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Background and aims The benefit of lateral pelvic lymph node dissection (LPLD) for locally advanced rectal cancer remains controversial. This meta-analysis aimed to evaluate the prognostic value of LPLD in patients with locally advanced rectal cancer. Methods We performed a systematic search in PubMed, Embase, and the Cochrane Library for publications comparing radical resection plus LPLD (LPLD group) with single radical resection (non-LPLD group) for locally advanced rectal cancer. A total of 15 studies satisfied our inclusion criteria and were assessed. Random-effects and fixed-effects meta-analytical models were used where indicated, and between-study heterogeneity was assessed. Results LPLD significantly increased grade 3-4 postoperative complications (odds ratio [OR]1.44, 95% CI 1.03-2.02; P = 0.03) compared with non-LPLD. There were no significant differences in 5-y overall survival (hazard ratio = 0.90, 95% CI 0.77-1.05; P = 0.17), 5-y disease-free survival (hazard ratio 1.12, 95% CI 0.60-2.09; P = 0.73), local recurrence (OR 0.89, 95% CI 0.53-1.51; P = 0.68) or distant recurrence (OR 0.85, 95% CI 0.64-1.12; P = 0.24). Conclusions We found that LPLD significantly increased grade 3-4 postoperative complications but did not increase 5-y overall survival or 5-y disease-free survival compared with single radical resection for locally advanced rectal cancer. Furthermore, it did not decrease the local recurrence or distant recurrence rates. Thus, more multicenter large-scale randomized controlled trials should be conducted to further explore whether the long-term survival benefits of LPLD truly exist.

Summary Background The optimal perioperative chemotherapeutic regimen for locally advanced gastric cancer remains undefined. We evaluated the efficacy and safety of perioperative and postoperative S-1 and oxaliplatin (SOX) compared with postoperative capecitabine and oxaliplatin (CapOx) in patients with locally advanced gastric cancer undergoing D2 gastrectomy. Methods We did this open-label, phase 3, superiority and non-inferiority, randomised trial at 27 hospitals in China. We recruited antitumour treatment-naive patients aged 18 years or older with historically confirmed cT4a N+ M0 or cT4b Nany M0 gastric or gastro-oesophageal junction adenocarcinoma, with Karnofsky performance score of 70 or more. Patients undergoing D2 gastrectomy were randomly assigned (1:1:1) via an interactive web response system, stratified by participating centres and Lauren classification, to receive adjuvant CapOx (eight postoperative cycles of intravenous oxaliplatin 130 mg/m2 on day one of each 21 day cycle plus oral capecitabine 1000 mg/m2 twice a day), adjuvant SOX (eight postoperative cycles of intravenous oxaliplatin 130 mg/m2 on day one of each 21 day cycle plus oral S-1 40–60 mg twice a day), or perioperative SOX (intravenous oxaliplatin 130 mg/m2 on day one of each 21 day plus oral S-1 40–60 mg twice a day for three cycles preoperatively and five cycles postoperatively followed by three cycles of S-1 monotherapy). The primary endpoint, assessed in the modified intention-to-treat population, 3-year disease-free survival to assess the superiority of perioperative-SOX compared with adjuvant-SOX and the non-inferiority (hazard ratio non-inferiority margin of 1·33) of adjuvant-SOX compared with adjuvant-CapOx. Safety analysis were done in patients who received at least one dose of the assigned treatment. This study is registered with ClinicalTrials.gov, NCT01534546. Findings Between Aug 15, 2012, and Feb 28, 2017, 1094 patients were screened and 1022 (93%) were included in the modified intention-to-treat population, of whom 345 (34%) patients were assigned to the adjuvant-CapOx, 340 (33%) patients to the adjuvant-SOX group, and 337 (33%) patients to the perioperative-SOX group. 3-year disease-free survival was 51·1% (95% CI 45·5–56·3) in the adjuvant-CapOx group, 56·5% (51·0–61·7) in the adjuvant-SOX group, and 59·4% (53·8–64·6) in the perioperative-SOX group. The hazard ratio (HR) was 0·77 (95% CI 0·61–0·97; Wald p=0·028) for the perioperative-SOX group compared with the adjuvant-CapOx group and 0·86 (0·68–1·07; Wald p=0·17) for the adjuvant-SOX group compared with the adjuvant-CapOx group. The most common grade 3–4 adverse events was neutropenia (32 [12%] of 258 patients in the adjuvant-CapOx group, 21 [8%] of 249 patients in the adjuvant-SOX group, and 30 [10%] of 310 patients in the perioperative-SOX group). Serious adverse events were reported in seven (3%) of 258 patients in adjuvant-CapOx group, two of which were related to treatment; eight (3%) of 249 patients in adjuvant-SOX group, two of which were related to treatment; and seven (2%) of 310 patients in perioperative-SOX group, four of which were related to treatment. No treatment-related deaths were reported. Interpretation Perioperative-SOX showed a clinically meaningful improvement compared with adjuvant-CapOx in patients with locally advanced gastric cancer who had D2 gastrectomy; adjuvant-SOX was non-inferior to adjuvant-CapOx in these patients. Perioperative-SOX could be considered a new treatment option for patients with locally advanced gastric cancer. Funding National Key Research and Development Program of China, Beijing Scholars Program 2018–2024, Peking University Clinical Scientist Program, Taiho, Sanofi-Aventis, and Hengrui Pharmaceutical. Translation For the Chinese translation of the abstract see Supplementary Materials section.

