3DDPDs: describing protein dynamics for proteochemometric bioactivity prediction. A case for (mutant) G protein-coupled receptors.

Proteochemometric (PCM) modelling is a powerful computational drug discovery tool used in bioactivity prediction of potential drug candidates relying on both chemical and protein information. In PCM features are computed to describe small molecules and proteins, which directly impact the quality of the predictive models. State-of-the-art protein descriptors, however, are calculated from the protein sequence and neglect the dynamic nature of proteins. This dynamic nature can be computationally simulated with molecular dynamics (MD). Here, novel 3D dynamic protein descriptors (3DDPDs) were designed to be applied in bioactivity prediction tasks with PCM models. As a test case, publicly available G protein-coupled receptor (GPCR) MD data from GPCRmd was used. GPCRs are membrane-bound proteins, which are activated by hormones and neurotransmitters, and constitute an important target family for drug discovery. GPCRs exist in different conformational states that allow the transmission of diverse signals and that can be modified by ligand interactions, among other factors. To translate the MD-encoded protein dynamics two types of 3DDPDs were considered: one-hot encoded residue-specific (rs) and embedding-like protein-specific (ps) 3DDPDs. The descriptors were developed by calculating distributions of trajectory coordinates and partial charges, applying dimensionality reduction, and subsequently condensing them into vectors per residue or protein, respectively. 3DDPDs were benchmarked on several PCM tasks against state-of-the-art non-dynamic protein descriptors. Our rs- and ps3DDPDs outperformed non-dynamic descriptors in regression tasks using a temporal split and showed comparable performance with a random split and in all classification tasks. Combinations of non-dynamic descriptors with 3DDPDs did not result in increased performance. Finally, the power of 3DDPDs to capture dynamic fluctuations in mutant GPCRs was explored. The results presented here show the potential of including protein dynamic information on machine learning tasks, specifically bioactivity prediction, and open opportunities for applications in drug discovery, including oncology.

The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide.

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.

Diode-side-pumped Alexandrite slab lasers.

We present the investigation of diode-side-pumping of Alexandrite slab lasers in a range of designs using linear cavity and grazing-incidence bounce cavity configurations. An Alexandrite slab laser cavity with double-pass side pumping produces 23.4 mJ free-running energy at 100 Hz rate with slope efficiency ~40% with respect to absorbed pump energy. In a slab laser with single-bounce geometry output power of 12.2 W is produced, and in a double-bounce configuration 6.5 W multimode and 4.5 W output in TEM00 mode is produced. These first results of slab laser and amplifier designs in this paper highlight some of the potential strategies for power and energy scaling of Alexandrite using diode-side-pumped Alexandrite slab architectures with future availability of higher power red diode pumping.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.

Diode-pumped Alexandrite lasers in Q-switched and cavity-dumped Q-switched operation.

We present the first study of Q-switched Alexandrite lasers under continuous-wave diode-pumping with operation up to 10 kHz repetition rates in TEM00, with spatial quality M2 1.15. With a pulsed-diode dual-end-pumped design, pulse energy is scaled to a record level of 3 mJ. We also demonstrate, for the first time, cavity-dumped Q-switching of diode-pumped Alexandrite lasers under continuous-wave and pulsed diode-pumping, up to 10 kHz. Pulse energy of 510 µJ is demonstrated with 3 ns pulse duration and 170 kW peak power, in TEM00 with M2 < 1.2. Second harmonic generation of the cavity-dumped Q-switched pulses was used to generate UV wavelength 379 nm with conversion efficiency of 47%.

Tissue factor expressed by circulating cancer cell-derived microparticles drastically increases the incidence of deep vein thrombosis in mice.

