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Pancreatic microcirculatory dysfunction emerged as a novel mechanism in the development of hypertension. However, the changes of pancreatic microcirculation profiles in hypertension remain unknown. Pancreatic microcirculatory blood distribution pattern and microvascular vasomotion of spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs) were determined by laser Doppler. Wavelet transform analysis was performed to convert micro-hemodynamic signals into time-frequency domains, based on which amplitude spectral scalograms were constructed. The amplitudes of characteristic oscillators were compared between SHRs and WKYs. The expression of eNOS was determined by immunohistochemistry, and plasma nitrite/nitrate levels were measured by Griess reaction. Additionally, endothelin-1, malondialdehyde, superoxide dismutase and interleukin-6 were determined by enzyme-linked immunosorbent assay. SHRs exhibited a lower scale blood distribution pattern with decreased average blood perfusion, frequency and amplitude. Wavelet transform spectral analysis revealed significantly reduced amplitudes of endothelial oscillators. Besides reduced expression of eNOS, the blood microcirculatory chemistry complements micro-hemodynamic profiles as demonstrated by an increase in plasma nitrite/nitrate, endothelin-1, malondialdehyde, interleukin-6 and a decrease of superoxide dismutase in SHRs. Here, we described abnormal pancreatic microcirculation profiles in SHRs, including disarranged blood distribution pattern, impaired microvascular vasomotion and reduced amplitudes of endothelial oscillators.

BACKGROUND Emerging evidence indicates that the pancreas serves as a major source of degrading protease activities and that uncontrolled proteolytic receptor cleavage occurs under hypertensive conditions, which leading to systemic dysfunction and end-organic damage. However, changes in pancreatic microcirculation profiles during the progression of hypertension remain unknown. METHODS Pancreatic microcirculatory blood distribution patterns and microvascular vasomotion of spontaneously hypertensive rats (SHRs) and normotensive control Wistar Kyoto rats at 5, 8, 13, and 18 weeks of age were determined. Wavelet transform analysis was performed to convert pancreatic microhemodynamic signals into time–frequency domains and construct 3-dimensional spectral scalograms. The amplitudes of characteristic oscillators including endothelial, neurogenic, myogenic, respiratory, and cardiac oscillators were compared among groups. Plasma nitrite/nitrate levels were measured using a Griess reaction. Additionally, endothelin-1, malondialdehyde, superoxide dismutase, and interleukin-6 levels were determined by enzyme-linked immunosorbent assay. RESULTS SHRs exhibited a reduced blood distribution pattern with progressively decreased average blood perfusion, amplitude, and frequency of microvascular vasomotion. Wavelet transform spectral analysis revealed significantly reduced amplitudes of endothelial oscillators from 8- to 18-week-old SHRs. Additionally, the blood microcirculatory chemistry complements explained the microhemodynamic profiles partially, as demonstrated by an increase in plasma nitrite/nitrate, endothelin-1, malondialdehyde, and interleukin-6 levels and a decreased superoxide dismutase level in SHRs. CONCLUSIONS Pancreatic microcirculation profiles are abnormal in the progression of hypertension in SHRs, including a disarranged blood distribution pattern, impaired microvascular vasomotion, and reduced amplitudes of endothelial oscillators., Graphical Abstract Graphical Abstract

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

The presence of asbestos in the environment has caused concern because exposure to asbestos can cause diseases such as stomach and pancreatic cancer. However, suitable up-to-date methods for quantitatively analyzing asbestos and assessing the toxicity of asbestos have not been developed. In this study, asbestos in drinking water was characterized using a stepwise multiple differential infra-red spectra method and a partial least squares method. The in vivo migration of ingested asbestos in mice was then investigated using the technique. The quantification limit of six kinds of asbestos by using inductively coupled plasma optical emission spectrometry and Fourier-transform infrared spectroscopy in water are respectively from 0.0468 to 0.0705 mg/L, from 0.0039 to 0.0064 mg/L. The relative standard deviations were respectively less than 2.85% and 3.81%. The recoveries of the test asbestos were respectively more than 95.10% and 95.38%. Asbestos was found mainly to accumulate in the livers of mice. The Fourier-transform infra-red spectroscopy inductively coupled plasma optical emission spectrometry method can be used to detect and precisely quantify asbestos in water samples and in animal tissues.

