NEW ADVANCES IN THE PROTECTIVE MECHANISMS OF ACIDIC pH AFTER ISCHEMIA: PARTICIPATION OF NO.

BACKGROUND: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. OBJECTIVE: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH=6.4) during the first 2 min of reperfusion (AR) in absence or in presence of L-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis RESULTS: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. L-NAME prevented the beneficial effects of AR CONCLUSIONS: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

Laccaria bicolor pectin methylesterases are involved in ectomycorrhiza development with Populus tremula × Populus tremuloides.

The development of ectomycorrhizal (ECM) symbioses between soil fungi and tree roots requires modification of root cell walls. The pectin-mediated adhesion between adjacent root cells loosens to accommodate fungal hyphae in the Hartig net, facilitating nutrient exchange between partners. We investigated the role of fungal pectin modifying enzymes in Laccaria bicolor for ECM formation with Populus tremula × Populus tremuloides. We combine transcriptomics of cell-wall-related enzymes in both partners during ECM formation, immunolocalisation of pectin (Homogalacturonan, HG) epitopes in different methylesterification states, pectin methylesterase (PME) activity assays and functional analyses of transgenic L. bicolor to uncover pectin modification mechanisms and the requirement of fungal pectin methylesterases (LbPMEs) for ECM formation. Immunolocalisation identified remodelling of pectin towards de-esterified HG during ECM formation, which was accompanied by increased LbPME1 expression and PME activity. Overexpression or RNAi of the ECM-induced LbPME1 in transgenic L. bicolor lines led to reduced ECM formation. Hartig Nets formed with LbPME1 RNAi lines were shallower, whereas those formed with LbPME1 overexpressors were deeper. This suggests that LbPME1 plays a role in ECM formation potentially through HG de-esterification, which initiates loosening of adjacent root cells to facilitate Hartig net formation.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment.

In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell-wall-degrading enzymes. Here, we characterize the biochemical properties of the symbiosis-induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance of LbGH28A was confirmed by generating RNA interference (RNAi)-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front. These findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.

Growth characterization and predictive behavior of Eurotium species in a feedstuff matrix.

Animal feeds are characterized by low water activity values. Nevertheless, fungal contamination with Eurotium species are quite common, causing nutritional depletion, spoilage and economic losses. The aim of this work was to assess Eurotium amstelodami, E. chevalieri, E. repens and E. rubrum growth in a feed matrix at different conditions of water activity (0.71-0.97) and temperature (5, 15, 25, 30 and 37°C). It was found that Eurotium species are able to grow in a wide range of water activity and temperature in a short period of time (7 days) and faster than in synthetic media. Rosso and probabilistic models were applied in order to determine the limiting and optimum growth conditions as well as growth probability at certain combinations of environmental factors. Both models provided an accurate fit to the cardinal parameters and good performance for growth/no growth cases. This is the first report assessing the growth parameters of Eurotium species directly in animal feed. Data obtained in the present study is useful to predict and avoid Eurotium species growth in animal feed.

CaMKII-dependent ryanodine receptor phosphorylation mediates sepsis-induced cardiomyocyte apoptosis.

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis-induced apoptosis. Wild-type (WT) CASP mice hearts showed an increase in apoptosis respect to WT-Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3-I) were protected against sepsis-induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca2+ release, prevented apoptosis in WT-CASP. To examine whether CaMKII-dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation-dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT-CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene-treated mice. We conclude that in sepsis, CaMKII-dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.

The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis.

To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes.

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis.

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small-Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

Soil lead pollution modifies the structure of arbuscular mycorrhizal fungal communities.

The impact of lead (Pb) pollution on native communities of arbuscular mycorrhizal fungi (AMF) was assessed in soil samples from the surroundings of an abandoned Pb smelting factory. To consider the influence of host identity, bulk soil surrounding plant roots soil samples of predominant plant species (Sorghum halepense, Bidens pilosa, and Tagetes minuta) growing in Pb-polluted soils and in an uncontaminated site were selected. Molecular diversity was assessed by sequencing the 18S rDNA region with primers specific to AMF (AMV4.5NF/AMDGR) using Illumina MiSeq. A total of 115 virtual taxa (VT) of AMF were identified in this survey. Plant species did not affect AMF diversity patterns. However, soil Pb content was negatively correlated with VT richness per sample. Paraglomeraceae and Glomeraceae were the predominant families while Acaulosporaceae, Ambisporaceae, Archaeosporaceae, Claroideoglomeraceae, Diversisporaceae, and Gigasporaceae were less abundant. Acaulosporaceae and Glomeraceae were negatively affected by soil Pb, but Paraglomeraceae relative abundance increased under increasing soil Pb content. Overall, 26 indicator taxa were identified; four of them were previously reported in Pb-polluted soils (VT060; VT222; VT004; VT380); and five corresponded to cultured spores of Scutellospora castaneae (VT041), Diversispora spp. and Tricispora nevadensis (VT060), Diversispora epigaea (VT061), Glomus proliferum (VT099), and Gl. indicum (VT222). Even though AMF were present in Pb-polluted soils, community structure was strongly altered via the differential responses of taxonomic groups of AMF to Pb pollution. These taxon-specific differences in tolerance to soil Pb content should be considered for future phytoremediation strategies based on the selection and utilization of native Glomeromycota.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted ß-1,4 endoglucanase that plays a key role in symbiosis development.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.

