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.

Is N-methylacetazolamide a possible new therapy against ischemia-reperfusion injury?

The increase of intracellular Ca2+ concentration, produced principally by its influx through the L-type Ca2+ channels, is one of the major contributors to the ischemia-reperfusion injury. The inhibition of those channels in different experimental models was effective to ameliorate the post-ischemic damage. However, at a clinical level, the results were contradictory. Recent results of our group obtained in an ¨ex vivo¨ heart model demonstrated that a chemical derived from acetazolamide, the N-methylacetazolamide (NMA) protected the heart against ischemia-reperfusion injury, diminishing the infarct size and improving the post-ischemic recovery of myocardial function and mitochondrial dynamic. A significant inhibitory action on L-type Ca2+ channels was also detected after NMA treatment, suggesting this action as responsible for the beneficial effects on myocardium exerted by this compound. Although these results were promising, the effectiveness of NMA in the treatment of ischemic heart disease in humans as well as the advantages or disadvantages in comparison to the classic calcium antagonists needs to be investigated.

Local intensity of artisanal gold mining drives mercury accumulation in neotropical oxbow lake fishes.

Driven by surges in global gold prices and additional socio-economic factors, artisanal small-scale gold mining (ASGM) in the Global South is increasing and driving emissions of significant quantities of mercury (Hg) into the air and freshwater. Hg can be toxic to animal and human populations and exacerbate the degradation of neotropical freshwater ecosystems. We examined drivers of Hg accumulation in fish that inhabit oxbow lakes of Peru's Madre de Dios, a region with high biodiversity value and increasing human populations that depend on ASGM. We hypothesized that fish Hg levels would be driven by local ASGM activities, by environmental Hg exposure, by water quality, and by fish trophic level. We sampled fish in 20 oxbow lakes spanning protected areas and areas subject to ASGM during the dry season. Consistent with previous findings, Hg levels were positively associated with ASGM activities, and were higher in larger, carnivorous fish and where water had lower dissolved oxygen levels. In addition, we found a negative relationship between fish mercury levels associated with ASGM and the occurrence of the piscivorous giant otter. The link between fine-scale quantification of spatial ASGM activity and Hg accumulation, as indicated by the result that in the lotic environment, localized effects of gold mining activities are stronger drivers (77 % model support) of Hg accumulation than environmental exposure (23 %) constitutes a novel contribution to a growing body of literature on Hg contamination. Our findings provide additional evidence of high Hg exposure risks to neotropical human and top carnivore populations subject to the impacts of ASGM, which depend on freshwater ecosystems undergoing gradual degradation. The documented spatial variation in Hg accumulation and increased Hg levels in carnivorous fish should serve as a warning to human communities in Madre de Dios to avoid the proximity of high-intensity gold mining areas and minimize local carnivorous fish consumption.

Quantum-resistance in blockchain networks.

The advent of quantum computing threatens blockchain protocols and networks because they utilize non-quantum resistant cryptographic algorithms. When quantum computers become robust enough to run Shor's algorithm on a large scale, the most used asymmetric algorithms, utilized for digital signatures and message encryption, such as RSA, (EC)DSA, and (EC)DH, will be no longer secure. Quantum computers will be able to break them within a short period of time. Similarly, Grover's algorithm concedes a quadratic advantage for mining blocks in certain consensus protocols such as proof of work. Today, there are hundreds of billions of dollars denominated in cryptocurrencies and other digital assets that rely on blockchain ledgers as well as thousands of blockchain-based applications storing value in blockchain networks. Cryptocurrencies and blockchain-based applications require solutions that guarantee quantum resistance in order to preserve the integrity of data and assets in these public and immutable ledgers. The quantum threat and some potential solutions are well understood and presented in the literature. However, most proposals are theoretical, require large QKD networks, or propose new quantum-resistant blockchain networks to be built from scratch. Our work, which is presented in this paper, is pioneer in proposing an end-to-end framework for post-quantum blockchain networks that can be applied to existing blockchain to achieve quantum-resistance. We have developed an open-source implementation in an Ethereum-based (i.e., EVM compatible) network that can be extended to other existing blockchains. For the implementation we have (i) used quantum entropy to generate post-quantum key pairs, (ii) established post-quantum TLS connections and X.509 certificates to secure the exchange of information between blockchain nodes over the internet without needing a large QKD network, (iii) introduced a post-quantum second signature in transactions using Falcon-512 post-quantum keys, and (iv) developed the first on-chain verification of post-quantum signatures using three different mechanisms that are compared and analyzed: Solidity smart-contracts run by the validators for each transaction, modified EVM Opcode, and precompiled smart contracts.