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species, {"references":["Ma L, Chen Z, Huang DW, Cissé OH, Rothenburger JL, Latinne A, Bishop L, Blair R, Brenchley JM, Chabé M, Deng X, Hirsch V, Keesler R, Kutty G, Liu Y, Margolis D, Morand S, Pahar B, Peng L, Van Rompay KKA, Song X, Song J, Sukura A, Thapar S, Wang H, Weissenbacher-Lang C, Xu J, Lee C-H, Jardine C, Lempicki RA, Cushion MT, Cuomo CA, Kovacs JA. 2020. Diversity and complexity of the large surface protein family in the compacted genomes of multiple Pneumocystis species. mBio 11:e02878-19. https://doi.org/10 .1128/mBio.02878-19."]}

The genus Henipavirus (HNVs) includes two fatal viruses, namely Nipah virus (NiV) and Hendra virus (HeV). Since 1994, NiV and HeV have been endemic to the Asia&ndash, Pacific region and responsible for more than 600 cases of infections. Two emerging HNVs, Ghana virus (GhV) and Mojiang virus (MojV), are speculated to be associated with unrecognized human diseases in Africa and China, respectively. Despite many efforts to develop vaccines against henipaviral diseases, there is presently no licensed human vaccine. As HNVs are highly pathogenic and diverse, it is necessary to develop universal vaccines to prevent future outbreaks. The attachment enveloped glycoprotein (G protein) of HNVs mediates HNV attachment to the host cell&rsquo, s surface receptors. G proteins have been used as a protective antigen in many vaccine candidates for HNVs. We performed quantitative studies on the antibody responses elicited by the G proteins of NiV, HeV, GhV, and MojV. We found that the G proteins of NiV and HeV elicited only a limited cross-reactive antibody response. Further, there was no cross-protection between MojV, GhV, and highly pathogenic HNVs. We then constructed a bivalent vaccine where the G proteins of NiV and HeV were fused with the human IgG1 Fc domain. The immunogenicity of the bivalent vaccine was compared with that of monovalent vaccines. Our results revealed that the Fc-based bivalent vaccine elicited a potent antibody response against both NiV and HeV. We also constructed a tetravalent Fc heterodimer fusion protein that contains the G protein domains of four HNVs. Immunization with the tetravalent vaccine elicited broad antibody responses against NiV, HeV, GhV, and MojV in mice, indicating compatibility among the four antigens in the Fc-fusion protein. These data suggest that our novel bivalent and tetravalent Fc-fusion proteins may be efficient candidates to prevent HNV infection.

The Rift Valley fever virus (RVFV) is an arthropod-borne virus that can not only cause severe disease in domestic animals but also in humans. However, the licensed vaccines or available therapeutics for humans do not exist. Here, we report two Gn-specific neutralizing antibodies (NAbs), isolated from a rhesus monkey immunized with recombinant human adenoviruses type 4 expressing Rift Valley fever virus Gn and Gc protein (rHAdV4-GnGcopt). The two NAbs were both able to protect host cells from RVFV infection. The interactions between NAbs and Gn were then characterized to demonstrate that these two NAbs might preclude RVFV glycoprotein rearrangement, hindering the exposure of fusion loops in Gc to endosomal membranes after the virus invades the host cell. The target region for the two NAbs is located in the Gn domain III, implying that Gn is a desired target for developing vaccines and neutralizing antibodies against RVFV.

