Introduction. Very few studies have examined the neuropsychological performance of HIV-positive women, and even fewer have attempted a comparison of cognitive functioning by gender. The aim of this study was to describe the nature of the neuropsychological performance of HIV seropositive patients by gender. Methods. A clinical sample made up of 151 subjects was recruited to participate in this study. All of the subjects underwent the same assessment process, consisting of a neuropsychological evaluation and an interview to gather sociodemographic, toxicological, and clinical data. Results and Discussion. Despite the fact that men obtained higher scores in visual memory, attention/psychomotor speed, and abstract reasoning/verbal intelligence, these differences were not statistically significant. In contrast, significant differences were found depending on subjects' serological status. Seropositive participants' neuropsychological performance was significantly lower than that of the seronegative participants in all of the areas assessed as follows: (1) visual memory; (2) attention/psychomotor speed; (3) abstract reasoning/verbal intelligence; (4) verbal memory for texts; (5) verbal memory for digits and words. Conclusions. The results from this study reveal no significant gender differences in the cognitive performance of patients infected with HIV-1.
A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.
Our evaluation study identifies facilitators and barriers to
participation among families participating in the treatment arm of Stanford
ECHALE. This culturally tailored obesity prevention trial consisted of a
combined intervention with two main treatment components: 1) a folkloric dance
program; and 2) a screen time reduction curriculum designed for 7–11
year old Latinas and their families. We conducted 83 interviews (40 parents and
43 girls) in participant homes after 6 months of enrollment in the ECHALE trial.
The Spradley ethnographic method and NVivo 8.0 were used to code and analyze
narrative data. Three domains emerged for understanding participation: 1) family
cohesiveness; 2) perceived gains; and 3) culturally relevant program structure.
Two domains emerged for non-participation: program requirements and perceived
discomforts. Non-parametric, Spearman’s rank correlation coefficients
were calculated to assess the relationships with participant attendance data.
Sustained participation was most strongly influenced by the domain perceived
gains when parents reported better self-esteem, confidence, improved attitude,
improved grades, etc. (Spearman r=.45,
P=.003). Alternatively, under the domain, perceived
discomforts, with subthemes such as child bullying, participation in the
combined intervention was inversely associated with attendance (Spearman
r=.38, P=.02). Family-centered,
school-based, community obesity prevention programs that focus on tangible
short-term gains for girls may generate greater participation rates, enhance
social capital, and promote community empowerment. These factors can be
emphasized in future obesity prevention program design and implementation.
(Ethn Dis. 2013; 23:452–461)
Obesity Prevention; Behavioral Health Interventions; Girls; Latinos
The course of human immunodeficiency virus type-1 (HIV-1) infection is influenced by a complex interplay between viral and host factors. HIV infection stimulates several proinflammatory genes, such as cyclooxigense-2 (COX-2), which leads to an increase in prostaglandin (PG) levels in the plasma of HIV-1-infected patients. These genes play an indeterminate role in HIV replication and pathogenesis. The effect of prostaglandin E2 (PGE2) on HIV infection is quite controversial and even contradictory, so we sought to determine the role of PGE2 and the signal transduction pathways involved in HIV infection to elucidate possible new targets for antiretrovirals.
Our results suggest that PGE2 post-infection treatment acts in the late stages of the viral cycle to reduce HIV replication. Interestingly, viral protein synthesis was not affected, but a loss of progeny virus production was observed. No modulation of CD4 CXCR4 and CCR5 receptor expression, cell proliferation, or activation after PGE2 treatment was detected. Moreover, PGE2 induced an increase in intracellular cAMP (cyclic AMP) levels through the EP2/EP4 receptors. PGE2 effects were mimicked by dbcAMP and by a specific Epac (exchange protein directly activated by cyclic AMP) agonist, 8-Cpt-cAMP. Treatment with PGE2 increased Rap1 activity, decreased RhoA activity and subsequently reduced the polymerization of actin by approximately 30% compared with untreated cells. In connection with this finding, polarized viral assembly platforms enriched in Gag were disrupted, altering HIV cell-to-cell transfer and the infectivity of new virions.
