Molecular Insights into the Regulatory Landscape of Protein Kinase C-Related Kinase-2 (PRK2/PKN2) Using Targeted Small Compounds.

The protein kinase C-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the PIF-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Quantifying thermal transport in buried semiconductor nanostructures via cross-sectional scanning thermal microscopy.

Managing thermal transport in nanostructures became a major challenge in the development of active microelectronic, optoelectronic and thermoelectric devices, stalling the famous Moore's law of clock speed increase of microprocessors for more than a decade. To find the solution to this and linked problems, one needs to quantify the ability of these nanostructures to conduct heat with adequate precision, nanoscale resolution, and, essentially, for the internal layers buried in the 3D structure of modern semiconductor devices. Existing thermoreflectance measurements and "hot wire" 3ω methods cannot be effectively used at lateral dimensions of a layer below a micrometre; moreover, they are sensitive mainly to the surface layers of a relatively high thickness of above 100 nm. Scanning thermal microscopy (SThM), while providing the required lateral resolution, provides mainly qualitative data of the layer conductance due to undefined tip-surface and interlayer contact resistances. In this study, we used cross-sectional SThM (xSThM), a new method combining scanning probe microscopy compatible Ar-ion beam exit nano-cross-sectioning (BEXP) and SThM, to quantify thermal conductance in complex multilayer nanostructures and to measure local thermal conductivity of oxide and semiconductor materials, such as SiO2, SiGex and GeSny. By using the new method that provides 10 nm thickness and few tens of nm lateral resolution, we pinpoint crystalline defects in SiGe/GeSn optoelectronic materials by measuring nanoscale thermal transport and quantifying thermal conductivity and interfacial thermal resistance in thin spin-on materials used in extreme ultraviolet lithography (eUV) fabrication processing. The new capability of xSThM demonstrated here for the first time is poised to provide vital insights into thermal transport in advanced nanoscale materials and devices.

Photonic-plasmonic mode coupling in nanopillar Ge-on-Si PIN photodiodes.

Incorporating group IV photonic nanostructures within active top-illuminated photonic devices often requires light-transmissive contact schemes. In this context, plasmonic nanoapertures in metallic films can not only be realized using CMOS compatible metals and processes, they can also serve to influence the wavelength-dependent device responsivities. Here, we investigate crescent-shaped nanoapertures in close proximity to Ge-on-Si PIN nanopillar photodetectors both in simulation and experiment. In our geometries, the absorption within the devices is mainly shaped by the absorption characteristics of the vertical semiconductor nanopillar structures (leaky waveguide modes). The plasmonic resonances can be used to influence how incident light couples into the leaky modes within the nanopillars. Our results can serve as a starting point to selectively tune our device geometries for applications in spectroscopy or refractive index sensing.

Weak localization and weak antilocalization in doped Ge1-y Sn y layers with up to 8% Sn.

Low-temperature magnetoresistance measurements of n- and p-doped germanium-tin (Ge1-y Sn y ) layers with Sn concentrations up to 8% show contributions arising from effects of weak localization for n-type and weak antilocalization for p-type doped samples independent of the Sn concentration. Calculations of the magnetoresistance using the Hikami-Larkin-Nagaoka model for two-dimensional transport allow us to extract the phase-coherence length for all samples as well as the spin-orbit length for the p-type doped samples. For pure Ge, we find phase-coherence lengths as long as (349.0 ± 1.4) nm and (614.0 ± 0.9) nm for n-type and p-type doped samples, respectively. The phase-coherence length decreases with increasing Sn concentration. From the spin-orbit scattering length, we determine the spin-diffusion scattering length in the range of 20-30 nm for all highly degenerate p-type doped samples irrespective of Sn concentration. These results show that Ge1-y Sn y is a promising material for future spintronic applications.

Ge(Sn) nano-island/Si heterostructure photodetectors with plasmonic antennas.

We report on photodetection in deep subwavelength Ge(Sn) nano-islands on Si nano-pillar substrates, in which self-aligned nano-antennas in the Al contact metal are used to enhance light absorption by means of local surface plasmon resonances. The impact of parameters such as substrate doping and device geometry on the measured responsivities are investigated and our experimental results are supported by simulations of the three-dimensional distribution of the electromagnetic fields. Comparatively high optical responsivities of about 0.1 A W-1 are observed as a consequence of the excitation of localized surface plasmons, making our nano-island photodetectors interesting for applications in which size reduction is essential.

