Molecular dissection of the domain architecture and catalytic activities of human PrimPol

PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication

A New Integrative and Mobilizable Element Is a Major Contributor to Tetracycline Resistance in Streptococcus dysgalactiae subsp. equisimilis

Tetracycline resistance in streptococci is mainly due to ribosomal protection mediated by the tet(M) gene that is usually located in the integrative and conjugative elements (ICEs) of the Tn916-family. In this study, we analyzed the genes involved in tetracycline resistance and the associated mobile genetic elements (MGEs) in Streptococcus dysgalactiae subsp. equisimilis (SDSE) causing invasive disease. SDSE resistant to tetracycline collected from 2012 to 2019 in a single hospital and from 2018 in three other hospitals were analyzed by whole genome sequencing. Out of a total of 84 SDSE isolates, 24 (28.5%) were resistant to tetracycline due to the presence of tet(M) (n = 22), tet(W) (n = 1), or tet(L) plus tet(W) (n = 1). The tet(M) genes were found in the ICEs of the Tn916-family (n = 10) and in a new integrative and mobilizable element (IME; n = 12). Phylogenetic analysis showed a higher genetic diversity among the strains carrying Tn916 than those having the new IME, which were closely related, and all belonged to CC15. In conclusion, tetracycline resistance in SDSE is mostly due to the tet(M) gene associated with ICEs belonging to the Tn916-family and a new IME. This new IME is a major cause of tetracycline resistance in invasive Streptococcus dysgalactiae subsp. equisimilis in our settings

Real-time quantification of microRNAs by stem–loop RT–PCR

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency

On a model-based approach to improve intranasal spray targeting for respiratory viral infections

The nasopharynx, at the back of the nose, constitutes the dominant initial viral infection trigger zone along the upper respiratory tract. However, as per the standard recommended usage protocol (“Current Use”, or CU) for intranasal sprays, the nozzle should enter the nose almost vertically, resulting in sub-optimal nasopharyngeal drug deposition. Through the Large Eddy Simulation technique, this study has replicated airflow under standard breathing conditions with 15 and 30 L/min inhalation rates, passing through medical scan-based anatomically accurate human airway cavities. The small-scale airflow fluctuations were resolved through use of a sub-grid scale Kinetic Energy Transport Model. Intranasally sprayed droplet trajectories for different spray axis placement and orientation conditions were subsequently tracked via Lagrangian-based inert discrete phase simulations against the ambient inhaled airflow field. Finally, this study verified the computational projections for the upper airway drug deposition trends against representative physical experiments on sprayed delivery performed in a 3D-printed anatomic replica. The model-based exercise has revealed a new “Improved Use” (or, IU) spray usage protocol for viral infections. It entails pointing the spray bottle at a shallower angle (with an almost horizontal placement at the nostril), aiming slightly toward the cheeks. From the conically injected spray droplet simulations, we have summarily derived the following inferences: (a) droplets sized between 7–17 μm are relatively more efficient at directly reaching the nasopharynx via inhaled transport; and (b) with realistic droplet size distributions, as found in current over-the-counter spray products, the targeted drug delivery through the IU protocol outperforms CU by a remarkable 2 orders-of-magnitude

On a model-based approach to improve intranasal spray targeting for respiratory viral infections

The nasopharynx, at the back of the nose, constitutes the dominant initial viral infection trigger zone along the upper respiratory tract. However, as per the standard recommended usage protocol (“Current Use”, or CU) for intranasal sprays, the nozzle should enter the nose almost vertically, resulting in sub-optimal nasopharyngeal drug deposition. Through the Large Eddy Simulation technique, this study has replicated airflow under standard breathing conditions with 15 and 30 L/min inhalation rates, passing through medical scan-based anatomically accurate human airway cavities. The small-scale airflow fluctuations were resolved through use of a sub-grid scale Kinetic Energy Transport Model. Intranasally sprayed droplet trajectories for different spray axis placement and orientation conditions were subsequently tracked via Lagrangian-based inert discrete phase simulations against the ambient inhaled airflow field. Finally, this study verified the computational projections for the upper airway drug deposition trends against representative physical experiments on sprayed delivery performed in a 3D-printed anatomic replica. The model-based exercise has revealed a new “Improved Use” (or, IU) spray usage protocol for viral infections. It entails pointing the spray bottle at a shallower angle (with an almost horizontal placement at the nostril), aiming slightly toward the cheeks. From the conically injected spray droplet simulations, we have summarily derived the following inferences: (a) droplets sized between 7–17 μm are relatively more efficient at directly reaching the nasopharynx via inhaled transport; and (b) with realistic droplet size distributions, as found in current over-the-counter spray products, the targeted drug delivery through the IU protocol outperforms CU by a remarkable 2 orders-of-magnitude