Cardioprotection of benzolamide in a regional ischemia model: Role of eNOS/NO.

BACKGROUND: Recent studies from our laboratory show the cardioprotective action of benzolamide (BZ, carbonic anhydrase inhibitor) against ischemia-reperfusion injury. However, the mechanisms involved have not been fully elucidated. OBJECTIVE: To examine the participation of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) in the effects of BZ in a model of regional ischemia. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of coronary artery occlusion followed by 60 min of reperfusion (IC). Other hearts received BZ during the first 10 min of reperfusion in absence or presence of L-NAME, NOS inhibitor. The infarct size (IS) and the post-ischemic recovery of myocardial function were measured. Oxidative/nitrosative damage were assessed by reduced glutathione (GSH) content, thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine levels. The expression of phosphorylated forms of Akt, p38MAPK and eNOS, and the concentration of inducible nitric oxide synthase (iNOS) were also determined. RESULTS: BZ significantly decreased IS (6.2 ±â€¯0.5% vs. 34 ±â€¯4%), improved post-ischemic contractility, preserved GSH levels and diminished TBARS and 3-nitrotyrosine. In IC hearts, P-Akt, P-p38MAPK and P-eNOS decreased and iNOS increased. After BZ addition the levels of P-kinases and P-eNOS increased and iNOS decreased. Except for P-Akt, P-p38MAPK and iNOS, the effects of BZ were abolished by L-NAME. CONCLUSIONS: Our data demonstrate that the treatment with BZ at the onset of reperfusion was effective to reduce cell death, contractile dysfunction and oxidative/nitrosative damage produced by coronary artery occlusion. These BZ-mediated beneficial actions appear mediated by eNOS/NO-dependent pathways.

Protected area coverage of threatened vertebrates and ecoregions in Peru: Comparison of communal, private and state reserves.

Protected areas (PAs) are a conservation mainstay and arguably the most effective conservation strategy for species protection. As a 'megadiverse' country, Peru is a priority for conservation actions. Peruvian legislation allows for the creation of state PAs and private/communal PAs. Using publicly available species distribution and protected area data sets we evaluated the coverage of Threatened terrestrial vertebrate species distributions and ecoregions provided by both kinds of PA in Peru. Peru's state PA system covers 217,879 km2 and private/communal PAs cover 16,588 km2. Of the 462 species of Threatened and Data Deficient species we evaluated, 75% had distributions that overlapped with at least one PA but only 53% had ≥10% of their distributions within PAs, with inclusion much reduced at higher coverage targets. Of the species we evaluated, 118 species are only found in national PAs and 29 species only found in private/communal PAs. Of the 17 terrestrial ecoregions found in Peru all are represented in PAs; the national PA system included coverage of 16 and private/communal PAs protect 13. One ecoregion is only protected in private/communal PAs, whereas four are only covered in national PAs. Our results show the important role private/communal PAs can play in the protection of ecological diversity.

Cyclosporine-A mimicked the ischemic pre- and postconditioning-mediated cardioprotection in hypertensive rats: Role of PKCε.

Our aim was to assess the action of cyclosporine-A (CsA) against reperfusion injury in spontaneously hypertensive rats (SHR) compared to the effects of ischemic pre- (IP) and postconditioning (IPC), examining the role played by PKCε. Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI) and 1h reperfusion (R); IP: a cycle of 5 min GI and 10 min of R prior to 45 min-GI; and IPC: three cycles of 30s-GI/30s-R at the start of R. Other hearts of the IC, IP and IPC groups received CsA (mitochondrial permeability transition pore inhibitor) or chelerythrine (Che, non-selective PKC inhibitor). Infarct size (IS) was assessed. TBARS and reduced glutathione (GSH) content - as parameters of oxidative damage, the expression of P-Akt, P-GSK-3ß, P-PKCε and cytochrome c (Cyc) release - as an index of mitochondrial permeability and the response of isolated mitochondria to Ca(2+) were also measured. IS similarly decreased in preconditioned, postconditioned and CsA treated heart showing the highest values in the combinations IP+CsA and IPC+CsA. TBARS decreased and GSH was partially preserved after all interventions. The content of P-Akt, P-GSK-3ß and P-PKCε increased in cytosol and decreased in mitochondria after IP and IPC. In CsA treated hearts these enzymes increased in both fractions reaching the highest values. Cyc release was attenuated and the response of mitochondria to Ca(2+) was improved by the interventions. The beneficial effects of IP and IPC were annulled when PKC was inhibited with Che. A PKCε/VDAC association was also detected. These data show that, in SHR, the CsA treatment mimicked and reinforced the cardioprotective action afforded by IP and IPC in which PKCε-mediated attenuation of mitochondrial permeability appears as the main mechanism involved.

