Thyrotropin Screening of Newborns: Before or After 72 Hours of Life? Before Discharge or at Home?

Objective: Until November 2019 in Belgium, dried blood spot (DBS) sampling was performed between 72 and 120 hours of life, when a majority of newborns had already been discharged from the maternity. In November 2019, the policy for newborn screening in South Belgium changed to allow sampling as soon as 48 hours of life, with the objective to accelerate the process and to allow more sampling during the hospital stay. Our objective was to evaluate the impact of this policy modification and, in particular, to assess the effectiveness of screening for hypothyroidism based on sampling before or after 72 hours of life, as well as to compare the effectiveness of DBS collection before discharge or at home. Methods: This retrospective study included live births ≥37 weeks of gestation, screened by the Université Libre de Bruxelles Newborn Screening Center between January 2019 and December 2021. To evaluate the efficiency of early sampling, we compared thyrotropin (TSH) results for screening <72 hours and screening ≥72 hours. We also compared TSH results of DBS performed before discharge with those performed at home. Results: A total of 53,794 newborns were included. The results of 24,816 healthy newborns screened before 72 hours of life and of 28,978 healthy newborns screened between 72 and 144 hours of life were compared. The median TSH level was similar (1.50 and 1.20 mU/L, respectively). The percentage of false positives was similar (0.08% and 0.07%, respectively). Earlier sampling, before 72 hours, allowed treatment of positive cases at 6 days rather than 8.5 days. DBS sampling at home resulted in longer delay for transferring the sample to the laboratory (a median of 3.0 days for hospital sampling vs. 5.0 days for home sampling). A poorer quality of home blood sampling was observed, with 0.27% unusable samples compared with 0.06% unusable samples for hospital sampling (p < 0.001). Conclusions: In term newborns, TSH screening before discharge, as early as 48 hours of life, is a valid strategy. It allows earlier treatment of positive cases, does not increase the percentage of false positives, and results in fewer unusable samples.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Three years pilot of spinal muscular atrophy newborn screening turned into official program in Southern Belgium.

Three new therapies for spinal muscular atrophy (SMA) have been approved by the United States Food and Drug Administration and the European Medicines Agency since 2016. Although these new therapies improve the quality of life of patients who are symptomatic at first treatment, administration before the onset of symptoms is significantly more effective. As a consequence, newborn screening programs have been initiated in several countries. In 2018, we launched a 3-year pilot program to screen newborns for SMA in the Belgian region of Liège. This program was rapidly expanding to all of Southern Belgium, a region of approximately 55,000 births annually. During the pilot program, 136,339 neonates were tested for deletion of exon 7 of SMN1, the most common cause of SMA. Nine SMA cases with homozygous deletion were identified through this screen. Another patient was identified after presenting with symptoms and was shown to be heterozygous for the SMN1 exon 7 deletion and a point mutation on the opposite allele. These ten patients were treated. The pilot program has now successfully transitioned into the official neonatal screening program in Southern Belgium. The lessons learned during implementation of this pilot program are reported.

A Water Molecule Triggers Guest Exchange at a Mono-Zinc Centre Confined in a Biomimetic Calixarene Pocket: A Model for Understanding Ligand Stability in Zn Proteins.

Invited for the cover of this issue are Kristin Bartik, Olivia Reinaud and co-workers at the Université libre de Bruxelles and Université Paris Descartes. The image depicts a Zn protein and highlights the role that a single water molecule can play in catalysing ligand exchange. Read the full text of the article at 10.1002/chem.202102184.

A Water Molecule Triggers Guest Exchange at a Mono-Zinc Centre Confined in a Biomimetic Calixarene Pocket: a Model for Understanding Ligand Stability in Zn Proteins.

