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The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells., The mechanism of cell invasion by bacterial toxins is still poorly understood. The adenylate cyclase (CyaA) toxin from Bordetella pertussis, the causative agent of whooping cough, directly translocates its catalytic domain across plasma membranes. The membrane‐permeabilizing segment (yellow) of CyaA translocates across the plasma membrane and binds calmodulin (red), which assists the entry of the catalytic domain (blue) into host cells while the hydrophobic and acylation domains (green) interact with the membrane and the C‐terminal Repeat‐in‐Toxin domain (grey) remains in the extra‐cellular milieu.

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin fromBordetella pertussisdisplays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454–484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

Purpose: To elucidate the role of inter-effort recovery in shuttle running by comparing the metabolic profiles of the 30-15 Intermittent Fitness Test (30-15IFT) and the corresponding continuous version (30-15IFT-CONT). Methods: Sixteen state-level handball players (age = 23 [3] y, height = 185 [7] cm, weight = 85 [14] kg) completed the 30-15IFT and 30-15IFT-CONT, and speed at the last completed stage (in kilometers per hour) and time to exhaustion (in seconds) were assessed. Furthermore, oxygen uptake (in milliliters per kilogram per minute) and blood lactate were obtained preexercise, during exercise, and until 15 minutes postexercise. Metabolic energy (in kilojoules), metabolic power (in Watts per kilogram), and relative (in percentage) energy contribution of the aerobic (WAER, WAERint), anaerobic lactic (WBLC, WBLCint), and anaerobic alactic (WPCr, WPCrint) systems were calculated by PCr-La-O2 method for 30-15IFT-CONT and 30-15IFT. Results: No difference in peak oxygen uptake was found between 30-15IFT and 30-15IFT-CONT (60.6 [6.6] vs 60.5 [5.1] mL·kg−1·min−1, P = .165, d = 0.20), whereas speed at the last completed stage was higher in 30-15IFT (18.3 [1.4] vs 16.1 [1.0] km·h−1, P d = 1.17). Metabolic energy was also higher in 30-15IFT (1224.2 [269.6] vs 772.8 [63.1] kJ, P d = 5.60), and metabolic profiles differed substantially for aerobic (30-15IFT = 67.2 [5.2] vs 30-15IFT-CONT = 85.2% [2.5%], P d = −4.01), anaerobic lactic (30-15IFT = 4.4 [1.4] vs 30-15IFT-CONT = 6.2% [1.8%], P d = −1.04), and anaerobic alactic (30-15IFT = 28.4 [4.7] vs 30-15IFT-CONT = 8.6% [2.1%], P d = 5.43) components. Conclusions: Both 30-15IFT and 30-15IFT-CONT are mainly fueled by aerobic energy, but their metabolic profiles differ substantially in both aerobic and anaerobic alactic energy contribution. Due to the presence of inter-effort recovery, intermittent shuttle runs rely to a higher extent on anaerobic alactic energy and a fast, aerobic replenishment of PCr during the short breaks between shuttles.

Purpose: To evaluate the metabolic relevance of type of locomotion in anaerobic testing by analyzing and comparing the metabolic profile of the Bosco Continuous Jumping Test (CJ30) with the corresponding profile of the Wingate Anaerobic Test (WAnT). Methods: A total of 11 well-trained, male team-sport athletes (age = 23.7 [2.2] y, height = 184.1 [2.8] cm, weight = 82.4 [6.4] kg) completed a CJ30 and WAnT each. During the WAnT, power data and revolutions per minute were recorded, and during the CJ30, jump height and jumping frequency were recorded. In addition, oxygen uptake and blood lactate concentration were assessed, and metabolic profiles were determined via the PCr-LA-O2 method. Results: In the CJ30, metabolic energy was lower (109.3 [18.0] vs 143.0 [13.1] kJ, P d = −2.302), while peak power (24.8 [4.4] vs 11.8 [0.5] W·kg−1, P d = 3.59) and mean power (20.8 [3.6] vs 9.1 [0.5] W·kg−1, P d = 4.14) were higher than in the WAnT. The metabolic profiles of the CJ30 (aerobic energy = 20.00% [4.7%], anaerobic alactic energy [WPCr] = 45.6% [4.5%], anaerobic lactic energy = 34.4% [5.2%]) and the WAnT (aerobic energy = 16.0% [3.0%], anaerobic alactic WPCr = 34.5% [5.0%], anaerobic lactic energy = 49.5% [3.3%]) are highly anaerobic. Absolute energy contribution for the CJ30 and WAnT was equal in WPCr (49.9 [11.1] vs 50.2 [11.2] kJ), but anaerobic lactic energy (37.7 [7.7] vs 69.9 [5.3] kJ) and aerobic energy (20.6 [5.7] vs 23.0 [4.0] kJ) were higher in the WAnT. Mechanical efficiency was substantially higher in the CJ30 (37.9% [4.5%] vs 15.6% [1.0%], P d = 6.86), while the fatigue index was lower (18.5% [3.8%] vs 23.2% [3.1%], P d = −1.38) than in the WAnT. Conclusions: Although the anaerobic share in both tests is similar and predominant, the CJ30 primarily taxes the WPCr system, while the WAnT more strongly relies on the glycolytic pathway. Thus, the 2 tests should not be used interchangeably, and the type of locomotion seems crucial when choosing an anaerobic test for a specific sport.

