Long-Term Monitoring of Aphid-Transmitted Viruses in Melon and Zucchini Crops: Genetic Diversity and Population Structure of Cucurbit Aphid-Borne Yellows Virus and Watermelon Mosaic Virus.

Understanding the emergence and prevalence of viral diseases in crops requires the systematic epidemiological monitoring of viruses, as well as the analysis of how ecological and evolutionary processes combine to shape viral population dynamics. Here, we extensively monitored the occurrence of six aphid-transmitted viruses in melon and zucchini crops in Spain for 10 consecutive cropping seasons between 2011 and 2020. The most prevalent viruses were cucurbit aphid-borne yellows virus (CABYV) and watermelon mosaic virus (WMV), found in 31 and 26% of samples with yellowing and mosaic symptoms. Other viruses, such as zucchini yellow mosaic virus, cucumber mosaic virus, Moroccan watermelon mosaic virus, and papaya ring spot virus, were detected less frequently (<3%) and mostly in mixed infections. Notably, our statistical analysis showed a significant association between CABYV and WMV in melon and zucchini hosts, suggesting that mixed infections might be influencing the evolutionary epidemiology of these viral diseases. We then carried out a comprehensive genetic characterization of the full-length genome sequences from CABYV and WMV isolates by using the Pacific Biosciences single-molecule real-time (PacBio) high-throughput technology to assess the genetic variation and structure of their populations. Our results showed that the CABYV population displayed seven codons under positive selection, and although most isolates clustered in the Mediterranean clade, a subsequent analysis of molecular variance revealed a significant, fine-scale temporal structure, which was in part explained by the level of the variance between isolates from single and mixed infections. In contrast, the WMV population genetic analysis showed that most of the isolates grouped into the Emergent clade, with no genetic differentiation and under purifying selection. These results underlie the epidemiological relevance of mixed infections for CABYV and provide a link between genetic diversity and CABYV dynamics at the whole-genome level.

ß-Pix directs collective migration of anterior visceral endoderm cells in the early mouse embryo.

Collective epithelial migration is important throughout embryonic development. The underlying mechanisms are poorly understood but likely involve spatially localized activation of Rho GTPases. We previously reported that Rac1 is essential for generating the protrusive activity that drives the collective migration of anterior visceral endoderm (AVE) cells in the early mouse embryo. To identify potential regulators of Rac1, we first performed an RNAi screen of Rho family exchange factors (guanine nucleotide exchange factor [GEF]) in an in vitro collective epithelial migration assay and identified ß-Pix. Genetic deletion of ß-Pix in mice disrupts collective AVE migration, while high-resolution live imaging revealed that this is associated with randomly directed protrusive activity. We conclude that ß-Pix controls the spatial localization of Rac1 activity to drive collective AVE migration at a critical stage in mouse development.

Long-Term Cocirculation of Two Strains of Pepino Mosaic Virus in Tomato Crops and Its Effect on Population Genetic Variability.

Mixed viral infections are common in plants, and the evolutionary dynamics of viral populations may differ depending on whether the infection is caused by single or multiple viral strains. However, comparative studies of single and mixed infections using viral populations in comparable agricultural and geographical locations are lacking. Here, we monitored the occurrence of pepino mosaic virus (PepMV) in tomato crops in two major tomato-producing areas in Murcia (southeastern Spain), supporting evidence showing that PepMV disease-affected plants had single infections of the Chilean 2 (CH2) strain in one area and the other area exhibited long-term (13 years) coexistence of the CH2 and European (EU) strains. We hypothesized that circulating strains of PepMV might be modulating the differentiation between them and shaping the evolutionary dynamics of PepMV populations. Our phylogenetic analysis of 106 CH2 isolates randomly selected from both areas showed a remarkable divergence between the CH2 isolates, with increased nucleotide variability in the geographical area where both strains cocirculate. Furthermore, the potential virus-virus interaction was studied further by constructing six full-length infectious CH2 clones from both areas, and assessing their viral fitness in the presence and absence of an EU-type isolate. All CH2 clones showed decreased fitness in mixed infections and although complete genome sequencing indicated a nucleotide divergence of those CH2 clones by area, the magnitude of the fitness response was irrespective of the CH2 origin. Overall, these results suggest that although agroecological cropping practices may be particularly important for explaining the evolutionary dynamics of PepMV in tomato crops, the cocirculation of both strains may have implications on the genetic variability of PepMV populations.

Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2.

Delamination of neural crest (NC) cells is a bona fide physiological model of epithelial-to-mesenchymal transition (EMT), a process that is influenced by Wnt/ß-catenin signalling. Using two in vivo models, we show that Wnt/ß-catenin signalling is transiently inhibited at the time of NC delamination. In attempting to define the mechanism underlying this inhibition, we found that the scaffold proteins Dact1 and Dact2, which are expressed in pre-migratory NC cells, are required for NC delamination in Xenopus and chick embryos, whereas they do not affect the motile properties of migratory NC cells. Dact1/2 inhibit Wnt/ß-catenin signalling upstream of the transcriptional activity of T cell factor (TCF), which is required for EMT to proceed. Dact1/2 regulate the subcellular distribution of ß-catenin, preventing ß-catenin from acting as a transcriptional co-activator to TCF, yet without affecting its stability. Together, these data identify a novel yet important regulatory element that inhibits ß-catenin signalling, which then affects NC delamination.

Seeds of Locally Aligned Motion and Stress Coordinate a Collective Cell Migration.

We find how collective migration emerges from mechanical information transfer between cells. Local alignment of cell velocity and mechanical stress orientation-a phenomenon dubbed "plithotaxis"-plays a crucial role in inducing coordinated migration. Leader cells at the monolayer edge better align velocity and stress to migrate faster toward the open space. Local seeds of enhanced motion then generate stress on neighboring cells to guide their migration. Stress-induced motion propagates into the monolayer as well as along the monolayer boundary to generate increasingly larger clusters of coordinately migrating cells that move faster with enhanced alignment of velocity and stress. Together, our analysis provides a model of long-range mechanical communication between cells, in which plithotaxis translates local mechanical fluctuations into globally collective migration of entire tissues.

Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord.

BMP activity is essential for many steps of neural development, including the initial role in neural induction and the control of progenitor identities along the dorsal-ventral axis of the neural tube. Taking advantage of chick in ovo electroporation, we show a novel role for BMP7 at the time of neurogenesis initiation in the spinal cord. Using in vivo loss-of-function experiments, we show that BMP7 activity is required for the generation of three discrete subpopulations of dorsal interneurons: dI1-dI3-dI5. Analysis of the BMP7 mouse mutant shows the conservation of this activity in mammals. Furthermore, this BMP7 activity appears to be mediated by the canonical Smad pathway, as we demonstrate that Smad1 and Smad5 activities are similarly required for the generation of dI1-dI3-dI5. Moreover, we show that this role is independent of the patterned expression of progenitor proteins in the dorsal spinal cord, but depends on the BMP/Smad regulation of specific proneural proteins, thus narrowing this BMP7 activity to the time of neurogenesis. Together, these data establish a novel role for BMP7 in primary neurogenesis, the process by which a neural progenitor exits the cell cycle and enters the terminal differentiation pathway.

LMO4 is an essential cofactor in the Snail2-mediated epithelial-to-mesenchymal transition of neuroblastoma and neural crest cells.

Neuroblastoma is an embryonic tumor derived from cells of the neural crest. Taking advantage of a newly developed neural crest lineage tracer and based on the hypothesis that the molecular mechanisms that mediate neural crest delamination are also likely to be involved in the spread of neuroblastoma, we were able to identify genes that are active both in neural crest development and neuroblastoma tumor formation. A subsequent search of the neuroblastoma gene server for human orthologues of genes differentially expressed in the chick embryo neural crest screen retrieved the LIM domain only protein 4 (LMO4), which was expressed in both cell types analyzed. Functional experiments in these two model systems revealed that LMO4 activity is required for neuroblastoma cell invasion and neural crest delamination. Moreover, we identified LMO4 as an essential cofactor in Snail2-mediated cadherin repression and in the epithelial-to-mesenchymal transition of both neural crest and neuroblastoma cells. Together, our results suggest that the association of high levels of LMO4 with aggressive neuroblastomas is dependent on LMO4 regulation of cadherin expression and hence, tumor invasiveness.

