The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Potential Development of Vitrified Immature Human Oocytes: Influence of the Culture Medium and the Timing of Vitrification.

How does the in vitro maturation (IVM) medium and the vitrification procedure affect the survival of germinal vesicle (GV) oocytes obtained from stimulated cycles and their development to the blastocyst stage? In total, 1085 GV human oocytes were obtained after women underwent a cycle of controlled ovarian stimulation, and these oocytes were subjected to IVM before or after their vitrification. IVM was carried out in two commercial culture media not specifically designed for maturation. MII oocytes were then activated and embryo development until day 6 was evaluated. According to the results, a higher percentage of oocytes reach the MII stage if they are vitrified before they undergo IVM. Nevertheless, the medium used and the sample size determine whether these differences become significant or not. Similar survival rates and development to blastocysts were observed in all the conditions studied.

Impact of Maturation and Vitrification Time of Human GV Oocytes on the Metaphase Plate Configuration.

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration.

Characterization of a Novel Splicing Variant in Acylglycerol Kinase (AGK) Associated with Fatal Sengers Syndrome.

Mitochondrial functional integrity depends on protein and lipid homeostasis in the mitochondrial membranes and disturbances in their accumulation can cause disease. AGK, a mitochondrial acylglycerol kinase, is not only involved in lipid signaling but is also a component of the TIM22 complex in the inner mitochondrial membrane, which mediates the import of a subset of membrane proteins. AGK mutations can alter both phospholipid metabolism and mitochondrial protein biogenesis, contributing to the pathogenesis of Sengers syndrome. We describe the case of an infant carrying a novel homozygous AGK variant, c.518+1G>A, who was born with congenital cataracts, pielic ectasia, critical congenital dilated myocardiopathy, and hyperlactacidemia and died 20 h after birth. Using the patient's DNA, we performed targeted sequencing of 314 nuclear genes encoding respiratory chain complex subunits and proteins implicated in mitochondrial oxidative phosphorylation (OXPHOS). A decrease of 96-bp in the length of the AGK cDNA sequence was detected. Decreases in the oxygen consumption rate (OCR) and the OCR:ECAR (extracellular acidification rate) ratio in the patient's fibroblasts indicated reduced electron flow through the respiratory chain, and spectrophotometry revealed decreased activity of OXPHOS complexes I and V. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts and describe the possible molecular mechanism underlying the pathogenicity of this novel AGK variant. Experimental validation using in vitro analysis allowed an accurate characterization of the disease-causing variant.

Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation.

The development of an effective program that combines in vitro maturation (IVM) and cryopreservation for immature oocytes would represent a novel advance for in vitro fertilization (IVF), especially as a means to preserve the fertility of women in unique situations. The aim of this study was to analyze the ultrastructural characteristics of human oocytes, obtained after controlled ovarian stimulation, to determine whether IVM is best performed before or after vitrification. To this end, we analyzed the following features in a total of 22 MII oocytes: size, zona pellucida and perivitelline space, mitochondria number, M-SER (mitochondria-smooth endoplasmic reticulum) aggregates and M-V (mitochondria-vesicle) complexes, the number of cortical granules and microvilli, and the presence of vacuolization using transmission electron microscopy (TEM). Each oocyte presented a rounded shape, with an intact oolemma, and was surrounded by a continuous zona pellucida and perivitelline space. Statistical analysis comparing oocytes vitrified before or after IVM indicated that there were no significant differences between examined characteristics.

Characterization of the plasma proteomic profile of Fabry disease: Potential sex- and clinical phenotype-specific biomarkers.