Kinases are frequently studied in the context of anticancer drugs. Their involvement in cell responses, such as proliferation, differentiation, and apoptosis, makes them interesting subjects in multitarget drug design. In this study, a workflow is presented that models the bioactivity spectra for two panels of kinases: (1) inhibition of RET, BRAF, SRC, and S6K, while avoiding inhibition of MKNK1, TTK, ERK8, PDK1, and PAK3, and (2) inhibition of AURKA, PAK1, FGFR1, and LKB1, while avoiding inhibition of PAK3, TAK1, and PIK3CA. Both statistical and structure-based models were included, which were thoroughly benchmarked and optimized. A virtual screening was performed to test the workflow for one of the main targets, RET kinase. This resulted in 5 novel and chemically dissimilar RET inhibitors with remaining RET activity of 50 value of 5.1 for the most active compound. The experimental validation of inhibitors for RET strongly indicates that the multitarget workflow is able to detect novel inhibitors for kinases, and hence, this workflow can potentially be applied in polypharmacology modeling. We conclude that this approach can identify new chemical matter for existing targets. Moreover, this workflow can easily be applied to other targets as well.

Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development. (C) 2020 Published by Elsevier Ltd.

For food emulsions containing enzymatically modified egg yolk, the conventional Folch extraction does not fully recover the polar lysophospholipids. This can be overcome by repeated methanol extractions. After solvent evaporation, the extracted (lyso)phospholipids are solubilized into mixed micelles with cholate as a detergent. The solubilized (lyso)phospholipids can be accurately quantified by 31P NMR with recoveries ranging between 96% and 108%. Detection at a high (16.4 T) relative to a mainstream (9.4 T) magnetic field strength did not offer a significant advantage since the slow molecular tumbling of the mixed micelles increased line widths. This was due to field-strength-dependent chemical shift anisotropy relaxation. Method precision is similar at 9.4 and 16.4 T, with within-laboratory reproducibilities of 7-22% and 12-25%, respectively. The method can be implemented as a routine analytical procedure at 9.4 T (400 MHz NMR spectrometer), and the limits of detection and quantification are adequate for the verification of the standard of identity of a mayonnaise prepared with enzymatically modified egg yolk.

Reported are the syntheses, crystal structures, and electronic structures of six rare-earth metal-lithium stannides with the general formulas RE3Li4- xSn4+ x (RE = La-Nd, Sm) and Eu7Li8- xSn10+ x. These new ternary compounds have been synthesized by high-temperature reactions of the corresponding elements. Their crystal structures have been established using single-crystal X-ray diffraction methods. The RE3Li4- xSn4+ x phases crystallize in the orthorhombic body-centered space group Immm (No. 71) with the Zr3Cu4Si4 structure type (Pearson code oI22), and the Eu7Li8- xSn10+ x phase crystallizes in the orthorhombic base-centered space group Cmmm (No. 65) with the Ce7Li8Ge10 structure type (Pearson code oC50). Both structures can be consdered as part of the [RESn2] n[RELi2Sn] m homologous series, wherein the structures are intergrowths of imaginary RESn2 (AlB2-like structure type) and RELi2Sn (MgAl2Cu-like structure type) fragments. Close examination the structures indicates complex occupational Li-Sn disorder, apparently governed by the drive of the structure to achieve an optimal number of valence electrons. This conclusion based on experimental results is supported by detailed electronic structure calculations, carried out using the tight-binding linear muffin-tin orbital method.