AGC kinases, mechanisms of regulation and innovative drug development.

The group of AGC kinases consists of 63 evolutionarily related serine/threonine protein kinases comprising PDK1, PKB/Akt, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, and YANK, while two other families, Aurora and PLK, are the most closely related to the group. Eight of these families are physiologically activated downstream of growth factor signalling, while other AGC kinases are downstream effectors of a wide range of signals. The different AGC kinase families share aspects of their mechanisms of inhibition and activation. In the present review, we update the knowledge of the mechanisms of regulation of different AGC kinases. The conformation of the catalytic domain of many AGC kinases is regulated allosterically through the modulation of the conformation of a regulatory site on the small lobe of the kinase domain, the PIF-pocket. The PIF-pocket acts like an ON-OFF switch in AGC kinases with different modes of regulation, i.e. PDK1, PKB/Akt, LATS and Aurora kinases. In this review, we make emphasis on how the knowledge of the molecular mechanisms of regulation can guide the discovery and development of small allosteric modulators. Molecular probes stabilizing the PIF-pocket in the active conformation are activators, while compounds stabilizing the disrupted site are allosteric inhibitors. One challenge for the rational development of allosteric modulators is the lack of complete structural information of the inhibited forms of full-length AGC kinases. On the other hand, we suggest that the available information derived from molecular biology and biochemical studies can already guide screening strategies for the identification of innovative mode of action molecular probes and the development of selective allosteric drugs for the treatment of human diseases.

Camptothecin and its analog SN-38, the active metabolite of irinotecan, inhibit binding of the transcriptional regulator and oncoprotein FUBP1 to its DNA target sequence FUSE.

The transcriptional regulator FUSE Binding Protein 1 (FUBP1) is overexpressed in more than 80% of all human hepatocellular carcinomas (HCCs) and other solid tumor entities including prostate and colorectal carcinoma. FUBP1 expression is required for HCC tumor cell expansion, and it functions as an important pro-proliferative and anti-apoptotic oncoprotein that binds to the single-stranded DNA sequence FUSE to regulate the transcription of a variety of target genes. In this study, we screened an FDA-approved drug library and discovered that the Topoisomerase I (TOP1) inhibitor camptothecin (CPT) and its derivative 7-ethyl-10-hydroxycamptothecin (SN-38), the active irinotecan metabolite that is used in the clinics in combination with other chemotherapeutics to treat carcinoma, inhibit FUBP1 activity. Both molecules prevent in vitro the binding of FUBP1 to its single-stranded target DNA FUSE, and they induce deregulation of FUBP1 target genes in HCC cells. Our results suggest the interference with the FUBP1/FUSE interaction as a further molecular mechanism that, in addition to the inactivation of TOP1, may contribute to the therapeutic potential of CPT/SN-38. Targeting of FUBP1 in HCC therapy with SN-38/irinotecan could be a particularly interesting option because of the high FUBP1 levels in HCC cells and their dependency on FUBP1 expression.

An Allosteric Inhibitor Scaffold Targeting the PIF-Pocket of Atypical Protein Kinase C Isoforms.

There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.

Bidirectional Allosteric Communication between the ATP-Binding Site and the Regulatory PIF Pocket in PDK1 Protein Kinase.

Allostery is a phenomenon observed in many proteins where binding of a macromolecular partner or a small-molecule ligand at one location leads to specific perturbations at a site not in direct contact with the region where the binding occurs. The list of proteins under allosteric regulation includes AGC protein kinases. AGC kinases have a conserved allosteric site, the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF) pocket, which regulates protein ATP-binding, activity, and interaction with substrates. In this study, we identify small molecules that bind to the ATP-binding site and affect the PIF pocket of AGC kinase family members, PDK1 and Aurora kinase. We describe the mechanistic details and show that although PDK1 and Aurora kinase inhibitors bind to the conserved ATP-binding site, they differentially modulate physiological interactions at the PIF-pocket site. Our work outlines a strategy for developing bidirectional small-molecule allosteric modulators of protein kinases and other signaling proteins.

Multidrug-resistant organisms in refugees: prevalences and impact on infection control in hospitals.

