Prognosticators of survival in patients with metastatic pancreatic cancer and ascites.

BACKGROUND: Identification of factors associated with survival after ascites diagnosis in metastatic pancreatic cancer (mPC) patients may guide treatment decisions and help to maintain quality of life in this highly symptomatic patient collective. PATIENTS AND METHODS: All patients treated for mPC at the Medical University of Vienna between 2010 and 2019 developing ascites throughout their course of disease were identified by retrospective chart review. General risk factors, metastatic sites, systemic inflammation and liver function parameters, as well as type of treatment after ascites diagnosis were investigated for associations with survival. RESULTS: One hundred and seventeen mPC patients with ascites were included in this study. Median time from mPC to ascites diagnosis was 8.9 months (range 0-99 months) and median overall survival (OS) after ascites diagnosis was 27.4 days (range 21.3-42.6 days). Identified prognostic factors at ascites diagnosis independently associated with an impaired OS were presence of liver metastases [hazard ratio (HR): 2.07, 95% confidence interval (CI) 1.13-3.79, P = 0.018), peritoneal carcinomatosis (HR: 1.74, 95% CI 1.11-2.71, P = 0.015), and portal vein obstruction (HR: 2.52, 95% CI 1.29-4.90, P = 0.007). Compared with best supportive care, continuation of systemic therapy after ascites diagnosis was independently associated with survival (HR: 0.35, 95% CI 0.20-0.61, P < 0.001) with a median OS of 62 days (95% CI 51-129 days, P < 0.001) versus 16 days (95% CI 11-24 days), respectively. CONCLUSIONS: Liver and peritoneal metastases as well as portal vein obstruction were found to be prognostic factors after ascites diagnosis in mPC patients. Continuation of systemic therapy after ascites diagnosis was associated with a longer OS, which needs to be evaluated in larger clinical trials including quality-of-life assessment.

Clinical risk factors for ascites in metastatic pancreatic cancer.

BACKGROUND: Malignant ascites is common in metastatic pancreatic cancer (mPC) and its management still remains a clinical challenge. Early identification of patients at risk for ascites development may support and guide treatment decisions. MATERIALS AND METHODS: Data of patients treated for mPC at the Medical University of Vienna between 2010 and 2019 were collected by retrospective chart review. Ascites was defined as clinically relevant accumulation of intraperitoneal fluid diagnosed by ultrasound or computer tomography scan of the abdomen. We investigated the association between general risk factors, metastatic sites, liver function, systemic inflammation as well as portal vein obstruction (PVO) and ascites development. RESULTS: Among 581 patients with mPC included in this study, 122 (21.0%) developed ascites after a median of 8.7 months after diagnosis of metastatic disease. The occurrence of ascites led to an 8.9-fold increased risk of death [confidence interval (CI) 7.2-11, P < 0.001] with a median overall survival of 1 month thereafter. Clinical risk factors for ascites were male sex [hazard ratio (HR) 1.71, CI 1.00-2.90, P = 0.048], peritoneal carcinomatosis (HR 6.79, CI 4.09-11.3, P < 0.001), liver metastases (HR 2.16, CI 1.19-3.91, P = 0.011), an albumin-bilirubin (ALBI) score grade 3 (HR 6.79, CI 2.11-21.8, P = 0.001), PVO (HR 2.28, CI 1.15-4.52, P = 0.019), and an elevated C-reactive protein (CRP) (HR 4.19, CI 1.58-11.1, P = 0.004). CONCLUSIONS: Survival after diagnosis of ascites is very limited in mPC patients. Male sex, liver and peritoneal metastases, impaired liver function, PVO, as well as systemic inflammation were identified as independent risk factors for ascites development in this uniquely large real-life patient cohort.

Bakuchiol, an antibacterial component of Psoralidium tenuiflorum.

Bioactivity guided fractionation of an EtOAc extract from Psoralidium tenuiflorum (Pursh) Rydb. (family : Fabaceae) yielded the known compound bakuchiol as a racemic mixture. This compound was purified by liquid-liquid partitioning and chromatography followed by NMR and GCMS characterisation. This compound exhibited Staphylococcus epidermidis cytotoxicity. COSY and HETCOR spectra of the isolated bakuchiol indicated two disagreements with previously reported NMR structural assignments; reassignments are described herein. Long-term storage of bakuchiol resulted in degradation to 4-hydroxybenzaldehyde, which was purified and characterised.

High throughput crystallography of TB drug targets.

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.

The treatment of spasticity with Delta9-tetrahydrocannabinol in persons with spinal cord injury.