Background The influence of positive microscopic margin (R1) resection on the prognosis of gastrointestinal stromal tumors (GISTs) is controversial. Tumor rupture is significantly associated with the occurrence of R1 resection and may be a confounder of R1 resection in GISTs. The present meta-analysis evaluated the real influence of R1 resection on the prognosis of GISTs by excluding the confounding effect of tumor rupture. Methods The PubMed, Embase, Web of Science, Cochrane Central Register of Controlled Trials (CENTRAL), and ClinicalTrials.gov databases were searched. Studies that compared R1 with negative microscopic margin (R0) resection in GIST patients and reported the time-to-event data of recurrence-free survival (RFS) or disease-free survival (DFS) were eligible for inclusion. The quality of the observational studies was assessed using the Newcastle–Ottawa scale. Results Of the 4896 records screened, 23 retrospective studies with 6248 participants were selected. In the overall analysis, R1 resection resulted in a significantly shorter RFS/DFS than R0 resection for GISTs (HR = 1.80, 95% CI = 1.54–2.10, P Conclusions This meta-analysis shows that R1 resection did not influence the survival outcome of GISTs. Reresection may not be necessary when positive microscopic margins exist. This analysis could provide high-quality evidence for the development of guidelines.

Treatment for de novo or recurrent tumors of liver transplantation (LT) recipients is challenging and immune checkpoint inhibitor (ICI) is recently well developed and could be a potentially effective option for this population. There remains limited evidence on the safety and efficacy of ICI therapy in LT recipients.A systematic literature search was conducted on PubMed database through April 1, 2022, to identify publications reporting ICI treatment for malignant tumors in LT recipients. We summarized the allograft rejection, mortality, and tumor response of ICI treatment.24 articles with 41 LT recipients were identified. The age of LT recipients ranged from 14 to 78, 76.2% were male, 56.1% had recurrent HCC, and 87.8% received anti-PD-1 therapy. Allograft rejection occurred in 31.7% of patients, death was reported in 46.3% and 6 cases died secondary to allograft rejection. Progressive disease rate of this population was 48.8% and 10 patients responded to immunotherapy. Half of recipients with positive PD-L1 staining (4/8) experienced allograft rejection.ICI therapy has potential therapeutic value on malignant tumors for LT recipients, accompanied by a high rate of allograft rejection and mortality. PD-L1 expression, type of ICI, and immunosuppression agent should be taken into consideration before initiation of immunotherapy. Further studies are needed to optimize this anticancer treatment approach in these patients.