INTRODUCTION: The refugee crisis is a great challenge to the social and healthcare system in European countries, especially in Germany. An abundance of data has been published on the refugees' health problems (infections as well as physical diseases and psychiatric problems) and their prevention (i.e., sanitary and vaccination programs). However, data on prevalences of multidrug-resistant organisms (MDRO) in refugees are scarce, although it is known that most refugees are from or travelled through countries with high prevalences of MDRO. This paper presents current data on MDRO colonization of refugees admitted to hospitals, and the impact of screening upon admission and infection control in hospitals is discussed. METHODS: Anonymous data obtained by screening upon hospital admission were reported by hospitals in the Rhine-Main region of Germany to the local public health department. Screening and microbiological analyses were performed from December 2015 to March 2016 according to standardized and validated methods. RESULTS: 9.8% of the refugees screened (32/325) exhibited colonization with methicillin-resistant Staphylococcus aureus (MRSA), and 23.3% of the refugees (67/290) were colonized with Gram-negative bacteria with extended spectrum beta-lactamases, and/or enterobacteria with resistance against 3 or 4 groups of antibacterials, so-called 3MRGN (multidrug-resistant Gram-negative bacteria with resistance against penicillins, cephalosporins and quinolones) and 4MRGN (with additional resistance against carbapenems). Carbapenem-resistant Gram-negative bacteria (CRGN) were detected in 2.1% (6/290) of the refugees. CONCLUSION: The data confirms the studies published between 2014 and 2016, encompassing refugees tested in Germany, the Netherlands and Israel, with prevalences of MRSA and CRGN up to 13.5% and 5.6%. The MDRO prevalences are higher than those of "risk groups" for MRSA, such as hemodialysis patients and patients depending on outpatient home-nursing care or residing in nursing homes. Therefore, screening and special infection control in hospitals is strongly suggested when refugees are admitted to hospitals, in order to ensure best medical practice and safety for all hospital patients regardless of their country of origin.

Electrical detection of spin transport in Si two-dimensional electron gas systems.

Spin transport in a semiconductor-based two-dimensional electron gas (2DEG) system has been attractive in spintronics for more than ten years. The inherent advantages of high-mobility channel and enhanced spin-orbital interaction promise a long spin diffusion length and efficient spin manipulation, which are essential for the application of spintronics devices. However, the difficulty of making high-quality ferromagnetic (FM) contacts to the buried 2DEG channel in the heterostructure systems limits the potential developments in functional devices. In this paper, we experimentally demonstrate electrical detection of spin transport in a high-mobility 2DEG system using FM Mn-germanosilicide (Mn(Si0.7Ge0.3)x) end contacts, which is the first report of spin injection and detection in a 2DEG confined in a Si/SiGe modulation doped quantum well structure (MODQW). The extracted spin diffusion length and lifetime are l sf = 4.5 µm and [Formula: see text] at 1.9 K respectively. Our results provide a promising approach for spin injection into 2DEG system in the Si-based MODQW, which may lead to innovative spintronic applications such as spin-based transistor, logic, and memory devices.

Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Lipid regulators of Pkh2 in Candida albicans, the protein kinase ortholog of mammalian PDK1.

Pkh is the yeast ortholog of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Pkh phosphorylates the activation loop of Ypks, Tpks, Sch9 and also phosphorylates the eisosome components Lsp1 and Pil1, which play fundamental roles upstream of diverse signaling pathways, including the cell wall integrity and sphingosine/long-chain base (LCB) signaling pathways. In S. cerevisiae, two isoforms, ScPkh1 and ScPkh2, are required for cell viability, while only one ortholog exists in C. albicans, CaPkh2. In spite of the extensive information gathered on the role of Pkh in the LCB signaling, the yeast Pkh kinases are not known to bind lipids and previous studies did not identify PH domains in Pkh sequences. We now describe that the C-terminal region of CaPkh2 is required for its intrinsic kinase activity. In addition, we found that the C-terminal region of CaPkh2 enables its interaction with structural and signaling lipids. Our results further show that phosphatidylserine, phosphatidic acid, phosphatidylinositol (3,4 and 4,5)-biphosphates, and phosphatidylinositol (3,4,5)-trisphosphate inhibit Pkh activity, whereas sulfatide binds with high affinity but does not affect the intrinsic activity of CaPkh2. Interestingly, we identified that its human ortholog PDK1 also binds to sulfatide. We propose a mechanism by which lipids and dihydrosphingosine regulate CaPkh2 kinase activity by modulating the interaction of the C-terminal region with the kinase domain, while sulfatide-like lipids support localization CaPkh2 mediated by a C-terminal PH domain, without affecting kinase intrinsic activity.