Our results demonstrate that PGE2, through Epac and Rap activation, alters the transport of newly synthesized HIV-1 components to the assembly site, reducing the release and infectivity of new cell-free virions and cell-to-cell HIV-1 transfer.
HIV testing rates remain very low in Colombia, with only 20% of individuals at risk ever tested. In order to tackle this issue, the Corporacion de Lucha Contra el Sida (CLS) has implemented a multidisciplinary, provider-initiated, population-based HIV testing/counselling strategy named BAFI. In this report, we describe the experience of CLS at reaching populations from low socioeconomic backgrounds in 2008-2009. Two different approaches were used: one led by CLS and local health care providers (BAFI-1) and the other by CLS and community leaders (BAFI-2). Both approaches included the following: consented HIV screening test, a demographic questionnaire, self-reported HIV knowledge and behaviour questionnaires, pre- and posttest counselling, confirmatory HIV tests, clinical follow-up, access to comprehensive care and antiretroviral treatment. A total of 2085 individuals were enrolled in BAFI-1 and 363 in BAFI-2. The effectiveness indicators for BAFI-1 and BAFI-2, respectively, were HIV positive-confirmed prevalence = 0.29% and 3.86%, return rate for confirmatory results = 62.5% and 93.7%, return rate for comprehensive care = 83.3% and 92.8%, and ART initiation rate = 20% and 76.9%. Although more people were reached with BAFI-1, the community-led BAFI-2 was more effective at reaching individuals with a higher prevalence of behavioural risk factors for HIV infection.
We here report a 3.02-Mbp annotated draft assembly of the Lactobacillus casei 5b genome. The sequence of this biogenic amine-degrading dairy isolate may help identify the mechanisms involved in the catabolism of biogenic amines and perhaps shed light on ways to reduce the presence of these toxic compounds in food.
The latest technological advances and information support systems
for clinics and hospitals produce a wide range of possibilities in the
storage and retrieval of an ever-growing amount of clinical information
as well as in detection and diagnosis. In this work, an Electronic
Health Record (EHR) combined with a Computer Aided Detection
(CADe) system for breast cancer diagnosis has been implemented.
Our objective is to provide to radiologists a comprehensive working
environment that facilitates the integration, the image visualization,
and the use of aided tools within the EHR. For this reason, a development
methodology based on hardware and software system features
in addition to system requirements must be present during the whole
development process. This will lead to a complete environment for displaying, editing, and reporting results not only for the patient information
but also for their medical images in standardised formats
such as DICOM and DICOM-SR. As a result, we obtain a CADe system
which helps in detecting breast cancer using mammograms and
is completely integrated into an EHR.
The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.
Here, we report a 3.2-Mbp draft assembly for the genome of Lactobacillus plantarum IPLA 88. The sequence of this sourdough isolate provides insight into the adaptation of this versatile species to different environments.
At seed maturity, quinoa (Chenopodium quinoa Willd.) perisperm consists of uniform, non-living, thin-walled cells full of starch grains. The objective of the present study was to study quinoa perisperm development and describe the programme of cell death that affects the entire tissue. A number of parameters typically measured during programmed cell death (PCD), such as cellular morphological changes in nuclei and cytoplasm, endoreduplication, DNA fragmentation, and the participation of nucleases and caspase-like proteases in nucleus dismantling, were evaluated; morphological changes in cytoplasm included subcellular aspects related to starch accumulation. This study proved that, following fertilization, the perisperm of quinoa simultaneously accumulates storage reserves and degenerates, both processes mediated by a programme of developmentally controlled cell death. The novel findings regarding perisperm development provide a starting point for further research in the Amaranthaceae genera, such as comparing seeds with and without perisperm, and specifying phylogeny and evolution within this taxon. Wherever possible and appropriate, differences between quinoa perisperm and grass starchy endosperm—a morphologically and functionally similar, although genetically different tissue—were highlighted and discussed.
Amaranthaceae; basal body; caspase-like protease; DNA fragmentation; endoreduplication; nucleases; quinoa; perisperm; programmed cell death (PCD); starch accumulation; TUNEL.
Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses.
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively.
Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.
