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The emergence and spread of azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern and endangers the "One Health" approach in our fight against antifungal resistance (AFR) in this pathogen. A major challenge to combat AFR in A. fumigatus is the massive aerial dispersal ability of its asexual spores. Our recent fine-scale survey of greenhouse populations of A. fumigatus near Kunming, Yunnan, China, suggested that the use of azole fungicides for plant protection was likely a major driver of the high-frequency azole-resistant A. fumigatus (ARAF) in greenhouses. Here, we investigated the potential spread of those ARAF and the structure of geographic populations of A. fumigatus by analyzing 452 isolates from 19 geographic locations across Yunnan. We found lower frequencies of ARAF in these outdoor populations than those in greenhouses near Kunming, but there were abundant new alleles and new genotypes, including those associated with azole resistance, consistent with multiple independent origins of ARAF across Yunnan. Interestingly, among the four ecological niches, the sediments of a large lake near Kunming were found to have the highest frequency of ARAF (~ 43%). While most genetic variations were observed within the 19 local populations, statistically significant genetic differentiations were found between many subpopulations within Yunnan. Furthermore, similar to greenhouse populations, these outdoor populations of A. fumigatus in Yunnan were significantly different from those in other parts of the world. Our results call for increased attention to local and regional studies of this fungal pathogen to help develop targeted control strategies against ARAF.

This research aims to find out how the application of S}araf in reading yellow book using qualitative descriptive research. The subjects of the study were lecturers of science s}araf, lecturers of Qiraah and Mut}alaáh as well as students of Arabic program at IAIN Bone. Data were collected through observation, interview and documentation techniques. Data analysis was carried out in three stages continuously including data reduction, data display and conclusion/verification withdrawal stage. The results shows that S}araf learning materials are divided into three stages; S}araf 1, S}araf2 and S}araf 3 by using various methods, including lecturing, discussions, demontation of bags }rif and tadrib. Tadrib/exercise was carried out with bag exercise }rif, practicing to make sentences, analyzing the function and form of words and giving harakat to a reading. The application of s}araf in reading the yellow book was began by giving harakat to a text to determine its meaning. S}araf, that has a study on the formation of the word is very instrumental in this case. Mastery of the bag will make it easier to read a reading, whether it has harakat or not. The understanding of an Arabic text is not limited to the mastery of any existing mufradat, but rather must pay attention to the form of the word used.

The arabinogalactan of Corynebacterianeae is a critical heteropolysaccharide that tethers outer membrane mycolic acids to peptidoglycan thus forming the characteristic cell wall core of these prokaryotes. An essential α-(1→5)-arabinosyltransferase, AftA, is responsible for the transfer of the first arabinofuranosyl (Araf) unit of the arabinan domain to the galactan backbone of arabinogalactan, but the number and precise position at which Araf residue(s) is/are added in mycobacteria remain ill-defined. Using membrane preparations from Mycobacterium smegmatis overexpressing aftA, farnesyl-phospho-arabinose as an Araf donor, and a series of synthetic galactan acceptors of various lengths, we here show that a single priming arabinosyl residue substitutes the C-5 position of a precisely positioned internal 6-linked galactofuranosyl residue of the galactan acceptors, irrespective of their length. This unexpected result suggests that, like the structurally related mycobacterial lipoarabinomannans, the arabinogalactan of mycobacteria may in fact harbor a single arabinan chain.

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. Experimental Design: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.

Increasing numbers of azole-resistant Aspergillus fumigatus (ARAf) in the environment have become a global public health issue. We surveyed tulip bulbs that were imported from the Netherlands and found that 6.3-15.8% of bulbs were contaminated by ARAf with a tandem-repeat mutation in the promoter region of the cyp51A gene. We also showed that fungicide treatment of the tulip bulbs by benomyl or prochloraz effectively reduced the rate of isolation. This is the first report demonstrating a method of eliminating human fungal pathogens from plant bulbs.

