A new biostimulant derived from soybean by-products enhances plant tolerance to abiotic stress triggered by ozone.

BACKGROUND: Tropospheric ozone is an air pollutant that causes negative effects on vegetation, leading to significant losses in crop productivity. It is generated by chemical reactions in the presence of sunlight between primary pollutants resulting from human activity, such as nitrogen oxides and volatile organic compounds. Due to the constantly increasing emission of ozone precursors, together with the influence of a warming climate on ozone levels, crop losses may be aggravated in the future. Therefore, the search for solutions to mitigate these losses becomes a priority. Ozone-induced abiotic stress is mainly due to reactive oxygen species generated by the spontaneous decomposition of ozone once it reaches the apoplast. In this regard, compounds with antioxidant activity offer a viable option to alleviate ozone-induced damage. Using enzymatic technology, we have developed a process that enables the production of an extract with biostimulant properties from okara, an industrial soybean byproduct. The biostimulant, named as OEE (Okara Enzymatic Extract), is water-soluble and is enriched in bioactive compounds present in okara, such as isoflavones. Additionally, it contains a significant fraction of protein hydrolysates contributing to its functional effect. Given its antioxidant capacity, we aimed to investigate whether OEE could alleviate ozone-induced damage in plants. For that, pepper plants (Capsicum annuum) exposed to ozone were treated with a foliar application of OEE. RESULTS: OEE mitigated ozone-induced damage, as evidenced by the net photosynthetic rate, electron transport rate, effective quantum yield of PSII, and delayed fluorescence. This protection was confirmed by the level of expression of genes associated with photosystem II. The beneficial effect was primarily due to its antioxidant activity, as evidenced by the lipid peroxidation rate measured through malondialdehyde content. Additionally, OEE triggered a mild oxidative response, indicated by increased activities of antioxidant enzymes in leaves (catalase, superoxide dismutase, and guaiacol peroxidase) and the oxidative stress index, providing further protection against ozone-induced stress. CONCLUSIONS: The present results support that OEE protects plants from ozone exposure. Taking into consideration that the promotion of plant resistance against abiotic damage is an important goal of biostimulants, we assume that its use as a new biostimulant could be considered.

Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Biostimulant Capacity of an Enzymatic Extract From Rice Bran Against Ozone-Induced Damage in Capsicum annum.

Ozone is a destructive pollutant, damaging crops, and decreasing crop yield. Therefore, there is great interest in finding strategies to alleviate ozone-induced crop losses. In plants, ozone enters leaves through the stomata and is immediately degraded into reactive oxygen species (ROS), producing ROS stress in plants. ROS stress can be controlled by ROS-scavenging systems that include enzymatic or non-enzymatic mechanisms. Our research group has developed a product from rice bran, a by-product of rice milling which has bioactive molecules that act as an antioxidant compound. This product is a water-soluble rice bran enzymatic extract (RBEE) which preserves all the properties and improves the solubility of proteins and the antioxidant components of rice bran. In previous works, the beneficial properties of RBEE have been demonstrated in animals. However, to date, RBEE has not been used as a protective agent against oxidative damage in agricultural fields. The main goal of this study was to investigate the ability of RBEE to be used as a biostimulant by preventing oxidative damage in plants, after ozone exposure. To perform this investigation, pepper plants (Capsicum annuum) exposed to ozone were treated with RBEE. RBEE protected the ozone-induced damage, as revealed by net photosynthetic rate and the content of photosynthetic pigments. RBEE also decreased the induction of antioxidant enzyme activities in leaves (catalase, superoxide dismutase, and ascorbate peroxidase) due to ozone exposure. ROS generation is a common consequence of diverse cellular traumas that also activate the mitogen-activated protein kinase (MAPK) cascade. Thus, it is known that the ozone damages are triggered by the MAPK cascade. To examine the involvement of the MAPK cascade in the ozone damage CaMPK6-1, CaMPK6-2, and CaMKK5 genes were analyzed by qRT-PCR. The results showed the involvement of the MAPK pathway in both, not only in ozone damage but especially in its protection by RBEE. Taken together, these results support that RBEE protects plants against ozone exposure and its use as a new biostimulant could be proposed.

