Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo.

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

The Nicotiana tabacum UGT89A2 enzyme catalyzes the glycosylation of di- and trihydroxylated benzoic acid derivatives.

Glycosyltransferases catalyze the transfer of a glycoside group to a wide range of acceptor compounds to produce glycoconjugates with diverse biological and pharmacological activities. The present work reports the identification and biochemical characterization of Nicotiana tabacum UGT89A2 glycosyltransferase (NtUGT89A2). The enzyme is a monomer in solution that catalyzes the O-ß-glucosylation of di- and tri-hydroxylated and chlorinated derivatives of benzoic acid. NtUGT89A2 has a preference for 2,5-dihydroxybenzoic acid (2,5-DHBA) over 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,4-dihydroxybenzoic acid (2,4-DHBA). Other substrates that can be used by NtUGT89A2 include 3,4,5-trihydroxybenzoic acid and chlorinated derivatives such as 2-chloro-5-hydroxybenzoic acid (2-Cl-5-HBA). The substrates of NtUGT89A2 were identified by thermal stability experiments, where we observed a maximum increase of the thermal denaturation midpoint (Tm) of 10 °C in the presence of 2,5-DHBA and UDP-glucose. On the other hand, the highest specific activity was obtained with 2,5-DHBA (225 ± 1.7 nkat/mg). Further characterization revealed that the enzyme has a micromolar affinity for its substrates. Notably, the enzyme retains full activity after incubation at 70 °C for 1 h. These results provide a basis for future functional and structural studies of NtUGT89A2.

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Designer TALEs enable discovery of cell death-inducer genes.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

Introduction of a terminal electron sink in chloroplasts decreases leaf cell expansion associated with higher proteasome activity and lower endoreduplication.

Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

Toward a greener multifunctional pharmaceutical excipient: in vivo safety evaluation of nanofibrillated cellulose from tobacco stalk.

Tobacco stalk is a cellulose-rich material and a sustainable alternative to be applied as a plant-based nanofibrillated cellulose (NFC) source. NFC use has garnered attention in the development of oral pharmaceutical forms, despite concerns about its safety due to the adverse effects of nicotine on health. Therefore, we aimed at establishing the safety of NFC derived from tobacco stalk for its potential use as a novel pharmaceutical excipient, exploring its potential functions for tablet production. We conducted acute and subchronic oral toxicity tests in adult female Wistar rats. Initially, individual animals received sequential doses (175-5,000 mg·kg-1) for 24 hours followed by a careful observation of any toxic effects. Subsequently, 20 rats were divided into four groups for a subchronic assay, evaluating toxicity signs, body weight changes, hematological, biochemical, and histopathological parameters. No deaths or other clinical toxicity signs were observed in either the acute or the subchronic assays. We noticed a significant reduction in body weight gain (p < 0.05) after 14 days. We found statistical differences for hematological and biochemical parameters, unrelated to dosage. There were no observed toxic effects, and tobacco stalk ingestion did not adversely affect organ morphology in the histopathological evaluation. The oral administration of NFC at 5,000 mg·kg-1 per day for 28 days was well-tolerated by treated rats, with no reported deaths. In conclusion, NFC derived from tobacco stalk has shown to be a sustainable and safe alternative for use as an excipient at experimental doses, demonstrating compatibility with its proposed applications.

Aphelenchoides besseyi Parasitizing Tobacco Cultivars in Brazil.

Foliar nematodes (Aphelenchoides spp.) are known to be parasites of tobacco in restricted areas, but symptoms caused by A. besseyi in tobacco are not well characterized, despite the great importance of this nematode worldwide. This study aimed to evaluate the host reaction of four Nicotiana tabacum cultivars (Comum, Xanthi, Samsun, and TNN) and N. benthamiana cultivar Comum to A. besseyi and to characterize the symptoms and the parasitism of this nematode. Two experiments were conducted under greenhouse conditions with controlled humidity and temperature, in which the plants were inoculated with 600 A. besseyi. At 30 days after inoculation (DAI), nematodes present in the soil, roots, and shoot parts were extracted, and roots and shoot tissues were stained with acid fuchsin. A high number of A. besseyi was obtained per gram of shoot tissues (125 to 2,169 nematodes), and severe symptoms were observed in leaves and inflorescences of all cultivars. The symptoms included foliar distortion and deformation, necrotic spots delimited by the veins, flower abortion, and poor development of plants. In addition, A. besseyi was observed to penetrate tobacco roots at 30 DAI, and nematodes were also observed in the foliar mesophyll, inflorescences, and stems, a parasitism that has not been previously reported in tobacco plants. The disease caused by A. besseyi in tobacco could be a concern for growers in southern and northeastern Brazil because this nematode can cause severe damage to the marketable leaves of tobacco, reducing its commercial value.

