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The transmission dynamics and burden of SARS-CoV-2 in many regions of the world is still largely unknown due to the scarcity of epidemiological analyses and lack of testing to assess the prevalence of disease. In this work, we develop a quantitative framework based on excess mortality data to reconstruct SARS-CoV-2 transmission dynamics and assess the level of underreporting in infections and deaths. Using weekly all-cause mortality data from Iran, we are able to show a strong agreement between our attack rate estimates and seroprevalence measurements in each province and find significant heterogeneity in the level of exposure across the country with 11 provinces reaching near 100% attack rates. Despite having a young population, our analysis reveals that incorporating limited access to medical services in our model, coupled with undercounting of COVID-19-related deaths, leads to estimates of infection fatality rate in most provinces of Iran that are comparable to high-income countries., (© 2022. The Author(s).)

Cell lines allow studying various biological processes that may not be easily tractable in whole organisms. Here, we have established the first male-specific cell line from the African malaria mosquito, Anopheles gambiae. The cells, named AgMM and derived from the sex-sorted neonate larvae, were able to undergo spontaneous contractions for a number of passages following establishment, indicating their myoblast origin. Comparison of their transcriptome to the transcriptome of an A. gambiae-derived Sua5.1 hemocyte cells revealed distinguishing molecular signatures of each cell line, including numerous muscle-related genes that were highly and uniquely expressed in the AgMM cells. Moreover, the AgMM cells express the primary sex determiner gene Yob and support male sex determination and dosage compensation pathways. Therefore, the AgMM cell line represents a valuable tool for molecular and biochemical in vitro studies of these male-specific processes. In a broader context, a rich transcriptomic data set generated in this study contributes to a better understanding of transcribed regions of the A. gambiae genome and sheds light on the biology of both cell types, facilitating their anticipated use for various cell-based assays., (© 2022. The Author(s).)

In the last decades, the interest in biological activity of natural compounds has been growing. In plant protection, essential oils have been reported to exhibit antiviral, antimycotic, and antiparasitic activities, and are regarded as promising for the formulation of safe antimicrobial agents. Attention has also been focused on hydrosols, the by-products of hydro-distillation of essential oils. Their production is easy, fast, and cheap, and they seem to arise less concern for human health than essential oils. Plant viruses represent a major concern for agricultural crops since no treatment compound is available for virus control. This work was aimed at evaluating the antiphytoviral effectiveness of treatments with three essential oils and corresponding hydrosols extracted from Origanum vulgare , Thymus vulgaris , and Rosmarinus officinalis on Cucurbita pepo plants infected by zucchini yellow mosaic virus or tomato leaf curl New Delhi virus. Treatments were applied either concurrently or after virus inoculation to ascertain an inhibition or curative activity, respectively. Symptoms were observed and samplings were performed weekly. Virus titer and expression levels of phenylalanine ammonia lyase gene (PAL) were measured on treated and untreated infected plants by real-time PCR. PAL gene plays an important role in plant defense response as it is involved in tolerance/resistance to phytopathogens. Results indicated that treatments were effective against tomato leaf curl New Delhi virus whether applied simultaneously with the inoculation or after. A major inhibition was observed with O. vulgare essential oil and hydrosol, resulting in 10 -4 -fold decrease of virus titer 3 weeks after treatment. Curative activity gave maximum results with all three essential oils and T. vulgaris and R. officinalis hydrosols, recording from 10 -2 -fold decrease to virus not detected 4 weeks after treatment. An induction of PAL gene expression was recorded at 12 d.p.i. and then was restored to the levels of untreated control. This allows to hypothesize an early plant defense response to virus infection, possibly boosted by treatments. Plant extracts' composition was characterized by gas chromatography-mass spectrometry. Phenols were largely main components of O. vulgare and T. vulgaris extracts (carvacrol and thymol, respectively), while extracts from R. officinalis were based on monoterpene hydrocarbons (essential oil) and oxygenated monoterpenes (hydrosol)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Taglienti, Donati, Ferretti, Tomassoli, Sapienza, Sabatino, Di Massimo, Fiorentino, Vecchiarelli, Nota and Ragno.)