Introduction Inducible T cell co-stimulator (ICOS) is a co-stimulatory receptor that is important for promoting immune activation and function. Despite reported clinical activity and a wide range of non-clinical studies supporting a role for ICOS in lymphocyte activation, proliferation and pro-inflammatory cytokine secretion, little is known regarding the potential of monoclonal agonist antibody-mediated ICOS signaling to drive cytotoxic T cell infiltration into tumors. Feladilimab (GSK3359609) is a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials. Here, we used PET/CT imaging to evaluate CD8+ T cell infiltration following treatment with a rodent surrogate of feladilimab (7E.17G9 mouse [m] IgG1) alone or in combination with anti-PD-1 mAb (RMP-14 rat IgG2a) in a syngeneic model of breast cancer (EMT6). Method Female BALB/c mice with established tumors (~150 mm3) received 10µg, IP of either IgG control mAbs, ICOS mAb, or ICOS + PD-1 mAbs on day 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 & 48 hrs post IV dose of 89Zr labeled CD8 minibody (IAB42M1-14, ImaginAb, CA) on day 0, 3, 5, 9, or 14. In addition to the CD8 minibody uptake in tumor & tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images. Top ranked features were used for hierarchical clustering to identify treatment effects. Results Tumor size regressed in all treated groups relative to IgG control, with a number of mice clearing tumors (ICOS: 4 mice, ICOS + PD-1: 9 mice). The in vivo uptake of CD8 minibody in TDLN was significantly higher in the ICOS + PD-1 group on day 4, 6, & 7 relative to IgG control P Conclusions Herein, we demonstrated for the first time that treatment of tumor-bearing mice with ICOS agonist mAb alone or in combination with PD-1 blockade can increase CD8+ T cell infiltration into tumors & TDLN, and is correlated with reduced tumor burden. Notably, radiomics features predicted an effect of treatment on CD8+ T cell infiltration earlier than the detection of absolute changes in the CD8 minibody uptake in tumor & TDLN. Overall, these data support the ongoing pivotal investigation of feladilimab. Moreover, this translational imaging method may be a useful tool to non-invasively monitor CD8+ T cell in response to immunotherapies and understand the temporal relationship between CD8+ T cell flux in tumor and in TDLN. Citation Format: Hasan Alsaid, Shih-Hsun Cheng, Meixia Bi, Mary V. Rambo, Tinamarie Skedzielewski, Bao Hoang, Sunish Mohanan, Andrew Gehman, Chih-Yang Hsu, Minh Doan, Fang Xie, M. Reid Groseclose, Christopher Hopson, Sara Brett, Ian A. Wilson, Andrew Nicholls, Marc Ballas, Jeremy D. Waight, Beat M. Jucker, Axel Hoos. Immuno-PET monitoring of CD8+ T cell infiltration post anti-ICOS agonist antibody treatment alone and in combination with PD-1 blocking antibody using a 89Zr anti-CD8+ mouse minibody in EMT 6 syngeneic tumor mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2816.

In recent years, antibody-based immunotherapies targeting immune checkpoint proteins cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have revolutionized the treatment of cancer. Despite durable clinical responses in many indications, a significant number of patients fail to yield meaningful benefit, emphasizing the need to identify novel and/or complementary therapeutic interventions (e.g., alternative immune checkpoints, cytotoxic agents, etc.). Inducible T cell Co-Stimulator (ICOS/CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells shortly after T cell receptor (TCR) activation. A growing body of non-clinical and clinical evidence supports the concept of antibody-mediated induction of ICOS signaling as a means to promote durable antitumor responses, particularly in collaboration with other checkpoint immunotherapies. To support ongoing and future clinical studies for feladilimab (GSK3359609), a non-depleting IgG4 ICOS agonist antibody currently being evaluated in pivotal clinical trials, we conducted a series of in vivo studies using a rodent surrogate (7E.17G9 mouse [m]IgG1; analogous non-depleting Fc) in ICOS-sensitive (EMT6 breast carcinoma) and ICOS-insensitive (CT26 colon carcinoma) tumor models. Female BALB/c mice with pre-established EMT6 tumors, exhibited partial responses to GSK'609 surrogate/tool antibody (7E.17G9 mIgG1), with ~20% (range 0-40%) of mice demonstrating tumor clearance. Concurrent administration of anti-PD-1 (RMP1-14 rat [r]IgG2a) and CTLA-4 (9D9 mIgG2b) dramatically improved tumor growth inhibition and survival. Notably, a triplet of TIM-3 blockade (RMT3-23 rIgG2a) in concert with ICOS and PD-1 mAbs further enhanced the survival of EMT6 tumor-bearing mice, suggesting complementary therapeutic mechanisms. In addition to combination with immune checkpoint blockade, we also explored the potential of ICOS co-stimulation with different focal irradiation (FRT) doses and frequencies in both EMT6 and CT26 tumor models. Intriguingly, we observed dramatic differences in response to concurrent FRT and ICOS mAb depending on the tumor model and associated FRT dose and frequency. Further, we identified a FRT dose threshold required for ICOS agonist appreciable combination responses, enabling exploration of additional immunotherapeutic combinations on top of ICOS/FRT. Collectively, these data support the combination of ICOS co-stimulation with different checkpoint immunotherapies (doublets and triplets), several of which are currently being evaluated in clinical studies. Moreover, these data demonstrate potential of ICOS co-stimulation with cytotoxic strategies, such as FRT - providing various dosing considerations and a framework future therapeutic intervention strategies. Citation Format: Jeremy D. Waight, Meixia Bi, Chris Hopson, Sapna Yadavilli, Kenneth W. Hance, Marc Ballas, Axel Hoos. ICOS co-stimulation in combination immune checkpoint blockade and/or dose-optimized focal irradiation results in enhanced tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1849.