Mesenchymal stem cell; Bovine fetuses; Differentiation potential; Multipotency
During the last decade, microsatellites (short tandem repeats or STRs) have been successfully used for animal genetic identification, traceability and paternity, although in recent year single nucleotide polymorphisms (SNPs) have been increasingly used for this purpose. An efficient SNP identification system requires a marker set with enough power to identify individuals and their parents. Genetic diagnostics generally include the analysis of related animals. In this work, the degree of information provided by SNPs for a consanguineous herd of cattle was compared with that provided by STRs. Thirty-six closely related Angus cattle were genotyped for 18 STRs and 116 SNPs. Cumulative SNPs exclusion power values (Q) for paternity and sample matching probability (MP) yielded values greater than 0.9998 and 4.32E−42, respectively. Generally 2–3 SNPs per STR were needed to obtain an equivalent Q value. The MP showed that 24 SNPs were equivalent to the ISAG (International Society for Animal Genetics) minimal recommended set of 12 STRs (MP ∼ 10−11). These results provide valuable genetic data that support the consensus SNP panel for bovine genetic identification developed by the Parentage Recording Working Group of ICAR (International Committee for Animal Recording).
microsatellite; single nucleotide polymorphism; exclusion probability; genetic identification; bovine
The Community Cancer Control Outreach Program (CCCOP) is a community-academic partnership aimed at developing and implementing a cancer control outreach, research, and training program in Puerto Rico. The CCCOP surveyed 56 partners to assess their awareness, training needs, and use of resources related to evidence-based programs (EBPs). Despite relatively high levels (70%) of confidence in adopting EBPs, there were low levels of awareness (37%) and use (25%) of existing EBPs resources. Respondents’ who had used EBPs resources were more likely to have positive beliefs about EBPs than nonusers (p<0.05). Training needs were high among respondents and no significant differences were found between those who had and had not used existing EBPs resources. These findings can guide the development of training tools and technical assistance to increase the use of EBPs for Latino audiences.
Evidence-based programs; Cancer control; Dissemination; Implementation; Puerto Rico
Infections caused by multidrug-resistant Acinetobacter baumannii constitute a major life-threatening problem worldwide, and early adequate antibiotic therapy is decisive for success. For these reasons, rapid detection of antibiotic susceptibility in this pathogen is a clinical challenge. Two variants of the Micromax kit were evaluated for a rapid detection in situ of susceptibility or resistance to meropenem or ciprofloxacin, separately, in 322 clinical isolates. Release of the nucleoid is the criterion of susceptibility to the beta-lactams (carbapenems), whereas diffusion of DNA fragments emerging from the nucleoid characterizes the quinolone activity. All the susceptible and resistant strains were correctly categorized in 100 min according to the MIC results and CLSI criteria. Thus, our technology is a promising tool for rapid identification of carbapenem and quinolone resistance of A. baumannii strains in hospital settings.
Stem cell therapy can promote good recovery from stroke. Several studies have demonstrated that mesenchymal stem cells (MSC) are safe and effective. However, more information regarding appropriate cell type is needed from animal model. This study was targeted at analyzing the effects in ischemic stroke of acute intravenous (i.v.) administration of allogenic bone marrow- (BM-MSC) and adipose-derived-stem cells (AD-MSC) on functional evaluation results and brain repair markers.
Allogenic MSC (2 × 106 cells) were administered intravenously 30 minutes after permanent middle cerebral artery occlusion (pMCAO) to rats. Infarct volume and cell migration and implantation were analyzed by magnetic resonance imaging (MRI) and immunohistochemistry. Function was evaluated by the Rogers and rotarod tests, and cell proliferation and cell-death were also determined. Brain repair markers were analyzed by confocal microscopy and confirmed by western blot.
Compared to infarct group, function had significantly improved at 24 h and continued at 14 d after i.v. administration of either BM-MSC or AD-MSC. No reduction in infarct volume or any migration/implantation of cells into the damaged brain were observed. Nevertheless, cell death was reduced and cellular proliferation significantly increased in both treatment groups with respect to the infarct group. At 14 d after MSC administration vascular endothelial growth factor (VEGF), synaptophysin (SYP), oligodendrocyte (Olig-2) and neurofilament (NF) levels were significantly increased while those of glial fiibrillary acid protein (GFAP) were decreased.
i.v. administration of allogenic MSC - whether BM-MSC or AD-MSC, in pMCAO infarct was associated with good functional recovery, and reductions in cell death as well as increases in cellular proliferation, neurogenesis, oligodendrogenesis, synaptogenesis and angiogenesis markers at 14 days post-infarct.