Creep and relaxation of knee cartilage and meniscus have been extensively studied at the tissue level with constitutive laws well established. At the joint level, however, both experimental and model studies have been focused on either elastic or kinematic responses of the knee, where the time-dependent response is typically neglected for simplicity. The objectives of this study were to quantify the in-vivo creep behavior of human knee joints produced by the cartilaginous tissues and to use the relevant data to validate a previously proposed poromechanical model. Two participants with no history of leg injury volunteered for 3T magnetic resonance imaging (MRI) of their unloaded right knees and for biplanar video-radiography (BVR) of the same knees during standing on an instrumented treadmill for 10 min. Approximately 550 temporal data points were obtained for the in-vivo displacement of the right femur relative to the tibia of the knee. Models of the bones and soft tissues were derived from the MRI. The bone models were used to reconstruct the 3D bone kinematics measured using BVR. Ground reaction forces were simultaneously recorded for the right leg, which were used as input for the subjectspecific finite element knee models. Cartilaginous tissues were modeled as fluid-saturated fibril-reinforced materials. In-vivo creep of the knee was experimentally observed for both participants, i.e., the joint displacement increased with time while the reaction forces at the foot were approximately constant. The creep displacements obtained from the finite element models compared well with the experimental data when the tissue properties were calibrated (Pearson correlation coefficient = 0.99). The results showed the capacity of the poromechanical knee model to capture the creep response of the joint. The combined experimental and model study may be used to understand the fluid-pressure load support and contact mechanics of the joint using material properties calibrated from the displacement data, which enhance the fidelity of model results. WOS:000712529500002 2-s2.0-85117732602 PubMed: 34700107

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

Gastric cancer (GC) is a major threat to human health, and its prognosis is poor due to the lack of appropriate biomarkers. LncRNAs are a group of non-protein-coding RNAs that regulate gene expression at the transcriptional or posttranscriptional level. LncRNAs play essential roles in GC initiation and development in the same way as oncogenes or tumour suppressor genes. Recent investigations have revealed that lncRNAs are often aberrantly expressed in GC; are involved in cell proliferation, apoptosis, migration and invasion; and correlate with the malignant phenotype of GC. LncRNAs, especially the lncRNAs present in the blood and gastric juice, show potential value as biomarkers for the diagnosis of GC or for determining disease prognosis. However, there are still many challenges to be faced before lncRNAs can be used in clinical applications. In this review, we summarise lncRNAs as the potential biomarkers for GC and the current challenges associated with the clinical application.

This Letter describes the discovery of a number of functionalized bisimidazoles bearing a cyclohexylphenyl, piperidylphenyl, or bicyclo[2,2,2]octylphenyl motif as HCV NS5A inhibitors. Compounds 2c, 4b and 6 have demonstrated low single-digit nM potency in gt-1a replicon and double-digit pM potency in gt-1b replicon, respectively. Moreover, both 4b and 6 have, respectively, exhibited good oral bioavailability in rats with a favorable liver/plasma ratio of the drug concentration.

The elastic response of the knee joint in various loading and pathological conditions has been investigated using anatomically accurate geometry. However, it is still challenging to predict the poromechanical response of the knee in realistic loading conditions. In the present study, a viscoelastic, poromechanical model of the knee joint was developed for soft tissues undergoing large deformation. Cartilages and menisci were modeled as fibril-reinforced porous materials and ligaments were considered as fibril-reinforced hyperelastic solids. Quasi-linear viscoelasticty was formulated for the collagen network of these tissues and nearly incompressible Neo-Hookean hyperelasticity was used for the non-fibrillar matrix. The constitutive model was coded with a user defined FORTRAN subroutine, in order to use ABAQUS for the finite element analysis. Creep and stress relaxation were investigated with large compression of the knee in full extension. The contact pressure distributions were found similar in creep and stress relaxation. However, the load transfer in the joint was completely different in these two loading scenarios. During creep, the contact pressure between cartilages decreased but the pressure between cartilage and meniscus increased with time. This led to a gradual transfer of some loading from the central part of cartilages to menisci. During stress relaxation, however, both contact pressures decreased monotonically.