Purpose: To analyze the energetic profiles of the Yo-Yo Intermittent Recovery Tests 1 and 2 (YYIR1 and YYIR2). Methods: Intermittent running distance (IR1D and IR2D), time to exhaustion (IR1T and IR2T), and total recovery time between shuttles (IR1R and IR2R) were measured in 10 well-trained male athletes (age 24.4 [2.0] y, height 182 [1] cm, weight 75.8 [7.9] kg). Respiratory gases and blood lactate (BLC) were obtained preexercise, during exercise, and until 15 min postexercise. Metabolic energy, average metabolic power , and energy share (percentage of aerobic [WAER], anaerobic lactic [WBLC], and anaerobic alactic energy system [WPCr]) were calculated using the PCr-La-O2 method. Results: Peak oxygen consumption was possibly higher in YYIR2 (60.3 [5.1] mL·kg−1·min−1) than in YYIR1 (P = .116, 57.7 [4.5] mL·kg−1·min−1, d = −0.58). IR1D, IR1T, and IR1R were very likely higher than IR2D, IR2T, and IR2R, respectively (P d = −2.83; P d = −3.03; and P d = −2.83). Metabolic energy was most likely lower in YYIR2 than in YYIR1 (P d = 3.24). Average metabolic power was most likely higher in YYIR2 than in YYIR1 (P −1, d = 3.54). When considering aerobic phosphocreatine restoration during breaks between shuttles, WAER (P = .693, 49% [10%] vs 48% [5%], d = −0.16) was similar, WPCr (P = .165, 47% [11%] vs 42% [6%], d = −0.54) possibly higher, and WBLC (P d = 1.95) almost certainly lower in YYIR1 than in YYIR2. Conclusions: WAER and WPCr are predominant in YYIR1 and YYIR2 with almost identical WAER. Higher IR1D and IR1T in YYIR1 result in higher metabolic energy but lower average metabolic power and slightly lower peak oxygen consumption. Higher IR1R allows for higher reliance on WPCr in YYIR1, while YYIR2 requires a higher fraction of WBLC.

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

Summary Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

Malaria, a disease endemic in many tropical and subtropical regions, is caused by infection of the erythrocyte by the apicomplexan parasite Plasmodium. Host-cell invasion is a complex process but two Plasmodium proteins, Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck protein complex (RON), play a key role. AMA1, present on the surface of the parasite, binds tightly to the RON2 component of the RON protein complex, which is inserted into the erythrocyte membrane during invasion. Blocking the AMA1-RON2 interaction with antibodies or peptides inhibits invasion, underlining its importance in the Plasmodium life cycle and as a target for therapeutic strategies. We describe the crystal structure of the complex formed between AMA1 from P. vivax (PvAMA1) and a peptide derived from the externally exposed region of P. vivax RON2 (PvRON2sp1), and of the heterocomplex formed between P. falciparum AMA1 (PfAMA1) and PvRON2sp1. Binding studies show that the affinity of PvRON2sp1 for PvAMA1 is weaker than that previously reported for the PfRON2sp1-PfAMA1 association. Moreover, while PvRON2sp1 shows strong cross-reactivity with PfAMA1, PfRON2sp1 displays no detectable interaction with PvAMA1. The structures show that the equivalent residues PvRON2-Thr2055 and PfRON2-Arg2041 largely account for this pattern of reactivity.

Although fresh frozen tissue is the optimal source of nucleic acids for sequencing, obtaining such tissue on a population scale is cost and time prohibitive; however, DNA and RNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue has been problematic, yielding low quantity and quality DNA and RNA. Through a pilot program, the National Cancer Institute is developing a Surveillance, Epidemiology and End Results (SEER)-linked Virtual Tissue Repository (VTR) for obtaining deidentified FFPE tissue and clinical history. One such pancreatic ductal adenocarcinoma (PDAC) pilot project requested FFPE tumor and normal tissue (duodenum, lymph node, or spleen) and clinical history data on 24 matched pairs, comparing PDAC patients who survived at least five years since diagnosis with patients who died within two years through six SEER registries. A pancreatic pathologist annotated H&E stained slides for tissue dissection of corresponding unstained tumor (DNA and RNA) and normal (DNA only) tissue slides. More than 30% of tumor and normal tissue specimens had DNA with high quality for sequencing (≥20% amplifiable DNA), and the majority of DNA samples had medium to high quality (amplifiable DNA ≥ 10%) for sequencing. Although less DNA (quantity) was obtained from tumor tissue than normal, the percent amplifiable DNA was similar. DNA quantity was independent of tissue age (4 to 18 years); however, DNA integrity number (DIN) was significantly decreased in older tissue. Although RNA quantity was independent of tissue age, RNA quality (DV200) was significantly decreased in older tissue. There was no significant association between RNA quantity and quality. From the 24 pairs, sufficient DNA was obtained for whole genome sequencing (WGS) for 14 pairs, whole exome sequencing (WES) for eight pairs, and methylation studies on three pairs. Four pairs that had cases with Citation Format: Yao Yuan, Alison Van Dyke, Valentina Petkov, Aatur Singhi, William Hoos, Lola Rahib, Lynn Matrisian, Lynne Penberthy. DNA and RNA from formalin-fixed, paraffin-embedded pancreatic cancer tissue for sequencing: A pilot study of the SEER-linked Virtual Tissue Repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-177.

