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Abstract Deoxynucleoside 5-monophosphate N -glycosidase, DNPH1 is a member of the nucleoside 2-deoxyribosyltransferase (NDT) family. This enzyme catalyzes the hydrolysis of deoxynucleoside monophosphates into free nucleobase moieties and 2-deoxyribose 5-phosphates. The DNPH1 enzymatic activity was first demonstrated in rats and then in human s. Subsequently the DNPH1 gene was identified in a variety of organisms, mainly in Metazoa. Herein, we demonstrate that despite DNPH1 genes being distributed in almost all metazoans, the occurrence of DNPH1genes is mosaic. For example, they cannot be found anywhere in the entire clade of Sauropsida or anywhere in the whole phyla of Arthropoda and Ctenophora. Even among mammals, there are organisms without functional DNPH1 protein (Camelidae and most likely Cetacea). By our knowledge, the DNPH1 gene is missing in plants, fungi and in majority of protists. Accordingly, the enzyme is apparently not of vital importance in all the branches of the Tree of Life. Surprisingly the DNPH1 gene may be found in archaea as well as in bacteria. This refers to the origin of the gene from the period before the archaea branched off from other bacteria. We show that the genomic and protein primary structures of DNPH1 are highly conserved and any modification in such a structure would result in conversion to a pseudogene, which could possibly be eliminated from the genome. Highlights • The DNPH1 gene structure has been conserved in all metazoans where present. • A majority of metazoans have a single functional DNPH1 gene. • The homolog of the DNPH1 gene is missing in many evolutionary branches. • DNPH1 is not a vital gene as many organisms can subsist without it. • DNPH1 in some archaea and bacteria is indistinguishable from that in Metazoa. [ABSTRACT FROM AUTHOR]

Deoxynucleoside 5-monophosphate N-glycosidase or DNPH1 (former name Rcl) is a nucleotide hydrolase whose expression in mammalian cancer tissues has been associated with its tumorigenic potential. Therefore, the enzyme has been studied principally in rat and human models. We found the corresponding gene also in the freshwater sponge Ephydatia muelleri, an animal phylogenetically very distant from mammals. Here we report the expression and characterization of the recombinant DNPH1 from E. muelleri . The ancient homolog of mammalian enzyme in a sponge showed the substrate specificity and catalytic efficiency similar to that in higher animals. E. muelleri DNPH1 is inhibited by the purine nucleotides with different numbers of 5′-phosphate groups (n = 1–4). Our results demonstrate that GTP but also dGTP are the best inhibitors, followed by all other purine nucleotides that were tested. Hence, the functioning of DNPH1 in cells where the natural ATP and GTP concentrations are much higher than those of the substrates, dNMPs, should normally be downregulated. We demonstrate for the first time the existence of biologically relevant natural inhibitors of DNPH1, namely ATP and GTP. [ABSTRACT FROM AUTHOR]

Drug-checking is an ensemble of different harm reduction techniques providing people the ability to test illegally purchased drugs for strength, the presence of particular substances, and possible adulterants. Drug-checking research has primarily focused on nightlife and festival communities of people who use drugs and has overlooked how it functions as a knowledge forming process, particularly by people whose drug use is more stigmatized. The implementation of Fourier-Transform Infrared Spectroscopy (FTIR) in Vancouver, Canada's Downtown Eastside in response to the overdose crisis has made it possible for people who use drugs to receive information about the drugs that they are consuming. Using insights developed from the 'ontological turn' and approaches to co-production from public health and science and technology studies, we explore the multiple relations that come to produce and contest drug-checking knowledge in this setting. We look at how knowledge is produced by and for people who use drugs, including people who use drugs operating the FTIR. Using rapid ethnographic assessment and semi-structured interviews, participants were recruited from a low-barrier supervised injection facility to explore their experience of drug-checking. Data were coded in NVivo 12 using an initial coding scheme, as well as an iterative coding scheme as the data were explored. We find that the traditional demarcation between lay and expert, or peer and professional, which co-production idioms often rely on, creates barriers to seeing the different knowledge formations of drug-checking knowledge, and instead offer up a new idiom, trans-production, to explore how knowledge and harm reduction services are mutually enacted. • Drug-checking research has overlooked how it functions as a knowledge forming process. • Explores how drug-checking knowledge is produced by and for people who use drugs (PWUD). • PWUD extended the reach of the data collected, to make meaning of the results. • PWUD have other ways of knowing that help explain certain excipients or adulterants. [ABSTRACT FROM AUTHOR]