A series of spiropiperidine carbazoles were synthesized and evaluated as MCHR2 antagonists using a FLIPR assay. The pharmacokinetic properties of selected compounds have also been studied. This effort led to the discovery of potent and specific MCHR2 antagonists. Compound 38 demonstrated good pharmacokinetic properties across rat, beagle dog and rhesus monkey and had a favorable selectivity profile against a number of other receptors. These MCHR2 antagonists are considered appropriate tool compounds for study of the function of MCHR2 in vivo.

A new class of MCHR1 antagonists was discovered via a high-throughput screen. Optimization of the lead structure resulted in the identification of indole 10e. This compound possesses good pharmacokinetic properties across preclinical species and is efficacious in reducing food consumption in an MCH cannulated rat model and a cynomolgus monkey food consumption model.

Piperazine-bisamide analogs were discovered as partial agonists of human growth hormone secretagogue receptor (GHSR) in a high throughput screen. The partial agonists were optimized for potency and converted into antagonists through structure–activity relationship (SAR) studies. The efforts also led to the identification of potent antagonist with favorable PK profile suitable as a tool compound for in vivo studies.

Estrogen enables uterine proliferation, which depends on synthesis of the IGF1 growth factor. This proliferation and IGF1 synthesis requires the estrogen receptor (ER), which binds directly to target DNA sequences (estrogen-responsive elements or EREs), or interacts with other transcription factors, such as AP1, to impact transcription. We observe neither uterine growth nor an increase in Igf1 transcript in a mouse with a DNA-binding mutated ER alpha (KIKO), indicating that both Igf1 regulation and uterine proliferation require the DNA binding function of the ER. We identified several potential EREs in the Igf1 gene, and chromatin immunoprecipitation analysis revealed ER alpha binding to these EREs in wild type but not KIKO chromatin. STAT5 is also reported to regulate Igf1; uterine Stat5a transcript is increased by estradiol (E(2)), but not in KIKO or alpha ERKO uteri, indicating ER alpha- and ERE-dependent regulation. ER alpha binds to a potential Stat5a ERE. We hypothesize that E(2) increases Stat5a transcript through ERE binding; that ER alpha, either alone or together with STAT5, then acts to increase Igf1 transcription; and that the resulting lack of IGF1 impairs KIKO uterine growth. Treatment with exogenous IGF1, alone or in combination with E(2), induces proliferation in wild type but not KIKO uteri, indicating that IGF1 replacement does not rescue the KIKO proliferative response. Together, these observations suggest in contrast to previous in vitro studies of IGF-1 regulation involving AP1 motifs that direct ER alpha-DNA interaction is required to increase Igf1 transcription. Additionally, full ER alpha function is needed to mediate other cellular signals of the growth factor for uterine growth.

This work reports the investigation of two coronal mass ejections (CME) observed in white light, H alpha, EUV and X-ray by various instruments both in space and on ground on February 18, 2003 and January 19, 2005, respectively. The white light coronal images show that the first CME began with the rarefaction of a region above the solar limb and was followed by the formation of its leading edge at the boundary of the rarefying region at altitude of 0.46 R-circle dot from the solar surface. The rarefaction coincided the slow rising phase of the filament eruption, and the CME leading edge was observed to form as the filament eruption started to accelerate apparently. In the early stage of the second CME, a bright loop was first observed above the solar limb with height of 0.37 R-circle dot in EUV images. We found that the more gradual CMEs initial process, the larger the timing difference between CMEs and their associated flares. The lower part of the filament brightened in H alpha images as the filament rose to a certain height. These brightenings imply that the filament may be heated by magnetic reconnection below the filament in the early stage of the eruption. We suggest that the possible mechanism which led to the formation of the CME leading edge and cavity is magnetic reconnection which occurred under the filament when it reached a certain height. (c) 2007 COSPAR. Published by Elsevier Ltd. All rights reserved.