Background: GSK3359609 (GSK609) is an inducible T cell co-stimulator (ICOS) agonist antibody and tremelimumab (treme) is a cytotoxic T-lymphocyte-antigen 4 (CTLA-4) antagonist antibody. Nonclinical and clinical studies have shown that ICOS expression on T cells is upregulated after anti-CTLA-4 treatment (tx). ICOS+ T cells have a positive association with overall survival (OS) in treme treated chemotherapy-resistant malignant mesothelioma and melanoma patients (pts). Therefore, the combination of these 2 drugs may provide greater antitumoral response than either drug alone (Coutzac C: AACR 2019 meeting abstract). Methods: This is a Phase I/II, open-label, 2-part study. In Part 1, N=~24 pts will be enrolled. The study is designed to assess treme starting at 75 mg followed by GSK609 starting at 8 mg. At dose level (DL) 1, one pt who will receive the lowest doses of both drugs will be enrolled. If no dose limiting toxicity (DLT) is observed in the DLT observation period of 28 days, then 1-3 pts will be enrolled in DL2 - DL6 in a zone-based fashion (zones include the DLs with the next highest dose of one of the two drugs - 2DL are included in each zone in this study) with different dose combinations of the drugs being evaluated. The dose of 1 of the 2 drugs will be escalated with each new DL up to the highest dose of each. If ≥ grade 2 tx-related AEs occur at DL1 or 2, additional pts will be enrolled to collect more safety information. If DLTs are observed, the bivariate Continual Reassessment Method (CRM) model will be used to guide dose recommendations. The primary objective of Part 1 will be to assess safety. Additional pts may be enrolled in Part 1 to further evaluate pharmacokinetics/pharmacodynamics (PK/PD). Part 2 is a randomized expansion enrolling recurrent/metastatic head and neck squamous cell carcinoma pts who have disease progression after platinum chemotherapy and anti-programmed death receptor protein-1 (PD-1)/anti-programmed death-ligand 1 (PD-L1) therapy, in combination or separately. Pts may not have had ≥4 lines of prior tx in either Part 1 or 2. In Part 2, pts will be randomized 2:1 to the doses of GSK609 and treme (n=60) selected from Part 1 or to the Investigator’s choice of SOC single-agent therapy (paclitaxel, docetaxel or cetuximab) (n=30). The primary objective in Part 2 is to evaluate overall survival and the primary analysis is to perform Bayesian predictive probability of success for a future hypothetical Phase III study based on survival data. Secondary endpoints include safety, ORR, PFS, DOR, and PK while pharmacodynamic and other biomarker analyses constitute exploratory endpoints. Pts will be treated for up to 2 years with survival follow-up for up to 2 years post-tx. This study has enrolled 1 pt. Study NCT03693612 funded by GSK. Citation Format: Aaron Hansen, Raghad Abdul-Karim, Naiyer Rizvi, Danny Rischen, John Hilton, Zujun Li, Patrick Ott, Natalie Karpinich-Fedoriw, Sapna Yadavilli, Xiaowei Wang, Laurel Adams, Melody Wyres, Charles Ferte, Marc Ballas, Axel Hoos, Daniel Zandberg. A Phase I/II, open-label, two part study of GSK3359609 in combination with tremelimumab in participants with selected, advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT166.

Traditional laser-matter spectroscopy techniques fail to accurately analyze photoelectrons and ions from ultrahigh intensity studies with terawatt and petawatt laser systems. We present a magnetic deflection, photoelectron spectrometer for ultrahigh intensity laser interactions with atoms and molecules in the single atom/molecule limit. Spectrometer fabrication and calibration, and noise background are presented as well as example photoelectron spectra for argon and chloromethane over an energy range from 20 keV to 2 MeV.

Transition-state mimicry is increasingly important both to understand enzyme mechanism and to direct the synthesis of putative therapeutic agents. X-ray crystallography is able to provide vital information on the interactions between an enzyme and the potential inhibitor. Here we report the structures, at approximately 2 A resolution, of a family GH1 beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus, in complex with both covalently (derived from 2-fluoro-glycosides) and noncovalently (hydroximolactam) bound inhibitors. The enzyme has broad specificity, accommodating both gluco- and galacto-configured substrates, and the crystallographic data demonstrate that the only difference in the way these ligands bind lies in the interactions between Gln18, Glu432, and Trp433, and the hydroxyl group at the O3 and O4 positions. Inhibition by the differently configured ligands was also shown to be extremely similar, with K(i) values of 1.04 and 1.08 microM for the gluco and galacto epimers, respectively. The noncovalently bound inhibitors have a trigonal anomeric carbon, adopt a (4)H(3) (half-chair) conformation, and an interaction is formed between O2 and the catalytic nucleophile, all of which contribute to (partial) mimicry of the oxocarbenium-ion-like transition state. The inhibition of the beta-glycosidase from S. solfataricus by hydroximolactams is discussed in light of the emerging work on family GH1 glycosidase inhibition by a spectrum of putative transition-state mimics.

