Membrane Lipids and Osmolytes in the Response of the Acidophilic Basidiomycete Phlebiopsis gigantea to Heat, Cold, and Osmotic Shocks.

Previously, we found for the first time the participation of osmolytes in adaptation to acidic conditions in three acidophilic fungi. Because trehalose can protect membranes, we hypothesized a relationship between osmolyte and membrane systems in adaptation to stressors. In the mycelium of Phlebiopsis gigantea, the level of osmolytes reaches 8% of the dry mass, while trehalose and arabitol make up 60% and 33% of the sum, respectively. Cold shock does not change the composition of osmolytes, heat shock causes a twofold increase in the trehalose level, and osmotic shock leads to a marked increase in the amount of trehalose and arabitol. Predominance of phospholipids (89% of the sum) and low proportions of sterols and sphingolipids are characteristic features of the membrane lipids' composition. Phosphatidic acids, along with phosphatidylethanolamines and phosphatidylcholines, are the main membrane lipids. The composition of the membrane lipids remains constant under all shocks. The predominance of linoleic (75% of the sum) and palmitic (20%) acids in phospholipids results in a high degree of unsaturation (1.5). Minor fluctuations in the fatty acid composition are observed under all shocks. The results demonstrate that maintaining or increasing the trehalose level provides stability in the membrane lipid composition during adaptation.

Determination of age-dependent bone marrow normocellularity.

OBJECTIVES: Determination of bone marrow cellularity is a key part of bone marrow examination because it provides a small window into a patient's current state of hematopoietic well-being. Traditionally, bone marrow cellularity is estimated semiquantitatively through microscopic examination of core biopsy specimens harvested from the iliac crest of the pelvic bone. Bone marrow cellularity is then designated as hypercellular, normocellular, or hypocellular based on the patient's age. This assessment can have significant clinical impact, but the variation in the age-adjusted normocellularity range is not sufficiently characterized because of a lack of study data, especially in older patients (those older than 70 years of age). This study further established the normal range of bone marrow cellularity, particularly in older adults. METHODS: In this study, 570 benign staging and healthy donor bone marrows from patients 1 year to 93 years of age were analyzed for cellularity. RESULTS: Linear regression modeling demonstrates that cellularity in adults declines approximately 3% per decade, including after the seventh decade of life. The 90% reference interval for normocellularity in United States is 30% to 75% for those aged 18 to 90 years. CONCLUSIONS: The findings revealed a more stable and slower rate of decline in cellularity with age in adults than the widely used linear model of "100% minus the patient age in decades." Normocellularity is better modeled based on age group. In those younger than 20 years of age, normocellularity ranges from 45% to 85% (mean [SD], 65% [20%]), as defined by Friebert et al in 1998. Based on our study finding of a little less than 3% decline per decade of age, the following is our recommendation for normocellularity range: For individuals 20 to 40 years of age, it ranges from 40% to 70% (mean [SD], 55% [15%]); for individuals 40 to 60 years of age, it ranges from 35% to 65% (mean [SD], 50% [15%]); and for individuals older than 60 years of age, it ranges from 30% to 60% (mean [SD], 45% [15%]). Interestingly, those older than 70 years of age do not show a significant decrease from those aged 60 to 69 years.

One Step Closer to Enigmatic USCα Methanotrophs: Isolation of a Methylocapsa-like Bacterium from a Subarctic Soil.

The scavenging of atmospheric trace gases has been recognized as one of the lifestyle-defining capabilities of microorganisms in terrestrial polar ecosystems. Several metagenome-assembled genomes of as-yet-uncultivated methanotrophic bacteria, which consume atmospheric CH4 in these ecosystems, have been retrieved in cultivation-independent studies. In this study, we isolated and characterized a representative of these methanotrophs, strain D3K7, from a subarctic soil of northern Russia. Strain D3K7 grows on methane and methanol in a wide range of temperatures, between 5 and 30 °C. Weak growth was also observed on acetate. The presence of acetate in the culture medium stimulated growth at low CH4 concentrations (~100 p.p.m.v.). The finished genome sequence of strain D3K7 is 4.15 Mb in size and contains about 3700 protein-encoding genes. According to the result of phylogenomic analysis, this bacterium forms a common clade with metagenome-assembled genomes obtained from the active layer of a permafrost thaw gradient in Stordalen Mire, Abisco, Sweden, and the mineral cryosol at Axel Heiberg Island in the Canadian High Arctic. This clade occupies a phylogenetic position in between characterized Methylocapsa methanotrophs and representatives of the as-yet-uncultivated upland soil cluster alpha (USCα). As shown by the global distribution analysis, D3K7-like methanotrophs are not restricted to polar habitats but inhabit peatlands and soils of various climatic zones.