The mitogen‐activated protein kinase (MAPK) signaling pathway plays a significant role in mediating cellular physiological activities, such as proliferation, differentiation, apoptosis, and senescence. This signaling pathway is composed of several major proto‐oncogenes of RAS/RAF/MEK/ERK, among which the BRAF proto‐oncogene, as one of the three members of the RAF family, has a higher mutation rate than ARAF and CRAF and has attracted extensive attention. Regarding the BRAF mutation, approximately 95% of BRAF mutations belong to the BRAF V600E mutation, which can enhance the expression of the MAPK signaling pathway and is thus related to the occurrence and development of various malignant tumors and has been successfully identified as a therapeutic target. Moreover, drug resistance to BRAF inhibitor treatment also appears to be an important issue. Considering the successful use of BRAF inhibitors in melanoma, we provide a brief overview of the BRAF mutations, including their basic structures and activation mechanisms, and the new classification method for BRAF mutations. Most importantly, we summarize the results of BRAF inhibitor treatment in different sarcomas. To overcome drug resistance to BRAF inhibitor treatment, we also outline the different mechanisms of drug resistance to BRAF inhibitor treatment and introduce the combination strategy of BRAF inhibitors with other targeted therapies., BRAF structures and their activation mechanisms.

Urinary stone disease has afflicted humankind since antiquity[1]. It remains a common urological condition worldwide, including in our environment[2]. Although open surgery was previously the main option for stone removal, advances in technology mean that treatment is now largely through minimally invasive surgery, as recommended by a number of urological guidelines[3,4]. Unfortunately, at our centre, we still treat urinary stones solely through open surgery, mostly because we lack endoscopic equipment. In addition, most of our patients, even if referred to facilities where endoscopic management is possible, cannot afford the cost of treatment.

Azole-treated plant bulbs have already been evoked as a potential explanation of the worldwide spread of azole-resistant Aspergillus fumigatus (ARAf). We previously pointed out the presence of a high rate of ARAf (71% of A. fumigatus detected on azole-supplemented media) in flower beds containing azole-treated bulbs at the hospital's surroundings. We show here that planting organic bulbs can be a solution to reduce ARAf burden (from 71% rate to below 3%). The results suggest that replacing treated bulbs with organic bulbs may be sufficient to regain a population that is predominantly susceptible in just 1 year. Lay Summary Antifungal resistance is increasingly observed in fungal pathogens. This study argues that planting organic bulbs in hospitals’ outdoor surroundings could be a good alternative to continue to beautify green spaces, without the risk of dissipating antifungal-resistant fungal pathogens.

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 μM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 ± 1.05 µM and 0.18 ± 0.01 µM, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked α-GalpA, 1, 4-linked α-GalpAOMe and 1, 2, 4-linked α-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked β-Galp, terminal (T)-linked β-Galp, 1, 5-linked α-Araf, T-linked α-Araf, 4-linked α-GalpA and/or 4-linked β-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

Heterosis is a common biological phenomenon that can be used to optimize yield and quality of crops. Using heterosis breeding, hybrids with suitable nicotine content have been applied to tobacco leaf production. However, the molecular mechanism of the formation of nicotine heterosis has never been explained from the perspective of protein. The DIA proteomics technique was used to compare the differential proteomics of the hybrid Va116 × Basma, showing strong heterosis in nicotine content from its parent lines Va116 and Basma. Proteomics analysis indicated that 65.2% of DEPs showed over-dominant expression patterns, and these DEPs included QS, BBL, GS, ARAF and RFC1 which related to nicotine synthesis. In addition, some DEPs (including GST, ABCE2 and ABCF1 and SLY1) that may be associated with nicotinic transport exhibited significant heterosis over the parental lines. These findings demonstrated that the efficiency of the synthesis and transport of nicotine in hybrids was significantly higher than that in the parent lines, and the accumulation of over-dominant expression proteins may be the cause of heterosis of nicotinic content in hybrids.