An in vitro model of neuronal ensembles.

Advances in 3D neuronal cultures, such as brain spheroids and organoids, are allowing unprecedented in vitro access to some of the molecular, cellular and developmental mechanisms underlying brain diseases. However, their efficacy in recapitulating brain network properties that encode brain function remains limited, thereby precluding development of effective in vitro models of complex brain disorders like schizophrenia. Here, we develop and characterize a Modular Neuronal Network (MoNNet) approach that recapitulates specific features of neuronal ensemble dynamics, segregated local-global network activities and a hierarchical modular organization. We utilized MoNNets for quantitative in vitro modelling of schizophrenia-related network dysfunctions caused by highly penetrant mutations in SETD1A and 22q11.2 risk loci. Furthermore, we demonstrate its utility for drug discovery by performing pharmacological rescue of alterations in neuronal ensembles stability and global network synchrony. MoNNets allow in vitro modelling of brain diseases for investigating the underlying neuronal network mechanisms and systematic drug discovery.

Implications of mixed viral infections on plant disease ecology and evolution.

Mixed viral infections occur more commonly than would be expected by chance in nature. Virus-virus interactions may affect viral traits and leave a genetic signature in the population, and thus influence the prevalence and emergence of viral diseases. Understanding about how the interactions between viruses within a host shape the evolutionary dynamics of the viral populations is needed for viral disease prevention and management. Here, we first synthesize concepts implied in the occurrence of virus-virus interactions. Second, we consider the role of the within-host interactions of virus-virus and virus-other pathogenic microbes, on the composition and structure of viral populations. Third, we contemplate whether mixed viral infections can create opportunities for the generation and maintenance of viral genetic diversity. Fourth, we attempt to summarize the evolutionary response of viral populations to mixed infections to understand how they shape the spatio-temporal dynamics of viral populations at the individual plant and field scales. Finally, we anticipate the future research under the reconciliation of molecular epidemiology and evolutionary ecology, drawing attention to the need of adding more complexity to future research in order to gain a better understanding about the mechanisms operating in nature.

A miniaturized multi-clamp CMOS amplifier for intracellular neural recording.

Intracellular electrophysiology is a foundational method in neuroscience and uses electrolyte-filled glass electrodes and benchtop amplifiers to measure and control transmembrane voltages and currents. Commercial amplifiers perform such recordings with high signal-to-noise ratios (SNRs) but are often expensive, bulky, and not easily scalable to many channels due to reliance on board-level integration of discrete components. Here, we present a monolithic complementary-metal-oxide-semiconductor (CMOS) multi-clamp amplifier integrated circuit capable of recording both voltages and currents with performance exceeding that of commercial benchtop instrumentation. Miniaturization enables high-bandwidth current mirroring, facilitating the synthesis of large-valued active resistors with lower noise than their passive equivalents. This enables the realization of compensation modules that can account for a wide range of electrode impedances. We validate the amplifier's operation electrically, in primary neuronal cultures, and in acute slices, using both high-impedance sharp and patch electrodes. This work provides a solution for low-cost, high-performance and scalable multi-clamp amplifiers.

Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration.

Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell-cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and ß-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype-based clustering of knockdown behaviors identified RHOA-ARHGEF18 and ARHGEF3-ARHGEF28-ARHGEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication.

Ventricular septal defect in a world class runner.

We report the case of an elite male, East African endurance runner (18 years old) who ranked in the top 15 in the World Cross Country Championships (sub 21 year old category) despite having a ventricular septal defect (VSD; width: 0.22 cm) that was diagnosed 2 weeks after this event with echocardiographic evaluation. This athlete was a moderate altitude native ( approximately 3000 m). Cardiac dimensions were within normal limits and no significant pathological signs were observed. His Vo(2max) was relatively low given his performance level (67.9 ml kg(-1) min(-1)). Despite his limited training background (only 1 year), his running economy was, however, better than the values reported in our laboratory for Caucasian runners of the same age. Further cardiological follow up might confirm that the VSD causes no pathological effects or any performance detriment in future years.

