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Abstract  Apomixis is a route of asexual reproduction through seeds, that progresses in the absence of meiosis and fertilization to generate maternal clonal progenies. Gametophytic apomicts are usually polyploid and probably arose from sexual ancestors through a limited number of mutations in the female reproductive pathway. A differential display analysis was carried out on immature inflorescences of sexual and apomictic tetraploid genotypes of Paspalum notatum, in order to identify genes associated with the emergence of apospory. Analysis of ∼10,000 transcripts led to the identification of 94 high-quality differentially expressed sequences. Assembling analysis, plus validation, rendered 65 candidate unigenes, organized as 14 contigs and 51 singletons. Thirty-four unigenes were isolated from apomictic plants and 31 from sexual ones. A total of 45 (69.2%) unigenes were functionally categorized. While several of the differentially expressed sequences appeared to be components of an extracellular receptor kinase (ERK) signal transduction cascade, others seemed to participate in a variety of central cellular processes like cell-cycle control, protein turnover, intercellular signalling, transposon activity, transcriptional regulation and endoplasmic reticulum-mediated biosynthesis. In silico mapping revealed that a particular group of five genes silenced in apomictic plants clustered in a rice genomic area syntenic with the region governing apospory in Paspalum notatum and Brachiaria brizantha. Two of these genes mapped within the set of apo-homologues in P. notatum. Four genes previously reported to be controlled by ploidy were identified among those expressed differentially between apomictic and sexual plants. In situ hybridization experiments were performed for selected clones. [ABSTRACT FROM AUTHOR]

Abstract  The molecular nature of gene expression during the initiation and progress of diplosporous apomixis is still unknown. Moreover, the basis of the close correlation between diplospory and polyploidy is not clarified yet. A comparative expression analysis was performed based on expressed sequence tags (ESTs) sequencing and differential display in an Eragrostis curvula diplosporous tetraploid genotype (T, 4x apo), a sexual diploid derivative obtained from tissue culture (D, 2x sex) and an artificial sexual tetraploid obtained from the diploid seeds after colchicine treatment (C, 4x sex). From a total of 8,884 unigenes sequenced from inflorescence-derived libraries, 112 (1.26%) showed significant differential expression in individuals with different ploidy level and/or variable reproductive mode. Independent comparisons between plants with different reproductive mode (same ploidy) or different ploidy level (same reproductive mode) allowed the identification of genes modulated in response to diplosporous development or polyploidization, respectively. Surprisingly, a group of genes (Group 3) were differentially expressed or silenced only in the 4x sex plant, presenting similar levels of expression in the 4x apo and the 2x sex genotypes. A group of randomly selected differential genes was validated by QR-PCR. Differential display analysis showed that in general the 4x apo and 4x sex expression profiles were more related and different from the 2x sex one, but confirmed the existence of Group 3-type genes, in both inflorescences and leaves. The possible biological significance for the occurrence of this particular group of genes is discussed. In silico mapping onto the rice genome was used to identify candidates mapping to the region syntenic to the diplospory locus. [ABSTRACT FROM AUTHOR]

The objective of this work was to determine the type of inheritance (disomic/polysomic) in tetraploid (2n=4x=40) Paspalum notatum and investigate the transmission pattern of the chromosome region associated with apospory. An F1 family segregating for the reproductive mode (aposporous vs non-aposporous) was generated by crossing a tetraploid sexual plant as female parent with an apomictic individual as pollen donor. Pollen mother cells from both parental plants were examined to ascertain chromosome-pairing behavior at meiosis. The high rate of quadrivalent chromosome associations indicated an autotetraploid origin of the species, although bivalent pairing and occasional univalents were detected. The observation of a lagging bivalent, a bridge of chromatin, or two aligned laggards in the aposporous parent suggested a chromosome inversion in this strain. Segregation ratios of AFLP markers and the proportion of linkages in repulsion versus coupling phase denoted tetrasomic inheritance, but markers displaying disomic ratios were also observed. Preferential chromosome pairing (disomic inheritance) in the chromosome segment related to apospory was detected. The possible relationship between a chromosome rearrangement and the inheritance of apospory is discussed. [ABSTRACT FROM AUTHOR]