RANK links senescence to stemness in the mammary epithelia, delaying tumor onset but increasing tumor aggressiveness.

Rank signaling enhances stemness in mouse and human mammary epithelial cells (MECs) and mediates mammary tumor initiation. Mammary tumors initiated by oncogenes or carcinogen exposure display high levels of Rank and Rank pathway inhibitors have emerged as a new strategy for breast cancer prevention and treatment. Here, we show that ectopic Rank expression in the mammary epithelia unexpectedly delays tumor onset and reduces tumor incidence in the oncogene-driven Neu and PyMT models. Mechanistically, we have found that ectopic expression of Rank or exposure to Rankl induces senescence, even in the absence of other oncogenic mutations. Rank leads to DNA damage and senescence through p16/p19. Moreover, RANK-induced senescence is essential for Rank-driven stemness, and although initially translates into delayed tumor growth, eventually promotes tumor progression and metastasis. We uncover a dual role for Rank in the mammary epithelia: Rank induces senescence and stemness, delaying tumor initiation but increasing tumor aggressiveness.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer.

BACKGROUND: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. METHODS: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. RESULTS: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. CONCLUSIONS: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells.

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.

Rhizospheric Organic Acids as Biostimulants: Monitoring Feedbacks on Soil Microorganisms and Biochemical Properties.

The biostimulant potential of three different organic acids (OAs) present in the rhizosphere, specifically lactic, oxalic, and citric acids, have been studied. The results showed a rapid and complete metabolism of these three acids with soil microorganisms using them as a source of carbon and energy. Biostimulation was confirmed by soil biochemical studies which showed an increase in enzymatic activities, such as dehydrogenase and phosphatase, lactic and citric acids being those that produced the greatest biostimulation. With regard to microbiota composition, amplicon sequencing of the 16S rRNA gene showed changes in the structure of soil microbial communities. Applying OAs produced a decrease in richness and diversity indices, inducing specific changes in the structure of the microbiological communities. Applying lactic acid induced rapid changes in microbiota composition at both phylum and family taxonomic levels, favoring the proliferation of microorganisms involved in its degradation and soil fertility, such as the genus Bacillus and the family Micrococcaceae. Once the lactic acid was degraded, the biodiversity tended to return to similar phyla, but specific distinctive families and genera remained, leaving a pattern of induction of taxa described as plant growth-promoting bacteria (PGPB), such as the Sinorhizobium and Lysobacter genera, and the Pseudomonaceae family. Similar behavior was found with citric acid, which favored the proliferation and dominance of microorganisms of the Clostridiaceae family, involved in its degradation, as well as microorganisms of both the Micrococcaceae and Pseudomonadaceae families which were found on day 7, leaving a similar pattern of induction as that found after the mineralization of lactic acid. On the other hand, oxalic acid induced long-lasting changes in the bacterial community composition. This was characterized by an increase in the proportion of the Burkholderiales order, which includes microorganisms involved in the degradation of this acid and microorganisms described as PGPB. This study presents evidence supporting the use of OAs as potential soil fertility inducers, due both to their effects in enhancing the dominance of taxa described as PGPB and to their stimulating soil microbial activity.

Insights into the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in Leishmania parasites and characterization of a choline kinase from Leishmania infantum.

The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a Vmax of 3.52µmol/min/mg and an apparent Km value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent Km for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the Vmax/Km decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited.

Synthesis of Novel Quaternary Ammonium Salts and Their in Vitro Antileishmanial Activity and U-937 Cell Cytotoxicity.

