Cytomolecular diversity among Vigna Savi (Leguminosae) subgenera.

The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.

Translocations and inversions: major chromosomal rearrangements during Vigna (Leguminosae) evolution.

KEY MESSAGE: Inversions and translocations are the major chromosomal rearrangements involved in Vigna subgenera evolution, being Vigna vexillata the most divergent species. Centromeric repositioning seems to be frequent within the genus. Oligonucleotide-based fluorescence in situ hybridization (Oligo-FISH) provides a powerful chromosome identification system for inferring plant chromosomal evolution. Aiming to understand macrosynteny, chromosomal diversity, and the evolution of bean species from five Vigna subgenera, we constructed cytogenetic maps for eight taxa using oligo-FISH-based chromosome identification. We used oligopainting probes from chromosomes 2 and 3 of Phaseolus vulgaris L. and two barcode probes designed from V. unguiculata (L.) Walp. genome. Additionally, we analyzed genomic blocks among the Ancestral Phaseoleae Karyotype (APK), two V. unguiculata subspecies (V. subg. Vigna), and V. angularis (Willd.) Ohwi & Ohashi (V. subg. Ceratotropis). We observed macrosynteny for chromosomes 2, 3, 4, 6, 7, 8, 9, and 10 in all investigated taxa except for V. vexillata (L.) A. Rich (V. subg. Plectrotropis), in which only chromosomes 4, 7, and 9 were unambiguously identified. Collinearity breaks involved with chromosomes 2 and 3 were revealed. We identified minor differences in the painting pattern among the subgenera, in addition to multiple intra- and interblock inversions and intrachromosomal translocations. Other rearrangements included a pericentric inversion in chromosome 4 (V. subg. Vigna), a reciprocal translocation between chromosomes 1 and 5 (V. subg. Ceratotropis), a potential deletion in chromosome 11 of V. radiata (L.) Wilczek, as well as multiple intrablock inversions and centromere repositioning via genomic blocks. Our study allowed the visualization of karyotypic patterns in each subgenus, revealing important information for understanding intrageneric karyotypic evolution, and suggesting V. vexillata as the most karyotypically divergent species.

A Novel Genetic Variant in MBD5 Associated with Severe Epilepsy and Intellectual Disability: Potential Implications on Neural Primary Cilia.

Disruptions in the MBD5 gene have been linked with an array of clinical features such as global developmental delay, intellectual disability, autistic-like symptoms, and seizures, through unclear mechanisms. MBD5 haploinsufficiency has been associated with the disruption of primary cilium-related processes during early cortical development, and this has been reported in many neurodevelopmental disorders. In this study, we describe the clinical history of a 12-year-old child harboring a novel MBD5 rare variant and presenting psychomotor delay and seizures. To investigate the impact of MBD5 haploinsufficiency on neural primary cilia, we established a novel patient-derived cell line and used CRISPR-Cas9 technology to create an isogenic control. The patient-derived neural progenitor cells revealed a decrease in the length of primary cilia and in the total number of ciliated cells. This study paves the way to understanding the impact of MBD5 haploinsufficiency in brain development through its potential impact on neural primary cilia.

Determination by ICP-MS of Essential and Toxic Trace Elements in Gums and Carrageenans Used as Food Additives Commercially Available in the Portuguese Market.

Gums and carrageenans are food additives widely used in food preparations to improve texture and as viscosifiers. Although they are typically added in small amounts, nowadays people tend to use more and more pre-prepared food. In this work, the content of a wide panel of trace elements in commercial products were analyzed. Carrageenans and gums (n = 13) were purchased in the Portuguese market and were from European suppliers. Samples were solubilized by closed-vessel microwave-assisted acid digestion and analyzed by ICP-MS. Globally, the content of essential trace elements decreased in the following order: Fe (on average, on the order of several tens of µg/g) > Mn > Zn > Cr > Cu > Co > Se > Mo (typically < 0.1 µg/g), while the content of non-essential/toxic trace elements decreased in the following order: Al > Sr > Rb > As > Li > Cd > Pb > Hg. The consumption of these food additives can significantly contribute to the daily requirements of some essential trace elements, namely Cr and Mo. The toxic trace elements Cd, As, Pb, and Hg were below the EU regulatory limits in all analyzed samples. Additional research is needed to define the potential risk of introducing toxic trace elements into food products through the use of these additives.

Generation and Characterization of Novel iPSC Lines from a Portuguese Family Bearing Heterozygous and Homozygous GRN Mutations.