Fabry disease (FD) is a X-linked rare lysosomal storage disorder caused by deficient α-galactosidase A (α-GalA) activity. Early diagnosis and the prediction of disease course are complicated by the clinical heterogeneity of FD, as well as by the frequently inconclusive biochemical and genetic test results that do not correlate with clinical course. We sought to identify potential biomarkers of FD to better understand the underlying pathophysiology and clinical phenotypes. We compared the plasma proteomes of 50 FD patients and 50 matched healthy controls using DDA and SWATH-MS. The >30 proteins that were differentially expressed between the 2 groups included proteins implicated in processes such as inflammation, heme and haemoglobin metabolism, oxidative stress, coagulation, complement cascade, glucose and lipid metabolism, and glycocalyx formation. Stratification by sex revealed that certain proteins were differentially expressed in a sex-dependent manner. Apolipoprotein A-IV was upregulated in FD patients with complications, especially those with chronic kidney disease, and apolipoprotein C-III and fetuin-A were identified as possible markers of FD with left ventricular hypertrophy. All these proteins had a greater capacity to identify the presence of complications in FD patients than lyso-GB3, with apolipoprotein A-IV standing out as being more sensitive and effective in differentiating the presence and absence of chronic kidney disease in FD patients than renal markers such as creatinine, glomerular filtration rate and microalbuminuria. Identification of these potential biomarkers can help further our understanding of the pathophysiological processes that underlie the heterogeneous clinical manifestations associated with FD.

Improving the analysis of Y-SNP haplogroups by a single highly informative 16 SNP multiplex PCR-minisequencing assay.

The SNP haplogroups of the Y-chromosome are nonrandomly distributed among human populations. They are used for tracing the phylogeographical history of paternal lineages of male individuals and can be a useful tool for approaching the patrilineal bio-geographic ancestry of unknown forensic evidences. With the aim of facilitating the inference of the principal informative worldwide Y-SNP haplogroups, we have selected the minimum possible number of key Y-SNPs to be amplified in a sensitive single multiplex PCR and detected by minisequencing. This assay, that includes 16 Y-SNPs, was tested for male human specificity, sensitivity, and reproducibility. Its effectiveness was assessed in a set of degraded DNA samples and in a panel of male individuals from different worldwide populations. All these tests demonstrated the convenience of this assay for assigning the major Y haplogroups to forensic evidences by one single PCR-minisequencing reaction.

High medium-term incidence of major cardiovascular events in discharged patients with unstable angina.

The introduction of high-sensitivity troponin (hsTn) assays has reduced the diagnosis of unstable angina (UA) in favor of non-ST elevation myocardial infarction (NSTEMI) in the context of non-ST elevation acute coronary syndrome (NSTEACS). It is unclear whether the detection of these hsTn levels affects the prognosis and therefore whether a different therapeutic approach is warranted. This study aims to determine whether using hsTn results in medium-term prognostic differences in patients with UA and NSTEMI. Methods: This multicenter, prospective registry study included consecutive patients who underwent hsTn assays and were discharged with a diagnosis of NSTEACS. Patients were followed for two years. Outcomes were the occurrence of major adverse cardiovascular events (MACE: cardiovascular death, non-fatal myocardial infarction, and non-fatal ischemic stroke), major bleeding, and all-cause mortality. Results: Patients with UA and NSTEMI did not show differences in terms of the invasive interventions received, the coronary artery disease diagnosed, the type of revascularization performed, or the proportion presenting MACE (UA 18.1% vs. NSTEMI 18.9%; p = 0.79). However, patients with NSTEMI had higher cardiovascular mortality at two years (UA 4% vs. NSTEMI 9.2%; p = 0.012), as well as, all-cause mortality (UA vs. 7.9% vs. NSTEMI 16.4%; p = 0.002). Conclusions: Medium-term incidence of MACE was similar in patients with UA and NSTEMI, but cardiovascular and all-cause mortality in NSTEMI patients was over twice that of patients with UA.

Y-STR variation in the Basque diaspora in the Western USA: evolutionary and forensic perspectives.

Individuals of Basque origin migrated in large numbers to the Western USA in the second half of the nineteenth century, and the flow continued with less intensity during the last century. The European source population, that of the Basque Country, has long been a cultural and geographical isolate. Previous studies have demonstrated that Y-STR frequencies of Basques are different from those of other Spanish and European populations [1]. The Basque diaspora in the Western USA is a recent migration, but the founder effect and the incorporation of new American Y chromosomes into the paternal genetic pool of the Basque diaspora could have influenced its genetic structure and could thus have practical implications for forensic genetics. To check for genetic substructure among the European source and Basque diaspora populations and determine the most suitable population database for the Basque diaspora in the Western USA, we have analysed the haplotype distribution of 17 Y-STRs in both populations. We have found that the Basque diaspora in the Western USA largely conserve the Y chromosome lineage characteristic of the autochthonous European Basque population with no statistically significant differences. This implies that a common 17 Y-STR Basque population database could be used to calculate identification or kinship parameters regardless of whether the Basque individuals are from the European Basque Country or from the Basque diaspora in the Western USA.