This work describes the synthesis of a series of quaternary ammonium salts and the assessment of their in vitro antileishmanial activity and cytotoxicity. A preliminary discussion on a structure-activity relationship of the compounds is also included. Three series of quaternary ammonium salts were prepared: (i) halomethylated quaternary ammonium salts (series I); (ii) non-halogenated quaternary ammonium salts (series II) and (iii) halomethylated choline analogs (series III). Assessments of their in vitro cytotoxicity in human promonocytic cells U-937 and antileishmanial activity in axenic amastigotes of L. (Viannia) panamensis (M/HOM/87/UA140-pIR-eGFP) were carried out using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) micromethod. Antileishmanial activity was also tested in intracellular amastigotes of L. (V) panamensis using flow cytometry. High toxicity for human U937 cells was found with most of the compounds, which exhibited Lethal Concentration 50 (LC50) values in the range of 9 to 46 µg/mL. Most of the compounds evidenced antileishmanial activity. In axenic amastigotes, the antileishmanial activity varied from 14 to 57 µg/mL, while in intracellular amastigotes their activity varied from 17 to 50 µg/mL. N-Chloromethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (1a), N-iodomethyl-N,N-dimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (2a), N,N,N-trimethyl-N-(4,4-diphenylbut-3-en-1-yl)ammonium iodide (3a) and N,N,N-trimethyl-N-(5,5-diphenylpent-4-en-1-yl)ammonium iodide (3b) turned out to be the most active compounds against intracellular amastigotes of L. (V) panamensis, with EC50 values varying between 24.7 for compound 3b and 38.4 µg/mL for compound 1a. Thus, these compounds represents new "hits" in the development of leishmanicidal drugs.

2-phenylethynesulphonamide (PFT-µ) enhances the anticancer effect of the novel hsp90 inhibitor NVP-AUY922 in melanoma, by reducing GSH levels.

Heat shock proteins (HSPs), are molecular chaperones that assist the proper folding of nascent proteins. This study aims to evaluate the antitumour effects of the hsp90 inhibitor NVP-AUY922 in melanoma, both in vitro and in vivo. Our results show that NVP-AUY922 inhibits melanoma cell growth in vitro, with down regulation of multiple signalling pathways involved in melanoma progression such as NF-ĸB and MAPK/ERK. However, NVP-AUY922 was unable to limit tumour growth in vivo. Cotreatment of A375M xenografts with NVP-AUY922 and PFT-µ, a dual inhibitor of both hsp70 and autophagy, induced a synergistic increase of cell death in vitro, and delayed tumour formation in A375M xenografts. PFT-µ depleted cells from the reduced form of glutathione (GSH) and increased oxidative stress. The oxidative stress induced by PFT-µ further enhanced NVP-AUY922-induced cytotoxic effects. These data suggest a potential therapeutic role for NVP-AUY922 used in combination with PFT-µ, in melanoma.

Diagnóstico clínico bucal de los pacientes VIH positivos, pertenecientes a la fundación nacional para la prevención, educación y acompañamiento de la persona VIH/SIDA / Oral clinical diagnosis of HIV positive patients, belonging to the national foundation for the prevention, education and support of the HIV / AIDS person

Las lesiones bucales que se presentan en pacientes PVVS son un signo muy fundamental para detectar la enfermedad en su etapa inicial, así como también conocer si la enfermedad ha avanzando a la etapa de SIDA. Esta investigación trata sobre el diagnóstico clínico bucal en pacientes PVVS, que se realizó en las instalaciones de FUNDASIDA, durante los meses de febrero a abril de dos mil diez, contando con la participación de treinta pacientes de ambos sexos entre las edades de dieciséis a setenta años, la mayoría están bajo tratamiento antirretroviral y cada uno de ellos lleva un chequeo médico donde se les evalúa los niveles de CD4 y carga viral. Posterior a la recolección de todos los datos, estos se procesaron a través del programa estadístico SPSS por sus siglas en ingles (Statistical Package for the Social Sciences versión Pasw18), realizando un análisis de frecuencia con variables. El objetivo de esta investigación estaba dirigido al diagnóstico clínico bucal en los pacientes PVVS, pertenecientes a FUNDASIDA, donde se observó la presencia de lesiones fúngicas en la que se destaca la Candidiasis Eritematosa en un 20%, al igual que la Queilitis Angular20%, y la Candidiasis Pseudomembranosa con él 6.6% y entre las lesiones de tipo vírales se encontró que el Herpes Simple Tipo I en un 6.6% al igual que la Leucoplasia Vellosa en un 6.6% y las manifestaciones de tipo bacterianas se encontró la Gingivitis que se dio en un 53.3% que no precisamente están asociadas a la infección de VIH/SIDA, al igual que la Caries Dental en un 93.3 %, seguido de Xerostomía 66.6% que en la mayoría de los casos está relacionada al tratamiento antirretroviral, entre otras manifestaciones se encontró la Hiperqueratosis causado portraumatismos en 3.3%, al igual que la Estomatitis Aftosa Recidivante, Estomatitis Aftosa Menor en un 3.3%