Using H-alpha, EUV, photospheric magnetic field and white-light coronagraph data, we study the eruption of an active-region H-alpha filament and associated partial-halo type coronal mass ejection (CME) occurring in NOAA AR 9616 on 2001 September 17. Accompanied by an M1.5 flare, the small active-region filament quickly erupted, a quiet-sun region outside the active region intensively darkened so a remote coronal dimming region was formed, and then a long H,, surge developed from the eruption site. During the eruption, remote EUV brightening appeared along the dimming boundary, leaving behind huge EUV loops connecting the eruption source region and the remote EUV brightening. Finally, as a definite indication of the start of CME, a large-scale dark rim appeared to span the eruption region. These observations indicate that a much larger-scale rearrangement of the coronal magnetic fields, eventually represented by the occurrence of the CME, was involved in the eruption of the filament with small spatial scale. The relationship between the filament eruption, flare and coronal dimming is also discussed. (c) 2006 Elsevier B.V. All rights reserved.

Background It has been suggested that the early onset of cartilage degeneration might be detected with a handheld indentation probe during knee arthroscopy, prior to any visible change on the articular surface. Collagen degradation has been considered as the first sign of cartilage degeneration. Therefore, it is important to consider the collagen network as a distinct constituent in the study of arthroscopic evaluation of cartilage degeneration. Methods The tip of an arthroscopic probe (indenter) was modeled as rigid and in contact with a cartilage/bone disk of sufficiently large radius to simulate an indentation in a joint. A fibril-reinforced model of cartilage, including streaming potentials and distinct constitutive laws for the proteoglycan matrix and collagen network, was used to determine the contact mechanics of indenter and cartilage. The finite element package ABAQUS was employed to obtain numerical solutions. Findings A spherical indenter produces a relatively uniform deformation in cartilage, but can easily slide on the articular surface. In contrast, a cylindrical indenter produces great deformation gradients for quick compression rates, but does not slide as easily on the articular surface as the spherical indenter. Small porous and large solid indenters should be used to evaluate the properties of the proteoglycan matrix and collagen network, respectively, in order to minimize or maximize the fluid pressure in the corresponding case. When the collagen network is substantially degraded, the gradients of fluid pressure and deformation are greatly reduced regardless of indenter geometry. Interpretation The indenter geometry including its porosity is important to the material safety of articular cartilage in indentation and precise evaluation of cartilage degeneration.

In this paper, the 3-dimensional configurations of the non-adiabatic region and the magnetic field lines (MFLs) in the magnetosphere are studied based on the Tsyganenko magnetic field model. According to the parameter δ = dB/B, the magnetosphere can be divided into the adiabatic and non-adiabatic region. A thin non-adiabatic region has been found in the near tail region, which moves toward to the Earth during the geomagnetic disturbance. Pitch angle scattering occurs when particles go through the non-adiabatic region, which is expected as the source region of the precipitating particles. Moreover, by investigating the 3-dimensional configurations of MFLs, we find that MFLs remain in the noon meridian plane if their foot prints are in the noon meridian plane; but leave the foot print plane and are swept tailward if their foot prints are not in the noon meridian plane. The characteristics of the MFLs suggest that the precipitation region is almost in the nightside region, centered near midnight.

The morphology of the plasmasphere during a geomagnetic storm is simulated by considering the two dimensional Ex B drift motion of plasmaspheric charged particles in the equatorial plane. assuming a time-independent dipolar magnetic field and a corotation electric field plus, a spatially uniform dawn-dusk convection electric field varying with K-p index, the spatial distributions of charged particles at different time during a geomagnetic storm are obtained. Our results show that if K-p increases with time, some particles inside the original plasmapause will convect into the magnetopause, forming a long tail that stretches from the plasmasphere to the magnetopause in the afternoon region. The particle convection weakens as K-p decreases, and as K-p returns to its not-mal value, the plasmasphere develops a thin tail that wraps around the Earth.