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 10 4 and 10 5 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences. We used the local branching index (LBI) as a proxy for transmission fitness. We found that LBI is more sensitive to differences in infectiousness than to differences in the duration of the infectious state. By analysing subtype-B samples from the Bridging the Evolution and Epidemiology of HIV in Europe project, we inferred a significant positive relationship between SPVL and LBI up to approximately 10 5 copies/ml, with some evidence for a peak around this value of SPVL. This is evidence of selection against low values of SPVL in HIV-1 subtype-B strains, likely related to lower infectiousness, and perhaps a peak in the transmission fitness in the expected range of SPVL. The less prominent signatures of selection against higher SPVL could be explained by an inherent limit of the method or the deployment of antiretroviral therapy., (© The Author(s) 2022. Published by Oxford University Press.)

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3'NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10 -5 -10 -6 -fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the first genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difficult to detect.

Background: During the Neolithic expansion, cattle accompanied humans and spread from their domestication centres to colonize the ancient world. In addition, European cattle occasionally intermingled with both indicine cattle and local aurochs resulting in an exclusive pattern of genetic diversity. Among the most ancient European cattle are breeds that belong to the so-called Podolian trunk, the history of which is still not well established. Here, we used genome-wide single nucleotide polymorphism (SNP) data on 806 individuals belonging to 36 breeds to reconstruct the origin and diversification of Podolian cattle and to provide a reliable scenario of the European colonization, through an approximate Bayesian computation random forest (ABC-RF) approach., Results: Our results indicate that European Podolian cattle display higher values of genetic diversity indices than both African taurine and Asian indicine breeds. Clustering analyses show that Podolian breeds share close genomic relationships, which suggests a likely common genetic ancestry. Among the simulated and tested scenarios of the colonization of Europe from taurine cattle, the greatest support was obtained for the model assuming at least two waves of diffusion. Time estimates are in line with an early migration from the domestication centre of non-Podolian taurine breeds followed by a secondary migration of Podolian breeds. The best fitting model also suggests that the Italian Podolian breeds are the result of admixture between different genomic pools., Conclusions: This comprehensive dataset that includes most of the autochthonous cattle breeds belonging to the so-called Podolian trunk allowed us not only to shed light onto the origin and diversification of this group of cattle, but also to gain new insights into the diffusion of European cattle. The most well-supported scenario of colonization points to two main waves of migrations: with one that occurred alongside with the Neolithic human expansion and gave rise to the non-Podolian taurine breeds, and a more recent one that favoured the diffusion of European Podolian. In this process, we highlight the importance of both the Mediterranean and Danube routes in promoting European cattle colonization. Moreover, we identified admixture as a driver of diversification in Italy, which could represent a melting pot for Podolian cattle.

The COVID-19 pandemic has seen the emergence of digital contact tracing to help to prevent the spread of the disease. A mobile phone app records proximity events between app users, and when a user tests positive for COVID-19, their recent contacts can be notified instantly. Theoretical evidence has supported this new public health intervention 1-6 , but its epidemiological impact has remained uncertain 7 . Here we investigate the impact of the National Health Service (NHS) COVID-19 app for England and Wales, from its launch on 24 September 2020 to the end of December 2020. It was used regularly by approximately 16.5 million users (28% of the total population), and sent approximately 1.7 million exposure notifications: 4.2 per index case consenting to contact tracing. We estimated that the fraction of individuals notified by the app who subsequently showed symptoms and tested positive (the secondary attack rate (SAR)) was 6%, similar to the SAR for manually traced close contacts. We estimated the number of cases averted by the app using two complementary approaches: modelling based on the notifications and SAR gave an estimate of 284,000 (central 95% range of sensitivity analyses 108,000-450,000), and statistical comparison of matched neighbouring local authorities gave an estimate of 594,000 (95% confidence interval 317,000-914,000). Approximately one case was averted for each case consenting to notification of their contacts. We estimated that for every percentage point increase in app uptake, the number of cases could be reduced by 0.8% (using modelling) or 2.3% (using statistical analysis). These findings support the continued development and deployment of such apps in populations that are awaiting full protection from vaccines.

