombinant and purified from human plasma using different protocols. Structural and functional analyses were conducted using orthogonal techniques, namely, electron microscopy, size-exclusion chromatography, single-molecule Förster resonance energy transfer, and microfluidic diffusional sizing. RESULTS: Electron microscopy and size-exclusion chromatography showed that the structure of ß2GPI produced recombinantly and purified from plasma is elongated, even when subjected to conditions previously reported to favor circularization. Single-molecule Förster resonance energy transfer analyses of ß2GPI labeled at positions 88 in DII and 278 in DV showed that these residues are located >90 Å apart, consistent with an elongated form. They also documented that the distance between these 2 residues did not change when the protein was reconstituted in human plasma. Microfluidic diffusional sizing documented that ß2GPI binds with moderate affinity to a prototypical anti-DI antibody targeting the epitope G40-R43 despite being elongated. CONCLUSION: Circulating ß2GPI is elongated and, therefore, fully capable of binding to anti-DI antibodies. Binding of ß2GPI to negatively charged phospholipids drives autoantibody recognition by increasing the local concentration of the antigen and not by dramatically changing its conformation. These findings clarify the structural properties of ß2GPI, which have important implications for understanding APS pathogenesis and the development of APS diagnostics and therapeutics.

The PALB2 DNA-binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) tumor suppressor is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency. The PALB2 DNA-binding domain (PALB2-DBD) supports DNA strand exchange, a complex multistep reaction supported by only a few protein families such as RecA-like recombinases or Rad52. The mechanisms of PALB2 DNA binding and strand exchange are unknown. We performed circular dichroism, electron paramagnetic spectroscopy, and small-angle X-ray scattering analyses and determined that PALB2-DBD is intrinsically disordered, even when bound to DNA. The intrinsically disordered nature of this domain was further supported by bioinformatics analysis. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome and have many important biological functions. The complexity of the strand exchange reaction significantly expands the functional repertoire of IDPs. The results of confocal single-molecule FRET indicated that PALB2-DBD binding leads to oligomerization-dependent DNA compaction. We hypothesize that PALB2-DBD uses a chaperone-like mechanism to aid formation and resolution of complex DNA and RNA multichain intermediates during DNA replication and repair. Since PALB2-DBD alone or within the full-length PALB2 is predicted to have strong liquid-liquid phase separation (LLPS) potential, protein-nucleic acids condensates are likely to play a role in complex functionality of PALB2-DBD. Similar DNA-binding intrinsically disordered regions may represent a novel class of functional domains that evolved to function in eukaryotic nucleic acid metabolism complexes.

Mutations in atypical hemolytic uremic syndrome provide evidence for the role of calcium in complement factor I.

Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy. Genetic variants in complement proteins are found in ≈60% of patients. Of these patients, ≈15% carry mutations in complement factor I (CFI). Factor I (FI) is a multidomain serine protease that cleaves and thereby inactivates C3b and C4b in the presence of cofactor proteins. Crystal structures have shown that FI possesses 2 calcium-binding domains, low-density lipoprotein receptor class A (LDLRA) 1 and LDLRA2. Yet, the role of calcium in FI is unknown. We determined that 9 genetic variants identified in aHUS (N151S, G162D, G188A, V230E, A240G, G243R, C247G, A258T, and Q260D) cluster around the calcium-binding site of LDLRA1. Using site-directed mutagenesis, we established that the synthesis of all, except A258T, was impaired, implying defective protein folding, perhaps due to loss of calcium binding. To further explore this possibility, we generated 12 alanine mutants that coordinate with the calcium in LDLRA1 and LDLRA2 (K239A, D242A, I244A, D246A, D252A, E253A, Y276A, N279A, E281A, D283A, D289A, and D290A) and are expected to perturb calcium binding. Except for K239A and Y276A, none of the mutants was secreted. These observations suggest that calcium ions play key structural and functional roles in FI.

The neonatal DAV-expert algorithm: a GAVeCeLT/GAVePed consensus for the choice of the most appropriate venous access in newborns.