The Role of Osmolytes and Membrane Lipids in the Adaptation of Acidophilic Fungi.

Acidophiles maintain near-neutral intracellular pH using proton pumps. We have suggested the protective role of osmolytes and membrane lipids in the adaptation to an acidic environment. Previously we have observed, for the first time, high levels of trehalose in acidophilic basidiomycete Sistotrema brinkmannii. Here, we have studied the composition of both osmolytes and membrane lipids of two more acidophilic fungi. Trehalose and polyols were among the main osmolytes during growth under optimal conditions (pH 4.0) in basidiomycete Phlebiopsis gigantea and ascomycete Mollisia sp. Phosphatidic acids, phosphatidylethanolamines, phosphatidylcholines, and sterols, were predominant membrane lipids in both fungi. P. gigantea had a narrow optimum of growth at pH 4.0, resulting in a sharp decline of growth rate at pH 2.6 and 5.0, accompanied by a decrease in the number of osmolytes and significant changes in the composition of membrane lipids. In contrast, Mollisia sp. had a broad optimal growth range (pH 3.0-5.0), and the number of osmolytes either stayed the same (at pH 6.0) or increased (at pH 2.6), while membrane lipids composition remained unchanged. Thus, the data obtained indicate the participation of osmolytes and membrane lipids in the adaptation of acidophilic fungi.

Acquired thermotolerance, membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock.

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.

Methylomonas rapida sp. nov., a novel species of fast-growing, carotenoid-producing obligate methanotrophs with high biotechnological potential.

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1T, which are phylogenetically distinct from the currently described Methylomonas species (94.0-97.3 % 16S rRNA gene sequence similarity). Strains 12 and MP1T were isolated from freshwater sediments collected in Moscow and Krasnodar regions, respectively. Cells of these strains are Gram-negative, red-pigmented, highly motile thick rods that contain a type I intracytoplasmic membrane system and possess a particulate methane monooxygenase (pMMO) enzyme. These bacteria grow between 8 and 45 °C (optimum 35 °C) in a relatively narrow pH range of 5.5-7.3 (optimum pH 6.6-7.2). Major carotenoids synthesized by these methanotrophs are 4,4'-diaplycopene-4,4'-dioic acid, 1,1'-dihydroxy-3,4-didehydrolycopene and 4,4'-diaplycopenoic acid. High biomass yield, of up to 3.26 g CDW/l, is obtained during continuous cultivation of MP1T on natural gas in a bioreactor at a dilution rate of 0.22 h-1. The complete genome sequence of strain MP1T is 4.59 Mb in size; the DNA G + C content is 52.8 mol%. The genome encodes four rRNA operons, one pMMO operon and 4,216 proteins. The genome sequence displays 82-85 % average nucleotide identity to those of earlier described Methylomonas species. We propose to classify these bacteria as representing a novel species of the genus Methylomonas, M. rapida sp. nov., with the type strain MP1T (=KCTC 92586T = VKM B-3663T).

Epinephrine extensively changes the biofilm matrix composition in Micrococcus luteus C01 isolated from human skin.

The importance of the impact of human hormones on commensal microbiota and microbial biofilms is established in lots of studies. In the present investigation, we continued and extended the research of epinephrine effects on the skin commensal Micrococcus luteus C01 and its biofilms, and also the matrix changes during the biofilm growth. Epinephrine in concentration 4.9 × 10-9 M which is close to normal blood plasma level increased the amount of polysaccharides and extracellular DNA in the matrix, changed extensively its protein, lipid and polysaccharide composition. The Ef-Tu factor was one of the most abundant proteins in the matrix and its amount increased in the presence of the hormone. One of the glucose-mannose polysaccharide was absent in the matrix in presence of epinephrine after 24 h of incubation. The matrix phospholipids were also eradicated by the addition of the hormone. Hence, epinephrine has a great impact on the M. luteus biofilms and their matrix composition, and this fact opens wide perspectives for the future research.

Current status of phosphoinotiside-3 kinase inhibitors in blood cancers.