Background: In May 2020, the UK National Health Service (NHS) Test and Trace programme was launched in England in response to the COVID-19 pandemic. The programme was first rolled out on the Isle of Wight and included version 1 of the NHS contact tracing app. The aim of the study was to make a preliminary assessment of the epidemiological impact of the Test and Trace programme using publicly available data., Methods: We used COVID-19 daily case data from Public Health England to infer incidence of new infections and estimate the reproduction number (R) for each of the 150 Upper-Tier Local Authorities (UTLAs) in England and nationally, before and after the launch of the Test and Trace programme on the Isle of Wight. We used Bayesian and maximum-likelihood methods to estimate R and compared the Isle of Wight with other UTLAs using a synthetic control method., Findings: We observed significant decreases in incidence and R on the Isle of Wight immediately after the launch of the Test and Trace programme. The Isle of Wight had a marked reduction in R, from 1·3 before the Test and Trace programme to 0·5 after by one of our measures, and went from having the third highest R before the Test and Trace programme, to the twelfth lowest afterwards compared with other UTLAs., Interpretation: Our results show that the epidemic on the Isle of Wight was controlled quickly and effectively after the launch of Test and Trace. Our findings highlight the need for further research to determine the causes of the reduction in the spread of the disease, as these could be translated into local and national non-pharmaceutical intervention strategies in the period before a treatment or vaccination for COVID-19 becomes available., Funding: Li Ka Shing Foundation and UK Economic and Social Research Council., (© 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution.

Doppel, a prion-like protein, is a GPI-membrane anchored protein generally not expressed in the Central Nervous System (CNS) of different mammalian species, including human. Nevertheless, in astrocytomas, a particular kind of glial tumors, the doppel encoding gene (PRND) is over-expressed and the corresponding protein product (Dpl) is ectopically localized in the cytoplasm of the tumor cells. In this study we have analysed the sub-cellular localization of Dpl using double-immunofluorescence staining and confocal microscopy examinations in two astrocytoma-derived human cell lines (IPDDC-A2 and D384-MG). Our results confirmed that Dpl is localized in the cytoplasm of the astrocytoma cells and indicated that it is mostly associated with Lamp-1 and Limp-2 positive lysosomal vesicles and, marginally, to the Golgi apparatus and other cellular organelles. Noticeably, none of the examined tumor cells showed a membrane-Dpl localization. The membrane-associated Dpl expression was restored after the transfection of the astrocytoma cells with mutated Dpl-expression vectors in its glycosylation sites. Additionally, Dpl showed altered expression and traffic using the acidotropic agent ammonium chloride, leading to the accumulation of Dpl in nascent exocytic vesicles. Altogether, these results indicated that in the astrocytic tumor cells Dpl has an altered biosynthetic trafficking, likely derived from abnormal post-translational processes: these modifications do not permit the localization of Dpl in correspondence of the plasma membrane and lead to its intracellular accumulation in the lysosomes. In these proteolytic compartments, the astrocytic tumor cells might provide to the degradation of the excess of a potentially cytotoxic Dpl product.

The speciation study of the Zn(2+)/glutathione (GSH, H(3)G) and Zn(2+)/N-acetylcysteinylglycine (NAcCG, H(2)L) was performed in aqueous solution by means of potentiometry and ESI mass spectrometry. The ligand N-acetylcysteinylglycine was synthesized by protection/activation strategies. (1)H NMR data for the Zn(2+)/NAcCG system at different pH were also collected, to gain insight in the coordination modes for the ligand. The information collected for the NAcCG model ligand were used to propose the structure in solution for the Zn(2+)/GSH complexes. Dinuclear complexes of GSH with Zn(2+), which have never been proposed previously in the literature, were identified in solution and a model of their structure was proposed. Moreover, the Zn(2+) promoted deprotonation of the cysteinyl peptidic NH with formation of five membered (S,N(Cys)(-)) chelating rings was evidenced. The speciation study of the ternary Zn(2+)/GSH/NAcCG system was also performed, showing that the Zn(2+) does not bind preferentially to GSH in presence of NAcCG. The (1)H NMR protonation studies of both GSH and NAcCG were also performed, and a novel proton dissociation microconstant calculation procedure has been proposed and applied to GSH equilibria.