Purpose As a subgroup of lung cancer, small cell lung cancer (SCLC) is characterized by a short tumor doubling time, high rates of early occurred distant cancer spread, and poor outcomes. Despite its exquisite sensitivity to chemotherapy and radiotherapy, acquired drug resistance and tumor progression are typical. This study aimed to develop a robust signature based on immune‐related genes to predict the outcome of patients with SCLC. Methods The expression data of 77 SCLC patients from George's cohort were divided into training set and testing set, and 1534 immune‐related genes from ImmPort database were used to generate and validate the signature. Cox proportional hazards and the Kaplan–Meier analysis were used for developing and testing the prognostic signature. Single‐sample gene set enrichment analysis was used to determine immune cell infiltration phenotypes. Results A 10‐gene model comprising NR3C1, NR1D2, TANK, ARAF, HDGF, INHBE, LRSAM1, PLXNA1, PML, and SP1 with the highest frequency after 1000 interactions, was chosen to construct immune‐related signature. This signature showed robust predictive value for SCLC patients’ survival in both training and testing sets. This signature was weakly associated with the clinic pathological values like TNM stage. Furthermore, patients with low risk presented with activation of immune signal pathways, and specific immune cell infiltration with high levels of CD56bright NK cells but low levels of CD8+ T cells, mast cells, and helper T cells. Conclusion The present study developed immune‐related signature that may help predict the prognosis of SCLC patients, which reflects an unappreciated level of heterogeneity of immunophenotype associated with diverse prognosis for specific subsets in this highly lethal cancer type., Here, we constructed a prognostic immune‐related signature for predicting small cell lung cancer (SCLC) patients’ survival. The 10‐gene signature was enriched in growth factor activity and immune‐related TGF‐β signaling pathway. Furthermore, increased CD56dim NK cells and reduced plasmacytoid dendritic cells infiltration were significantly associated with survival prolongment. However, SCLC patients with low risk presents more CD56bright NK cells but less CD8+ T cells, mast cells, and helper T cells infiltration compared to those with high risk. Our findings indicate that this immune‐related signature may help predict the prognosis of SCLC patients, which reflects an unappreciated level of heterogeneity of immunophenotype associated with diverse prognosis for specific subsets in this highly lethal cancer type.

We experienced two cases of postoperative iatrogenic aorto-right atrial fistula (ARAF) after tricuspid valve repair (TVR) using minimally invasive cardiac surgery (MICS) technique. In both the cases, the flow of ARAF passed through the sinus of Valsalva near the noncoronary cusp (NCC)/right coronary cusp (RCC) commissure or NCC to right atrium. The quality of the fine needle used in the MICS technique may be inferior to that used in conventional surgery; ARAF after TVR could be a unique pitfall with the MICS technique.

Background/aim Prostate cancer (PCa) is the second-most commonly occurring cancer among men, worldwide. Although the mechanisms associated with the progression of castration-resistant prostate cancer (CRPC) have been widely studied, the mechanism associated with more distant metastases from the bone remains unknown. This study aimed to characterize potential pathogenic kinases associated with highly metastatic PCa, that may regulate phosphorylation in extensively involved and diverse signaling pathways that are associated with the development of various cancers. Materials and methods A mass spectrometry (MS)-based comparative phosphoproteome strategy was utilized to identify differentially expressed kinases between the highly aggressive PCa cell-lines PC-3 and PC-3M. Results Among 2,968 phosphorylation sites in PCa cells, 151 differently expressed phosphoproteins were identified. Seven motifs: -SP-, -SxxE-, -PxS-, -PxSP-, -SxxK-, -SPxK-, and -SxxxxxP- were found to be highly expressed in PC-3M cells. Based on these motifs, the kinases p21-activated kinase (PAK)2, Ste20-like kinase (SLK), mammalian Ste20-like kinase (MST)4, mitogen-activated kinase kinase (MAP2K)2, and A-Raf proto-oncogene serine/threonine kinase (ARAF) were up-regulated in PC-3M cells. Conclusion PAK2, SLK, MST4, MAP2K2, and ARAF are kinases that are potentially associated with the progression of increased migration in PC-3M cells and may represent molecule regulators or drug targets for highly metastatic PCa therapy.