STUDY DESIGN: Open label study to determine drug dose for a randomized double-blind placebo-controlled parallel study. OBJECTIVES: To assess the efficacy and side effects of oral Delta(9)-tetrahydrocannabinol (THC) and rectal THC-hemisuccinate (THC-HS) in SCI patients. SETTING: REHAB Basel, Switzerland. METHOD: Twenty-five patients with SCI were included in this three-phase study with individual dose adjustment, each consisting of 6 weeks. Twenty-two participants received oral THC open label starting with a single dose of 10 mg (Phase 1, completed by 15 patients). Eight subjects received rectal THC-HS (Phase 2, completed by seven patients). In Phase 3, six patients were treated with oral THC and seven with placebo. Major outcome parameters were the spasticity sum score (SSS) using the Modified Ashworth Scale (MAS) and self-ratings of spasticity. RESULTS: Mean daily doses were 31 mg with THC and 43 mg with THC-HS. Mean SSS for THC decreased significantly from 16.72 (+/-7.60) at baseline to 8.92 (+/-7.14) on day 43. Similar improvement was seen with THC-HS. We observed a significant improvement of SSS with active drug (P=0.001) in the seven subjects who received oral THC in Phase 1 and placebo in Phase 3. Major reasons for drop out were increase of pain and psychological side effects. CONCLUSION: THC is an effective and safe drug in the treatment of spasticity. At least 15-20 mg per day were needed to achieve a therapeutic effect.

Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis.

Histone acetylation affects many nuclear processes including transcription, chromatin assembly, and DNA damage repair. Acetylation of histone H3 lysine 56 (H3 K56ac) in budding yeast occurs during mitotic S phase and persists during DNA damage repair. Here, we show that H3 K56ac is also present during premeiotic S phase and is conserved in fission yeast. Furthermore, the H3 K56ac modification is not observed in the absence of the histone chaperone Asf1. asf1delta and H3 K56R mutants exhibit similar sensitivity to DNA damaging agents. Mutational analysis of Asf1 demonstrates that DNA damage sensitivity correlates with (i) decreased levels of H3 K56ac and (ii) a region implicated in histone binding. In contrast, multiple asf1 mutants that are resistant to DNA damage display WT levels of K56ac. These data suggest that maintenance of H3 K56 acetylation is a primary contribution of Asf1 to genome stability in yeast.

Recent advances in understanding structure-function relationships in the type II topoisomerase mechanism.

DNA topos (topoisomerases) are complex, multisubunit enzymes that remodel DNA topology. Members of the type II topo family function by passing one segment of duplex DNA through a transient break in another, a process that consumes two molecules of ATP and requires the co-ordinated action of multiple domains. Recent structural data on type II topo ATPase regions, which activate and enforce the directionality of DNA strand passage, have highlighted how ATP physically controls the catalytic cycle of the enzyme. Structural and biochemical studies of specialized DNA-binding domains in two paralogous bacterial type IIA topos (DNA gyrase and topo IV) show how these enzymes selectively negatively supercoil or decatenate DNA. Taken together, these findings expand our understanding of how disparate functional elements work together to co-ordinate the type II topo mechanism.

Two bioactive saponins from Albizia subdimidiata from the Suriname rainforest.

Bioassay-guided fractionation of a methanol extract of Albizia subdimidiata using the engineered yeast strains 1138, 1140, 1353, and Sc7 of Saccharomyces cerevisiae as the bioassay tool resulted in the isolation of the two active saponins 1 and 2; one of these, albiziatrioside A (1), is described for the first time. The structures of 1 and 2 were established on the basis of HRMS, 1D and 2D NMR spectral data, and GC--MS analysis of the sugar units. Both isolated compounds showed significant cytotoxicity against the A2780 cell line.

Isolation, synthesis, and structure-activity relationships of bioactive benzoquinones from Miconia lepidota from the Suriname rainforest.

Bioactivity-directed fractionation of an EtOAc extract from the leaves of Miconia lepidota afforded the two benzoquinones 2-methoxy-6-heptyl-1,4-benzoquinone (1) and 2-methoxy-6-pentyl-1,4-benzoquinone (primin) (2). This is the first reported isolation of 1. Both quinones 1 and 2 exhibited activity toward mutant yeast strains based on Saccharomyces cerevisiae, indicative of their cytotoxicity and potential anticancer activity. A number of previously synthesized and new analogues were prepared and tested in the same strains. Compounds 1, 2, 2-methoxy-6-butyl-1,4-benzoquinone (5), and 2-methoxy-6-decyl-1,4-benzoquinone (6) were tested in two cytotoxicity assays. In the M109 tumor cell lines, quinones 1, 2, and 6 had an IC(50) value of 10 microg/mL. In the A2780 cell line, compounds 1, 2 and 5 had IC(50) values of 7.9, 2.9, and 3.2 microg/mL, respectively.

Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI.

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.

Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control.

Cdc6/Cdc18 is a conserved and essential component of prereplication complexes. The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe, reveals novel aspects of Cdc6/Cdc18 function. Two domains of Cdc6 form an AAA+-type nucleotide binding fold that is observed bound to Mg.ADP. A third domain adopts a winged-helix fold similar to known DNA binding modules. Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo. Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel.

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

DNA replication at high resolution.

Several decades of research have delineated the roles of many proteins central to DNA replication. Here we present a structural perspective of this work spanning the past 15 years and highlight several recent advances in the field.

Structure of the RNA polymerase domain of E. coli primase.

All cellular organisms use specialized RNA polymerases called "primases" to synthesize RNA primers for the initiation of DNA replication. The high-resolution crystal structure of a primase, comprising the catalytic core of the Escherichia coli DnaG protein, was determined. The core structure contains an active-site architecture that is unrelated to other DNA or RNA polymerase palm folds, but is instead related to the "toprim" fold. On the basis of the structure, it is likely that DnaG binds nucleic acid in a groove clustered with invariant residues and that DnaG is positioned within the replisome to accept single-stranded DNA directly from the replicative helicase.

Ketamine-fentanyl-midazolam infusion for the control of symptoms in terminal life care.

In this report, we describe nine terminally ill patients with metastatic cancer who were treated with an intravenous infusion consisting of ketamine (2 mg/ml)/fentanyl (5 micrograms/ml)/midazolam (0.1 mg/ml) (K/F/M) to control pain after traditional analgesic therapies were unsuccessful. In addition to pain, all patients exhibited some symptoms of cognitive compromise and agitation. After initiation of the K/F/M infusion, all patients exhibited some degree of qualitative improvement in these symptoms as well as in overall pain control. We feel that these observations warrant reporting of the efficacy of this infusion for the treatment of uncontrolled pain and agitation in terminally ill patients when the traditional methods of pain control are inadequate.

Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

Enzymes that push DNA around.

DNA topoisomerases are proteins that regulate DNA topology in cells through selective cycles of DNA cleavage, manipulation, and religation. Two papers describe an ensemble of different protein conformations and nucleotide-protein complexes of Escherichia coli topoisomerase. These results lead to new insights about how this enzyme recognizes DNA and catalyzes supercoil relaxation.

Crystal structure of the N-terminal domain of the DnaB hexameric helicase.

BACKGROUND: The hexameric helicase DnaB unwinds the DNA duplex at the Escherichia coli chromosome replication fork. Although the mechanism by which DnaB both couples ATP hydrolysis to translocation along DNA and denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerization of the N-terminal domain has been observed and may occur during the enzymatic cycle. This N-terminal domain is required both for interaction with other proteins in the primosome and for DnaB helicase activity. Knowledge of the structure of this domain may contribute to an understanding of its role in DnaB function. RESULTS: We have determined the structure of the N-terminal domain of DnaB crystallographically. The structure is globular, highly helical and lacks a close structural relative in the database of known protein folds. Conserved residues and sites of dominant-negative mutations have structurally significant roles. Each asymmetric unit in the crystal contains two independent and identical copies of a dimer of the DnaB N-terminal domain. CONCLUSIONS: The large-scale domain or subunit reorientation that is seen in DnaB by electron microscopy might result from the formation of a true twofold symmetric dimer of N-terminal domains, while maintaining a head-to-tail arrangement of C-terminal domains. The N-terminal domain of DnaB is the first region of a hexameric DNA replicative helicase to be visualized at high resolution. Comparison of this structure to the analogous region of the Rho RNA/DNA helicase indicates that the N-terminal domains of these hexameric helicases are structurally dissimilar.

The structural basis for terminator recognition by the Rho transcription termination factor.

The E. coli Rho protein disengages newly transcribed RNA from its DNA template, helping terminate certain transcripts. We have determined the X-ray crystal structure of the RNA-binding domain of Rho complexed to an RNA ligand. Filters that screen both ligand size and chemical functionality line the primary nucleic acid-binding site, imparting sequence specificity to a generic single-stranded nucleic acid-binding fold and explaining the preference of Rho for cytosine-rich RNA. The crystal packing reveals two Rho domain protomers bound to a single RNA with a single base spacer, suggesting that the strong RNA-binding sites of Rho may arise from pairing of RNA-binding modules. Dimerization of symmetric subunits on an asymmetric ligand is developed as a model for allosteric control in the action of the intact Rho hexamer.