Differential tissue immune stimulation through immersion in bacterial and viral agonists in the Antarctic Notothenia rossii.

The genome evolution of Antarctic notothenioids has been modulated by their extreme environment over millennia and more recently by human-caused constraints such as overfishing and climate change. Here we investigated the characteristics of the immune system in Notothenia rossii and how it responds to 8 h immersion in viral (Poly I:C, polyinosinic: polycytidylic acid) and bacterial (LPS, lipopolysaccharide) proxies. Blood plasma antiprotease activity and haematocrit were reduced in Poly I:C-treated fish only, while plasma protein, lysozyme activity and cortisol were unchanged with both treatments. The skin and duodenum transcriptomes responded strongly to the treatments, unlike the liver and spleen which had a mild response. Furthermore, the skin transcriptome responded most to the bacterial proxy (cell adhesion, metabolism and immune response processes) and the duodenum (metabolism, response to stress, regulation of intracellular signal transduction, and immune system responses) to the viral proxy. The differential tissue response to the two proxy challenges is indicative of immune specialisation of the duodenum and the skin towards pathogens. NOD-like and C-type lectin receptors may be central in recognising LPS and Poly I:C. Other antimicrobial compounds such as iron and selenium-related genes are essential defence mechanisms to protect the host from sepsis. In conclusion, our study revealed a specific response of two immune barrier tissue, the skin and duodenum, in Notothenia rossii when exposed to pathogen proxies by immersion, and this may represent an adaptation to pathogen infective strategies.

Portuguese Neonatal Screening Program: A Cohort Study of 18 Years Using MS/MS.

The Portuguese Neonatal Screening Program (PNSP) conducts nationwide screening for rare diseases, covering nearly 100% of neonates and screening for 28 disorders, including 24 inborn errors of metabolism (IEMs). The study's purpose is to assess the epidemiology of the screened metabolic diseases and to evaluate the impact of second-tier testing (2TT) within the PNSP. From 2004 to 2022, 1,764,830 neonates underwent screening using tandem mass spectrometry (MS/MS) to analyze amino acids and acylcarnitines in dried blood spot samples. 2TT was applied when necessary. Neonates with profiles indicating an IEM were reported to a reference treatment center, and subsequent biochemical and molecular studies were conducted for diagnostic confirmation. Among the screened neonates, 677 patients of IEM were identified, yielding an estimated birth prevalence of 1:2607 neonates. The introduction of 2TT significantly reduced false positives for various disorders, and 59 maternal cases were also detected. This study underscores the transformative role of MS/MS in neonatal screening, emphasizing the positive impact of 2TT in enhancing sensitivity, specificity, and positive predictive value. Our data highlight the efficiency and robustness of neonatal screening for IEM in Portugal, contributing to early and life-changing diagnoses.

Theoretical Prediction of Core-Level Binding Energies: Analysis of Unexpected Errors.

The analysis of the C(1s) and O(1s) core-level binding energies (CLBEs) of selected molecules computed by means of total energy Hartree-Fock (ΔSCF-HF) differences shows that in some cases, the calculated values for the C(1s) are larger than the experiment, which is unexpected. The origin of these unexpected errors of the Hartree-Fock ΔSCF BEs is shown to arise from static, nondynamical, electron correlation effects which are larger for the ion than for the neutral system. Once these static correlation effects are included by using complete active space self-consistent field (CASSCF) wave functions that include internal correlation terms, the resulting ΔSCF BEs are, as expected, smaller than measured values.

Comparison of Computational Strategies for the Calculation of the Electronic Coupling in Intermolecular Energy and Electron Transport Processes.

Electronic couplings in intermolecular electron and energy transfer processes calculated by six different existing computational techniques are compared to nonorthogonal configuration interaction for fragments (NOCI-F) results. The paper addresses the calculation of the electronic coupling in diketopyrrolopyrol, tetracene, 5,5'-difluoroindigo, and benzene-Cl for hole and electron transport, as well as the local exciton and singlet fission coupling. NOCI-F provides a rigorous computational scheme to calculate these couplings, but its computational cost is rather elevated. The here-considered ab initio Frenkel-Davydov (AIFD), Dimer projection (DIPRO), transition dipole moment coupling, Michl-Smith, effective Hamiltonian, and Mulliken-Hush approaches are computationally less demanding, and the comparison with the NOCI-F results shows that the NOCI-F results in the couplings for hole and electron transport are rather accurately predicted by the more approximate schemes but that the NOCI-F exciton transfer and singlet fission couplings are more difficult to reproduce.