In this study, the ligand exchange mechanism at a biomimetic ZnII centre, embedded in a pocket mimicking the possible constrains induced by a proteic structure, is explored. The residence time of different guest ligands (dimethylformamide, acetonitrile and ethanol) inside the cavity of a calix[6]arene-based tris(imidazole) tetrahedral zinc complex was probed using 1D EXchange SpectroscopY NMR experiments. A strong dependence of residence time on water content was observed with no exchange occurring under anhydrous conditions, even in the presence of a large excess of guest ligand. These results advocate for an associative exchange mechanism involving the transient exo-coordination of a water molecule, giving rise to 5-coordinate ZnII intermediates, and inversion of the pyramid at the ZnII centre. Theoretical modelling by DFT confirmed that the associative mechanism is at stake. These results are particularly relevant in the context of the understanding of kinetic stability/lability in Zn proteins and highlight the key role that a single water molecule can play in catalysing ligand exchange and controlling the lability of ZnII in proteins.

Oxidative Stress, Glutathione Metabolism, and Liver Regeneration Pathways Are Activated in Hereditary Tyrosinemia Type 1 Mice upon Short-Term Nitisinone Discontinuation.

Hereditary tyrosinemia type 1 (HT1) is an inherited condition in which the body is unable to break down the amino acid tyrosine due to mutations in the fumarylacetoacetate hydrolase (FAH) gene, coding for the final enzyme of the tyrosine degradation pathway. As a consequence, HT1 patients accumulate toxic tyrosine derivatives causing severe liver damage. Since its introduction, the drug nitisinone (NTBC) has offered a life-saving treatment that inhibits the upstream enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD), thereby preventing production of downstream toxic metabolites. However, HT1 patients under NTBC therapy remain unable to degrade tyrosine. To control the disease and side-effects of the drug, HT1 patients need to take NTBC as an adjunct to a lifelong tyrosine and phenylalanine restricted diet. As a consequence of this strict therapeutic regime, drug compliance issues can arise with significant influence on patient health. In this study, we investigated the molecular impact of short-term NTBC therapy discontinuation on liver tissue of Fah-deficient mice. We found that after seven days of NTBC withdrawal, molecular pathways related to oxidative stress, glutathione metabolism, and liver regeneration were mostly affected. More specifically, NRF2-mediated oxidative stress response and several toxicological gene classes related to reactive oxygen species metabolism were significantly modulated. We observed that the expression of several key glutathione metabolism related genes including Slc7a11 and Ggt1 was highly increased after short-term NTBC therapy deprivation. This stress response was associated with the transcriptional activation of several markers of liver progenitor cells including Atf3, Cyr61, Ddr1, Epcam, Elovl7, and Glis3, indicating a concreted activation of liver regeneration early after NTBC withdrawal.

Towards the Development of Photo-Reactive Ruthenium(II) Complexes Targeting Telomeric G-Quadruplex DNA.

The design and characterization of new ruthenium(II) complexes aimed at targeting G-quadruplex DNA is reported. Importantly, these complexes are based on oxidizing 1,4,5,8-tetraazaphenanthrene (TAP) ancillary ligands known to favour photo-induced electron transfer (PET) with DNA. The photochemistry of complexes 1-4 has been studied by classical methods, which revealed two of them to be capable of photo-abstracting an electron from guanine. From studies of the interactions with DNA through luminescence, circular dichroism, bio-layer interferometry, and surface plasmon resonance experiments, we have demonstrated the selectivity of these complexes for telomeric G-quadruplex DNA over duplex DNA. Preliminary biological studies of these complexes have been performed: two of them showed remarkable photo-cytotoxicity towards telomerase-negative U2OS osteosarcoma cells, whereas very low mortality was observed in the dark at the same photo-drug concentration.

Submerging a Biomimetic Metallo-Receptor in Water for Molecular Recognition: Micellar Incorporation or Water Solubilization? A Case Study.

