Heterogenous NECTIN4 expression in urothelial high-risk non-muscle-invasive bladder cancer.

High-grade non-muscle-invasive bladder cancer (HG NMIBC) patients are at high risk (HR) of progression to muscle-invasion. Bladder-preserving therapies for this patient subgroup are limited, and additional treatments are desirable. Recently, enfortumab vedotin, targeting cancer-associated NECTIN4, has been approved for the treatment of advanced urothelial carcinoma. However, data on the expression of NECTIN4 and its therapeutic potential for HR NMIBC are scarce. Here, NECTIN4 was immunohistochemically analyzed in urothelial HG NMIBC by studying cohorts of carcinoma in situ (CIS)/T1HG (N = 182 samples), HG papillary tumors from mixed-grade lesions (mixed TaHG) (N = 87) and papillary HG tumors without a history of low-grade disease (pure TaHG/T1HG) (N = 98) from overall 225 patients. Moreover, inter-lesional NECTIN4 heterogeneity in multifocal HG NMIBC tumors was determined. A high prevalence of NECTIN4 positivity was noted across HG NMIBC subgroups (91%, N = 367 samples), with 77% of samples showing moderate/strong expression. Heterogenous NECTIN4 levels were observed between HG NMIBC subgroups: non-invasive areas of CIS/T1HG and pure TaHG/T1HG samples showed NECTIN4 positivity in 96% and 99%, with 88% and 83% moderate/strong expressing specimens, respectively, whereas significantly lower NECTIN4 levels were detected in mixed TaHG lesions (72% positivity, 48% of samples with moderate/strong NECTIN4 expression). Moreover, higher NECTIN4 heterogeneity was observed in patients with multifocal mixed TaHG tumors (22% of patients) compared to patients with multifocal CIS/T1HG and pure TaHG/T1HG tumors (9% and 5%). Taken together, NECTIN4-directed antibody-drug conjugates might be promising for the treatment of HR NMIBC patients, especially for those exhibiting CIS/T1HG and pure TaHG/T1HG tumors without a history of low-grade disease.

Surface-Mediated Ring-Opening and Porphyrin Deconstruction via Conformational Distortion.

The breakdown of macrocyclic compounds is of utmost importance in manifold biological and chemical processes, usually proceeding via oxygenation-induced ring-opening reactions. Here, we introduce a surface chemical route to selectively break a prototypical porphyrin species, cleaving off one pyrrole unit and affording a tripyrrin derivative. This pathway, operational in an ultrahigh vacuum environment at moderate temperature is enabled by a distinct molecular conformation achieved via the specific interaction between the porphyrin and its copper support. We provide an atomic-level characterization of the surface-anchored tripyrrin, its reaction intermediates, and byproducts by bond-resolved atomic force microscopy, unequivocally identifying the molecular skeletons. The ring-opening is rationalized by the distortion reducing the macrocycle's stability. Our findings open a route to steer ring-opening reactions by conformational design and to study intriguing tetrapyrrole catabolite analogues on surfaces.

Intratumoral heterogeneity of surrogate molecular subtypes in urothelial carcinoma in situ of the urinary bladder: implications for prognostic stratification of high-risk non-muscle-invasive bladder cancer.

Reliable factors predicting the disease course of non-muscle-invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) are unavailable. Molecular subtypes have potential for prognostic stratification of muscle-invasive bladder cancer, while their value for CIS patients is unknown. Here, the prognostic impact of both clinico-pathological parameters, including CIS focality, and immunohistochemistry-based surrogate subtypes was analyzed in a cohort of high-risk NMIBC patients with CIS. In 128 high-risk NMIBC patients with CIS, luminal (KRT20, GATA3, ERBB2) and basal (KRT5/6, KRT14) surrogate markers as well as p53 were analyzed in 213-231 biopsies. To study inter-lesional heterogeneity of CIS, marker expression in independent CIS biopsies from different bladder localizations was analyzed. Clinico-pathological parameters and surrogate subtypes were correlated with recurrence-free (RFS), progression-free (PFS), cancer-specific (CSS), and overall survival (OS). Forty-six and 30% of CIS patients exhibited a luminal-like (KRT20-positive, KRT5/6-negative) and a null phenotype (KRT20-negative, KRT5/6-negative), respectively. A basal-like subtype (KRT20-negative, KRT5/6-positive) was not observed. A significant degree of inter-lesional CIS heterogeneity was noted, reflected by 23% of patients showing a mixed subtype. Neither CIS surrogate subtype nor CIS focality was associated with patient outcome. Patient age and smoking status were the only potentially independent prognostic factors predicting RFS, PFS, OS, and PFS, respectively. In conclusion, further clarification of heterogeneity of surrogate subtypes in HR NMIBC and their prognostic value is of importance with regard to potential implementation of molecular subtyping into clinical routine. The potential prognostic usefulness of patient age and smoking status for high-risk NMIBC patients with CIS needs further validation.

Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals.

Recent years have seen an increasing interest in laccase enzymes. Due to their ability of oxidizing various substrates, they are nowadays applied in multiple industrial fields including pulp delignification, textile dye bleaching, and bioremediation. In contrast to laccase production from native sources, with its generally low yield and high cost, heterologous laccase expression is far better suited to meet the growing industrial demands. TVLCC5 gene encoding Trametes versicolor laccase 5 was overexpressed in Arxula adeninivorans using the strong constitutive TEF1 promoter. Recombinant Tvlcc5 protein was purified by immobilized-metal ion affinity chromatography and biochemically characterized using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate for standard activity assays. The enzyme showed the highest activity at 50 °C between pH 4.5-5.5. The half-life of Tvlcc5 at 60 °C was around 20 min. The negative effect of chloride anions on enzyme activity was demonstrated. A fed-batch cultivation of Tvlcc5 producing strain A. adeninivorans G1212/YRC102-TEF1-TVLCC5-6H was performed and resulted in a laccase activity of 4986.3 U L-1. To improve the expression level of recombinant laccase in A. adeninivorans, cultivation conditions were optimized by single factor experiments. Recombinant Tvlcc5 proved to be a promising agent for degradation of pharmaceuticals that are an important source of environmental pollution. Concentration of diclofenac and sulfamethoxazole decreased to 46.8% and 51.1% respectively after 24 h incubation with Tvlcc5. When 1 mM redox mediator ABTS was added complete degradation was obtained within 1 h.

Selection of the Optimal Yeast Host for the Synthesis of Recombinant Enzymes.

Yeasts, like Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Yarrowia lipolytica, and Saccharomyces cerevisiae are frequently used producers of recombinant enzymes, particularly when posttranslational modifications are mandatory to obtain full functionality. The wide-range transformation/expression platform presented in this chapter can be used to select the optimal yeast host for high-level synthesis of the desired enzyme with favorable biochemical properties. This platform is composed of a selection marker and up to four expression modules in a linearized cassette. Here we describe the protocols for the assembly as well as the transformation of yeast strains with the respective cassettes, screening of transformants, the isolation and biochemical characterization of the enzymes, and finally a simple fermentation strategy to achieve maximal yields of the chosen recombinant enzyme.

Exploration of Interfacial Porphine Coupling Schemes and Hybrid Systems by Bond-Resolved Scanning Probe Microscopy.

The templated synthesis of porphyrin-based oligomers and heterosystems is of considerable interest for materials with tunable electronic gaps, photovoltaics, or sensing device elements. In this work, temperature-induced dehydrogenative coupling between unsubstituted free-base porphine units and their attachment to graphene nanoribbons on a well-defined Ag(111) support are scrutinized by bond-resolved scanning probe microscopy techniques. The detailed inspection of covalently fused porphine dimers obtained by in vacuo on-surface synthesis clearly reveals atomistic details of coupling motifs, whereby also putative reaction intermediates are identified. Moreover, the covalent attachment of porphines at preferred locations of atomically precise armchair-type graphene nanoribbons is demonstrated.

Fusing tetrapyrroles to graphene edges by surface-assisted covalent coupling.

Surface-assisted covalent linking of precursor molecules enables the fabrication of low-dimensional nanostructures, which include graphene nanoribbons. One approach to building functional multicomponent systems involves the lateral anchoring of organic heteromolecules to graphene. Here we demonstrate the dehydrogenative coupling of single porphines to graphene edges on the same metal substrate as used for graphene synthesis. The covalent linkages are visualized by scanning probe techniques with submolecular resolution, which directly reveals bonding motifs and electronic features. Distinct configurations are identified that can be steered towards entities predominantly fused to graphene edges through two pyrrole rings by thermal annealing. Furthermore, we succeeded in the concomitant metallation of the macrocycle with substrate atoms and the axial ligation of adducts. Such processes combined with graphene-nanostructure synthesis has the potential to create complex materials systems with tunable functionalities.

Direct Identification and Determination of Conformational Response in Adsorbed Individual Nonplanar Molecular Species Using Noncontact Atomic Force Microscopy.