A 'small-world-like' model for comparing interventions aimed at preventing and controlling influenza pandemics

BACKGROUND: With an influenza pandemic seemingly imminent, we constructed a model simulating the spread of influenza within the community, in order to test the impact of various interventions. METHODS: The model includes an individual level, in which the risk of influenza virus infection and the dynamics of viral shedding are simulated according to age, treatment, and vaccination status; and a community level, in which meetings between individuals are simulated on randomly generated graphs. We used data on real pandemics to calibrate some parameters of the model. The reference scenario assumes no vaccination, no use of antiviral drugs, and no preexisting herd immunity. We explored the impact of interventions such as vaccination, treatment/prophylaxis with neuraminidase inhibitors, quarantine, and closure of schools or workplaces. RESULTS: In the reference scenario, 57% of realizations lead to an explosive outbreak, lasting a mean of 82 days (standard deviation (SD) 12 days) and affecting 46.8% of the population on average. Interventions aimed at reducing the number of meetings, combined with measures reducing individual transmissibility, would be partly effective: coverage of 70% of affected households, with treatment of the index patient, prophylaxis of household contacts, and confinement to home of all household members, would reduce the probability of an outbreak by 52%, and the remaining outbreaks would be limited to 17% of the population (range 0.8%–25%). Reactive vaccination of 70% of the susceptible population would significantly reduce the frequency, size, and mean duration of outbreaks, but the benefit would depend markedly on the interval between identification of the first case and the beginning of mass vaccination. The epidemic would affect 4% of the population if vaccination started immediately, 17% if there was a 14-day delay, and 36% if there was a 28-day delay. Closing schools when the number of infections in the community exceeded 50 would be very effective, limiting the size of outbreaks to 10% of the population (range 0.9%–22%). CONCLUSION: This flexible tool can help to determine the interventions most likely to contain an influenza pandemic. These results support the stockpiling of antiviral drugs and accelerated vaccine development

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A multilayered post-GWAS assessment on genetic susceptibility to pancreatic cancer

Funder: Fundación Científica Asociación Española Contra el Cáncer (ES)Funder: Cancer Focus Northern Ireland and Department for Employment and LearningFunder: Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, USAAbstract: Background: Pancreatic cancer (PC) is a complex disease in which both non-genetic and genetic factors interplay. To date, 40 GWAS hits have been associated with PC risk in individuals of European descent, explaining 4.1% of the phenotypic variance. Methods: We complemented a new conventional PC GWAS (1D) with genome spatial autocorrelation analysis (2D) permitting to prioritize low frequency variants not detected by GWAS. These were further expanded via Hi-C map (3D) interactions to gain additional insight into the inherited basis of PC. In silico functional analysis of public genomic information allowed prioritization of potentially relevant candidate variants. Results: We identified several new variants located in genes for which there is experimental evidence of their implication in the biology and function of pancreatic acinar cells. Among them is a novel independent variant in NR5A2 (rs3790840) with a meta-analysis p value = 5.91E−06 in 1D approach and a Local Moran’s Index (LMI) = 7.76 in 2D approach. We also identified a multi-hit region in CASC8—a lncRNA associated with pancreatic carcinogenesis—with a lowest p value = 6.91E−05. Importantly, two new PC loci were identified both by 2D and 3D approaches: SIAH3 (LMI = 18.24), CTRB2/BCAR1 (LMI = 6.03), in addition to a chromatin interacting region in XBP1—a major regulator of the ER stress and unfolded protein responses in acinar cells—identified by 3D; all of them with a strong in silico functional support. Conclusions: This multi-step strategy, combined with an in-depth in silico functional analysis, offers a comprehensive approach to advance the study of PC genetic susceptibility and could be applied to other diseases

Conception et mise en oeuvre d’une approche bioinformatique dédiée à l’identification des ICE, IME et éléments composites dans les génomes de Firmicutes