PURPOSE OF REVIEW: Treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) underwent paradigm shifts, with targeted agents rapidly displacing chemotherapy. Phosphoinotiside-3 kinase (PI3K) is essential for survival and proliferation of neoplastic B cells and has proven a tractable target in NHL, with four agents receiving FDA approval in the last decade. This review summarizes key data and challenges associated with use of PI3K inhibitors in routine practice. RECENT FINDINGS: Idelalisib and duvelisib are active in CLL and indolent NHL, including in patients with high-risk features. Despite differential targeting of PI3K isoforms, they exhibit comparable efficacy and adverse event profile including autoimmune events (transaminitis, colitis, pneumonitis), mediated by Treg/Th17 imbalance. Although copanlisib, a pan-PI3K inhibitor, is associated with a distinct safety profile (hyperglycemia, hypertension), preclinical studies indicate that umbralisib, a dual inhibitor of PI3Kδ and casein kinase 1ε, may have less effect on Tregs. However, both drugs may still cause immune-mediated toxicities. SUMMARY: With close monitoring and management of adverse events, PI3K inhibitors continue to have a role in therapy of R/R CLL and NHL. Strategies to mitigate adverse events and increase efficacy of PI3K inhibitors include time-limited combination approaches, intermittent dosing schedules.

Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma.

Aberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.

Peat-Inhabiting Verrucomicrobia of the Order Methylacidiphilales Do Not Possess Methanotrophic Capabilities.

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5-5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83-86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.

Membrane lipid and osmolyte readjustment in the alkaliphilic micromycete Sodiomyces tronii under cold, heat and osmotic shocks.

Previously, we showed for the first time that alkaliphilic fungi, in contrast to alkalitolerant fungi, accumulated trehalose under extremely alkaline conditions, and we have proposed its key role in alkaliphilia. We propose that high levels of trehalose in the mycelium of alkaliphiles may promote adaptation not only to alkaline conditions, but also to other stressors. Therefore, we studied changes in the composition of osmolytes, and storage and membrane lipids under the action of cold (CS), heat (HS) and osmotic (OS) shocks in the obligate alkaliphilic micromycete Sodiomyces tronii. During adaptation to CS, an increase in the degree of unsaturation of phospholipids was observed while the composition of osmolytes, membrane and storage lipids remained the same. Under HS conditions, a twofold increase in the level of trehalose and an increase in the proportion of phosphatidylethanolamines were observed against the background of a decrease in the proportion of phosphatidic acids. OS was accompanied by a decrease in the amount of membrane lipids, while their ratio remained unchanged, and an increase in the level of polyols (arabitol and mannitol) in the fungal mycelium, which suggests their role for adaptation to OS. Thus, the observed consistency of the composition of membrane lipids suggests that trehalose can participate in adaptation not only to extremely alkaline conditions, but also to other stressors - HS, CS and OS. Taken together, the data obtained indicate the adaptability of the fungus to the action of various stressors, which can point to polyextremotolerance.

Expanding Characterized Diversity and the Pool of Complete Genome Sequences of Methylococcus Species, the Bacteria of High Environmental and Biotechnological Relevance.

The bacterial genus Methylococcus, which comprises aerobic thermotolerant methanotrophic cocci, was described half-a-century ago. Over the years, a member of this genus, Methylococcus capsulatus Bath, has become a major model organism to study genomic and metabolic basis of obligate methanotrophy. High biotechnological potential of fast-growing Methylococcus species, mainly as a promising source of feed protein, has also been recognized. Despite this big research attention, the currently cultured Methylococcus diversity is represented by members of the two species, M. capsulatus and M. geothermalis, while finished genome sequences are available only for two strains of these methanotrophs. This study extends the pool of phenotypically characterized Methylococcus strains with good-quality genome sequences by contributing four novel isolates of these bacteria from activated sludge, landfill cover soil, and freshwater sediments. The determined genome sizes of novel isolates varied between 3.2 and 4.0Mb. As revealed by the phylogenomic analysis, strains IO1, BH, and KN2 affiliate with M. capsulatus, while strain Mc7 may potentially represent a novel species. Highest temperature optima (45-50°C) and highest growth rates in bioreactor cultures (up to 0.3h-1) were recorded for strains obtained from activated sludge. The comparative analysis of all complete genomes of Methylococcus species revealed 4,485 gene clusters. Of these, pan-genome core comprised 2,331 genes (on average 51.9% of each genome), with the accessory genome containing 846 and 1,308 genes in the shell and the cloud, respectively. Independently of the isolation source, all strains of M. capsulatus displayed surprisingly high genome synteny and a striking similarity in gene content. Strain Mc7 from a landfill cover soil differed from other isolates by the high content of mobile genetic elements in the genome and a number of genome-encoded features missing in M. capsulatus, such as sucrose biosynthesis and the ability to scavenge phosphorus and sulfur from the environment.

NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells.

Peripheral T-cell lymphoma (PTCL) is characterized by poor outcomes. We and others have shown that targeting the NEDD8-activating enzyme (NAE) with an investigational inhibitor pevonedistat deregulates cell cycle and mitosis in lymphoma and leukemia. Here, we report that PTCL is characterized by increased rate of chromosomal instability. NAE inhibition promotes cell cycle arrest and induces multipolar anaphases in T-cell lymphoma cell lines, resulting in apoptosis, also observed in primary malignant PTCL cells treated with pevonedistat. We identified p27Kip1 as a mediator of anaphase catastrophe in these cells. Targeting neddylation with pevonedistat may be a promising approach to treatment of PTCL.

Pharmacologic Targeting of Mcl-1 Induces Mitochondrial Dysfunction and Apoptosis in B-Cell Lymphoma Cells in a TP53- and BAX-Dependent Manner.

PURPOSE: Bcl-2 has been effectively targeted in lymphoid malignancies. However, resistance is inevitable, and novel approaches to target mitochondrial apoptosis are necessary. AZD5991, a selective BH3-mimetic in clinical trials, inhibits Mcl-1 with high potency. EXPERIMENTAL DESIGN: We explored the preclinical activity of AZD5991 in diffuse large B-cell lymphoma (DLBCL) and ibrutinib-resistant mantle cell lymphoma (MCL) cell lines, MCL patient samples, and mice bearing DLBCL and MCL xenografts using flow cytometry, immunoblotting, and Seahorse respirometry assay. Cas9 gene editing and ex vivo functional drug screen assays helped identify mechanisms of resistance to Mcl-1 inhibition. RESULTS: Mcl-1 was expressed in DLBCL and MCL cell lines and primary tumors. Treatment with AZD5991 restricted growth of DLBCL cells independent of cell of origin and overcame ibrutinib resistance in MCL cells. Mcl-1 inhibition led to mitochondrial dysfunction as manifested by mitochondrial membrane depolarization, decreased mitochondrial mass, and induction of mitophagy. This was accompanied by impairment of oxidative phosphorylation. TP53 and BAX were essential for sensitivity to Mcl-1, and oxidative phosphorylation was implicated in resistance to Mcl-1 inhibition. Induction of prosurvival proteins (e.g., Bcl-xL) in stromal conditions that mimic the tumor microenvironment rendered protection of primary MCL cells from Mcl-1 inhibition, while BH3-mimetics targeting Bcl-2/xL sensitized lymphoid cells to AZD5991. Treatment with AZD5991 reduced tumor growth in murine lymphoma models and prolonged survival of MCL PDX mice. CONCLUSIONS: Selective targeting Mcl-1 is a promising therapeutic approach in lymphoid malignancies. TP53 apoptotic network and metabolic reprogramming underlie susceptibility to Mcl-1 inhibition.

First Report of Fusarium proliferatum Causing Garlic clove Rot in Russian Federation.