Ageing and prostate: age-related changes in androgen receptors of epithelial cells from benign hypertrophic glands compared with cancer.

Total and nuclear androgen receptors (AR) were studied from epithelial cells in internal and external prostatic zones in 51- to 86-year-old individuals with benign prostatic hyperplasia (BPH) (n = 68) and prostatic cancer (n = 9). We focussed on the role played by androgens on those processes, despite the fact that at these ages, its secretion has normally decreased. In BPH, the nuclear AR do not change, but total measured androgen receptors rise with age (r = 0.5, P < 0.01). Total or nuclear AR do not correlate with gland volume, despite its increase with age (r = 0.8, P < 0.05). In prostates less than 180 cc in volume, there is a significant correlation between size, serum total testosterone level (r = 0.53, P < 0.05) and prostatic specific antigen (PSA) (r = 0.63, P < 0.05). The amount of nuclear AR in cells from the external zone (infiltrated by cancer or healthy) is two times greater than in those from the internal region. Total receptor content of the external zone cells is also high, but the sample is too small to demonstrate an age dependence. The results suggest that ageing is accompanied by an accumulation of non-nuclear AR in the cytosol, that does not play a role in the development of BPH because the amount of nuclear receptors remains unaltered. The enrichment in nuclear receptors of the external zone cells, independently of the presence of cancer, points to a greater androgen dependence in these cells than in cells of the internal region.

[Guidelines of the Spanish Society of Cardiology for clinical practice in exercise testing]. / Guías de práctica clínica de la Sociedad Española de Cardiología en pruebas de esfuerzo.

Most exercise testing is performed in adults with known or suspected ischemic heart disease. In the last few years cardiac imaging techniques have been applied in this field, improving the information obtained with the procedure. However, the exceptions to this rule are emerging rapidly not only in healthy people (asymptomatic individuals, athletes, handicapped people) but also in cardiac patients (advanced congestive heart failure, hypertension, rhythm disorders, congenital heart disease, etc.). All the-se issues justify the need for a multidisciplinary consensus document in Spain. This paper reviews and updates the methodological aspects of the stress test, including those related to oxygen consumption measurements. The main aim of this review was to determine the role of exercise testing in the evaluation of ischemic heart disease as well as the applications of imaging stress testing. The usefulness of this test in other non-ischemic cardiac disorders and in selected subsets of healthy people is also reviewed.

[Benign fibrous histiocytoma of the female urethra]. / Histiocitoma fibroso benigno de uretra femenina.

Presentation of one case of benign fibrous histiocytoma of the female urethra in a 49-year old woman. Investigation of the various clinical and diagnostic aspects. Vaginal tumorectomy was curative, and no recurrence was observed 14 months after the control.

[Bladder neoplasms in patients under 40 years of age]. / Tumores vesicales en pacientes menores de cuarenta años.

The incidence, presentation and clinical evolution of vesical tumours diagnosed in our Centre over the last 10 years (1986-1995) in patients under forty are examined. This retrospective study included 19 of the 643 neoplasias treated during that period; 7 of these in patients under 30 and 12 in patients over 31. Haematuria was the most frequent reason for visiting the Centre. At the time of diagnosis, all cases were surface tumours and none progressed to the infiltrant stage. Tumours in patients over 30 are characterised by a higher histological grade and greater relapse rate versus those in patients under 30. Therefore, age may be a favourable prognostic factor in patients under 30 versus the older group who follow a course more similar to the remainder of usual patients. Diagnosis and treatment of these neoplasias should be just the same as for tumours in older patients.

[The index of PSA age vs PSA density. 2 new instruments in the detection of prostatic carcinoma]. / Indice de edad de PSA VS. densidad de PSA. Dos nuevos instrumentos en la detección del carcinoma prostático.

