When enough is enough: Optimising monitoring effort for large-scale wolf population size estimation in the Italian Alps.

The ongoing expansion of wolf (Canis lupus) populations in Europe has led to a growing demand for up-to-date abundance estimates. Non-invasive genetic sampling (NGS) is now widely used to monitor wolves, as it allows individual identification and abundance estimation without physically capturing individuals. However, NGS is resource-intensive, partly due to the elusive behaviour and wide distribution of wolves, as well as the cost of DNA analyses. Optimisation of sampling strategies is therefore a requirement for the long-term sustainability of wolf monitoring programs. Using data from the 2020-2021 Italian Alpine wolf monitoring, we investigate how (i) reducing the number of samples genotyped, (ii) reducing the number of transects, and (iii) reducing the number of repetitions of each search transect impacted spatial capture-recapture population size estimates. Our study revealed that a 25% reduction in the number of transects or, alternatively, a 50% reduction in the maximum number of repetitions yielded abundance estimates comparable to those obtained using the entire dataset. These modifications would result in a 2046 km reduction in total transect length and 19,628 km reduction in total distance searched. Further reducing the number of transects resulted in up to 15% lower and up to 17% less precise abundance estimates. Reducing only the number of genotyped samples led to higher (5%) and less precise (20%) abundance estimates. Randomly subsampling genotyped samples reduced the number of detections per individual, whereas subsampling search transects resulted in a less pronounced decrease in both the total number of detections and individuals detected. Our work shows how it is possible to optimise wolf monitoring by reducing search effort while maintaining the quality of abundance estimates, by adopting a modelling framework that uses a first survey dataset. We further provide general guidelines on how to optimise sampling effort when using spatial capture-recapture in large-scale monitoring programmes.

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution.

It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere's ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

An insight into the sialome, mialome and virome of the horn fly, Haematobia irritans.

BACKGROUND: The horn fly (Haematobia irritans) is an obligate blood feeder that causes considerable economic losses in livestock industries worldwide. The control of this cattle pest is mainly based on insecticides; unfortunately, in many regions, horn flies have developed resistance. Vaccines or biological control have been proposed as alternative control methods, but the available information about the biology or physiology of this parasite is rather scarce. RESULTS: We present a comprehensive description of the salivary and midgut transcriptomes of the horn fly (Haematobia irritans), using deep sequencing achieved by the Illumina protocol, as well as exploring the virome of this fly. Comparison of the two transcriptomes allow for identification of uniquely salivary or uniquely midgut transcripts, as identified by statistically differential transcript expression at a level of 16 x or more. In addition, we provide genomic highlights and phylogenetic insights of Haematobia irritans Nora virus and present evidence of a novel densovirus, both associated to midgut libraries of H. irritans. CONCLUSIONS: We provide a catalog of protein sequences associated with the salivary glands and midgut of the horn fly that will be useful for vaccine design. Additionally, we discover two midgut-associated viruses that infect these flies in nature. Future studies should address the prevalence, biological effects and life cycles of these viruses, which could eventually lead to translational work oriented to the control of this economically important cattle pest.

Retrospective analysis: reproducibility of interblastomere differences of mRNA expression in 2-cell stage mouse embryos is remarkably poor due to combinatorial mechanisms of blastomere diversification.