Garlic (Allium sativum L.) is a widely consumed bulbous crop both worldwide and in Russia. About 200,000 tons of garlic is produced in Russia annually (https://rosstat.gov.ru/). Significant pre- and post-harvest losses of garlic regularly occur due to Fusarium sp. (Taylor et al., 2013). Since September 2018, rotting has been observed in Russia during garlic bulb storage (data of the Federal Scientific Vegetable Center, FSVC, Moscow Region). The outer bulb surface looked healthy, but underneath the integumentary scales, the cloves had light brown and brown spots. When grown, diseased plants were characterized by root and bulb disruption and leaf drying; for some cultivars, up to 100% of plants died. In January 2020, cv. Strelets and Dubkovsky bulbs, collected in July 2019, with rot symptoms, were taken from the FSVC storage. Necrotic clove tissue fragments (0.2-0.5 cm) were cut, sanitized with 70% ethanol for 3 min, rinsed with sterile water, and incubated on potato dextrose agar (PDA) with 1 mg/ml ampicillin at 22°C in the dark. Four single-spore cultures were obtained from four diseased bulbs. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. All isolates (Dubkovsky, Dubkovsky 2, Strelets, and Strelets 2) produced numerous oval unicellular microconidia without septa, 4.1 to 11.6 × 1.3 to 3.4 µm (n = 50) and very few macroconidia with 3-4 septa (21 to 26 × 3 to 4 µm (n = 30)), narrowed at both ends. The cultural and conidial characteristics of the isolates corresponded to Fusarium species (Leslie and Summerell 2006). To determine the species, DNA was extracted from four isolates, and the internal transcribed spacer (ITS), and genes of translation elongation factor 1α (EF1α) and subunits 1 and 2 of DNA-directed RNA polymerase II (RPB1 and RPB2) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 1998a), RPB1-F5/RPB1-R8 (O'Donnell et al. 2010) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained sequences were identical for all four isolates. The isolate Strelets sequences were deposited in NCBI GenBank (MW149129 (ITS), MW161161 (EF1α), MW413302 (RPB1) and MW413303 (RPB2)); their analysis in MLST (http://fusarium.mycobank.org) showed 98.8-99.8% similarity to F. proliferatum (NRRL 13582, 13598 and others), which is part of the F. fujikuroi complex (O'Donnell et al. 1998b). The test on pathogenicity was performed two times according to (Leyronas et al. 2018). For this, three replicates of 10 cloves (cv. Strelets) were soaked in a conidial suspension (~106 conidia/ml; Strelets isolate) for 24 h. Ten control cloves were soaked in sterile water. The cloves were incubated on Petri dishes (5 cloves on a dish; on filter paper wettened with sterile water) in the dark at 23°C. After 5 days, brown lesions and white mycelium developed on the surface of the treated cloves. The taxonomic status of the fungus isolated from necrotic tissue was determined as F. proliferatum according to the ITS, EF1α, RPB1 and RPB2 analysis. Garlic basal and bulb rot is known to be caused by F. oxysporum f. sp. cepae and F. proliferatum (Snowdon 1990). This study is the first report of F. proliferatum causing rot of garlic bulbs during storage in Russia. F. proliferatum produces a variety of mycotoxins during bulb infestation, and our findings are important for diagnosing a Fusarium disease and the use of garlic crop in culinary and medicine. Funding The reported study was funded by Russian Foundation for Basic Research, project number 20-316-70009. References: Leslie, J. F., and Summerell, B. A. 2006. Page 224 in: The Fusarium Laboratory Manual. Blackwell, Oxford, UK. https://doi.org/10.1002/9780470278376 Leyronas, C., et al. 2018. Plant Dis. 102:2658 https://doi.org/10.1094/PDIS-06-18-0962-PDN Liu, Y.J. et al. 1999. Mol. Biol. Evol. 16: 1799 https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K, et al. 1998a. Proc Natl Acad Sci USA. 95(5):2044. https://doi.org/10.1073/pnas.95.5.2044. O'Donnell, et al. 1998b. Mycologia 90:465 O'Donnell, K., et al. 2010. J. Clin. Microbiol., 48: 3708 https://doi.org/10.1128/JCM.00989-10 Snowdon, A. L. Pages 250-252 in: A Color Atlas of Post-Harvest Diseases and Disorders of Fruits and Vegetables. Vol. 1. 1990. Wolfe Scientific, London. Taylor, A, et al. 2013. Plant Pathol. 62:103. https://doi.org/10.1111/j.1365-3059.2012.02624.x White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells.

Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.

Rokubacteria in Northern Peatlands: Habitat Preferences and Diversity Patterns.

Rokubacteria is a phylogenetic clade of as-yet-uncultivated prokaryotes, which are detected in diverse terrestrial habitats and are commonly addressed as members of the rare biosphere. This clade was originally described as a candidate phylum; however, based on the results of comparative genome analysis, was later defined as the order-level lineage, Rokubacteriales, within the phylum Methylomirabilota. The physiology and lifestyles of these bacteria are poorly understood. A dataset of 16S rRNA gene reads retrieved from four boreal raised bogs and six eutrophic fens was examined for the presence of the Rokubacteriales; the latter were detected exclusively in fens. Their relative abundance varied between 0.2 and 4% of all bacteria and was positively correlated with pH, total nitrogen content, and availability of Ca and Mg. To test an earlier published hypothesis regarding the presence of methanotrophic capabilities in Rokubacteria, peat samples were incubated with 10% methane for four weeks. No response to methane availability was detected for the Rokubacteriales, while clear a increase in relative abundance was observed for the conventional Methylococcales methanotrophs. The search for methane monooxygenase encoding genes in 60 currently available Rokubacteriales metagenomes yielded negative results, although copper-containing monooxygenases were encoded by some members of this order. This study suggests that peat-inhabiting Rokubacteriales are neutrophilic non-methanotrophic bacteria that colonize nitrogen-rich wetlands.