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1–3 d pre‐anthesis; at anthesis; and 2, 4 and 6 d post‐anthesis. Cytoembryological analyses indicated that approx. 17 % of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety‐five oligonucleotides were assayed on the progenitors in order to search for male‐specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3·4 % before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off‐type plants (16·5 %), of which only one was a BIII hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed. [ABSTRACT FROM PUBLISHER]

The common races of bahiagrass, Paspalum notatum, are tetraploid (2n=4x=40) and reproduce by aposporous apomixis. Paspalum notatum var. saurae is the corresponding diploid (2n=2x=20) sexual race that outbreeds due to self-incompatibility. Chromosome doubling was induced by colchicine treatments in three individual plants from a natural diploid population. Embryological studies demonstrated that one of the induced autotetraploid plants reproduced sexually. The other two autotetraploids were facultative apomicts. These results indicate that an unexpressed gene(s) for apomixis exists at the diploid level. The expression of the trait is ploidy-dependent. The ploidy dependency may act either on the locus controlling apomixis through some transcription factors or via a secondary locus which requires a higher allele dosage to affect the expression of the main locus. [ABSTRACT FROM AUTHOR]

Simple Summary: The therapy of Acute Lymphoblastic Leukemia (ALL) is based on Escherichia coli (E. coli) L-asparaginase, which is a very effective drug in most cases. However, its side effects sometimes prevent its usage or impose its interruption. The main issues derive from its bacterial origin, which elicits a strong immune response in the patient, and from its generalized action on all body compartments. In this work, we describe how we generated a fully active and miniaturized form of L-asparaginase starting from a camel single domain antibody, a class of antibodies known to have a very limited immunogenicity in humans. We also targeted it onto tumor cells by attaching it to an antibody fragment directed onto the CD19 B-cell surface receptor, expressed on ALL cells. We named this new molecule "Targeted Catalytic Nanobody" (T-CAN). The T-CAN is active and successfully binds to CD19 expressing cells in vitro. Thanks to its reduced immunogenic potential, it represents a new tool which deserves further development. E. coli L-asparaginase is an amidohydrolase (EC 3.5.1.1) which has been successfully used for the treatment of Acute Lymphoblastic Leukemia for over 50 years. Despite its efficacy, its side effects, and especially its intrinsic immunogenicity, hamper its usage in a significant subset of cases, thus limiting therapeutic options. Innovative solutions to improve on these drawbacks have been attempted, but none of them have been truly successful so far. In this work, we fully replaced the enzyme scaffold, generating an active, miniaturized form of L-asparaginase by protein engineering of a camel single domain antibody, a class of antibodies known to have a limited immunogenicity in humans. We then targeted it onto tumor cells by an antibody scFv fragment directed onto the CD19 B-cell surface receptor expressed on ALL cells. We named this new type of nanobody-based antibody-drug conjugate "Targeted Catalytic Nanobody" (T-CAN). The new molecule retains the catalytic activity and the binding capability of the original modules and successfully targets CD19 expressing cells in vitro. Thanks to its theoretically reduced immunogenic potential compared to the original molecule, the T-CAN can represent a novel approach to tackle current limitations in L-asparaginase usage. [ABSTRACT FROM AUTHOR]

Unlabelled: Anticardiolipin antibodies were determined in 29 diabetic children and adolescents, aged 3.9-26.8 years, with disease duration from 1 month to 19 years. Anti-islet cell antibodies (ICA-IgG and CF-ICA), anti-insulin antibodies (IAA), antithyroid antibodies and non organ-specific (NOSA) antibodies were also determined. Patients were grouped according to insulin-dependent diabetes mellitus (IDDM) duration: group I (n = 11) < 6 months, and group II (n = 18) > 5 years. Eleven of group II patients showed precocious signs of micro-angiopathic complications. Forty-two age- and sex-matched healthy subjects served as controls. IgG and IgM anticardiolipin antibodies were evaluated by ELISA and their results expressed as arbitrary units (AU). IgG anticardiolipin antibodies were found in 7 patients (24%), while IgM anticardiolipin antibodies were absent in all. IgG anticardiolipin antibodies were more frequent in IDDM patients than in controls (P < 0.005) and group I (in 6 out of 11 patients; 54.5%) than in group II (in 1 out of 18 patients; 5.5%) (P < 0.025). In five out of six group I patients with IgG anticardiolipin antibodies, ICA-IgG and/or CF-ICA were also found. No correlation was observed between anticardiolipin and other auto-antibodies, micro-angiopathic complications, and HLA typing.Conclusion: Anticardiolipin antibodies may reflect an abnormal immunological response in the early stage of diabetes mellitus and represent a transient auto-immune phenomenon. [ABSTRACT FROM AUTHOR]

