Studies on the biological activities of actinomycins Z1 and Z5

The biological activities of actinomycins Z₁, Z₅, and IV (D) were compared. No consistent pattern was observed between the in vitro and in vivo results. Inhibition of E. coli DNA-dependent RNA polymerase in vitro followed the sequence IV > Z₁ > Z₅; however, for inhibition of RNA synthesis in B. subtilis and in HeLa cells the sequence was IV > Z₅ > Z₁ and the latter relationship was seen in the antimicrobial activity also. Physicochemical data did not agree with any of these findings. Difference spectra obtained with B. subtilis and M. lysodeikticus DNA followed the sequence Z₁ > IV > Z₅, while the relative thermal denaturation profiles were the exact opposite, Z₅ > IV > Z₁. These physicochemical criteria appear to be less reliable quantitative guides to biological activity. The relative ineffectiveness of actinomycin Z₁in vivo is probably the result of permeability differences associated with the presence of an hydroxylated proline residue (3-hydroxy-4-oxo-5-methylproline).     

C3 deposition in cholesterol-induced atherosclerosis in rabbits: a possible etiologic role for complement in atherogenesis.

Hypercholesterolemia was induced in rabbits by feeding Purina Chow supplemented with cholesterol (5 g/kg body weight/day). The serum cholesterol levels of these rabbits increased progressively and after 3 to 5 months were 4 to 9-fold greater than those of the control animals. Decrease in total hemolytic complement was not apparent during the feeding regimen. Morphologic examination of aortae of these hypercholesterolemic rabbits showed typical atherosclerotic intimal plaques. Immunofluorescent microscopy with fluorescein (F)-labeled anti-rabbit C3 showed deposition of C3 in the intimal and inner medial layers as early as 3 months on high cholesterol diet. C3 deposits were also observed in the renal glomeruli and in the walls of coronary arteries. However, fluorescent studies failed to demonstrate the presence of IgG, IgM, and C4 at these sites. Tissues from control animals fed normal diets were negative for immunoglobulins, C3, and C4. These results suggest that the complement system may be implicated in the pathogenesis of cholesterol-induced atherosclerosis in rabbits.     

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences ( KY468817 – KY468826 ) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).     

Synthetic biology advances and applications in the biotechnology industry: a perspective

Synthetic biology is a logical extension of what has been called recombinant DNA (rDNA) technology or genetic engineering since the 1970s. As rDNA technology has been the driver for the development of a thriving biotechnology industry today, starting with the commercialization of biosynthetic human insulin in the early 1980s, synthetic biology has the potential to take the industry to new heights in the coming years. Synthetic biology advances have been driven by dramatic cost reductions in DNA sequencing and DNA synthesis; by the development of sophisticated tools for genome editing, such as CRISPR/Cas9; and by advances in informatics, computational tools, and infrastructure to facilitate and scale analysis and design. Synthetic biology approaches have already been applied to the metabolic engineering of microorganisms for the production of industrially important chemicals and for the engineering of human cells to treat medical disorders. It also shows great promise to accelerate the discovery and development of novel secondary metabolites from microorganisms through traditional, engineered, and combinatorial biosynthesis. We anticipate that synthetic biology will continue to have broadening impacts on the biotechnology industry to address ongoing issues of human health, world food supply, renewable energy, and industrial chemicals and enzymes.     

Genetic contributions to precocity traits in racing Thoroughbreds

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high‐density SNP array data, additive SNP heritability () was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome‐wide association studies (GWAS) for age at first race and age at best race, a 1.98‐Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10^?12 and P = 1.89 × 10^?14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10^?5). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.     

Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1

The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To increase metabolic instability in plasma we synthesised esters described as specific substrates for paraoxonase and butyrylcholinesterases, esterases present in human plasma.     

Dysbiotic drift and biopsychosocial medicine: how the microbiome links personal,public and planetary health

The emerging concept of planetary health emphasizes that the health of human civilization is intricately connected to the health of natural systems within the Earth’s biosphere; here, we focus on the rapidly progressing microbiome science - the microbiota-mental health research in particular - as a way to illustrate the pathways by which exposure to biodiversity supports health. Microbiome science is illuminating the ways in which stress, socioeconomic disadvantage and social polices interact with lifestyle and behaviour to influence the micro and macro-level biodiversity that otherwise mediates health. Although the unfolding microbiome and mental health research is dominated by optimism in biomedical solutions (e.g. probiotics, prebiotics), we focus on the upstream psychosocial and ecological factors implicated in dysbiosis; we connect grand scale biodiversity in the external environment with differences in human-associated microbiota, and, by extension, differences in immune function and mental outlook. We argue that the success of planetary health as a new concept will be strengthened by a more sophisticated understanding of the ways in which individuals develop emotional connections to nature (nature relatedness) and the social policies and practices which facilitate or inhibit the pro-environmental values that otherwise support personal, public and planetary health.     

Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue

Background

There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material.

Methods

Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel? microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model.

Results

The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9–98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and???0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50?ng and 3.5?μg respectively was conservative.

Conclusion

The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

     

Urinary Thromboxane B2 in Cardiac Transplant Patients as a Screening Method of Rejection

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A₂ (TXA₂) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA₂ metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B₂ (i-TXB₂) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB₂ was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 ± 372 vs. 526 ± 57 pg/mL). The urine i-TXB₂ (704 ± 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB₂ were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 ± 11 vs. 39 ± 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB₂ is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB₂ may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.