Fruit Morphology and Ripening-Related QTLs in a Newly Developed Introgression Line Collection of the Elite Varieties 'Védrantais' and 'Piel de Sapo'.

Melon is an economically important crop with widely diverse fruit morphology and ripening characteristics. Its diploid sequenced genome and multiple genomic tools make this species suitable to study the genetic architecture of fruit traits. With the development of this introgression line population of the elite varieties 'Piel de Sapo' and 'Védrantais', we present a powerful tool to study fruit morphology and ripening traits that can also facilitate characterization or pyramidation of QTLs in inodorous melon types. The population consists of 36 lines covering almost 98% of the melon genome, with an average of three introgressions per chromosome and segregating for multiple fruit traits: morphology, ripening and quality. High variability in fruit morphology was found within the population, with 24 QTLs affecting six different traits, confirming previously reported QTLs and two newly detected QTLs, FLQW5.1 and FWQW7.1. We detected 20 QTLs affecting fruit ripening traits, six of them reported for the first time, two affecting the timing of yellowing of the rind (EYELLQW1.1 and EYELLQW8.1) and four at the end of chromosome 8 affecting aroma, abscission and harvest date (EAROQW8.3, EALFQW8.3, ABSQW8.3 and HARQW8.3). We also confirmed the location of several QTLs, such as fruit-quality-related QTLs affecting rind and flesh appearance and flesh firmness.

Modulating climacteric intensity in melon through QTL stacking.

Fruit ripening is one of the main processes affecting fruit quality and shelf life. In melon there are both climacteric and non-climacteric genotypes, making it a suitable species to study fruit ripening. In the current study, in order to fine tune ripening, we have pyramided three climacteric QTLs in the non-climacteric genotype "Piel de Sapo": ETHQB3.5, ETHQV6.3 and ETHQV8.1. The results showed that the three QTLs interact epistatically, affecting ethylene production and ripening-related traits such as aroma profile. Each individual QTL has a specific role in the ethylene production profile. ETHQB3.5 accelerates the ethylene peak, ETHQV6.3 advances the ethylene production and ETHQV8.1 enhances the effect of the other two QTLs. Regarding aroma, the three QTLs independently activated the production of esters changing the aroma profile of the fruits, with no significant effects in fruit firmness, soluble solid content and fruit size. Understanding the interaction and the effect of different ripening QTLs offers a powerful knowledge for candidate gene identification as well as for melon breeding programs, where fruit ripening is one of the main objectives.

The maternal legacy of Basques in northern navarre: New insights into the mitochondrial DNA diversity of the Franco-Cantabrian area.

Autochthonous Basques are thought to be a trace from the human population contraction that occurred during the Last Glacial Maximum, based mainly on the salient frequencies and coalescence ages registered for haplogroups V, H1, and H3 of mitochondrial DNA in current Basque populations. However, variability of the maternal lineages still remains relatively unexplored in an important fraction of the Iberian Basque community. In this study, mitochondrial DNA diversity in Navarre (North Spain) was addressed for the first time. To that end, HVS-I and HVS-II sequences from 110 individuals were examined to identify the most relevant lineages, including analysis of coding region SNPs for the refinement of haplogroup assignment. We found a prominent frequency of subhaplogroup J1c (11.8%) in Navarre, coinciding with previous studies on Basques. Subhaplogroup H2a5, a putative autochthonous Basque lineage, was also observed in Navarre, pointing to a common origin of current Basque geographical groups. In contrast to other Basque subpopulations, comparative analyses at Iberian and European scales revealed a relevant frequency of subhaplogroup H3 (10.9%) and a frequency peak for U5b (15.5%) in Navarre. Furthermore, we observed low frequencies for maternal lineages HV0 and H1 in Navarre relative to other northern Iberian populations. All these findings might be indicative of intense genetic drift episodes generated by population fragmentation in the area of the Franco-Cantabrian refuge until recent times, which could have promoted genetic microdifferentiation between the different Basque subpopulations.

A novel introgression line collection to unravel the genetics of climacteric ripening and fruit quality in melon.