Summary The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.

Garcia, Ana Maria, Anne B. Hoos, and Silvia Terziotti, 2011. A Regional Modeling Framework of Phosphorus Sources and Transport in Streams of the Southeastern United States. Journal of the American Water Resources Association (JAWRA) 47(5):991-1010. DOI: 10.1111/j.1752-1688.2010.00517.x Abstract: We applied the SPARROW model to estimate phosphorus transport from catchments to stream reaches and subsequent delivery to major receiving water bodies in the Southeastern United States (U.S.). We show that six source variables and five land-to-water transport variables are significant (p

Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α.

We demonstrate a compact and cost-effective setup to generate broadband THz radiation. As pump source we use a diode-pumped solid-state femtosecond oscillator or a femtosecond fiber laser system, partially in combination with an optical parametric oscillator. For the THz generation we utilize optical rectification in gallium phosphide (GaP) and gallium arsenide (GaAs). The THz power is on the order of 1 μW and we demonstrate imaging and spectral measurements with this setup.

Accumulation of amyloid β-protein (Aβ) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aβ peptides readily form β-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aβ peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952–9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720–4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aβ and inhibited Aβ fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aβ fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aβ is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aβ fibrillogenesis. Under conditions comparable with those used for Aβ, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aβ in primary cortical neurons. Our findings suggest that inhibition of Aβ fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.

G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1−/− mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1−/− mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1−/− mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1−/− mice on HFD displayed increased hepatic steatosis when compared with Gpbar1+/+ males and Gpbar1−/− females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.

The performance of many contemporary detergent products critically depends on polymers. Water-soluble polycarboxylates represent an important class of detergent polymers, and their quantitative assessment in detergent matrices stands as a considerable challenge. The presence of high levels of surfactants is a major complication, due to the strong tendency of surfactants to form micelles and to interact with the polymers. First, we addressed critical steps in the subsequent combined use of liquid extraction and off-line size exclusion chromatography-nuclear magnetic resonance (SEC-NMR) for identification and quantification of polycarboxylates in detergent products. Next, the different steps in the off-line SEC-NMR procedure were optimized with respect to precision and accuracy. This resulted in recoveries of more than 80% for maleic acid/acrylic acid copolymers; in detergent products a proportional bias of 30% is achieved. The method showed good precision with a relative standard deviation of within-laboratory reproducibility between 5% and 14%.

The deposition of amyloid beta-protein (Abeta) fibrils into plaques within the brain parenchyma and along cerebral blood vessels is a hallmark of Alzheimer's disease. Abeta peptides are produced through the successive cleavage of the Abeta precursor protein by beta- and gamma-secretase, producing peptides between 39 and 43 amino acids in length. The most common of these are Abeta40 (the most abundant) and Abeta42. Abeta42 is more fibrillogenic than Abeta40 and has been implicated in early Abeta plaque deposition. Our previous studies determined that myelin basic protein (MBP) was capable of inhibiting fibril formation of a highly fibrillogenic Abeta peptide containing both E22Q (Dutch) and D23N (Iowa) mutations associated with familial forms of cerebral amyloid angiopathy [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961]. In this study, we show through a combination of biochemical and ultrastructural techniques that MBP is also capable of inhibiting the beta-sheet fibrillar assembly of the normal Abeta42 peptide. These findings suggest that MBP may play a role in regulating the deposition of Abeta42 and thereby also may regulate the early formation of amyloid plaques in Alzheimer's disease.

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 °C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 μmol/L of modified HSA could be obtained by applying 10 μL of NAPQI-HSA sample. © 2007 Elsevier B.V. All rights reserved.

Deposition of fibrillar amyloid beta-protein (Abeta) in the brain is a prominent pathological feature of Alzheimer disease and related disorders, including familial forms of cerebral amyloid angiopathy (CAA). Mutant forms of Abeta, including Dutch- and Iowa-type Abeta, which are responsible for familial CAA, deposit primarily as fibrillar amyloid along the cerebral vasculature and are either absent or present only as diffuse non-fibrillar plaques in the brain parenchyma. Despite the lack of parenchymal fibril formation in vivo, these CAA mutant Abeta peptides exhibit a markedly increased rate and extent of fibril formation in vitro compared with wild-type Abeta. Based on these conflicting observations, we sought to determine whether brain parenchymal factors that selectively interact with and modulate CAA mutant Abeta fibril assembly exist. Using a combination of immunoaffinity chromatography and mass spectrometry, we identified myelin basic protein (MBP) as a prominent brain parenchymal factor that preferentially binds to CAA mutant Abeta compared with wild-type Abeta. Surface plasmon resonance measurements confirmed that MBP bound more tightly to Dutch/Iowa CAA double mutant Abeta than to wild-type Abeta. Using a combination of biochemical and ultrastructural techniques, we found that MBP inhibited the fibril assembly of CAA mutant Abeta. Together, these findings suggest a possible role for MBP in regulating parenchymal fibrillar Abeta deposition in familial CAA.