Molecular recognition in water is an important topic, but a challenging task due to the very competitive nature of the medium. The focus of this study is the comparison of two different strategies for the water solubilization of a biomimetic metallo-receptor based on a poly(imidazole) resorcinarene core. The first relies on a new synthetic path for the introduction of hydrophilic substituents on the receptor, at a remote distance from the coordination site. The second involves the incorporation of the organosoluble metallo-receptor into dodecylphosphocholine (DPC) micelles, which mimic the proteic surrounding of the active site of metallo-enzymes. The resorcinarene ligand can be transferred into water through both strategies, in which it binds ZnII over a wide pH window. Quite surprisingly, very similar metal ion affinities, pH responses, and recognition properties were observed with both strategies. The systems behave as remarkable receptors for small organic anions in water at near-physiological pH. These results show that, provided the biomimetic site is well structured and presents a recognition pocket, the micellar environment has very little impact on either metal ion binding or guest hosting. Hence, micellar incorporation represents an easy alternative to difficult synthetic work, even for the binding of charged species (metal cations or anions), which opens new perspectives for molecular recognition in water, whether for sensing, transport, or catalysis.

Synthesis and photophysical studies of a multivalent photoreactive RuII-calix[4]arene complex bearing RGD-containing cyclopentapeptides.

Photoactive ruthenium-based complexes are actively studied for their biological applications as potential theragnostic agents against cancer. One major issue of these inorganic complexes is to penetrate inside cells in order to fulfil their function, either sensing the internal cell environment or exert a photocytotoxic activity. The use of lipophilic ligands allows the corresponding ruthenium complexes to passively diffuse inside cells but limits their structural and photophysical properties. Moreover, this strategy does not provide any cell selectivity. This limitation is also faced by complexes anchored on cell-penetrating peptides. In order to provide a selective cell targeting, we developed a multivalent system composed of a photoreactive ruthenium(II) complex tethered to a calix[4]arene platform bearing multiple RGD-containing cyclopentapeptides. Extensive photophysical and photochemical characterizations of this Ru(II)-calixarene conjugate as well as the study of its photoreactivity in the presence of guanosine monophosphate have been achieved. The results show that the ruthenium complex should be able to perform efficiently its photoinduced cytotoxic activity, once incorporated into targeted cancer cells thanks to the multivalent platform.

Ultrafast charge transfer excited state dynamics in trifluoromethyl-substituted iridium(iii) complexes.

Time-resolved spectroscopy was exploited to gain new insights into the nature and dynamics of charge transfer excited states of bis-cyclometalated Ir(iii) complexes. We showed that its dynamics is strongly influenced by the nature of the diimine ligand due to the existence of a ligand-ligand charge transfer process in the picosecond timescale. All the results are supported by DFT/TD-DFT calculations and spectroelectrochemistry.

Ter-Ionic Complex that Forms a Bond Upon Visible Light Absorption.

A "ter-ionic complex" composed of a tetracationic Ru(II) complex and two iodide ions was found to yield a covalent I-I bond upon visible light excitation in acetone solution. 1H NMR, visible absorption and DFT studies revealed that one iodide was associated with a ligand while the other was closer to the Ru metal center. Standard Stern-Volmer quenching of the excited state by iodide revealed upward curvature with a novel saturation at high concentrations. The data were fully consistent with a mechanism in which the Ru metal center in the excited state accepts an electron from iodide to form an iodine atom and, within 70 ns, that atom reacts with the iodide associated with the ligand to yield I2•-. This rapid formation of an I-I bond was facilitated by the supramolecular assembly of the three reactant ions necessary for this ter-ionic reaction that is relevant to solar fuel production.

Converging Energy Transfer in Polynuclear Ru(II) Multiterpyridine Complexes: Significant Enhancement of Luminescent Properties.

Ruthenium-based complexes are widely used as photocatalysts, as photosensitizers, or as building blocks for supramolecular assemblies. In the field of solar energy conversion, building light harvesting antenna is of prime interest. Nevertheless, collecting light is mandatory but not sufficient; once collected and transferred, the exciton has to be long-lived enough to be transferred to a catalytic site. If Ru(II) terpyridine complexes are prime building blocks for structural reasons, the short lifetime of their excited state prevents their use as a harvesting center in light antennae. In this paper, we present new polynuclear assemblies, based on Ru(II)-terpyridine units where delocalization of the excited state is combined with an antenna effect. As a consequence, complexes C1-C3 display long-lived excited states compared to [Ru(tpy)2]2+, making them promising efficient antenna building blocks to be connected to a final acceptor or a catalytic center.