BACKGROUND: The risk of thrombotic complications such as deep vein thrombosis (DVT) during tumor development is well known. Tumors release into the circulation procoagulant microparticles (MPs) that can participate in thrombus formation following vessel injury. The importance of this MP tissue factor (TF) in the initiation of cancer-associated DVT remains uncertain. OBJECTIVE: To investigate how pancreatic cancer MPs promote DVT in vivo. METHODS: We combined a DVT mouse model in which thrombosis is induced by flow restriction in the inferior vena cava with one of subcutaneous pancreatic cancer in C57BL/6J mice. We infused high-TF and low-TF tumor MPs to determine the importance of TF in experimental cancer-associated DVT. RESULTS: Both tumor-bearing mice and mice infused with tumor MPs subjected to 3 h of partial flow restriction developed an occlusive thrombus; fewer than one-third of the control mice did. We observed that MPs adhered to neutrophil extracellular traps (NETs), which are functionally important players during DVT, whereas neither P-selectin nor glycoprotein Ib were required for MP recruitment in DVT. The thrombotic phenotype induced by MP infusion was suppressed by hirudin, suggesting the importance of thrombin generation. TF carried by tumor MPs was essential to promote DVT, as mice infused with low-TF tumor MPs had less thrombosis than mice infused with high-TF tumor MPs. CONCLUSIONS: TF expressed on tumor MPs contributes to the increased incidence of cancer-associated venous thrombosis in mice in vivo. These MPs may adhere to NETs formed at the site of thrombosis.

Investigation of a versatile pulsed laser source based on a diode seed and ultra-high gain bounce geometry amplifiers.

We present an investigation of a versatile pulsed laser source using a low power, gain-switched diode laser with independently variable repetition rate and pulse duration to seed an ultra-high gain Nd:YVO4 bounce geometry amplifier system at 1064nm. Small-signal gain as high as 50dB was demonstrated in a bounce geometry pre-amplifier from just 24W pumping, with good preservation of TEM00 beam quality. The single amplifier is shown to be limited by amplified spontaneous emission. Study is made of further scaling with a second power amplifier, achieving average output power of ~14W for a pulsed diode seed input of 188µW. This investigation provides some guidelines for using the bounce amplifier to obtain flexible pulse amplification of low-power seed sources to reach scientifically and commercially useful power levels.

Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants.

Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding.

Neutrophil extracellular traps promote deep vein thrombosis in mice.

BACKGROUND: Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be prothrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). OBJECTIVE: To explore the source and role of extracellular chromatin in DVT. METHODS: We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). RESULTS: We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared with sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. CONCLUSIONS: Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development.

Passively Q-switched Nd:YVO4 laser with greater than 11 W average power.

This paper presents the highest average power passively Q-switched Nd:vanadate laser, to the best of our knowledge. A maximum average output power greater than 11 W was demonstrated using a Nd:YVO4 bounce geometry laser operating at 1064 nm in TEM00 mode, with a spatially stigmatic design. Pulse energies greater than 58 µJ and peak powers in excess of 1.9 kW were obtained; the maximum repetition rate recorded was 190 kHz, close to the upper limit achievable with Cr4+:YAG saturable absorbers.

Excitotoxicity through Ca2+-permeable AMPA receptors requires Ca2+-dependent JNK activation.

The GluA4-containing Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (Ca-AMPARs) were previously shown to mediate excitotoxicity through mechanisms involving the activator protein-1 (AP-1), a c-Jun N-terminal kinase (JNK) substrate. To further investigate JNK involvement in excitotoxic pathways coupled to Ca-AMPARs we used HEK293 cells expressing GluA4-containing Ca-AMPARs (HEK-GluA4). Cell death induced by overstimulation of Ca-AMPARs was mediated, at least in part, by JNK. Importantly, JNK activation downstream of these receptors was dependent on the extracellular Ca(2+) concentration. In our quest for a molecular link between Ca-AMPARs and the JNK pathway we found that the JNK interacting protein-1 (JIP-1) interacts with the GluA4 subunit of AMPARs through the N-terminal domain. In vivo, the excitotoxin kainate promoted the association between GluA4 and JIP-1 in the rat hippocampus. Taken together, our results show that the JNK pathway is activated by Ca-AMPARs upon excitotoxic stimulation and suggest that JIP-1 may contribute to the propagation of the excitotoxic signal.

Nonlinear mirror modelocking of a bounce geometry laser.

We present the investigation of nonlinear mirror modelocking (NLM) of a bounce amplifier laser. This technique, a potential rival to SESAM modelocking, uses a nonlinear crystal and a dichroic mirror to passively modelock a Nd:GdVO(4) slab bounce amplifier operating at 1063nm. At 11.3W, we present the highest power achieved using the NLM technique, using type-II phase-matched KTP, with a pulse duration of 57ps. Using type-I phase-matched BiBO, modelocking was achieved with a shorter pulse duration of 5.7ps at an average power of 7.1W.