There have been several studies assessing the epidemiology and effects of tobacco smoke in the cystic fibrosis (CF) population, but few address the efforts of smoking cessation interventions. Our objective is to present one tobacco prevention and cessation programme targeting patients with CF in the Mediterranean region of Murcia (Spain).
All registered patients in the Regional CF unit (n=105) in 2008 were included in a cross-sectional and prospective uncontrolled study of tobacco use and exposure in CF patients using a baseline and one-year follow-up. Target population includes both patients and other family members living at home. The study included an initial telephone questionnaire, measurement of lung function, urinary cotinine levels, and several telephone counselling calls and/or personalized smoking cessation services.
Of the 97 contacted patients, 59.8% (n=58) were exposed to environmental tobacco smoke (ETS), 12.4% (n=12) had smoked at one time, and 14.3% (n=8) of patients over the age of 15 actively smoked. The mean age was 31.13 (range: 19-45). Of the non-smokers (n=89), 56.2% reported ETS and 26.9% live with at least one smoker at home. 49.2% had urinary cotinine levels >10 ng/ml. The correlation found between patients’ cotinine levels and their reported tobacco exposure was (0.77, p<0.0001). Active smoking by mothers during pregnancy was associated with significantly lower lung function in young CF patients (-0,385, p= 0.04). At the one-year follow-up, 13 individuals made attempts to stop smoking, 6 of which are now ex-smokers (12.5% of all smokers).
Smoking during pregnancy adversely affects lung function in individuals with CF. Tobacco he targeted tobacco prevention and cessation programmes are an effective and vital component for CF disease management. The trained professionals in prevention and smoking cessation services could provide patients with adequate follow-up, integrating an environmental health approach into CF patients’ healthcare
Environmental tobacco smoke; cystic fibrosis; smoking prevention
Many terrestrial ecosystems are changing due to extensive land use and habitat fragmentation, posing a major threat to biodiversity. In this study, the effects of patch size, isolation, and edge/interior localization on the ground dwelling insect communities in the Chaco Serrano woodland remnants in central Argentina were examined. Sampling was carried out in December 2003 and March 2004 in nine remnants (0.57 to 1000 hectares) using pitfall traps. In total, 7071 individuals representing 12 orders and 79 families were recorded. The taxonomic composition of these communities was linked to remnant size. Insect abundance increased (as did their richness, albeit marginally) as remnant area decreased, with no significant effects of isolation or edge/interior localization on abundance, richness, or diversity. No differential area effects were observed when abundance and richness of predators, scavengers, and herbivores were compared. Thus, ground insect communities in fragmented Chaco Serrano seem to respond mainly to patch level, rather than to within-patch (edge effects) or landscape (isolation) level variables. These results suggest that small Chaco Serrano remnants, by supporting larger ground-dwelling insect assemblages, may play an important role from a conservation viewpoint.
epigaeic insects; feeding guilds; habitat fragmentation; subtropical forest
The present study reports an easy and efficient method for obtaining adult mesenchymal precursors from different adult mouse tissues.
Materials and Methods
We describe the isolation and expansion of mesenchymal precursors from skin and lung by a non-enzymatic method. Skin and lung mesenchymal precursors isolated by a modified explant technique were characterized in vitro by defined morphology and by a specific gene expression profile and surface markers.
Results and Conclusions
Our results show that these precursors express stem cell and mesenchymal surface markers as well as epithelial markers. However, they are negative for markers of endothelium, cardiac and skeletal muscle or adipose tissue, indicating that they have initiated commitment to the tissues from which were isolated. These precursors can migrate without any stimulus and in response to stimuli as SDF1, MCP1 and TNFα and can be differentiated into epithelial lineages. Based on the properties of these precursors from adult tissues, we propose their use as tools for regenerative biomedicine.
