Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the ...European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival).
Clinically annotated, resected stage I–III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ~150 (13 genes) mutations covered in the multiplex test.
Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases.
Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I–III NSCLC cohort, none of the mutations characterized showed prognostic significance.
The official classification of newly discovered or long-known unassigned viruses by the International Committee on Taxonomy of Viruses (ICTV) requires the deposition of coding-complete or ...-near-complete virus genome sequences in GenBank to fulfill a requirement of the taxonomic proposal (TaxoProp) process. However, this requirement is fairly new; thus, genomic sequence information is fragmented or absent for many already-classified viruses. As a result, taxon-wide modern phylogenetic analyses are often challenging, if not impossible. This problem is particularly eminent among viruses with segmented genomes, such as bunyavirals, which were frequently classified solely based on single-segment sequence information. To solve this issue for one bunyaviral family,
, we call on the community to provide additional sequence information for incompletely sequenced classified viruses by mid-June 2023. Such sequence information may be sufficient to prevent their possible declassification during the ongoing efforts to establish a coherent, consistent, and evolution-based hantavirid taxonomy.
Thick
c-plane unintentional doped and iron-doped GaN substrates were grown by hydride vapor phase epitaxial technique on sapphire substrates. The morphology and crystalline quality of the ...freestanding samples show no evident degradation due to iron doping. Low-temperature photoluminescence measurements show reduction of the exciton-bound to neutral impurities band intensities with iron doping increase. Near-infrared photoluminescence studies confirm the incorporation and activation of iron impurities. Variable temperature resistivity measurements verified that the iron-doped films are semi-insulating.
Shallow-etch mesa isolation (SEMI) of graded-bandgap “W”-structured type II superlattice (GGW) infrared photodiodes provides a powerful means for reducing excess dark currents due to surface and bulk ...junction related processes, and it is particularly well suited for focal-plane array fabrication. In the
n
-on-
p
GGW photodiode structure the energy gap is increased in a series of steps from that of the lightly
p
-type infrared-absorbing region to a value typically two to three times larger. The wider gap levels off about 10 nm short of the doping-defined junction, and continues for another 0.25
μ
m into the heavily
n
-doped cathode before the structure is terminated by an
n
+
-doped InAs top cap layer. The increased bandgap in the high-field region near the junction helps to strongly suppress both bulk tunneling and generation–recombination (G–R) current by imposing a much larger tunneling barrier and exponentially lowering the intrinsic carrier concentration. The SEMI approach takes further advantage of the graded structure by exposing only the widest-gap layers on etched surfaces. This lowers surface recombination and trap-assisted tunneling in much the same way as the GGW suppresses these processes in the bulk. Using SEMI, individual photodiodes are defined using a shallow etch that typically terminates only 10 nm to 20 nm past the junction, which is sufficient to isolate neighboring pixels while leaving the narrow-gap absorber layer buried 100 nm to 200 nm below the surface. This provides for separate optimization of the photodiode’s electrical and optical area. The area of the junction can be reduced to a fraction of that of the pixel, lowering bulk junction current, while maintaining 100% optical fill factor with the undisturbed absorber layer. Finally, with the elimination of deep, high-aspect-ratio trenches, SEMI simplifies array fabrication. We report herein results from SEMI-processed GGW devices, including large-area discrete photodiodes, mini-arrays, and a focal-plane array. Current–voltage data show strong suppression of side-wall leakage relative to that for more deeply etched devices, as well as scaling of dark current with junction area without loss of quantum efficiency.
-Hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) is a microbial group A flavoprotein monooxygenase that catalyzes the
-hydroxylation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate with the ...stoichiometric consumption of NAD(P)H and oxygen. PHBH and related enzymes lack a canonical NAD(P)H-binding domain and the way they interact with the pyridine nucleotide coenzyme has remained a conundrum. Previously, we identified a surface exposed protein segment of PHBH from
involved in NADPH binding. Here, we report the first amino acid sequences of NADH-preferring PHBHs and a phylogenetic analysis of putative PHBHs identified in currently available bacterial genomes. It was found that PHBHs group into three clades consisting of NADPH-specific, NAD(P)H-dependent and NADH-preferring enzymes. The latter proteins frequently occur in
. To validate the results, we produced several putative PHBHs in
and confirmed their predicted coenzyme preferences. Based on phylogeny, protein energy profiling and lifestyle of PHBH harboring bacteria we propose that the pyridine nucleotide coenzyme specificity of PHBH emerged through adaptive evolution and that the NADH-preferring enzymes are the older versions of PHBH. Structural comparison and distance tree analysis of group A flavoprotein monooxygenases indicated that a similar protein segment as being responsible for the pyridine nucleotide coenzyme specificity of PHBH is involved in determining the pyridine nucleotide coenzyme specificity of the other group A members.
Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic ...properties of OYERo2a from
1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by
-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (
) while showing increased
values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but
was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted
-phenyl-2-methylmaleimide equally well (81-84%) with an enantiomeric excess (
) of more than 99% for the
-product. With cyclic ketones, the highest conversion (89%) and
(>99%) was observed for the reaction of WT with
-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.
Asymmetric hydrogenation of activated alkenes catalysed by ene-reductases from the old yellow enzyme family (OYEs) leading to chiral products is of potential interest for industrial processes. OYEs’ ...dependency on the pyridine nucleotide coenzyme can be circumvented through established artificial hydride donors such as nicotinamide coenzyme biomimetics (NCBs). Several OYEs were found to exhibit higher reduction rates with NCBs. In this review, we describe a new classification of OYEs into three main classes by phylogenetic and structural analysis of characterized OYEs. The family roots are linked with their use as chiral catalysts and their mode of action with NCBs. The link between bioinformatics (sequence analysis), biochemistry (structure–function analysis), and biocatalysis (conversion, enantioselectivity and kinetics) can enable an early classification of a putative ene-reductase and therefore the indication of the binding mode of various activated alkenes.
Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by ...genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of 'thermophilic-like' OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg(-1)) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.
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