STUDY QUESTION: What is the prevalence, reproducibility and biological significance of transcriptomic differences between sister blastomeres of the mouse 2-cell embryo? SUMMARY ANSWER: Sister 2-cell stage blastomeres are distinguishable from each other by mRNA analysis, attesting to the fact that differentiation starts mostly early in the mouse embryo; however, the interblastomere differences are poorly reproducible and invoke the combinatorial effects of known and new mechanisms of blastomere diversification. WHAT IS KNOWN ALREADY: Transcriptomic datasets for single blastomeres in mice have been available for years but have never been systematically analysed together, although such an analysis may shed light onto some unclarified topics of early mammalian development. Two unknowns that remain are at which stage embryonic blastomeres start to diversify from each other and what is the molecular origin of that difference. At the earliest postzygotic stage, the 2-cell stage, opinions differ regarding the answer to these questions; one group claims that the first zygotic division yields two equal blastomeres capable of forming a full organism (totipotency) and another group claims evidence for interblastomere differences reminiscent of the prepatterning found in embryos of lower taxa. Regarding the molecular origin of interblastomere differences, there are four prevalent models which invoke (1) oocyte anisotropy, (2) sperm entry point, (3) partition errors of the transcript pool and (4) asynchronous embryonic genome activation in the two blastomeres. STUDY DESIGN, SIZE, DURATION: Seven transcriptomic studies published between 2011 and 2017 were eligible for retrospective analysis, since both blastomeres of the mouse 2-cell embryo had been analysed individually regarding the original pair associations and since the datasets were made available in public repositories. Five of these studies, encompassing a total of 43 pairs of sister blastomeres, were selected for further analyses based on high interblastomere correlations of mRNA levels. A double cut-off was used to select mRNAs that had robust interblastomere differences both within and between embryos (hits). The hits of each study were compared and contrasted with the hits of the other studies using Venn diagrams. The hits shared by at least four of five studies were analysed further by bioinformatics. PARTICIPANTS/MATERIALS, SETTING, METHODS: PubMed was systematically examined for mRNA expression profiles of single 2-cell stage blastomeres in addition to publicly available microarray datasets (GEO, ArrayExpress). Based on the original normalizations, data from seven studies were screened for pairwise sample correlation at the gene level (Spearman), and the top five datasets with the highest correlation were subjected to hierarchical cluster analysis. Interblastomere differences of gene expression were expressed as a ratio of the higher to the lower mRNA level for each pair of blastomeres. A double cut-off was used to make the call of interblastomere difference, accepting genes with mRNA ratios above 2 when observed in at least 50% of the pairs, and discarding the other genes. The proportion of interblastomere differences common to at least four of the five datasets was calculated. Finally, the corresponding gene, pathway and enrichment analyses were performed utilizing PANTHER and GORILLA platforms. MAIN RESULTS AND THE ROLE OF CHANCE: An average of 17% of genes within the datasets are differently expressed between sister blastomeres, a proportion which falls to 1% when considering the differences that are common to at least four of the five studies. Housekeeping mRNAs were not included in the 17% and 1% gene lists, suggesting that the interblastomere differences do not occur simply by chance. The 1% of shared interblastomere differences comprise 100 genes, of which 35 are consistent with at least one of the four prevalent models of sister blastomere diversification. Bioinformatics analysis of the remaining 65 genes that are not consistent with the four models suggests that at least one more mechanism is at play, potentially related to the endomembrane system. Although there are many dimensions to the issue of reproducibility (biological, experimental, analytical), we consider that the sister blastomeres are poised to escape high interblastomere correlations of mRNA levels, because at least five sources of diversity superimpose on each other, accounting for at least 25 = 32 different states. As a result, interblastomere mRNA differences of a given 2-cell embryo are necessarily difficult to reproduce in another 2-cell embryo. LARGE SCALE DATA: Data were as provided by the original studies (GSE21688, GSE22182, GSE27396, GSE45719, GSE57249, E-MTAB-3321, GSE94050). LIMITATIONS, REASONS FOR CAUTION: The original studies present similarities (e.g. fertilization in vivo after ovarian stimulation) as well as differences (e.g. mouse strains, method and timing of blastomere separation). We identified robust mRNA differences between the sister blastomeres, but these differences are underestimated because our double cut-off method works with thresholds and affords more protection against false positives than false negatives. Regarding the false negatives, transcriptome analysis may have captured only part of the interblastomere differences due to: (1) the 2-fold cut-off not being sensitive enough to detect the remaining part of the interblastomere differences, (2) the detection limit of the transcriptomic methods not being sufficient, or (3) interblastomere differences being oblivious to transcriptomic identification because transcriptional changes are oscillatory or because differences are mediated non-transcriptionally or post-transcriptionally. Regarding the false positives, it seems unlikely that a difference was found just by chance for the same group of transcripts due to the same technical error, given that different laboratories produced the data. WIDER IMPLICATIONS OF THE FINDINGS: It is clear that the sister blastomeres are distinguishable from each other by mRNA analysis even at the 2-cell stage; however, efforts to identify large stable patterns may be in vain. This elicits thoughts about the wisdom of adding new transcriptomic datasets to the ones that already exist; if all transcriptomic datasets produced so far show a reproducibility of 1%, then any future study would probably face the same issue again. Possibly, a solid identification of the 'large stable pattern that should be there but was not found' requires an even larger dataset than the sum of the seven datasets considered here. Conversely, small stable patterns may be easier to identify, but their biological relevance is less obvious. Alternatively, interblastomere differences may not be mediated by nucleic acids but by other cellular components. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Deutsche Forschungsgemeinschaft (grant DFG BO 2540-4-3 to M.B. and grant NO 413/3-3 to V.N.). The authors declare that they have no competing financial interests.

Totipotency segregates between the sister blastomeres of two-cell stage mouse embryos.

Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) - a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres' and blastocysts' transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

Cytotoxicity of 1,4-diamino-2-butanone, a putrescine analogue, to RKO cells: mechanism and redox imbalance.

α-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. In vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive α-oxoaldehyde and H2O2. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05-10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC50 ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 µM) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.

Governing cell lineage formation in cloned mouse embryos.