Antiretroviral treatment (ART) has contributed to increased life expectancy of HIV-1 infected children. In developed countries, an increasing number of children reaching adulthood are transferred to adult units. The objectives were to describe the demographic and clinical features, ART history, antiviral drug resistance and drug susceptibility in HIV-1 perinatally infected adolescents transferred to adult care units in Spain from the Madrid Cohort of HIV-1 infected children.
Clinical, virological and immunological features of HIV-1 vertically infected patients in the Madrid Cohort of HIV-infected children were analyzed at the time of transfer. Pol sequences from each patient were recovered before transfer. Resistance mutations according to the InternationaI AIDS Society 2011 list were identified and interpreted using the Stanford algorithm. Results were compared to the non-transferred HIV-1 infected pediatric cohort from Madrid.
One hundred twelve infected patients were transferred to adult units between 1997 and 2011. They were mainly perinatally infected (93.7%), with a mean nadir CD4+-T-cells count of 10% and presented moderate or severe clinical symptoms (75%). By the time of transfer, the mean age was 18.9 years, the mean CD4+T-cells count was 627.5 cells/ml, 64.2% presented more than 350 CD4+T-cells/ml and 47.3% had ≤200 RNA-copies/ml. Most (97.3%) were ART experienced receiving Highly Active ART (HAART) (84.8%). Resistance prevalence among pretreated was 50.9%, 76.9% and 36.5% for Protease Inhibitors (PI), Nucleoside Reverse Transcriptase Inhibitors (NRTI) and Non-NRTI (NNRTI), respectively. Resistance mutations were significantly higher among transferred patients compared to non-transferred for the PI+NRTI combination (19% vs. 8.4%). Triple resistance was similar to non-transferred pediatric patients (17.3% vs. 17.6%).
Despite a good immunological and virological control before transfer, we found high levels of resistance to PI, NRTI and triple drug resistance in HIV-1 infected adolescents transferred to adult units.
A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke.
A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously.
This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research.
Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions.
An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, was used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells.
L. brevis IOEB 9809 can produce tyramine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.
Biogenic amines; Lactic acid bacteria; Putrescine; Tyramine; Food safety; Food toxicity
The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis
rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.
The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an “NLS” of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.
The conserved heterodimeric endonuclease Mus81–Eme1/Mms4 plays an important role in the maintenance of genomic integrity in eukaryotic cells. Here, we show that budding yeast Mus81–Mms4 is strictly regulated during the mitotic cell cycle by Cdc28 (CDK)- and Cdc5 (Polo-like kinase)-dependent phosphorylation of the non-catalytic subunit Mms4. The phosphorylation of this protein occurs only after bulk DNA synthesis and before chromosome segregation, and is absolutely necessary for the function of the Mus81–Mms4 complex. Consistently, a phosphorylation-defective mms4 mutant shows highly reduced nuclease activity and increases the sensitivity of cells lacking the RecQ-helicase Sgs1 to various agents that cause DNA damage or replicative stress. The mode of regulation of Mus81–Mms4 restricts its activity to a short period of the cell cycle, thus preventing its function during chromosome replication and the negative consequences for genome stability derived from its nucleolytic action. Yet, the controlled Mus81–Mms4 activity provides a safeguard mechanism to resolve DNA intermediates that may remain after replication and require processing before mitosis.
The biofertilization of crops with plant-growth-promoting microorganisms is currently considered as a healthy alternative to chemical fertilization. However, only microorganisms safe for humans can be used as biofertilizers, particularly in vegetables that are raw consumed, in order to avoid sanitary problems derived from the presence of pathogenic bacteria in the final products. In the present work we showed that Rhizobium strains colonize the roots of tomato and pepper plants promoting their growth in different production stages increasing yield and quality of seedlings and fruits. Our results confirmed those obtained in cereals and alimentary oil producing plants extending the number of non-legumes susceptible to be biofertilized with rhizobia to those whose fruits are raw consumed. This is a relevant conclusion since safety of rhizobia for human health has been demonstrated after several decades of legume inoculation ensuring that they are optimal bacteria for biofertilization.