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.

Combination venetoclax and selinexor effective in relapsed refractory multiple myeloma with translocation t(11;14).

Patients with multiple myeloma-bearing translocation t(11;14) have recently been shown to benefit from the apoptosis-inducing drug venetoclax; however, the drug lacks FDA approval in multiple myeloma thus far due to a potential safety signal in the overall patient population. Selinexor is an inhibitor of nuclear export that is FDA-approved for patients with multiple myeloma refractory to multiple lines of therapy. Here, we report that in four patients with multiple myeloma with t(11;14), the concomitant administration of venetoclax and selinexor was safe and associated with disease response. Moreover, the combination was synergistic in t(11;14) multiple myeloma cell lines and caused decreased levels of Cyclin D1 (which is overexpressed due to the CCND1-IGH fusion) when given in combination as compared to single agents. These data suggest that the combination of venetoclax and selinexor is effective and t(11;14) may serve as a therapeutic marker for response and target for future clinical trials.

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.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current.

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 µM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aß were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aß decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.

Linking Chronic Otitis Media and Nasal Obstruction: A CFD Approach.

OBJECTIVES: To investigate a possible relationship between altered nasal flow and chronic otitis media (COM) using computational fluid dynamics (CFD). STUDY DESIGN: Retrospective case series. METHODS: Retrospective cohort sample of CT scans from patients with COM and controls without COM to compare the results of various nasal airflow parameters determined by CFD between a group of patients with COM (N = 60) and a control group of subjects without any evidence of ear disease (N = 81). The CT were subjected to various procedures to carry out CFD studies, determining the resistance to nasal flow, the proportion of flow through the right and left nasal cavity, and two nondimensional estimators. The results of CFD studies between patients with COM and controls were compared. RESULTS: Whereas only 12.3% of the controls had CFD alteration (10 out of 81), 43.3% of the patients suffering COM displayed alterations of our nondimensional parameters R-ϕ (26 out of 60). CONCLUSIONS: According to our results, the incidence of alterations in nasal airflow by studying with CFD is significantly higher in patients with COM than in controls. To our knowledge, this is the first article linking nasal cavity and COM using a CFD approach. Our results support the hypothesis that nasal flow alterations could be implicated in the etiopathogenesis of the COM. LEVEL OF EVIDENCE: 4 Laryngoscope, 132:1224-1230, 2022.

Chronic GPER activation prevents ischemia/reperfusion injury in ovariectomized rats.

During menopause women are exposed to an increase in cardiovascular risk. G protein-coupled estrogen receptor (GPER) is known to mediate several of the protective effects of such hormones. G1 was described as a selective and synthetic agonist for GPER. The aim of the present research is to evaluate the effect of a chronic treatment with G1 in ovariectomized (OVX) rats exposed to ischemia/reperfusion (I/R). Considering the hypothesis that an impaired mitochondrial state could be involved in the alterations produced in OVX rats, other objective of this study was to investigate it in an isolated preparation. Three months old rats were assigned to undergo either bilateral ovariectomy or sham operation. The OVX rats were randomly treated during one month with either G1 or vehicle. Cardiac mitochondria from OVX rats showed a depolarized membrane potential and a decreased calcium retention capacity in comparison with Sham rats, which were prevented by chronic G1 treatment. I/R caused a higher decrease of left ventricular developed pressure and a higher increase of left ventricular end diastolic pressure in OVX compared to Sham hearts. These altered mechanical parameters were prevented by G1. The induced infarct size was significantly higher in OVX, which was reduced by G1 treatment. These results indicate that the mitochondrial state in OVX rats is impaired, accompanied by an altered mechanical response after ischemia and reperfusion injury, which was effectively prevented with chronic treatment with G1. The present study may provide further insights for the potential development of a therapy based on the GPER modulation.

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.

Antifungal activity of silver nanoparticles and clotrimazole against Candida spp

Abstract The aim of present study was calculate the Minimum inhibitory concentrations (MICs) of silver nanoparticles and clotrimazole for Candida species and their interaction by the adaptation of standarized methods. The MICs values of clotrimazole were 9 E-04-3 E-03 ug/ml, 0.1-0.6 ug/ml, 3 E-03- 0.1 ug/ml and 3 E-03-0.3 ug/ml for Candida albicans susceptible to fluconazole, Candida albicans resistance to fluconazole, Candida krusei and Candida parapsilosis respectively. The MICs values of silver nanoparticles were 26.50- 53 ug/ml; 26.50-106 ug/ml; 106-212 ug/ ml and 26.50- 53 ug/ml for Candida albicans susceptible to fluconazole, Candida albicans resistance to fluconazole, Candida krusei and Candida parapsilosis respectively. Synergism between clotrimazole and silver nanoparticles was measured by checkerboard BMD (broth microdilution) test and shown only for C. albicans susceptible to fluconazole because the fractional inhibitory concentrations (FICs) values were 0.07 - 0.15 ug/ml. Indifference was shown for the other species tested because the FICs values were between 0.5 - 2- 3.06 ug/ml. The results suggest synergistic activity depending on the fungus species analysed, however we recommend the incorporation of others measurement methodologies to confirm our results. As for measurement methodologies of MICs of silver nanoparticles and clotrimazole international normative were respected to guarantee reproducible and comparable results.