Methane-Oxidizing Communities in Lichen-Dominated Forested Tundra Are Composed Exclusively of High-Affinity USCα Methanotrophs.

Upland soils of tundra function as a constant sink for atmospheric CH4 but the identity of methane oxidizers in these soils remains poorly understood. Methane uptake rates of -0.4 to -0.6 mg CH4-C m-2 day-1 were determined by the static chamber method in a mildly acidic upland soil of the lichen-dominated forested tundra, North Siberia, Russia. The maximal CH4 oxidation activity was localized in an organic surface soil layer underlying the lichen cover. Molecular identification of methanotrophic bacteria based on retrieval of the pmoA gene revealed Upland Soil Cluster Alpha (USCα) as the only detectable methanotroph group. Quantification of these pmoA gene fragments by means of specific qPCR assay detected ~107pmoA gene copies g-1 dry soil. The pmoA diversity was represented by seven closely related phylotypes; the most abundant phylotype displayed 97.5% identity to pmoA of Candidatus Methyloaffinis lahnbergensis. Further analysis of prokaryote diversity in this soil did not reveal 16S rRNA gene fragments from well-studied methanotrophs of the order Methylococcales and the family Methylocystaceae. The largest group of reads (~4% of all bacterial 16S rRNA gene fragments) that could potentially belong to methanotrophs was classified as uncultivated Beijerinckiaceae bacteria. These reads displayed 96-100 and 95-98% sequence similarity to 16S rRNA gene of Candidatus Methyloaffinis lahnbergensis and "Methylocapsa gorgona" MG08, respectively, and were represented by eight species-level operational taxonomic units (OTUs), two of which were highly abundant. These identification results characterize subarctic upland soils, which are exposed to atmospheric methane concentrations only, as a unique habitat colonized mostly by USCα methanotrophs.

Osmolytes and membrane lipids in the adaptation of micromycete Emericellopsis alkalina to ambient pH and sodium chloride.

The accumulation of low molecular weight cytoprotective compounds (osmolytes) and changes in the membrane lipids composition are of key importance for the adaptation to stress impacts. However, the reason behind the wide variety of osmolytes present in the cell remains unclear. We suggest that specific functions of osmolytes can be revealed by studying the adaptation mechanisms of the mycelial fungus Emericellopsis alkalina (Hypocreales, Ascomycota) that is resistant to both alkaline pH values and high sodium chloride concentrations. It has been established that the fungus uses different osmolytes to adapt to ambient pH and NaCl concentration. Arabitol was predominant osmolyte in alkaline conditions, while mannitol prevailed in acidic conditions. On the salt-free medium mannitol was the main osmolyte; under optimal conditions (pH 10.2; 0.4 M NaCl) arabitol and mannitol were both predominant. Higher NaCl concentrations (1.0-1.5 M) resulted in the accumulation of low molecular weight polyol - erythritol, which amounted up to 12-14%, w/w. On the contrary, changes in the composition of membrane lipids were limited under pH and NaCl impacts; only higher NaCl concentrations led to the increase in the degree of unsaturation of membrane lipids. Results obtained indicated the key role of the osmolytes in the adaptation to the ambient pH and osmotic impacts.

Osmolytes and membrane lipids in adaptive response of thermophilic fungus Rhizomucor miehei to cold, osmotic and oxidative shocks.

In contrast to mesophiles, in which levels of trehalose and phosphatidic acids (PA) increased only under heat shock (HS), in thermophiles trehalose and PA were predominant under optimal growth conditions. To study the role of trehalose protection in the adaptation of thermophiles to various stressors, the composition of osmolytes and membrane lipids in the thermophilic fungus Rhizomucor miehei was studied under cold (CS), osmotic (OS) and oxidative (OxS) shocks. CS resulted in no accumulation of glycerol in the mycelium, while the amount of trehalose decreased. The main lipid changes were the increase in the PA proportion with simultaneous decrease of sterols (St), the increase of the unsaturation degree of polar lipids and the decrease of the ergosterol proportion in total St. OS did not cause changes in the lipid composition, but led to the decrease of ergosterol proportion too. Despite the low ability of Mucorales to produce polyols, increase in the level of arabitol and glycerol was observed under OS. OxS led to the decrease of trehalose level and had no effect on the lipid composition. Thus, our results show the similarity (OS) and the difference (CS and OxS) between adaptation mechanisms of thermophiles and mesophiles.