Trifluoromethyl-Substituted Iridium(III) Complexes: From Photophysics to Photooxidation of a Biological Target.

Photodynamic therapeutic agents are of key interest in developing new strategies to develop more specific and efficient anticancer treatments. In comparison to classical chemotherapeutic agents, the activity of photodynamic therapeutic compounds can be finely controlled thanks to the light triggering of their photoreactivity. The development of type I photosensitizing agents, which do not rely on the production of ROS, is highly desirable. In this context, we developed new iridium(III) complexes which are able to photoreact with biomolecules; namely, our Ir(III) complexes can oxidize guanine residues under visible light irradiation. We report the synthesis and extensive photophysical characterization of four new Ir(III) complexes, [Ir(ppyCF3)2(N^N)]+ [ppyCF3 = 2-(3,5-bis(trifluoromethyl)phenyl)pyridine) and N^N = 2,2'-dipyridyl (bpy); 2-(pyridin-2-yl)pyrazine (pzpy); 2,2'-bipyrazine (bpz); 1,4,5,8-tetraazaphenanthrene (TAP)]. In addition to an extensive experimental and theoretical study of the photophysics of these complexes, we characterize their photoreactivity toward model redox-active targets and the relevant biological target, the guanine base. We demonstrate that photoinduced electron transfer takes place between the excited Ir(III) complex and guanine which leads to the formation of stable photoproducts, indicating that the targeted guanine is irreversibly damaged. These results pave the way to the elaboration of new type I photosensitizers for targeting cancerous cells.

Selective recognition of quaternary ammonium ions and zwitterions by using a biomimetic bis-calix[6]arene-based receptor.

Artificial receptors able to recognize efficiently chemical species bearing a quaternary ammonium group have potential applications in the fields of biological and environmental analyses. A possible biomimetic strategy for the elaboration of such receptors consists of associating in close proximity a polyaromatic cavity with a polar binding site. Herein, we show that bis-calix[6]arene 1 behaves as a heteroditopic receptor that can bind biologically relevant quaternary ammonium ions and zwitterions. This host can selectively extract carbamylcholine G3 from water, opening the way for the sensing of this acetylcholine agonist. In some cases, a kinetic selectivity is observed for the shorter guests whereas kinetically stable host-guest complexes can be detected under conditions where they are thermodynamically disfavoured. These results can be rationalized by the unique mode of entrance and escape of the guests into bis-calix[6]arene 1. All these binding properties strongly differ from those reported for related calixarene-based receptors.

New Ruthenium-Based Probes for Selective G-Quadruplex Targeting.

Telomeric regions containing G-quadruplex (G4) structures play a pivotal role in the development of cancers. The development of specific binders for G4s is thus of great interest in order to gain a deeper understanding of the role of these structures, and to ultimately develop new anticancer drug candidates. For several years, RuII complexes have been studied as efficient probes for DNA. Interest in these complexes stems mainly from the tunability of their structures and properties, and the possibility of using light excitation as a tool to probe their environment or to selectively trigger their reaction with a biological target. Herein, we report on the synthesis and thorough study of new RuII complexes based on a novel dipyrazino[2,3-a:2',3'-h]phenazine ligand (dph), obtained through a Chichibabin-like reaction. Luminescence experiments, surface plasmon resonance (SPR), and computational studies have demonstrated that these complexes behave as selective probes for G-quadruplex structures.

A Ruthenium(II) Complex as a Luminescent Probe for DNA Mismatches and Abasic Sites.