Cytogenotoxicity effects in addicts with multidrug consumption.

Drug abuse is considered a global health problem with serious social impact. In recent decades, changes in drug consumption patterns have shown a clear rising trend in the use of multiple drugs. Although the buccal micronucleus cytome (BMCyt) assay has evaluated cytotoxicity in drug abuse, there has not been an approach that takes into account this pattern of multiple drug use. Therefore, in this study, we evaluate for the first time the cytogenotoxic effects in multidrug users, and its correlation with the amount consumed and years of abuse. This study was conducted on 166 individuals by the BMCyt assay. A total of 83 individuals with a history of multiple licit (alcohol and tobacco) and at least one illicit drug abuse (marijuana, methamphetamines, cocaine, and/or inhalants), and 83 healthy individuals, non-drug abusers were analyzed. The results showed that drug abusers had higher frequencies of nuclear abnormalities nuclear buds, binucleated cells, pyknotic nuclei (PNs), karyorrhexis (KX), and abnormally condensed chromatin when compared with healthy controls. Moreover, results suggests that the use of licit and illicit drugs is related to cytogenotoxic damage, as was shown by an upward trend in the frequency of nuclear abnormalities identified in groups 1 (alcohol + tobacco + at least one illicit drug) and 2 (tobacco + at least one illicit drug). Furthermore, a positive correlation was found in the different groups, between the years and the amount of consumption of some drugs (alcohol, methamphetamine, and tobacco) with cytotoxicity markers such as KL, KX, and PNs.

Exploring Potential Surrogate Systems for Studying the Early Steps of the Sporisorium scitamineum Pathogenesis.

Despite its global importance as a primary source of table sugar and bioethanol, sugarcane faces a significant threat to its production due to diseases. One of these diseases, sugarcane smut, involves the emergence of a whip-like structure from the host apical shoot. The slow onset of this pathogenesis is the most substantial challenge for researchers to investigate the molecular events leading to resistance or susceptibility. In this study, we explored the early interaction between the smut fungus Sporisorium scitamineum and foliar tissues of the model plants Arabidopsis thaliana and Nicotiana benthamiana. Upon inoculation with the fungus, A. thaliana showed a compatible reaction, producing lesions during fungus colonization, whereas N. benthamiana showed signs of nonhost resistance. In addition, we propose a sugarcane detached leaf assay using plants cultivated in vitro to reveal sugarcane smut response outcomes. We used two sugarcane genotypes with known contrasting reactions to smut in the field. Although there is no evidence of sugarcane smut fungus infecting host leaves naturally, the sugarcane detached leaf assay enabled a rapid assessment of disease outcomes. Different symptoms in the detached leaves after inoculation distinguished smut-susceptible and smut-resistant sugarcane genotypes. Microscopic observations and gene expression analysis of S. scitamineum candidate effectors confirmed the fungal growth and its restriction on the compatible and incompatible interactions, respectively. These findings offer new prospects into the disease phenotyping of S. scitamineum, which could greatly expedite the comprehension of the initial stages of the pathogenesis and predict smut resistance in sugarcane genotypes.

Association of genetic polymorphism of glutathione S-transferases with colorectal cancer susceptibility in snuff (Naswar) addicts / Associação de polimorfismo genético de glutationa S-transferase com suscetibilidade ao câncer colorretal em viciados em rapé (Naswar)