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

INTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). METHODS: From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. RESULTS/CASE REPORT: A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. CONCLUSION: This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Glutaric Acidemia Type 1: Diagnosis, Clinical features, and Outcome in a Portuguese Cohort.

INTRODUCTION: Glutaric acidemia type 1 (GA1) is a rare autosomal recessive disorder characterized by a deficiency of glutaryl-CoA dehydrogenase, resulting in the accumulation of glutaric acid (GA), 3-hydroxyglutaric acid, and glutarylcarnitine, especially in the brain. GA1-affected children are clinically characterized by macrocephaly. Neurological abnormalities usually appear between 6 and 18 months of age, often triggered by a catabolic event. On the other hand, several biochemically affected individuals may remain asymptomatic or experience an insidious onset of mild neurological abnormalities. METHODS: Retrospective study of GA1 patients followed at a Portuguese Hereditary Metabolic Disease Center, to characterize the phenotypic and genotypic variations associated with GA1. Therefore, we analyzed the clinical, neuroradiological, biochemical, and genetic information from 14 patients. RESULTS: 14 patients (four months-27 years old) were identified in the last 26 years, 9 were male, 1 was from a consanguineous family. 11 were diagnosed by newborn screening (NBS), and 3 identified following clinical symptoms (later diagnosed, LD). There were 3 phenotypic presentations: 6 asymptomatic, 3 with a motor disability after encephalopathic crisis (EC), and 5 with insidious onset. Acute EC occurred in 1/3 of the LD patients and in 2/11 NBS-identified patients. About urinary GA concentrations: 5 were low excretors (LE), 9 were high excretors (HE). All LE showed symptoms, and 2 had EC. Concerning HE, 3 showed symptoms and 1 had EC. GCDH analysis showed: 6 compound heterozygotes and 8 homozygotes. most frequent variant was c.1204C>T (p.R402W). All of them received appropriate therapy from the time of diagnosis, with a mean age of 23.3 months in LD patients and 13.3 days in NBS-identified patients. CONCLUSION: The outcomes were different between the two groups: all the LD patients presented motor dysfunction however in the NBS-identified patients only 5 developed this symptom. Patients identified by NBS had better outcomes showing that NBS enables an early diagnosis, and treatment, and consequently improves the clinical outcomes for these patients. No correlation was observed with clinical phenotype between LE and HE, as both groups can suffer the most severe neurological manifestations. These conclusions are in agreement with previous cohorts described in the literature.

Pilot Study on Newborn Screening for Spinal Muscular Atrophy.

INTRODUCTION: Newborn screening (NBS) in Portugal is a significant public health measure to provide early detection for specific disorders so that early treatment is possible. Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that causes degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neurons. Its incidence is estimated in 1.6000-11.800 live births. A pilot study on 100.000 newborns is being carried out at the neonatal screening laboratory with the aim of determining the specificity, sensitivity, and feasibility of the SMA screening at the NBS laboratory in Portugal. METHODS: The study presented here was based on data obtained from neonatal screening, involving the analysis of 25.000 newborns. SMA screening is performed by a qualitative detection of exon 7 of the SMN1 gene. The assay was performed using a commercially available real-time PCR, the Eonis SMN1, TREC, and KREC kit. RESULTS/CASE REPORT: The dried blood spots of a total of 25.000 newborns were tested; among these newborns, two were diagnosed as having SMA with survival motor neuron 1 (SMN1) deletion. These two SMA-positive samples were sent to a specialized clinical centre and a peripheral blood sample was sent to the reference laboratory for confirmation of the exon 7 deletion and determination of the SMN2 copy number. CONCLUSION: Early diagnosis and intervention are important for SMA treatment to be effective; the treatment should be started at the pre-symptomatic stage of SMA. Thus, newborn screening for SMA is strongly recommended. Currently, targeted therapies for SMA are available, and attempts are being made worldwide to include SMA screening in newborns.

Toward a Rigorous Theoretical Description of Photocatalysis Using Realistic Models.

