A cornucopia of diversity-Ranunculales as a model lineage.

The Ranunculales are a hyperdiverse lineage in many aspects of their phenotype, including growth habit, floral and leaf morphology, reproductive mode, and specialized metabolism. Many Ranunculales species, such as opium poppy and goldenseal, have a high medicinal value. In addition, the order includes a large number of commercially important ornamental plants, such as columbines and larkspurs. The phylogenetic position of the order with respect to monocots and core eudicots and the diversity within this lineage make the Ranunculales an excellent group for studying evolutionary processes by comparative studies. Lately, the phylogeny of Ranunculales was revised, and genetic and genomic resources were developed for many species, allowing comparative analyses at the molecular scale. Here, we review the literature on the resources for genetic manipulation and genome sequencing, the recent phylogeny reconstruction of this order, and its fossil record. Further, we explain their habitat range and delve into the diversity in their floral morphology, focusing on perianth organ identity, floral symmetry, occurrences of spurs and nectaries, sexual and pollination systems, and fruit and dehiscence types. The Ranunculales order offers a wealth of opportunities for scientific exploration across various disciplines and scales, to gain novel insights into plant biology for researchers and plant enthusiasts alike.

Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

A MITE insertion abolishes the AP3-3 self-maintenance regulatory loop in apetalous flowers of Nigella damascena.

MADS-box transcription factors are important regulators of floral organ identity through their binding to specific motifs, termed CArG, in the promoter of their target genes. Petal initiation and development depend on class A and B genes, but MADS-box genes of the APETALA3 (AP3) clade are key regulators of this process. In the early diverging eudicot Nigella damascena, an apetalous [T] morph is characterized by the lack of expression of the NdAP3-3 gene, with its expression being petal-specific in the wild-type [P] morph. All [T] morph plants are homozygous for an NdAP3-3 allele with a Miniature Inverted-repeat Transposable Element (MITE) insertion in the second intron of the gene. Here, we investigated to which extent the MITE insertion impairs regulation of the NdAP3-3 gene. We found that expression of NdAP3-3 is initiated in the [T] morph, but the MITE insertion prevents its positive self-maintenance by affecting the correct splicing of the mRNA. We also found specific CArG features in the promoter of the NdAP3-3 genes with petal-specific expression. However, they are not sufficient to drive expression only in petals of transgenic Arabidopsis, highlighting the existence of Nigella-specific cis/trans-acting factors in regulating AP3 paralogs.

Evolutionary analyses and expression patterns of TCP genes in Ranunculales.

TCP transcription factors play a role in a large number of developmental processes and are at the crossroads of numerous hormonal biosynthetic and signaling pathways. The complete repertoire of TCP genes has already been characterized in several plant species, but not in any species of early diverging eudicots. We focused on the order Ranunculales because of its phylogenetic position as sister group to all other eudicots and its important morphological diversity. Results show that all the TCP genes expressed in the floral transcriptome of Nigella damascena (Ranunculaceae) are the orthologs of the TCP genes previously identified from the fully sequenced genome of Aquilegia coerulea. Phylogenetic analyses combined with the identification of conserved amino acid motifs suggest that six paralogous genes of class I TCP transcription factors were present in the common ancestor of angiosperms. We highlight independent duplications in core eudicots and Ranunculales within the class I and class II subfamilies, resulting in different numbers of paralogs within the main subclasses of TCP genes. This has most probably major consequences on the functional diversification of these genes in different plant clades. The expression patterns of TCP genes in Nigella damascena were consistent with the general suggestion that CIN and class I TCP genes may have redundant roles or take part in same pathways, while CYC/TB1 genes have more specific actions. Our findings open the way for future studies at the tissue level, and for investigating redundancy and subfunctionalisation in TCP genes and their role in the evolution of morphological novelties.

Frailty as a predictor of clinical problems and events that require elderly patients with heart failure to use health resources.

BACKGROUND: The clinical management of elderly patients with heart failure (HF) is not firmly established. Decision-making should be individualized depending on the biological deterioration of each patient, from aggressive management to a palliative approach. Frailty can serve as the basis for this comprehensive individualized management. Our objective was to evaluate the importance of the main clinical problems, as well as the events that required the use of health resources, based the degree of frailty, in elderly patients with HF. METHODS AND RESULTS: Retrospective observational cohort study. Frailty was defined according to the deficit accumulation construct. A total of 546 patients hospitalized for acute HF were included. The median age (Q1-Q3) was 82 (78-86) years. A total of 454 patients (83%) showed some degree of frailty: 221 (48.7%) mild, 207 (45.6%) moderate and 26 (5.7%) advanced. There was a significant tendency towards polypharmacy from no to severe frailty. Hospital events were recorded for 4 (1-6) patients with mild frailty, 4 (2-6) patients with moderate frailty and 2 ((1-4) patients with advanced frailty (p = 0.045). A total of 204 patients (37.4%) died during follow-up. The median time to death was 11.4 (4-16.8), 6.7 (3.3-11.6), 6.5 (3.4-12.2) and 4.1 (0.8-7.7) months for patients with no, mild, moderate, or advanced frailty, respectively (p = 0.006). CONCLUSIONS: Frailty due to deficit accumulation is a good predictor of clinical problems and events that require the use of health resources; therefore, it can serve as a basis for the management of HF in the elderly.

Transcriptome Analysis Reveals Putative Target Genes of APETALA3-3 During Early Floral Development in Nigella damascena L.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

Updated guidelines for predictive biomarker testing in advanced non-small-cell lung cancer: a National Consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology.

In 2011 the Spanish Society of Medical Oncology (SEOM) and the Spanish Society of Pathology (SEAP) started a joint project to establish guidelines on biomarker testing in patients with advanced non-small-cell lung cancer (NSCLC) based on current evidence. As this field is constantly evolving, these guidelines have been updated, previously in 2012 and 2015 and now in 2019. Current evidence suggests that the mandatory tests to conduct in all patients with advanced NSCLC are for EGFR and BRAF mutations, ALK and ROS1 rearrangements and PD-L1 expression. The growing need to study other emerging biomarkers has promoted the routine use of massive sequencing (next-generation sequencing, NGS). The coordination of every professional involved and the prioritisation of the most suitable tests and technologies for each case remains a challenge.

Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae.

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression.

Phylogenetic analysis of ORF5 and ORF7 of porcine reproductive and respiratory syndrome (PRRS) virus and the frequency of wild-type PRRS virus in México.

Porcine reproductive and respiratory syndrome (PRRS) is caused by a genetically diverse RNA virus and is an economically significant disease in the swine industry. In this study, a total of 8,126 serum samples were obtained from 275 technified and semi-technified farms belonging to 30 of the 32 states of Mexico and representative of the eight regions of the country. Anti-PRRSv antibodies against the PRRS vaccine and an isolated wild Mexican virus were tested by ELISA. Antibodies were found in 15%-49% of the tested sera, with 2.4%-9.8% against the vaccine and 7.7%-26% against the wild virus. The PRRSv virus was detected by RT-PCR in 77 of the 1,630 pooled samples tested, representing seven of the eight geographic regions into which the Mexican Republic is divided. The complete sequences of open reading frames 5 and 7 from 20 PRRSv-positive samples were determined. The analysis of the sequences together with the previously published sequences of historic strains revealed that all the strains belonged to the one, five and eight lineages of the PRRSV2. Striking differences, particularly in ORF5 and ORF7, were found between sequences of the strains and the reference virus, due to insertions and substitutions in positions that play key roles in the recognition, structure and function of the virus. Overall, these results established the magnitude of PRRS virus genetic diversity, and the most frequent virus strain that predominates in Mexico. The PRRSV2 is presented in the porcine population of Mexico; the circulating strains have important changes in ORF5 and ORF7, which probably explain the results obtained in the serological analysis of the wild virus and vaccine strains.

Autologous stem cell transplantation (ASCT) in patients with mantle cell lymphoma: a retrospective study of the Spanish lymphoma group (GELTAMO).

Guidelines recommend autologous stem cell transplantation (ASCT) consolidation in first complete or partial response after regimens including rituximab (R) and high-dose AraC (HDAC), but its use beyond that response is questioned. We present a retrospective analysis of 268 patients with MCL who received ASCT. With a median follow-up for survival patients of 54 months, progression-free survival and overall survival for the whole series were 38 and 74 months, respectively, and for patients transplanted in first CR 49 and 97 months, respectively. Patients without CR before transplant were analyzed separately, those who achieved CR after transplantation had better PFS (48 vs 0.03 months, p < 0.001) and OS (92 vs 16 months, p < 0.001) than the remaining. In univariate analysis, first CR at transplant (p = 0.01) and prior rituximab (p = 0.02) were the variables associated with PFS. For OS, the same variables resulted significant (p = 0.03 and p < 0.001, respectively). In multivariate analysis, only the status at transplant (first CR) remained significant. This retrospective study concludes that ASCT consolidation in first CR induces high survival rates. In other stages of disease, the need of ASCT as consolidation may be questioned.

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis.

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA-deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.

Toxicity and efficacy of busulfan and fludarabine myeloablative conditioning for HLA-identical sibling allogeneic hematopoietic cell transplantation in AML and MDS.

The safety and efficacy of a 4-day myeloablative conditioning (MAC) regimen consisting of Bu 3.2 mg/kg and fludarabine 40 mg/m(2)/day for HLA-identical sibling allogeneic hematopoietic cell transplantation (HCT) in myeloid malignancies was investigated in 133 patients (median age, 47 years; range 19-74 years) with de novo AML (60%), secondary AML (20%) or myelodysplastic syndrome (20%). All patients engrafted. Hepatic veno-occlusive disease occurred in five patients (4%), and severe toxicities, mostly mucositis, occurred in twenty-three (17%) patients. The non-relapse mortality (NRM) at 100 days was 1.5%. The incidences of acute GVHD grade 2-4 and grade 3-4 were 32 and 13%, respectively. At a median follow-up of 38 months, the cumulative incidence of chronic GVHD was 67%. The relapse incidence was 30% (27 and 31%, respectively, in patients with early- and late-stage disease), and the overall NRM was 15%. The actuarial 4-year disease-free survival (DFS) and overall survival (OS) were 54 and 62%, respectively. Patients aged <50 years had better outcomes compared with older patients (DFS 64 vs 42%, P=0.006; OS 73 vs 47%, P<0.001, respectively).

Experience with anidulafungin in patients with allogeneic hematopoietic stem cell transplantation and graft-versus-host disease.

BACKGROUND: It is well known that both acute and chronic graft-versus-host disease (GVHD) are associated with invasive fungal disease (IFD). Because the galactomannan antigen diagnostic test has low specificity and sensitivity outside of the neutropenic period, many institutions use posaconazole or voriconazole for IFD prophylaxis during GVHD treatment. Moreover, several factors, mainly hepatic impairment, can limit the use of extended spectrum azoles, both in prophylaxis or treatment. METHODS: We retrospectively analyzed 25 patients with allogeneic hematopoietic stem cell transplantation (HSCT) and GVHD - grade III-IV acute GHVD (n = 15), progressive chronic GVHD (n = 7), and "overlap" GVHD (n = 3) - who received intravenous anidulafungin (200 mg on day 1, followed by 100 mg once daily). If necessary, anidulafungin treatment was followed by oral administration of 200 mg voriconazole twice a day or 200 mg posaconazole 3 times daily until patients were considered not at risk for IFD. RESULTS: Twenty-one patients (85%) received anidulafungin as prophylaxis and 5 patients (15%) received it as treatment. Median duration of intravenous anidulafungin administration was 8 days (range 6-17). Seven patients (28%) presented mild adverse effects, with no significant interactions with calcineurin inhibitors. Sequentially, 4 patients received voriconazole and 6 posaconazole. Two patients (8%) developed IFD after anidulafungin withdrawal: 1 with Candida albicans and the other with Mucor, 8 and 5 days after withdrawal, respectively. CONCLUSIONS: Our results are of interest owing to the absence of data in the literature on anidulafungin use in HSCT patients with GVHD, and suggest that anidulafungin, because of its spectrum, pharmacological profile, low toxicity, and absence of interactions with immunosuppressants, could be a drug of choice in this setting.

Interaction between affordance and handedness recognition: a chronometric study

The visualization of tools and manipulable objects activates motor-related areas in the cortex, facilitating possible actions toward them. This pattern of activity may underlie the phenomenon of object affordance. Some cortical motor neurons are also covertly activated during the recognition of body parts such as hands. One hypothesis is that different subpopulations of motor neurons in the frontal cortex are activated in each motor program; for example, canonical neurons in the premotor cortex are responsible for the affordance of visual objects, while mirror neurons support motor imagery triggered during handedness recognition. However, the question remains whether these subpopulations work independently. This hypothesis can be tested with a manual reaction time (MRT) task with a priming paradigm to evaluate whether the view of a manipulable object interferes with the motor imagery of the subject's hand. The MRT provides a measure of the course of information processing in the brain and allows indirect evaluation of cognitive processes. Our results suggest that canonical and mirror neurons work together to create a motor plan involving hand movements to facilitate successful object manipulation.

Interaction between affordance and handedness recognition: a chronometric study.

The visualization of tools and manipulable objects activates motor-related areas in the cortex, facilitating possible actions toward them. This pattern of activity may underlie the phenomenon of object affordance. Some cortical motor neurons are also covertly activated during the recognition of body parts such as hands. One hypothesis is that different subpopulations of motor neurons in the frontal cortex are activated in each motor program; for example, canonical neurons in the premotor cortex are responsible for the affordance of visual objects, while mirror neurons support motor imagery triggered during handedness recognition. However, the question remains whether these subpopulations work independently. This hypothesis can be tested with a manual reaction time (MRT) task with a priming paradigm to evaluate whether the view of a manipulable object interferes with the motor imagery of the subject's hand. The MRT provides a measure of the course of information processing in the brain and allows indirect evaluation of cognitive processes. Our results suggest that canonical and mirror neurons work together to create a motor plan involving hand movements to facilitate successful object manipulation.

A randomized 12-week clinical comparison of an oscillating-rotating toothbrush to a new sonic brush in the reduction of gingivitis and plaque.

OBJECTIVE: To evaluate the efficacy of a marketed oscillating-rotating (O-R) power toothbrush (Oral-B Triumph with SmartGuide and FlossAction brush head, D34/EB25) to a new sonic toothbrush (Sonicare FlexCare Platinum) in the reduction of gingivitis and plaque over a 12-week test period. METHODS: This was a single center, randomized, open label, examiner-blind, two-treatment, parallel group study. Subjects who met the entrance criteria were enrolled in the study and randomly assigned to either the O-R or sonic treatment group. Subjects brushed with their assigned toothbrush and a marketed fluoride dentifrice for two minutes twice daily at home for 12 weeks. Gingivitis and plaque were evaluated at Baseline, Week 6, and Week 12. Gingivitis was assessed using the Modified Gingival Index (MGI) and Gingival Bleeding Index (GBI), and plaque was assessed using the Rustogi Modified Navy Plaque Index (RMNPI). Data were analyzed using an Analysis of Covariance (ANCOVA) with Baseline as the covariate. RESULTS: In total, 130 subjects (65 per group) were randomized to treatment and 127 subjects completed the study. Both brushes produced statistically significant (p < 0.001) reductions in gingivitis and plaque measures relative to Baseline. At Week 12, the O-R brush demonstrated significantly greater reductions than the sonic brush in whole mouth gingivitis measures (p = 0.007). Additionally, the O-R brush presented significantly fewer bleeding sites (p < 0.007) and significantly greater reductions in whole mouth plaque measures (p < or = 0.035) at Weeks 6 and 12 versus the sonic brush. The benefit for the O-R brush versus the sonic brush at Week 12 was 11.7% for gingivitis, 19.8% for number of bleeding sites, and 12.2% for whole mouth plaque. There were no adverse events reported or observed for either brush. CONCLUSION: The oscillating-rotating toothbrush demonstrated statistically significantly greater reductions in whole mouth plaque at Weeks 6 and 12, as well as significantly greater gingivitis reductions over the long-term (12 weeks), compared to the new sonic toothbrush.