The current study aimed to investigate the relationship between polymorphisms in detoxifying (GSTM1, GSTT1, and GSTP1) genes and their association with colorectal cancer (CRC) in tobacco addicts of Pashtun ethnicity. Polymorphisms in the selected genes were genotyped in a case-control study consisting of 100 histologically confirmed male CRC patients and 100 birth-year and gender-matched healthy controls using the PCR−RFLP method. The GSTM1 null, and GSTT1 null genotypes were significantly contributed to the risk of CRC in the cases (OR= 3.131, 95% CI: 1.451−6.758, P = 0.004, and OR= 3.541, 95% CI: 1.716−7.306, P = 0.001, respectively), whereas the association observed for GSTP1 Val/Val (1.139, 95% CI: 0.356−3.644, P = 0.826) did not show statistical significance. The combined GSTM1 null and GSTT1 null showed a 41-fold increased risk (95% CI: 4.945−351.950, P = 0.001), while, the combined GSTM1 null and GSTP1 Ile/Val or Val/Val variant genotypes exhibited about 3-fold (95% CI: 1.196−7.414, P = 0.019) increased risk to CRC. Similarly, the combined GSTT1 null and GSTP1 Ile/Val or Val/Val variant genotypes showed about a 3-fold (95% CI: 1.285−8.101, P = 0.013) increased risk of CRC. In the combination of three GST genotypes, the GSTM1 null, GSTT1 null, and GSTP1 Ile/Val or Val/Val variant genotypes demonstrated a more than a 22-fold (95% CI: 2.441−212.106, P = 0.006) increased risk of CRC. Our findings suggest that GSTM1 and GSTT1 polymorphism and its combination with GSTP1 may be associated with CRC susceptibility in the Naswar addicted Pashtun population of Khyber Pakhtunkhwa, Pakistan.

O presente estudo teve como objetivo investigar a relação entre polimorfismos em genes desintoxicantes (GSTM1, GSTT1 e GSTP1) e sua associação com câncer colorretal (CCR) em tabagistas da etnia pashtun. Os polimorfismos nos genes selecionados foram genotipados em um estudo de caso-controle composto por 100 pacientes do sexo masculino com CCR, confirmados histologicamente, e 100 controles saudáveis, ​​pareados por ano de nascimento e sexo usando o método PCR-RFLP. Os genótipos GSTM1 nulo e GSTT1 nulo contribuíram significativamente para o risco de CCR nos casos (OR = 3,131, IC 95%: 1,451-6,758, P = 0,004; OR = 3,541, IC 95%: 1,716-7,306, P = 0,001, respectivamente), enquanto a associação observada para GSTP1 Val/Val (1,139, IC 95%: 0,356-3,644, P = 0,826) não apresentou significância estatística. O GSTM1 nulo e o GSTT1 nulo combinados mostraram um risco 41 vezes maior (IC 95%: 4,945-351,950, P = 0,001) para CCR, enquanto os genótipos GSTM1 nulo e GSTP1 Ile/Val ou Val/Val combinados apresentaram risco cerca de 3 vezes maior (IC 95%: 1,196-7,414, P = 0,019) para CCR. Da mesma forma, os genótipos combinados GSTT1 nulo e GSTP1 Ile/Val ou Val/Val tiveram um risco para CRC cerca de 3 vezes maior (95% CI: 1,285-8,101, P = 0,013). Na combinação de três genótipos GST, os genótipos GSTM1 nulo, GSTT1 nulo e GSTP1 Ile/Val ou Val/Val apresentaram um risco 22 vezes maior (IC 95%: 2,441-212,106, P = 0,006) para CRC. Nossos achados sugerem que o polimorfismo GSTM1 e GSTT1 e sua combinação com GSTP1 podem estar associados à suscetibilidade ao CRC da população pashtun de Khyber Pakhtunkhwa, Paquistão, viciada em Naswar.

Production and characterization of egg yolk antibodies against the ZIKV NS2B expressed in Nicotiana benthamiana.

The emergence of Zika virus (ZIKV) and its associated neonatal and congenital complications pose a threat to global health, particularly in tropical and subtropical regions with co-circulation of related flaviviruses and intense vector proliferation. Diagnosis of ZIKV by RT-PCR is limited to the viraemic phase and is not always accessible in low-income tropical settings, while serological tests often show cross-reactivity with other flaviviruses. Given the similarity of ZIKV symptoms to those of other arboviruses, but the different prognosis and risks, it is important to develop specific and accessible diagnostic tools. Egg yolk antibodies (IgY) were obtained from Leghorn laying hens immunized with recombinant ZIKV NS2B protein produced in agroinfiltrated Nicotiana benthamiana. After three immunizations, total IgY was recovered from the eggs by the 20% ammonium sulfate precipitation method. After characterisation by SDS-PAGE, dot blotting and ELISA, the IgY was adsorbed to dengue virus (DENV) from cell culture supernatants and tested for its ability to specifically detect ZIKV-positive sera samples. High yield and purity were observed on SDS-PAGE for polyclonal IgY, which reacted with NS2B at high titres in ELISA and detected both NS2B and ZIKV in dot blotting. However, a cross-reaction with DENV was observed and the anti-NS2B IgY was unable to discriminate ZIKV from DENV positive sera samples, even after adsorption with DENV. This is probably due to the phylogenetic relationship of the viruses and the shared identity of their proteins.