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare form of autosomal recessive combined primary immunodeficiency caused by a enzyme defect leading to the accumulation of inosine, 2'-deoxy-inosine (dIno), guanosine, and 2'-deoxy-guanosine (dGuo) in all cells, especially lymphocytes. Treatments are available and curative for PNP deficiency, but their efficacy depends on the early approach. PNP--combined immunodeficiency complies with the criteria for inclusion in a newborn screening program. Objective: This study evaluate whether mass spectrometry can identify metabolite abnormalities in dried blood spots (DBSs) from affected patients, with the final goal of individuating the disease at birth during routine newborn screening. Methods: DBS samples from 9 patients with genetically confirmed PNP-combined immunodeficiency, 10,000 DBS samples from healthy newborns, and 240 DBSs from healthy donors of different age ranges were examined. Inosine, dIno, guanosine, and dGuo were tested by using tandem mass spectrometry (TMS). T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) levels were evaluated by using quantitative RT-PCR only for the 2 patients (patients 8 and 9) whose neonatal DBSs were available. Results: Mean levels of guanosine, inosine, dGuo, and dIno were 4.4,133.3, 3.6, and 3.8 µmol/L, respectively, in affected patients. No indeterminate or false-positive results were found. In patient 8 TREC levels were borderline and KREC levels were abnormal; in patient 9 TRECs were undetectable, whereas KREC levels were normal. Conclusion: TMS is a valid method for diagnosis of PNP deficiency on DBSs of affected patients at a negligible cost. TMS identifies newborns with PNP deficiency, whereas TREC or KREC measurement alone can fail. [ABSTRACT FROM AUTHOR]

Background: Adenosine deaminase (ADA)–severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. Objective: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. Methods: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2′-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. Results: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value, <1.5 μmol/L) and 2′-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value, <0.07 μmol/L); the mean levels of adenosine and 2′-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. Conclusion: Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency. [Copyright &y& Elsevier]

Abstract: In this study we consider a population subdivided in groups. Each group is connected to two neighboring groups by means of migration channels. The migration channels are used with different probability, according to a gradient that gives a preferential direction of colonization. Within groups we have a fast population dynamics modeled with a Moran process. Between groups we have a process of colonization selection at low migration rate, based on the propagule model. We consider resident and invading individuals, with the invading individuals having a relative fitness r. Groups composed only of invaders will also have a relative group fitness R. We show that, independently of the value of r, a strong gradient reduces the advantage of groups with large fitness and thus works against group selection. [Copyright &y& Elsevier]

• Replication and tropism of SARS-CoV-2 in cattle, sheep, and pigs using EVOCs, were investigated. • Respiratory tissues of cattle and sheep, but not those of pigs, are able to sustain viral replication. • A SARS-CoV-2 isolate harbouring mutation D614 G in the S protein has greater replication capabilities. • SARS-CoV-2 binds to ACE2-expressing cells of the respiratory tract of cattle and sheep. There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus. [ABSTRACT FROM AUTHOR]

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Å resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an 15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages usecommonparticle-opening mechanisms but have specialized into different infection niches. [ABSTRACT FROM AUTHOR]