This Perspective aims at providing a road map to computational heterogeneous photocatalysis highlighting the knowledge needed to boost the design of efficient photocatalysts. A plausible computational framework is suggested focusing on static and dynamic properties of the relevant excited states as well of the involved chemistry for the reactions of interest. This road map calls for explicitly exploring the nature of the charge carriers, the excited-state potential energy surface, and its time evolution. Excited-state descriptors are introduced to locate and characterize the electrons and holes generated upon excitation. Nonadiabatic molecular dynamics simulations are proposed as a convenient tool to describe the time evolution of the photogenerated species and their propagation through the crystalline structure of photoactive material, ultimately providing information about the charge carrier lifetime. Finally, it is claimed that a detailed understanding of the mechanisms of heterogeneously photocatalyzed reactions demands the analysis of the excited-state potential energy surface.

Toll-Like Receptor Evolution: Does Temperature Matter?

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head-kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

XPS binding energy shifts as a function of bond distances: a case study of CO.

The O(1s) and C(1s) XPS core-level binding energies. BEs, have been studied as a function of the C-O internuclear distance for a large range of distances. The BE(r) for both BEs show considerable variation over the distances studied which is, however, different for the O(1s) and C(1s) BEs. The origin of the dependence on C-O distance is established and shown to involve more than the electric field generated because of the charge separation within CO being C+qand O-q. Furthermore, the BE(r) is shown to be different for Hartree-Fock and correlated wavefunctions indicating that the BE(r) can provide evidence of how electron correlation modifies the valence charge distribution. The difference between the O(1s) and C(1s) BEs is examined and it is proposed that this difference can be used as a measure of the accuracy of theoretically predicted BEs. It is believed that the features found for CO may be representative for the BE variations with geometry for other systems; an effect that has been mostly overlooked.

Role of RNA in Molecular Diagnosis of MADD Patients.

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) encodes the ETF-ubiquinone oxidoreductase (ETF-QO) and has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). In this study, we present the clinical and molecular diagnostic challenges, at the DNA and RNA levels, involved in establishing the genotype of four MADD patients with novel ETFDH variants: a missense variant, two deep intronic variants and a gross deletion. RNA sequencing allowed the identification of the second causative allele in all studied patients. Simultaneous DNA and RNA investigation can increase the number of MADD patients that can be confirmed following the suggestive data results of an expanded newborn screening program. In clinical practice, accurate identification of pathogenic mutations is fundamental, particularly with regard to diagnostic, prognostic, therapeutic and ethical issues. Our study highlights the importance of RNA studies for a definitive molecular diagnosis of MADD patients, expands the background of ETFDH mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.

Galanin isoforms by alternative splicing: Structure, expression, and immunohistochemical location in the gonads of European sea bass.

Galanin (Gal) is a neuropeptide with multiple functions that is widely expressed in the central and peripheral nervous systems of vertebrates. Anatomical and functional evidence suggests a possible role in regulating reproduction in fishes. To test this possibility, we have isolated and characterized two gal alternative transcripts in European sea bass (Dicentrarchus labrax) that encode two prepropeptides, respectively of 29 (gal_MT853221) and 53 (gal_MT853222) amino acids. The two gal transcripts are highly expressed in brain, pituitary and gonads, and appear to be differentially regulated in males and females. In males, gal_MT853222 in the hypothalamus and gal_MT853221 in the pituitary were downregulated with the progression of spermatogenesis (stages I-III). Both transcripts are downregulated in testicles of 1-year (precocious) and 2-year spermiating males compared to immature fish of the same age. Gal peptides and receptors are expressed throughout ovarian development in the hypothalamic-pituitary-gonadal (HPG) axis of females. In the testis, immunoreactive Gal-29 and Gal-53 peptides were detected in blood vessels and Leydig cells during the spermatogenesis stages I-III but Gal immunostaining was barely undetected in more advanced stages. In the ovary, both peptides localized in interstitial cells and blood vessels and in theca cells surrounding the maturing oocytes. The immunolocalization of galanin in Leydig and theca cells suggests a possible role in steroid production regulation. The different pattern of gal expression and Gal localization in the testis and ovary may suggest the possibility that androgens and estrogens may also regulate Gal gene transcription and translation. Altogether, this study showed evidence for the possible involvement of locally produced Gal in gametogenesis and that its production is differentially regulated in male and female gonads.

[Portuguese Newborn Screening Program.] / Programa portugués de cribado neonatal.