The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.

The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.

Postoperative concurrent chronomodulated 5-fluorouracil/leucovorin infusion and pelvic radiotherapy for squamous cell carcinoma of the ovary arising from mature cystic teratoma.

Primary squamous cell carcinoma (SCC) of the ovary arising from mature cystic teratoma (MCT) is rare. Survival is very poor. Although different postoperative treatments have been tried, none appears to have influenced outcomes. A 44-year-old patient with FIGO stage IIB SCC of the ovary arising in MCT had exploratory laparotomy and left salpingo-oophorectomy, leaving macroscopic residual pelvic disease. Postoperative treatment consisted of a continuous concurrent chronomodulated infusion of 5-fluorouracil 150 mg/m2/day and leucovorin 10 mg/m2/day with 5 weeks of pelvic radiotherapy. Three years after initial surgery, she remains disease and complication free. Given this patient's unusually long disease-free survival and the efficacy of concurrent chemotherapy and radiotherapy in other SCCs, the concurrent circadian treatment approach used for this patient should be explored in other cases of SCC of the ovary.

Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells.

Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca(2+) and guanosine 5'-[gamma-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20-30 min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25 microM ATP can extend responsiveness significantly; this effect is maximal at 50 microM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPalpha and PITPbeta have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C(16)), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C(16) suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.

Preoperative chemo-radiation for carcinoma of the vulva with N2/N3 nodes: a gynecologic oncology group study.

PURPOSE: To determine if patients with carcinoma of the vulva, with N2/N3 lymph nodes, could undergo resection of the lymph nodes and primary tumor following preoperative chemo-radiation. METHODS AND MATERILAS: Fifty-two patients were entered in the study, but six patients did not meet the criteria of the protocol and were excluded. The remaining 46 patients are the subject of this report. Patients underwent a split course of radiation, 4760 cGy to the primary and lymph nodes, with concurrent chemotherapy, cisplatin/5-FU, followed by surgery. RESULTS: Four patients did not complete the chemo-radiation, because three expired and one refused to complete the treatment. Four patients who completed chemo-radiation did not undergo surgery, because two of them died of non-cancer-related causes, and in the other two patients, the nodes remained unresectable. Following chemo-radiation, the disease in the lymph nodes became resectable in 38/40 patients. Two patients who completed the course of chemo-radiation did not undergo surgery as per protocol because of pulmonary metastasis. One underwent radical vulvectomy and unilateral node dissection and the other radical vulvectomy only. The specimen of the lymph nodes was histologically negative in 15/37 patients. Nineteen patients developed recurrent and/or metastatic disease. The sites of failure were as follows: primary area only, 9; lymph node area only, 1; primary area and distant metastasis, 1; distant metastasis only, 8. Local control of the disease in the lymph nodes was achieved in 36/37 and in the primary area in 29/38 of the patients. Twenty patients are alive and disease-free, and five have expired without evidence of recurrence or metastasis. Two patients died of treatment-related complications. CONCLUSION: High resectability and local control rates of the lymph nodes were obtained in patients with carcinoma of the vulva with N2/N3 nodes treated preoperatively with chemo-radiation.

Concurrent chemotherapy and radiation for locally advanced cervical cancer: the new standard of care.

Radical pelvic irradiation has constituted the definitive therapy for patients with large cervical cancers. No substantial improvements have been made in treatment outcomes. In the past year, however, a series of large, well-conducted randomized trials has evaluated the role of concurrent chemotherapy with pelvic irradiation in cervical cancer. These trials include definitive treatment of patients with stage IB2 to IVA disease and adjuvant treatment after radical surgery in stage IB2-IIA disease. Five trials have shown a consistent 30% to 50% reduction in the risk of death from disease when concurrent chemotherapy is used. Questions still remain as to what constitutes the best chemotherapy dose and schedule. In all of the positive trials, cisplatin was used, but three also used 5-fluorouracil. The level of survival improvement that occurs when chemotherapy is added to optimal irradiation and whether patients with stage IIIB and IVA benefit are also unclear. Improvements in survival rates for patients with solid tumors occur slowly. Based on the evidence, it is likely that concurrent chemotherapy with radiation will become the new standard of care for bulky and advanced cervix cancer.