Abstract Previously we had discovered unusual enzymatic activity in the marine sponge Axinella polypoides , ATP N -glycosidase (Reintamm et al., 2003). We show here that the Ephydatia muelleri mRNA encoding protein with PNP_UDP_1 (phosphorylase superfamily) signature is the secreted ATP N -glycosidase. The functionality of the protein was established by recombinant expression in Pichia pastoris. In addition to the enzymatic domain, the full-length protein contains the N-terminal cysteine-rich domain belonging to the subfamily SCP_HrTT-1 (cd05559) of the SCP (sperm coating protein) superfamily (cl00133). Highlights • The first protein with ATP N -glycosidase activity was identified. • The protein is a distant member of the PNP_UDP_1 superfamily. • The full-length protein from Ephydatia muelleri has an additional N-terminal SCP domain. • The ATP N -glycosidase from E. muelleri is a secreted protein. • The expression of ATP N -glycosidase in Pichia pastoris induces massive adenine production. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUV-primed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNV E-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses. • USUV ip infection stimulates protective immunity in CD-1 and BALB/c mice against lethal WNV challenges. • LD USUV-primed mice, although showing no detectable neutralizing antibodies, are largely protected from lethal WNV challenges. • Immunization with WNV E-USUV protects adult CD-1 mice against a lethal WNV L2 challenge. [ABSTRACT FROM AUTHOR]

Highlights: [•] Streptococcus pneumoniae is present in the nasopharyngeal swab of the majority of children 0–5 years. [•] Streptococcus pneumoniae is present in the nasopharyngeal swab of children vaccinated with 7-valent pneumococcal conjugate vaccine. [•] within the short term (1 year) from vaccination, the carriage of the 7 vaccine serotypes of Streptococcus pneumoniae decreases and increases in the following years. [Copyright &y& Elsevier]

Abstract: Background and aim of the work: Since the introduction of the 7-valent vaccine, invasive pneumococcal disease have greatly decreased; however, changes in the distribution of pneumococcal serotypes have recently highlighted the need for vaccines with wider coverage. The aim of the work was to assess the potential serotype coverage of three pneumococcal conjugate vaccines (7-, 10- and 13-valent) against bacteremic pneumococcal pneumonia and meningitis/sepsis in Italian children. Patients and methods: We determined pneumococcal serotypes in immunocompetent patients who had been admitted to hospital with suspicion of invasive bacterial disease and had confirmed bacteremic pneumococcal pneumonia or meningitis/sepsis determined by molecular detection of Streptococcus pneumoniae in a normally sterile site. Positive samples were serotyped using Realtime-PCR. Results: Between April 2008 and March 2011, a total of 144 patients (age median 4.1 years; Interquartile range 1.8–5.6) with pneumococcal meningitis/sepsis (n =43) or pneumonia (n =101) from 83 participating centers located in 19 of 20 Italian regions were serotyped. The 10 most prevalent serotypes were 1 (29.9%), 3 (16.0%), 19A (13.2%), 7F (8.3%), 5 (4.2%), 14 (4.2%), 6A (3.5%), 6B (3.5%), 18C (3.5%), 19F (3.5%). Overall, serotype coverage for PCV-7, -10 and -13 were respectively 19.4%, 61.8% and 94.4% with no statistical difference between pneumonia and meningitis/sepsis. Potential coverage was similar for children 0–2 or 2–5 years of age. Cultures resulted positive in 35/99 (35.4%) samples simultaneously obtained for both culture and RT-PCR. Conclusion: These findings indicate that increasing the potential serotype coverage by introducing PCV13 in the vaccination schedule for infancy could provide substantial added benefit for protection from pneumococcal pneumonia or meningitis/sepsis in Italy in children below 2 years as well in older children. The importance of molecular methods for diagnosis and serotyping of invasive pneumococcal disease was confirmed. [Copyright &y& Elsevier]