The Portuguese Newborn Screening Program is a public health program that started in 1979, screening for PKU, being totally supported by public funds. It's a non-mandatory well implemented program that testes about 99.9% of Portuguese newborns. In the actual screening panel encompasses 26 disorders, including inborn errors of metabolism, congenital hypothyroidism and cystic fibrosis. Sample collection is advised to be made at 3rd day of life and treatment begins in average by the 10th day. Every testes are performed in one single national laboratory, that processes about 88,000 samples/year. In the 41 years of program existence, more than 3,800,000 newborns were screened and 2,130 affected newborns detected, reflecting the positive impact of the Program in the population. Future perspectives include the increase of program value in terms of public health by optimizing the screening of the disorders already screened and evaluation the possibility of adding others.

El Programa Portugués de Cribado Neonatal es un programa de Salud Pública a nivel nacional, que tuvo su inicio en 1979 con el cribado de la fenilcetonuria y es financiado totalmente por el Estado portugués. Se trata de un programa no obligatorio, con una tasa de cobertura del 99,8%, en el que hoy en día se criban veintiséis enfermedades, incluyendo metabolopatías, hipotiroidismo congénito y fibrosis quística. La toma de muestra se hace al 3er día de vida y el tratamiento de los neonatos afectos empieza en torno al 10º día. Todos los análisis están centralizados en un único laboratorio, que procesa aproximadamente 88.000 muestras al año. En los últimos cuarenta y un años se cribaron más de 3.800.000 neonatos y se detectaron 2.130 niños afectados, lo que es un indicador del impacto del programa en la población. Los desafíos futuros incluyen la búsqueda de nuevas estrategias para incrementar el valor del programa, donde se evalúen nuevas enfermedades a cribar y la optimización del cribado actual.

Phenylketonuria in Portugal: Genotype-phenotype correlations using molecular, biochemical, and haplotypic analyses.

BACKGROUND: The impairment of the hepatic enzyme phenylalanine hydroxylase (PAH) causes elevation of phenylalanine levels in blood and other body fluids resulting in the most common inborn error of amino acid metabolism (phenylketonuria). Persistently high levels of phenylalanine lead to irreversible damage to the nervous system. Therefore, early diagnosis of the affected individuals is important, as it can prevent clinical manifestations of the disease. METHODS: In this report, the biochemical and genetic findings performed in 223 patients diagnosed through the Portuguese Neonatal Screening Program (PNSP) are presented. RESULTS: Overall, the results show that a high overlap exists between different types of variants and phenylalanine levels. Molecular analyses reveal a wide mutational spectrum in our population with a total of 56 previously reported variants, most of them found in compound heterozygosity (74% of the patients). Intragenic polymorphic markers were used to assess the haplotypic structure of mutated chromosomes for the most frequent variants found in homozygosity in our population (p.Ile65Thr, p.Arg158Gln, p.Leu249Phe, p.Arg261Gln, p.Val388Met, and c.1066-11G>A). CONCLUSION: Our data reveal high heterogeneity at the biochemical and molecular levels and are expected to provide a better understanding of the molecular basis of this disease and to provide clues to elucidate genotype-phenotype correlations.

Clinical, biochemical and molecular findings of 24 Brazilian patients with glutaric acidemia type 1: 4 novel mutations in the GCDH gene.

Glutaric aciduria type 1 (GA-1) is a rare but treatable inherited disease caused by deficiency of glutaryl-CoA dehydrogenase activity due to GCDH gene mutations. In this study, we report 24 symptomatic GA-1 Brazilian patients, and present their clinical, biochemical, and molecular findings. Patients were diagnosed by high levels of glutaric and/or 3-hydroxyglutaric and glutarylcarnitine. Diagnosis was confirmed by genetic analysis. Most patients had the early-onset severe form of the disease and the main features were neurological deterioration, seizures and dystonia, usually following an episode of metabolic decompensation. Despite the early symptomatology, diagnosis took a long time for most patients. We identified 13 variants in the GCDH gene, four of them were novel: c.91 + 5G > A, c.167T > G, c.257C > T, and c.10A > T. The most common mutation was c.1204C > T (p.R402W). Surprisingly, the second most frequent mutation was the new mutation c.91 + 5G > A (IVS1 ds G-A + 5). Our results allowed a complete characterization of the GA-1 Brazilian patients. Besides, they expand the mutational spectrum of GA-1, with the description of four new mutations. This work reinforces the importance of awareness of GA-1 among doctors in order to allow early diagnosis and treatment in countries like Brazil where the disease has not been included in newborn screening programs.

Limitations of the equivalent core model for understanding core-level spectroscopies.

