The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus.

Karyotyping in Agaricus bisporus is crucial for both the isolation of homokaryotic strains and the confirmation of dikaryon establishment. For the verification of the karyotype, the A mating type loci of two homokaryotic strains, H39 and H97, were analyzed through comparative sequence analysis. The two loci showed major differences in two sequence regions designated as Region 1 and Region 2. H97 had a putative DNA transposon in Region 1 that had target site duplications (TSDs), terminal inverted repeats (TIRs), and a loop sequence, in contrast to H39, which only had the insertional target sequence. Homologous sequences of the transposon were discovered in the two different chromosomes of H97 and in one of H39, all of which have different TSDs but share high sequence homology in TIR. Region 2 shared three consensus sequences between H97 and H39. However, it was only from H97 that a large insertional sequence of unknown origin was discovered between the first and second consensus sequences. The difference in length in Region 1, employed for the verification of the A mating type, resulted in the successful verification of mating types in the heterokaryotic and homokaryotic strains. This length difference enables the discrimination between homo- and heterokaryotic spores by PCR. The present study suggests that the A mating type locus in A. bisporus H97 has evolved through transposon insertion, allowing the discrimination of the mating type, and thus the nuclear type, between A. bisporus H97 and H39.

Comparative structural analysis on the mitochondrial DNAs from various strains of Lentinula edodes.

The evolution of mitochondria through variations in mitochondrial DNA (mtDNA) is one of the intriguing questions in eukaryotic cells. In order to assess the causes of the variations in mitochondria, the mtDNAs of the 21 strains of Lentinula edodes were assembled for this study, and analyzed together with four published mtDNA sequences. The mtDNAs were within the sizes of 117 kb ~ 122 kb. The gene number was observed consistent except for two mtDNAs, which carry a duplicated trnG1-trnG2 unit or a putative gene deletion. The size variation was largely attributed to the number of introns, repeated sequences, transposable elements (TEs), and plasmid-related sequences. Intron loss and gain were found from cox1, rnl, and rns of three mtDNAs. Loss of two introns in cox1 of KY217797.1 reduced its size by 2.7 kb, making it the smallest cox1 gene (8.4 kb) among the cox1s of the 25 mtDNAs, whereas gain of a Group II intron (2.65 kb) and loss of a Group I intron (1.7 kb) in cox1 of MF774813.1 resulted in the longest cox1 (12 kb). In rnl of L. edodes, we discovered four intron insertion consensus sequences which were unique to basidiomycetes but not ascomycetes. Differential incorporation of introns was the primary cause of the rnl size polymorphism. Homing endonucleases (HEGs) were suggestively involved in the mobilization of the introns because all of the introns have HEG genes of the LAGRIDADG or GIY-YIG families with the conserved HEG cleavage sites. TEs contributed to 11.04% of the mtDNA size in average, of which 7.08% was LTR-retrotransposon and 3.96% was DNA transposon, whereas the repeated sequences covered 4.6% of the mtDNA. The repeat numbers were variable in a strain-dependent manner. Both the TEs and repeated sequences were mostly found in the intronic and intergenic regions. Lastly, two major deletions were found in the plasmid-related sequence regions (pol2-pol3 and pol1-atp8) in the five mtDNAs. Particularly, the 6.8 kb-long deletion at pol2-pol3 region made MF774813.1 the shortest mtDNA of all. Our results demonstrate that mtDNA is a dynamic molecule that persistently evolves over a short period of time by insertion/deletion and repetition of DNA segments at the strain level.

Mitochondrial Effects on the Physiological Characteristics of Lentinula edodes.

In the mating of filamentous basidiomycetes, dikaryotic mycelia are generated through the reciprocal movement of nuclei to a monokaryotic cytoplasm where a nucleus of compatible mating type resides, resulting in the establishment of two different dikaryotic strains having the same nuclei but different mitochondria. To better understand the role of mitochondria in mushrooms, we created four sets of dikaryotic strains of Lentinula edodes, including B2 × E13 (B2 side) and B2 × E13 (E13 side), B5 × E13 (B5 side) and B5 × E13 (E13 side), E8 × H3 (E8 side) and E8 × H3 (H3 side), and K3 × H3 (K3 side) and K3 × H3 (H3 side). The karyotypes and mitochondrial types of the dikaryotic strains were successfully identified by the A mating type markers and the mitochondrial variable length tandem repeat markers, respectively. Comparative analyses of the dikaryotic strains on the mycelial growth, substrate browning, fruiting characteristics, and mitochondrial gene expression revealed that certain mitochondria are more effective in the mycelial growth and the production of fruiting body, possibly through the activated energy metabolism. Our findings indicate that mitochondria affect the physiology of dikaryotic strains having the same nuclear information and therefore a selection strategy aimed at mitochondrial function is needed in the development of new mushroom strain.

Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation.

Mushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma, and Formitella were explored in terms of mycelial growth characteristics for the application of mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials. Among the 64 strains of Polyporales, G. lucidum LBS5496GL was selected as the best candidate because it showed fast mycelial growth with high mycelial strength in both the sawdust-based solid medium and the potato dextrose liquid plate medium. Some of the Polyporales in this study have shown good mycelial growth, however, they mostly formed mycelial mat of weak physical strength. The higher physical strength of mycelial mat by G. lucidum LBS5496GL was attributed to its thick hyphae with the diameter of 13 µm as revealed by scanning electron microscopic analysis whereas the hyphae of others exhibited less than 2 µm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL as a carbon and a nitrogen source, respectively.

Investigation of Mating Pheromone-Pheromone Receptor Specificity in Lentinula edodes.

The B mating-type locus of Lentinula edodes, a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (RCBs) and the mating pheromones (PHBs) in both the Bα and Bß subloci. The complexity of the B mating-type locus, five Bα subloci with five alleles of RCB1 and nine PHBs and three Bß subloci with 3 alleles of RCB2 and five PHBs, has led us to investigate the specificity of the PHB-RCB interaction because the interaction plays a key role in non-self-recognition. In this study, the specificities of PHBs to RCB1-2 and RCB1-4 from the Bα sublocus and RCB2-1 from the Bb sublocus were investigated using recombinant yeast strains generated by replacing STE2, an endogenous yeast mating pheromone receptor, with the L. edodesRCBs. Fourteen synthetic PHBs with C-terminal carboxymethylation but without farnesylation were added to the recombinant yeast cells and the PHB-RCB interaction was monitored by the expression of the FUS1 gene-a downstream gene of the yeast mating signal pathway. RCB1-2 (Bα2) was activated by PHB1 (4.3-fold) and PHB2 (2.1-fold) from the Bα1 sublocus and RCB1-4 (Bα4) was activated by PHB5 (3.0-fold) and PHB6 (2.7-fold) from the Bα2 sublocus and PHB13 (3.0-fold) from the Bα5 sublocus. In particular, PHB3 from Bß2 and PHB9 from Bß3 showed strong activation of RCB2-1 of the Bß1 sublocus by 59-fold. The RCB-PHB interactions were confirmed in the monokaryotic S1-10 strain of L. edodes by showing increased expression of clp1, a downstream gene of the mating signal pathway and the occurrence of clamp connections after the treatment of PHBs. These results indicate that a single PHB can interact with a non-self RCB in a sublocus-specific manner for the activation of the mating pheromone signal pathways in L. edodes.

Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.

BACKGROUND: In this first-in-human, Phase 1 study of a microRNA-based cancer therapy, the recommended Phase 2 dose (RP2D) of MRX34, a liposomal mimic of microRNA-34a (miR-34a), was determined and evaluated in patients with advanced solid tumours. METHODS: Adults with various solid tumours refractory to standard treatments were enrolled in 3 + 3 dose-escalation cohorts and, following RP2D determination, expansion cohorts. MRX34, with oral dexamethasone premedication, was given intravenously daily for 5 days in 3-week cycles. RESULTS: Common all-cause adverse events observed in 85 patients enrolled included fever (% all grade/G3: 72/4), chills (53/14), fatigue (51/9), back/neck pain (36/5), nausea (36/1) and dyspnoea (25/4). The RP2D was 70 mg/m2 for hepatocellular carcinoma (HCC) and 93 mg/m2 for non-HCC cancers. Pharmacodynamic results showed delivery of miR-34a to tumours, and dose-dependent modulation of target gene expression in white blood cells. Three patients had PRs and 16 had SD lasting ≥4 cycles (median, 19 weeks, range, 11-55). CONCLUSION: MRX34 treatment with dexamethasone premedication demonstrated a manageable toxicity profile in most patients and some clinical activity. Although the trial was closed early due to serious immune-mediated AEs that resulted in four patient deaths, dose-dependent modulation of relevant target genes provides proof-of-concept for miRNA-based cancer therapy. CLINICAL TRIAL REGISTRATION: NCT01829971.

Variable Number Tandem Repeats in the Mitochondrial DNA of Lentinula edodes.