The depth dependence of material properties of articular cartilage, known as the zonal differences, is incorporated into a nonlinear fibril-reinforced poroelastic model developed previously in order to explore the significance of material heterogeneity in the mechanical behavior of cartilage. The material variations proposed are based on extensive observations. The collagen fibrils are modeled as a distinct constituent which reinforces the other two constituents representing proteoglycans and water. The Young's modulus and Poisson's ratio of the drained nonfibrillar matrix are so determined that the aggregate compressive modulus for confined geometry fits the experimental data. Three nonlinear factors are considered, i.e. the effect of finite deformation, the dependence of permeability on dilatation and the fibril stiffening with its tensile strain. Solutions are extracted using a finite element procedure to simulate unconfined compression tests. The features of the model are then demonstrated with an emphasis on the results obtainable only with a nonhomogeneous model, showing reasonable agreement with experiments. The model suggests mechanical behaviors significantly different from those revealed by homogeneous models: not only the depth variations of the strains which are expected by qualitative analyses, but also, for instance, the relaxation-time dependence of the axial strain which is normally not expected in a relaxation test. Therefore, such a nonhomogeneous model is necessary for better understanding of the mechanical behavior of cartilage.

The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.

The finite element method is used for those fluid-saturated poroelastic rods in which diffusion is possible only in the axial direction as a result of the microgeometry of the solid skeleton material. Variational principles are developed first for this purpose. Two types of variables, the displacements and pore pressure, are involved in the time dependent functionals. The method of Lagrange multipliers is employed in order to include the flow equations (generalized Darcy’s law) into the Euler–Lagrange equations of the functionals. A mixed finite element scheme is then presented based on one of the variational functionals obtained. Numerical solutions for both types of variables are found to coincide well with the existing analytical solutions. Some interesting results are demonstrated which are not available by analytical methods.

We thank the North Carolina Supercomputing Center, the Pittsburgh Supercomputing Center and the National Cancer Institute Supercomputing Center for access to resources. LGP thanks the National Institutes of Health for HL-06350 and the National Institute of Environmental Health Sciences (NIEHS) for access to their facilities.

Persistent expression of transgene is most desirable for treatment of genetic diseases. It is primarily controlled by genetic elements in the promoter region where transcription factors bind. The objective of this study is to assess the role of the transcription factor binding sites (TFBS) present in the most commonly used CMV and albumin promoters containing the promoter core sequence and several TFBS for ATF1, NFκB, SRF, AP1, Sp1, NF1α, CEBPA, HNF4α, and HNF1 in determining persistency of transgene expression in mice. Secreted embryonic alkaline phosphatase (SEAP) and interleukin 10 (IL-10) were used as a reporter to monitor transgene expression. Variousdeletion mutation plasmids at the promoter region were made to examine the function of each of the TFBS in regulating reporter gene expression. Similarly, a series of new plasmids containing only one TFBS inserted at the 5’ end of the TATA-box region was made to assess its unique activity in affecting transgene expression. Our results showed that TFBSs are essential for effective transgene expression in mouse liver after a hydrodynamic tail vein gene delivery. Plasmid with a TATA-box with all TFBS deleted resulted in minimal level of gene product, 700-fold lower than the original plasmids. Reporter gene expression appears transient reaching the background level in less than 2 weeks. The more TFBS the plasmid has, the higher level of gene product was seen. Plasmid containing a single TFBS either NFκB, ATF1, CEBPA, HNF4α, or HNF1 exhibits persistent transgene expression for more than 2 months, at which animals injected with plasmids containing NFkB, ATF1, CEBPA, HNF4α, or HNF1 exhibited gene product level 60, 130, 1020, 2250, and 2076 folds higher than that of animals injected with plasmids without TFBS, respectively. Mechanism study employing real time PCR, Western Blotting, and ChIP reveals involvement of histone acetylation and the binding of acetylated histones to plasmid DNA sequences in TFBS-mediated enhancement in persistency of reporter gene expression. TFBSs for HNF4α appear most potent in recruiting H4AC and TBP proteins to the plasmids. Our results suggest that TFBSs for NFkB, ATF1, CEBPA, HNF4α, and HNF1 are important elements to include into the promoter for achieving longer-term gene expression. In addition, histone acetylation on plasmid is a probable pathway that TFBS regulate the duration of transgene expression.