Introgression lines are valuable germplasm for scientists and breeders, since they ease genetic studies such as QTL interactions and positional cloning as well as the introduction of favorable alleles into elite varieties. We developed a novel introgression line collection in melon using two commercial European varieties with different ripening behavior, the climacteric cantalupensis 'Védrantais' as recurrent parent and the non-climacteric inodorus 'Piel de Sapo' as donor parent. The collection contains 34 introgression lines, covering 99% of the donor genome. The mean introgression size is 18.16 Mb and ~ 3 lines were obtained per chromosome, on average. The high segregation of these lines for multiple fruit quality traits allowed us to identify 27 QTLs that modified sugar content, altered fruit morphology or were involved in climacteric ripening. In addition, we confirmed the genomic location of five major genes previously described, which control mainly fruit appearance, such as mottled rind and external color. Most of the QTLs had been reported before in other populations sharing parental lines, while three QTLs (EAROQP11.3, ECDQP11.2 and FIRQP4.1) were newly detected in our work. These introgression lines would be useful to perform additional genetic studies, as fine mapping and gene pyramiding, especially for important complex traits such as fruit weight and climacteric ripening.

Spontaneous Reporting of Adverse Drug Reactions in a Pediatric Population in a Tertiary Hospital.

The pediatric population is a vulnerable group for adverse drug reactions (ADRs), and data on spontaneous reporting of ADRs in the hospital setting are scarce. We conducted a retrospective analysis of ADRs in pediatric patients spontaneously reported by health care professionals to a Pharmacovigilance Program in a tertiary hospital between 2010 and 2020, and we compared characteristics of ADRs between pediatric age subgroups. From 1787 spontaneously reported ADRs in an 11-year period, 103 (5.85%) were pediatric ADRs. The median age of patients with ADRs was 8.4 years (range 1 day-17 years) and 57.3% were male. The most frequent ADRs reported were nervous system disorders (13.6%) and the most frequently involved drugs were antineoplastics and immunodulators (32.4%). A 59.2% of the ADRs were serious and 55.3% were classified as being type B reactions. Medication errors were involved in 7.8% of the ADRs and 11.9% of the suspected drugs were used off-label. Spontaneous reports of ADRs in newborns, infants, and toddlers were more serious and less often described in the product data sheet than in children and adolescents (p < 0.001 and p = 0.004 respectively). Medication errors were more frequent in patients under two years of age. These results should be interpreted with caution due to under-reporting and biases in spontaneous reporting of ADRs.

Mitochondrial DNA diversity in a population from Santa Catarina (Brazil): predominance of the European input.

The state of Santa Catarina (Brazil) is known to have represented a cultural crossroads in South America due to several historic migrations mainly from Europe and Africa. We set out to scrutinize whether the genetic imprint of these migrations could be traced through analysis of the matrilineal gene pool of the Catarinenses. The entire control region of the mitochondrial DNA was studied in 80 healthy and maternally unrelated individuals. The analysis of haplogroup distribution revealed that this population is extremely heterogeneous, showing the coexistence of matrilineal lineages with three different phylogeographic origins. European lineages are the most frequent due mainly to the impact of relatively recent migratory waves from Europe. In spite of this, Native American lineages and African lineages incorporated with the slave trade are also present in noticeable proportions. The strikingly high variability generated by intense gene flow is mirrored in a high sequence diversity (0.9930) and power of discrimination (0.9806). Thus, analysis of the entire mitochondrial DNA control region emerges as a valuable tool for forensic genetic purposes in this highly admixed population, an attribute common to several present-day Latin American populations.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line.

BACKGROUND: The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. RESULTS: Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. CONCLUSION: The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell.

Characterization of the Iberian Y chromosome haplogroup R-DF27 in Northern Spain.

The European paternal lineage R-DF27 has been proposed as a haplogroup of Iberian origin due to its maximum frequencies in the Iberian Peninsula. In this study, the distribution and structure of DF27 were characterized in 591 unrelated male individuals from four key populations of the north area of the Iberian Peninsula through the analysis of 12 Y-SNPs that define DF27 main sublineages. Additionally, Y-SNP allele frequencies were also gathered from the reference populations in the 1000 Genomes Project to compare and obtain a better landscape of the distribution of DF27. Our results reveal frequencies over 35% of DF27 haplogroup in the four North Iberian populations analyzed and high frequencies for its subhaplogroups. Considering the low frequency of DF27 and its sublineages in most populations outside of the Iberian Peninsula, this haplogroup seems to have geographical significance; thus, indicating a possible Iberian patrilineal origin of vestiges bearing this haplogroup. The dataset presented here contributes with new data to better understand the complex genetic variability of the Y chromosome in the Iberian Peninsula, that can be applied in Forensic Genetics.

