Application of Artificial Intelligence/Machine Vision & Learning for the Development of a Live Single-cell Phenotypic Biomarker Test to Predict Prostate Cancer Tumor Aggressiveness

To assess the usefulness and applications of machine vision (MV) and machine learning (ML) techniques that have been used to develop a single cell-based phenotypic (live and fixed biomarkers) platform that correlates with tumor biological aggressiveness and risk stratification, 100 fresh prostate samples were acquired, and areas of prostate cancer were determined by post-surgery pathology reports logged by an independent pathologist. The prostate samples were dissociated into single-cell suspensions in the presence of an extracellular matrix formulation. These samples were analyzed via live-cell microscopy. Dynamic and fixed phenotypic biomarkers per cell were quantified using objective MV software and ML algorithms. The predictive nature of the ML algorithms was developed in two stages. First, random forest (RF) algorithms were developed using 70% of the samples. The developed algorithms were then tested for their predictive performance using the blinded test dataset that contained 30% of the samples in the second stage. Based on the ROC (receiver operating characteristic) curve analysis, thresholds were set to maximize both sensitivity and specificity. We determined the sensitivity and specificity of the assay by comparing the algorithm-generated predictions with adverse pathologic features in the radical prostatectomy (RP) specimens. Using MV and ML algorithms, the biomarkers predictive of adverse pathology at RP were ranked and a prostate cancer patient risk stratification test was developed that distinguishes patients based on surgical adverse pathology features. The ability to identify and track large numbers of individual cells over the length of the microscopy experimental monitoring cycles, in an automated way, created a large biomarker dataset of primary biomarkers. This biomarker dataset was then interrogated with ML algorithms used to correlate with post-surgical adverse pathology findings. Algorithms were generated that predicted adverse pathology with >0.85 sensitivity and specificity and an AUC (area under the curve) of >0.85. Phenotypic biomarkers provide cellular and molecular details that are informative for predicting post-surgical adverse pathologies when considering tumor biopsy samples. Artificial intelligence ML-based approaches for cancer risk stratification are emerging as important and powerful tools to compliment current measures of risk stratification. These techniques have capabilities to address tumor heterogeneity and the molecular complexity of prostate cancer. Specifically, the phenotypic test is a novel example of leveraging biomarkers and advances in MV and ML for developing a powerful prognostic and risk-stratification tool for prostate cancer patients.     

Quirks of dye nomenclature. 3. Trypan blue

Trypan blue is colorant from the 19^th century that has an association with Africa as a chemotherapeutic agent against protozoan (Trypanosomal) infections, which cause sleeping sickness. The dye still is used for staining biopsies, living cells and organisms, and it also has been used as a colorant for textiles.     

Prediction and course of symptoms and lung function around an exacerbation in chronic obstructive pulmonary disease

Background

Frequent exacerbations induce a high burden to Chronic Obstructive Pulmonary Disease (COPD). We investigated the course of exacerbations in the published COSMIC study that investigated the effects of 1-year withdrawal of fluticasone after a 3-month run-in treatment period with salmeterol/fluticasone in patients with COPD.

Methods

In 373 patients, we evaluated diary cards for symptoms, Peak Expiratory Flow (PEF), and salbutamol use and assessed their course during exacerbations.

Results

There were 492 exacerbations in 224 patients. The level of symptoms of cough, sputum, dyspnea and nocturnal awakening steadily increased from 2 weeks prior to exacerbation, with a sharp rise during the last week. Symptoms of cough, sputum, and dyspnea reverted to baseline values at different rates (after 4, 4, and 7 weeks respectively), whereas symptoms of nocturnal awakening were still increased after eight weeks. The course of symptoms was similar around a first and second exacerbation. Increases in symptoms and salbutamol use and decreases in PEF were associated with a higher risk to develop an exacerbation, but with moderate predictive values, the areas under the receiver operating curves ranging from 0.63 to 0.70.

Conclusions

Exacerbations of COPD are associated with increased symptoms that persist for weeks and the course is very similar between a first and second exacerbation. COPD exacerbations are preceded by increased symptoms and salbutamol use and lower PEF, yet predictive values are too low to warrant daily use in clinical practice.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA.

The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that gamma complex hydrolyzes two ATP molecules sequentially when placing beta around DNA. The first ATP is hydrolyzed fast, at 25-30 s(-1), while the second ATP hydrolysis is limited to the steady-state rate of 2 s(-1). This step-wise reaction depends on both primed DNA and beta. DNA alone promotes rapid hydrolysis of two ATP molecules, while beta alone permits hydrolysis of only one ATP. These results suggest that beta inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of beta from the gamma complex. This implies that DNA-dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp-assembly pathway. We also discuss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.     

A Cytogenetic Analysis of the Punch-Tudor Region of Chromosome 2r in Drosophila Melanogaster

Eleven chromosomal deficiencies and several rearrangements in the Pu-tud region of chromosome 2R have been generated and examined cytologically. The Pu locus has been localized to chromosome bands 57C5-6 and tud to 57C7-8. Mutagenesis within the region defined by the deletion intervals has resulted in the isolation of 92 new lethal mutations. Seventy-six of these mutations have been separated into 16 complementation groups that have been ordered and placed cytologically by deletion mapping. All new alleles fully complement tud for both lethal and grandchildless phenotypes. The largest number of new mutations, a total of 25, are Pu alleles.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Chemical Selection of Mutants That Affect Alcohol Dehydrogenase in Drosophila II. Use of 1–PENTYNE–3–Ol

We describe a procedure for the selection of alcohol dehydrogenase negative mutants in Drosophila. The method consists of exposing eggs and larvae to low concentrations of 1-pentyne-3-ol dissolved in the culture medium. Only those flies with greatly reduced levels of alcohol dehydrogenase activity survive. In addition, genotypically negative flies die if their mothers are alcohol dehydrogenase positive. Using this procedure and formaldehyde to generate mutants, we were able to detect seven alcohol dehydrogenase negative mutants out of 350,000 individuals subjected to selection. At least five of the mutants contain small deletions that include the alcohol dehydrogenase locus.     

Studies on an extract of rat testicular microsomal fraction that catalyses the transformation of progesterone into 17-hydroxyprogesterone and androgens

The supernatant obtained by centrifugation of Triton N-101-treated freeze-dried rat testicular microsomal fraction at 105000g(av.) for 2h transformed progesterone into testosterone via 17-hydroxypregn-4-ene-3,20-dione and androst-4-ene-3,17-dione. Hydroxylation at C-17 of 3beta-hydroxypregn-5-en-20-one and deoxycorticosterone was not observed. Non-haem iron protein, cytochrome P-450 and material with NADPH dehydrogenase activity were precipitated by 40% saturation of the supernatant with ammonium sulphate; however, it was not possible to establish the participation of these substances in the 17alpha-hydroxylase and side-chain-cleavage activities also present in the precipitate. The results of gel-filtration chromatography indicated that the Triton N-101 extract consisted primarily of a suspension of small particles of microsomes and that the progesterone 17-hydroxylase and the 17-hydroxypregn-4-ene-3,20-dione side-chain-cleavage enzyme were not in true solution.