TFF3 has been identified as a novel biomarker to distinguish between lung adenocarcinoma (ADC) and lung squamous-cell carcinoma (SCC). Herein, we determined the oncogenic functions of TFF3 and demonstrated the potential of pharmacological inhibition of TFF3 in lung ADC using a novel small-molecule inhibitor of TFF3 dimerization (AMPC). Forced expression of TFF3 in lung ADC cells enhanced cell proliferation and survival, increased anchorage-independent growth, cancer stem cell behavior, growth in 3D Matrigel, and cell migration and invasion. In contrast, depleted expression of TFF3 suppressed these cellular functions. Mechanistically, TFF3 exerted its oncogenic function through upregulation of ARAF and hence enhanced downstream activation of MEK1/2 and ERK1/2. Pharmacological inhibition of TFF3 by AMPC, resulted in markedly decreased cell survival, proliferation, 3D growth and foci formation, and impaired tumor growth in a xenograft mouse model. Moreover, the combination of various MEK1/2 inhibitors with AMPC exhibited synergistic inhibitory effects on lung ADC cell growth. In conclusion, this study provides the first evidence that TFF3 is a potent promoter of lung ADC progression. Targeting TFF3 with a novel small-molecule inhibitor alone or in combination with conventional MEK1/2 inhibitors are potential strategies to improve the outcome of lung ADC.

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 μg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 μg/mL/>51.4 and 80.5 μg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 μg/mL. Synthesis of viral protein showed a dose–dependent response and the nucleic acid synthesis was inhibited up to 25 μg/mL, by P1 and P1S and up to 50 μg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in the domestic environment of patients at risk for aspergillosis. Here, we assessed the mother's and father's homes of an 18-year-old cystic fibrosis patient harbouring chronic colonisation with H285Y CYP51A azole-resistant isolate, in order to explore the link between environmental exposure and ARAF infection. In one dwelling, a very high overall contamination level was found (710–7.240 CFU/m3), with a predominance of A. fumigatus (640–6.490 CFU/m3), and ARAF showing the TR34/L98H mutation was isolated. Mycological follow-up of the patient showed the persistence of H285Y isolates, but no acquisition of TR34/L98H isolates was observed. This could be due to the low proportion of TR34/L98H isolates (

A novel acidic polysaccharide fraction (PZMP2-2) was isolated from Ziziphus Jujuba cv. Muzao fruit via hot water extraction and purified by DEAE-Sepharose FF and Sephacryl S-300 column chromatography. PZMP2-2 consisted of rhamnose, arabinose, xylose, galactose, and galacturonic acid in a ratio of 1.18: 5.23: 0.22: 2.68: 2.20, with a molecular weight of 62.73 kDa. Structural features of PZMP2-2 were investigated using FT-IR, SEM, AFM, GC–MS analysis and NMR spectroscopy. Methylation and GC–MS analysis revealed that the main glycosidic bonds in PZMP2-2 comprised 1-linked Galp, 1,3-linked Araf, 1,2,4-linked Rhap, 1,3-linked Galp, 1,4-linked GalpA and 1,3,5-linked Araf. NMR analysis indicate that PZMP 2–2 has a linear backbone of (1 → 4)-linked GlcpA and (1 → 2,4)-linked Rhap residues, with branches at the O-4 position, consisting of Araf and Galp residues.

A novel polysaccharide DG2 was obtained from the Dendrobium officinale endophytic fungus Fusarium solani DO7. The structural characterization and immunological activity of DG2 were investigated. Results showed that DG2 with the molecular weight of 152.8 kDa was composed of Galp, Manp, Glcp and Araf at a molar ratio of 4.3:1.9:2.8: 1.0. The main linkage types of DG2 were (1 → )-β-D-Glc, (1 → 4)-β-D-Glc, (1 → 3,6)-β-D-Gal and (1 → 6)-β-D-Gal, which demonstrated DG2 was a novel galactoglucan and different from other fungal and botanic polysaccharide. Furthermore, DG2 did not contain deoxynivalenol, and showed no direct toxicity to human embryonic kidney cells and RAW 264.7 cells. Meanwhile, DG2 could increase the TNF-α, IL-6 and iNOs mRNA expression levels of RAW264.7 cells, and further promote the production of TNF-α, IL-6 and NO. These results suggested DG2 could enhance macrophage-mediated immune responses and have the potential to be an attractive functional food supplement candidate for the hypoimmunity therapies.