The solution equilibria of gamma-aminobutanehydroxamic acid (GABAha) with H+ and Cu2+ were investigated by potentiometry, titration calorimetry, spectrophotometry, NMR spectroscopy, and ESI-MS. The thermodynamic parameters of the CuII [12]metallacrown-4 obtained for GABAha were compared with those of the corresponding complexes of (S)-alpha-Alaha and beta-Alaha. The stability (-DeltaG0) sequence was beta-Alaha>>alpha-Alaha>GABAha, whereas the order of formation enthalpies (-DeltaH0) was beta-Alaha>>GABAha>alpha-Alaha. These data were interpreted on the basis of the dimensions of the chelate rings and the planarity of the metallamacrocycles. The CuII [12]metallacrown-4 ([12]MC-4) complex of GABAha was isolated and its crystal structure, which is the first reported for a [12]MC-4 of a gamma-aminohydroxamic acid, fully supports the structural features interpreted from the thermodynamic data.

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

SPRN is a new prion-like gene coding for Sho, a protein with significant similarity to PrP. SPRN was initially described in zebrafish; however, the strong evolutionary conservation led to the hypothesis that SPRN might be the ancestral prion-like gene. We mapped SPRN in Bos taurus by comparative analysis of the locus and of the predicted flanking genes. BACs, spanning the whole SPRN genomic locus, were assigned to BTA26q23 by radiation hybrid mapping and fluorescent in situ hybridization (FISH). Sequencing of five genes flanking SPRN, namely, ECHS1, PAOX, MTG1, SPRN, and CYP2E1, high-resolution FISH on mechanically stretched chromosomes, and combed BAC DNA allowed us to establish their order and reciprocal orientation. The results confirmed that BTA26q23 corresponds to HSA10q24.3-26.3, which is the site where the human SPRN is located. The gene order in Bos taurus is the same as in man, cen-ECHS1-PAOX-MTG1-SPRN-CYP2E1-tel, but PAOX has a different orientation in the two species. SPRN has the typical two-exon PRNP arrangement, with the CDS fully contained within exon 2; furthermore, it codes for a 143-amino-acid protein with 74.8% identity and 84.7% similarity with the human PRNP. RT-PCR and Northern blot analysis showed that SPRN is expressed at high levels in brain and less in testis and lung.

Background: Doppel (Dpl) is a homologue of the prion protein (PrPC). In contrast to PrP(C), Dpl is dispensable for prion disease, but appears to have an essential function in male spermatogenesis. Recently, Dpl has been found to be aberrantly expressed in astrocytic and leukaemic tumor specimens, showing a peculiar cytosolic cellular localization. The aim of this study was to clarify some of the putative Dpl interacting proteins., Materials and Methods: A yeast two hybrid system was employed and the results were verified by co-immunoprecipitation using transfected cells., Results: Several potential Dpl-binding candidates were identified and, among them, the receptor for activated C-kinase (RACK1) protein was further investigated. RACK1 deletion mutants showed that some of its WD containing domains were directly involved in the binding with Dpl. Our data showed that Dpl interacts with RACK1 by means of its structured globular carboxyl-terminal region., Conclusion: This new Dpl interacting partner might suggest functional hypotheses about the role of this protein in an astrocytoma context where Dpl was found ectopically expressed.