Background: Protein S (PS), a vitamin K-dependent plasma glycoprotein, functions as a cofactor for the anticoagulants activated protein C (APC) and tissue factor (TF) pathway inhibitor alpha (TFPIa), which inhibit factors Va (FVa) and Xa (FXa), respectively. Although it is unclear which of these functions is/are important in vivo, homozygous deficiency of PS is associated with life-threatening thrombosis shortly after birth. FVa and FXa form the prothrombinase complex, which generates thrombin, suggesting that PS has a role in the direct inhibition of thrombin production. However, neither the PS/APC nor PS/TFPIα system alone is effective at inhibiting thrombin generation by prothrombinase. In addition to its role in regulating coagulation, PS also functions as an inhibitor of the complement system. Approximately 60% of plasma PS circulates bound to complement factor C4bp, which blocks its anticoagulant activity. We sought to determine the impact of PS/APC on TFPIα function, and vice versa, using purified protein and plasma-based systems. Methods and Results: To assess the effect of the combined PS/APC and PS/TFPIα systems on thrombin generation, we supplemented plasma with thrombomodulin, which promotes APC activation. In the absence of thrombomodulin, 5nM TFPIα decreased peak thrombin by 55.1% (33.1±1.9 nM in the presence of TFPIα vs. 73.7±39.9 nM in the absence) and endogenous thrombin potential (ETP) by 35.4% (475±42 nM*min vs. 735±189nM*min). In the presence of thrombomodulin, TFPIα decreased these parameters by 65.7% (11.4 ± 2.6 nM) and 77.5% (107±22 nM*min), respectively, suggesting that APC makes TFPIα a more potent inhibitor of thrombin generation. We next sought to study each of these PS functions in a purified protein system and in plasma. To study the effect of PS/APC on TFPIα function, we produced a recombinant protein consisting of the first two epidermal growth factor-like domains of PS (EGF1-2), which contain the putative APC binding sites. In a purified protein assay, APC inhibited the rate of thrombin activation by prothrombinase by 19.62±0.01% in the absence of PS and by 34.96±0.02% in the presence of 50nM PS. EGF1-2 dose-dependently reversed the effect of PS, with 75% reversal achieved with the addition of 200nM EGF1-2. Unexpectedly, EGF1-2 had the opposite effect in plasma thrombin generation assays and potently inhibited TF-initiated thrombin generation either in the presence or absence of thrombomodulin. We hypothesize that the EGF1-2 domains also form part of the C4bp binding site, and that addition of EGF1-2 protein resulted in release of PS from C4bp and an increase in the anticoagulant PS pool. In support of this hypothesis, EGF1-2 had no effect on thrombin generation in PS-depleted plasma, which is also depleted of C4bp. When the PS-depleted plasma was supplemented with 150nM PS, EGF1-2 had the expected procoagulant activity (increasing peak thrombin from 50.4±19.9 nM to 90.4±6.0 nM). Notably, even with a saturating concentration of EGF1-2, thrombomodulin and PS significantly decreased thrombin generation, suggesting that PS-TFPIα-mediated FXa inhibition promotes APC-mediated FVa degradation, even if PS cannot directly bind APC. We similarly assessed the impact of the PS-TFPI function, using a protein from the saliva of black flies, "black fly protease inhibitor" (BFPI), which contains the TFPIα domain that inhibits FXa but lacks the domain that binds PS. BFPI inhibits free FXa similarly to TFPIα, but PS does not promote this inhibition. Like TFPIα, BFPI is a poor inhibitor of thrombin generation by prothrombinase containing thrombin-activated FVa (5 nM BFPI had no impact on thrombin generation in the presence or absence of PS). However, in the presence of APC and PS, 5nM BFPI decreased the maximum rate of thrombin generation by 17.3±3.3%. These data suggest that PS/APC-mediated degradation of FVa promotes TFPIα-mediated inhibition of FXa, regardless of whether PS is able to bind TFPIα. Conclusions: Our data suggest that the PS-APC and PS-TFPIα systems cooperatively regulate thrombin generation by prothrombinase. While maximal inhibition requires that PS act as a cofactor for both APC and TFPIα, PS-APC independently promotes TFPIα function, and PS-TFPIα separately promotes APC. Based on these data, we propose a model in which PS-APC-mediated inhibition of FVa renders FXa susceptible to TFPIα and vice versa. Disclosures No relevant conflicts of interest to declare.