International audience; The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and β-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-β-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-β-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.

Valorization of argi-waste polymers into value-added materials is essential for sustainable development of polymeric industry. Reported herein is a 1-step and 2-step strategy for fabrication of flexible and stretchable thermoplastics prepared by compression molding from two structurally different arabinoxylans (AX). The synthesis was accomplished using n-butyl glycidyl ether whose epoxide ring opened on hydroxyl group and resulted in introduction of alkoxide sidechains for the 1-step synthesis. AX was preactivated by periodate oxidation as 1st step for the 2-step synthesis. Two structurally different AXs, i.e. wheat bran extracted arabinoxylan (AXWB, araf/xylp=3/4) and barley husk extracted arabinoxylan (AXBH, araf/xylp=1/4) were used to understand the effects of the araf/xylp on thermoplastic properties because melt processability has been rare for low araf/xylp AXs. AXBH-derived samples demonstrated melt compression processability. AXWB and AXBH derived thermoplastics featured dual and single glass transition (Tg) characteristics respectively as confirmed by DSC and DMA, but AXBH derived thermoplastics had lower stretchability (maximum 160%) compared to AXWB samples (maximum 300 %). Higher araf/xylp and thus in turn longer alkoxide side chains in AXWB derived thermoplastics explained differences in stretchability.

Despite the frequent detection of KRAS driver mutations in patients with colorectal cancer (CRC), no effective treatments that target mutant KRAS proteins have been introduced into clinical practice. In this study, we identified potential effector molecules, based on differences in gene expression between CRC patients carrying wild-type KRAS (n = 390) and those carrying KRAS mutations in codon 12 (n = 240). CRC patients with wild-type KRAS harboring mutations in HRAS, NRAS, PIK3CA, PIK3CD, PIK3CG, RALGDS, BRAF, or ARAF were excluded from the analysis. At least 11 promising candidate molecules showed greater than two-fold change between the KRAS G12 mutant and wild-type and had a Benjamini-Hochberg-adjusted P value of less than 1E-08, evidence of significantly differential expression between these two groups. Among these 11 genes examined in cell lines transfected with KRAS G12 mutants, BMP4, PHLDA1, and GJB5 showed significantly higher expression level in KRAS G12A, G12D, and G12V transfected cells than in the wild-type transfected cells. We expect that this study will lead to the development of novel treatments that target signaling molecules functioning with KRAS G12-driven CRC.

Background: Wood chipping has been described as a potential hotspot for the selection of azole-resistant Aspergillus fumigatus (ARAf). We previously reported ARAf isolates in sawmills (Eastern France), most of which contained the TR34/L98H mutation. Methods: To study genotypic relatedness, microsatellite genotyping (short tandem repeat for A. fumigatus (STRAf)) was performed on 41 azole-susceptible A. fumigatus (ASAf) and 23 ARAf isolated from 18 sawmills and two clinical A. fumigatus (sensitive and resistant) isolated from a sinus sample of a woodworker. Results: Fifty-four unique multilocus genotypes (MLGs) were described among the 66 isolates: 13/24 ARAf and 41/42 ASAf. Allelic diversity was higher for ASAf than for ARAf. Among the 24 ARAf, five isolates had their own MLGs. Thirteen ARAf (54%) belonged to the same group, composed of four close MLGs, defined using Bruvo&rsquo, s distance. Thirty-two of the 42 ASAf (76%) had their own MLGs and could not be grouped with the Bruvo&rsquo, s distance cutoff used (0.2). Conclusion: Thus, at a regional scale and in the particular environment of the wood industry, common but also different distinct genotypes, even in the same sawmill, were identified. This suggests that the hypothesis of ARAf clonal expansion from a common strain is probably insufficient to explain genotype emergence and distribution.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1–3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors. The development, characterization and phase I clinical testing of the RAF inhibitor belvarafenib in cancer and a new resistance mechanism mediated by ARAF mutations are described.