The solution structure of the Ca2+-loaded conantokin-T (con-T), a gamma-carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-CONH2,gam ma = Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy. An end-to-end alpha-helix was the dominant conformation in solution, similar to that of apo-con-T, except that reorientation of several side chains occurred in the Ca2+-coordinated complex. The most notable examples of this were those of Gla10 and Gla14, which were more optimally positioned for complexation with Ca2+. In addition to the stabilization offered to the alpha-helix by Ca2+ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the alpha-helical conformation. Docking of Ca2+ in the con-T structure was accomplished using genetic algorithm-molecular dynamics simulation approaches. The results showed that one Ca2+ ion is most likely coordinated by four side chain oxygen atoms, two each from Gla10 and Gla14. Another bound Ca2+ ion has as its donor sites three oxygen atoms, two from Gla3 and one from Gln6. To examine the functional roles of the individual Gla residues, a series of variant peptides have been synthesized with Ala substituted for each Gla residue, and several properties of the resulting variants have been examined. The data obtained demonstrated the importance of Gla10 and Gla14 in stabilizing binding of the highest affinity Ca2+ site and in governing the conformational change induced by Ca2+. The critical nature of Gla3 and Gla4 in inhibition of the spermine-induced potentiation of the binding of MK-801 to open ion channels of the N-methyl-D-aspartate receptor was established, as well as the role of Gla4 in stabilizing the apo-con-T alpha-helical conformation.

A theory is developed for fluid-saturated poroelastic plates made of a material for which diffusion is possible in the in-plane directions only. Both bending and in-plane loading are considered. The plates are isotropic in the plate plane and obey the Kirchhoff hypotheses. Biot's constitutive law is adopted and Darcy's law is used to describe the fluid flow in pores. Quasistatic and transverse vibration problems are investigated. Example solutions are presented and observations are made on the types of deflection/vibration patterns exhibited.

A series of novel, azacyclic ureas which are highly potent inhibitors of the HIV-1 protease (IC50 = 4.1 to0.5 nM) were synthesized. Aqueous solubilities of this series of compounds were improved by incorporating polar functional groups at the P1' P2 and P2' positions. These compounds also possess good anti-viral activity by inhibition of the cytopathic effect of HIV-13B in MT-4 cells in vitro.

The synthesis of 9-deoxotaxol (9) ,7,9-dideoxy and 7,9,10-trideoxygenated analogs is described starting from 13-acetyl-9(R)-dihydrobaccatin III.

C-3′-N-Acyl analogs of 9(R)-dihydrotaxol were synthesized from 7-triethylsilyl-9(R)-dihydrobaccatin III and the corresponding (3R,4S)-N-acyl-3-(1-ethoxyethoxy)-4-phenylazetidin-2-ones. The analogs were tested in a microtubule assembly assay, and in an invitro cytotoxicity assay. The highest activities observed were for the alkylcarbamate substitutions.

Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes. However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system. Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation. Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation. The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E. coli. The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E. coli. The E. coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme. The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme. The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.

Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.

Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.

(2 + 1) resonance-enhanced multiphoton ionization studies are performed on the D 2 Π(ν=2) state of CH. Anomalous line intensities are shown to be due to an accidental near resonance with the C 2 Σ + state at the one-photon level. The sudden onset of predissociation for N ⩾ 12 of this level is investigated and is attributed to complex interactions among three 2 Π states in this energy region.

