Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-ribosomal Peptide Synthetase Module

Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on themultifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique nonribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues

CD28 expression in sentinel node biopsies from breast cancer patients in comparison with CD3-ζ chain expression

BACKGROUND: Immunosuppression is documented in several malignant diseases, including breast cancer. Subsequently, future therapeutic concepts might include immunological approaches. However, detailed knowledge about tumor immunogenicity and host immunoreactivity, and how to assess these adequately, is still limited. We studied CD28 and CD3-ζ expression in sentinel node biopsies (SNB) from breast cancer patients to analyze tumor-related changes in T cell activity. METHOD: 25 women underwent surgery for primary breast cancer, including SNB. Frozen sections from 21 sentinel nodes could be analyzed with a double-staining technique. CD28 expression was studied in CD4+ and CD8+ T-lymphocyte subsets and compared with CD3-ζ expression in three specified nodal regions. RESULTS: The degree of CD28 expression varied between the different lymph node areas. The lowest degree of CD28 expression was observed in CD4+ T-lymphocytes in the paracortex and germinal centers. Here, a good agreement with CD3-ζ expression was found. A higher CD28 expression was noted in CD4+ T-cells in the primary follicles, where concordance with CD3-ζ expression was weaker. The CD8+ T-lymphocyte subset displayed generally a higher degree of CD28 expression than the CD4+ subset. CONCLUSION: Sentinel lymph nodes from breast cancer patients displayed local immunosuppression of varying extent. In the areas with the lowest degree of CD28 expression an accordingly low CD3-ζ expression was found. The SNB might prove an important diagnostic tool for the evaluation of interactions between tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy

Response of the Esophageal Epithelium to Concomitant Cis-Dichlorodiammineplatinum(II) and Radiation Treatment. An Electron Microscopic Study in Rabbits

The rabbit esophageal mucosa was irradiated with daily fractions of 2 Gy up to an accumulated dose of 20 Gy (total dose 2, 6, 10. 16 or 20 Gy). Fifteen to forty-five minutes before the start of each irradiation 0.3 mg Cis-dichlorodiammineplatinum (cis-DDP, cisplatinum) was given by intraperitoneal injection to each rabbit. Examinations were carried out 1-10 days after each fractionation schedule, when specimens were taken for morphological investigations. Scanning electron microscope (SEM) examination showed a gradual development of damage with cell loss and structural disarrangement of the microridges and whorls on the surface. However, with further treatment the esophageal mucosa exposed to cis-DDP and radiation normalized faster and more complete compared to the esophageal part exposed to cis-DDP alone. The difference may depend on an accelerated proliferation in the part of the trachea that is exposed to a combined treatment

The Response of the Tracheal Epithelium to Concomitant Cis-Diamminedichloroplatinum (II) and Radiation. An Electron Microscopic Study in Rabbits

The ciliated epithelium of the rabbit trachea was irradiated with daily fractions of 2 Gy up to an accumulated dose of 20 Gy (total dose: 2, 6, 10, 16, or 20 Gy). Fifteen to forty-five minutes before the start of each irradiation 0.3 mg Cis-diamminedichloroplatinum (cis-DDP) was given by intraperitoneal injection to each rabbit. Examinations were carried out 1-10 days after each fractionation schedule, when specimens were taken for morphological investigations. Scanning electron microscope (SEM) examination showed a gradual development of ciliary damage, from blebs on the cilia to swollen tips, broken and bent cilia and finally an epithelial injury with areas free from cilia, and a surface covered with microvilli-like structures. SEM also showed cell loss, and remnants of dead cells on the surface together with detritus. By transmission electron microscopy (TEM), ciliary damage, cell death and cell loss of the ciliated cell layer, as well as exfoliation of portions of goblet-like cells on the surface, could be confirmed. Scoring of SEM and TEM micro graphs showed that for the tracheal part treated with cis-DDP and radiation, the maximal damage was expressed in the dose group 10 Gy, and above this no further increase in the average reaction occurred. For the part of the trachea only exposed to cis-DDP, the damage increased with the dose. The difference observed speaks for an accelerated proliferation exerted by the radiation

The Effect of Ionizing Irradiation on Type I Collagen of the Tail in Growing Mice: A Histology and Electron Microscopy Study