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 μmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 μl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures. © 2005 Elsevier B.V. All rights reserved.

We describe a new algorithm for design of strand sets, for use in DNA computations or universal microarrays. Our algorithm can design sets that satisfy any of several thermodynamic and combinatorial constraints, which aim to maximize desired hybridizations between strands and their complements, while minimizing undesired cross-hybridizations. To heuristically search for good strand sets, our algorithm uses a conflict-driven stochastic local search approach, which is known to be effective in solving comparable search problems. The PairFold program of Andronescu et al. [M. Andronescu, Z. C. Zhang and A. Condon (2005) J. Mol. Biol., 345, 987-1001; M. Andronescu, R. Aguirre-Hernandez, A. Condon, and H. Hoos (2003) Nucleic Acids Res., 31, 3416-3422.] is used to calculate the minimum free energy of hybridization between two mismatched strands. We describe new thermodynamic measures of the quality of strand sets. With respect to these measures of quality, our algorithm consistently finds, within reasonable time, sets that are significantly better than previously published sets in the literature.

An algorithm is presented for the generation of sets of non-interacting DNA sequences, employing existing thermodynamic models for the prediction of duplex stabilities and secondary structures. A DNA 'word' structure is employed in which individual DNA 'words' of a given length (e.g. 12mer and 16mer) may be concatenated into longer sequences (e.g. four tandem words and six tandem words). This approach, where multiple word variants are used at each tandem word position, allows very large sets of non-interacting DNA strands to be assembled from combinations of the individual words. Word sets were generated and their figures of merit are compared to sets as described previously in the literature (e.g. 4, 8, 12, 15 and 16mer). The predicted hybridization behavior was experimentally verified on selected members of the sets using standard UV hyperchromism measurements of duplex melting temperatures (T(m)s). Additional experimental validation was obtained by using the sequences in formulating and solving a small example of a DNA computing problem.

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.

The syntheses and in vitro antimalarial screening of 50 bridged, bicyclic endoperoxides of types 9-13 are reported. In contrast to antimalarial trioxanes of the artemisinin family, but like yingzhaosu A and arteflene, the peroxide function of compounds 9-13 is contained in a 2,3-dioxabicyclo[3.3.1]nonane system 6. Peroxides 9 and 10 (R(1) = OH) are readily available through a multicomponent, sequential, free-radical reaction involving thiol-monoterpenes co-oxygenation (a TOCO reaction). beta-Sulfenyl peroxides 9 and 10 (R(1) = OH) are converted into beta-sulfinyl and beta-sulfonyl peroxides of types 11-13 by controlled S-oxidation and manipulation of the tert-hydroxyl group through acylation, alkylation, or dehydration followed by selective hydrogenation. Ten enantiopure beta-sulfonyl peroxides of types 12 and 13 exhibit in vitro antimalarial activity comparable to that of artemisinin (IC(50) = 6-24 nM against Plasmodium falciparum NF54). In vivo testing of a few selected peroxides against Plasmodium berghei N indicates that the antimalarial efficacies of beta-sulfonyl peroxides 39a, 46a, 46b, and 50a are comparable to those of some of the best antimalarial drugs and are higher than artemisinin against chloroquine-resistant Plasmodium yoelii ssp. NS. In view of the nontoxicity of beta-sulfonyl peroxides 39a, 46a, and 46b in mice, at high dosing, these compounds are regarded as promising antimalarial drug candidates.

Ezetimibe (1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4-hydroxyphenyl)-2-azetidinone) and its analog SCH48461 are potent and selective cholesterol absorption inhibitors that inhibit the transport of cholesterol across the intestinal wall, thereby lowering plasma cholesterol. After a dose response for ezetimibe in rats was established, experiments were conducted to determine whether acute administration could alter hepatic or intestinal cholesterol synthesis. To determine whether this class of intestinal cholesterol absorption inhibitors could discriminate between newly synthesized cholesterol in the intestine versus exogenously administered cholesterol, rats were intraduodenally dosed with 14C-cholesterol and 3H-mevalonate, and mesenteric lymph was analyzed for radiolabeled cholesterol and cholesteryl ester content. Ezetimibe attenuated diet-induced hypercholesterolemia 60–94% at doses of 0.1–3 mg kg−1 in rats. A single administration of ezetimibe did not have a direct effect on intestinal or hepatic cholesterol synthesis, while ketoconazole significantly inhibited cholesterol synthesis after a single dose. The ezetimibe analog, SCH48461, inhibited the movement of exogenously administered cholesterol into lymph, but did not affect the appearance of newly synthesized cholesterol into lymph. These data suggest that this class of cholesterol absorption inhibitors does discriminate by blocking the movement of exogenous cholesterol in the enterocyte before it reaches the intracellular cholesterol pool to be incorporated into intestinal lipoproteins, without affecting the incorporation of newly synthesized cholesterol into intestinal lipoproteins. British Journal of Pharmacology (2003) 138, 1459–1464. doi:10.1038/sj.bjp.0705187

