A framework for clinical cancer subtyping from nucleosome profiling of cell-free DNA.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.

Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aß) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aß is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aß-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale. METHODS: We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aß or LPS and INFγ. RESULTS: We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aß does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aß formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain. CONCLUSIONS: These results suggest that synthetic Aß treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.

Mutation and selection in a large population.

In this paper we study a large, but finite population, in which mutation and selection occur at a single genetic locus in a diploid organism. We provide theoretical results for the equilibrium allele frequencies, their variances and covariances and their equilibrium distribution, when the population size is larger than the reciprocal of the mean allelic mutation rate. We are also able to infer that the equilibrium distribution of allele frequencies takes the form of a constrained multivariate Gaussian distribution. Our results provide a rapid way of obtaining useful information in the case of complex mutation and selection schemes when the population size is large. We present numerical simulations to test the applicability of our theoretical formulations. The results of these simulations are in very reasonable agreement with the theoretical predictions.

Adaptive combinatorial design to explore large experimental spaces: approach and validation.

Systems biology requires mathematical tools not only to analyse large genomic datasets, but also to explore large experimental spaces in a systematic yet economical way. We demonstrate that two-factor combinatorial design (CD), shown to be useful in software testing, can be used to design a small set of experiments that would allow biologists to explore larger experimental spaces. Further, the results of an initial set of experiments can be used to seed further 'Adaptive' CD experimental designs. As a proof of principle, we demonstrate the usefulness of this Adaptive CD approach by analysing data from the effects of six binary inputs on the regulation of genes in the N-assimilation pathway of Arabidopsis. This CD approach identified the more important regulatory signals previously discovered by traditional experiments using far fewer experiments, and also identified examples of input interactions previously unknown. Tests using simulated data show that Adaptive CD suffers from fewer false positives than traditional experimental designs in determining decisive inputs, and succeeds far more often than traditional or random experimental designs in determining when genes are regulated by input interactions. We conclude that Adaptive CD offers an economical framework for discovering dominant inputs and interactions that affect different aspects of genomic outputs and organismal responses.

Role of zymogen and activated factor X as scaffolds for the inhibition of the blood coagulation factor VIIa-tissue factor complex by recombinant nematode anticoagulant protein c2.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.

Kinetic studies of the branched chain amino acid preferring peptidase activity of the 20S proteasome: development of a continuous assay and inhibition by tripeptide aldehydes and clasto-lactacystin beta-lactone.

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.

Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells.

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

The rising prevalence of HIV-1 infection in patients attending an inner city accident and emergency department.

The recently published findings of the unlinked anonymous HIV prevalence study in England and Wales showed unchanging HIV prevalence in groups such as homo/bisexual men, and declining rates in non-injecting heterosexual men attending genitourinary medicine clinics. However, this multicentre study did detect a significant rise in seroprevalence rates in pregnant women in England and Wales and sentinel groups within hospitals in London, warning that changing patterns of HIV infection might account for these variable results. In 1992-1993 a seroprevalence study of adult patients attending the accident and emergency department at St. Mary's Hospital in West Central London showed a rate of HIV-1 infection of 1 in 77. We have repeated the seroprevalence study over the same calendar months in 1994-1995 to gain further information about HIV positive patients attending the department and to see whether a change in the patterns of HIV infection in the population served by St Mary's Hospital had occurred.

Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone.

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.

Comparison of metabolic responses to laparoscopic and minilaparotomy cholecystectomy.

This randomized study compared the metabolic responses to laparoscopic cholecystectomy (n = 10) and minilaparotomy cholecystectomy with a 5-7-cm incision (n = 10). Venous blood samples were taken before operation and at 3, 6, 9, 12, 18, 24, 48, 72 and 168 h after incision and analysed for levels of C-reactive protein, interleukin 6, cortisol, albumin, transferrin, iron, fibrinogen, fibrin degradation products and polymorphonuclear elastase, and for neutrophil and lymphocyte counts. Urine samples (24 h) were analysed for urea, creatinine, 3-methylhistidine and catecholamines. The magnitude of the metabolic changes from baseline levels was quantified by calculating areas under each individual curve. A significant metabolic response with a similar time course and magnitude of changes occurred after laparoscopic and minilaparotomy cholecystectomy but with wide variation in magnitude between individuals.

Continuous coeliac plexus blockade plus intermittent wound infiltration with bupivacaine following upper abdominal surgery: a double-blind randomised study.

In this double-blind trial, we observed the effect of intermittent wound infiltration with local anaesthetic plus continuous coeliac plexus blockade on postoperative pain relief, pulmonary function, the neuroendocrine and acute phase protein response following upper abdominal surgery. In Group A (n = 10) patients received bupivacaine intermittently into the wound and continuously into the coeliac plexus following an initial bolus. A total of 862.5 mg of bupivacaine was used over 12 h with no observed toxicity. Group B (n = 10) received equal volumes of saline. Although pain relief was poor in both groups, the bupivacaine group used less morphine postoperatively and had lower pain scores than the saline group 4 h after operation (P less than 0.05). Pulmonary function was significantly reduced in both groups with no statistical difference between the two. Significant reductions in serum glucose and cortisol were achieved (P less than 0.05), suggesting that afferent neural blockade was partially effective in attenuating the neuroendocrine response. However, the postoperative rise in interleukin-6 was not affected by this technique. It is concluded that total afferent neural blockade cannot be achieved with peripheral wound and coeliac plexus administration of relatively large doses of local anaesthetic during upper abdominal surgery.

Adenoid cystic carcinoma: the results of radical surgery.

A personal series of 267 salivary tumours seen in a 20-year period was reviewed. Thirty-six patients with a previously untreated histologically proven adenoid cystic carcinoma of the major and minor salivary glands were submitted to radical surgery. The 5-year survival was 73%; no patient died of disease or suffered a recurrence of disease beyond the 5-year mark, suggesting that radical surgery achieves good results and largely pre-empts the notorious pattern of repeated recurrence. Only 8% of the patients died solely of primary recurrence, suggesting that the place of supraradical surgery is likely to be very limited.