Variable number tandem repeats (VNTRs) in mitochondrial DNA (mtDNA) of Lentinula edodes are of interest for their role in mtDNA variation and their application as genetic marker. Sequence analysis of three L. edodes mtDNAs revealed the presence of VNTRs of two categories. Type I VNTRs consist of two types of repeat units in a symmetric distribution, whereas Type II VNTRs contain tandemly arrayed repeats of 7- or 17-bp DNA sequences. The number of repeat units was variable depending on the mtDNA of different strains. Using the variations in VNTRs as a mitochondrial marker and the A mating type as a nuclear type marker, we demonstrated that one of the two nuclei in the donor dikaryon preferentially enters into the monokaryotic cytoplasm to establish a new dikaryon which still retains the mitochondria of the monokaryon in the individual mating. Interestingly, we found 6 VNTRs with newly added repeat units from the 22 mates, indicating that elongation of VNTRs occurs during replication of mtDNA. This, together with comparative analysis of the repeating pattern, enables us to propose a mechanistic model that explains the elongation of Type I VNTRs through reciprocal incorporation of basic repeat units, 5'-TCCCTTTAGGG-3' and its complementary sequence (5'-CCCTAAAGGGA-3').

Diversity of A mating type in Lentinula edodes and mating type preference in the cultivated strains.

Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5' region of HD2-intergenic region-5' region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.

Activation of the Mating Pheromone Response Pathway of Lentinula edodes by Synthetic Pheromones.

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of Lentinula edodes was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene (znf2) expression and by pseudoclamp formation in a monokaryotic strain S1-11 of L. edodes. Treatment with synthetic PHBs activated the expression of homeodomain genes (HDs) residing in the A mating type locus, and of A-regulated genes, including znf2, clp1, and priA, as well as genes in the B mating type locus, including pheromone (phb) and receptor (rcb) genes. The synthetic PHBs failed to discriminate self from non-self RCBs. PHBs of the B4 mating type (B4 PHBs) were able to activate the mating pheromone response pathway in both monokaryotic S1-11 and S1-13 strains, whose B mating types were B4 (self) and B12 (non-self), respectively. The same was true for B12 PHBs in the B4 (non-self) and B12 (self) mating types. The synthetic PHBs also promoted the mating of two monokaryotic strains carrying B4-common incompatible mating types (A5B4 × A1B4). However, the dikaryon generated by this process exhibited abnormally high content of hyphal branching and frequent clamp connections and, more importantly, was found to be genetically unstable due to overexpression of mating-related genes such as clp1. Although synthetic PHBs were unable to discriminate self from non-self RCBs, they showed a higher affinity for non-self RCBs, through which the mating pheromone response pathway in non-self cells may be preferentially activated.

Genetic incorporation of Nε-acetyllysine reveals a novel acetylation-sumoylation switch in yeast.

The lysine acetylation of proteins plays a key role in regulating protein functions, thereby controlling a wide range of cellular processes. Despite the prevalence and significance of lysine acetylation in eukaryotes, however, its systematic study has been challenged by the technical limitations of conventional approaches for selective lysine acetylation in vivo. Here, we report the in vivo study of lysine acetylation via the genetic incorporation of Nε-acetyllysine in yeast. We demonstrate that a newly discovered acetylation-sumoylation switch precisely controls the localization and cellular function of the yeast septin protein, Cdc11, during the cell cycle. This approach should facilitate the comprehensive in vivo study of lysine acetylation across a wide range of proteins in eukaryotic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of Lentinula edodes.

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.

Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors.