A prospective study was designed to get to know the reliability of PSA density and PSA age index as tools in the diagnostic approach for prostate carcinoma. It was noted that PSA age index is more susceptible than PSA density to diagnose prostate cancer. We noted also that up to 80% of patients with prostate cancer and intermediate PSA levels have PSA density < 0.15 and that, according to current protocols biopsy should not have been performed. It was concluded that the value of PSA age index is at least equal to PSA density, but it eradicates the subjectivity introduced by this data because of the need of measuring prostate volume. We believe that in the future it may be indicated to perform biopsies at PSA levels above 4 ng/ml, and it may be necessary to decrease PSa normal density levels down to 0.1 or less.

Identification of a putative transcriptome signature common to neuroblastoma and neural crest cells.

Neuroblastoma, the most common extracranial tumor in children, is caused by genetic lesions in neural crest precursors of the peripheral nervous system. However, since neural crest cells are neither present after birth and nor are they readily accessible for analysis, very little is known about the genetic networks they might share with neuroblastoma cells during their development, despite their common embryonic origin. Here we have developed a novel resource for lineage tracing and for the isolation of neural crest cells in the chick embryo, enabling us to perform a genome-wide expression screen in neural crest progenitors. In this analysis, we efficiently retrieved known neural crest specific genes that validate our screening strategy and we identified new genes that participate in diverse cell activities, yet with a strong representation of genes associated to cell signaling and cell mobility, two hallmarks of migratory cells. We crossed this transcriptome data with that in the neuroblastoma gene server to search for the human orthologues of these genes associated with neuroblastoma. Accordingly, we retrieved 54 genes expressed strongly in both populations, from which we were able to validate a total of 27 genes expressed in the neural crest that are relevant to neuroblastoma formation. We propose that neural crest and neuroblastoma tumor cells share a common genetic signature that might serve to characterize neuroblastoma cancer stem cells, thereby contributing to the identification of specific targets against which new therapeutic strategies can be designed.

Sonic hedgehog signaling switches the mode of division in the developing nervous system.

The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis, and these regulatory processes are subverted in tumor formation. Here, we developed markers that provided the single-cell resolution necessary to quantify the three modes of division taking place in the developing nervous system in vivo: self-expanding, PP; self-replacing, PN; and self-consuming, NN. Using these markers and a mathematical model that predicts the dynamics of motor neuron progenitor division, we identify a role for the morphogen Sonic hedgehog in the maintenance of stem cell identity in the developing spinal cord. Moreover, our study provides insight into the process linking lineage commitment to neurogenesis with changes in cell-cycle parameters. As a result, we propose a challenging model in which the external Sonic hedgehog signal dictates stem cell identity, reflected in the consequent readjustment of cell-cycle parameters.

Jagged2 controls the generation of motor neuron and oligodendrocyte progenitors in the ventral spinal cord.

In the developing spinal cord, motor neurons (MNs) and oligodendrocytes arise sequentially from a common pool of progenitors. However, the genetic network responsible for this neurogenesis to gliogenesis switch is largely unknown. A transcriptome analysis identified the Notch ligand Jagged2 (JAG2) as a Sonic hedgehog-regulated factor transiently expressed in MN progenitors (pMNs). In vivo loss- and gain-of-function experiments show that JAG2 schedules the differentiation of the pMN progenitors. At early developmental stages, Olig2 expressing pMN progenitors that enter the differentiation pathway exclusively generate MNs. At these times, the activation of the Notch pathway by JAG2 maintains selected pMN progenitors in an undifferentiated state by two mechanisms; first it inhibits MN generation by reducing Olig2 proteins levels, and second it directly inhibits the premature generation of oligodendrocyte progenitors (OLPs) by maintaining high levels of Hes5. Later, extinction of JAG2 from the pMN results in the loss of Hes5 expression, heralding the gliogenic phase of pMN progenitors. Strikingly, downregulation of JAG2 in pMN progenitors is sufficient to promote the precocious generation of OLPs. Together these data provide evidence that JAG2 is a key regulator of the timely and ordered generation of two of the defining cell types in the spinal cord, MNs and OLPs.