Growth and characterization of SiGeSn quantum well photodiodes.

We report on the fabrication and electro-optical characterization of SiGeSn multi-quantum well PIN diodes. Two types of PIN diodes, in which two and four quantum wells with well and barrier thicknesses of 10 nm each are sandwiched between B- and Sb-doped Ge-regions, were fabricated as single-mesa devices, using a low-temperature fabrication process. We discuss measurements of the diode characteristics, optical responsivity and room-temperature electroluminescence and compare with theoretical predictions from band structure calculations.

Discovery of a Potent Allosteric Kinase Modulator by Combining Computational and Synthetic Methods.

The rational design of allosteric kinase modulators is challenging but rewarding. The protein kinase PDK1, which lies at the center of the growth-factor signaling pathway, possesses an allosteric regulatory site previously validated both in vitro and in cells. ANCHOR.QUERY software was used to discover a potent allosteric PDK1 kinase modulator. Using a recently published PDK1 compound as a template, several new scaffolds that bind to the allosteric target site were generated and one example was validated. The inhibitor can be synthesized in one step by multicomponent reaction (MCR) chemistry when using the ANCHOR.QUERY approach. Our results are significant because the outlined approach allows rapid and efficient scaffold hopping from known molecules into new easily accessible and biologically active ones. Based on increasing interest in allosteric-site drug discovery, we foresee many potential applications for this approach.

Electroluminescence of GeSn/Ge MQW LEDs on Si substrate.

Multi-quantum well light-emitting diodes, consisting of ten alternating GeSn/Ge-layers, were grown by molecular beam epitaxy on Si. The Ge barriers were 10 nm thick, and the GeSn wells were grown with 7% Sn and thicknesses between 6 and 12 nm. The electroluminescence spectra measured at 300 and 80 K yield a broad and intensive luminescence band. Deconvolution revealed three major lines produced by the GeSn wells that can be interpreted in terms of quantum confinement. We interpret that the three lines represent two direct lines, formed by transitions with the light and heavy hole band, respectively, and an indirect line. Biaxial compressive strain causes a splitting of light and heavy holes in the GeSn wells. This interpretation is supported by an effective mass band structure calculation.

Molecular mechanism of regulation of the atypical protein kinase C by N-terminal domains and an allosteric small compound.

Protein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kinases have a pocket in the catalytic domain, the PDK1-interacting fragment (PIF)-pocket, which is a key mediator of the activation. We show here that helix αC within the PIF-pocket of atypical protein kinase C (aPKC) is the target of the interaction with its inhibitory N-terminal domains. We also provide structural evidence that the small compound PS315 is an allosteric inhibitor that binds to the PIF-pocket of aPKC. PS315 exploits the physiological dynamics of helix αC for its binding and allosteric inhibition. The results will support research on allosteric mechanisms and selective drug development efforts against PKC isoforms.

GeSn-on-Si normal incidence photodetectors with bandwidths more than 40 GHz.

GeSn (Sn content up to 4.2%) photodiodes with vertical pin structures were grown on thin Ge virtual substrates on Si by a low temperature (160 °C) molecular beam epitaxy. Vertical detectors were fabricated by a double mesa process with mesa radii between 5 µm and 80 µm. The nominal intrinsic absorber contains carrier densities from below 1 · 10(16) cm(-3) to 1 · 10(17) cm(-3) for Ge reference detectors and GeSn detectors with 4.2% Sn, respectively. The photodetectors were investigated with electrical and optoelectrical methods from direct current up to high frequencies (40 GHz). For a laser wavelength of 1550 nm an increasing of the optical responsivities (84 mA/W -218 mA/W) for vertical incidence detectors with thin (300 nm) absorbers as function of the Sn content were found. Most important from an application perspective all detectors had bandwidth above 40 GHz at enough reverse voltage which increased from zero to -5 V within the given Sn range. Increasing carrier densities (up to 1 · 10(17) cm(-3)) with Sn contents caused the depletion of the nominal intrinsic absorber at increasing reverse voltages.

Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction.

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7ß1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.