Paspalum is an important genus of the family Gramineae that includes several valuable forage grasses. Many of the species are polyploid and either obligate or facultative apomicts. Cyto-embryological observations of several tetraploid genotypes of P. notatum were performed to determine their mode of reproduction. Afterwards, selfed progenies of the genotypes F131, Q3664 and Q4117 were analysed using RFLP and RAPD genetic fingerprints to identify maternal and non-maternal (aberrant) plants, and to establish the degree of apomictic reproduction. Five maize clones and six primers were used for detecting genetic deviations from the maternal profile. Maize clones umc379, umc384 and umc318 and primers OPG10 and OPI4 were the most informative for discriminating between maternal and aberrant individuals within the progenies of F131 and Q3664. The combined results of three RFLP clones or 4–6 RAPD primers were necessary to ascertain the mode of reproduction in plants F131 and Q3664. The results obtained with the RFLP and RAPD markers were in agreement with the cyto-embryological studies in ascertaining the mode and degree of apomictic reproduction. Plant F131 showed a completely sexual reproductive behaviour, Q3664 an elevated expression of sexuality, while Q4117 was highly apomictic. A fingerprint analysis of an outcrossing population, aimed at the identification of hybrid plants, was also performed. Maize clones um318 and umc379 and primers OPC2 and OPC9 were used. The presence of specific bands belonging to the male parent permitted a rapid and easy detection of hybrids. The methodology described here can be applied both for the characterisation of P. notatum populations and to identify hybrid progenies in Paspalum breeding programs. [ABSTRACT FROM AUTHOR]

A bulked segregant analysis using RFLPs and RAPDs was carried out to identify molecular markers co-segregating with apomixis in a Brachiaria F1 population. The test population used was a cross between sexual B. ruziziensis R44 and the aposporous apomictic Brachiaria brizantha cv Marandu. The Brachiaria genome was systematically scanned using 61 cDNA and genomic maize clones detecting 65 loci located at 40 cM, on average, one from each other in the maize genome. The finding of a clone that presented a polymorphic band co-segregating with apomixis (umc147) led to the identification of another marker within the same area (umc72). The clones belong to a duplicated linkage group that maps to the distal part of maize chromosome-1 long arm and chromosome-5 short arm. RAPD analysis using 184 primers from Operon sets yielded one more marker (OPC4) significantly linked to the trait mapping the same locus. OPC4 had been previously reported as a potential marker for apospory in Pennisetum. A map of the region was constructed using additional clones that belong to the same maize linkage group. Since that was the only genomic region that presented an apomixis-linked polymorphism our observations support the existence of a single locus directing apospory in Brachiaria. [ABSTRACT FROM AUTHOR]

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukaemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca[sub2+], a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca([sub2+])-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer is one of the main causes of death worldwide and early detection is crucial for effective treatment. Scientists have therefore focused on the identification of circulating cancer-specific molecules, so-called markers, that can be detected with non- or less-invasive techniques. One attractive emerging marker is represented by circulating antibodies against molecules that are specific for the tumor cells and not produced by normal cells. Many such auto-antibodies have been discovered, but still not much is known about their significance and role in either promoting or inhibiting tumor growth. The aim of this review is to summarize the current knowledge on auto-antibodies in different cancer types and on their current or possible utilization for effective cancer management. Reference to autoimmune diseases will also be made, as they share with cancer the presence of such auto-antibodies. Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body's defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community. [ABSTRACT FROM AUTHOR]

In the past decades, the grasses of the Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme. [ABSTRACT FROM AUTHOR]

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses. [ABSTRACT FROM AUTHOR]