Problemas do sono em adultos assistidos na Estratégia de Saúde da Família / Sleep problems in adults assisted in the Family Health Strategy

Objetivo: Estimar a prevalência e investigar fatores associados aos problemas do sono em adultos cadastrados na Estratégia de Saúde da Família em 2016.Métodos: Estudo transversal, amostral, com 791 indivíduos, com idade igual ou superior a 18 anos, realizado entre 2017 e 2018. Os problemas de sono foram mensurados pelo Mini Sleep Questionnaire, questionário validado no Brasil.Resultados: A prevalência de problemas de sono foi de 49,6%. Destes, 10,4% tiveram alterações leves, 7,6% moderadas e 31,6% graves. Observou-se associação estatisticamente significante entre não ter uma atividade de trabalho remunerada, não ter atividade de lazer, hábito de fumar, presença de doença crônica e autoavaliação negativa da saúde com os problemas do sono.Conclusão: Observou-se elevada prevalência de problemas de sono. Esses resultados podem contribuir para uma melhor compreensão dos problemas do sono na população adulta e, assim, colaborar com a adoção de medidas mais eficazes para o enfrentamento desse problema

Objective: To estimate the prevalence and investigate factors associated with sleep problems in adults enrolled in the Family Health Strategy in 2016. Methods: Cross-sectional sample study with 791 individuals aged 18 years or over, carried out between 2017 and 2018. Sleep problems were measured using the Mini Sleep Questionnaire, a questionnaire validated in Brazil. Results: The prevalence of sleep problems was 49.6%. Of these, 10.4% had mild, 7.6% moderate, and 31.6% severe alterations. There was a statistically significant association between not having a paid work activity, not having leisure activities, smoking, chronic disease, and negative self-assessment of health with sleep problems. Conclusion: There was a high prevalence of sleep problems. These results can contribute to a better understanding of sleep problems in the adult population and, thus, collaborate with adopting more effective measures to treat them

A NanoLuc-Based Transactivation Assay in Plants.

Protein-DNA interactions are determinant of the regulation of gene expression in living organisms. Luminescence studies have been used in a wide range of techniques to identify how gene transcription can be regulated by proteins such as transcription factors (TFs). Despite the great advances in the use of luciferases as reporters in the performance of this mechanism, some of them still have disadvantages that have been tried to be solved by the generation of new luciferases that induce a more stable and perfectly visualizable reaction. NanoLuc is a recently described luciferase that has been characterized by its efficient, stable, and powerful luminescence. These qualities have been considered to create a new and efficient reporter system to detect protein-DNA interactions. In this chapter, we take advantage of NanoLuc and describe its use in a reliable procedure to detect protein-DNA interactions in Nicotiana benthamiana extracts and entire leaves.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem.

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

Long-term e-cigarette aerosol exposure causes pulmonary emphysema in adult female and male mice.

This study aimed to evaluate long-term exposure to conventional cigarette smoke (CC) and electronic cigarette (EC) aerosol in adult male and female C57BL/6 mice. Forty-eight C57BL/6 mice were used, male (n = 24) and female (n = 24), both were divided into three groups: control, CC and EC. The CC and EC groups were exposed to cigarette smoke or electronic cigarette aerosol, respectively, 3 times a day for 60 consecutive days. Afterwards, they were maintained for 60 days without exposure to cigarettes or electronic cigarette aerosol. Both cigarettes promoted an influx of inflammatory cells to the lung in males and females. All animals exposed to CC and EC showed an increase in lipid peroxidation and protein oxidation. There was an increase of IL-6 in males and females exposed to EC. The IL-13 levels were higher in the females exposed to EC and CC. Both sexes exposed to EC and CC presented tissue damage characterized by septal destruction and increased alveolar spaces compared to control. Our results demonstrated that exposure to CC and EC induced pulmonary emphysema in both sexes, and females seem to be more susceptible to EC.

Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Effect of humic compost on the adsorption of cadmium (II) in aqueous medium.

The humic compost obtained from the treatment of tobacco from smuggled cigarettes (SCT) and industrial sewage sludge (ISS) was evaluated as adsorbent for Cd (II) in aqueous solution, for possible decontamination of water resources. Optimum conditions were found at pH 5 and a 3 g/L adsorbent concentration, which presented 92% Cd (II) removal and maximum adsorption capacity of 28.546 mg/g. The pseudo-second-order kinetic model presented the best fit, pointing 120 min as the time needed to attain a steady state. FTIR and EDX results suggest the formation of coordinated Cd (II) bonds by functional groups between the compost and the solution. The results obtained in real samples showed that, even under different environmental conditions, the Cd (II) adsorption varied between 80.05 and 91.61%. The results indicated that the compost evaluated can be used for remediation of Cd (II)-contaminated water resources.

Effectiveness of a Mobile App to Increase Risk Perception of Tobacco, Alcohol, and Marijuana Use in Mexican High School Students: Quantitative Study.

BACKGROUND: Young people have the highest rate of drug use worldwide. Recent data from Mexico in this population show that the prevalence of illicit drug use doubled between 2011 and 2016 (2.9%-6.2%), with marijuana being the one with the highest increase (2.4%-5.3%), but also point out that alcohol and tobacco use have remained steady or decreased. Mexican adolescents are at high risk for drug use owing to a low perception of risk and the availability of drugs. Adolescence is an ideal period to reduce or prevent risky behaviors using evidence-based strategies. OBJECTIVE: In this study, we aimed to test the short-term effectiveness of a mobile intervention app ("What Happens if you Go Too Far?" ["¿Qué pasa si te pasas?"]) that seeks to increase risk perception of tobacco, alcohol, and marijuana use in a sample of Mexican high school students. METHODS: A nonexperimental evaluation based on pretest-posttest design was used to measure the effectiveness of a preventive intervention using a mobile app, "What Happens If You Go Too Far?" The dimensions analyzed were knowledge of drugs and their effects, life skills, self-esteem, and risk perception. The intervention was conducted on a high school campus with 356 first-year students. RESULTS: The sample included 359 first-year high school students (mean 15, SD 0.588 years; women: 224/359, 62.4% men: 135/359, 37.6%). The intervention increased the overall risk perception of tobacco (χ24=21.6; P<.001) and alcohol use (χ24=15.3; P<.001). There was no significant difference in the perception that it is dangerous to smoke 5 cigarettes, and there was a marginal difference in the perception that it is very dangerous to smoke 1 cigarette or to use alcohol or marijuana. We used a generalized estimating equation method to determine the impact of the variables on risk perception. The results showed that knowledge about smoking increased the risk perception of smoking 1 cigarette (odds ratio [OR] 1.1065, 95% CI 1.013-1.120; P=.01), and that knowledge about marijuana use (OR 1.109, 95% CI 1.138-1.185; P=.002) and self-esteem (OR 1.102, 95% CI 1.007-1.206; P=.04) produced significant increases in the risk perception of consuming 5 cigarettes. Resistance to peer pressure and assertiveness also increased the perceived risk of using tobacco and alcohol. CONCLUSIONS: The intervention has the potential to increase the perception of risk toward drug use in high school students by providing knowledge about the effects and psychosocial risks of drug use and by strengthening life skills that are associated with increased risk perception. The use of mobile technologies in intervention processes may broaden the scope of preventive work for adolescents.

Nicotiana hairy roots for recombinant protein expression, where to start? A systematic review.

BACKGROUND: Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their benefits for the recombinant expression of valuables proteins; it includes simplified downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a promising technology. METHODS AND RESULTS: In the current paper, we propose the first step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. CONCLUSION: The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to appraisal the literature status.

Break-induced replication is the primary recombination pathway in plant somatic hybrid mitochondria: a model for mitochondrial horizontal gene transfer.

Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, we described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid between N. tabacum and Physochlaina orientalis, and re-analyzed the somatic hybrids that we previously generated. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them (~80%) were asymmetrical, consistent with the break-induced replication pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. Break-induced replication is the main pathway of interparental recombination in somatic hybrid mitochondria. Findings of this study are relevant to mitogenome editing assays and to mechanistic aspects of DNA integration following mitochondrial DNA horizontal transfer events.