Kinases form the major part of the druggable genome and their selective inhibition in human cancers has had reasonable clinical success. In contrast to tumorigenesis, the role of kinases in mediating immune responses is poorly understood. However, synergistic therapeutic regimens combining targeted therapy and immune therapy have been found to increase the median survival of tumor patients. In this context, we uncovered that RAF and MEK1/2 kinases, which are the integral parts of the classical MAPK cascade, have unique roles in driving DC differentiation and activation. RAF kinases are stabilized in their protein levels during DC differentiation and are obligatory for normal functioning of DCs. But, the targeting of MEK1/2 kinases with specific inhibitors did not phenocopy the effects observed with RAF inhibitors suggesting that RAF and MEK1/2 kinases may have specific and unique roles in driving immune responses, which deserves further studies to successfully administer these inhibitors in clinics.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) among chronic pulmonary aspergillosis (CPA) patients treated with azoles in Japan is unknown. The aim of this study was to determine the detection rate of ARAF in isolates from CPA patients who were treated with azoles for varying durations. The potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104) between February 2012 and February 2019, at National Hospital Organization Tokyo National Hospital. The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF among all isolates was 8.3% (n = 10). Of the 10 resistant isolates, eight were ITCZ-resistant and five were VRCZ-resistant. Among 47 isolates obtained from 36 CPA patients who were treated with ITCZ (for an average of 256 days) and/or VRCZ (for an average of 29 days), the resistance rates were 17.0% and 10.6%, respectively. In addition, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients, particularly in those with ongoing long-term azole treatment, at the time of azole antifungal therapy failure.Aspergillus fumigatus can acquire azole resistance during long-term treatment with azole drugs in patients with chronic pulmonary aspergillosis (CPA). The aim of this study was to determine the detection rate of azole-resistant A. fumigatus (ARAF) in isolates from CPA patients who had been treated with azoles. In addition, a potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104). The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF from all isolates was 8.3% (n = 10). Greater than 10% of the 47 isolates obtained from 36 CPA patients who had been treated with azoles exhibited resistance. Furthermore, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients undergoing long-term azole treatment at the time of antifungal therapy failure.

PURPOSE OF REVIEW: This review summarizes current treatment options for echinocandin-resistant Candida spp. (ERC) and azole-resistant Aspergillus fumigatus (ARAF), emphasizing recent in vitro/in vivo data, clinical reports, and consensus statements. RECENT FINDINGS: Advances in ERC and ARAF treatment are limited to specific antifungal combinations and dose optimization but remain reliant on amphotericin products. Although novel antifungals may provide breakthroughs in the treatment of resistant fungi, these agents are not yet available. Early identification and appropriate treatment remain a paramount, albeit elusive, task. SUMMARY: When either ERC or ARAF are suspected or proven, amphotericin products remain the cornerstone of initial therapy. For ERC, azoles are de-escalation options for susceptible isolates in stable patients to avoid amphotericin toxicities. Although combination echinocandin with high-dose salvage posaconazole or isavuconazole may be attempted in ARAF, it requires careful consideration following patient stabilization. Future research defining optimal therapies and early identification of ERC and ARAF is of extreme importance.

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

Aspergillus fumigatus is a ubiquitous saprophytic fungus. Inhalation of A. fumigatus spores can lead to Invasive Aspergillosis (IA) in people with weakened immune systems. The use of triazole antifungals with the demethylation inhibitor (DMI) mode of action to treat IA is being hampered by the spread of DMI-resistant “ARAf” (azole-resistant Aspergillus fumigatus) genotypes. DMIs are also used in the environment, for example, as fungicides to protect yield and quality in agronomic settings, which may lead to exposure of A. fumigatus to DMI residues. An agronomic setting can be a “hotspot” for ARAf if it provides a suitable substrate and favourable conditions for the growth of A. fumigatus in the presence of DMI fungicides at concentrations capable of selecting ARAf genotypes at the expense of the susceptible wild-type, followed by the release of predominantly resistant spores. Agronomic settings that do not provide these conditions are considered “coldspots". Identifying and mitigating hotspots will be key to securing the agronomic use of DMIs without compromising their use in medicine. We provide a review of studies of the prevalence of ARAf in various agronomic settings and discuss the mitigation options for confirmed hotspots, particularly those relating to the management of crop waste.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities., National Cancer Institute (U.S.) (Grant R01CA180006)