The cellular prion protein (PrP(C)) is a highly conserved protein throughout the evolution of mammals and therefore is thought to play important cellular functions. Despite decades of intensive researches, the physiological function of PrP(C) remains enigmatic. Differently, in particular pathological contexts, generally referred as transmissible spongiform encephalopathies, a conformational isoform of PrP(C), i.e., PrP(Sc), is considered the causative agent of these diseases. In this study, we investigated putative PrP(C) cellular functions through the identification of PrP(C) protein interactants. Using a bacterial two-hybrid approach, we identified a novel interaction between PrP(C) and a two-pore potassium channel protein, TREK-1. This interaction was further verified in transfected eukaryotic cells using co-immunoprecipitation and confocal microscopic analysis of the fluorescent transfected proteins. Importantly, in the cerebellar cortex, the endogenous PrP(C) and TREK-1 proteins exhibited co-localization signals in correspondence of the Purkinje cells. Furthermore, a deletion mapping study defined the carboxyl-terminal regions of the two proteins as the possible determinants of the PrP(C)-TREK-1 interaction. Our results indicated a novel PrP(C) interacting protein and suggested that this complex might be relevant in modulating a variety of electrophysiological-dependent cellular responses.

Background: The Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus., Results: The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs., Conclusion: A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of the urinary bladder, and will be the basis to define the molecular structure of the bovine homologue of FRA3B, the major common fragile site of the human genome.

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

The genomic structure of the caprine Doppel gene (PRND) was determined using the ovine sequence as a scaffold to generate PCR fragments that were aligned with a cDNA sequence obtained from testicular mRNA. The caprine gene contains two exons, 89 and >2291 bp long, separated by a 1689-bp intron. Two mRNA isoforms of 3.2 and 4.8 kb were identified in the testis, as well as the exact transcription start site by fluorescently labeled oligonucleotide extension (FLOE). Like in sheep and cattle, the open reading frame (ORF) (537 bp) lies within exon 2 and is very much conserved in sheep (99.3%) and cattle (97%). The intronic sequence is also highly conserved (95.3%) compared with sheep, with the only exception of a 47-bp insertion. The PRND ORF was sequenced in 47 healthy and 17 TSE-affected goats of the Italian Ionica breed. Seven nucleotide positions showed variation: T28C, C65T, A151G, G286A, C385G, T451C, and T528C. Five were commonly represented polymorphisms: T28C, T451C, and T528C are silent mutations at codons L10, L151, and I176, respectively, while A151G and C385G determine a T51A and L129V amino acid change, respectively. The two remaining variants, C65T and G286A, were rare, leading to the amino acid substitutions S22F and E96K, respectively. None of the polymorphisms was significantly relatable to the TSE status, and the same result was obtained by the analysis of the combined haplotypes at the five major polymorphic sites, namely, T28C, C65T, A151G, G286A, and C385G.

Background: The doppel protein (Dpl) is a newly recognized cellular prion protein (PrP(C))-like molecule encoded by a novel gene locus, PRND, located on the same chromosomal region of the PrP(C) coding gene. Recently, Dpl was shown to be aberrantly expressed in astrocytic tumor specimens and in astrocytoma-derived cell lines, showing a peculiar cytoplasmic localization. Here, Dpl interactions with some of the prion-interacting proteins were studied. In particular, whether the tumor astrocytic environment is suitable for doppel interaction with GFAP and Grb2 proteins, as well as with the PrPC protein itself was investigated., Materials and Methods: In order to verify our hypothesis, an innovative mammalian two-hybrid system and co-immunoprecipitation assays were employed., Results: The results reported the absence of protein interactions. Our findings provided evidence that, in our astrocytoma cell-based model, Dpl does not share with PrP(C) the ability to interact with GFAP and Grb2., Conclusion: Identifying Dpl ligands may provide new insights into the involvement of Dpl in astrocytoma tumor progression.

The PRND gene encodes Doppel (Dpl), a protein that is strongly expressed in testis and at much lower levels in other tissues. Despite the recent discovery of Dpl involvement in spermiogenesis and in apoptotic death of cerebellar neurons, respectively in wild type and transgenic mice, the physiological role of this prion-like protein remains unknown. To better understand which factors may contribute to the modulation of PRND activity, a study of the bovine promoter region was performed. First, the transcription start site of PRND mRNA was identified using an innovative fluorescently labelled oligonucleotide extension (FLOE) method. The initiation site mapped 129 nt upstream of the protein coding sequence and represents a refinement of a previous assignment based on RACE. Second, deletion mutants of the 4530 nt encompassing 2704 nt 5' of the bovine PRND, exon 1, intron 1, and the first 6 nt of exon 2, have been investigated with CAT-reporter assays in order to identify critical elements for the activation of the gene. The results showed that the region -323/+32 (+1 is the transcription start site mapped by FLOE) represents the promoter region and contains positive cis-acting elements (CCAAT and E box) confirming previous reports with the mouse gene. Additional regulatory elements, including binding sites for repressor molecules, have been identified upstream of that region and in the first portion of intron 1, suggesting a complex tissue-specific regulation of Doppel gene expression.