[Ru(bpy)2(BNIQ)]2+ (BNIQ = Benzo[c][1,7]naphthyridine-1-isoquinoline), which incorporates the sterically expansive BNIQ ligand, is a highly selective luminescent probe for DNA mismatches and abasic sites, possessing a 500-fold higher binding affinity toward these destabilized regions relative to well-matched base pairs. As a result of this higher binding affinity, the complex exhibits an enhanced steady-state emission in the presence of DNA duplexes containing a single base mismatch or abasic site compared to fully well-matched DNA. Luminescence quenching experiments with Cu(phen)22+ and [Fe(CN)6]3- implicate binding of the complex to a mismatch from the minor groove via metalloinsertion. The emission response of the complex to different single base mismatches, binding preferentially to the more destabilized mismatches, is also consistent with binding by metalloinsertion. This work shows that high selectivity toward destabilized regions in duplex DNA can be achieved through the rational design of a complex with a sterically expansive aromatic ligand.

Photochemistry of ruthenium(ii) complexes based on 1,4,5,8-tetraazaphenanthrene and 2,2'-bipyrazine: a comprehensive experimental and theoretical study.

Polyazaaromatic ruthenium(ii) complexes have been largely studied over the last decades, particularly in the scope of the biological applications, for the development of new diagnostic and phototherapeutic agents. In this context, Ru(ii) complexes able to react with biomolecules upon excitation are of great interest. Photo-oxidizing Ru(ii) complexes based on π-deficient ligands, such as bpz (2,2'-bypyrazine) and TAP (1,4,5,8-tetraazaphenathrene), were designed to allow a photo-induced electron transfer (PET) to take place in presence of biomolecules, thanks to their highly photo-oxidizing 3MLCT state. This PET can occur from either a guanine moiety (G) or a tryptophan residue (Trp) to the excited complex and can ultimately lead to the formation of a photoadduct, i.e. the formation of a covalent bond between the Ru(ii) complex and the G or Trp moieties of a biomolecule. Here, we report the synthesis of two new photo-oxidizing Ru(ii) complexes, [Ru(TAP)2bpz]2+ and [Ru(bpz)2TAP]2+, and the study of their photophysical and electrochemical properties. The influence of the structure of the ligand bpz/TAP on the photophysical and electrochemical properties of the four resulting complexes has been precisely determined thanks to the experimental and theoretical data obtained for to these new complexes.

Photocontrolled Supramolecular Assembling of Azobenzene-Based Biscalix[4]arenes upon Starting and Stopping Laser Trapping.

Laser trapping in chemistry covers various studies ranging from single molecules, nanoparticles, and quantum dots to crystallization and liquid-liquid phase separation of amino acids. In this work, a supramolecular assembly of azobenzene-based biscalix[4]arene is generated in ethyl acetate using laser trapping; its nucleation and growth are elucidated. No trapping behavior was observed when a 1064 nm laser beam was focused inside of the solution; however, interesting assembling phenomena were induced when it was shined at the air/solution interface. A single disk having two layers was first prepared at the focal point of ∼1 µm and then expanded to the size of a few tens of micrometers, although no optical force was exerted outside of the focal volume. Upon switching the trapping laser off, needles were generated at the outer layer of the assembly, giving a stable sea urchin-like morphology to the generated assembly. At a 30-50% dilution of the initial solution in ethyl acetate, a mushroom-like morphology was also observed. Laser trapping-induced assembly of azobenzene-based biscalix[4]arene was quite different from the sharp-ellipsoidal aggregates obtained by the spontaneous evaporation of the solution. These trapping phenomena were specifically observed for biscalix[4]arene in the trans conformation of azo-benzene moiety but not for the cis-form, suggesting that the laser trapping of this azobenzene-based biscalix[4]arene is photocontrollable. Dynamics and mechanism of the supramolecular assembling are considered, referring to laser trapping-induced nucleation and liquid-liquid phase separation of amino acids.

A nano-sized container for specific encapsulation of isolated water molecules.

A calix[4]arene-based molecular box was synthesized. Its properties were characterized through XRD and extensive NMR studies. This receptor is able to encapsulate specifically two isolated water molecules in both non-protic and protic solvents. This is a consequence of high size, geometric and electronic complementarity between the host and the water molecules.