Rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells

Timely characterization of a cancer's evolution is required to predict treatment efficacy and to detect resistance early. High content analysis of single Circulating Tumor Cells (CTCs) enables sequential characterization of genotypic, morphometric and protein expression alterations in real time over the course of cancer treatment. This concept was investigated in a patient with castrate-resistant prostate cancer progressing through both chemotherapy and targeted therapy. In this case study, we integrate across four timepoints 41 genome-wide copy number variation (CNV) profiles plus morphometric parameters and androgen receptor (AR) protein levels. Remarkably, little change was observed in response to standard chemotherapy, evidenced by the fact that a unique clone (A), exhibiting highly rearranged CNV profiles and AR+ phenotype was found circulating before and after treatment. However, clinical response and subsequent progression after targeted therapy was associated with the drastic depletion of clone A, followed by the sequential emergence of two distinct CTC sub-populations that differed in both AR genotype and expression phenotype. While AR- cells with flat or pseudo-diploid CNV profiles (clone B) were identified at the time of response, a new tumor lineage of AR+ cells (clone C) with CNV altered profiles was detected during relapse. We showed that clone C, despite phylogenetically related to clone A, possessed a unique set of somatic CNV alterations, including MYC amplification, an event linked to hormone escape. Interesting, we showed that both clones acquired AR gene amplification by deploying different evolutionary paths. Overall, these data demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the multi-scale analysis of fluid biopsies to quantify and monitor disease evolution in individual patients

Multiplex Accurate Sensitive Quantitation (MASQ) With Application to Minimal Residual Disease in Acute Myeloid Leukemia

Measuring minimal residual disease in cancer has applications for prognosis, monitoring treatment and detection of recurrence. Simple sequence-based methods to detect nucleotide substitution variants have error rates (about 10-3) that limit sensitive detection. We developed and characterized the performance of MASQ (multiplex accurate sensitive quantitation), a method with an error rate below 10-6. MASQ counts variant templates accurately in the presence of millions of host genomes by using tags to identify each template and demanding consensus over multiple reads. Since the MASQ protocol multiplexes 50 target loci, we can both integrate signal from multiple variants and capture subclonal response to treatment. Compared to existing methods for variant detection, MASQ achieves an excellent combination of sensitivity, specificity and yield. We tested MASQ in a pilot study in acute myeloid leukemia (AML) patients who entered complete remission. We detect leukemic variants in the blood and bone marrow samples of all five patients, after induction therapy, at levels ranging from 10-2 to nearly 10-6. We observe evidence of sub-clonal structure and find higher target variant frequencies in patients who go on to relapse, demonstrating the potential for MASQ to quantify residual disease in AML

Biological properties of aerococci and bacilli as a component of new associate-probiotic complex

Dysbioses of the gastrointestinal tract are common among people of all ages and genders. Development of this pathology is associated with a number of complications, from indigestion to occurrence of malignant disease. Therefore, there is a need in development of measures of their prevention and correction. Probiotics are used as drugs against dysbiosis. Most of the presently known probiotics contain bacterial cells of one species, although combination preparations feature higher efficiency. At the same time, there are difficulties in construction of these drugs, primarily due to incompatibility of physiological properties of microorganisms and mutually antagonistic action of their components. The aim was to examine the compatibility of Bacillus subtilis and Aerococcus viridans in a single preparation, their antagonistic activity against different strains of test-cultures and general antagonism directed on different groups of bacteria for subsequent formation of associative probiotic complex. Properties of aerococci strains were studied and A. viridans 167 strain was selected for inclusion into the probiotic preparation. The tested strain showed the highest indicators of production of hydrogen peroxide, which is one of the mechanisms of antagonistic effect against opportunistic pathogens. General study of biological properties of aerococci strains showed that producing of hydrogen peroxide and superoxide radical in them was conditioned by functioning of NAD-independent lactatoxidase. It has been determined that antioxidant defense of aerococci from the action of endogenous and active excretable forms of oxygen was provided by activity of superoxide-dismutase and GSH-peroxidase. The method of deferred antagonism found no depressing mutual action between probiotic strains of B. subtilis 3 and A. viridans 167 at their joint cultivation. Inhibition of growth at the joint application of A. viridans 167 and B. subtilis 3 strains was recorded for both museum and clinical strains of test-cultures Escherichia coli, Proteus vulgaris, Klebsiella ozaenae, Citrobacter freundii, Pseudomonas aeruginosa, Staphylococcus aureus, S. epidermidis, Candida albicans. Separate application of A. viridans 167 or B. subtilis 3 against strains of these opportunistic pathogens was characterized by relatively less antagonistic effect of each of strains under study. The results allow us to recommend the studied strains of B. subtilis 3 and A. viridans 167 for use as the components to construct a new associative probiotic preparation

Pyogenic spinal infections warrant a total spine MRI

Study design: retrospective case series. Objective: the presenting clinical symptoms of spinal infections are often nonspecific and a delay in diagnosis can lead to adverse patient outcomes. The morbidity and mortality of patients with multifocal spinal infections is significantly higher compared to unifocal infections. The purpose of the current study was to analyse the risk factors for multifocal spinal infections. Methods: we conducted a retrospective review of all pyogenic non-tuberculous spinal infections treated surgically at a single tertiary care medical center from 2006–2020. The medical records, imaging studies, and laboratory data of 43 patients during this time period were reviewed and analysed after receiving Institutional Review Board approval. Univariate and multivariate analyses were performed to identify factors associated with a multifocal spinal infection. Results: 15 patients (35 %) had multifocal infections. In univariate analysis, there was a significant association with chronic kidney disease (p=0.040), gender (p=0.003), a white blood cell count (p=0.011), and cervical (p&lt;0.001) or thoracic (p&lt;0.001) involvement. In multivariate analysis, both cervical and thoracic involvement remained statistically significant (p=0.001 and p&lt;0.001, respectively). Conclusions: patients with infections in the thoracic or cervical region are more likely to have a multifocal infection. Multifocal pyogenic spinal infections remain a common entity and a total spine MRI should be performed to aid in prompt diagnosis.</p

Inter-laboratory reproducibility of fast gas chromatography–electron impact–time of flight mass spectrometry (GC–EI–TOF/MS) based plant metabolomics

The application of gas chromatography–mass spectrometry (GC–MS) to the ‘global’ analysis of metabolites in complex samples (i.e. metabolomics) has now become routine. The generation of these data-rich profiles demands new strategies in data mining and standardisation of experimental and reporting aspects across laboratories. As part of the META-PHOR project’s (METAbolomics for Plants Health and OutReach: http://www.meta-phor.eu/) priorities towards robust technology development, a GC–MS ring experiment based upon three complex matrices (melon, broccoli and rice) was launched. All sample preparation, data processing, multivariate analyses and comparisons of major metabolite features followed standardised protocols, identical models of GC (Agilent 6890N) and TOF/MS (Leco Pegasus III) were also employed. In addition comprehensive GC×GC–TOF/MS was compared with 1 dimensional GC–TOF/MS. Comparisons of the paired data from the various laboratories were made with a single data processing and analysis method providing an unbiased assessment of analytical method variants and inter-laboratory reproducibility. A range of processing and statistical methods were also assessed with a single exemplary dataset revealing near equal performance between them. Further investigations of long-term reproducibility are required, though the future generation of global and valid metabolomics databases offers much promise

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD +  dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate

LKR/SDH Plays Important Roles throughout the Tick Life Cycle Including a Long Starvation Period

BACKGROUND:Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS:We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE:Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods