Neurodevelopmental disorder associated with gene ARF3: A case report.

We present a case study of a patient exhibiting acquired microcephaly along with global developmental delay and drug-resistant epilepsy. Brain magnetic resonance imaging revealed distinctive features, including a Z-shaped morphology of the brainstem, volumetric reduction of white matter, diffuse thinning of the corpus callosum, and partial fusion of the cerebellar hemispheres at their most cranial portion. Whole-exome sequencing uncovered a pathogenic variant in the ARF3 gene c.200A>T, p.(Asp67Val). The neurodevelopmental disorder associated with the ARF3 gene is exceptionally rare, with only two previously documented cases in the literature. This disorder is characterized by global developmental delay and brain malformations, particularly affecting the white matter, cerebellum, and brainstem. It can also manifest as acquired microcephaly and epilepsy. These phenotypic characteristics align with Golgipathies, underscoring the significance of considering this group of conditions in relevant clinical contexts. In cases where a Z-shaped morphology of the brainstem is observed, ARF3-associated disorder should be included in the list of differential diagnoses.

Blood Serum Stimulates the Virulence Potential of Mucorales through Enhancement in Mitochondrial Oxidative Metabolism and Rhizoferrin Production.

This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of Galleria mellonella. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against Caenorhabditis elegans, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of Mucor lusitanicus that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of M. lusitanicus and Rhizopus arrhizus that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.

PIF4 enhances the expression of SAUR genes to promote growth in response to nitrate.

Nitrate supply is fundamental to support shoot growth and crop performance, but the associated increase in stem height exacerbates the risks of lodging and yield losses. Despite their significance for agriculture, the mechanisms involved in the promotion of stem growth by nitrate remain poorly understood. Here, we show that the elongation of the hypocotyl of Arabidopsis thaliana, used as a model, responds rapidly and persistently to upshifts in nitrate concentration, rather than to the nitrate level itself. The response occurred even in shoots dissected from their roots and required NITRATE TRANSPORTER 1.1 (NRT1.1) in the phosphorylated state (but not NRT1.1 nitrate transport capacity) and NIN-LIKE PROTEIN 7 (NLP7). Nitrate increased PHYTOCHROME INTERACTING FACTOR 4 (PIF4) nuclear abundance by posttranscriptional mechanisms that depended on NRT1.1 and phytochrome B. In response to nitrate, PIF4 enhanced the expression of numerous SMALL AUXIN-UP RNA (SAUR) genes in the hypocotyl. The growth response to nitrate required PIF4, positive and negative regulators of its activity, including AUXIN RESPONSE FACTORs, and SAURs. PIF4 integrates cues from the soil (nitrate) and aerial (shade) environments adjusting plant stature to facilitate access to light.

Dietary polyphenols and their relationship to the modulation of non-communicable chronic diseases and epigenetic mechanisms: A mini-review.

Chronic Non-Communicable Diseases (NCDs) have been considered a global health problem, characterized as diseases of multiple factors, which are developed throughout life, and regardless of genetics as a risk factor of important relevance, the increase in mortality attributed to the disease to environmental factors and the lifestyle one leads. Although the reactive species (ROS/RNS) are necessary for several physiological processes, their overproduction is directly related to the pathogenesis and aggravation of NCDs. In contrast, dietary polyphenols have been widely associated with minimizing oxidative stress and inflammation. In addition to their antioxidant power, polyphenols have also drawn attention for being able to modulate both gene expression and modify epigenetic alterations, suggesting an essential involvement in the prevention and/or development of some pathologies. Therefore, this review briefly explained the mechanisms in the development of some NCDs, followed by a summary of some evidence related to the interaction of polyphenols in oxidative stress, as well as the modulation of epigenetic mechanisms involved in the management of NCDs.

Incidence, etiology, sociodemographic and clinical characterization of acute respiratory failure in pediatric patients at a high-altitude city: A multicenter cohort study.

Background: Acute respiratory failure is a life-threatening medical condition, associated with a variety of conditions and risk factors, including acute respiratory diseases which are a frequent cause of pediatric morbidity and mortality worldwide. In Colombia, the literature related to ARF is scarce. Objective: To determine the incidence, causes, and sociodemographic and clinical characteristics of ARF in three hospitals in Bogota, a high-altitude city located in Colombia, during the COVID-19 pandemic. Methods: A multicenter prospective cohort study called the FARA cohort was developed between April 2020 - December 2021. Patients older than one month and younger than 18 years with respiratory distress who developed ARF were included. Results: 685 patients with respiratory distress were recruited in 21 months. The incidence density of ARF was found to be 41.7 cases per 100 person-year CI 95%, (37.3-47.7). The median age was 4.5 years.. Most of the patients consulted during the first 72 h after the onset of symptoms. Upon admission, 67.2% were potentially unstable. The most frequent pathologies were asthma, bronchiolitis, pneumonia, and sepsis. At admission, 75.6% of the patients required different oxygen delivery systems, 29,5% a low-flow oxygen system, 36,8% a high-flow oxygen system, and 9,28% invasive mechanical ventilation. SARS-COV-2, respiratory syncytial virus, rhinovirus/enterovirus, and adenovirus were the most frequently isolated viral agents. The coinfection cases were scarce. Conclusions: This multicenter study, the FARA cohort, developed at 2,600 meters above sea level, shows the first data on incidence, etiology, sociodemographic and clinical characterization in a pediatric population with ARF that also concurs with the COVID-19 pandemic. These results, not only have implications for public health but also contribute to the scientific and epidemiological literature on a disease developed at a high altitude.

Noninvasive ventilation in patients with COVID-19 from the perspective of the risk of contamination: a narrative review.

INTRODUCTION: noninvasive ventilation (NIV) can be a useful resource to treat acute respiratory failure (ARF), which occurs in patients with COVID-19. However, it is important to consider that there are still no clinical studies that have verified the safety of its use in increase of contamination. AREAS COVERED: Given the potential benefits and simultaneous concerns over the use of NIV in patients with COVID-19, further inquiry is necessary to reach a clinical consensus and provide recommendations for safe use, avoiding contamination. In this context, this narrative review, which included articles published in the Embase, SciELO, PEDro, PubMed and Cochrane up to August 2021, is focused to evaluate available studies related to interfaces, types of circuits, recommended filters, cares for the environment and protective factors for NIV use in patients with COVID-19. EXPERT OPINION: The studies analyzed recommend that the use of NIV can be safe: 1) with equipment that allows the use of the helmet as a safer interface; 2) with double circuit and antimicrobial filter in the expiratory branch; 3) in an environment that allows negative pressure, reducing the dispersion of aerosol particles in the environment; 4) the health team must use the recommended PPE to avoid contamination.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

Transcript pattern analysis of Arf-family genes in the phytopathogen Fusarium oxysporum f. sp. lycopersici reveals the role of Arl3 in the virulence.

Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as a critical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.

The Small GTPases in Fungal Signaling Conservation and Function.

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1-G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus-plant interactions is reviewed as well.

Pinning Control for the p53-Mdm2 Network Dynamics Regulated by p14ARF.

p53 regulates the cellular response to genotoxic damage and prevents carcinogenic events. Theoretical and experimental studies state that the p53-Mdm2 network constitutes the core module of regulatory interactions activated by cellular stress induced by a variety of signaling pathways. In this paper, a strategy to control the p53-Mdm2 network regulated by p14ARF is developed, based on the pinning control technique, which consists into applying local feedback controllers to a small number of nodes (pinned ones) in the network. Pinned nodes are selected on the basis of their importance level in a topological hierarchy, their degree of connectivity within the network, and the biological role they perform. In this paper, two cases are considered. For the first case, the oscillatory pattern under gamma-radiation is recovered; afterward, as the second case, increased expression of p53 level is taken into account. For both cases, the control law is applied to p14ARF (pinned node based on a virtual leader methodology), and overexpressed Mdm2-mediated p53 degradation condition is considered as carcinogenic initial behavior. The approach in this paper uses a computational algorithm, which opens an alternative path to understand the cellular responses to stress, doing it possible to model and control the gene regulatory network dynamics in two different biological contexts. As the main result of the proposed control technique, the two mentioned desired behaviors are obtained.

tasiR-ARFs Production and Target Regulation during In Vitro Maize Plant Regeneration.

During in vitro maize plant regeneration somatic cells change their normal fate and undergo restructuring to generate pluripotent cells able to originate new plants. Auxins are essential to achieve such plasticity. Their physiological effects are mediated by auxin response factors (ARFs) that bind auxin responsive elements within gene promoters. Small trans-acting (ta)-siRNAs, originated from miR390-guided TAS3 primary transcript cleavage, target ARF3/4 class (tasiR-ARFs). Here we found that TAS3b precursor as well as derived tasiR-ARFbD5 and tasiR-ARFbD6 display significantly lower levels in non-embryogenic callus (NEC), while TAS3g, miR390 and tasiR-ARFg are more abundant in the same tissue. However, Argonaute (AGO7) and leafbladeless 1 (LBLl) required for tasiR-ARF biogenesis showed significantly higher transcript levels in EC suggesting limited tasiR-ARF biogenesis in NEC. The five maize ARFs targeted by tasiR-ARFs were also significantly enriched in EC and accompanied by higher auxin accumulation with punctuate patterns in this tissue. At hormone half-reduction and photoperiod implementation, plant regeneration initiated from EC with transient TAS3g, miR390 and tasiR-ARFg increase. Upon complete hormone depletion, TAS3b became abundant and derived tasiR-ARFs gradually increased at further regeneration stages. ZmARF transcripts targeted by tasiR-ARFs, as well as AGO7 and LBL1 showed significantly lower levels during regeneration than in EC. These results indicate a dynamic tasiR-ARF mediated regulation throughout maize in vitro plant regeneration.

Arf-like proteins (Arl1 and Arl2) are involved in mitochondrial homeostasis in Mucor circinelloides.

Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(-) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(-) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose. To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.

Methylation profiling in promoter sequences of ATM and CDKN2A (p14ARF/p16INK4a ) genes in blood and cfDNA from women with impalpable breast lesions.

The objective of the present study was to evaluate the epigenetic changes occurring in early stages of breast cancer. The present study investigated the methylation profile of the ATM, p14ARF and p16INK4a promoters in total blood and plasma cell-free DNA (cfDNA) from women with impalpable breast lesions compared with in total blood of a control cohort of women without breast lesions. The samples were evaluated using the methylation-specific PCR method. The Fisher's exact test was used to evaluate statistical significance between the methylation and clinical variables. A total of 111 women were evaluated, including 56 women with impalpable breast cancer (39/56 also had paired plasma cfDNA) and 55 women in the control cohort (55 blood DNA). For blood DNA from women with malignant impalpable breast lesions, p16INK4a exhibited the greatest percentage of methylation (48%), followed by ATM (37.5%) and p14ARF (27%) promoters, regardless of age variation. For plasma cfDNA, the methylation rates for ATM, p14ARF and p16INK4a were 26, 26 and 10%, respectively. The methylation rates for the blood DNA of controls were the lowest for ATM (9%), p14ARF (7%) and p16INK4a (7%). The women with impalpable breast lesions (benign and malignant lesions) exhibited the highest methylation rate, regardless of age, compared with the paired plasma cfDNA and controls. This epigenetic change was statistically significant for the promoters of ATM (P=0.009) and p16INK4a (P=0.001) (impalpable breast lesions vs. control). The present study demonstrated that epigenetic changes occurring in the ATM and CDKN2A genes detectable in liquid biopsy were associated with the development of impalpable breast lesions.

The Recycling Endosome in Nerve Cell Development: One Rab to Rule Them All?

Endocytic recycling is an intracellular process that returns internalized molecules back to the plasma membrane and plays crucial roles not only in the reuse of receptor molecules but also in the remodeling of the different components of this membrane. This process is required for a diversity of cellular events, including neuronal morphology acquisition and functional regulation, among others. The recycling endosome (RE) is a key vesicular component involved in endocytic recycling. Recycling back to the cell surface may occur with the participation of several different Rab proteins, which are master regulators of membrane/protein trafficking in nerve cells. The RE consists of a network of interconnected and functionally distinct tubular subdomains that originate from sorting endosomes and transport their cargoes along microtubule tracks, by fast or slow recycling pathways. Different populations of REs, particularly those formed by Rab11, Rab35, and Arf6, are associated with a myriad of signaling proteins. In this review, we discuss the cumulative evidence suggesting the existence of heterogeneous domains of REs, controlling different aspects of neurogenesis, with a particular focus on the commonalities and singularities of these REs and their contribution to nerve development and differentiation in several animal models.

AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots.

In Arabidopsis, lateral root (LR) development is mainly controlled by several known auxin-regulated transcription factors (TFs). Here, we show that AtHB23 (a homeodomain-leucine zipper I TF) participates in this intricate network. Our study of the expression pattern of AtHB23 revealed that it is transcriptionally activated in the early stages of secondary LR primordium (LRP). We found that AtHB23 directly limits the expression of LBD16, a key factor in LR initiation, and also directly induces the auxin transporter gene LAX3. We propose that this HD-Zip I mediates the regulation of LAX3 by ARF7/19. Furthermore, AtHB23 plays distinct roles during the formation of secondary and tertiary roots, exhibiting differential expression patterns. ATHB23 is expressed throughout the tertiary root primordium, whereas it is restricted to early stages in secondary primordia, likely later repressing LBD16 in tertiary LR development and further inhibiting root emergence. Our results suggest that different genetic programs govern the formation of LRP from the main or secondary roots, thereby shaping the global dynamic architecture of the root system.

Distinct Roles of Direct Transduction Versus Exposure to the Tumor Secretome on Murine Endothelial Cells After Melanoma Gene Therapy with Interferon-ß and p19Arf.

Tumor vasculature plays a central role in tumor progression, making it an attractive therapeutic target. In this study, we explore the antiangiogenic potential of our melanoma gene therapy approach combining interferon ß (IFNß) and p19Arf gene transfer. Since these proteins are modulators of tumor vasculature, we explore the impact of IFNß and p19Arf gene transfer on murine endothelial cells (tEnd). Adenovirus-mediated gene transfer of p19Arf to tEnd cells inhibited proliferation, tube formation, migration, and led to increased expression of genes related to the p53 cell death pathway, yet IFNß gene transfer had no significant impact on tEnd viability. Alternatively, tEnd cells were exposed to the factors generated by transduced B16 (mouse melanoma) cells using either coculture or conditioned medium. In either case, transduction of B16 cells with the IFNß vector, whether alone or in combination with p19Arf, resulted in endothelial cell death. Strikingly, treatment of tEnd cells with recombinant IFNß did not induce death, demonstrating that additional factors produced by B16 cells contributed to the demise of tEnd cells. In this work, we have shown that our melanoma gene therapy strategy produces desirable negative effects on endothelial cells, possibly correlating with antiangiogenic activity.

Small GTPases in plant biotic interactions.

The superfamily of small monomeric GTPases originated in a common ancestor of eukaryotic multicellular organisms and, since then, it has evolved independently in each lineage to cope with the environmental challenges imposed by their different life styles. Members of the small GTPase family function in the control of vesicle trafficking, cytoskeleton rearrangements and signaling during crucial biological processes, such as cell growth and responses to environmental cues. In this review, we discuss the emerging roles of these small GTPases in the pathogenic and symbiotic interactions established by plants with microorganisms present in their nearest environment, in which membrane trafficking is crucial along the different steps of the interaction, from recognition and signal transduction to nutrient exchange.

Comparative phylogenetic and expression analysis of small GTPases families in legume and non-legume plants.

BACKGROUND: Small monomeric GTPases act as molecular switches in several processes that involve polar cell growth, participating mainly in vesicle trafficking and cytoskeleton rearrangements. This gene superfamily has largely expanded in plants through evolution as compared with other Kingdoms, leading to the suggestion that members of each subfamily might have acquired new functions associated to plant-specific processes. Legume plants engage in a nitrogen-fixing symbiotic interaction with rhizobia in a process that involves polar growth processes associated with the infection throughout the root hair. To get insight into the evolution of small GTPases associated with this process, we use a comparative genomic approach to establish differences in the Ras GTPase superfamily between legume and non-legume plants. RESULTS: Phylogenetic analyses did not show clear differences in the organization of the different subfamilies of small GTPases between plants that engage or not in nodule symbiosis. Protein alignments revealed a strong conservation at the sequence level of small GTPases previously linked to nodulation by functional genetics. Interestingly, one Rab and three Rop proteins showed conserved amino acid substitutions in legumes, but these changes do not alter the predicted conformational structure of these proteins. Although the steady-state levels of most small GTPases do not change in response to rhizobia, we identified a subset of Rab, Rop and Arf genes whose transcript levels are modulated during the symbiotic interaction, including their spatial distribution along the indeterminate nodule. CONCLUSIONS: This study provides a comprehensive study of the small GTPase superfamily in several plant species. The genetic program associated to root nodule symbiosis includes small GTPases to fulfill specific functions during infection and formation of the symbiosomes. These GTPases seems to have been recruited from members that were already present in common ancestors with plants as distant as monocots since we failed to detect asymmetric evolution in any of the subfamily trees. Expression analyses identified a number of legume members that can have undergone neo- or sub-functionalization associated to the spatio-temporal transcriptional control during the onset of the symbiotic interaction.

Control of morphology and virulence by ADP-ribosylation factors (Arf) in Mucor circinelloides.

Mucor circinelloides is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor (Arf)-encoding genes (arf1-arf4) and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in M. circinelloides. Transcription analysis revealed that certain arf genes are affected by dimorphism of M. circinelloides, such as the arf2 transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four arf genes to evaluate their function in dimorphism and virulence. We found that both arf1 and arf2 are required for sporulation, but these genes also perform distinct functions; arf2 participates in yeast development, whereas arf1 is involved in aerobic growth. Conversely, arf3 and arf4 play only minor roles during aerobic growth. Moreover, we observed that all single arf-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the arf3 mutant being most virulent. Lastly, arf1/arf2 and arf3/arf4 double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in M. circinelloides such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.

p14 expression differences in ovarian benign, borderline and malignant epithelial tumors.

BACKGROUND: Abnormalities in tumor suppressors p14, p16 and p53 are reported in several human cancers. In ovarian epithelial carcinogenesis, p16 and p53 show higher immunohistochemical staining frequencies in malignant tumors and are associated with poor prognoses. p14 was only analyzed in carcinomas, with conflicting results. There are no reports on its expression in benign and borderline tumors. This study aims to determine p14, p16 and p53 expression frequencies in ovarian benign, borderline and malignant tumors and their associations with clinical parameters. METHODS: A cross-sectional study utilizing immunohistochemistry was performed on paraffin-embedded ovarian epithelial tumor samples. Clinical data were collected from medical records. Fisher's exact test and the Bonferroni correction were performed for frequency associations. Survival comparisons utilized Kaplan-Meier and log rank testing. Associations were considered significant when p < 0.05. RESULTS: p14 absent expression was associated with malignant tumors (60 % positive) (p = 0.000), while 93 % and 94 % of benign and borderline tumors, respectively, were positive. p16 was positive in 94.6 % of carcinomas, 75 % of borderline and 45.7 % of benign tumors (p = 0.000). p53 negative staining was associated with benign tumors (2.9 % positive) (p = 0.016) but no difference was observed between borderline (16.7 %) and malignant tumors (29.7 %) (p = 0.560). No associations were found between expression rates, disease-free survival times or clinical variables. Carcinoma subtypes showed no difference in expression. CONCLUSIONS: This is the first description of p14 expression in benign and borderline tumors. It remains stable in benign and borderline tumors, while carcinomas show a significant absence of staining. This may indicate that p14 abnormalities occur later in carcinogenesis. p16 and p53 frequencies increase from benign to borderline and malignant tumors, similarly to previous reports, possibly reflecting the accumulation of inactive mutant protein. The small sample size may have prevented statistically significant survival analyses and clinical correlations. Future studies should investigate genetic abnormalities in p14 coding sequences and include all types of ovarian epithelial tumors. Bigger sample sizes may be needed for significant associations.