Purpose Naturally occurring tumor suppressor microRNA-34a (miR-34a) downregulates the expression of >30 oncogenes across multiple oncogenic pathways, as well as genes involved in tumor immune evasion, but is lost or under-expressed in many malignancies. This first-in-human, phase I study assessed the maximum tolerated dose (MTD), safety, pharmacokinetics, and clinical activity of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumors. Patients and Methods Adult patients with solid tumors refractory to standard treatment were enrolled in a standard 3 + 3 dose escalation trial. MRX34 was given intravenously twice weekly (BIW) for three weeks in 4-week cycles. Results Forty-seven patients with various solid tumors, including hepatocellular carcinoma (HCC; n = 14), were enrolled. Median age was 60 years, median prior therapies was 4 (range, 1-12), and most were Caucasian (68%) and male (57%). Most common adverse events (AEs) included fever (all grade %/G3%: 64/2), fatigue (57/13), back pain (57/11), nausea (49/2), diarrhea (40/11), anorexia (36/4), and vomiting (34/4). Laboratory abnormalities included lymphopenia (G3%/G4%: 23/9), neutropenia (13/11), thrombocytopenia (17/0), increased AST (19/4), hyperglycemia (13/2), and hyponatremia (19/2). Dexamethasone premedication was required to manage infusion-related AEs. The MTD for non-HCC patients was 110 mg/m2, with two patients experiencing dose-limiting toxicities of G3 hypoxia and enteritis at 124 mg/m2. The half-life was >24 h, and Cmax and AUC increased with increasing dose. One patient with HCC achieved a prolonged confirmed PR lasting 48 weeks, and four patients experienced SD lasting ≥4 cycles. Conclusion MRX34 treatment with dexamethasone premedication was associated with acceptable safety and showed evidence of antitumor activity in a subset of patients with refractory advanced solid tumors. The MTD for the BIW schedule was 110 mg/m2 for non-HCC and 93 mg/m2 for HCC patients. Additional dose schedules of MRX34 have been explored to improve tolerability.

Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca(2+)-independent, Mg(2+)-dependent DNase I activity.

HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0-8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells.

Differential Expression of Laccase Genes in Pleurotus ostreatus and Biochemical Characterization of Laccase Isozymes Produced in Pichia pastoris.

In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at 15℃ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and 5℃ higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and 50℃, which may reflect the physiological conditions at the primordiation stage.

Axitinib versus sorafenib as a second-line therapy in Asian patients with metastatic renal cell carcinoma: results from a randomized registrational study.

BACKGROUND: This registrational trial evaluated the efficacy, safety, and patient-reported outcomes of axitinib versus sorafenib as a second-line treatment in Asian patients with clear-cell metastatic renal cell carcinoma (mRCC). METHODS: In this open-label, multicenter study, previously treated Asian patients with clear-cell mRCC were stratified by Eastern Cooperative Oncology Group performance status and prior therapy and randomized in a 2:1 ratio to receive axitinib (5 mg twice daily) or sorafenib (400 mg twice daily). The primary end point was progression-free survival (PFS) assessed by a masked independent review committee. RESULTS: A total of 204 Asian patients received axitinib (n=135) or sorafenib (n=69). Median PFS (95% confidence interval [CI]) was 6.5 (4.7-9.1) months with axitinib versus 4.8 (3.0-6.5) months with sorafenib (hazard ratio, 0.731; 95% CI, 0.506-1.058; one-sided P=0.0531). The objective response rate (95% CI) was 23.7% (16.8%-31.8%) with axitinib versus 10.1% (4.2%-19.8%) with sorafenib. Common, grade ≥3, all-causality adverse events were hypertension (19.3%), weight decrease (5.2%), and proteinuria (5.2%) with axitinib and hypertension (8.7%) and palmar-plantar erythrodysesthesia (7.2%) with sorafenib. In a time-to-deterioration composite end point of death, progression, and worsening of Functional Assessment of Cancer Therapy Kidney Symptom Index score, patients treated with axitinib demonstrated a 17%-24% risk reduction compared with sorafenib-treated patients. CONCLUSION: Axitinib is clinically active and well tolerated in previously treated Asian patients with mRCC, consistent with the results from the global Phase III trial. These results establish axitinib as a second-line treatment option for Asian patients with mRCC.

Phase II study of axitinib with doublet chemotherapy in patients with advanced squamous non-small-cell lung cancer.

BACKGROUND: Axitinib is an orally active and potent tyrosine kinase inhibitor of vascular endothelial growth factor receptors 1, 2 and 3. This phase II study assessed the efficacy and safety of axitinib combined with cisplatin/gemcitabine in chemotherapy-naïve patients with advanced/metastatic (stage IIIB/IV) squamous non-small-cell lung cancer (NSCLC). METHODS: Axitinib (starting dose 5 mg twice daily [bid]; titrated up or down to 2-10 mg bid) was administered orally on a continuous schedule with cisplatin (80 mg/m(2) intravenously [i.v.] every 3 weeks) and gemcitabine (1,250 mg/m(2) i.v. on days 1 and 8 of each 3-week cycle), and was continued as monotherapy after completion of six cycles (maximum) of chemotherapy. The primary study endpoint was objective response rate, as defined by Response Evaluation Criteria in Solid Tumours. RESULTS: Of the 38 patients treated, one (2.6%) patient achieved a complete response and 14 (36.8%) patients had a partial response; nine (23.7%) patients showed stable disease and three (7.9%) patients had disease progression. Median progression-free survival was 6.2 months, and median overall survival was 14.2 months. The estimated probability of survival at 12 months and 24 months was 63.2% and 30.8%, respectively. The most frequent grade ≥3 toxicities were neutropaenia and hypertension (13.2% each). Three (7.9%) patients experienced haemoptysis, of which one case (2.6%) was fatal. CONCLUSIONS: Treatment with the combination of axitinib and cisplatin/gemcitabine demonstrated anti-tumour activity in patients with advanced/metastatic squamous NSCLC and the fatal haemoptysis rate was low. However, without a reference arm (cisplatin/gemcitabine alone), it is not conclusive whether the combination is better than chemotherapy alone. This study was registered at ClinicalTrials.gov, registration # NCT00735904, on August 13, 2008.