Blastomeres of the pre-implantation mouse embryo form trophectoderm and inner cell mass via a process that requires the transcription factors Tead4, Cdx2, Oct4 and Nanog. In mouse morulae cloned by somatic cell nuclear transfer, we observed that the trophectoderm transcription factor Cdx2 is expressed very differently at the protein level compared to time- and stage-matched fertilized counterparts. Protein levels of Cdx2 in cloned embryos appear 'erratic,' i.e. are widely distributed, when plotted as histograms. In contrast to Cdx2, protein levels of the upstream factor Tead4 and of inner cell mass transcription factors Oct4 and Nanog are similar in cloned and fertilized embryos. These observations suggest that trophectoderm formation is initiated but not maintained correctly in cloned mouse morulae, which is consistent with cloned blastocysts' limited implantation and post-implantation success. Because a cell's ability to differentiate is greatly enhanced if it is surrounded by more cells differentiating the same way, a concept designated community effect by Gurdon, we reasoned that the insufficient cell numbers often observed in cloned embryos might lead to premature Cdx2 expression and differentiation of blastomeres into trophectoderm. Therefore, we created larger cloned embryos by aggregating them at the 4-cell stage. Homologous aggregation stimulates expression of multiple signaling pathways' components and results in cloned embryos with levels of Cdx2 similar to fertilized embryos. Most of the resultant morulae and blastocysts consist of cells of all three founders, indicating that aggregation increases stability of all of the individual components. We conclude that the induction of pluripotency in cloned embryos is more efficient than previously assumed, and we propose that a minimum cell number is necessary to stabilize pluripotency and inhibit premature expression of Cdx2 in cloned mouse embryos.

o-Nitroanilines as major metabolic products of anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans in microsomal and cytosolic fractions of rat hepatocytes and in whole parasitic cells.

The metabolism of six anti-Trypanosoma cruzi 5-phenylethenylbenzofuroxans (PhEBfx) was studied in vitro using rat hepatic microsomal and cytosolic fractions as a mammalian model and whole cells of T. cruzi as a parasitic model. Some of the expected metabolites were synthesized to provide authentic chromatographic standards. The metabolites were identified using high-performance liquid chromatography (HPLC) in comparison with the authentic standards and their proportions were determined. Their structures were confirmed using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The behaviour of the six PhEBfx in the three different systems was similar. The main metabolites, formed by reductive processes, were the corresponding o-nitroanilines. Two of the test compounds were studied for extended time periods in the rat liver preparations and their terminal metabolites were identified as o-phenylendiamine derivatives.

Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol.

Culturing embryos in different media is a useful approach to characterize their nature in regard to "memory" of the donor nucleus and its "reprogramming" after somatic cell nuclear transfer (SCNT). However, efforts to elucidate the mechanisms of reprogramming are seriously undermined when embryo culture conditions are not completely defined. Using recombinant human albumin (rHA) is a step toward establishing defined culture conditions for mouse cloning. Recombinant HA supports blastocyst formation of cumulus cell-derived clones at a rate comparable with two types of bovine serum albumin (BSA); following transfer of blastocysts to the genital tract, rates of development to midgestation (10.5 dpc) were indistinguishable. rHA also supports the derivation of germline competent embryonic stem (ES) cells from SCNT blastocysts at a substantial rate compared with BSA counterparts and with zygotic blastocysts. Unlike the developmental parameters, the gene expression patterns of clones cultured in rHA or BSA were not superimposed; identical patterns were observed for zygotic blastocysts in the two albumins. In summary, the present study demonstrates that (1) rHA can replace BSA, proving a defined protein source for SCNT in mice; (2) although using rHA is similar to BSA, it is not equal (rHA leaves a mark on gene expression of clones but not zygotes). Future studies that investigate reprogramming after SCNT will need to consider not only the implications of culture media for cloning but also the supplement choice.

Furoxan derivatives as cytotoxic agents: preliminary in vivo antitumoral activity studies.

Furoxan derivatives with in vitro cytotoxic activity were investigated as antitumoral agents in vivo. The compounds were tested in murine models of both CCRFS-180 II sarcoma and mammary adenocarcinoma. Two of the furoxan derivatives considered here, 3-formyl-4-phenyl-1,2,5-oxadiazole N2-oxide and 3-carbonitrile-4-phenyl-1,2,5-oxadiazole N2-oxide, present in vivo antitumoral activity. They were able to produce more than 90% of tumoral necrosis under the experimental protocol of administration and posology employed. NO-releasing capacity of furoxans may explain the anti-neoplastic activity of these compounds.

1,2,5-Oxadiazole N-oxide derivatives as potential anti-cancer agents: synthesis and biological evaluation. Part IV.

Several new 1,2,5-oxadiazole N-oxide derivatives and some deoxygenated analogues were synthesized to be tested as potential selective hypoxic cell cytotoxins. Compounds prepared were designed in order to gain insight into the mechanism of action of this kind of cytotoxin. Compounds were tested in oxia and hypoxia and they proved to be non-selective. 3-Cyano-N(2)-oxide-4-phenyl-1,2,5-oxadiazole showed the best cytotoxic activity in oxia. The cytotoxicity observed for these derivatives could be explained in terms of the electronic characteristics of the 1,2,5-oxadiazole substituents. Electrochemical and ESR studies were performed on the more cytotoxic derivative.

Gene expression and chromatin organization during mouse oocyte growth.

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.

Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post-hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division.