We investigated the expression patterns and distribution of Doppel (Dpl), the product of the prion-like gene PRND, in the leukaemic cell lines HL-60 and K562 and in bone marrow cells from 44 patients with acute myeloid leukaemia (AML) and 63 patients with myelodysplastic syndrome (MDS). Whereas normal samples were negative or showed very weak expression that was restricted to CD34(+) cells, Dpl was detected in both cell lines and in most AML and MDS cases by immunocytochemistry and Western blotting. Quantitative reverse transcription polymerase chain reaction revealed variable mRNA levels in almost all AML and MDS cases, but barely detectable levels in normal bone marrow. These differences were confirmed by in situ hybridization. PRND expression was higher in advanced compared with early MDS (P = 0.01), but Dpl levels did not predict disease progression. In AML there was no correlation between Dpl levels and clinical or laboratory findings. In conclusion, this is the first time that the expression of PRND has been demonstrated in human bone marrow. The molecular mechanism of the observed overexpression is unknown; however, the differential Dpl distribution in AML and MDS versus healthy subjects makes it a possible leukaemia-associated antigen that could be useful for diagnostic and therapeutic purposes.

The expression of the prion (PRNP) and prion like-doppel (PRND) genes and the presence of the proteins prion (PrP) and doppel (Dpl) were investigated in human gliomas. The PRNP and PRND expression profiles were evaluated by real-time reverse transcription-quantitative PCR in low- and high-grade astrocytomas, in glioblastoma-derived cell lines and in non-glial tumor specimens. The presence of PrP and Dpl proteins and their cellular localization were evaluated by Western blot and immunohistochemistry. High levels of PRNP expression were found in all tumoral samples studied. Unlike the non-tumoral controls, PRND was aberrantly expressed in glioblastoma multiforme and in two glioblastoma multiforme-derived cell lines, even in the absence of the PRND gene amplification. PRND expression was directly related to malignancy of the tumor: highest in glioblastoma multiforme, lower in anaplastic astrocytoma and even lower in the low-grade astrocytoma samples. High levels of PRND were also found in non-glial malignant tumor samples, such as gastric adenocarcinoma and anaplastic meningioma. Western blot analysis confirmed the PrP and Dpl expression, displaying variability in the electrophoretic patterns. Immunohistochemical analysis revealed a diffuse cytoplasmatic Dpl distribution in different astrocytic neoplastic cells, in infiltrating lymphocytes and in blood vessel endothelial cells. Of note, Dpl reactivity was different from that of the PrP, since PrP showed typical Golgi and membrane localised staining. Our findings suggest that the PRND gene might be a useful molecular marker in astrocytoma progression and in tumor grade definition. Understanding of the mechanisms of PRND increased expression might provide insight into the regulatory pathways of glioma development.

A 3000-rad radiation hybrid panel was constructed for cattle and used to build outline RH maps for all 29 autosomes and the X and Y chromosomes. These outline maps contain about 1200 markers, most of which are anonymous microsatellite loci. Comparisons between the RH chromosome maps, other published RH maps, and linkage maps allow regions of chromosomes that are poorly mapped or that have sparse marker coverage to be identified. In some cases, mapping ambiguities can be resolved. The RH maps presented here are the starting point for mapping additional loci, in particular genes and ESTs that will allow detailed comparative maps between cattle and other species to be constructed. Radiation hybrid cell panels allow high-density genetic maps to be constructed, with the advantage over linkage mapping that markers do not need to be polymorphic. A large quantity of DNA has been prepared from the cells forming the RH panel reported here and is publicly available for mapping large numbers of loci.