In most NICUs, the choice of the venous access device currently relies upon the operator's experience and preferences. However, considering the high failure rate of vascular devices in the neonatal population, such clinical choice has a critical relevance and should preferably be based on the best available evidence. Though some algorithms have been published over the last 5 years, none of them seems in line with the current scientific evidence. Thus, the GAVePed-which is the pediatric interest group of the most important Italian group on venous access, GAVeCeLT-has developed a national consensus about the choice of the venous access device in the neonatal population. After a systematic review of the available evidence, the panel of the consensus (which included Italian neonatologists specifically experts in this area) has provided structured recommendations answering four sets of questions regarding (1) umbilical venous catheters, (2) peripheral cannulas, (3) epicutaneo-cava catheters, and (4) ultrasound-guided centrally and femorally inserted central catheters. Only statements reaching a complete agreement were included in the final recommendations. All recommendations were also structured as a simple visual algorithm, so as to be easily translated into clinical practice.  Conclusion: The goal of the present consensus is to offer a systematic set of recommendations on the choice of the most appropriate vascular access device in Neonatal Intensive Care Unit.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

BACKGROUND: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. OBJECTIVES: We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. METHODS: Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. RESULTS: PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. CONCLUSION: PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Probing the conformational dynamics of thiol-isomerases using non-canonical amino acids and single-molecule FRET.

Disulfide bonds drive protein correct folding, prevent protein aggregation, and stabilize three-dimensional structures of proteins and their assemblies. Dysregulation of this activity leads to several disorders, including cancer, neurodegeneration, and thrombosis. A family of 20+ enzymes, called thiol-isomerases (TIs), oversee this process in the endoplasmic reticulum of human cells to ensure efficacy and accuracy. While the biophysical and biochemical properties of cysteine residues are well-defined, our structural knowledge of how TIs select, interact and process their substrates remains poorly understood. How TIs structurally and functionally respond to changes in redox environment and other post-translational modifications remain unclear, too. We recently developed a workflow for site-specific incorporation of non-canonical amino acids into protein disulfide isomerase (PDI), the prototypical member of TIs. Combined with click chemistry, this strategy enabled us to perform single-molecule biophysical studies of PDI under various solution conditions. This paper details protocols and discusses challenges in performing these experiments. We expect this approach, combined with other emerging technologies in single-molecule biophysics and structural biology, to facilitate the exploration of the mechanisms by which TIs carry out their fascinating but poorly understood roles in humans, especially in the context of thrombosis.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors.

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Reduction of protein disulfide isomerase results in open conformations and stimulates dynamic exchange between structural ensembles.

Human protein disulfide isomerase (PDI) is an essential redox-regulated enzyme required for oxidative protein folding. It comprises four thioredoxin domains, two catalytically active (a, a') and two inactive (b, b'), organized to form a flexible abb'a' U-shape. Snapshots of unbound oxidized and reduced PDI have been obtained by X-ray crystallography. Yet, how PDI's structure changes in response to the redox environment and inhibitor binding remains controversial. Here, we used multiparameter confocal single-molecule FRET to track the movements of the two catalytic domains with high temporal resolution. We found that at equilibrium, PDI visits three structurally distinct conformational ensembles, two "open" (O1 and O2) and one "closed" (C). We show that the redox environment dictates the time spent in each ensemble and the rate at which they exchange. While oxidized PDI samples O1, O2, and C more evenly and in a slower fashion, reduced PDI predominantly populates O1 and O2 and exchanges between them more rapidly, on the submillisecond timescale. These findings were not expected based on crystallographic data. Using mutational analyses, we further demonstrate that the R300-W396 cation-π interaction and active site cysteines dictate, in unexpected ways, how the catalytic domains relocate. Finally, we show that irreversible inhibitors targeting the active sites of reduced PDI did not abolish these protein dynamics but rather shifted the equilibrium toward the closed ensemble. This work introduces a new structural framework that challenges current views of PDI dynamics, helps rationalize its multifaceted role in biology, and should be considered when designing PDI-targeted therapeutics.

Post-Transplant Thrombotic Microangiopathy due to a Pathogenic Mutation in Complement Factor I in a Patient With Membranous Nephropathy: Case Report and Review of Literature.