Mutations in granulin (GRN) have been associated with neurodegenerative diseases, such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). In Portugal, GRN mutations account for around half of all FTLD cases with known genetic origin. Here, we describe the generation and characterization of three human-induced pluripotent stem cell (hiPSC) lines from a Portuguese family harboring heterozygous and homozygous GRN mutation. hiPSCs were reprogrammed from human dermal fibroblasts by episomal nucleofection of the Yamanaka factors. The new generated lines were positive for pluripotency markers, could be further differentiated to cells expressing all trilineage markers, and presented a normal karyotype. They were also capable of differentiating into 3D brain organoids and presented a significant decrease in progranulin protein levels. Hence, these cell lines constitute suitable new tools to elucidate the pathophysiological mechanisms associated with the GRN mutations in the context of FTLD.

BAC- and oligo-FISH mapping reveals chromosome evolution among Vigna angularis, V. unguiculata, and Phaseolus vulgaris.

Cytogenomic resources have accelerated synteny and chromosome evolution studies in plant species, including legumes. Here, we established the first cytogenetic map of V. angularis (Va, subgenus Ceratotropis) and compared this new map with those of V. unguiculata (Vu, subgenus Vigna) and P. vulgaris (Pv) by BAC-FISH and oligopainting approaches. We mapped 19 Vu BACs and 35S rDNA probes to the 11 chromosome pairs of Va, Vu, and Pv. Vigna angularis shared a high degree of macrosynteny with Vu and Pv, with five conserved syntenic chromosomes. Additionally, we developed two oligo probes (Pv2 and Pv3) used to paint Vigna orthologous chromosomes. We confirmed two reciprocal translocations (chromosomes 2 and 3 and 1 and 8) that have occurred after the Vigna and Phaseolus divergence (~9.7 Mya). Besides, two inversions (2 and 4) and one translocation (1 and 5) have occurred after Vigna and Ceratotropis subgenera separation (~3.6 Mya). We also observed distinct oligopainting patterns for chromosomes 2 and 3 of Vigna species. Both Vigna species shared similar major rearrangements compared to Pv: one translocation (2 and 3) and one inversion (chromosome 3). The sequence synteny identified additional inversions and/or intrachromosomal translocations involving pericentromeric regions of both orthologous chromosomes. We propose chromosomes 2 and 3 as hotspots for chromosomal rearrangements and de novo centromere formation within and between Vigna and Phaseolus. Our BAC- and oligo-FISH mapping contributed to physically trace the chromosome evolution of Vigna and Phaseolus and its application in further studies of both genera.

Regeneration of pulp-dentin complex using human stem cells of the apical papilla: in vivo interaction with two bioactive materials.

OBJECTIVES: To compare the regenerative properties of human stem cells of the apical papilla (SCAPs) embedded in a platelet-rich plasma (PRP) scaffold, when implanted in vivo using an organotypic model composed of human root segments, with or without the presence of the bioactive cements - ProRoot MTA or Biodentine. MATERIAL AND METHODS: SCAPs were isolated from third molars with incomplete rhizogenesis and expanded and characterized in vitro using stem cell and surface markers. The pluripotency of these cells was also assessed using adipogenic, chondrogenic, and osteogenic differentiation protocols. SCAPs together with a scaffold of PRP were added to the root segment lumen and the organotypic model implanted on the dorsal region of immunodeficient rats for a period of 4 months. RESULTS: Presence of SCAPs induced de novo formation of dentin-like and pulp-like tissue. A barrier of either ProRoot MTA or Biodentine did not significantly affect the fraction of sections from roots segments observed to contain deposition of hard material (P > 0.05). However, the area of newly deposited dentin was significantly greater in segments containing a barrier of Biodentine compared with ProRoot MTA (P < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: SCAPs offer a viable alternative to other dental stem cells (DSCs) in their regenerative properties when enclosed in the microenvironment of human tooth roots. The present study also suggests that the presence of bioactive materials does not hinder or impede the formation of new hard tissues, but the presence of Biodentine may promote greater mineralized tissue deposition.

Breaks of macrosynteny and collinearity among moth bean (Vigna aconitifolia), cowpea (V. unguiculata), and common bean (Phaseolus vulgaris).