Current technologies and related issues for mushroom transformation.

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Isolation and Characterization of Monokaryotic Strains of Lentinula edodes Showing Higher Fruiting Rate and Better Fruiting Body Production.

The effects of monokaryotic strains on fruiting body formation of Lentinula edodes were examined through mating and cultivation of the mated dikaryotic mycelia in sawdust medium. To accomplish this, monokaryotic strains of L. edodes were isolated from basidiospores of the commercial dikaryotic strains, Chamaram (Cham) and Sanjo701 (SJ701). A total of 703 matings (538 self-matings and 165 outcrosses) were performed, which generated 133 self-mates and 84 outcross mates. The mating rate was 25% and 50% for self-mating and outcross, respectively. The bipolarity of the outcross indicated the multi-allelic nature of the mating type genes. The mating was only dependent on the A mating type locus, while the B locus showed no effect, implying that the B locus is multi-allelic. Next, 145 selected dikaryotic mates were cultivated in sawdust medium. The self-mated dikaryotic progenies showed 51.3% and 69.5% fruiting rates for Cham and SJ701, respectively, while the fruiting rate of the outcross mates was 63.2%. The dikaryotic mates generated by mating with one of the monokaryotic strains, including A20, B2, E1, and E3, showed good fruiting performance and tended to yield high fruiting body production, while many of the monokaryotic strains failed to form fruiting bodies. Overall, these findings suggest that certain monokaryotic strains have traits enabling better mating and fruiting.

Genotype Correlations With Blood Pressure and Efficacy From a Randomized Phase III Trial of Second-Line Axitinib Versus Sorafenib in Metastatic Renal Cell Carcinoma.

BACKGROUND: In the phase III axitinib second-line (AXIS) trial, axitinib significantly prolonged progression-free survival (PFS) versus sorafenib in patients with previously treated metastatic renal cell carcinoma (mRCC). Analyses of associations between germline single-nucleotide polymorphisms (SNPs) and outcomes are reported. PATIENTS AND METHODS: DNA samples from blood were genotyped using TaqMan allelic discrimination. Logistic/Cox regression analyses were used to evaluate association of 15 SNPs in vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)1, VEGFR2, or hypoxia-inducible factor (HIF)-1α with outcomes for blood pressure (BP; Grade ≥ 3 hypertension, diastolic BP > 90 mm Hg, and increase ≥ 15 mm Hg from baseline) and efficacy (independent review committee-assessed objective response rate and PFS, and overall survival [OS]). Multivariate analyses assessed SNPs and baseline characteristics as potential predictors of PFS and OS. RESULTS: Genotype data were available for 305 (42.7%) of 714 patients; 159 received axitinib and 146 sorafenib. After Bonferroni adjustment, no SNP was associated with BP outcomes. In axitinib-treated patients, VEGF-A rs699947 (A/A vs. C/C) and rs833061 (C/C vs. T/T) were associated with longer OS (27.0 vs. 13.4 months; hazard ratio [HR], 0.39; Padjusted = .015). In sorafenib-treated patients, VEGFR2 rs2071559 (G/G vs. A/A) was associated with longer OS (26.8 vs. 13.8 months; HR, 0.41; Padjusted = .030). In multivariate analyses, no SNP predicted axitinib efficacy; VEGFR2 rs2071559 predicted PFS (P = .0053) and OS (P = .0027) for sorafenib. Sensitivity/specificity of VEGFR2 rs2071559 for OS was < 80%. CONCLUSION: No SNP predicted axitinib outcomes. Although VEGFR2 rs2071559 predicted sorafenib efficacy in patients with mRCC, sensitivity/specificity limitations preclude its use for selecting individual patients for sorafenib treatment.