Thrombotic microangiopathy (TMA) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and organ injury occurring due to endothelial cell damage and microthrombi formation in small vessels. TMA is primary when a genetic or acquired defect is identified, as in atypical hemolytic uremic syndrome (aHUS) or secondary when occurring in the context of another disease process such as infection, autoimmune disease, malignancy or drugs. Differentiating between a primary complement-mediated process and one triggered by secondary factors is critical to initiate timely treatment but can be challenging for clinicians, especially after a kidney transplant due to presence of multiple confounding factors. Similarly, primary membranous nephropathy is an immune-mediated glomerular disease associated with circulating autoantibodies (directed against the M-type phospholipase A2 receptor (PLA2R) in 70% cases) while secondary membranous nephropathy is associated with infections, drugs, cancer, or other autoimmune diseases. Complement activation has also been proposed as a possible mechanism in the etiopathogenesis of primary membranous nephropathy; however, despite complement being a potentially common link, aHUS and primary membranous nephropathy have not been reported together. Herein we describe a case of aHUS due to a pathogenic mutation in complement factor I that developed after a kidney transplant in a patient with an underlying diagnosis of PLA2R antibody associated-membranous nephropathy. We highlight how a systematic and comprehensive analysis helped to define the etiology of aHUS, establish mechanism of disease, and facilitated timely treatment with eculizumab that led to recovery of his kidney function. Nonetheless, ongoing anti-complement therapy did not prevent recurrence of membranous nephropathy in the allograft. To our knowledge, this is the first report of a patient with primary membranous nephropathy and aHUS after a kidney transplant.

Functional analysis of rare genetic variants in complement factor I in advanced age-related macular degeneration.

Factor I (FI) is a serine protease inhibitor of the complement system. Heterozygous rare genetic variants in complement factor I (CFI) are associated with advanced age-related macular degeneration (AMD). The clinical impact of these variants is unknown since a majority have not been functionally characterized and are classified as 'variants of uncertain significance' (VUS). This study assessed the functional significance of VUS in CFI. Our previous cross-sectional study using a serum-based assay demonstrated that CFI variants in advanced AMD can be categorized into three types. Type 1 variants cause a quantitative deficiency of FI. Type 2 variants demonstrate a qualitative deficiency. However, Type 3 variants consist of VUS that are less dysfunctional than Types 1 and 2 but are not as biologically active as wild type (WT). In this study, we employed site-directed mutagenesis followed by expression of the recombinant variant and a comprehensive set of functional assays to characterize nine Type 3 variants that were identified in 37 individuals. Our studies establish that the expression of the recombinant protein compared with WT is reduced for R202I, Q217H, S221Y and G263V. Further, G362A and N536K, albeit expressed normally, have significantly less cofactor activity. These results led to re-categorization of CFI variants R202I, Q217H, S221Y and G263V as Type 1 variants and to reclassification of N536K and G362A as Type 2. The variants K441R, Q462H and I492L showed no functional defect and remained as Type 3. This study highlights the utility of an in-depth biochemical analysis in defining the pathologic and clinical implications of complement variants underlying AMD.

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding.

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds' reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.

The care of critically ill infants and toddlers in neonatal intensive care units across Italy and Europe: our proposal for healthcare organization.

Numerous studies have shown that critically ill infants and toddlers admitted to paediatric intensive care units (PICUs) have a lower mortality than those admitted to adult ICUs. In 2014, there were only 23 registered PICUs in Italy, most of which were located in the north. For this reason, in Italy and elsewhere in Europe, some neonatal ICUs (NICUs) have begun managing critically ill infants and toddlers. Our proposal for healthcare organization is to establish "extended NICUs" in areas where paediatric intensive care beds are lacking. While some countries have opted for a strict division between neonatal and paediatric intensive care units, the model of "extended NICUs" has already been set up in Italy and in Europe. In this instance, the management of critically ill infants and toddlers undoubtedly falls upon neonatologists, who, however, must gain specific knowledge and technical skills in paediatric critical care medicine (PCCM). Postgraduate residencies in paediatrics need to include periods of specific training in neonatology and PCCM. The Italian Society of Neonatology's Early Childhood Intensive Care Study Group is supporting certified training courses for its members involving both theory and practice. CONCLUSION: Scientific societies should promote awareness of the issues involved in the intensive management of infants and toddlers in NICUs and the training of all health workers involved. These societies include the Italian Society of Neonatology, the European Society of Paediatric and Neonatal Intensive Care, and the Union of European Neonatal and Perinatal Societies. They should also act in concert with the governmental institutional bodies to establish the standards for the "extended NICUs." WHAT IS KNOWN: • The mortality of critically ill infants and toddlers admitted to PICUs is lower than that for those admitted to adult ICUs. • In Italy, there are only a handful of PICUs, located mainly in the north. WHAT IS NEW: • Critically ill infants and small toddlers can be managed in "extended NICUs" in areas with a lack of paediatric intensive care beds. • "Extended NICUs" is our proposal for healthcare organization to compensate for the paucity of paediatric intensive care beds, but neonatologists must be trained to provide them with specific knowledge and technical skills in PCCM.