Over-expression of histone H3K4 demethylase gene JMJ15 enhances salt tolerance in Arabidopsis.

Histone H3 lysine 4 trimethylation (H3K4me3) has been shown to be involved in stress-responsive gene expression and gene priming in plants. However, the role of H3K4me3 resetting in the processes is not clear. In this work we studied the expression and function of Arabidopsis H3K4 demethylase gene JMJ15. We show that the expression of JMJ15 was relatively low and was limited to a number of tissues during vegetative growth but was higher in young floral organs. Over-expression of the gene in gain-of-function mutants reduced the plant height with accumulation of lignin in stems, while the loss-of-function mutation did not produce any visible phenotype. The gain-of-function mutants showed enhanced salt tolerance, whereas the loss-of-function mutant was more sensitive to salt compared to the wild type. Transcriptomic analysis revealed that over-expression of JMJ15 down-regulated many genes which are preferentially marked by H3K4me3 and H3K4me2. Many of the down-regulated genes encode transcription regulators involved in stress responses. The data suggest that increased JMJ15 levels may regulate the gene expression program that enhances stress tolerance.

The basis for evolution of DNA-binding specificity of the Aft1 transcription factor in yeasts.

The Saccharomyces cerevisiae Aft1 and Kluyveromyces lactis KlAft are orthologous yeast transcription activators that regulate the expression of the same group of iron-uptake genes but bind to the different DNA sites: TGCACCC for Aft1 and PuCACCC for KlAft. To establish whether the DNA-binding mechanisms of Aft1 and KlAft have diverged during the evolution of the Aft-type transcription factor, we examined the function of a nonconserved region in their DNA-binding domains. A large part of this region is composed of a sequence predicted to be disordered in structure and potentially phosphorylated. We show with deletion mutant analyses that this sequence is essential for the binding of Aft1 to its DNA site and for the iron uptake and growth of S. cerevisiae under iron-limited conditions. We constructed hybrid proteins by exchanging the nonconserved regions of Aft1 and KlAft. We show that the Aft1 region is necessary and sufficient for KlAft to bind efficiently to the Aft1 DNA site in S. cerevisiae and to complement the iron-dependent phenotype of the aft1Δaft2Δ mutant. This demonstrates that the changes in the nonconserved region of the Aft-type DNA-binding domain have led to changes in the DNA-binding specificity and have major consequences for the regulation of iron homeostasis. The combination of bioinformatic and experimental analyses indicates that the sequence TGCACCC is the most probable ancestral Aft-type element. Our findings suggest that the changes in the nonconserved region of the DNA-binding domain are responsible for the evolution of the TGCACCC sequence toward PuCACCC in the K. lactis species.

Lung adenocarcinoma in the era of targeted therapies: histological classification, sample priorization, and predictive biomarkers

The arrival of targeted therapies has presented both a conceptual and a practical challenge in the treatment of patients with advanced non-small cell lung carcinomas (NSCLCs). The relationship of these treatments with specific histologies and predictive biomarkers has made the handling of biopsies the key factor for success. In this study, we highlight the balance between precise histological diagnosis and the practice of conducting multiple predictive assays simultaneously. This can only be achieved where there is a commitment to multidisciplinary working by the tumor board to ensure that a sensible protocol is applied. This proposal for prioritizing samples includes both recent technological advances and the some of the latest discoveries in the molecular classification of NSCLCs (AU)