In order to examine the effect of radiation on growing tissue, especially the fibroblasts and their end-product, the collagen fibres, tails from 24 mice were irradiated at an age of 8 days with 20 Gy and 30 Gy (\u3e°Co). Tails from 18 animals served as controls. Six mice from each group were sacrificed on day 8, 20 and 30. Transmission electron microscopy was used to examine the fibroblasts and the collagen fibrils. Non-irradiated fibroblasts had a nucleus rich in chromatin and an abundant endoplasmic reticulum with cistemae and condensing vacuoles. On day 20, approximately 50 % , and on day 30, 25 % of the fibroblasts irradiated with 30 Gy had a sparse endoplasmic reticulum pointing to a reduction of protein synthesis. While, on day 20, the fibrils irradiated with 20 Gy and with 30 Gy had significantly larger diameters compared to the controls, on day 30, the irradiated fibrils had a notably smaller diameter compared to the controls; 30 Gy-fibrils were larger than the 20 Gy-fibrils on both days. On day 20, the binding mean value of the 30 Gy-fibrils exceeded that of the controls and was significantly higher than that of the 20 Gy-fibrils, which was lower, though not significantly, than the controls. On day 30, the banding mean value of the 30 Gy-fibrils was notably lower than the control; and the value of the 20 Gy-fibrils was significantly lower than that of the 30 Gy-fibrils. The results are explained as an edema together with an inhibitory effect on the protein synthesis of the fibroblasts caused by the irradiation. This deduction 1s further supported by light microscopy of the tails

Scanning Electron Microscopy of Human Esophageal Mucosa in Patients with Carcinoma of the Esophagus

Specimens taken at surgery from 15 patients with carcinoma of the esophagus were examined with scanning electron microscopy. Nine patients were treated with chemotherapy (cisplatin + 5-fluorouracil), surgery and radiotherapy; one received preoperative radiotherapy only; and the remaining five primary surgery only. Scanning electron microscopy was performed on specimens of both tumor tissue and the mucosa at least 5 cm from the tumor. In adjacent non-tumor tissue, damage due to treatment was observed in the form of changes in microridges and increased cell loss. In tumor tissue, the degree of damage was correlated to tumor response to treatment. For patients with no residual tumor after treatment, the ultrastructure was normalized with a low tumor score, while for patients with residual tumor, the score was high

Collisional Penrose Process near the Horizon of Extreme Kerr Black Holes

Collisions of particles in black hole ergospheres may result in an arbitrarily large center-of-mass energy. This led recently to the suggestion [M. Banados, J. Silk, and S. M. West, Phys. Rev. Lett. 103, 111102 (2009)] that black holes can act as ultimate particle accelerators. If the energy of an outgoing particle is larger than the total energy of the infalling particles, the energy excess must come from the rotational energy of the black hole and hence, a Penrose process is involved. However, while the center-of-mass energy diverges, the position of the collision makes it impossible for energetic particles to escape to infinity. Following an earlier work on collisional Penrose processes [T. Piran and J. Shaham, Phys. Rev. D 16, 1615 (1977)], we show that even under the most favorable idealized conditions the maximal energy of an escaping particle is only a modest factor above the total initial energy of the colliding particles. This implies that one should not expect collisions around a black hole to act as spectacular cosmic accelerators

Vaccines : A rapidly evolving technology - Are the hurdles being addressed?

AbstractVaccination usually works in infectious disease, why not in Cancer? Differences in the potency of microbial and cancer antigens, poor initiation of an immune response due to inadequate expression of tumour associated antigens, weak antigens or tolerance induction and local immune suppression were considered. There is a big difference between a therapeutic and a prophylactic vaccine.The opinion of the expert group was that an improved therapeutic efficacy can hardly be expected by further variation of types of vaccines, schedules, routes of administration and adjuvants alone. A major hurdle for developing therapeutic cancer vaccines is the need to effectively monitor the immune response and to be able to use this in an adaptive trial approach.End-points of assessment should be different from standard treatments as complete response or partial responses are usually low, unless combined with other therapies.In order to focus resources to overcome the hurdles of enhancing the therapeutic efficacy of cancer vaccines the Cancer Vaccine Clinical Trial Working Group, representing academia and the pharmaceutical and biotechnology industries has in a consensus process defined 'A clinical development paradigm for cancer vaccines and related biologics'