In recent years atomic force microscopy (AFM) at highest resolution was widely applied to mostly planar molecules, while its application toward exploring species with structural flexibility and a distinct 3D character remains a challenge. Herein, the scope of noncontact AFM is widened by investigating subtle conformational differences occurring in the well-studied reference systems 2H-TPP and Cu-TPP on Cu(111). Different saddle-shape conformations of both species can be recognized in conventional constant-height AFM images. To unambiguously identify the behavior of specific molecular moieties, we extend data acquisition to distances that are inaccessible with constant-height measurements by introducing vertical imaging, that is, AFM mapping in a plane perpendicular to the sample surface. Making use of this novel technique the vertical displacement of the central Cu atom upon tip-induced conformational switching of Cu-TPP is quantified. Further, for 2H-TPP two drastically different geometries are observed, which are systematically characterized. Our results underscore the importance of structural flexibility in adsorbed molecules with large conformational variability and, consequently, the objective to characterize their geometry at the single-molecule level in real space.

Tailoring Large Pores of Porphyrin Networks on Ag(111) by Metal-Organic Coordination.

The engineering of nanoarchitectures to achieve tailored properties relevant for macroscopic devices is a key motivation of organometallic surface science. To this end, understanding the role of molecular functionalities in structure formation and adatom coordination is of great importance. In this study, the differences in formation of Cu-mediated metal-organic coordination networks based on two pyridyl- and cyano-bearing free-base porphyrins on Ag(111) are elucidated by use of low-temperature scanning tunneling microscopy (STM). Distinct coordination networks evolve via different pathways upon codeposition of Cu adatoms. The cyano-terminated module directly forms 2D porous networks featuring fourfold-coordinated Cu nodes. By contrast, the pyridyl species engage in twofold coordination with Cu and a fully reticulated 2D network featuring a pore size exceeding 3 nm2 only evolves via an intermediate structure based on 1D coordination chains. The STM data and complementary Monte Carlo simulations reveal that these distinct network architectures originate from spatial constraints at the coordination centers. Cu adatoms are also shown to form two- and fourfold monoatomic coordination nodes with monotopic nitrogen-terminated linkers on the very same metal substrate-a versatility that is not achieved by other 3d transition metal centers but consistent with 3D coordination chemistry. This study discloses how specific molecular functionalities can be applied to tailor coordination architectures and highlights the potential of Cu as coordination center in such low-dimensional structures on surfaces.

Iron phthalocyanine on Cu(111): Coverage-dependent assembly and symmetry breaking, temperature-induced homocoupling, and modification of the adsorbate-surface interaction by annealing.

We have examined the geometric and electronic structures of iron phthalocyanine assemblies on a Cu(111) surface at different sub- to mono-layer coverages and the changes induced by thermal annealing at temperatures between 250 and 320 °C by scanning tunneling microscopy, x-ray photoelectron spectroscopy, and x-ray absorption spectroscopy. The symmetry breaking observed in scanning tunneling microscopy images is found to be coverage dependent and to persist upon annealing. Further, we find that annealing to temperatures between 300 and 320 °C leads to both desorption of iron phthalocyanine molecules from the surface and their agglomeration. We see clear evidence of temperature-induced homocoupling reactions of the iron phthalocyanine molecules following dehydrogenation of their isoindole rings, similar to what has been observed for related tetrapyrroles on transition metal surfaces. Finally, spectroscopy indicates a modified substrate-adsorbate interaction upon annealing with a shortened bond distance. This finding could potentially explain a changed reactivity of Cu-supported iron phthalocyanine in comparison to that of the pristine compound.

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications.

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6× His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml(-1) could be achieved even with a nonoptimized cultivation procedure.

Surface-assisted dehydrogenative homocoupling of porphine molecules.

The templated synthesis of porphyrin dimers, oligomers, and tapes has recently attracted considerable interest. Here, we introduce a clean, temperature-induced covalent dehydrogenative coupling mechanism between unsubstituted free-base porphine units yielding dimers, trimers, and larger oligomers directly on a Ag(111) support under ultrahigh-vacuum conditions. Our multitechnique approach, including scanning tunneling microscopy, near-edge X-ray absorption fine structure and photoelectron spectroscopy complemented by theoretical modeling, allows a comprehensive characterization of the resulting nanostructures and sheds light on the coupling mechanism. We identify distinct coupling motifs and report a decrease of the electronic gap and a modification of the frontier orbitals directly associated with the formation of triply fused dimeric species. This new on-surface homocoupling protocol yields covalent porphyrin nanostructures addressable with submolecular resolution and provides prospective model systems towards the exploration of extended oligomers with tailored chemical and physical properties.