Hydraulic conductivity and aquaporin transcription in roots of trembling aspen (Populus tremuloides) seedlings colonized by Laccaria bicolor.

Ectomycorrhizal fungi have been reported to increase root hydraulic conductivity (L pr) by altering apoplastic and plasma membrane intrinsic protein (PIP)-mediated cell-to-cell water transport pathways in associated roots, or to have little effect on root water transport, depending on the interacting species and imposed stresses. In this study, we investigated the water transport properties and PIP transcription in roots of aspen (Populus tremuloides) seedlings colonized by the wild-type strain of Laccaria bicolor and by strains overexpressing a major fungal water-transporting aquaporin JQ585595. Inoculation of aspen seedlings with L. bicolor resulted in about 30 % colonization rate of root tips, which developed dense mantle and the Hartig net that was restricted in the modified root epidermis. Transcript abundance of the aspen aquaporins PIP1;2, PIP2;1, and PIP2;2 decreased in colonized root tips. Root colonization by JQ585595-overexpressing strains had no significant impact on seedling shoot water potentials, gas exchange, or dry mass; however, it led to further decrease in transcript abundance of PIP1;2 and PIP2;3 and the significantly lower L pr than in non-inoculated roots. These results, taken together with our previous study that showed enhanced root water hydraulics of L. bicolor-colonized white spruce (Picea glauca), suggest that the impact of L. bicolor on root hydraulics varies by the ectomycorrhiza-associated tree species.

Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings.

The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water-transporting aquaporin in Laccaria bicolor. Picea glauca was inoculated with wild-type (WT), mock transgenic or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants.

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides).

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700-fold higher in the hyphae within the root than in the free-living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock-down strains, a non-mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H2 O2 and CO2 transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.

Mycotoxins and mycotoxigenic fungi in poultry feed for food-producing animals.

Moulds are capable of reducing the nutritional value of feedstuff as well as elaborating several mycotoxins. Mycotoxin-contaminated feed has adverse effects on animal health and productivity. Also, mycotoxins may be carried over into meat and eggs when poultry are fed with contaminated feed. In a point prevalence study feedstuff used for poultry nutrition in Argentina was analyzed for fungal flora, natural incidence of selected mycotoxins, and nutritional quality. Ten mould genera were recovered, six of them known to be mycotoxigenic. More than 28 species were determined. Fumonisins were detected in all the samples (median 1,750 ppb). Forty-four out of 49 samples (90%) were contaminated with DON (median 222 ppb) and OTA (median 5 ppb). Also, 44 out of 49 samples were contaminated with aflatoxins (median 2.685 ppb), 42 samples (86%) with ZEA (median 50 ppb), and 38 samples (78%) with T2-toxin (median 50 ppb). Ninety percent of the samples had at least one type of nutritional deficiency. This study indicates the need for continuous assessment of the mycological status of animal feed production, in order to feed animals for optimal performance ensuring food safety.

Evaluation of arbuscular mycorrhizal fungi capacity to alleviate abiotic stress of olive (Olea europaea L.) plants at different transplant conditions.

The capacity of roots to sense soil physicochemical parameters plays an essential role in maintaining plant nutritional and developmental functions under abiotic stress. These conditions generate reactive oxygen species (ROS) in plant tissues causing oxidation of proteins and lipids among others. Some plants have developed adaptive mechanisms to counteract such adverse conditions such as symbiotic association with arbuscular mycorrhizal fungi (AMF). AMF enhance plant growth and improve transplant survival by protecting host plants against environmental stresses. The aim of this study was to evaluate the alleviation of transplanting stress by two strains of Rhizophagus irregularis (GC2 and GA5) in olive. Our results show that olive plants have an additional energetic expense in growth due to an adaptative response to the growing stage and to the mycorrhizal colonization at the first transplant. However, at the second transplant the coinoculation improves olive plant growth and protects against oxidative stress followed by the GA5-inoculation. In conclusion, a combination of two AMF strains at the beginning of olive propagation produces vigorous plants successfully protected in field cultivation even with an additional cost at the beginning of growth.

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells.

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.