Yersinia pestis (Y. pestis) has caused an alarming number of deaths throughout recorded human history, and novel prophylactics and therapeutics are necessary given its potential as a bioweapon. Only one monoclonal antibody has been identified to date that provides complete protection against Y. pestis. Here, we describe a second novel murine monoclonal antibody (F2H5) that provided complete protection against Y. pestis 141 infection when administered prophylactically to Balb/c mice (100 μg intravenously). We humanized F2H5, characterized its ability to bind to the Y. pestis F1 protein and further characterized the neutralizing epitope using computational and experimental approaches. While Western blot results suggested a linear epitope, peptide mapping using ELISA failed to identify an epitope, suggesting a conformational epitope instead. We adopted a computational approach based on Residue Contact Frequency to predict the site of antigen-antibody interaction and defined the F2H5/F1 binding site computationally. Based on computational approach, we determined that residues G104E105N106 in F1 were critical to F2H5 binding and that CDRH2 and CDRH3 of F2H5 interacted with F1. Our results show that combining computational approach and experimental approach can effectively identify epitopes.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses., Pneumocystis jirovecii is a fungus that can cause life-threatening pneumonia in immunocompromised patients. Here, the authors sequence the genomes of P. jirovecii and two other Pneumocystis species, and show the unexpected absence of chitin (a near universal fungal cell wall component).

We present calculations on the ratio between double and single ionization of helium by a strong laser pulse at a wavelength of 780 nm using the quantitative rescattering (QRS) model. According to this model, the yield for the doubly charged ion $\mathrm{He}^{2+}$ can be obtained by multiplying the returning electron wave packet (RWP) with the total cross sections (TCSs) for electron impact ionization and electron impact excitation of $\mathrm{He}^{+}$ in the singlet spin channel. The singlet constraint was imposed since the interaction of the helium atom with the laser and the recollision processes both preserve the total spin of the system. An $R$-matrix (close-coupling) code is used to obtain accurate TCSs, while the RWPs, according to the QRS, are calculated by the strong-field approximation for high-energy photoelectrons. The laser field, which lowers the required energy for the electron to escape from the nucleus at the time of recollision, is also taken into account. The simulated results are in good agreement with the measured $\mathrm{He}^{2+}\text{/}\mathrm{He}^{+}$ ratio over a broad range of laser intensities. The result demonstrates that the QRS approach based on the rescattering model is fully capable of quantitatively interpreting nonsequential double ionization processes.

Three main types of cocoa tea derived from Camellia ptilophylla, white cocoa tea (WCT), green cocoa tea (GCT) and black cocoa tea (BCT) were prepared. A comparison between cocoa tea and commonly consumed tea Biluochun cha (BLC), Longjing cha (Lj), Qimenhong cha (Qmh) and Yinghong 9# (Yh9) was studied. Levels of catechins and purine alkaloids were determined by HPLC. Total polyphenol contents were determined by ferrous tartrate assay. Antioxidative activity was evaluated using ABTS decolorization assay, DPPH radical assay and ferric thiocyanate (FTC) method. The results showed that WCT contained the highest polyphenol content and showed the highest antioxidative activity. The radical scavenging ability of the tea extracts followed this order: WCT > GCT > Blc > Lj > BCT > Yh9 > Qmh. Tea extracts measured by the linoleic acid system also showed the same patterns of activity as the ABTS and DPPH method. A high correlation between polyphenol content and antioxidative activity of tea extracts was observed.