Analysis of the R1b-DF27 haplogroup shows that a large fraction of Iberian Y-chromosome lineages originated recently in situ.

Haplogroup R1b-M269 comprises most Western European Y chromosomes; of its main branches, R1b-DF27 is by far the least known, and it appears to be highly prevalent only in Iberia. We have genotyped 1072 R1b-DF27 chromosomes for six additional SNPs and 17 Y-STRs in population samples from Spain, Portugal and France in order to further characterize this lineage and, in particular, to ascertain the time and place where it originated, as well as its subsequent dynamics. We found that R1b-DF27 is present in frequencies ~40% in Iberian populations and up to 70% in Basques, but it drops quickly to 6-20% in France. Overall, the age of R1b-DF27 is estimated at ~4,200 years ago, at the transition between the Neolithic and the Bronze Age, when the Y chromosome landscape of W Europe was thoroughly remodeled. In spite of its high frequency in Basques, Y-STR internal diversity of R1b-DF27 is lower there, and results in more recent age estimates; NE Iberia is the most likely place of origin of DF27. Subhaplogroup frequencies within R1b-DF27 are geographically structured, and show domains that are reminiscent of the pre-Roman Celtic/Iberian division, or of the medieval Christian kingdoms.

New clues to the evolutionary history of the main European paternal lineage M269: dissection of the Y-SNP S116 in Atlantic Europe and Iberia.

The dissection of S116 in more than 1500 individuals from Atlantic Europe and the Iberian Peninsula has provided important clues about the controversial evolutionary history of M269. First, the results do not point to an origin of M269 in the Franco-Cantabrian refuge, owing to the lack of sublineage diversity within M269, which supports the new theories proposing its origin in Eastern Europe. Second, S116 shows frequency peaks and spatial distribution that differ from those previously proposed, indicating an origin farther west, and it also shows a high frequency in the Atlantic coastline. Third, an outstanding frequency of the DF27 sublineage has been found in Iberia, with a restricted distribution pattern inside this peninsula and a frequency maximum in the area of the Franco-Cantabrian refuge. This entire panorama indicates an old arrival of M269 into Western Europe, because it has generated at least two episodes of expansion in the Franco-Cantabrian area. This study demonstrates the importance of continuing the dissection of the M269 lineage in different European populations because the discovery and study of new sublineages can adjust or even completely revise the theories about European peopling, as has been the case for the place of origin of M269.

"On the job" learning: A bioinformatics course incorporating undergraduates in actual research projects and manuscript submissions.

The sequencing of whole genomes and the analysis of genetic information continues to fundamentally change biological and medical research. Unfortunately, the people best suited to interpret this data (biologically trained researchers) are commonly discouraged by their own perceived computational limitations. To address this, we developed a course to help alleviate this constraint. Remarkably, in addition to equipping our undergraduates with an informatic toolset, we found our course design helped prepare our students for collaborative research careers in unexpected ways. Instead of simply offering a traditional lecture- or laboratory-based course, we chose a guided inquiry method, where an instructor-selected research question is examined by students in a collaborative analysis with students contributing to experimental design, data collection, and manuscript reporting. While students learn the skills needed to conduct bioinformatic research throughout all sections of the course, importantly, students also gain experience in working as a team and develop important communication skills through working with their partner and the class as a whole, and by contributing to an original research article. Remarkably, in its first three semesters, this novel computational genetics course has generated 45 undergraduate authorships across three peer-reviewed articles. More importantly, the students that took this course acquired a positive research experience, newfound informatics technical proficiency, unprecedented familiarity with manuscript preparation, and an earned sense of achievement. Although this course deals with analyses of genetic systems, we suggest the basic concept of integrating actual research projects into a 16-week undergraduate course could be applied to numerous other research-active academic fields.