The fructose-2,6-bisphosphatase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to be structurally and functionally homologous to phosphoglycerate mutase. Both enzymes catalyze their reactions via phosphoenzyme intermediates which utilize an active site histidine as a nucleophilic phosphoacceptor and another histidine as a proton donor to the leaving group. Glu327 in the bisphosphatase domain of the rat liver bifunctional enzyme is conserved in all phosphoglycerate mutase structures and is postulated, by modelling studies, to be located in the active site. Glu327 was mutated to Ala, Gln, or Asp. The mutant and wild-type enzymes were expressed in Escherichia coli with a T-7 RNA polymerase-based expression system and purified to homogeneity by substrate elution from phosphocellulose. The Glu327 mutants had apparent molecular weights of 110,000 by gel filtration and had unaltered 6-phosphofructo-2-kinase activity. Circular dichroism showed that the secondary structure of the Glu327 mutant enzyme forms was the same as the wild-type enzyme. The maximal velocity of the fructose-2,6-bisphosphatase of the Glu327----Ala, Glu327----Gln, and Glu327----Asp mutants was 4, 2, and 20%, respectively, that of the wild-type enzyme, but the rate of phosphoenzyme formation of the mutants was reduced by at least a factor of 1000. In addition, the rate constants of phosphoenzyme hydrolysis for the Glu372----Ala and Glu327----Gln mutants were 2.7 and 1.3%, respectively, of the wild type, whereas the rate constant for the Glu327----Asp mutant was 60% of the wild-type value. Glu327 was not a substrate or product binding site determinant since the Km for fructose-2,6-bisphosphate and Ki for fructose-6-phosphate of the mutants were not appreciably changed. The results implicate Glu327 as part of a catalytic triad in fructose-2,6-bisphosphatase and suggest that it influences the protonation state of the active site histidine residues during phosphoenzyme formation and/or acts as a base catalyst to enhance the nucleophilic attack of water on the phosphoenzyme intermediate.

Resonance-enhanced multiphoton dissociation (REMPD) of CH + yields C + H + products. By monitoring the H + signal as a function of either ω 1 (the laser frequency used to carry out REMPI of CH via the E′ (3pσ) Rydberg state) or ω 2 (the frequency used for REMPD of CH + produced by ω 1 ), we are able to determine rotational state distributions of CH + ions. The observed distributions are in accord with theoretical propensity rules and serve to verify the configuration and symmetry of the Rydberg state. The technique used in this work can serve as an alternative to laser-induced fluorescence when the latter is impractical.

Corrigendum to ‘‘Phenylalanine derivatives as GPR142 agonists for the treatment of Type II diabetes’’ [Bioorg. Med. Chem. Lett. 22 (2012) 6218–6223] Xiaohui Du a,⇑, Yong-Jae Kim , SuJen Lai , Xi Chen , Mike Lizarzaburu , Simon Turcotte , Zice Fu , Qingxiang Liu , Ying Zhang , Alykhan Motani , Kozo Oda , Ryo Okuyama , Futoshi Nara , Michiko Murakoshi , Angela Fu , Jeff D. Reagan , Peter Fan , Yumei Xiong , Wang Shen , Leping Li , Jonathan Houze , Julio C. Medina a

(2+1) MPI spectra of the v ′=0–3 levels of the 3dπ 1 Σ g + Rydberg state have been obtained, assigned and analyzed. Rotational line intensities are accounted for by invoking heterogeneous predissociation due to the 1 1 Π g valence state.

We report a study of the 3 + 1 MPI spectrum of acetylene in the 74450–90100 cm 1 energy range. Many Rydberg and some valence states are observed, most of which are stable on the time scale of the experiment Only in a region with high density of valence states is there evidence for non-radiative decay. We also report non-linear optical processes in C 2 H 2 , particularly third-harmonic generation (THG). At pressures above a few Torr, THG effectively quenches the MPI signal for all states of the correct symmetry. This observation explains the absence of most Rydberg structure in a previous 3+1 MPI study of C 2 H 2 , and demonstrates the need to work at very low densities.

State-selected hot chlorine atoms are cleanly produced in the 2P 3 2 by photodissociation of Cl2 in the wavelength region between 323.6 and 331.0 nm. Ionization (by 3+1 MPI) with mass spectral analysis has been used to probe the atomic Cl state. The identification of the repulsive state is determined from a state correlation diagram based on Mulliken's work and from Franck-Condon considerations based upon potential energy curves calculated by Peyerimhoff and Buenker. The results are consistent with adiabatic dissociation of the photoexcited chlorine molecules.