Synthesis of β-sulfenyl endoperoxides 9 was achieved by a four component sequential free radical reaction based on the application of the thiol-olefin-co-oxygenation reaction to monoterpenes, followed by in situ treatment with triphenylphosphine. β-Sulfenyl endoperoxides 9 were oxidized with m-CPBA to β-sulfonyl endoperoxides 10 . This process provides an efficient method for the preparation of peroxides containing the 2,3-dioxabicyclo[3.3.1]nonane system ( 2 ) characteristic of antimalarial agents of the yingzhaosu A ( 3 ) family. A simple NMR diagnostic tool for the identification of stereoisomers is described.

Ln7Zn21+xSi2-x (Ln: Ce with x = 0.95, Pr with x = 0.09, Nd with x = 0.53) are prepared by melting of the elements (evacuated silica tube, 1073 K, 3 d).

Background Mouse models are used in the study of human disease. Despite well-known homologies, the difference in immune response between mice and humans impacts the application of data derived from mice to human disease outcomes. Nitric oxide synthase-2 (NOS2) is a key gene that displays species-specific outcomes via altered regulation of the gene promoter and via post-transcriptional mechanisms in humans that are not found in mice. The resulting levels of NO produced by activation of human NOS2 are different from the levels of NO produced by mouse Nos2. Since both tissue redox environment and immune responsiveness are regulated by the level of NO and its interactions, we investigated the significance of mouse and human differences on brain oxidative stress and on immune activation in HuNOS2tg/mNos2-/- mice that express the entire human NOS2 gene and that lack a functional mNos2 compared to wild type (WT) mice that express normal mNos2. Methods/results Similarly to human, brain tissue from HuNOS2tg/mNos2-/- mice showed the presence of a NOS2 gene 3′UTR binding site. We also identified miRNA-939, the binding partner for this site, in mouse brain lysates and further demonstrated reduced levels of nitric oxide (NO) typical of the human immune response on injection with lipopolysaccharide (LPS). HuNOS2tg/mNos2-/- brain samples were probed for characteristic differences in redox and immune gene profiles compared to WT mice using gene arrays. Selected genes were also compared against mNos2-/- brain lysates. Reconstitution of the human NOS2 gene significantly altered genes that encode multiple anti-oxidant proteins, oxidases, DNA repair, mitochondrial proteins and redox regulated immune proteins. Expression levels of typical pro-inflammatory, anti-inflammatory and chemokine genes were not significantly different with the exception of increased TNFα and Ccr1 mRNA expression in the HuNOS2tg/mNos2-/- mice compared to WT or mNos2-/- mice. Conclusions NO is a principle factor in establishing the tissue redox environment and changes in NO levels impact oxidative stress and immunity, both of which are primary characteristics of neurodegenerative diseases. The HuNOS2tg/mNos2-/- mice provide a potentially useful mechanism to address critical species- specific immune differences that can impact the study of human diseases.

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

Ezetimibe (SCH58235) is a potent, selective, cholesterol absorption inhibitor. The objective of this study was to determine whether ezetimibe reduces plasma cholesterol and inhibits atherogenesis in apolipoprotein E knockout (apoE−/−) mice. Cholesterol absorption was inhibited by >90% at doses of ezetimibe >3 mg/kg in apoE−/− mice. Atherosclerosis and lipoprotein changes were determined in apoE−/− mice fed a high-fat (0.15% cholesterol) “western” diet, a low-fat (0.15% cholesterol) diet, or a semisynthetic cholesterol-free diet with or without ezetimibe (5 mg/kg per day) for 6 months. Ezetimibe reduced plasma cholesterol levels from 964 to 374 mg/dL, from 726 to 231 mg/dL, and from 516 to 178 mg/dL in the western, low-fat, and cholesterol-free diet groups, respectively. The reductions occurred in the very low density and low density lipoprotein fractions, whereas high density lipoprotein cholesterol levels were increased by ezetimibe treatment. Ezetimibe reduced aortic atherosclerotic lesion surface area from 20.2% to 4.1% in the western diet group and from 24.1% to 7.0% in the low-fat cholesterol diet group. Ezetimibe reduced carotid artery atherosclerotic lesion cross-sectional area by 97% in the western and low-fat cholesterol groups and by 91% in the cholesterol-free group. Ezetimibe inhibits cholesterol absorption, reduces plasma cholesterol, increases high density lipoprotein levels, and inhibits the progression of atherosclerosis under western, low-fat, and cholesterol-free dietary conditions in apoE−/− mice. Although apoE−/− mice are more hypercholesterolemic than are humans and low density lipoprotein reductions with ezetimibe are not as pronounced clinically, ezetimibe may inhibit atherogenesis in individuals consuming restricted-fat or western diets.