Control of molecular organization and energy level alignment by an electronically nanopatterned boron nitride template.

Suitable templates to steer the formation of nanostructure arrays on surfaces are indispensable in nanoscience. Recently, atomically thin sp(2)-bonded layers such as graphene or boron nitride (BN) grown on metal supports have attracted considerable interest due to their potential geometric corrugation guiding the positioning of atoms, metallic clusters or molecules. Here, we demonstrate three specific functions of a geometrically smooth, but electronically corrugated, sp(2)/metal interface, namely, BN/Cu(111), qualifying it as a unique nanoscale template. As functional adsorbates we employed free-base porphine (2H-P), a prototype tetrapyrrole compound, and tetracyanoquinodimethane (TCNQ), a well-known electron acceptor. (i) The electronic moirons of the BN/Cu(111) interface trap both 2H-P and TCNQ, steering self-organized growth of arrays with extended molecular assemblies. (ii) We report an effective decoupling of the trapped molecules from the underlying metal support by the BN, which allows for a direct visualization of frontier orbitals by scanning tunneling microscopy (STM). (iii) The lateral molecular positioning in the superstructured surface determines the energetic level alignment; i.e., the energy of the frontier orbitals, and the electronic gap are tunable.

How surface bonding and repulsive interactions cause phase transformations: ordering of a prototype macrocyclic compound on Ag(111).

We investigated the surface bonding and ordering of free-base porphine (2H-P), the parent compound of all porphyrins, on a smooth noble metal support. Our multitechnique investigation reveals a surprisingly rich and complex behavior, including intramolecular proton switching, repulsive intermolecular interactions, and density-driven phase transformations. For small concentrations, molecular-level observations using low-temperature scanning tunneling microscopy clearly show the operation of repulsive interactions between 2H-P molecules in direct contact with the employed Ag(111) surface, preventing the formation of islands. An increase of the molecular coverage results in a continuous decrease of the average intermolecular distance, correlated with multiple phase transformations: the system evolves from an isotropic, gas-like configuration via a fluid-like phase to a crystalline structure, which finally gives way to a disordered layer. Herein, considerable site-specific molecule-substrate interactions, favoring an exclusive adsorption on bridge positions of the Ag(111) lattice, play an important role. Accordingly, the 2D assembly of 2H-P/Ag(111) layers is dictated by the balance between adsorption energy maximization while retaining a single adsorption site counteracted by the repulsive molecule-molecule interactions. The long-range repulsion is associated with a charge redistribution at the 2H-P/Ag(111) interface comprising a partial filling of the lowest unoccupied molecular orbital, resulting in long-range electrostatic interactions between the adsorbates. Indeed, 2H-P molecules in the second layer that are electronically only weakly coupled to the Ag substrate show no repulsive behavior, but form dense-packed islands.

Boron nitride on Cu(111): an electronically corrugated monolayer.

Ultrathin films of boron nitride (BN) have recently attracted considerable interest given their successful incorporation in graphene nanodevices and their use as spacer layers to electronically decouple and order functional adsorbates. Here, we introduce a BN monolayer grown by chemical vapor deposition of borazine on a single crystal Cu support, representing a model system for an electronically patterned but topographically smooth substrate. Scanning tunneling microscopy and spectroscopy experiments evidence a weak bonding of the single BN sheet to Cu, preserving the insulating character of bulk hexagonal boron nitride, combined with a periodic lateral variation of the local work function and the surface potential. Complementary density functional theory calculations reveal a varying registry of the BN relative to the Cu lattice as origin of this electronic Moiré-like superstructure.

A surface-anchored molecular four-level conductance switch based on single proton transfer.

The development of a variety of nanoscale applications requires the fabrication and control of atomic or molecular switches that can be reversibly operated by light, a short-range force, electric current or other external stimuli. For such molecules to be used as electronic components, they should be directly coupled to a metallic support and the switching unit should be easily connected to other molecular species without suppressing switching performance. Here, we show that a free-base tetraphenyl-porphyrin molecule, which is anchored to a silver surface, can function as a molecular conductance switch. The saddle-shaped molecule has two hydrogen atoms in its inner cavity that can be flipped between two states with different local conductance levels using the electron current through the tip of a scanning tunnelling microscope. Moreover, by deliberately removing one of the hydrogens, a four-level conductance switch can be created. The resulting device, which could be controllably integrated into the surrounding nanoscale environment, relies on the transfer of a single proton and therefore contains the smallest possible atomistic switching unit.