// Jun Gong 1 , May Cho 1 , Marvin Sy 1 , Ravi Salgia 1 and Marwan Fakih 1 1 Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA, USA Correspondence to: Marwan Fakih, email: mfakih@coh.org Keywords: metastatic colorectal cancer, comprehensive genomic profiling, next-generation sequencing, FoundationOne, retrospective Received: September 06, 2016 Accepted: January 16, 2017 Published: February 02, 2017 ABSTRACT Background: Recent molecular characterization of colorectal tumors has identified several molecular alterations of interest that are considered targetable in metastatic colorectal cancer (mCRC). Methods: We conducted a single-institution, retrospective study based on comprehensive genomic profiling of tumors from 138 patients with mCRC using next-generation sequencing (NGS) via FoundationOne. Results: Overall, RAS mutations were present in 51.4% and RAF mutations were seen in 7.2% of mCRC patients. We found a novel KRAS R68S1 mutation associated with an aggressive phenotype. RAS amplifications (1.4% KRAS and 0.7% NRAS ), MET amplifications (2.2%), BRAF L597R alterations (0.7%), ARAF S214F alterations (0.7%), and concurrent RAS + RAF (1.4%), BRAF + RAF1 (0.7%), and rare PTEN-PIK3CA-AKT pathway mutations were identified and predominantly associated with poor prognosis. ERBB2 ( HER2 ) amplified tumors were identified in 5.1% and all arose from the rectosigmoid colon. Three cases (2.2%) were associated with a hypermutated profile that was corroborated with findings of high tumor mutational burden (TMB): 2 cases with MSI-H and 1 case with a POLE mutation. Conclusions: Comprehensive genomic profiling can uncover alterations beyond the well-characterized RAS / RAF mutations associated with anti-EGFR resistance. ERBB2 amplified tumors commonly originate from the rectosigmoid colon, are predominantly RAS / BRAF wild-type, and may predict benefit to HER2-directed therapy. Hypermutant tumors or tumors with high TMB correlate with MSI-H status or POLE mutations and may predict a benefit from anti-PD-1 therapy.

To understand the characterizations of azole resistance in Aspergillus fumigatus from potting soil samples in the hospital, a total of 58 samples were collected. Among 106 A. fumigatus samples obtained, 5 isolates from 4 soil samples located in the gerontology department were identified as azole-resistant A. fumigatus (ARAF). Four ARAF isolates harbored the TR(34)/L98H allele, and the other one had no mutation in the cyp51A gene. Among 174 A. fumigatus samples selected for genotyping, TRESPERG typing obtained a close discriminatory power (Simpson’s index of diversity [DI], 0.9941; 95% confidence interval [CI], 0.9913 to 0.9968) compared with the short tandem repeat (STR) typing (DI, 0.9997; 95% CI, 0.9976 to 1.0000). Genotyping showed that the TR(34)/L98H isolates in the hospital had a close genetic relationship with ARAF isolates from China and many other countries. In conclusion, this study indicated the presence of ARAF in potting soil samples from the hospital, which might pose a risk of causing ARAF infection in patients.

Aspergillus fumigatus is the primary agent of invasive aspergillosis (IA) causing high morbidity and mortality in immunocompromised patients. Triazole resistance in A. fumigatus and its sources have gained wide attention. For several years, environmental fungicides use has been proposed as the major cause for triazole resistance in A. fumigatus. However, there are few studies on azole-resistant A. fumigatus (ARAF) selected by triazole fungicides in agricultural systems. We studied the possible emergence of ARAF in the field after exposure to triazole fungicide tebuconazole. Our results showed that exposure to tebuconazole in soil selects for resistance to triazoles in A. fumigatus. The probability of ARAF developing in soils depends upon the concentrations of tebuconazole after application. We suggest that tebuconazole applications should be minimized to reduce selective pressure for the generation of ARAFs.