The equivalent core model, or the Z + 1 approximation, has been used to interpret the binding energy, BE, shifts observed in X-ray photoelectron spectroscopy, XPS; in particular to relate these shifts to their origin in the electronic structure of the system. Indeed, a recent paper has claimed that the equivalent core model provides an intuitive chemical view of XPS BE shifts. In the present paper, we present a detailed comparison of the electronic structure provided from rigorous core-hole theory and from the equivalent core model to assess the validity and the utility of the use of the equivalent core model. This comparison shows that the equivalent core model provides a qualitative view of the different properties of initial and core-hole electronic structure. It is also shown that a very serious limitation of the equivalent core model is that it fails to distinguish between initial and final state contributions to the shifts of BEs which seriously reduces the utility of the information obtained with the equivalent core model. Indeed, there is a danger of making an incorrect assignment of the importance of relaxation because the equivalent core model appears to stress the role of final state effects. Given the importance of the distinction of initial and final state effects, we provide rigorous definitions of these two effects and we discuss an example where an incorrect interpretation was made based on the use of the equivalent core model.

LPS Modulates the Expression of Iron-Related Immune Genes in Two Antarctic Notothenoids.

The non-specific immunity can induce iron deprivation as a defense mechanism against potential bacterial pathogens, but little information is available as to its role in Antarctic fish. In this study the response of iron metabolism related genes was evaluated in liver and head kidney of the Antarctic notothenoids Notothenia coriiceps and Notothenia rossii 7 days after lipopolysaccharide (LPS) injection. Average plasma Fe2+ concentration was unaffected by treatment in any of the species. The gene expression response to LPS varied between tissues and species, being stronger in N. coriiceps and more prominent in the head kidney than liver. The reaction to LPS was marked by increased individual variability in most genes analyzed, even when the change in expression was not statistically significant, suggesting different individual sensitivity and coping responses in these wild fish. We found that iron related genes had an attenuated and homogenous response to LPS but there was no detectable relationship between plasma Fe2+ and gene expression. However, overall in both tissues and species LPS exposure set a multilevel response that concur to promote intracellular accumulation of iron, an indication that Antarctic Notothenoids use innate nutritional immunity as a resistance mechanism against pathogens.

Effect of electron correlation in the decomposition of core level binding energy shifts into initial and final state contributions.

The influence of electron correlation into the decomposition of core level binding energy shifts, measured by X-ray photoelectron spectroscopy (XPS), into initial and final effects is analysed for a series of molecules where these effects are noticeable. Moreover, the series of molecules is chosen in such a way that electron delocalization and increasing number of electrons may provide a large screening of the core hole. A detailed analysis shows that the Hartree-Fock decomposition is biased whereas a physically meaningful decomposition is obtained when electron correlation effects are taken into account. The results show that in this case, trends in core level binding energy shifts are driven by initial state effects thus providing further support to the use of these observable quantities to interpret changes in the chemical bond in the neutral molecule rather than on the core ionized cation. Consequences for the theoretical interpretation of XPS data in materials and surface science are discussed.

Deactivation of Excited States in Transition-Metal Complexes: Insight from Computational Chemistry.

Investigation of the excited-state decay dynamics of transition-metal systems is a crucial step for the development of photoswitchable molecular based materials with applications in growing fields as energy conversion, data storage, or molecular devices. The photophysics of these systems is an entangled problem arising from the interplay of electronic and geometrical rearrangements that take place on a short time scale. Several factors play a role in the process: various electronic states of different spin and chemical character are involved, the system undergoes important structural variations and several nonradiative processes can occur. Computational chemistry is a useful tool to get insight into the microscopic description of the photophysics of these materials, since it provides unique information about the character of the electronic spin states involved, the energetics and time evolution of the system. In this review article, we present an overview of the state of the art methodologies available to address the several aspects that have to be incorporated to properly describe the deactivation of excited states in transition-metal complexes. The most recent developments in theoretical methods are discussed and illustrated with examples.

On the prediction of core level binding energies in molecules, surfaces and solids.

Core level binding energies, directly measured by X-ray photoelectron spectroscopy (XPS), provide unique information regarding the chemical environment of atoms in a given system. However, interpretation of XPS in extended systems may not be straightforward and requires assistance from theory. The different state-of-the-art theoretical methods commonly used to approach core level binding energies and their shifts with respect to a given reference are reviewed and critically assessed with special emphasis on recently developed theoretical methods and with a focus on future applications in materials and surface sciences.