Myocardial and mitochondrial effects of the anhydrase carbonic inhibitor ethoxzolamide in ischemia-reperfusion.

We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 µM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.

Nucleus-directed fluorescent reporter system for promoter studies in the ectomycorrhizal fungus Laccaria bicolor.

Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.

Growth characterization and predictive behavior of Eurotium species in a feedstuff matrix / Caracterización del crecimiento y comportamiento predictivo de especies de Eurotium en matriz de alimento balanceado

ABSTRACT Animal feeds are characterized by low water activity values. Nevertheless, fungal contamination withEurotiumspecies are quite common, causing nutritional depletion, spoilage and economic losses. The aim of this work was to assessEurotiumamstelodami,E. chevalieri,E. repensandE. rubrumgrowth 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 thatEurotiumspecies 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 ofEurotiumspecies directly in animal feed. Data obtained in the present study is useful to predict and avoidEurotiumspecies growth in animal feed.

RESUMEN Los alimentos balanceados se caracterizan por tener bajos valores de actividad de agua. Sin embargo, la contaminación por hongos con especies deEurotiumes bastante común y causa agotamiento nutricional, deterioro y pérdidas económicas. El objetivo de este trabajo fue evaluar el crecimiento deEurotiumamstelodami,E. chevalieri,E. repensyE. rubrumen una matriz de alimento balanceado a diferentes condiciones de actividad de agua (0,71-0,97) y temperatura (5, 15, 25, 30 y 37°C). Se determinó que las especies deEurotiumpueden crecer en un amplio rango de actividades de agua y temperatura en un corto período de tiempo (7 días), y a mayor velocidad que en medio sintético. Se utilizaron los modelos de Rosso y probabilísticos para determinar las condiciones de crecimiento limitantes y óptimas, así como la probabilidad de crecimiento en ciertas combinaciones de factores ambientales. Ambos modelos proporcionaron un ajuste preciso a los parámetros cardinales y una buenaperformancepara los casos de crecimiento/sin crecimiento. Este es el primer trabajo que evalúa los parámetros de crecimiento de las especies deEurotiumdirectamente en alimento balanceado. Los datos obtenidos en el presente estudio son útiles para predecir y evitar el crecimiento de especies deEurotiumen este tipo de alimentos.

Mycorrhizal stress alleviation in Senecio bonariensis Hook & Arn growing in urban polluted soils.

Loss of biodiversity and accumulation of contaminants in urban soils and water bodies cause serious issues in metropolitan areas. The Matanza-Riachuelo river basin (metropolitan area of Buenos Aires, Argentina) is one of the most environmentally degraded regions in the world. Senecio bonariensis Hook & Arn (Asteraceae) grows in the periodically flooded soils of this wetland. This plant concentrates potentially toxic trace elements (PTEs) in its tissues and establishes symbiosis with arbuscular mycorrhizal (AM) fungi that collaborate with PTE phytostabilization in soils. The objective of this work was to evaluate tolerance and stress alleviation of AM-colonized S. bonariensis when transplanting and exposing to highly polluted environmental conditions of the river basin. Plants were initially inoculated with different AM strains and maintained in greenhouse conditions. After 6 mo, they were transplanted to the field. These plants showed a more equal distribution between shoot and root biomass production in comparison to field spontaneous S. bonaerensis plants. Plants in earlier contact with native soil inoculum showed positive correlation with phosphorus content and a significant increase of vesicle frequency. Plants belatedly contacted with native inoculum in the field (control) showed a higher catalase level that was positively correlated with the total colonization frequency and chlorophyll content. The ability to establish symbiosis with Rhizophagus intraradices (strain GC3), commonly used in the formulation of biofertilizers, was also analyzed. Plants inoculated with GC3 at the beginning of the assay showed lower colonization and were less efficient in the field. The preservation of spontaneous native plants with ornamental value and bioaugmentation of their associated microbiome can contribute to the stabilization of contaminants in soils.

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.

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.

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.

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.