Cocoa tea is a new natural decaffeinated tea. Three types of cocoa tea (green, oolong and black) were produced and their sensory evaluation scores accessed. Main components and their dissolution rates in ten infusions of the three teas were analyzed by HPLC and spectrophotometry. The comprehensive score of sensory evaluations was 85.1, 86.8 and 94.4, respectively. Upon fermentation the contents of water extract (46.67%, 41.8% and 40%), tea polyphenols (38.58%, 30.41% and 23.6%) and total catechins (23.51%, 17.68% and 4.02%) in green, oolong and black teas decreased gradually, and theaflavins (0.11%, 0.11% and 0.17%), thearubigins (4.29%, 5.00% and 9.71%), theabrownins (2.75%, 4.90% and 13.52%), water-soluble carbohydrates, flavonoid glycoside and gallic acid in the three teas increased gradually. Theobromine (3.52%, 3.43% and 3.71%) was the alkaloid present in cocoa tea and its content did not change upon fermentation. In the three teas, 9–25% of the main components dissolved in the first 10 s-infusion, more than 80% of most components dissolved in five infusions. A recommendation for consuming cocoa tea is to infuse tea leaves (g) with 50 times boiling water (ml) for 3 min, the first infusion should not be discarded and five infusions were sufficient.

Summary In the present study, we employed high performance liquid chromatography with an amide-C16 column to determine the eighteen major active ingredients in black tea, including theanine, gallic acid, four purine alkaloids, eight catechins and four theaflavins. The method was successfully used to analyse two new kinds of black teas from the leaves of Camellia ptilophylla and Camellia kucha in China and several other world- famous black teas. Forty percentage ethanol was chosen as the extraction solvent for preparing tea extracts. All of the eighteen compounds could be separated within 86 min with a gradient elution system. Excellent linearity was observed for all the standard calibration curves, and correlation coefficients were above 0.9991. The developed method is accurate and sensitive enough for the determination of active components in black tea.

A simple and fast HPLC analysis method for phenolic compounds, purine alkaloids and theanine in various Camellia species was developed. Using an amide-C16 column, catechins, gallic acid, caffeine, theobromine, theacrine, theophyline and theanine could be rapidly separated within 45 min with a gradient elution system. Excellent linearity was observed for all 14 compounds in the range studied, with correlation coefficients between 0.9994 and 0.9999. Limit of detection and limit of quantification of the 14 compounds varied from 0.0001 to 0.072 ng/μL and 0.0004 to 0.24 ng/μL, respectively. Four kinds of Camellia species were analyzed using this method. In traditional cultivated tea trees, Camellia sinensis and Camellia assamica, the main purine alkaloid was caffeine and main phenolic compound was (−)-epigallocatechin gallate (EGCG). The main purine alkaloids in Camellia ptilophylla and C. assamica var. kucha were theobromine and theacrine; their contents were 4.001±0.1184% dry weight and 2.116±0.0270% dry weight, respectively. The main phenolic compounds in C. ptilophylla and C. assamica var. kucha were (−)-gallocatechin gallate (GCG) and EGCG respectively. The content of theanine in the four Camellia species samples ranged from 0.136±0.0026% to 1.485±0.0491% dry weight.

Purpose The aim of this study was to explore role of PTEN gene in chemosensitivity to cisplatin in human ovarian cancer cells and related mechanisms. Method A PTEN-targeted short hairpin RNA (shRNA) expression vector and a wild-type sense PTEN plasmid were constructed, human ovarian cisplatin-sensitive cancer cell line OV2008 and its resistant variant C13∗ cells were transfected with PTEN shRNA or wild-type PTEN plasmid, respectively, and cells were then treated with cisplatin. Next, AKT activity was regulated with co-transfection of antisense or sense AKT plasmid in OV2008 /PTENshRNA cells or C13∗/p-PTEN cells, respectively. Effects of transfection of above vectors on cell growth, apoptosis and expression of PTEN and AKT were evaluated. Results Expression of PTEN in OV2008 cells was significantly higher than that in C13∗ cells. Transfection of PTEN shRNA into OV2008 cells remarkably down-regulated expression of PTEN and up-regulated expression of phospho-AKT protein, with transfected cells being resistant to cisplatin. Overexpression of PTEN by transfection with sense PTEN obviously enhanced cisplatin-induced apoptosis of C13∗ cells. Furthermore, decreased AKT activity could increase cisplatin-induced apoptosis in OV2008/PTENshRNA cells; while, transfection of pcDNA3.1-AKT plasmid into C13∗/p-PTEN cells resulted in increased activity of AKT, with cisplatin-induced apoptosis being inhibited significantly. Conclusions PTEN might reverse chemoresistance to cisplatin in human ovarian cancer cells through inactivation of the PI3K/AKT cell survival pathway and may serve as a potential molecular target for the treatment of chemoresistant ovarian cancer.