ICEs (Integrative Conjugative Elements) and IMEs (Integrative Mobilizable Elements) are bacterial mobile elements that play a key role in horizontal transfers. They have the capacity to integrate and transfer by conjugation from one bacterium to another. These elements are widespread in bacterial genomes but are still poorly understood. Their automatic identification is a challenge and they are generally not annotated in genomes. So far, only two bioinformatic approaches allow the detection of ICEs and IMEs, but their reliability remains highly variable, particularly among Firmicutes. Moreover, neither of these approaches can accurately detect composite elements consisting of nested or accreted ICEs and IMEs, which are frequently observed in bacterial genomes. We have developed a strategy and a tool called ICEscreen to identify ICEs and IMEs in the genomes of Firmicutes, including nested or accreted elements. Our tool starts with the detection of four signature proteins (SPs) that are essential to the functioning of these elements and then carries out the detection and typing of the elements based on the colocalization of the SPs and the characterisation of their content. Our tool uses a dedicated algorithm to solve the structure of the elements whether they are composite or not. To perform these steps, we have built a bank of ICEs and IMEs signature proteins from a list of genes known to be involved in the dynamics of these elements in streptococci and also public HMM profiles and HMM profiles constructed especially for this study. To validate the ICEscreen results, we built a dataset, FirmiData, consisting of 40 genomes of Firmicutes for which the ICEs and IMEs were annotated semi-manually and curated. We then compared the results of ICEscreen with those of two reference tools: CONJscan and ICEfinder. ICEscreen detects almost all the elements of the reference (96%) making it a more powerful tool than CONJscan (58%) and especially ICEfinder (53%) on our dataset. ICEscreen is thus a tool for the annotation and discovery of ICE and IME in the genomes of Firmicutes, which can help to better characterize their contribution to horizontal gene transfers, particularly during the transmission of antibiotic resistance, with which they are frequently associated.Les ICE (Éléments intégratifs conjugatifs) et les IME (Éléments intégratifs mobilisables) sont des éléments mobiles bactériens qui jouent un rôle clé dans les transferts horizontaux. Ils ont la capacité de s'intégrer et de se transférer par conjugaison d'une bactérie à une autre. Ces éléments sont très répandus dans les génomes bactériens mais sont encore mal connus. Leur identification automatique est un défi et ils ne sont en général pas annotés dans les génomes. Jusqu'à présent, seules deux approches bioinformatiques permettent la détection des ICE et la détection des IME, mais leur fiabilité reste très variable, en particulier chez les Firmicutes. De plus, aucune de ces approches ne permet de détecter avec précision les éléments composites constitués d’ICE et d’IME emboîtés ou en accrétions, qui sont fréquemment observés dans des génomes bactériens. Nous avons développé une stratégie et un outil nommé ICEscreen permettant d’identifier les ICE et IME dans les génomes des Firmicutes, y compris les éléments emboîtés ou en accrétions. Notre outil, commence par la détection de quatre protéines signatures (SP) indispensables au fonctionnement de ces éléments puis effectue la détection et le typage des éléments à partir de la colocalisation des SP et de la caractérisation de leur contenu. Notre outil utilise un algorithme dédié permettant de résoudre la structure des éléments qu'ils soient composites ou non. Pour réaliser ces étapes nous avons construit une banque de protéines signatures d’ICE et d’IME de référence à partir d’une liste de gènes connus pour être impliqués dans la dynamique de ces éléments chez les streptocoques ainsi que de profils HMM publics ou construits pour cette étude. Pour valider les résultats d’ICEscreen nous avons construit un jeu de données, FirmiData, constitué de 40 génomes de Firmicutes pour lesquels les ICE et IME ont été annotés semi-manuellement et expertisés. Nous avons ensuite comparé les résultats de ICEscreen avec ceux de deux outils de référence : CONJscan et ICEfinder. ICEscreen détecte la quasi-totalité des éléments de la référence (96 %) ce qui en fait un outil plus performant que CONJscan (58 %) et surtout ICEfinder (53 %) sur notre jeu de données. ICEscreen est ainsi un outil d’aide à l’annotation et à la découverte d’ICE et d’IME dans les génomes de Firmicutes, ce qui peut aider à mieux caractériser leur contribution aux transferts horizontaux de gènes, notamment lors de la transmission de la résistance aux antibiotiques, auxquels ils sont fréquemment associés