Oral lesions that occur in PVVS patients are a very fundamental sign to detect the disease in its initial stage, as well as to know if the disease has progressed to the stage of AIDS. This research deals with the oral clinical diagnosis in PVVS patients, which was carried out at the FUNDASIDA facilities, during the months of February to April of two thousand and ten, with the participation of thirty patients of both sexes between the ages of sixteen and seventy years, most are under antiretroviral treatment and each one of them undergoes a medical check-up where their CD4 levels and viral load are evaluated. After collecting all the data, these were processed through the statistical program SPSS for its acronym in English (Statistical Package for the Social Sciences version Pasw18), performing a frequency analysis with variables. The objective of this research was directed to the oral clinical diagnosis in PLWHA patients, belonging to FUNDASIDA, where the presence of fungal lesions was observed in which Erythematous Candidiasis stands out in 20%, as well as Angular Cheilitis 20%, and Pseudomembranous Candidiasis with 6.6% and among the viral type lesions it was found that Herpes Simplex Type I in 6.6% as well as Vellous Leukoplakia in 6.6% and the bacterial manifestations Gingivitis was found that occurred in 53.3% that are not precisely associated with HIV / AIDS infection, like Dental Caries in 93.3%, followed by Xerostomia 66.6% that in most cases is related to antiretroviral treatment, among other manifestations it was found Hyperkeratosis caused by trauma in 3.3%, as well as Recurrent Aphthous Stomatitis, Minor Aphthous Stomatitis in 3.3%

Bleomicina: un modelo de fibrosis pulmonar / Bleomycin: a lung fibrosis animal model

La bleomicina es un glicopéptido utilizado para el tratamiento del cáncer cuyo potencial terapéutico está limitado por su toxicidad pulmonar. El efecto citotóxico depende de la dosis e involucra el desarrollo de neumonitis que progresa a fibrosis; las células epiteliales alveolares son el blanco principal del daño inducido por la bleomicina. Se considera que la muerte de células epiteliales alveolares por apoptosis es un evento clave en el inicio y la progresión de la fibrosis pulmonar (FP) que se caracteriza por el depósito excesivo de moléculas de la matriz extracelular, principalmente de colágenas fibrilares en el parénquima pulmonar. En la investigación básica de la FP, la bleomicina se ha utilizado como el principal agente fibrogénico en modelos animales. Durante los últimos años, el modelo de bleomicina desarrollado en ratones trasgénicos se ha empleado para elucidar in vivo el papel de un gran número de biomoléculas involucradas en la FP.

Bleomycin is a glycopeptide used for cancer treatment, but the therapeutic potencial of this drug is limited by its lung toxicity. The cytotoxic effect of bleomycin is dose-dependent and involves pneumonitis that proceeds to lung fibrosis (LF). Alveolar epithelial cells are the main target of bleomycin induced injury. Alveolar epithelial cell death by apoptosis is considered as a key event in the initiation and progression of LF, that is characterized by excessive deposition of extracellular matrix, mainly fibrilar collagens in the lung parenchyma. Bleomycin has been used as the main fibrogenic agent in animal models in LF basic research; in recent years, a bleomycin model developed in transgenic mice has been used to elucidate the in vivo role of a great number of biomolecules involved in LF.