Comparative cytogenetic mapping is a powerful approach to gain insights into genome organization of orphan crops, lacking a whole sequenced genome. To investigate the cytogenomic evolution of important Vigna and Phaseolus beans, we built a BAC-FISH (fluorescent in situ hybridization of bacterial artificial chromosome) map of Vigna aconitifolia (Vac, subgenus Ceratotropis), species with no sequenced genome, and compared with V. unguiculata (Vu, subgenus Vigna) and Phaseolus vulgaris (Pv) maps. Seventeen Pv BACs, eight Vu BACs, and 5S and 35S rDNA probes were hybridized in situ on the 11 Vac chromosome pairs. Five Vac chromosomes (Vac6, Vac7, Vac9, Vac10, and Vac11) showed conserved macrosynteny and collinearity between V. unguiculata and P. vulgaris. On the other hand, we observed collinearity breaks, identified by pericentric inversions involving Vac2 (Vu2), Vac4 (Vu4), and Vac3 (Pv3). We also detected macrosynteny breaks of translocation type involving chromosomes 1 and 8 of V. aconitifolia and P. vulgaris; 2 and 3 of V. aconitifolia and P. vulgaris; and 1 and 5 of V. aconitifolia and V. unguiculata. Considering our data and previous BAC-FISH studies, six chromosomes (1, 2, 3, 4, 5, and 8) are involved in major karyotype divergences between genera and five (1, 2, 3, 4, and 5) between Vigna subgenera, including mechanisms such as duplications, inversions, and translocations. Macrosynteny breaks between Vigna and Phaseolus suggest that the major chromosomal rearrangements have occurred within the Vigna clade. Our cytogenomic comparisons bring new light on the degree of shared macrosynteny and mechanisms of karyotype diversification during Vigna and Phaseolus evolution.

Genome composition and pollen viability of Jatropha (Euphorbiaceae) interspecific hybrids by Genomic In Situ Hybridization (GISH).

Interspecific hybridization is required for the development of Jatropha curcas L. improved cultivars, due to its narrow genetic basis. The present study aimed to analyze the parental genomic composition of F1 and BC1F1 generations derived from interspecific crosses (J. curcas/J. integerrima and J. curcas/J. multifida) by GISH (Genomic In Situ Hybridization), and the meiotic index and pollen viability of F1 hybrids. In F1 cells from both hybrids, 11 chromosomes of each parental was observed, as expected, but chromosome rearrangement events could be detected using rDNA chromosome markers, suggesting unbalanced cells. In the BC1F1, both hybrids had 22 chromosomes, suggesting that only n = 11 gametes were viable in the next generation. However, GISH allowed the identification of three and two alien chromosomes in J. curcas//J. integerrima and J. curcas//J. multifida BC1F1 hybrids, respectively, suggesting a preferential transmission of J. curcas chromosomes for both hybrids. Pollen viability in F1 hybrids derived from J. curcas/J. integerrima crosses were higher (82-83%) than those found for J. curcas/J. multifida (68%), showing post-meiotic problems in these last hybrids, with dyads, triads, polyads, and micronuclei as post-meiosis results. The here presented cytogenetic characterization of interspecific hybrids and their backcross progenies can contribute to the selection of the best genotypes for future assisted breeding of J. curcas.

miRNAs: New Biomarkers and Therapeutic Targets in Dementia.

BACKGROUND: Dementia is a complex pathological state that affects millions of individuals worldwide and is responsible for a huge socioeconomic burden, making it a major health concern of current times. Given the impact of dementia in both patients and caregivers, it is crucial to fully clarify the molecular mechanisms underlying dementia-associated disorders, since without this knowledge our ability to correctly diagnose and treat these diseases is severely hampered. METHODS: Epigenetic mechanisms, such as miRNA-mediated post-transcriptional regulation, have been reported to play a role in dementia pathogenesis. Given their ability to bind complementary mRNA sequences, miRNAs are able to induce temporary or permanent translation repression of their mRNA targets. RESULTS: Consequently, changes in miRNA levels may contribute to alterations in the expression of dementiarelated proteins, impacting the course of the disease. Conversely, studies have also reported that some of these proteins are able to regulate the biogenesis and transport of miRNAs, hinting at novel disease-related mechanisms that are now beginning to be explored. These findings have made miRNAs both interesting tools and promising targets in the design of novel therapeutic strategies. Moreover, the discovery of circulating miRNAs, which are released by cells of various tissues, including the brain, and travel in membrane-bound vesicles found in most biofluids, opened new possibilities concerning the usefulness of miRNAs as biomarkers of disease. CONCLUSION: In this context, the major aim of this review is to discuss the relevance of these small non-coding RNAs in dementia, focusing on their role as gene expression regulators, their potential as biomarkers of dementia subtype and stage, and the hypothesis of using miRNA modulation as an innovative therapeutic approach to treat dementia-related disorders.