Objectives We investigated the effects of oxymetazoline hydrochloride on the regulation of healthy human nasal ciliary beat frequency (CBF) and its influence on nasal mucociliary transport time (MTT). Methods Changes in (cultured) human nasal CBF in response to increasing concentrations of oxymetazoline within 20 minutes were quantified by use of high-speed digital microscopy. Moreover, the MTT before and after application of 0.05% oxymetazoline was determined by use of the saccharin test. Results Whereas no statistically significant difference was identified when compared to basal CBF at the concentration of 0.025% or 0.05%, both 0.10% and 0.20% oxymetazoline induced a significantly lower CBF at the end of the observation period. The decrement induced by 0.20% oxymetazoline appeared earlier. At concentrations ranging from 0.025% to 0.20%, the inhibitory effect was dependent on the concentration of oxymetazoline. In addition, the use of 0.05% oxymetazoline increased the mean (±SD) human nasal MTT from 474 ± 21 seconds to 572 ± 41 seconds (n = 29). Conclusions The clinical concentration of oxymetazoline, 0.05%, has no obvious inhibitory effect on human nasal CBF in vitro. The increased MTT caused by 0.05% oxymetazoline in vivo is within the normal range.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.

Structure-based design and synthesis of the 3,4'-bispyridinylethylene series led to the discovery of 3-isoquinolinylpyridine 13a as a potent PKB/Akt inhibitor with an IC(50) of 1.3nM against Akt1. Compound 13a shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to marginal selectivity against closely related kinases in the AGC and CMGC families. Moreover, 13a demonstrates potent cellular activity comparable to staurosporine, with IC(50) values of 0.42 and 0.59microM against MiaPaCa-2 and the Akt1 overexpressing FL5.12-Akt1, respectively. Inhibition of phosphorylation of the Akt downstream target GSK3 was also observed in FL5.12-Akt1 cells with an EC(50) of 1.5microM. The X-ray structures of 12 and 13a in complex with PKA in the ATP-binding site were determined.

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.

The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside GT1band neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects.

Two new resveratrol tetramers, cajyphenol A (1) and cajyphenol B (2), together with two known resveratrol dimers, quadrangularin A (3) and pallidol (4), and resveratrol (5) were isolated from the stems of Cayratia japonica (Thunb.) Gagn. Their structures were established by means of NMR (¹H,¹H-COSY, ¹H, ¹³C-HSQC, HMBC, and NOESY) and ESI-MS analyses. Compounds 1-5 showed fast-binding inhibitory activities on fatty acid synthase with the IC₅₀ values of 1.63±0.02, 1.49±0.03, 7.50±0.01, 11.1±0.01, and 10.2±0.01 μM, respectively. Compounds 1-5 also exhibited antioxidant activities in the ORAC assay.

We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation.

The anti-inflammatory and analgesic effects of theacrine (1, 3, 7, 9-tetramethyluric acid), a purine alkaloid which is abundantly present in Camellia kucha, were investigated. Xylene-induced ear edema, acetic acid-induced vascular permeability and lambda-carrageenan-induced paw edema were used to investigate anti-inflammatory activity, and acetic acid-induced writhing and hot-plate tests were used to determine analgesic effect. Oral administration of theacrine (8-32 mg/kg) induced dose-related anti-inflammatory and analgesic effects. On the other hand, oral caffeine administration (8-32 mg/kg) did not show an inhibitory effect on the inhibition of inflammatory response or cause analgesia. Additionally, the result of the acute toxicity test showed that the LD(50) of theacrine was 810.6 mg/kg (769.5-858.0mg/kg). The data obtained suggest theacrine possessed analgesic and anti-inflammatory activities.

PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Δ27LFn) and EFn. In a cell model of intoxication, fusion protein of Δ27LFn and EFn (Δ27LFn–EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Δ27LFn–EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Δ27LFn–EFn has the potential as a candidate therapeutic agent against anthrax. Structured summary MINT- 7014735 , MINT- 7014747 , MINT- 7014761 : PA63 (uniprotkb: P13423 ) and LF (uniprotkb: P15917 ) bind (MI: 0407 ) by surface plasmon resonance (MI: 0107 )

Overexpression of prosurvival proteins such as Bcl-2 and Bcl-X L has been correlated with tumorigenesis and resistance to chemotherapy, and thus, the development of antagonists of these proteins may provide a novel means for the treatment of cancer. We recently described the discovery of 1 (ABT-737), which binds Bcl-2, Bcl-X L, and Bcl-w with high affinity, shows robust antitumor activity in murine tumor xenograft models, but is not orally bioavailable. Herein, we report that targeted modifications at three key positions of 1 resulted in a 20-fold improvement in the pharmacokinetic/pharmacodynamic relationship (PK/PD) between oral exposure (AUC) and in vitro efficacy in human tumor cell lines (EC 50). The resulting compound, 2 (ABT-263), is orally efficacious in an established xenograft model of human small cell lung cancer, inducing complete tumor regressions in all animals. Compound 2 is currently in multiple phase 1 clinical trials in patients with small cell lung cancer and hematological malignancies.

Purpose Although multiple mechanisms have been implicated in paclitaxel (PTX)-induced resistance in ovarian cancer, recent evidence has suggested that Akt2 has an important role in the protection of cells from paclitaxel-induced apoptosis. In the present study, we investigated the role of the Akt2/survivin pathway in paclitaxel-induced resistance by a modified method to generate an effective shRNA vector. Methods We applied RNAi-mediated silencing techniques to investigate the mechanism of the Akt2/survivin pathway on PTX-induced resistance in ovarian cancer cells (A2780 and SKOV3). The expression of Akt2 and survivin mRNA and related protein levels were evaluated with semiquantitative real-time RT-PCR and western blot analysis, respectively. Inhibition of cell proliferation was determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, and the induction of apoptosis was examined through flow cytometry (FACS) and Hoechst staining. Results Akt2 down-regulation sensitized ovarian cancer cells to paclitaxel-induced apoptosis, and inhibited survivin expression. We further demonstrated that suppressing the inhibition of survivin expression can induce the drug-resistance to paclitaxel. We introduced a modified vector to generate shRNA to induce RNA interference, which contained three U6 promoters to express different shRNAs; it severely reduced Akt2 gene expression and showed good specificity. Conclusion Our findings will aid in understanding the molecular mechanism of paclitaxel-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.

We theoretically show that selection of a single quantum path in high-order harmonics generation can be realized in a few-optical-cycle regime with two-color schemes. We also demonstrate, in theory as well, the generation of spectrally smooth and ultrabroad extreme ultraviolet supercontinuum in argon gas which can produce single similar to 79 as pulses with currently available ultrafast laser sources. Our finding can be beneficial for generating isolated sub-100 as extreme ultraviolet pulses.

Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Based on lead compounds 2 and 3 a series of 3,5-disubstituted pyridines have been designed and evaluated for inhibition of AKT/PKB. Modifications at the 3 position of the pyridine ring led to a number of potent compounds with improved physical properties, resulting in the identification of 11g as a promising, orally active Akt inhibitor. The synthesis, structure–activity relationship studies, and pharmacokinetic data are presented in this paper.

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.