Abstract: A new 13-valent conjugated polysaccharide vaccine (PCV13) against Streptococcus pneumoniae infections, which replaced the 7-valent vaccine (PCV7) in the regional immunization programmes for newborns and children who started but not completed the 3 doses schedule of PCV7, is available in Italy since 2010. The opportunity of administering a further dose of PCV13 to children under 5 years of age who had already completed their vaccination with PCV7, with the aim of extending the serotype coverage, triggered an animated scientific debate. The purpose of this study was to perform a clinical/economic evaluation of the administration of a dose of PCV13, in a catch-up programme, for children under 5 years of age, who had already received 3 doses of PCV7. A mathematical model of the clinical/economic impact of the adoption of 4 catch-up strategies with PCV13 (children up to 24, 36, 48 and 60 months old) was set up, with a vaccination coverage of 80%, versus immunization with 3 doses of PCV7 without the catch-up programme. The time span covered by the simulation was 5.5 years. The following clinical outcomes of infection were evaluated: hospitalised meningitis/sepsis, hospitalised bacteraemic pneumonias (complicated and uncomplicated), hospitalised non-bacteraemic pneumonias, and non-hospitalised pneumonias. The administration of one dose of PCV13 to children up to 60 months of age significantly reduces the number of cases of pneumococcal diseases (especially, non-hospitalised pneumonias, 80% of all events prevented, and hospitalised cases of non-bacteraemic pneumococcal pneumonias, 15% of all events prevented) and, subsequently, the relative cost for medical treatment. This results in savings for medical costs amounting to more than 1,000,000 Euros when vaccinating children under 24 months of age (up to almost 3 million Euros for children up to 60 months). More than half of those savings are attributable to avoided hospitalised cases of non-bacteraemic pneumococcal pneumonias. Increasing the number of cohorts involved in the vaccination programme, the impact of immunization increases. The average cost per event avoided is 1674 Euros vaccinating children up to 24 months, and increases to 2522 Euros by vaccinating up to 60 months of age. The cost per year of life saved for different vaccination strategies is always acceptable (from 12,250 Euros to 22,093 Euros). The results of this study justify, even from the economic point of view, the recommendation of the Italian Ministry of Health to vaccinate children up to 24 months of life in a catch-up programme, as well as the administration of PCV13 children up to 36 months of age, already used in some Italian regions. Furthermore, a catch-up programme that provides the immunization of children under 60 months of age, is also justified from both the economic and clinical point of view. [Copyright &y& Elsevier]

• WNV RNA was identified in September 2016 in two chickens sera in Sejnene in the north of Tunisia. • LSTD (day time land surface temperature) and LSTN (night time Land Surface Temperature) values were lower in Sejnene compared to Tozeur and Moknine. • The Tunisian sequence was close to those of WNV strains detected in a pool of mosquitoes in 2016 and in organs of a sparrow hawk (Accipiter nisus) in 2017 in Italy. Tunisia has experienced various West Nile disease outbreaks. Notwithstanding the serological and molecular confirmations in humans, horses and birds, the human surveillance system can still be improved. Three sentinel chicken flocks were placed in different Tunisian endemic regions and followed up from September 2016 to January 2017. A total of 422 sera from Sejnene (north of Tunisia), 392 from Moknine (east coast of Tunisia) and 386 from Tozeur (south of Tunisia) were tested for West Nile-specific antibodies and viral RNA. The WNV elisa positive rate in sentinel chickens in Sejnene was 10.7% (95% CI: 5.08-21.52). No positive samples were detected in Moknine. In Tozeur, the overall serological elisa positive rate during the study period was 9.8% (95% CI:4.35-21.03). West Nile virus nucleic acid was detected in two chickens in Sejnene.Phylogenetic analysis of one of the detected partial NS3 gene sequences showed that recent Tunisian WNV strain belong to WNV lineage 1 and is closely related to Italian strains detected in mosquitoes in 2016 and in a sparrow hawk in 2017. This report showed the circulation, first molecular detection and sequencing of WNV lineage 1 in chickens in the north of Tunisia and highlights the use of poultry as a surveillance tool to detect WNV transmission in a peri-domestic area. Image, graphical abstract [ABSTRACT FROM AUTHOR]